Maria Grazia Roncarolo
George D. Smith Professor of Stem Cell and Regenerative Medicine, Emerita
Pediatrics - Stem Cell Transplantation
Bio
Maria Grazia Roncarolo, MD is the George D. Smith Professor in Stem Cell and Regenerative Medicine, Professor of Pediatrics and of Medicine, director of the Center for Definitive and Curative Medicine, and co-director of the Institute for Stem Cell Biology and Regenerative Medicine.
Dr. Roncarolo leads efforts to translate scientific discoveries in genetic diseases and regenerative medicine into novel patient therapies, including treatments based on stem cells and gene therapy.
A pediatric immunologist by training, she earned her medical degree at the University of Turin, Italy. She spent her early career in Lyon, France, where she focused on severe inherited metabolic and immune diseases, including severe combined immunodeficiency (SCID), better known as the "bubble boy disease." Dr. Roncarolo was a key member of the team that carried out the first stem cell transplants given before birth to treat these genetic diseases.
While studying inherited immune diseases, Dr. Roncarolo discovered a new class of T cells. These cells, called T regulatory type 1 cells, help maintain immune system homeostasis by preventing autoimmune diseases and assisting the immune system in tolerating transplanted cells and organs. Dr. Roncarolo completed the first clinical trial using T regulatory type 1 cells to prevent severe graft-versus-host disease in leukemia patients receiving blood-forming stem-cell transplants from donors who were not genetic matches.
Dr. Roncarolo worked for several years at DNAX Research Institute for Molecular and Cellular Biology in Palo Alto, where she contributed to the discovery of novel cytokines, cell-signaling molecules that are part of the immune response. She studied the role of cytokines in inducing immunological tolerance and in promoting stem cell growth and differentiation.
Dr. Roncarolo developed new gene-therapy approaches, which she pursued as director of the Telethon Institute for Cell and Gene Therapy at the San Raffaele Scientific Institute in Milan. She was the principal investigator leading the successful gene therapy trial for SCID patients who lack an enzyme critical to DNA synthesis, which is a severe life-threatening disorder. Based on the results of this trial, gene therapy for ADA-SCID has obtained Orphan drug status from both the FDA and EMEA and it was licensed to Glaxo Smith Klein, which has received European Commission approval to market under the name of Strimvelis. Under her direction, the San Raffaele Scientific Institute has been seminal in showing the efficacy of gene therapy for otherwise untreatable inherited metabolic diseases and primary immunodeficiencies.
Dr. Roncarolo established the Stanford Center for Definitive and Curative Medicine to cure patients with currently incurable diseases through the development of innovative stem cell-and gene-based therapies.
Academic Appointments
-
Honorary Faculty Emeritus, Pediatrics - Stem Cell Transplantation
-
Member, Bio-X
-
Member, Stanford Cancer Institute
Administrative Appointments
-
Professor, Departments of Pediatrics and Medicine, Stanford University (2014 - Present)
-
Director, Stanford Center for Definitive and Curative Medicine (CDCM) (2016 - 2022)
-
Co-Director, Institute for Stem Cell Biology and Regenerative Medicine, Stanford School of Medicine (2014 - 2022)
-
Division Chief, Pediatric Stem Cell Transplantation and Regenerative Medicine, Stanford School of Medicine (2014 - 2019)
-
Co-Director, Bass Center for Childhood Cancer and Blood Diseases, Lucile Packard Children's Hospital Stanford (2014 - 2019)
Honors & Awards
-
Outstanding Achievement Award, American Society of Gene & Cell Therapy (2017)
-
Knighthood "Commendatore dell'Ordine Al Merito della Repubblica Italiana", President of Italy (2014)
-
"Gold Apple" Prize for outstanding contribution to science, Marisa Bellisario Foundation (2013)
-
Elected Member, Austrian Academy of Sciences (2012)
-
Eurordis Scientific Award 2012 for outstanding contributions to the cure of genetic diseases, Eurordis (2012)
-
Outstanding Achievement Award for career and pioneering contributions to the field, European Society of Gene and Cell Therapy (2010)
-
Elected Member, Academia Europaea of Sciences (2005)
-
Nominated "Ufficiale dell'Ordine Al Merito della Repubblica Italiana", President of Italy (2000)
Boards, Advisory Committees, Professional Organizations
-
Member, Regulatory and Ethics Committee of European Society of Gene and Cell Therapy (ESGCT) (2006 - Present)
-
Member, Editorial Board for Current Gene Therapy (2006 - Present)
-
Charter Member, Eureka Institute for Translational Medicine (2008 - Present)
-
Member, European Group for the Bone and Marrow Transplantation (EBMT) Immunology Working Party (2008 - Present)
-
Member, Review Editorial Board of Frontiers in Immunological Tolerance (2010 - Present)
-
Member, Editorial Board for Molecular Therapy: Methods and Clinical Development (2013 - Present)
-
Member of the Editorial Board, Current Stem Cell Reports (2014 - Present)
-
Member, Scientific Advisory Board of BC Children's Hospital Research Institute (CHRI) (2014 - Present)
-
Member, Scientific Advisory Board of Spark Therapeutics (2015 - Present)
-
Co-Chair, Scientific Advisory Board of Glaxo Smith Kline Cell and Gene Therapy (CGT) (2016 - Present)
-
Member, Scientific Program Committee of the Federation of Clinical Immunology Societies (FOCIS) Meeting (2016 - Present)
-
Member, Editorial Board for Science Immunology (2016 - Present)
-
Member, External Immunology Board of Glaxo Smith Kline Immunology Network (2015 - 2016)
-
Member, Scientific Board of the Association “Festival della Scienza” (2013 - 2014)
-
Member, Scientific Advisory Board of the French Rare Diseases Foundation (2012 - 2014)
-
Member, Scientific Advisory Board of the Global Health Institute (GIH) Lausanne (2011 - 2014)
-
Member, Scientific Committee of the European Congress of Immunology (ECI) (2010 - 2012)
-
Member, Scientific Committee of the 2nd International Conference on Immune Tolerance (2010 - 2011)
-
Member, Organizing Committee of the Federation of Clinical Immunology Societies (FOCIS) Meeting (2010 - 2011)
-
Member, Nominating Committee of the American Society of Gene and Cell Therapy (ASGCT) (2010 - 2011)
-
Member, American Society of Hematology (ASH) Committee in Immunology and Host Defense (2009 - 2014)
-
Member, Scientific Program Committee of the International Congress of Immunology (ICI) (2009 - 2013)
-
Member, Organizing and Scientific Committees of the Federation of European Biochemical Societies (FEBS) (2008 - 2011)
-
Chair, Immunology of Gene Therapy Committee of the American Society of Gene and Cell Therapy (ASGCT) (2008 - 2009)
-
Member, Program Committee of the American Society of Gene and Cell Therapy (ASGCT) (2008 - 2009)
-
Member, Editorial Board for Italian Journal of Pediatrics (2007 - 2014)
-
Member, Editorial Board for Human Immunology (2007 - 2014)
-
Member, External Scientific Advisory Board of the Tumorzentrum L. Heilmeyer Comprehensive Cancer Center Freiburg (CCCF) (2007 - 2012)
-
Member, Membership Committee of the American Society of Gene and Cell Therapy (ASGCT) (2005 - 2011)
-
Member, Scientific Committee of the European School of Hematology (ESH) (2004 - 2014)
-
Member, Immunology of Gene Therapy Committee of the American Society of Gene and Cell Therapy (ASGCT) (2004 - 2010)
-
Member, Scientific Advisory Board of the University of Nantes's Institut de transplantation et de recherche en transplantation (ITERT) (2001 - 2009)
-
President, Genethon Scientific Advisory Board of the Association Française contre les Myopathies (AFM) (1999 - 2002)
-
Member, Scientific Advisory Board of Kinetix Pharmaceutical (1997 - 2000)
Professional Education
-
M.D., University of Turin, Italy, Medicine (1982)
-
Natl. Board, University of Turin, Italy, Pediatrics (1986)
-
Natl. Board, University of Milan, Italy, Clinical Immunology (1990)
Patents
-
Manuela Battaglia, Maria Grazia Roncarolo. "United States Patent 8562974 Method for expanding Cd4+ Cd25+ T regulator cells", Fondazione Telethon, Ospedale San Raffaele S.R.L., Oct 22, 2013
-
Manuela Battaglia, Maria-Grazia Roncarolo. "United States Patent 1869163 Method for expanding cd4+ cd25+ t regulatory cells", Fondazione Centro San Raffaele Del Monte Tabor, Fondazione Telethon, Sep 11, 2011
-
Frank Kolbinger, Herrera José M. Carballido, András Aszodi, José W. Saldanha, Bruce M. Hall, Silvia Gregori, Maria Grazia Roncarolo, Véronique Loux, Gregorio Aversa, Margit Jeschke. "United States Patent 1664122 Therapeutic humanised antibodies against cd45 isoforms", Novartis AG, Novartis Pharma GmbH, Mar 17, 2010
-
Manuela Battaglia, Maria-Grazia Roncarolo. "Australia Patent 2006217546 Method for expanding cd4+ cd25+ t regulatory cells", San Raffaele Centro Fond, Fond Telethon, Manuela Battaglia, Maria Grazia Roncarolo, Oct 29, 2009
-
Aszodi Andras, Aversa Gregorio, Carballido Herrera Jose M, Gregori Silvia, Hall Bruce M, Jeschke Margit, Kolbinger Frank, Loux Veronique, Roncarolo Maria Grazia, Saldanha Jose W. "Australia Patent 2004272289 Therapeutic binding molecules", Sep 18, 2008
-
Megan K. Levings, Rene De Waal Malefyt, Maria Grazia Roncarolo. "United States Patent 6,746,670 Regulatory T cells; methods", Schering Corporation, Jun 8, 2004
-
Maria-Grazia Roncarolo, Rene de Waal Malefyt, Rosa Bacchetta, Herve M. Groux, Jan E. de Vries. "United States Patent 6,277,635 Use of interleukin-10 to produce a population of suppressor cells", Schering Corporation, Aug 21, 2001
-
Maria-Grazia Roncarolo. "United States Patent 5,879,937 Cytokine-induced proliferation of amniotic t-cells", Schering Corporation, Mar 9, 1999
-
Maria-Grazia Roncarolo. "United States Patent 5,405,751 Prenatal diagnosis by cytokine induced proliferation of fetal T cells", Schering Corporation, Apr 11, 1995
Current Research and Scholarly Interests
Research Interests
Immunetolerance: Mechanisms underlying T-cell tolerance, induction of T-cell anergy and regulatory T cells; Immunomodulation: mAbs, proteins and low molecular weight compounds which can modulate T-cell activation; Primary immunodeficiencies: Characterization of molecular and immunological defects; Gene therapy: Gene transduction of hematopoietic cells for gene therapy in primary immunodeficiencies and metabolic diseases; Hematopoiesis: Mechanisms underlying growth and differentiation of hematopoietic stem cells; Transplantation: Immune reconstitution and T-cell tolerance after allogenic stem cell transplantation; Cytokines/Cytokine receptors: Role in regulation of immune and inflammatory responses
Clinical Interests
Primary Immunodeficiencies
Monogenic Autoimmune Disorders
Allogenic Bone Marrow Transplantation
Gene Therapy Clinical Trials
Cell Therapy Clinical Trials
Clinical Trials in Autoimmune Diseases and Organ Transplantation
Clinical Trials in Hemoglobinopathies
Clinical Trials
-
CD4^LVFOXP3 in Participants With IPEX
Recruiting
This first-in-human, Phase 1 clinical trial will test the feasibility of the manufacturing and the safety of the administration of CD4\^LVFOXP3 in up to 30 evaluable human participants with IPEX and evaluate the impact of the CD4\^LVFOXP3 infusion on the disease.
-
Stem Cell Transplant From Donors After Alpha Beta Cell Depletion in Children and Adults With T-allo10 Cells Addback
Recruiting
The purpose of this study is to determine the safety of a cell therapy, T-allo10, after αβdepleted-HSCT in the hopes that it will boost the adaptive immune reconstitution of the patient while sparing the risk of developing severe Graft-versus-Host Disease (GvHD). The primary objective of Phase 1a is to determine the recommended Phase 2 dose (RP2D) administered after infusion of αβdepleted-HSCT in children and young adults with hematologic malignancies. A Phase 1b extension will occur after dose escalation, enrolling at the RP2D for the T-allo10 cells determined in the Phase 1 portion to evaluate the safety and efficacy of infusion of T-allo10 after receipt of αβdepleted-HSCT. Additionally, Phase 1b aims to explore improvements in immune reconstitution. All participants on this study must be enrolled on another study: NCT04249830
2024-25 Courses
-
Independent Studies (15)
- Directed Reading in Immunology
IMMUNOL 299 (Aut, Win, Spr, Sum) - Directed Reading in Pediatrics
PEDS 299 (Aut, Win, Spr, Sum) - Directed Reading in Stem Cell Biology and Regenerative Medicine
STEMREM 299 (Aut, Win, Spr, Sum) - Early Clinical Experience
PEDS 280 (Aut, Win, Spr, Sum) - Early Clinical Experience in Immunology
IMMUNOL 280 (Aut, Win, Spr, Sum) - Graduate Research
IMMUNOL 399 (Aut, Win, Spr, Sum) - Graduate Research
PEDS 399 (Aut, Win, Spr, Sum) - Graduate Research
STEMREM 399 (Aut, Win, Spr, Sum) - Medical Scholars Research
PEDS 370 (Aut, Win, Spr, Sum) - Medical Scholars Research
STEMREM 370 (Aut, Win, Spr, Sum) - Out-of-Department Graduate Research
BIO 300X (Aut, Win, Spr) - Teaching in Immunology
IMMUNOL 290 (Aut, Win, Spr, Sum) - Undergraduate Directed Reading/Research
PEDS 199 (Aut, Win, Spr, Sum) - Undergraduate Research
IMMUNOL 199 (Aut, Win, Spr, Sum) - Undergraduate Research
STEMREM 199 (Aut, Win, Spr, Sum)
- Directed Reading in Immunology
All Publications
-
Identification of unstable regulatory and autoreactive effector T cells that are expanded in patients with FOXP3 mutations.
Science translational medicine
2023; 15 (727): eadg6822
Abstract
Studies of the monogenic autoimmune disease immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX) have elucidated the essential function of the transcription factor FOXP3 and thymic-derived regulatory T cells (Tregs) in controlling peripheral tolerance. However, the presence and the source of autoreactive T cells in IPEX remain undetermined. Here, we investigated how FOXP3 deficiency affects the T cell receptor (TCR) repertoire and Treg stability in vivo and compared T cell abnormalities in patients with IPEX with those in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED). To study Tregs independently of their phenotype and to analyze T cell autoreactivity, we combined Treg-specific demethylation region analyses, single-cell multiomic profiling, and bulk TCR sequencing. We found that patients with IPEX, unlike patients with APECED, have expanded autoreactive T cells originating from both autoreactive effector T cells (Teffs) and Tregs. In addition, a fraction of the expanded Tregs from patients with IPEX lost their phenotypic and functional markers, including CD25 and FOXP3. Functional experiments with CRISPR-Cas9-mediated FOXP3 knockout Tregs and Tregs from patients with IPEX indicated that the patients' Tregs gain a TH2-skewed Teff-like function, which is consistent with immune dysregulation observed in these patients. Analyses of FOXP3 mutation-carrier mothers and a patient with IPEX after hematopoietic stem cell transplantation indicated that Tregs expressing nonmutated FOXP3 prevent the accumulation of autoreactive Teffs and unstable Tregs. These findings could be directly used for diagnostic and prognostic purposes and for monitoring the effects of immunomodulatory treatments.
View details for DOI 10.1126/scitranslmed.adg6822
View details for PubMedID 38117899
-
A novel FOXP3 knockout-humanized mouse model for pre-clinical safety and efficacy evaluation of Treg-like cell products.
Molecular therapy. Methods & clinical development
2023; 31: 101150
Abstract
Forkhead box P3 (FOXP3) is an essential transcription factor for regulatory T cell (Treg) function. Defects in Tregs mediate many immune diseases including the monogenic autoimmune disease immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), which is caused by FOXP3 mutations. Treg cell products are a promising modality to induce allograft tolerance or reduce the use of immunosuppressive drugs to prevent rejection, as well as in the treatment of acquired autoimmune diseases. We have recently opened a phase I clinical trial for IPEX patients using autologous engineered Treg-like cells, CD4LVFOXP3. To facilitate the pre-clinical studies, a novel humanized-mouse (hu-mouse) model was developed whereby immune-deficient mice were transplanted with human hematopoietic stem progenitor cells (HSPCs) in which the FOXP3 gene was knocked out (FOXP3KO) using CRISPR-Cas9. Mice transplanted with FOXP3KO HSPCs had impaired survival, developed lymphoproliferation 10-12 weeks post-transplant and T cell infiltration of the gut, resembling human IPEX. Strikingly, injection of CD4LVFOXP3 into the FOXP3KO hu-mice restored in vivo regulatory functions, including control of lymphoproliferation and inhibition of T cell infiltration in the colon. This hu-mouse disease model can be reproducibly established and constitutes an ideal model to assess pre-clinical efficacy of human Treg cell investigational products.
View details for DOI 10.1016/j.omtm.2023.101150
View details for PubMedID 38027059
View details for PubMedCentralID PMC10679769
-
IPEX Syndrome from diagnosis to cure, learning along the way.
The Journal of allergy and clinical immunology
2023
Abstract
In the past two decades, a significant number of studies have been published describing the molecular and clinical aspects of Immune dysregulation Polyendocrinopathy Enteropathy X-linked Syndrome (IPEX). These studies have refined our knowledge of this rare yet prototypic genetic autoimmune disease, advancing the diagnosis, broadening the clinical spectrum, and improving our understanding of the underlying immunological mechanisms. Despite these advances, Forkhead box P3 (FOXP3) mutations have devastating consequences, and treating patients with IPEX remains a challenge even with safer strategies for hematopoietic stem cell transplantation, and gene therapy becoming a promising reality. The aim of this review is to highlight novel features of the disease to further advance awareness and improve the diagnosis and treatment of patients with IPEX Syndrome.
View details for DOI 10.1016/j.jaci.2023.11.021
View details for PubMedID 38040040
-
Radiation and Busulfan-Free Hematopoietic Stem Cell Transplantation Using Briquilimab (JSP191) Anti-CD117 Antibody-Conditioning, Transient Immunosuppression and TCR α β + T-Cell/CD19+B-Cell Depleted Haploidentical Grafts in Patients with Fanconi Anemia
AMER SOC HEMATOLOGY. 2023
View details for DOI 10.1182/blood-2023-178400
View details for Web of Science ID 001159306700230
-
T-ALLO10 INFUSION AFTER A.DEPLETED-HSCT IN CHILDREN AND YOUNG ADULTS WITH HEMATOLOGIC MALIGNANCIES: IMPROVED IMMUNE RECONSTITUTION IN THE ABSENCE OF SEVERE GVHD
SPRINGERNATURE. 2023: 232-234
View details for Web of Science ID 001110902800295
-
LENTIVIRAL-MEDIATED GENE THERAPY FOR PATIENTS WITH FANCONI ANEMIA [GROUP A]: UPDATED RESULTS FROM GLOBAL RP-L102 CLINICAL TRIALS
SPRINGERNATURE. 2023: 276-277
View details for Web of Science ID 001110902800355
-
LENTIVIRAL-MEDIATED GENE THERAPY FOR SEVERE PYRUVATE KINASE DEFICIENCY: RESULTS FROM AN ONGOING GLOBAL PHASE 1 STUDY
SPRINGERNATURE. 2023: 275-276
View details for Web of Science ID 001110902800354
-
LENTIVIRAL-MEDIATED GENE THERAPY FOR FANCONI ANEMIA [GROUP A]: RESULTS FROM RP-L102 CLINICAL TRIALS
WILEY. 2023: S136-S137
View details for Web of Science ID 001042987300270
-
LENTIVIRAL-MEDIATED GENE THERAPY FOR SEVERE PYRUVATE KINASE DEFICIENCY: GLOBAL PHASE 1 STUDY RESULTS
WILEY. 2023: S133-S134
View details for Web of Science ID 001042987300264
-
Discovery of Key Transcriptional Regulators of Alloantigen-Inducible Tregs Used for Cell Therapy
CELL PRESS. 2023: 370-371
View details for Web of Science ID 001045144201371
-
Global Phase 1 Study Results of Lentiviral Mediated Gene Therapy for Severe Pyruvate Kinase Deficiency
CELL PRESS. 2023: 118-119
View details for Web of Science ID 001045144200219
-
Lentiviral-Mediated Gene Therapy for Fanconi Anemia [Group A]: Results from Global RP-L102 Clinical Trials
CELL PRESS. 2023: 118
View details for Web of Science ID 001045144200218
-
SATB1 chromatin loops regulate Megakaryocyte/Erythroid Progenitor Expansion by facilitating HSP70 and GATA1 induction.
Stem cells (Dayton, Ohio)
2023
Abstract
Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome associated with severe anemia, congenital malformations and increased risk of developing cancer. The chromatin-binding SATB1 is downregulated in Megakaryocyte/Erythroid Progenitors (MEPs) in patients and cell models of DBA, leading to a reduction in MEP expansion. Here we demonstrate that SATB1 expression is required for the upregulation of the critical erythroid factors HSP70 and GATA1 that accompanies MEP differentiation. SATB1 binding to specific sites surrounding the HSP70 genes, promotes chromatin loops that are required for induction of HSP70, which in turn promotes GATA1 induction. This demonstrates that SATB1, although gradually downregulated during myelopoiesis, maintains a biological function in early myeloid progenitors.
View details for DOI 10.1093/stmcls/sxad025
View details for PubMedID 36987811
-
Hematopoietic and Immunological Assessment in Fanconi Anemia after Ex Vivo Lentiviral FANCA Gene Therapy with RP-L102
AMER SOC HEMATOLOGY. 2022: 7772-7773
View details for DOI 10.1182/blood-2022-157840
View details for Web of Science ID 000893230300348
-
Lentiviral-mediated Gene Therapy for Patients with Fanconi Anemia [Group A]: Updated Results from Global RP-L102 Clinical Trials
AMER SOC HEMATOLOGY. 2022: 10646-10647
View details for DOI 10.1182/blood-2022-168342
View details for Web of Science ID 000893230303287
-
Epigenetic and Immunological Indicators of IPEX Disease in subjects with FOXP3 gene mutation.
The Journal of allergy and clinical immunology
2022
Abstract
Forkhead-Box-Protein-3 (FOXP3) is the master transcription factor in CD4+CD25hiCD127lo regulatory T (Treg) cells. Mutations in FOXP3 result in IPEX (Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-linked) syndrome. Clinical presentation of IPEX syndrome is broader than initially described, challenging the understanding of the disease, its evolution and treatment choice.To study the type and extent of immunological abnormalities which remain ill-defined in IPEX, across genetic and clinical heterogeneity.We performed Treg-specific epigenetic quantification and immunological characterization of severe "typical" (n=6) and "atypical" or asymptomatic (n=9) IPEX patients.Increased number of cells with Treg-Specific Demethylated Region (TSDR) demethylation in FOXP3 is a consistent feature in IPEX patients, with i) highest values in those with typical IPEX, ii) increased values in subjects with pathogenic FOXP3 but still no symptoms, and iii) gradual increase over the course of disease progression. Large scale profiling using Luminex identified plasma inflammatory signature of macrophage activation and Th2 polarization, with cytokines previously not associated with IPEX pathology, including CCL22, CCL17, CCL15, and IL-13, and the inflammatory markers TNFα, IL-1A, IL-8, sFasL, and CXCL9. Similarly, both Treg and Teff compartments, studied by CyTOF, were skewed towards the Th2 compartment, especially in typical IPEX.Elevated TSDR demethylated cells, combined with elevation of plasmatic and cellular markers of a polarized Type 2 inflammatory immune response extends our understanding of IPEX diagnosis and heterogeneity.IPEX-specific epigenetic and immunologic changes provide invaluable tools that, complementing the genetic diagnosis, allow monitoring disease progression and enable early treatment interventions.
View details for DOI 10.1016/j.jaci.2022.09.013
View details for PubMedID 36152823
-
Downregulation of SATB1 by miRNAs Reduces Megakaryocyte/Erythroid Progenitor Expansion in pre-clinical models of Diamond Blackfan Anemia.
Experimental hematology
2022
Abstract
Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome that is associated with anemia, congenital anomalies, and cancer predisposition. It is categorized as a ribosomopathy, because over 80% or patients have haploinsufficiency of either a small or large subunit-associated ribosomal protein (RP). The erythroid pathology is predominantly due to a block and delay in early committed erythropoiesis with reduced Megakaryocyte/Erythroid Progenitors (MEPs). To understand the molecular pathways leading to pathogenesis of DBA, we performed RNA-seq on mRNA and miRNA from RPS19-deficient human hematopoietic stem and progenitor cells (HSPCs) and compared an existing database documenting transcript fluctuations across stages of early normal erythropoiesis. We determined the chromatin regulator, SATB1 was prematurely downregulated through the coordinated action of upregulated miR-34 and miR-30 during differentiation in ribosomal-insufficiency. Restoration of SATB1 rescued MEP expansion, leading to a modest improvement in erythroid and megakaryocyte expansion in RPS19-insufficiency. However, SATB1 expression did not impact expansion of committed erythroid progenitors, indicating ribosomal insufficiency impacts multiple stages during erythroid differentiation.
View details for DOI 10.1016/j.exphem.2022.04.005
View details for PubMedID 35460833
-
Unraveling Transcriptomic Profiles of Pediatric Acute Myeloid Leukemia Cells Sensitive or Resistant to Cytotoxic Killing by Engineered TR1-like Cells
CELL PRESS. 2022: 153
View details for Web of Science ID 000794043700315
-
Two is Better Than One: CRISPR/Cas9 Based Gene Editing with FOXP3 Isoforms for IPEX Therapy
CELL PRESS. 2022: 34
View details for Web of Science ID 000794043700064
-
Downregulation of SATB1 by miRNAs Reduces Megakaryocyte/Erythroid Progenitor Expansion in pre-clinical models of Diamond Blackfan Anemia
Experimental Hematology
2022
View details for DOI 10.1016/j.exphem.2022.04.005
-
The Women of FOCIS: Promoting Equality and Inclusiveness in a Professional Federation of Clinical Immunology Societies.
Frontiers in immunology
2022; 13: 816535
Abstract
The authors of this article, all women who have been deeply committed to the Federation of Clinical Immunology Societies (FOCIS), performed a retrospective analysis of gender equality practices of FOCIS to identify areas for improvement and make recommendations accordingly. Gender data were obtained and analyzed for the period from January 2010 to July 2021. Outcome measures included numbers of men and women across the following categories: membership enrollment, meeting and course faculty and attendees, committee and leadership composition. FOCIS' past and present leaders, steering committee members, FCE directors, individual members, as well as education, annual meeting scientific program and FCE committee members and management staff of FOCIS were surveyed by email questionnaire for feedback on FOCIS policies and practice with respect to gender equality and inclusion. Although women represent 50% of the membership, they have been underrepresented in all leadership, educational, and committee roles within the FOCIS organization. Surveying FOCIS leadership and membership revealed a growing recognition of disparities in female leadership across all FOCIS missions, leading to significant improvement in multiple areas since 2016. We highlight these changes and propose a number of recommendations that can be used by FOCIS to improve gender equality.
View details for DOI 10.3389/fimmu.2022.816535
View details for PubMedID 35444663
-
Type 1 regulatory T cell-mediated tolerance in health and disease.
Frontiers in immunology
2022; 13: 1032575
Abstract
Type 1 regulatory T (Tr1) cells, in addition to other regulatory cells, contribute to immunological tolerance to prevent autoimmunity and excessive inflammation. Tr1 cells arise in the periphery upon antigen stimulation in the presence of tolerogenic antigen presenting cells and secrete large amounts of the immunosuppressive cytokine IL-10. The protective role of Tr1 cells in autoimmune diseases and inflammatory bowel disease has been well established, and this led to the exploration of this population as a potential cell therapy. On the other hand, the role of Tr1 cells in infectious disease is not well characterized, thus raising concern that these tolerogenic cells may cause general immune suppression which would prevent pathogen clearance. In this review, we summarize current literature surrounding Tr1-mediated tolerance and its role in health and disease settings including autoimmunity, inflammatory bowel disease, and infectious diseases.
View details for DOI 10.3389/fimmu.2022.1032575
View details for PubMedID 36389662
-
Functional Immune Tolerance Induced By Sequential Hematopoietic Stem Cell-Solid Organ Transplantation
AMER SOC HEMATOLOGY. 2021: 1818-+
View details for DOI 10.1182/blood-2021-149920
View details for Web of Science ID 000736398807056
-
JSP191 As a Single-Agent Conditioning Regimen Results in Successful Engraftment, Donor Myeloid Chimerism, and Production of Donor Derived Naive Lymphocytes in Patients with Severe Combined Immunodeficiency (SCID)
AMER SOC HEMATOLOGY. 2021
View details for DOI 10.1182/blood-2021-153074
View details for Web of Science ID 000736398802095
-
Engineering Human Invariant Natural Killer T (iNKT) Cells to Overexpress Immunomodulatory Cytokines
AMER SOC HEMATOLOGY. 2021
View details for DOI 10.1182/blood-2021-153975
View details for Web of Science ID 000736413908013
-
Gene Therapy for Fanconi Anemia [Group A]: Interim Results of RP-L102 Clinical Trials
AMER SOC HEMATOLOGY. 2021
View details for Web of Science ID 000736413908091
-
Alloantigen-specific type 1 regulatory T cells suppress through CTLA-4 and PD-1 pathways and persist long-term in patients.
Science translational medicine
2021; 13 (617): eabf5264
Abstract
[Figure: see text].
View details for DOI 10.1126/scitranslmed.abf5264
View details for PubMedID 34705520
-
Development of beta-globin gene correction in human hematopoietic stem cells as a potential durable treatment for sickle cell disease.
Science translational medicine
2021; 13 (598)
Abstract
Sickle cell disease (SCD) is the most common serious monogenic disease with 300,000 births annually worldwide. SCD is an autosomal recessive disease resulting from a single point mutation in codon six of the beta-globin gene (HBB). Ex vivo beta-globin gene correction in autologous patient-derived hematopoietic stem and progenitor cells (HSPCs) may potentially provide a curative treatment for SCD. We previously developed a CRISPR-Cas9 gene targeting strategy that uses high-fidelity Cas9 precomplexed with chemically modified guide RNAs to induce recombinant adeno-associated virus serotype 6 (rAAV6)-mediated HBB gene correction of the SCD-causing mutation in HSPCs. Here, we demonstrate the preclinical feasibility, efficacy, and toxicology of HBB gene correction in plerixafor-mobilized CD34+ cells from healthy and SCD patient donors (gcHBB-SCD). We achieved up to 60% HBB allelic correction in clinical-scale gcHBB-SCD manufacturing. After transplant into immunodeficient NSG mice, 20% gene correction was achieved with multilineage engraftment. The long-term safety, tumorigenicity, and toxicology study demonstrated no evidence of abnormal hematopoiesis, genotoxicity, or tumorigenicity from the engrafted gcHBB-SCD drug product. Together, these preclinical data support the safety, efficacy, and reproducibility of this gene correction strategy for initiation of a phase 1/2 clinical trial in patients with SCD.
View details for DOI 10.1126/scitranslmed.abf2444
View details for PubMedID 34135108
-
Gene Therapy for Fanconi Anemia [Group A]: Preliminary Results of Ongoing RP-L102 Clinical Trials
CELL PRESS. 2021: 339
View details for Web of Science ID 000645188700687
-
LV.InsB9-23-Based Therapy to Arrest T1D and Suppress Its Recurrency Post Allo-Islets Transplant
CELL PRESS. 2021: 358-359
View details for Web of Science ID 000645188700726
-
Preclinical Safety and Efficacy Validation of CD4(LVFOXP3) Cells as an Innovative Cell-Based Gene Therapy Approach for IPEX Syndrome
CELL PRESS. 2021: 340
View details for Web of Science ID 000645188700689
-
Engineered Type 1 Regulatory T Cells Have a Cytotoxic Profile and Kill Pediatric Acute Myeloid Leukemia Cells
CELL PRESS. 2021: 317
View details for Web of Science ID 000645188700642
-
Lentiviral Mediated Gene Therapy for Pyruvate Kinase Deficiency: Updated Results of a Global Phase 1 Study for Adult and Pediatric Patients
CELL PRESS. 2021: 42-43
View details for Web of Science ID 000645188700083
-
Adoptively Transferred, In Vitro-Generated Alloantigen-Specific Type 1 Regulatory T (Tr1) Cells Persist Long-Term In Vivo
CELL PRESS. 2021: 73
View details for Web of Science ID 000645188700142
-
Hergen Spits-A legend at the top of his career.
Allergy
2021
View details for DOI 10.1111/all.14788
View details for PubMedID 33751599
-
BHLHE40 Regulates IL-10 and IFN-γ Production in T Cells but Does Not Interfere With Human Type 1 Regulatory T Cell Differentiation
Frontiers in Immunology
2021
View details for DOI 10.3389/fimmu.2021.683680
-
The Yin and Yang of Type 1 Regulatory T Cells: From Discovery to Clinical Application.
Frontiers in immunology
2021; 12: 693105
Abstract
Regulatory T cells are essential players of peripheral tolerance and suppression of inflammatory immune responses. Type 1 regulatory T (Tr1) cells are FoxP3- regulatory T cells induced in the periphery under tolerogenic conditions. Tr1 cells are identified as LAG3+CD49b+ mature CD4+ T cells that promote peripheral tolerance through secretion of IL-10 and TGF-beta in addition to exerting perforin- and granzyme B-mediated cytotoxicity against myeloid cells. After the initial challenges of isolation were overcome by surface marker identification, ex vivo expansion of antigen-specific Tr1 cells in the presence of tolerogenic dendritic cells (DCs) and IL-10 paved the way for their use in clinical trials. With one Tr1-enriched cell therapy product already in a Phase I clinical trial in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT), Tr1 cell therapy demonstrates promising results so far in terms of efficacy and safety. In the current review, we identify developments in phenotypic and molecular characterization of Tr1 cells and discuss the potential of engineered Tr1-like cells for clinical applications of Tr1 cell therapies. More than 3 decades after their initial discovery, Tr1 cell therapy is now being used to prevent graft versus host disease (GvHD) in allo-HSCT and will be an alternative to immunosuppression to promote graft tolerance in solid organ transplantation in the near future.
View details for DOI 10.3389/fimmu.2021.693105
View details for PubMedID 34177953
-
Pre-clinical development and molecular characterization of an engineered type 1 regulatory T-cell product suitable for immunotherapy.
Cytotherapy
2021
Abstract
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapeutic approach for many hematological disorders. However, allo-HSCT is frequently accompanied by a serious side effect: graft-versus-host disease (GVHD). The clinical use of allo-HSCT is limited by the inability of current immunosuppressive regimens to adequately control GvHD without impairing the graft-versus-leukemia effect (GvL) conferred by transplanted healthy immune cells. To address this, the authors have developed an engineered type 1 regulatory T-cell product called CD4IL-10 cells. CD4IL-10 cells are obtained through lentiviral transduction, which delivers the human IL10 gene into purified polyclonal CD4+ T cells. CD4IL-10 cells may provide an advantage over standard-of-care immunosuppressants because of the ability to suppress GvHD through continuous secretion of IL-10 and enhance the GvL effect in myeloid malignancies through targeted killing of malignant myeloid cells.Here the authors established a production process aimed at current Good Manufacturing Practice (cGMP) production for CD4IL-10 cells.The authors demonstrated that the CD4IL-10 cell product maintains the suppressive and cytotoxic functions of previously described CD4IL-10 cells. In addition, RNA sequencing analysis of CD4IL-10 identified novel transcriptome changes, indicating that CD4IL-10 cells primarily upregulate cytotoxicity-related genes. These include four molecules with described roles in CD8+ T and natural killer cell-mediated cytotoxicity: CD244, KLRD1, KLRC1 and FASLG. Finally, it was shown that CD4IL-10 cells upregulate IL-22, which mediates wound healing and tissue repair, particularly in the gut.Collectively, these results pave the way toward clinical translation of the cGMP-optimized CD4IL-10 cell product and uncover new molecules that have a role in the clinical application of CD4IL-10 cells.
View details for DOI 10.1016/j.jcyt.2021.05.010
View details for PubMedID 34404616
-
Co-Expression of FOXP3FL and FOXP3Δ2 Isoforms Is Required for Optimal Treg-Like Cell Phenotypes and Suppressive Function.
Frontiers in immunology
2021; 12: 752394
Abstract
FOXP3 is the master transcription factor in both murine and human FOXP3+ regulatory T cells (Tregs), a T-cell subset with a central role in controlling immune responses. Loss of the functional Foxp3 protein in scurfy mice leads to acute early-onset lethal lymphoproliferation. Similarly, pathogenic FOXP3 mutations in humans lead to immunodysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX) syndrome, which are characterized by systemic autoimmunity that typically begins in the first year of life. However, although pathogenic FOXP3 mutations lead to overlapping phenotypic consequences in both systems, FOXP3 in human Tregs, but not mouse, is expressed as two predominant isoforms, the full length (FOXP3FL) and the alternatively spliced isoform, delta 2 (FOXP3Δ2). Here, using CRISPR/Cas9 to generate FOXP3 knockout CD4+ T cells (FOXP3KOGFP CD4+ T cells), we restore the expression of each isoform by lentiviral gene transfer to delineate their functional roles in human Tregs. When compared to FOXP3FL or FOXP3Δ2 alone, or double transduction of the same isoform, co-expression of FOXP3FL and FOXP3Δ2 induced the highest overall FOXP3 protein expression in FOXP3KOGFP CD4+ T cells. This condition, in turn, led to optimal acquisition of Treg-like cell phenotypes including downregulation of cytokines, such as IL-17, and increased suppressive function. Our data confirm that co-expression of FOXP3FL and FOXP3Δ2 leads to optimal Treg-like cell function and supports the need to maintain the expression of both when engineering therapeutics designed to restore FOXP3 function in otherwise deficient cells.
View details for DOI 10.3389/fimmu.2021.752394
View details for PubMedID 34737751
View details for PubMedCentralID PMC8560788
-
InsB9-23 Gene Transfer To Hepatocytes-Based Combined Therapy Abrogates Recurrence of Type-1 Diabetes After Islet Transplantation.
Diabetes
2020
Abstract
The induction of antigen (Ag)-specific tolerance represents a therapeutic option for autoimmune diabetes. We demonstrated that administration of lentiviral vector enabling expression of insulinB9-23 (LV.InsB) in hepatocytes, arrests beta cell destruction in pre-diabetic NOD mice, by generating InsB9-23-specific FoxP3+T regulatory cells (Tregs). LV.InsB in combination with a suboptimal dose of anti-CD3 mAb (combined therapy, 1X5g CT5) reverts diabetes and prevents recurrence of autoimmunity following islets transplantation in 50% of NOD mice. We investigated whether CT optimization could lead to abrogation of recurrence of autoimmunity. Therefore, allo-islets were transplanted after optimized CT tolerogenic conditioning (1X25g CT25). Diabetic NOD mice conditioned with CT25 when glycaemia was <500mg/dL, remained normoglycaemic for 100 days after allo-islets transplantation, displayed reduced insulitis, but independently from the graft. Accordingly, cured mice showed T cell unresponsiveness to InsB9-23 stimulation and increased Tregs frequency in islets infiltration and pancreatic LN. Additional studies revealed a complex mechanism of Ag-specific immune regulation driven by CT25, in which both Tregs and PDL1 co-stimulation cooperate to control diabetogenic cells, while transplanted islets play a crucial role, although transient, recruiting diabetogenic cells. Therefore, CT25 before allo-islets transplantation represents an Ag-specific immunotherapy to resolve autoimmune diabetes in the presence of residual endogenous beta cell mass.
View details for DOI 10.2337/db19-1249
View details for PubMedID 33122392
-
Celebrating 20 years of FOCIS.
Science immunology
2020; 5 (52)
View details for DOI 10.1126/sciimmunol.abe8102
View details for PubMedID 33037068
-
Gene Therapy for Wiskott-Aldrich Syndrome: History, New Vectors, Future Directions.
The Journal of allergy and clinical immunology
2020
View details for DOI 10.1016/j.jaci.2020.06.018
View details for PubMedID 32623069
-
Engineered Type-1 Regulatory T Cells as Cellular Therapy for Treatment of Immune Mediated Diseases
AMER ASSOC IMMUNOLOGISTS. 2020
View details for Web of Science ID 000589972400598
-
A beta T-Cell/CD19 B-Cell Depleted Haploidentical Stem Cell Transplantation: A New Platform for Curing Rare and Monogenic Disorders
ELSEVIER SCIENCE INC. 2020: S288
View details for Web of Science ID 000516887900438
-
Regulatory Type 1 T Cell Infusion in Mismatched Related or Unrelated Hematopoietic Stem Cell Transplantation (HSCT) for Hematologic Malignancies
ELSEVIER SCIENCE INC. 2020: S272–S273
View details for Web of Science ID 000516887900410
-
Early Epigenetic Immune Quantification Following Alpha/Beta T-Cell/CD19 B-Cell Depleted Haploidentical Stem Cell Transplant Correlates with CD4+T Cell Recovery at Day+100
ELSEVIER SCIENCE INC. 2020: S305
View details for Web of Science ID 000516887900465
-
Human-engineered Treg-like cells suppress FOXP3-deficient T cells but preserve adaptive immune responses in vivo.
Clinical & translational immunology
2020; 9 (11): e1214
Abstract
Genetic or acquired defects in FOXP3+ regulatory T cells (Tregs) play a key role in many immune-mediated diseases including immune dysregulation polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. Previously, we demonstrated CD4+ T cells from healthy donors and IPEX patients can be converted into functional Treg-like cells by lentiviral transfer of FOXP3 (CD4LVFOXP3). These CD4LVFOXP3 cells have potent regulatory function, suggesting their potential as an innovative therapeutic. Here, we present molecular and preclinical in vivo data supporting CD4LVFOXP3 cell clinical progression.The molecular characterisation of CD4LVFOXP3 cells included flow cytometry, qPCR, RNA-seq and TCR-seq. The in vivo suppressive function of CD4LVFOXP3 cells was assessed in xenograft-versus-host disease (xeno-GvHD) and FOXP3-deficient IPEX-like humanised mouse models. The safety of CD4LVFOXP3 cells was evaluated using peripheral blood (PB) humanised (hu)- mice testing their impact on immune response against pathogens, and immune surveillance against tumor antigens.We demonstrate that the conversion of CD4+ T cells to CD4LVFOXP3 cells leads to specific transcriptional changes as compared to CD4+ T-cell transduction in the absence of FOXP3, including upregulation of Treg-related genes. Furthermore, we observe specific preservation of a polyclonal TCR repertoire during in vitro cell production. Both allogeneic and autologous CD4LVFOXP3 cells protect from xeno-GvHD after two sequential infusions of effector T cells. CD4LVFOXP3 cells prevent hyper-proliferation of CD4+ memory T cells in the FOXP3-deficient IPEX-like hu-mice. CD4LVFOXP3 cells do not impede in vivo expansion of antigen-primed T cells or tumor clearance in the PB hu-mice.These data support the clinical readiness of CD4LVFOXP3 cells to treat IPEX syndrome and other immune-mediated diseases caused by insufficient or dysfunctional FOXP3+ Tregs.
View details for DOI 10.1002/cti2.1214
View details for PubMedID 33304583
View details for PubMedCentralID PMC7688376
-
Engineered type 1 regulatory T cells designed for clinical use kill primary pediatric acute myeloid leukemia cells
Haematologica
2020
View details for DOI 10.3324/haematol.2020.263129
-
Genome editing of donor-derived T-cells to generate allogenic chimeric antigen receptor-modified T cells: Optimizing αβ T cell-depleted haploidentical hematopoietic stem cell transplantation.
Haematologica
2020
Abstract
Allogeneic hematopoietic stem cell transplantation is an effective therapy for high-risk leukemias. In children, graft manipulation based on the selective removal of αβ T cells and B cells has been shown to reduce the risk of acute and chronic graft-versus-host disease, thus allowing the use of haploidentical donors which expands the population that allogeneic hematopoietic stem cell transplantation can be used in. Leukemic relapse, however, remains a problem. T cells expressing chimeric antigen receptors can potently eliminate leukemia, including in the central nervous system. We hypothesized that by modifying donor αβ T cells to simultaneously express a CD19-specific chimeric antigen receptors and inactivating the T cell receptor by genome editing, we could create a therapy that enhances the anti-leukemic efficacy of the stem cell transplant without increasing the risk of graft-versus-host disease. Using genome editing with Cas9 ribonucleoprotein and adeno-associated virus serotype 6, we integrate a CD19-specific chimeric antigen receptor in-frame into the TRAC locus. Greater than 90% of cells lost TCR expression, while >75% expressed the CAR. The product was further purified to ultimately have less than 0.05% residual TCR+ cells. In vitro, the CAR T cells efficiently eliminated target cells and produced high cytokine levels when challenged with CD19+ leukemia cells. In vivo, the gene modified T cells eliminated leukemia without causing xenogeneic graft-versus-host disease in a xenograft model. Gene editing was highly specific with no evidence of off-target effects. These data support the concept that the addition of αβ T cell-derived, genome edited T cells expressing CD19-specific chimeric antigen receptors could enhance the anti-leukemic efficacy of αβ T cell-depleted haploidentical hematopoietic stem cell transplantation without increasing the risk of graft-versus-host disease.
View details for DOI 10.3324/haematol.2019.233882
View details for PubMedID 32241852
-
Alloantigen-specific Tr1 cells designed to prevent GvHD have a distinct molecular identity and suppress through CTLA-4 and PD-1
Society for Immunotherapy of Cancer’s (SITC) 35th Anniversary Annual Meeting
2020: A159–A159
View details for DOI 10.1136/jitc-2020-SITC2020.0146
-
Changing the Natural History of Fanconi Anemia Complementation Group-A with Gene Therapy: Early Results of US Phase I Study of Lentiviral-Mediated Ex-VivoFANCA Gene Insertion in Human Stem and Progenitor Cells
AMER SOC HEMATOLOGY. 2019
View details for DOI 10.1182/blood-2019-127352
View details for Web of Science ID 000577160408174
-
Non-Genotoxic Anti-CD117 Antibody Conditioning Results in Successful Hematopoietic Stem Cell Engraftment in Patients with Severe Combined Immunodeficiency
AMER SOC HEMATOLOGY. 2019
View details for DOI 10.1182/blood-2019-126239
View details for Web of Science ID 000518218500805
-
Gene therapy for primary immunodeficiency.
Human molecular genetics
2019
Abstract
Gene therapy is now being trialled as a therapeutic option for an expanding number of conditions, based primarily on the successful treatment over the past two decades of patients with specific primary immunodeficiencies (PIDs) including severe combined immunodeficiency (SCID) and Wiskott-Aldrich syndrome (WAS) and metabolic conditions such as leukodystrophy. The field has evolved from the use of gammaretroviral vectors to more sophisticated lentiviral platforms which offer an improved biosafety profile alongside greater efficiency for HSC gene transfer. Here we review more recent developments including licensing of gene therapies, use of gene corrected autologous T cells as an alternative strategy for some PIDs and the potential of targeted gene correction using various gene editing platforms. Given the promising results of recent clinical trials it is likely that autologous gene therapies will become standard of care for a number of devastating diseases in the coming decade.
View details for DOI 10.1093/hmg/ddz170
View details for PubMedID 31297531
-
Graft Engineering and Adoptive Immunotherapy: New Approaches to Promote Immune Tolerance After Hematopoietic Stem Cell Transplantation
FRONTIERS IN IMMUNOLOGY
2019; 10
View details for DOI 10.3389/fimmu.2019.01342
View details for Web of Science ID 000474769300001
-
REGULATORY TYPE 1 T CELL INFUSION IN MISMATCHED RELATED OR UNRELATED HEMATOPOIETIC STEM CELL TRANSPLANTATION (HSCT) FOR HEMATOLOGIC MALIGNANCIES
WILEY. 2019
View details for Web of Science ID 000490282100500
-
Lentiviral haemopoietic stem/progenitor cell gene therapy for treatment of Wiskott-Aldrich syndrome: interim results of a non-randomised, open-label, phase 1/2 clinical study
LANCET HAEMATOLOGY
2019; 6 (5): E239–E253
View details for DOI 10.1016/S2352-3026(19)30021-3
View details for Web of Science ID 000466131400007
-
Gene correction for SCID-X1 in long-term hematopoietic stem cells (vol 10, 1634, 2019)
NATURE COMMUNICATIONS
2019; 10
View details for DOI 10.1038/s41467-019-10080-9
View details for Web of Science ID 000465840000001
-
Engineered Type-1 Regulatory T Cells for Treatment of Graft-versus-Host Disease in Allogeneic Hematopoietic Stem Cell Transplant Recipients
CELL PRESS. 2019: 459
View details for Web of Science ID 000464381005080
-
Immunoregulatory Cell Therapy with Lentiviral-Mediated FOXP3 Converted CD4+T Cells into Treg Cells: Towards the Proof-of-Concept Application in IPEX Syndrome
CELL PRESS. 2019: 311
View details for Web of Science ID 000464381003119
-
Lentiviral haemopoietic stem/progenitor cell gene therapy for treatment of Wiskott-Aldrich syndrome: interim results of a non-randomised, open-label, phase 1/2 clinical study.
The Lancet. Haematology
2019
Abstract
BACKGROUND: Wiskott-Aldrich syndrome is a rare, life-threatening, X-linked primary immunodeficiency characterised by microthrombocytopenia, infections, eczema, autoimmunity, and malignant disease. Lentiviral vector-mediated haemopoietic stem/progenitor cell (HSPC) gene therapy is a potentially curative treatment that represents an alternative to allogeneic HSPC transplantation. Here, we report safety and efficacy data from an interim analysis of patients with severe Wiskott-Aldrich syndrome who received lentiviral vector-derived gene therapy.METHODS: We did a non-randomised, open-label, phase 1/2 clinical study in paediatric patients with severe Wiskott-Aldrich syndrome, defined by either WAS gene mutation or absent Wiskott-Aldrich syndrome protein (WASP) expression or a Zhu clinical score of 3 or higher. We included patients who had no HLA-identical sibling donor available or, for children younger than 5 years of age, no suitable 10/10 matched unrelated donor or 6/6 unrelated cord blood donor. After treatment with rituximab and a reduced-intensity conditioning regimen of busulfan and fludarabine, patients received one intravenous infusion of autologous CD34+ cells genetically modified with a lentiviral vector encoding for human WAS cDNA. The primary safety endpoints were safety of the conditioning regimen and safety of lentiviral gene transfer into HSPCs. The primary efficacy endpoints were overall survival, sustained engraftment of genetically corrected HSPCs, expression of vector-derived WASP, improved T-cell function, antigen-specific responses to vaccinations, and improved platelet count and mean platelet volume normalisation. This interim analysis was done when the first six patients treated had completed at least 3 years of follow-up. The planned analyses are presented for the intention-to-treat population. This trial is registered with ClinicalTrials.gov (number NCT01515462) and EudraCT (number 2009-017346-32).FINDINGS: Between April 20, 2010, and Feb 26, 2015, nine patients (all male) were enrolled of whom one was excluded after screening; the age range of the eight treated children was 1·1-12·4 years. At the time of the interim analysis (data cutoff April 29, 2016), median follow-up was 3·6 years (range 0·5-5·6). Overall survival was 100%. Engraftment of genetically corrected HSPCs was successful and sustained in all patients. The fraction of WASP-positive lymphocytes increased from a median of 3·9% (range 1·8-35·6) before gene therapy to 66·7% (55·7-98·6) at 12 months after gene therapy, whereas WASP-positive platelets increased from 19·1% (range 4·1-31·0) to 76·6% (53·1-98·4). Improvement of immune function was shown by normalisation of in-vitro T-cell function and successful discontinuation of immunoglobulin supplementation in seven patients with follow-up longer than 1 year, followed by positive antigen-specific response to vaccination. Severe infections fell from 2·38 (95% CI 1·44-3·72) per patient-year of observation (PYO) in the year before gene therapy to 0·31 (0·04-1·11) per PYO in the second year after gene therapy and 0·17 (0·00-0·93) per PYO in the third year after gene therapy. Before gene therapy, platelet counts were lower than 20 * 109 per L in seven of eight patients. At the last follow-up visit, the platelet count had increased to 20-50 * 109 per L in one patient, 50-100 * 109 per L in five patients, and more than 100 * 109 per L in two patients, which resulted in independence from platelet transfusions and absence of severe bleeding events. 27 serious adverse events in six patients occurred after gene therapy, 23 (85%) of which were infectious (pyrexia [five events in three patients], device-related infections, including one case of sepsis [four events in three patients], and gastroenteritis, including one case due to rotavirus [three events in two patients]); these occurred mainly in the first 6 months of follow-up. No adverse reactions to the investigational drug product and no abnormal clonal proliferation or leukaemia were reported after gene therapy.INTERPRETATION: Data from this study show that gene therapy provides a valuable treatment option for patients with severe Wiskott-Aldrich syndrome, particularly for those who do not have a suitable HSPC donor available.FUNDING: Italian Telethon Foundation, GlaxoSmithKline, and Orchard Therapeutics.
View details for PubMedID 30981783
-
Gene correction for SCID-X1 in long-term hematopoietic stem cells
NATURE COMMUNICATIONS
2019; 10
View details for DOI 10.1038/s41467-019-09614-y
View details for Web of Science ID 000463872400010
-
Gene correction for SCID-X1 in long-term hematopoietic stem cells.
Nature communications
2019; 10 (1): 1634
Abstract
Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl.
View details for PubMedID 30967552
-
Coexpression of CD163 and CD141 identifies human circulating IL-10-producing dendritic cells (DC-10).
Cellular & molecular immunology
2019
Abstract
Tolerogenic dendritic cells (DCs) are key players in maintaining immunological homeostasis, dampening immune responses, and promoting tolerance. DC-10, a tolerogenic population of human IL-10-producing DCs characterized by the expression of HLA-G and ILT4, play a pivotal role in promoting tolerance via T regulatory type 1 (Tr1) cells. Thus far, the absence of markers that uniquely identify DC-10 has limited in vivo studies. By in vitro gene expression profiling of differentiated human DCs, we identified CD141 and CD163 as surface markers for DC-10. The coexpression of CD141 and CD163 in combination with CD14 and CD16 enables the ex vivo isolation of DC-10 from the peripheral blood. CD14+CD16+CD141+CD163+ cells isolated from the peripheral blood of healthy subjects (ex vivo DC-10) produced spontaneously and upon activation of IL-10 and limited levels of IL-12. Moreover, in vitro stimulation of allogeneic naive CD4+ T cells with ex vivo DC-10 induced the differentiation of alloantigen-specific CD49b+LAG-3+ Tr1 cells. Finally, ex vivo DC-10 and in vitro generated DC-10 exhibited a similar transcriptional profile, which are characterized by an anti-inflammatory and pro-tolerogenic signature. These results provide new insights into the phenotype and molecular signature of DC-10 and highlight the tolerogenic properties of circulating DC-10. These findings open the opportunity to track DC-10 in vivo and to define their role in physiological and pathological settings.
View details for DOI 10.1038/s41423-019-0218-0
View details for PubMedID 30842629
-
Regulatory Type 1 T Cell Infusion in Mismatched Related or Unrelated Hematopoietic Stem Cell Transplantation (HSCT) for Hematologic Malignancies
ELSEVIER SCIENCE INC. 2019
View details for Web of Science ID 000540655500260
-
Toxicity-Free Hematopoietic Stem Cell Engraftment Achieved with Anti-CD117 Monoclonal Antibody Conditioning
ELSEVIER SCIENCE INC. 2019
View details for Web of Science ID 000540655500124
-
Chimerism Analysis in Pediatric Hematopoietic Stem Cell Transplantation for Non-Malignant Disorders
ELSEVIER SCIENCE INC. 2019
View details for Web of Science ID 000540655500467
-
Driving Medical Innovation Through Interdisciplinarity: Unique Opportunities and Challenges.
Frontiers in medicine
2019; 6: 35
View details for DOI 10.3389/fmed.2019.00035
View details for PubMedID 30863750
View details for PubMedCentralID PMC6400109
-
Driving Medical Innovation Through Interdisciplinarity: Unique Opportunities and Challenges
FRONTIERS IN MEDICINE
2019; 6
View details for DOI 10.3389/fmed.2019.00035
View details for Web of Science ID 000459743100001
-
Lentiviral Gene Therapy in HSCs Restores Lineage-Specific Foxp3 Expression and Suppresses Autoimmunity in a Mouse Model of IPEX Syndrome
CELL STEM CELL
2019; 24 (2): 309-+
View details for DOI 10.1016/j.stem.2018.12.003
View details for Web of Science ID 000458027300016
-
Author Correction: Gene correction for SCID-X1 in long-term hematopoietic stem cells.
Nature communications
2019; 10 (1): 5624
Abstract
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
View details for DOI 10.1038/s41467-019-13620-5
View details for PubMedID 31796738
-
Author Correction: Gene correction for SCID-X1 in long-term hematopoietic stem cells.
Nature communications
2019; 10 (1): 2021
Abstract
The original version of this Article omitted the following from the Acknowledgements: "G.B. acknowledges the support from the Cancer Prevention and Research Institute of Texas (RR140081 and RR170721)."This has now been corrected in both the PDF and HTML versions of the Article.
View details for PubMedID 31028274
-
Lentiviral Gene Therapy in HSCs Restores Lineage-Specific Foxp3 Expression and Suppresses Autoimmunity in a Mouse Model of IPEX Syndrome.
Cell stem cell
2018
Abstract
Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a devastating autoimmune disease caused by mutations in FoxP3, a transcription factor required for the development and function of regulatory Tcells (Treg cells). Allogeneic hematopoietic stem cell transplant (HSCT) can be curative, but suitable donors are often unavailable. Here, we demonstrate a strategy for autologous HSCT and gene therapy utilizing a lentiviral vector (LV) to restore FoxP3 expression under the control of endogenous human FOXP3 regulatory elements. Both murine transplant models and humanized mice engrafted with LV-modified HSCs show high levels of LV expression selective for CD4+CD25+FoxP3+ Treg cells. LV transduction of scurfy (FoxP3mut) HSCs restores development of functional FoxP3+ Treg cells that suppress Tcell proliferation invitro and rescue the scurfy autoimmune phenotype invivo. These findings demonstrate preclinical efficacy for the treatment of IPEX patients by autologous HSC transplant and may provide valuable insights into new cell therapies for autoimmunity.
View details for PubMedID 30639036
-
Molecular and functional heterogeneity of IL-10-producing CD4+ T cells.
Nature communications
2018; 9 (1): 5457
Abstract
IL-10 is a prototypicalanti-inflammatory cytokine, which is fundamental to the maintenance of immune homeostasis, especially in the intestine. There is an assumption that cells producing IL-10 have an immunoregulatory function. However, here we report that IL-10-producing CD4+ T cells are phenotypically and functionally heterogeneous. By combining single cell transcriptome and functional analyses, we identified a subpopulation of IL-10-producing Foxp3neg CD4+ T cells that displays regulatory activity unlike other IL-10-producing CD4+ T cells, which are unexpectedly pro-inflammatory. The combinatorial expression of co-inhibitory receptors is sufficient to discriminate IL-10-producing CD4+ T cells with regulatory function from others and to identify them across different tissues and disease models in mice and humans. These regulatory IL-10-producing Foxp3neg CD4+ T cells have a unique transcriptional program, which goes beyond the regulation of IL-10 expression. Finally, we found that patients with Inflammatory Bowel Disease demonstrate a deficiency in this specific regulatory T-cell subpopulation.
View details for DOI 10.1038/s41467-018-07581-4
View details for PubMedID 30575716
-
Tregopathies: Monogenic diseases resulting in regulatory T-cell deficiency.
The Journal of allergy and clinical immunology
2018; 142 (6): 1679–95
Abstract
Monogenic diseases of the immune system, also known as inborn errors of immunity, are caused by single-gene mutations resulting in immune deficiency and dysregulation. More than 350 diseases have been described to date, and the number is rapidly expanding, with increasing availability of next-generation sequencing facilitating the diagnosis. The spectrum of immune dysregulation is wide, encompassing deficiencies in humoral, cellular, innate, and adaptive immunity; phagocytosis; and the complement system, which lead to autoinflammation and autoimmunity. Multiorgan autoimmunity is a dominant symptom when genetic mutations lead to defects in molecules essential for the development, survival, and/or function of regulatory T (Treg) cells. Studies of "Tregopathies" are providing critical mechanistic information on Treg cell biology, the role of Treg cell-associated molecules, and regulation of peripheral tolerance in human subjects. The pathogenic immune networks underlying these diseases need to be dissected to apply and develop immunomodulatory treatments and design curative treatments using cell and gene therapy. Here we review the pathogenetic mechanisms, clinical presentation, diagnosis, and current and future treatments of major known Tregopathies caused by mutations in FOXP3, CD25, cytotoxic Tlymphocyte-associated antigen 4 (CTLA4), LPS-responsive and beige-like anchor protein (LRBA), and BTB domain and CNC homolog 2 (BACH2) and gain-of-function mutations in signal transducer and activator oftranscription 3 (STAT3). We also discuss deficiencies in genesencoding STAT5b and IL-10 or IL-10 receptor aspotential Tregopathies.
View details for DOI 10.1016/j.jaci.2018.10.026
View details for PubMedID 30527062
-
Engineering Regenerative Thymic Tissues to Restore Long-Term T Cell Lymphopoiesis
AMER SOC HEMATOLOGY. 2018
View details for DOI 10.1182/blood-2018-99-117499
View details for Web of Science ID 000454842805362
-
Reprogramming human T cell function and specificity with non-viral genome targeting
NATURE
2018; 559 (7714): 405-+
Abstract
Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.
View details for PubMedID 29995861
-
APVO210: A Bispecific Anti-CD86-IL-10 Fusion Protein (ADAPTIR (TM)) to Induce Antigen-Specific T Regulatory Type 1 Cells
FRONTIERS IN IMMUNOLOGY
2018; 9
View details for DOI 10.3389/fimmu.2018.00881
View details for Web of Science ID 000433143600001
-
APVO210: A Bispecific Anti-CD86-IL-10 Fusion Protein (ADAPTIR™) to Induce Antigen-Specific T Regulatory Type 1 Cells.
Frontiers in immunology
2018; 9: 881
Abstract
IL-10 is a potent immunosuppressive cytokine that promotes the differentiation of tolerogenic dendritic cells (DC-10), and the subsequent induction of antigen-specific T regulatory type 1 (Tr1) cells, which suppress immune responses. However, IL-10 acts on multiple cell types and its effects are not solely inhibitory, therefore, limiting its use as immunomodulant. APVO210 is a bispecific fusion protein composed of an anti-CD86 antibody fused with monomeric IL-10 (ADAPTIR™ from Aptevo Therapeutics). APVO210 specifically induces IL-10R signaling in CD86+ antigen-presenting cells, but not in T and B cells. In this study, we tested whether APVO210 promotes the differentiation of tolerogenic DC-10 and the differentiation of antigen-specific CD4+ Tr1 cells in vitro. We compared the effect of APVO210 with that of recombinant human (rh) IL-10 on the in vitro differentiation of DC-10, induction of alloantigen-specific anergic CD4+ T cells, enrichment in CD49b+LAG3+ Tr1 cells mediating antigen-specific suppression, and stability upon exposure to inflammatory cytokines. APVO210 induced the differentiation of tolerogenic DC (DC-A210) that produced high levels of IL-10, expressed CD86, HLA-G, and intermediate levels of CD14 and CD16. These DC-A210 induced alloantigen-specific anergic T-cell cultures (T-alloA210) that were enriched in CD49b+ LAG3+ Tr1 cells, produced high levels of IL-10, and had suppressive properties. The phenotype and high IL-10 production by DC-A210, and the alloantigen-specific anergy of T-alloA210 were preserved upon exposure to the inflammatory cytokines IL-1β, IL-6, and TNF-α. The effects of APVO210 were comparable to that of dimeric rh IL-10. In conclusion, our data demonstrate that APVO210 drives the differentiation of tolerogenic DC and functional alloantigen-specific Tr1 cells in vitro. Since APVO210 specifically targets CD86+ cells, we hypothesize that it will specifically target CD86+ DC to induce Tr1 cells in vivo, and mediate antigen-specific immunological tolerance by induction of tolerogenic DC and Tr1 cells.
View details for DOI 10.3389/fimmu.2018.00881
View details for PubMedID 29887861
View details for PubMedCentralID PMC5980965
-
Genome Editing for IL-10 Deficiency in Purified Hematopoietic Stem Cells
CELL PRESS. 2018: 237–38
View details for Web of Science ID 000435342203069
-
DEVELOPMENT OF PERSISTENT MIXED CHIMERISM (MC) IN ALLO-HSCT TRANSPLANTED BETA-THAL PATIENTS IS ASSOCIATED WITH LOW MC EARLY AFTER TRANSPLANT AND TR1 CELLS
WILEY. 2018: 399
View details for Web of Science ID 000430290800162
-
CRISPR-Based Therapy for IPEX Syndrome as a Model of Genetic Autoimmunity
CELL PRESS. 2018: 95–96
View details for Web of Science ID 000435342201075
-
Genome Editing of Long-Term Human Hematopoietic Stem Cells for X-Linked Severe Combined Immunodeficiency
SPRINGER/PLENUM PUBLISHERS. 2018: 365–66
View details for Web of Science ID 000431311600087
-
FOXP3 Gene Transfer in T cells and FOXP3 Gene Editing in HSC as Novel Treatment Options for IPEX Syndrome
SPRINGER/PLENUM PUBLISHERS. 2018: 427
View details for Web of Science ID 000431311600212
-
Gene Therapy for Adenosine Deaminase Deficiency: A Comprehensive Evaluation of Short- and Medium-Term Safety
MOLECULAR THERAPY
2018; 26 (3): 917–31
Abstract
Loss of adenosine deaminase activity leads to severe combined immunodeficiency (ADA-SCID); production and function of T, B, and natural killer (NK) cells are impaired. Gene therapy (GT) with an autologous CD34+-enriched cell fraction that contains CD34+ cells transduced with a retroviral vector encoding the human ADA cDNA sequence leads to immune reconstitution in most patients. Here, we report short- and medium-term safety analyses from 18 patients enrolled as part of single-arm, open-label studies or compassionate use programs. Survival was 100% with a median of 6.9 years follow-up (range, 2.3 to 13.4 years). Adverse events were mostly grade 1 or grade 2 and were reported by all 18 patients following GT. Thirty-nine serious adverse events (SAEs) were reported by 15 of 18 patients; no SAEs were considered related to GT. The most common adverse events reported post-GT include upper respiratory tract infection, gastroenteritis, rhinitis, bronchitis, oral candidiasis, cough, neutropenia, diarrhea, and pyrexia. Incidence rates for all of these events were highest during pre-treatment, treatment, and/or 3-month follow-up and then declined over medium-term follow-up. GT did not impact the incidence of neurologic/hearing impairments. No event indicative of leukemic transformation was reported.
View details for PubMedID 29433935
View details for PubMedCentralID PMC5910668
-
Long-Term Treatment Outcome in IPEX Syndrome Patients: An International Multicenter Retrospective Study
ELSEVIER SCIENCE INC. 2018: S81–S82
View details for Web of Science ID 000425476000088
-
Long-term follow-up of IPEX syndrome patients after different therapeutic strategies: An international multicenter retrospective study
JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY
2018; 141 (3): 1036-+
Abstract
Immunodysregulation polyendocrinopathy enteropathy x-linked (IPEX) syndrome is a monogenic autoimmune disease caused by FOXP3 mutations. Because it is a rare disease, the natural history and response to treatments, including allogeneic hematopoietic stem cell transplantation (HSCT) and immunosuppression (IS), have not been thoroughly examined.This analysis sought to evaluate disease onset, progression, and long-term outcome of the 2 main treatments in long-term IPEX survivors.Clinical histories of 96 patients with a genetically proven IPEX syndrome were collected from 38 institutions worldwide and retrospectively analyzed. To investigate possible factors suitable to predict the outcome, an organ involvement (OI) scoring system was developed.We confirm neonatal onset with enteropathy, type 1 diabetes, and eczema. In addition, we found less common manifestations in delayed onset patients or during disease evolution. There is no correlation between the site of mutation and the disease course or outcome, and the same genotype can present with variable phenotypes. HSCT patients (n = 58) had a median follow-up of 2.7 years (range, 1 week-15 years). Patients receiving chronic IS (n = 34) had a median follow-up of 4 years (range, 2 months-25 years). The overall survival after HSCT was 73.2% (95% CI, 59.4-83.0) and after IS was 65.1% (95% CI, 62.8-95.8). The pretreatment OI score was the only significant predictor of overall survival after transplant (P = .035) but not under IS.Patients receiving chronic IS were hampered by disease recurrence or complications, impacting long-term disease-free survival. When performed in patients with a low OI score, HSCT resulted in disease resolution with better quality of life, independent of age, donor source, or conditioning regimen.
View details for PubMedID 29241729
-
Engineered T Regulatory Type 1 Cells for Clinical Application
FRONTIERS IN IMMUNOLOGY
2018; 9: 233
Abstract
T regulatory cells, a specialized subset of T cells, are key players in modulating antigen (Ag)-specific immune responses in vivo. Inducible T regulatory type 1 (Tr1) cells are characterized by the co-expression of CD49b and lymphocyte-activation gene 3 (LAG-3) and the ability to secrete IL-10, TGF-β, and granzyme (Gz) B, in the absence of IL-4 and IL-17. The chief mechanisms by which Tr1 cells control immune responses are secretion of IL-10 and TGF-β and killing of myeloid cells via GzB. Tr1 cells, first described in peripheral blood of patients who developed tolerance after HLA-mismatched fetal liver hematopoietic stem cell transplantation, have been proven to modulate inflammatory and effector T cell responses in several immune-mediated diseases. The possibility to generate and expand Tr1 cells in vitro in an Ag-specific manner has led to their clinical use as cell therapy in patients. Clinical grade protocols to generate or to enrich and expand Tr1 cell medicinal products have been established. Proof-of-concept clinical trials with Tr1 cell products have demonstrated the safety and the feasibility of this approach and indicated some clinical benefit. In the present review, we provide an overview on protocols established to induce/expand Tr1 cells in vitro for clinical application and on results obtained in Tr1 cell-based clinical trials. Moreover, we will discuss a recently developed protocol to efficient convert human CD4+ T cells into a homogeneous population of Tr1-like cells by lentiviral vector-mediated IL-10 gene transfer.
View details for DOI 10.3389/fimmu.2018.00233
View details for Web of Science ID 000425152300001
View details for PubMedID 29497421
View details for PubMedCentralID PMC5818395
-
First Occurrence of Plasmablastic Lymphoma in Adenosine Deaminase-Deficient Severe Combined Immunodeficiency Disease Patient and Review of the Literature
FRONTIERS IN IMMUNOLOGY
2018; 9: 113
Abstract
Adenosine deaminase-deficient severe combined immunodeficiency disease (ADA-SCID) is a primary immune deficiency characterized by mutations in the ADA gene resulting in accumulation of toxic compounds affecting multiple districts. Hematopoietic stem cell transplantation (HSCT) from a matched donor and hematopoietic stem cell gene therapy are the preferred options for definitive treatment. Enzyme replacement therapy (ERT) is used to manage the disease in the short term, while a decreased efficacy is reported in the medium-long term. To date, eight cases of lymphomas have been described in ADA-SCID patients. Here we report the first case of plasmablastic lymphoma occurring in a young adult with ADA-SCID on long-term ERT, which turned out to be Epstein-Barr virus associated. The patient previously received infusions of genetically modified T cells. A cumulative analysis of the eight published cases of lymphoma from 1992 to date, and the case here described, reveals a high mortality (89%). The most common form is diffuse large B-cell lymphoma, which predominantly occurs in extra nodal sites. Seven cases occurred in patients on ERT and two after haploidentical HSCT. The significant incidence of immunodeficiency-associated lymphoproliferative disorders and poor survival of patients developing this complication highlight the priority in finding a prompt curative treatment for ADA-SCID.
View details for DOI 10.3389/fimmu.2018.00113
View details for Web of Science ID 000423990100001
View details for PubMedID 29456531
View details for PubMedCentralID PMC5801298
-
Minimum Information about T Regulatory Cells: A Step toward Reproducibility and Standardization
FRONTIERS IN IMMUNOLOGY
2018; 8: 1844
Abstract
Cellular therapies with CD4+ T regulatory cells (Tregs) hold promise of efficacious treatment for the variety of autoimmune and allergic diseases as well as posttransplant complications. Nevertheless, current manufacturing of Tregs as a cellular medicinal product varies between different laboratories, which in turn hampers precise comparisons of the results between the studies performed. While the number of clinical trials testing Tregs is already substantial, it seems to be crucial to provide some standardized characteristics of Treg products in order to minimize the problem. We have previously developed reporting guidelines called minimum information about tolerogenic antigen-presenting cells, which allows the comparison between different preparations of tolerance-inducing antigen-presenting cells. Having this experience, here we describe another minimum information about Tregs (MITREG). It is important to note that MITREG does not dictate how investigators should generate or characterize Tregs, but it does require investigators to report their Treg data in a consistent and transparent manner. We hope this will, therefore, be a useful tool facilitating standardized reporting on the manufacturing of Tregs, either for research purposes or for clinical application. This way MITREG might also be an important step toward more standardized and reproducible testing of the Tregs preparations in clinical applications.
View details for DOI 10.3389/fimmu.2017.01844
View details for Web of Science ID 000419897500001
View details for PubMedID 29379498
View details for PubMedCentralID PMC5775516
-
Peanut-specific type 1 regulatory T cells induced in vitro from allergic subjects are functionally impaired
JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY
2018; 141 (1): 202-+
Abstract
Peanut allergy (PA) is a life-threatening condition that lacks regulator-approved treatment. Regulatory T type 1 (TR1) cells are potent suppressors of immune responses and can be induced in vivo upon repeated antigen exposure or in vitro by using tolerogenic dendritic cells. Whether oral immunotherapy (OIT) leads to antigen-specific TR1 cell induction has not been established.We sought to determine whether peanut-specific TR1 cells can be generated in vitro from peripheral blood of patients with PA at baseline or during OIT and whether they are functional compared with peanut-specific TR1 cells induced from healthy control (HC) subjects.Tolerogenic dendritic cells were differentiated in the presence of IL-10 from PBMCs of patients with PA and HC subjects pulsed with the main peanut allergens of Arachis hypogaea, Ara h 1 and 2, and used as antigen-presenting cells for autologous CD4+ T cells (CD4+ T cells coincubated with tolerogenic dendritic cells pulsed with the main peanut allergens [pea-T10 cells]). Pea-T10 cells were characterized by the presence of CD49b+ lymphocyte-activation gene 3 (LAG3)+ TR1 cells, antigen-specific proliferative responses, and cytokine production.CD49b+LAG3+ TR1 cells were induced in pea-T10 cells at comparable percentages from HC subjects and patients with PA. Despite their antigen specificity, pea-T10 cells of patients with PA with or without OIT, as compared with those of HC subjects, were not anergic and had high TH2 cytokine production upon peanut-specific restimulation.Peanut-specific TR1 cells can be induced from HC subjects and patients with PA, but those from patients with PA are functionally defective independent of OIT. The unfavorable TR1/TH2 ratio is discussed as a possible cause of PA TR1 cell impairment.
View details for PubMedID 28689791
-
Identity and Diversity of Human Peripheral Th and T Regulatory Cells Defined by Single-Cell Mass Cytometry
JOURNAL OF IMMUNOLOGY
2018; 200 (1): 336–46
Abstract
Human CD3+CD4+ Th cells, FOXP3+ T regulatory (Treg) cells, and T regulatory type 1 (Tr1) cells are essential for ensuring peripheral immune response and tolerance, but the diversity of Th, Treg, and Tr1 cell subsets has not been fully characterized. Independent functional characterization of human Th1, Th2, Th17, T follicular helper (Tfh), Treg, and Tr1 cells has helped to define unique surface molecules, transcription factors, and signaling profiles for each subset. However, the adequacy of these markers to recapitulate the whole CD3+CD4+ T cell compartment remains questionable. In this study, we examined CD3+CD4+ T cell populations by single-cell mass cytometry. We characterize the CD3+CD4+ Th, Treg, and Tr1 cell populations simultaneously across 23 memory T cell-associated surface and intracellular molecules. High-dimensional analysis identified several new subsets, in addition to the already defined CD3+CD4+ Th, Treg, and Tr1 cell populations, for a total of 11 Th cell, 4 Treg, and 1 Tr1 cell subsets. Some of these subsets share markers previously thought to be selective for Treg, Th1, Th2, Th17, and Tfh cells, including CD194 (CCR4)+FOXP3+ Treg and CD183 (CXCR3)+T-bet+ Th17 cell subsets. Unsupervised clustering displayed a phenotypic organization of CD3+CD4+ T cells that confirmed their diversity but showed interrelation between the different subsets, including similarity between Th1-Th2-Tfh cell populations and Th17 cells, as well as similarity of Th2 cells with Treg cells. In conclusion, the use of single-cell mass cytometry provides a systems-level characterization of CD3+CD4+ T cells in healthy human blood, which represents an important baseline reference to investigate abnormalities of different subsets in immune-mediated pathologies.
View details for PubMedID 29180490
-
IL-10-Engineered Human CD4(+) Tr1 Cells Eliminate Myeloid Leukemia in an HLA Class I-Dependent Mechanism
MOLECULAR THERAPY
2017; 25 (10): 2254–69
Abstract
T regulatory cells (Tregs) play a key role in modulating T cell responses. Clinical trials showed that Tregs modulate graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, their ability to mediate anti-leukemic activity (graft-versus-leukemia [GvL]) is largely unknown. Enforced interleukin-10 (IL-10) expression converts human CD4+ T cells into T regulatory type 1 (Tr1)-like (CD4IL-10) cells that suppress effector T cells in vitro and xenoGvHD in humanized mouse models. In the present study, we show that CD4IL-10 cells mediate anti-leukemic effects in vitro and in vivo in a human leukocyte antigen (HLA) class I-dependent but antigen-independent manner. The cytotoxicity mediated by CD4IL-10 cells is granzyme B (GzB) dependent, is specific for CD13+ target cells, and requires CD54 and CD112 expression on primary leukemic target blasts. CD4IL-10 cells adoptively transferred in humanized mouse models directly mediate anti-tumor and anti-leukemic effects. In addition, when co-transferred with peripheral blood mononuclear cells (PBMCs), CD4IL-10 cells contribute to the GvL activity but suppress xenoGvHD mediated by the PBMCs. These findings provide for the first time a strong rationale for CD4IL-10 cell immunotherapy to prevent GvHD and promote GvL in allo-HSCT for myeloid malignancies.
View details for DOI 10.1016/j.ymthe.2017.06.029
View details for Web of Science ID 000412968200009
View details for PubMedID 28807569
View details for PubMedCentralID PMC5628869
-
gene therapy in Europe: paving the road for the next generation of advanced therapy medicinal products.
EMBO molecular medicine
2017; 9 (6): 737-740
View details for DOI 10.15252/emmm.201707573
View details for PubMedID 28396566
-
CRISPR-Based Gene Correction to Treat IPEX Syndrome
CELL PRESS. 2017: 168
View details for Web of Science ID 000401083600357
-
LV.InsulinB9-23/Anti-CD3 mAb Inhibits Recurrence of Autoimmunity in Diabetic NOD Mice After Islet Transplant
CELL PRESS. 2017: 96
View details for Web of Science ID 000401083600199
-
Lentiviral haemopoietic stem-cell gene therapy in early-onset metachromatic leukodystrophy: an ad-hoc analysis of a non-randomised, open-label, phase 1/2 trial.
Lancet
2016; 388 (10043): 476-487
Abstract
Metachromatic leukodystrophy (a deficiency of arylsulfatase A [ARSA]) is a fatal demyelinating lysosomal disease with no approved treatment. We aimed to assess the long-term outcomes in a cohort of patients with early-onset metachromatic leukodystrophy who underwent haemopoietic stem-cell gene therapy (HSC-GT).This is an ad-hoc analysis of data from an ongoing, non-randomised, open-label, single-arm phase 1/2 trial, in which we enrolled patients with a molecular and biochemical diagnosis of metachromatic leukodystrophy (presymptomatic late-infantile or early-juvenile disease or early-symptomatic early-juvenile disease) at the Paediatric Clinical Research Unit, Ospedale San Raffaele, in Milan. Trial participants received HSC-GT, which consisted of the infusion of autologous HSCs transduced with a lentiviral vector encoding ARSA cDNA, after exposure-targeted busulfan conditioning. The primary endpoints of the trial are safety (toxicity, absence of engraftment failure or delayed haematological reconstitution, and safety of lentiviral vector-tranduced cell infusion) and efficacy (improvement in Gross Motor Function Measure [GMFM] score relative to untreated historical controls, and ARSA activity, 24 months post-treatment) of HSC-GT. For this ad-hoc analysis, we assessed safety and efficacy outcomes in all patients who had received treatment and been followed up for at least 18 months post-treatment on June 1, 2015. This trial is registered with ClinicalTrials.gov, number NCT01560182.Between April, 2010, and February, 2013, we had enrolled nine children with a diagnosis of early-onset disease (six had late-infantile disease, two had early-juvenile disease, and one had early-onset disease that could not be definitively classified). At the time of analysis all children had survived, with a median follow-up of 36 months (range 18-54). The most commonly reported adverse events were cytopenia (reported in all patients) and mucositis of different grades of severity (in five of nine patients [grade 3 in four of five patients]). No serious adverse events related to the medicinal product were reported. Stable, sustained engraftment of gene-corrected HSCs was observed (a median of 60·4% [range 14·0-95·6] lentiviral vector-positive colony-forming cells across follow-up) and the engraftment level was stable during follow-up; engraftment determinants included the duration of absolute neutropenia and the vector copy number of the medicinal product. A progressive reconstitution of ARSA activity in circulating haemopoietic cells and in the cerebrospinal fluid was documented in all patients in association with a reduction of the storage material in peripheral nerve samples in six of seven patients. Eight patients, seven of whom received treatment when presymptomatic, had prevention of disease onset or halted disease progression as per clinical and instrumental assessment, compared with historical untreated control patients with early-onset disease. GMFM scores for six patients up to the last follow-up showed that gross motor performance was similar to that of normally developing children. The extent of benefit appeared to be influenced by the interval between HSC-GT and the expected time of disease onset. Treatment resulted in protection from CNS demyelination in eight patients and, in at least three patients, amelioration of peripheral nervous system abnormalities, with signs of remyelination at both sites.Our ad-hoc findings provide preliminary evidence of safety and therapeutic benefit of HSC-GT in patients with early-onset metachromatic leukodystrophy who received treatment in the presymptomatic or very early-symptomatic stage. The results of this trial will be reported when all 20 patients have achieved 3 years of follow-up.Italian Telethon Foundation and GlaxoSmithKline.
View details for DOI 10.1016/S0140-6736(16)30374-9
View details for PubMedID 27289174
-
In Vivo Tracking of Human Hematopoiesis Reveals Patterns of Clonal Dynamics during Early and Steady-State Reconstitution Phases.
Cell stem cell
2016; 19 (1): 107-119
Abstract
Hematopoietic stem/progenitor cells (HSPCs) are capable of supporting the lifelong production of blood cells exerting a wide spectrum of functions. Lentiviral vector HSPC gene therapy generates a human hematopoietic system stably marked at the clonal level by vector integration sites (ISs). Using IS analysis, we longitudinally tracked >89,000 clones from 15 distinct bone marrow and peripheral blood lineages purified up to 4 years after transplant in four Wiskott-Aldrich syndrome patients treated with HSPC gene therapy. We measured at the clonal level repopulating waves, populations' sizes and dynamics, activity of distinct HSPC subtypes, contribution of various progenitor classes during the early and late post-transplant phases, and hierarchical relationships among lineages. We discovered that in-vitro-manipulated HSPCs retain the ability to return to latency after transplant and can be physiologically reactivated, sustaining a stable hematopoietic output. This study constitutes in vivo comprehensive tracking in humans of hematopoietic clonal dynamics during the early and late post-transplant phases.
View details for DOI 10.1016/j.stem.2016.04.016
View details for PubMedID 27237736
View details for PubMedCentralID PMC4942697
-
Update on the safety and efficacy of retroviral gene therapy for immunodeficiency due to adenosine deaminase deficiency
BLOOD
2016; 128 (1): 45-54
Abstract
Adenosine deaminase (ADA) deficiency is a rare, autosomal-recessive systemic metabolic disease characterized by severe combined immunodeficiency (SCID). The treatment of choice for ADA-deficient SCID (ADA-SCID) is hematopoietic stem cell transplant from an HLA-matched sibling donor, although <25% of patients have such a donor available. Enzyme replacement therapy (ERT) partially and temporarily relieves immunodeficiency. We investigated the medium-term outcome of gene therapy (GT) in 18 patients with ADA-SCID for whom an HLA-identical family donor was not available; most were not responding well to ERT. Patients were treated with an autologous CD34(+)-enriched cell fraction that contained CD34(+) cells transduced with a retroviral vector encoding the human ADA complementary DNA sequence (GSK2696273) as part of single-arm, open-label studies or compassionate use programs. Overall survival was 100% over 2.3 to 13.4 years (median, 6.9 years). Gene-modified cells were stably present in multiple lineages throughout follow up. GT resulted in a sustained reduction in the severe infection rate from 1.17 events per person-year to 0.17 events per person-year (n = 17, patient 1 data not available). Immune reconstitution was demonstrated by normalization of T-cell subsets (CD3(+), CD4(+), and CD8(+)), evidence of thymopoiesis, and sustained T-cell proliferative capacity. B-cell function was evidenced by immunoglobulin production, decreased intravenous immunoglobulin use, and antibody response after vaccination. All 18 patients reported infections as adverse events; infections of respiratory and gastrointestinal tracts were reported most frequently. No events indicative of leukemic transformation were reported. Trial details were registered at www.clinicaltrials.gov as #NCT00598481.
View details for DOI 10.1182/blood-2016-01-688226
View details for Web of Science ID 000379249600012
View details for PubMedID 27129325
-
Gene Editing as a Therapeutic Approach to Treat IPEX Syndrome
NATURE PUBLISHING GROUP. 2016: S51
View details for DOI 10.1016/S1525-0016(16)32932-X
View details for Web of Science ID 000375264200124
-
LV.InsB9-23/Anti-CD3 mAb Inhibits Recurrence of Autoimmunity in NOD Mice After Islet Transplants
NATURE PUBLISHING GROUP. 2016: S23
View details for Web of Science ID 000375264200052
-
Targeting of Myeloid Leukemia by IL-10-Engineered Human CD4(+) Tr1 Cells
NATURE PUBLISHING GROUP. 2016: S252
View details for DOI 10.1016/S1525-0016(16)33445-1
View details for Web of Science ID 000375264200627
-
Concise Review: Cell-Based Therapies and Other Non-Traditional Approaches for Type 1 Diabetes
STEM CELLS
2016; 34 (4): 809-819
Abstract
The evolution of Type 1 diabetes (T1D) therapy has been marked by consecutive shifts, from insulin replacement to immunosuppressive drugs and targeted biologics (following the understanding that T1D is an autoimmune disease), and to more disease-specific or patient-oriented approaches such as antigen-specific and cell-based therapies, with a goal to provide efficacy, safety, and long-term protection. At the same time, another important paradigm shift from treatment of new onset T1D patients to prevention in high-risk individuals has taken place, based on the hypothesis that therapeutic approaches deemed sufficiently safe may show better efficacy if applied early enough to maintain endogenous β cell function, a concept supported by many preclinical studies. This new strategy has been made possible by capitalizing on a variety of biomarkers that can more reliably estimate the risk and rate of progression of the disease. More advanced ("omic"-based) biomarkers that also shed light on the underlying contributors of disease for each individual will be helpful to guide the choice of the most appropriate therapies, or combinations thereof. In this review, we present current efforts to stratify patients according to biomarkers and current alternatives to conventional drug-based therapies for T1D, with a special emphasis on cell-based therapies, their status in the clinic and potential for treatment and/or prevention.
View details for DOI 10.1002/stem.2290
View details for Web of Science ID 000374697700002
View details for PubMedID 26840009
-
LIPOPOLYSACCHARIDE-RESPONSIVE AND BEIGE-LIKE ANCHOR (LRBA) PROTEIN DEFICIENCY MANIFESTING WITH LYPODISTROPHY AND ALPS-LIKE PHENOTYPE TREATED WITH LEPTIN AND RAPAMYCIN
SPRINGER/PLENUM PUBLISHERS. 2016: 293–94
View details for Web of Science ID 000372165300149
-
From IPEX syndrome to FOXP3 mutation: a lesson on immune dysregulation.
Annals of the New York Academy of Sciences
2016
Abstract
Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a rare disorder that increasingly has gained attention as a model of genetic autoimmunity. Numerous papers documenting the key clinical and molecular characteristics of IPEX have provided a detailed understanding of this devastating disease. IPEX is a primary immunodeficiency caused by mutations in the gene FOXP3, which encodes an essential transcription factor required for maintenance of thymus-derived regulatory T (tTreg ) cells. tTreg cell dysfunction is the main pathogenic event leading to multiorgan autoimmunity in IPEX. In addition to the traditional clinical presentation (i.e., severe enteropathy, type 1 diabetes, and eczema), IPEX may encompass other variable and distinct clinical manifestations. As IPEX awareness and characterization have increased, so has identification of FOXP3 mutations, with at least 70 to date. Thus, while FOXP3 is the unifying gene, IPEX is a complex and diverse clinical continuum of disorders. Despite understanding IPEX pathogenesis, new treatment options have remained elusive, although early diagnosis led to hematopoietic stem cell transplantation (HSCT) and immunosuppression treatment and improved patient outcomes. Here, we review current knowledge about IPEX syndrome and highlight findings that could lead to novel targeted treatments.
View details for DOI 10.1111/nyas.13011
View details for PubMedID 26918796
-
Safety and Clinical Benefit of Lentiviral Hematopoietic Stem Cell Gene Therapy for Wiskott-Aldrich Syndrome
AMER SOC HEMATOLOGY. 2015
View details for Web of Science ID 000368019001011
-
Clinical Outlook for Type-1 and FOXP3(+) T Regulatory Cell-Based Therapy
FRONTIERS IN IMMUNOLOGY
2015; 6
Abstract
T regulatory cells (Tregs) are subsets of T lymphocytes specialized in modulating antigen-specific immune responses in vivo. Hence, Tregs represent an ideal therapeutic tool to control detrimental immune reactions. Based on solid pre-clinical results, investigators started testing the safety and efficacy of Treg-based therapies in humans. Despite promising results, a number of issues remain to be solved. We will discuss the results obtained from clinical trials and the challenges and risks we are facing in the further development of Treg-based therapies.
View details for DOI 10.3389/fimmu.2015.00593
View details for Web of Science ID 000366474100001
View details for PubMedID 26635807
View details for PubMedCentralID PMC4658444
-
Hurdles in therapy with regulatory T cells.
Science translational medicine
2015; 7 (304): 304ps18-?
Abstract
Improper activation of the immune system contributes to a variety of clinical conditions, including autoimmune and allergic diseases as well as solid organ and bone marrow transplantation. One approach to counteract this activation is through adoptive therapy with regulatory T cells (Tregs). Efforts to manufacture these cells have led to good maunfacturing practice-compliant protocols, and Treg products are entering early clinical trials. Here, we report the stance of the European Union Cooperation in Science and Technology Action BM1305, "Action to Focus and Accelerate Cell-based Tolerance-inducing Therapies-A FACTT," which identifies hurdles hindering Treg clinical applications in Europe and provides possible solutions.
View details for DOI 10.1126/scitranslmed.aaa7721
View details for PubMedID 26355029
-
B-cell reconstitution after lentiviral vector-mediated gene therapy in patients with Wiskott-Aldrich syndrome
JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY
2015; 136 (3): 692-?
View details for DOI 10.1016/j.jaci.2015.01.035
View details for Web of Science ID 000360913300022
-
Fatal autoimmunity in mice reconstituted with human hematopoietic stem cells encoding defective FOXP3
BLOOD
2015; 125 (25): 3886-3895
Abstract
Mice reconstituted with a human immune system provide a tractable in vivo model to assess human immune cell function. To date, reconstitution of murine strains with human hematopoietic stem cells (HSCs) from patients with monogenic immune disorders have not been reported. One obstacle precluding the development of immune-disease specific "humanized" mice is that optimal adaptive immune responses in current strains have required implantation of autologous human thymic tissue. To address this issue, we developed a mouse strain that lacks murine major histocompatibility complex class II (MHC II) and instead expresses human leukocyte antigen DR1 (HLA-DR1). These mice displayed improved adaptive immune responses when reconstituted with human HSCs including enhanced T-cell reconstitution, delayed-type hypersensitivity responses, and class-switch recombination. Following immune reconstitution of this novel strain with HSCs from a patient with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, associated with aberrant FOXP3 function, mice developed a lethal inflammatory disorder with multiorgan involvement and autoantibody production mimicking the pathology seen in affected humans. This humanized mouse model permits in vivo evaluation of immune responses associated with genetically altered HSCs, including primary immunodeficiencies, and should facilitate the study of human immune pathobiology and the development of targeted therapeutics.
View details for DOI 10.1182/blood-2014-12-618363
View details for Web of Science ID 000357283300011
View details for PubMedID 25833964
-
Insulin B chain 9-23 gene transfer to hepatocytes protects from type 1 diabetes by inducing Ag-specific FoxP3(+) T-regs
SCIENCE TRANSLATIONAL MEDICINE
2015; 7 (289)
Abstract
Antigen (Ag)-specific tolerance in type 1 diabetes (T1D) in human has not been achieved yet. Targeting lentiviral vector (LV)-mediated gene expression to hepatocytes induces active tolerance toward the encoded Ag. The insulin B chain 9-23 (InsB9-23) is an immunodominant T cell epitope in nonobese diabetic (NOD) mice. To determine whether auto-Ag gene transfer to hepatocytes induces tolerance and control of T1D, NOD mice were treated with integrase-competent LVs (ICLVs) that selectively target the expression of InsB9-23 to hepatocytes. ICLV treatment induced InsB9-23-specific effector T cells but also FoxP3(+) regulatory T cells (Tregs), which halted islet immune cell infiltration, and protected from T1D. Moreover, ICLV treatment combined with a single suboptimal dose of anti-CD3 monoclonal antibody (mAb) is effective in T1D reversal. Splenocytes from LV.InsB9-23-treated mice, but not from LV.OVA (ovalbumin)-treated control mice, stopped diabetes development, demonstrating that protection is Ag-specific. Depletion of CD4(+)CD25(+)FoxP3(+) T cells led to diabetes progression, indicating that Ag-specific FoxP3(+) Tregs mediate protection. Integrase-defective LVs (IDLVs).InsB9-23, which alleviate the concerns for insertional mutagenesis and support transient transgene expression in hepatocytes, were also efficient in protecting from T1D. These data demonstrate that hepatocyte-targeted auto-Ag gene expression prevents and resolves T1D and that stable integration of the transgene is not required for this protection. Gene transfer to hepatocytes can be used to induce Ag-specific tolerance in autoimmune diseases.
View details for DOI 10.1126/scitranslmed.aaa3032
View details for Web of Science ID 000355179800004
View details for PubMedID 26019217
-
Abnormalities of acid-base balance and predisposition to metabolic acidosis in Metachromatic Leukodystrophy patients
MOLECULAR GENETICS AND METABOLISM
2015; 115 (1): 48-52
Abstract
Metachromatic Leukodystrophy (MLD; MIM# 250100) is a rare inherited lysosomal storage disorder caused by the deficiency of Arylsulfatase A (ARSA). The enzymatic defect results in the accumulation of the ARSA substrate that is particularly relevant in myelin forming cells and leads to progressive dysmyelination and dysfunction of the central and peripheral nervous system. Sulfatide accumulation has also been reported in various visceral organs, although little is known about the potential clinical consequences of such accumulation. Different forms of MLD-associated gallbladder disease have been described, and there is one reported case of an MLD patient presenting with functional consequences of sulfatide accumulation in the kidney. Here we describe a wide cohort of MLD patients in whom a tendency to sub-clinical metabolic acidosis was observed. Furthermore in some of them we report episodes of metabolic acidosis of different grades of severity developed in acute clinical conditions of various origin. Importantly, we finally show how a careful acid-base balance monitoring and prompt correction of imbalances might prevent severe consequences of acidosis.
View details for DOI 10.1016/j.ymgme.2015.02.009
View details for Web of Science ID 000355031800008
View details for PubMedID 25796965
-
Insulin B9-23 LV-Driven Expression in Hepatocytes Combined With Suboptimal Dose of Anti-CD3 mAb Cures Type 1 Diabetes in NOD Mice
NATURE PUBLISHING GROUP. 2015: S69
View details for DOI 10.1016/S1525-0016(16)33778-9
View details for Web of Science ID 000359951700174
-
HLA-G expression levels influence the tolerogenic activity of human DC-10
HAEMATOLOGICA
2015; 100 (4): 551-560
View details for DOI 10.3324/haematol.2014.113803
View details for Web of Science ID 000354786300031
-
In vivo tracking of T cells in humans unveils decade-long survival and activity of genetically modified T memory stem cells
SCIENCE TRANSLATIONAL MEDICINE
2015; 7 (273)
Abstract
A definitive understanding of survival and differentiation potential in humans of T cell subpopulations is of paramount importance for the development of effective T cell therapies. In particular, uncovering the dynamics in vivo in humans of the recently described T memory stem cells (TSCM) would be crucial for therapeutic approaches that aim at taking advantage of a stable cellular vehicle with precursor potential. We exploited data derived from two gene therapy clinical trials for an inherited immunodeficiency, using either retrovirally engineered hematopoietic stem cells or mature lymphocytes to trace individual T cell clones directly in vivo in humans. We compared healthy donors and bone marrow-transplanted patients, studied long-term in vivo T cell composition under different clinical conditions, and specifically examined TSCM contribution according to age, conditioning regimen, disease background, cell source, long-term reconstitution, and ex vivo gene correction processing. High-throughput sequencing of retroviral vector integration sites (ISs) allowed tracing the fate of more than 1700 individual T cell clones in gene therapy patients after infusion of gene-corrected hematopoietic stem cells or mature lymphocytes. We shed light on long-term in vivo clonal relationships among different T cell subtypes, and we unveiled that TSCM are able to persist and to preserve their precursor potential in humans for up to 12 years after infusion of gene-corrected lymphocytes. Overall, this work provides high-resolution tracking of T cell fate and activity and validates, in humans, the safe and functional decade-long survival of engineered TSCM, paving the way for their future application in clinical settings.
View details for DOI 10.1126/scitranslmed.3010314
View details for Web of Science ID 000349700900002
View details for PubMedID 25653219
-
Sirolimus-based graft-versus-host disease prophylaxis promotes the in vivo expansion of regulatory T cells and permits peripheral blood stem cell transplantation from haploidentical donors
LEUKEMIA
2015; 29 (2): 396-405
Abstract
Hematopoietic stem cell transplantation (HSCT) from human leukocyte antigen (HLA) haploidentical family donors is a promising therapeutic option for high-risk hematologic malignancies. Here we explored in 121 patients, mostly with advanced stage diseases, a sirolimus-based, calcineurin-inhibitor-free prophylaxis of graft-versus-host disease (GvHD) to allow the infusion of unmanipulated peripheral blood stem cell (PBSC) grafts from partially HLA-matched family donors (TrRaMM study, Eudract 2007-5477-54). Conditioning regimen was based on treosulfan and fludarabine, and GvHD prophylaxis on antithymocyte globulin Fresenius (ATG-F), rituximab and oral administration of sirolimus and mycophenolate. Neutrophil and platelet engraftment occurred in median at 17 and 19 days after HSCT, respectively, and full donor chimerism was documented in patients' bone marrow since the first post-transplant evaluation. T-cell immune reconstitution was rapid, and high frequencies of circulating functional T-regulatory cells (Treg) were documented during sirolimus prophylaxis. Incidence of acute GvHD grade II-IV was 35%, and occurrence and severity correlated negatively with Treg frequency. Chronic GvHD incidence was 47%. At 3 years after HSCT, transpant-related mortality was 31%, relapse incidence 48% and overall survival 25%. In conclusion, GvHD prophylaxis with sirolimus-mycophenolate-ATG-F-rituximab promotes a rapid immune reconstitution skewed toward Tregs, allowing the infusion of unmanipulated haploidentical PBSC grafts.Leukemia advance online publication, 4 July 2014; doi:10.1038/leu.2014.180.
View details for DOI 10.1038/leu.2014.180
View details for Web of Science ID 000349445000016
View details for PubMedID 24897508
-
BAT2 and BAT3 polymorphisms as novel genetic risk factors for rejection after HLA-related SCT
BONE MARROW TRANSPLANTATION
2014; 49 (11): 1400-1404
Abstract
The genetic background of donor and recipient is an important factor determining the outcome of allogeneic hematopoietic SCT (allo-HSCT). We applied whole-genome analysis to investigate genetic variants-other than HLA class I and II-associated with negative outcome after HLA-identical sibling allo-HSCT in a cohort of 110 β-Thalassemic patients. We identified two single-nucleotide polymorphisms (SNPs) in BAT2 (A/G) and BAT3 (T/C) genes, SNP rs11538264 and SNP rs10484558, both located in the HLA class III region, in strong linkage disequilibrium between each other (R(2)=0.92). When considered as single SNP, none of them reached a significant association with graft rejection (nominal P<0.00001 for BAT2 SNP rs11538264, and P<0.0001 for BAT3 SNP rs10484558), whereas the BAT2/BAT3 A/C haplotype was present at significantly higher frequency in patients who rejected as compared to those with functional graft (30.0% vs 2.6%, nominal P=1.15 × 10(-8); and adjusted P=0.0071). The BAT2/BAT3 polymorphisms and specifically the A/C haplotype may represent a novel immunogenetic factor associated with graft rejection in patients undergoing allo-HSCT.Bone Marrow Transplantation advance online publication, 11 August 2014; doi:10.1038/bmt.2014.177.
View details for DOI 10.1038/bmt.2014.177
View details for Web of Science ID 000344927400010
View details for PubMedID 25111513
-
Mixed chimerism evolution is associated with T regulatory type 1 (Tr1) cells in a ß-thalassemic patient after haploidentical haematopoietic stem cell transplantation.
Chimerism
2014; 5 (3-4): 75-79
Abstract
In a cohort of β-Thalassemia (β-Thal) transplanted with haploidentical-HSCT we identified one transplanted patient characterized by persistent mixed chimerism (PMC) for several months after HSCT. In this unique β-Thal patient we assessed the donor engraftment overtime after transplantation, the potential loss of the non-shared HLA haplotype, and the presence of CD49b(+)LAG-3(+) T regulatory type 1 (Tr1) cells, previously demonstrated to be associated with PMC after HLA-related HSCT for β-Thal. The majority of the patient's erythrocytes were of donor origin, whereas T cells were initially mostly derived from the recipient, no HLA loss, but an increased frequency of circulating Tr1 cells were observed. For the first time, we showed that when the proportion of residual donor cells decreases, the frequency of CD49b(+)LAG-3(+) Tr1 cells declines, reaching the levels present in healthy subjects. These findings confirm previous results obtained in transplant related settings for β-Thal, and supported the central role of Tr1 cells in promoting and maintaining PMC after allo-HSCT.
View details for DOI 10.1080/19381956.2015.1103423
View details for PubMedID 26650878
-
B-cell development and functions and therapeutic options in adenosine deaminase-deficient patients
JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY
2014; 133 (3): 799-?
Abstract
Adenosine deaminase (ADA) deficiency causes severe cellular and humoral immune defects and dysregulation because of metabolic toxicity. Alterations in B-cell development and function have been poorly studied. Enzyme replacement therapy (ERT) and hematopoietic stem cell (HSC) gene therapy (GT) are therapeutic options for patients lacking a suitable bone marrow (BM) transplant donor.We sought to study alterations in B-cell development in ADA-deficient patients and investigate the ability of ERT and HSC-GT to restore normal B-cell differentiation and function.Flow cytometry was used to characterize B-cell development in BM and the periphery. The percentage of gene-corrected B cells was measured by using quantitative PCR. B cells were assessed for their capacity to proliferate and release IgM after stimulation.Despite the severe peripheral B-cell lymphopenia, patients with ADA-deficient severe combined immunodeficiency showed a partial block in central BM development. Treatment with ERT or HSC-GT reverted most BM alterations, but ERT led to immature B-cell expansion. In the periphery transitional B cells accumulated under ERT, and the defect in maturation persisted long-term. HSC-GT led to a progressive improvement in B-cell numbers and development, along with increased levels of gene correction. The strongest selective advantage for ADA-transduced cells occurred at the transition from immature to naive cells. B-cell proliferative responses and differentiation to immunoglobulin secreting IgM after B-cell receptor and Toll-like receptor triggering were severely impaired after ERT and improved significantly after HSC-GT.ADA-deficient patients show specific defects in B-cell development and functions that are differently corrected after ERT and HSC-GT.
View details for DOI 10.1016/j.jaci.2013.12.1043
View details for Web of Science ID 000332397600024
View details for PubMedID 24506932
-
Forkhead box P3: The Peacekeeper of the Immune System
INTERNATIONAL REVIEWS OF IMMUNOLOGY
2014; 33 (2): 129-145
Abstract
Ten years ago Forkhead box P3 (FOXP3) was discovered as master gene driving CD4(+)CD25(+) T cell regulatory (Treg) function. Since then, several layers of complexity have emerged in the regulation of its expression and function, which is not only exerted in Treg cells. While the mechanisms leading to the highly selective expression of FOXP3 in thymus-derived Treg cells still remain to be elucidated, we review here the current knowledge on the role of FOXP3 in the development of Treg cells and the direct and indirect consequences of FOXP3 mutations on multiple arms of the immune response. Finally, we summarize the newly acquired knowledge on the epigenetic regulation of FOXP3, still largely undefined in human cells.
View details for DOI 10.3109/08830185.2013.863303
View details for Web of Science ID 000332870100005
View details for PubMedID 24354325
-
Immunological outcome in haploicentical-HSC transplanted patients treated with IL-10-anergizec donor T Cells
FRONTIERS IN IMMUNOLOGY
2014; 5
Abstract
T-cell therapy after hematopoietic stem cell transplantation (HSCT) has been used alone or in combination with immunosuppression to cure hematologic malignancies and to prevent disease recurrence. Here, we describe the outcome of patients with high-risk/advanced stage hematologic malignancies, who received T-cell depleted (TCD) haploidentical-HSCT (haplo-HSCT) combined with donor T lymphocytes pretreated with IL-10 (ALT-TEN trial). IL-10-anergized donor T cells (IL-10-DLI) contained T regulatory type 1 (Tr1) cells specific for the host alloantigens, limiting donor-vs.-host-reactivity, and memory T cells able to respond to pathogens. IL-10-DLI were infused in 12 patients with the goal of improving immune reconstitution after haplo-HSCT without increasing the risk of graft-versus-host-disease (GvHD). IL-10-DLI led to fast immune reconstitution in five patients. In four out of the five patients, total T-cell counts, TCR-Vβ repertoire and T-cell functions progressively normalized after IL-10-DLI. These four patients are alive, in complete disease remission and immunosuppression-free at 7.2 years (median follow-up) after haplo-HSCT. Transient GvHD was observed in the immune reconstituted (IR) patients, despite persistent host-specific hypo-responsiveness of donor T cells in vitro and enrichment of cells with Tr1-specific biomarkers in vivo. Gene-expression profiles of IR patients showed a common signature of tolerance. This study provides the first indication of the feasibility of Tr1 cell-based therapy and paves way for the use of these Tr1 cells as adjuvant treatment for malignancies and immune-mediated disorders.
View details for DOI 10.3389/fimmu.2014.00016
View details for Web of Science ID 000354034700001
View details for PubMedCentralID PMC3907718
-
Tr1 cells and the counter-regulation of immunity: natural mechanisms and therapeutic applications.
Current topics in microbiology and immunology
2014; 380: 39-68
Abstract
T regulatory Type 1 (Tr1) cells are adaptive T regulatory cells characterized by the ability to secrete high levels of IL-10 and minimal amounts of IL-4 and IL-17. Recently, CD49b and LAG-3 have been identified as Tr1-cell-specific biomarkers in mice and humans. Tr1 cells suppress T-cell- and antigen-presenting cell- (APC) responses primarily via the secretion of IL-10 and TGF-β. In addition, Tr1 cells release granzyme B and perforin and kill myeloid cells. Tr1 cells inhibit T cell responses also via cell-contact dependent mechanisms mediated by CTLA-4 or PD-1, and by disrupting the metabolic state of T effector cells via the production of the ectoenzymes CD39 and CD73. Tr1 cells were first described in peripheral blood of patients who developed tolerance after HLA-mismatched fetal liver hematopoietic stem cell transplant. Since their discovery, Tr1 cells have been proven to be important in maintaining immunological homeostasis and preventing T-cell-mediated diseases. Furthermore, the possibility to generate and expand Tr1 cells in vitro has led to their utilization as cellular therapy in humans. In this chapter we summarize the unique and distinctive biological properties of Tr1 cells, the well-known and newly discovered Tr1-cell biomarkers, and the different methods to induce Tr1 cells in vitro and in vivo. We also address the role of Tr1 cells in infectious diseases, autoimmunity, and transplant rejection in different pre-clinical disease models and in patients. Finally, we highlight the pathological settings in which Tr1 cells can be beneficial to prevent or to cure the disease.
View details for DOI 10.1007/978-3-662-43492-5_3
View details for PubMedID 25004813
-
CD4(+) T Cells from IPEX Patients Convert into Functional and Stable Regulatory T Cells by FOXP3 Gene Transfer
SCIENCE TRANSLATIONAL MEDICINE
2013; 5 (215)
Abstract
In humans, mutations in the gene encoding for forkhead box P3 (FOXP3), a critically important transcription factor for CD4⁺CD25⁺ regulatory T (T(reg)) cell function, lead to a life-threatening systemic poly-autoimmune disease, known as immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. Severe autoimmunity results from the inborn dysfunction and instability of FOXP3-mutated T(reg) cells. Hematopoietic stem cell transplantation is the only current curative option for affected patients. We show here that when CD4⁺ T cells are converted into T(reg) cells after lentivirus-mediated FOXP3 gene transfer, the resulting CD4(FOXP3) T cell population displays stable phenotype and suppressive function, especially when naïve T cells are converted. We further demonstrate that CD4(FOXP3) T cells are stable in inflammatory conditions not only in vitro but also in vivo in a model of xenogeneic graft-versus-host disease. We therefore applied this FOXP3 gene transfer strategy for the development of a T(reg) cell-based therapeutic approach to restore tolerance in IPEX syndrome. IPEX-derived CD4(FOXP3) T cells mirrored T(reg) cells from healthy donors in terms of cellular markers, anergic phenotype, cytokine production, and suppressive function. These findings pave the way for the treatment of IPEX patients by adoptive cell therapy with genetically engineered T(reg) cells and are seminal for future potential application in patients with autoimmune disorders of different origin.
View details for DOI 10.1126/scitranslmed.3007320
View details for Web of Science ID 000328685500005
View details for PubMedID 24337481
-
Liver gene therapy by lentiviral vectors reverses anti-factor IX pre-existing immunity in haemophilic mice
EMBO MOLECULAR MEDICINE
2013; 5 (11): 1684-1697
Abstract
A major complication of factor replacement therapy for haemophilia is the development of anti-factor neutralizing antibodies (inhibitors). Here we show that liver gene therapy by lentiviral vectors (LVs) expressing factor IX (FIX) strongly reduces pre-existing anti-FIX antibodies and eradicates FIX inhibitors in haemophilia B mice. Concomitantly, plasma FIX levels and clotting activity rose to 50-100% of normal. The treatment was effective in 75% of treated mice. FIX-specific plasma cells (PCs) and memory B cells were reduced, likely because of memory B-cell depletion in response to constant exposure to high doses of FIX. Regulatory T cells displaying FIX-specific suppressive capacity were induced in gene therapy treated mice and controlled FIX-specific T helper cells. Gene therapy proved safer than a regimen mimicking immune tolerance induction (ITI) by repeated high-dose FIX protein administration, which induced severe anaphylactoid reactions in inhibitors-positive haemophilia B mice. Liver gene therapy can thus reverse pre-existing immunity, induce active tolerance to FIX and establish sustained FIX activity at therapeutic levels. These data position gene therapy as an attractive treatment option for inhibitors-positive haemophilic patients.
View details for DOI 10.1002/emmm.201302857
View details for Web of Science ID 000326463300004
View details for PubMedID 24106222
-
Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy.
Science
2013; 341 (6148): 1233158-?
Abstract
Metachromatic leukodystrophy (MLD) is an inherited lysosomal storage disease caused by arylsulfatase A (ARSA) deficiency. Patients with MLD exhibit progressive motor and cognitive impairment and die within a few years of symptom onset. We used a lentiviral vector to transfer a functional ARSA gene into hematopoietic stem cells (HSCs) from three presymptomatic patients who showed genetic, biochemical, and neurophysiological evidence of late infantile MLD. After reinfusion of the gene-corrected HSCs, the patients showed extensive and stable ARSA gene replacement, which led to high enzyme expression throughout hematopoietic lineages and in cerebrospinal fluid. Analyses of vector integrations revealed no evidence of aberrant clonal behavior. The disease did not manifest or progress in the three patients 7 to 21 months beyond the predicted age of symptom onset. These findings indicate that extensive genetic engineering of human hematopoiesis can be achieved with lentiviral vectors and that this approach may offer therapeutic benefit for MLD patients.
View details for DOI 10.1126/science.1233158
View details for PubMedID 23845948
-
Lentiviral Hematopoietic Stem Cell Gene Therapy Benefits Metachromatic Leukodystrophy
SCIENCE
2013; 341 (6148): 864-U58
View details for DOI 10.1126/science.1233158
View details for Web of Science ID 000323370600034
-
Lentiviral Hematopoietic Stem Cell Gene Therapy in Patients with Wiskott-Aldrich Syndrome
SCIENCE
2013; 341 (6148): 865-U71
View details for DOI 10.1126/science.1233151
View details for Web of Science ID 000323370600035
-
Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome.
Science
2013; 341 (6148): 1233151-?
Abstract
Wiskott-Aldrich syndrome (WAS) is an inherited immunodeficiency caused by mutations in the gene encoding WASP, a protein regulating the cytoskeleton. Hematopoietic stem/progenitor cell (HSPC) transplants can be curative, but, when matched donors are unavailable, infusion of autologous HSPCs modified ex vivo by gene therapy is an alternative approach. We used a lentiviral vector encoding functional WASP to genetically correct HSPCs from three WAS patients and reinfused the cells after a reduced-intensity conditioning regimen. All three patients showed stable engraftment of WASP-expressing cells and improvements in platelet counts, immune functions, and clinical scores. Vector integration analyses revealed highly polyclonal and multilineage haematopoiesis resulting from the gene-corrected HSPCs. Lentiviral gene therapy did not induce selection of integrations near oncogenes, and no aberrant clonal expansion was observed after 20 to 32 months. Although extended clinical observation is required to establish long-term safety, lentiviral gene therapy represents a promising treatment for WAS.
View details for DOI 10.1126/science.1233151
View details for PubMedID 23845947
-
Transplant Tolerance to Pancreatic Islets Is Initiated in the Graft and Sustained in the Spleen
AMERICAN JOURNAL OF TRANSPLANTATION
2013; 13 (8): 1963-1975
Abstract
The immune system is comprised of several CD4(+) T regulatory (Treg) cell types, of which two, the Foxp3(+) Treg and T regulatory type 1 (Tr1) cells, have frequently been associated with transplant tolerance. However, whether and how these two Treg-cell types synergize to promote allograft tolerance remains unknown. We previously developed a mouse model of allogeneic transplantation in which a specific immunomodulatory treatment leads to transplant tolerance through both Foxp3(+) Treg and Tr1 cells. Here, we show that Foxp3(+) Treg cells exert their regulatory function within the allograft and initiate engraftment locally and in a non-antigen (Ag) specific manner. Whereas CD4(+) CD25(-) T cells, which contain Tr1 cells, act from the spleen and are key to the maintenance of long-term tolerance. Importantly, the role of Foxp3(+) Treg and Tr1 cells is not redundant once they are simultaneously expanded/induced in the same host. Moreover, our data show that long-term tolerance induced by Foxp3(+) Treg-cell transfer is sustained by splenic Tr1 cells and functionally moves from the allograft to the spleen.
View details for DOI 10.1111/ajt.12333
View details for Web of Science ID 000322330000008
View details for PubMedID 23834659
-
Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells
NATURE MEDICINE
2013; 19 (6): 739-?
Abstract
CD4(+) type 1 T regulatory (Tr1) cells are induced in the periphery and have a pivotal role in promoting and maintaining tolerance. The absence of surface markers that uniquely identify Tr1 cells has limited their study and clinical applications. By gene expression profiling of human Tr1 cell clones, we identified the surface markers CD49b and lymphocyte activation gene 3 (LAG-3) as being stably and selectively coexpressed on mouse and human Tr1 cells. We showed the specificity of these markers in mouse models of intestinal inflammation and helminth infection and in the peripheral blood of healthy volunteers. The coexpression of CD49b and LAG-3 enables the isolation of highly suppressive human Tr1 cells from in vitro anergized cultures and allows the tracking of Tr1 cells in the peripheral blood of subjects who developed tolerance after allogeneic hematopoietic stem cell transplantation. The use of these markers makes it feasible to track Tr1 cells in vivo and purify Tr1 cells for cell therapy to induce or restore tolerance in subjects with immune-mediated diseases.
View details for DOI 10.1038/nm.3179
View details for Web of Science ID 000319981600023
View details for PubMedID 23624599
-
Regulatory T cells: recommendations to simplify the nomenclature.
Nature immunology
2013; 14 (4): 307-308
View details for DOI 10.1038/ni.2554
View details for PubMedID 23507634
-
HLA-G expressing DC-10 and CD4(+) T cells accumulate in human decidua during pregnancy
HUMAN IMMUNOLOGY
2013; 74 (4): 406-411
Abstract
Multiple mechanisms underlie the surprising willingness of mothers to tolerate the semi-allogeneic fetal tissues during pregnancy. Chief among these is the expression of the HLA-G molecules that has been largely demonstrated to be responsible for reprogramming the local maternal immune response towards tolerance. We recently identified a subset of tolerogenic dendritic cells, DC-10 that secrete high amounts of IL-10 and express high levels of HLA-G and its ligand ILT4. DC-10 are present in the peripheral blood and are essential in inducing adaptive regulatory T cells. We investigated the presence of DC-10 and HLA-G-expressing CD4(+) T cells in human decidua in the first trimester of pregnancy. Results showed that these cells are highly represented in human decidua as compared to the peripheral blood. This is the first report describing decidual DC-10 and CD4(+)HLA-G(+) T cells, strongly suggesting that they may accumulate or be induced at the fetal maternal interface to promote tolerance.
View details for DOI 10.1016/j.humimm.2012.11.031
View details for Web of Science ID 000317025100004
View details for PubMedID 23238214
-
Immune responses in liver-directed lentiviral gene therapy
TRANSLATIONAL RESEARCH
2013; 161 (4): 230-240
Abstract
The use of lentiviral vectors (LV)s for in vivo gene therapy is an ideal platform for treating many types of disease. Since LVs can transduce a wide array of cells, support long-term gene expression, and be modified to enhance cell targeting, LVs are a powerful modality to deliver life-long therapeutic proteins. A major limitation facing the use of LVs for in vivo gene therapy is the induction of immune responses, which can reduce the transduction efficiency of LV, eliminate the transduced cells, and inhibit the effect of the therapeutic protein. LV strategies designed to restrict transgene expression to the liver to exploit its naturally tolerogenic properties have proven to significantly reduce the induction of pathogenic immune responses and increase therapeutic efficacy. In this review, we outline the immunological hurdles facing in vivo LV gene therapy and highlight the advantages and limitations of using liver-directed LV gene therapy.
View details for DOI 10.1016/j.trsl.2012.12.018
View details for Web of Science ID 000316837400004
View details for PubMedID 23360745
-
Human IL2RA null mutation mediates immunodeficiency with lymphoproliferation and autoimmunity
CLINICAL IMMUNOLOGY
2013; 146 (3): 248-261
Abstract
Cell-surface CD25 expression is critical for maintaining immune function and homeostasis. As in few reported cases, CD25 deficiency manifests with severe autoimmune enteritis and viral infections. To dissect the underlying immunological mechanisms driving these symptoms, we analyzed the regulatory and effector T cell functions in a CD25 deficient patient harboring a novel IL2RA mutation. Pronounced lymphoproliferation, mainly of the CD8(+) T cells, was detected together with an increase in T cell activation markers and elevated serum cytokines. However, Ag-specific responses were impaired in vivo and in vitro. Activated CD8(+)STAT5(+) T cells with lytic potential infiltrated the skin, even though FOXP3(+) Tregs were present and maintained a higher capacity to respond to IL-2 compared to other T-cell subsets. Thus, the complex pathogenesis of CD25 deficiency provides invaluable insight into the role of IL2/IL-2RA-dependent regulation in autoimmunity and inflammatory diseases.
View details for DOI 10.1016/j.clim.2013.01.004
View details for Web of Science ID 000316241700010
View details for PubMedID 23416241
-
A novel function for FOXP3 in humans: intrinsic regulation of conventional T cells
BLOOD
2013; 121 (8): 1265-1275
Abstract
The role of forkhead box P3 (FOXP3) is well-established in T-regulatory cells, but the function of transient FOXP3 expression in activated human conventional T (Tconv) cells is unknown. In the present study, we used 2 approaches to determine the role of FOXP3 in human Tconv cells. First, we obtained Tconv clones from a female subject who is hemizygous for a null mutation in FOXP3, allowing the comparison of autologous T-cell clones that do or do not express FOXP3. Second, we knocked down activation-induced FOXP3 in Tconv cells from healthy donors with small interfering RNAagainst FOXP3. We found that FOXP3-deficient Tconv cells proliferate more and produce more cytokines than wild-type Tconv cells and have differential expression of 274 genes. We also investigated the role of FOXP3 in Th1 and Th17 cells and found that the expression of activation-induced FOXP3 was higher and more sustained in Th17 cells compared with Th1 cells. Knocking down FOXP3 expression in Th17 cells significantly increased the production of IFN-γ and decreased the expression of CCR4, but had no effect on IL-17 expression. These data reveal a novel function of FOXP3 in Tconv cells and suggest that expression of this protein is important in the function of multiple CD4(+) T-cell lineages.
View details for DOI 10.1182/blood-2012-05-431023
View details for Web of Science ID 000321750000011
View details for PubMedID 23169781
-
Preclinical Safety and Efficacy of Human CD34(+) Cells Transduced With Lentiviral Vector for the Treatment of Wiskott-Aldrich Syndrome
MOLECULAR THERAPY
2013; 21 (1): 175-184
Abstract
Gene therapy with ex vivo-transduced hematopoietic stem/progenitor cells may represent a valid therapeutic option for monogenic immunohematological disorders such as Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency associated with thrombocytopenia. We evaluated the preclinical safety and efficacy of human CD34(+) cells transduced with lentiviral vectors (LV) encoding WAS protein (WASp). We first set up and validated a transduction protocol for CD34(+) cells derived from bone marrow (BM) or mobilized peripheral blood (MPB) using a clinical grade, highly purified LV. Robust transduction of progenitor cells was obtained in normal donors and WAS patients' cells, without evidence of toxicity. To study biodistribution of human cells and exclude vector release in vivo, LV-transduced CD34(+) cells were transplanted in immunodeficient mice, showing a normal engraftment and differentiation ability towards transduced lymphoid and myeloid cells in hematopoietic tissues. Vector mobilization to host cells and transmission to germline cells of the LV were excluded by different molecular assays. Analysis of vector integrations showed polyclonal integration patterns in vitro and in human engrafted cells in vivo. In summary, this work establishes the preclinical safety and efficacy of human CD34(+) cells gene therapy for the treatment of WAS.
View details for DOI 10.1038/mt.2012.23
View details for Web of Science ID 000313035000020
View details for PubMedID 22371846
-
Cells Transduced With Lentiviral Vector for the Treatment of Wiskott-Aldrich Syndrome.
Molecular therapy : the journal of the American Society of Gene Therapy
2013; 21 (1): 175-184
Abstract
Gene therapy with ex vivo-transduced hematopoietic stem/progenitor cells may represent a valid therapeutic option for monogenic immunohematological disorders such as Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency associated with thrombocytopenia. We evaluated the preclinical safety and efficacy of human CD34(+) cells transduced with lentiviral vectors (LV) encoding WAS protein (WASp). We first set up and validated a transduction protocol for CD34(+) cells derived from bone marrow (BM) or mobilized peripheral blood (MPB) using a clinical grade, highly purified LV. Robust transduction of progenitor cells was obtained in normal donors and WAS patients' cells, without evidence of toxicity. To study biodistribution of human cells and exclude vector release in vivo, LV-transduced CD34(+) cells were transplanted in immunodeficient mice, showing a normal engraftment and differentiation ability towards transduced lymphoid and myeloid cells in hematopoietic tissues. Vector mobilization to host cells and transmission to germline cells of the LV were excluded by different molecular assays. Analysis of vector integrations showed polyclonal integration patterns in vitro and in human engrafted cells in vivo. In summary, this work establishes the preclinical safety and efficacy of human CD34(+) cells gene therapy for the treatment of WAS.
View details for DOI 10.1038/mt.2012.23
View details for PubMedID 28178602
-
Dendritic cell functional improvement in a preclinical model of lentiviral-mediated gene therapy for Wiskott-Aldrich syndrome
GENE THERAPY
2012; 19 (12): 1150-1158
Abstract
Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency caused by the defective expression of the WAS protein (WASP) in hematopoietic cells. It has been shown that dendritic cells (DCs) are functionally impaired in WAS patients and was(-/-) mice. We have previously demonstrated the efficacy and safety of a murine model of WAS gene therapy (GT), using stem cells transduced with a lentiviral vector (LV). The aim of this study was to investigate whether GT can correct DC defects in was(-/-) mice. As DCs expressing WASP were detected in the secondary lymphoid organs of the treated mice, we tested the in vitro and in vivo function of bone marrow-derived DCs (BMDCs). The BMDCs showed efficient in vitro uptake of latex beads and Salmonella typhimurium. When BMDCs from the treated mice (GT BMDCs) and the was(-/-) mice were injected into wild-type hosts, we found a higher number of cells that had migrated to the draining lymph nodes compared with mice injected with was(-/-) BMDCs. Finally, we found that ovalbumin (OVA)-pulsed GT BMDCs or vaccination of GT mice with anti-DEC205 OVA fusion protein can efficiently induce antigen-specific T-cell activation in vivo. These findings show that WAS GT significantly improves DC function, thus adding new evidence of the preclinical efficacy of LV-mediated WAS GT.
View details for DOI 10.1038/gt.2011.202
View details for Web of Science ID 000311997800004
View details for PubMedID 22189416
-
Enforced IL-10 Expression Confers Type 1 Regulatory T Cell (Tr1) Phenotype and Function to Human CD4(+) T Cells
MOLECULAR THERAPY
2012; 20 (9): 1778-1790
Abstract
Type 1 regulatory T (Tr1) cells are an inducible subset of CD4(+) Tr cells characterized by high levels of interleukin (IL)-10 production and regulatory properties. Several protocols to generate human Tr1 cells have been developed in vitro. However, the resulting population includes a significant fraction of contaminating non-Tr1 cells, representing a major bottleneck for clinical application of Tr1 cell therapy. We generated an homogeneous IL-10-producing Tr1 cell population by transducing human CD4(+) T cells with a bidirectional lentiviral vector (LV) encoding for human IL-10 and the marker gene, green fluorescent protein (GFP), which are independently coexpressed. The resulting GFP(+) LV-IL-10-transduced human CD4(+) T (CD4(LV-IL-10)) cells expressed, upon T-cell receptor (TCR) activation, high levels of IL-10 and concomitant low levels of IL-4, and markers associated with IL-10. Moreover, CD4(LV-IL-10) T cells displayed typical Tr1 features: the anergic phenotype, the IL-10, and transforming growth factor (TGF)-β dependent suppression of allogeneic T-cell responses, and the ability to suppress in a cell-to-cell contact independent manner in vitro. CD4(LV-IL-10) T cells were able to control xeno graft-versus-host disease (GvHD), demonstrating their suppressive function in vivo. These results show that constitutive over-expression of IL-10 in human CD4(+) T cells leads to a stable cell population that recapitulates the phenotype and function of Tr1 cells.
View details for DOI 10.1038/mt.2012.71
View details for Web of Science ID 000308519800015
View details for PubMedID 22692497
-
T Cells.
Molecular therapy : the journal of the American Society of Gene Therapy
2012; 20 (9): 1778-1790
Abstract
Type 1 regulatory T (Tr1) cells are an inducible subset of CD4(+) Tr cells characterized by high levels of interleukin (IL)-10 production and regulatory properties. Several protocols to generate human Tr1 cells have been developed in vitro. However, the resulting population includes a significant fraction of contaminating non-Tr1 cells, representing a major bottleneck for clinical application of Tr1 cell therapy. We generated an homogeneous IL-10-producing Tr1 cell population by transducing human CD4(+) T cells with a bidirectional lentiviral vector (LV) encoding for human IL-10 and the marker gene, green fluorescent protein (GFP), which are independently coexpressed. The resulting GFP(+) LV-IL-10-transduced human CD4(+) T (CD4(LV-IL-10)) cells expressed, upon T-cell receptor (TCR) activation, high levels of IL-10 and concomitant low levels of IL-4, and markers associated with IL-10. Moreover, CD4(LV-IL-10) T cells displayed typical Tr1 features: the anergic phenotype, the IL-10, and transforming growth factor (TGF)-β dependent suppression of allogeneic T-cell responses, and the ability to suppress in a cell-to-cell contact independent manner in vitro. CD4(LV-IL-10) T cells were able to control xeno graft-versus-host disease (GvHD), demonstrating their suppressive function in vivo. These results show that constitutive over-expression of IL-10 in human CD4(+) T cells leads to a stable cell population that recapitulates the phenotype and function of Tr1 cells.
View details for DOI 10.1038/mt.2012.71
View details for PubMedID 28157534
-
Genotypes and haplotypes in the 3' untranslated region of the HLA-G gene and their association with clinical outcome of hematopoietic stem cell transplantation for beta-thalassemia
TISSUE ANTIGENS
2012; 79 (5): 326-332
Abstract
Polymorphisms in the 3' untranslated region (3'UTR) of HLA-G, an important player in immunological tolerance, could be involved in post-transcriptional expression control, and their association with different clinical immune-related conditions including autoimmunity and transplantation is of mounting interest. Most studies have focused on a 14 base pair (bp) insertion/deletion (ins/del), while additional single-nucleotide polymorphisms (SNPs) in the HLA-G 3'UTR have been described but not extensively investigated for their clinical relevance. Here we have comparatively studied the association between 3'UTR haplotypes of HLA-G, or the 14 bp ins/del, with clinical outcome of HLA-identical sibling hematopoietic stem cell transplantation (HSCT) in 147 Middle Eastern beta-thalassemia patients. Sequence based typing of 3'UTR HLA-G polymorphisms in the patients and in 102 healthy Italian blood donors showed strong linkage disequilibrium between the 14 bp ins/del and five 3'UTR SNPs, which together could be arranged into eight distinct haplotypes based on expectation-maximization studies, with four predominant haplotypes (UTRs1-4). After HSCT, we found a moderate though not significant association between the presence of UTR-2 in double dose and protection from acute graft versus host disease (hazard ratio (HR) 0.45, 95% confidence intervals (CI): 0.14-1.45; P = 0.18), an effect that was also seen when the corresponding 14 bp ins/ins genotype was considered alone (HR 0.42, 95% CI: 0.16-1.06; P = 0.07). No association was found with rejection or survival. Taken together, our data show that there is no apparent added value of considering entire 3'UTR HLA-G haplotypes for risk prediction after allogeneic HSCT for beta-thalassemia.
View details for DOI 10.1111/j.1399-0039.2012.01862.x
View details for Web of Science ID 000302621200002
View details for PubMedID 22489942
-
Alterations in the adenosine metabolism and CD39/CD73 adenosinergic machinery cause loss of Treg cell function and autoimmunity in ADA-deficient SCID
BLOOD
2012; 119 (6): 1428-1439
Abstract
Adenosine acts as anti-inflammatory mediator on the immune system and has been described in regulatory T cell (Treg)-mediated suppression. In the absence of adenosine deaminase (ADA), adenosine and other purine metabolites accumulate, leading to severe immunodeficiency with recurrent infections (ADA-SCID). Particularly ADA-deficient patients with late-onset forms and after enzyme replacement therapy (PEG-ADA) are known to manifest immune dysregulation. Herein we provide evidence that alterations in the purine metabolism interfere with Treg function, thereby contributing to autoimmune manifestations in ADA deficiency. Tregs isolated from PEG-ADA-treated patients are reduced in number and show decreased suppressive activity, whereas they are corrected after gene therapy. Untreated murine ADA(-/-) Tregs show alterations in the plasma membrane CD39/CD73 ectonucleotidase machinery and limited suppressive activity via extracellular adenosine. PEG-ADA-treated mice developed multiple autoantibodies and hypothyroidism in contrast to mice treated with bone marrow transplantation or gene therapy. Tregs isolated from PEG-ADA-treated mice lacked suppressive activity, suggesting that this treatment interferes with Treg functionality. The alterations in the CD39/CD73 adenosinergic machinery and loss of function in ADA-deficient Tregs provide new insights into a predisposition to autoimmunity and the underlying mechanisms causing defective peripheral tolerance in ADA-SCID.
View details for DOI 10.1182/blood-2011-07-366781
View details for Web of Science ID 000300420900019
View details for PubMedID 22184407
-
Demethylation analysis of the FOXP3 locus shows quantitative defects of regulatory T cells in IPEX-like syndrome
JOURNAL OF AUTOIMMUNITY
2012; 38 (1): 49-58
Abstract
Immune dysregulation, Polyendocrinopathy, Enteropathy X-linked (IPEX) syndrome is a unique example of primary immunodeficiency characterized by autoimmune manifestations due to defective regulatory T (Treg) cells, in the presence of FOXP3 mutations. However, autoimmune symptoms phenotypically resembling IPEX often occur in the absence of detectable FOXP3 mutations. The cause of this "IPEX-like" syndrome presently remains unclear. To investigate whether a defect in Treg cells sustains the immunological dysregulation in IPEX-like patients, we measured the amount of peripheral Treg cells within the CD3(+) T cells by analysing demethylation of the Treg cell-Specific-Demethylated-Region (TSDR) in the FOXP3 locus and demethylation of the T cell-Specific-Demethylated-Region (TLSDR) in the CD3 locus, highly specific markers for stable Treg cells and overall T cells, respectively. TSDR demethylation analysis, alone or normalized for the total T cells, showed that the amount of peripheral Treg cells in a cohort of IPEX-like patients was significantly reduced, as compared to both healthy subjects and unrelated disease controls. This reduction could not be displayed by flow cytometric analysis, showing highly variable percentages of FOXP3(+) and CD25(+)FOXP3(+) T cells. These data provide evidence that a quantitative defect of Treg cells could be considered a common biological hallmark of IPEX-like syndrome. Since Treg cell suppressive function was not impaired, we propose that this reduction per se could sustain autoimmunity.
View details for DOI 10.1016/j.jaut.2011.12.009
View details for Web of Science ID 000301313400006
View details for PubMedID 22264504
-
The cellular and molecular mechanisms of immuno-suppression by human type 1 regulatory T cells.
Frontiers in immunology
2012; 3: 30-?
Abstract
The immuno-regulatory mechanisms of IL-10-producing type 1 regulatory T (Tr1) cells have been widely studied over the years. However, several recent discoveries have shed new light on the cellular and molecular mechanisms that human Tr1 cells use to control immune responses and induce tolerance. In this review we outline the well known and newly discovered regulatory properties of human Tr1 cells and provide an in-depth comparison of the known suppressor mechanisms of Tr1 cells with FOXP3(+) T(reg). We also highlight the role that Tr1 cells play in promoting and maintaining tolerance in autoimmunity, allergy, and transplantation.
View details for DOI 10.3389/fimmu.2012.00030
View details for PubMedID 22566914
-
The cellular and molecular mechanisms of immuno-suppression by human type 1 regulatory T cells
FRONTIERS IN IMMUNOLOGY
2012; 3
View details for DOI 10.3389/fimmu.2012.00030
View details for Web of Science ID 000209501300030
-
Health related quality of life in Middle Eastern children with beta-thalassemia.
BMC blood disorders
2012; 12: 6-?
Abstract
Thalassemia is a common disorder worldwide with a predominant incidence in Mediterranean countries, North Africa, the Middle East, India, Central Asia, and Southeast Asia. Whilst substantial progress has been made towards the improvement of Health related quality of life (HRQoL) in western countries, scarce evidence-based data exists on HRQol of thalassemia children and adolescents living in developing countries.We studied 60 thalassemia children from Middle Eastern countries with a median age of 10 years (range 5 to 17 years). HRQoL was assessed with the Pediatric Quality of Life Inventory (PedsQL) 4.0. The Questionnaire was completed at baseline by all patients and their parents. The agreement between child-self and parent-proxy HRQoL reports and the relationship between HRQoL profiles and socio-demographic and clinical factors were investigated.The scores of parents were generally lower than those of their children for Emotional Functioning (mean 75 vs 85; p = 0.002), Psychosocial Health Summary (mean 70.3 vs 79.1; p = 0.015) and the Total Summary Score (mean 74.3 vs 77.7 p = 0.047). HRQoL was not associated with ferritin levels, hepatomegaly or frequency of transfusions or iron chelation therapy. Multivariate analysis showed that a delayed start of iron chelation had a negative impact on total PedsQL scores of both children (p = 0.046) and their parents (p = 0.007).The PedsQL 4.0 is a useful tool for the measurement of HRQoL in pediatric thalassemia patients. This study shows that delayed start of iron chelation has a negative impact on children's HRQoL.
View details for DOI 10.1186/1471-2326-12-6
View details for PubMedID 22726530
-
Rapamycin Combined with Anti-CD45RB mAb and IL-10 or with G-CSF Induces Tolerance in a Stringent Mouse Model of Islet Transplantation
PLOS ONE
2011; 6 (12)
Abstract
A large pool of preexisting alloreactive effector T cells can cause allogeneic graft rejection following transplantation. However, it is possible to induce transplant tolerance by altering the balance between effector and regulatory T (Treg) cells. Among the various Treg-cell types, Foxp3(+)Treg and IL-10-producing T regulatory type 1 (Tr1) cells have frequently been associated with tolerance following transplantation in both mice and humans. Previously, we demonstrated that rapamycin+IL-10 promotes Tr1-cell-associated tolerance in Balb/c mice transplanted with C57BL/6 pancreatic islets. However, this same treatment was unsuccessful in C57BL/6 mice transplanted with Balb/c islets (classified as a stringent transplant model). We accordingly designed a protocol that would be effective in the latter transplant model by simultaneously depleting effector T cells and fostering production of Treg cells. We additionally developed and tested a clinically translatable protocol that used no depleting agent.Diabetic C57BL/6 mice were transplanted with Balb/c pancreatic islets. Recipient mice transiently treated with anti-CD45RB mAb+rapamycin+IL-10 developed antigen-specific tolerance. During treatment, Foxp3(+)Treg cells were momentarily enriched in the blood, followed by accumulation in the graft and draining lymph node, whereas CD4(+)IL-10(+)IL-4(-) T (i.e., Tr1) cells localized in the spleen. In long-term tolerant mice, only CD4(+)IL-10(+)IL-4(-) T cells remained enriched in the spleen and IL-10 was key in the maintenance of tolerance. Alternatively, recipient mice were treated with two compounds routinely used in the clinic (namely, rapamycin and G-CSF); this drug combination promoted tolerance associated with CD4(+)IL-10(+)IL-4(-) T cells.The anti-CD45RB mAb+rapamycin+IL-10 combined protocol promotes a state of tolerance that is IL-10 dependent. Moreover, the combination of rapamycin+G-CSF induces tolerance and such treatment could be readily translatable into the clinic.
View details for DOI 10.1371/journal.pone.0028434
View details for Web of Science ID 000298365700036
View details for PubMedID 22174806
-
Forkhead box protein 3 (FOXP3) mutations lead to increased T(H)17 cell numbers and regulatory T-cell instability
JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY
2011; 128 (6): 1376-U790
View details for DOI 10.1016/j.jaci.2011.09.010
View details for Web of Science ID 000298342700040
View details for PubMedID 22000569
-
Stability of human rapamycin-expanded CD4(+)CD25(+) T regulatory cells
HAEMATOLOGICA-THE HEMATOLOGY JOURNAL
2011; 96 (9): 1357-1365
Abstract
The clinical use of ex vivo-expanded T-regulatory cells for the treatment of T-cell-mediated diseases has gained increasing momentum. However, the recent demonstration that FOXP3(+) T-regulatory cells may contain interleukin-17-producing cells and that they can convert into effector cells once transferred in vivo raises significant doubts about their safety. We previously showed that rapamycin permits the ex vivo expansion of FOXP3(+) T-regulatory cells while impairing the proliferation of non-T-regulatory cells. Here we investigated the Th17-cell content and the in vivo stability of rapamycin-expanded T-regulatory cells as pertinent aspects of cell-based therapy.T-regulatory-enriched cells were isolated from healthy volunteers and were expanded ex vivo with rapamycin with a pre-clinical applicable protocol. T-regulatory cells cultured with and without rapamycin were compared for their regulatory activity, content of pro-inflammatory cells and stability.We found that CD4(+)CCR6(+)CD161(+) T cells (i.e., precursor/committed Th17 cells) contaminate the T-regulatory cells cultured ex vivo in the absence of rapamycin. In addition, Th17 cells do not expand when rapamycin-treated T-regulatory cells are exposed to a "Th17-favorable" environment. Rapamycin-expanded T-regulatory cells maintain their in vitro regulatory phenotype even after in vivo transfer into immunodeficient NOD-SCID mice despite being exposed to the irradiation-induced pro-inflammatory environment. Importantly, no additional rapamycin treatment, either in vitro or in vivo, is required to keep their phenotype fixed.These data demonstrate that rapamycin secures ex vivo-expanded human T-regulatory cells and provide additional justification for their clinical use in future cell therapy-based trials.
View details for DOI 10.3324/haematol.2011.041483
View details for Web of Science ID 000295219500019
View details for PubMedID 21565906
-
Killing of myeloid APCs via HLA class I, CD2 and CD226 defines a novel mechanism of suppression by human Tr1 cells
EUROPEAN JOURNAL OF IMMUNOLOGY
2011; 41 (6): 1652-1662
Abstract
IL-10-producing CD4(+) type 1 regulatory T (Tr1) cells, defined based on their ability to produce high levels of IL-10 in the absence of IL-4, are major players in the induction and maintenance of peripheral tolerance. Tr1 cells inhibit T-cell responses mainly via cytokine-dependent mechanisms. The cellular and molecular mechanisms underlying the suppression of APC by Tr1 cells are still not completely elucidated. Here, we defined that Tr1 cells specifically lyse myeloid APC through a granzyme B (GZB)- and perforin (PRF)-dependent mechanism that requires HLA class I recognition, CD54/lymphocyte function-associated antigen (LFA)-1 adhesion, and activation via killer cell Ig-like receptors (KIRs) and CD2. Notably, interaction between CD226 on Tr1 cells and their ligands on myeloid cells, leading to Tr1-cell activation, is necessary for defining Tr1-cell target specificity. We also showed that high frequency of GZB-expressing CD4(+) T cells is detected in tolerant patients and correlates with elevated occurrence of IL-10-producing CD4(+) T cells. In conclusion, the modulatory activities of Tr1 cells are not only due to suppressive cytokines but also to specific cell-to-cell interactions that lead to selective killing of myeloid cells and possibly bystander suppression.
View details for DOI 10.1002/eji.201041120
View details for Web of Science ID 000291559700016
View details for PubMedID 21469116
-
In vivo T-cell dynamics during immune reconstitution after hematopoietic stem cell gene therapy in adenosine deaminase severe combined immune deficiency
JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY
2011; 127 (6): 1368-U95
Abstract
Gene therapy (GT) with hematopoietic stem cells is a promising treatment for inherited immunodeficiencies.Limited information is available on the relative contribution of de novo thymopoiesis and peripheral expansion to T-cell reconstitution after GT as well as on the potential effects of gene transfer on hematopoietic stem cells and lymphocyte replicative lifespan. We studied these issues in patients affected by adenosine deaminase severe combined immune deficiency after low-intensity conditioning and reinfusion of retrovirally transduced autologous CD34(+) cells.Immunophenotype, proliferative status, telomere length, and T-cell receptor excision circles were investigated at early and late time points (up to 9 years) after GT treatment. Control groups consisted of pediatric healthy donors and patients undergoing allogeneic bone marrow transplantation (BMT).We observed no telomere shortening in the bone marrow compartment and in granulocytes, whereas peripheral blood naive T cells from both GT and BMT patients showed a significant reduction in telomere length compared with healthy controls. This was in agreement with the presence of a high fraction of actively cycling naive and memory T cells and lower T-cell receptor excision circles.These data indicate that T-cell homeostatic expansion contributes substantially to immune reconstitution, like BMT, and is not associated with senescence in the stem cell compartment.
View details for DOI 10.1016/j.jaci.2011.03.004
View details for Web of Science ID 000291048500007
View details for PubMedID 21477850
-
Prospective Assessment of Health-Related Quality of Life in Pediatric Patients with Beta-Thalassemia following Hematopoietic Stem Cell Transplantation
BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION
2011; 17 (6): 861-866
Abstract
Although hematopoietic stem cell transplantation (HSCT) has been widely used to treat pediatric patients with beta-thalassemia major, evidence showing whether this treatment improves health-related quality of life (HRQoL) is lacking. We used child-self and parent-proxy reports to prospectively evaluate HRQoL in 28 children with beta-thalassemia from Middle Eastern countries who underwent allogeneic HSCT in Italy. The PedsQL 4.0 Generic Core Scales were administered to patients and their parents 1 month before and 3, 6, and 18 months after transplantation. Two-year overall survival, thalassemia-free survival, mortality, and rejection were 89.3%, 78.6%, 10.9% and 14.3%, respectively. The cumulative incidence of acute and chronic graft-versus-host disease (GVHD) was 36% and 18%, respectively. Physical functioning declined significantly from baseline to 3 months after HSCT (median PedsQL score, 81.3 vs 62.5; P = .02), but then increased significantly up to 18 months after HSCT (median score, 93.7; P = .04). Agreement between child-self and parent-proxy ratings was high. Chronic GVHD was the most significant factor associated with lower HRQoL scores over time (P = .02). The child-self and parent-proxy reports showed improved HRQoL in the children with beta-thalassemia after HSCT. Overall, our study provides preliminary evidence-based data to further support clinical decision making in this area.
View details for DOI 10.1016/j.bbmt.2010.09.011
View details for Web of Science ID 000291194000009
View details for PubMedID 20870029
-
Lentiviral-mediated gene therapy leads to improvement of B-cell functionality in a murine model of Wiskott-Aldrich syndrome
JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY
2011; 127 (6): 1376-U109
Abstract
Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency characterized by thrombocytopenia, eczema, infections, autoimmunity, and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical donors is curative, but it is not available to all patients. We have developed a gene therapy (GT) approach for WAS by using a lentiviral vector encoding for human WAS promoter/cDNA (w1.6W) and demonstrated its preclinical efficacy and safety.To evaluate B-cell reconstitution and correction of B-cell phenotype in GT-treated mice.We transplanted Was(-/-) mice sublethally irradiated (700 rads) with lineage marker-depleted bone marrow wild-type cells, Was(-/-) cells untransduced or transduced with the w1.6W lentiviral vector and analyzed B-cell reconstitution in bone marrow, spleen, and peritoneum.Here we show that WAS protein(+) B cells were present in central and peripheral B-cell compartments from GT-treated mice and displayed the strongest selective advantage in the splenic marginal zone and peritoneal B1 cell subsets. After GT, splenic architecture was improved and B-cell functions were restored, as demonstrated by the improved antibody response to pneumococcal antigens and the reduction of serum IgG autoantibodies.WAS GT leads to improvement of B-cell functions, even in the presence of a mixed chimerism, further validating the clinical application of the w1.6W lentiviral vector.
View details for DOI 10.1016/j.jaci.2011.03.030
View details for Web of Science ID 000291048500008
View details for PubMedID 21531013
-
Immune intervention with T regulatory cells: Past lessons and future perspectives for type 1 diabetes
SEMINARS IN IMMUNOLOGY
2011; 23 (3): 182-194
Abstract
In type 1 diabetes (T1D), insulin-producing pancreatic β-cells are attacked and destroyed by the immune system. Although man-made insulin is life-saving, it is not a cure and it cannot prevent long-term complications. In addition, most T1D patients would do almost anything to achieve release from the burden of daily glucose monitoring and insulin injection. Despite the formation of very large and promising clinical trials, a means to prevent/cure T1D in humans remains elusive. This has led to an increasing interest in the possibility of using T cells with regulatory properties (Treg cells) as a biological therapy to preserve and restore tolerance to self-antigens. In the present review we will attempt to consolidate learning from the past and to describe what we now believe could in the future become a successful Treg-cell based immune intervention in T1D.
View details for DOI 10.1016/j.smim.2011.07.007
View details for Web of Science ID 000295393300005
View details for PubMedID 21831659
-
Hepatocyte-Targeted Expression by Integrase-Defective Lentiviral Vectors Induces Antigen-Specific Tolerance in Mice with Low Genotoxic Risk
HEPATOLOGY
2011; 53 (5): 1696-1707
Abstract
Lentiviral vectors are attractive tools for liver-directed gene therapy because of their capacity for stable gene expression and the lack of preexisting immunity in most human subjects. However, the use of integrating vectors may raise some concerns about the potential risk of insertional mutagenesis. Here we investigated liver gene transfer by integrase-defective lentiviral vectors (IDLVs) containing an inactivating mutation in the integrase (D64V). Hepatocyte-targeted expression using IDLVs resulted in the sustained and robust induction of immune tolerance to both intracellular and secreted proteins, despite the reduced transgene expression levels in comparison with their integrase-competent vector counterparts. IDLV-mediated and hepatocyte-targeted coagulation factor IX (FIX) expression prevented the induction of neutralizing antibodies to FIX even after antigen rechallenge in hemophilia B mice and accounted for relatively prolonged therapeutic FIX expression levels. Upon the delivery of intracellular model antigens, hepatocyte-targeted IDLVs induced transgene-specific regulatory T cells that contributed to the observed immune tolerance. Deep sequencing of IDLV-transduced livers showed only rare genomic integrations that had no preference for gene coding regions and occurred mostly by a mechanism inconsistent with residual integrase activity.IDLVs provide an attractive platform for the tolerogenic expression of intracellular or secreted proteins in the liver with a substantially reduced risk of insertional mutagenesis.
View details for DOI 10.1002/hep.24230
View details for Web of Science ID 000289956100030
View details for PubMedID 21520180
-
Clinical tolerance in allogeneic hematopoietic stem cell transplantation
IMMUNOLOGICAL REVIEWS
2011; 241: 145-163
Abstract
Allogeneic hematopoietic stem cell transplantation (HSCT) has been a curative therapeutic option for a wide range of immune hematologic malignant and non-malignant disorders including genetic diseases and inborn errors. Once in the host, allogeneic transplanted cells have not only to ensure myeloid repopulation and immunological reconstitution but also to acquire tolerance to host human leukocyte antigens via central or peripheral mechanisms. Peripheral tolerance after allogeneic HSCT depends on several regulatory mechanisms aimed at blocking alloimmune reactivity while preserving immune responses to pathogens and tumor antigens. Patients transplanted with HSCT represent an ideal model system in humans to identify and characterize the key cellular and molecular players underlying these mechanisms. The knowledge gained from these studies has allowed the development of novel therapeutic strategies aimed at inducing long-term peripheral tolerance, which can be applicable not only in allogeneic HSCT but also in autoimmune diseases and solid-organ transplantation. In the present review, we describe Type 1 regulatory T cells, initially discovered and characterized in chimeric patients transplanted with human leukocyte antigen-mismatched HSCT, and how their presence correlates to tolerance induction and maintenance. Furthermore, we summarize different cell therapy approaches with regulatory T cells, designed to facilitate tolerance induction, minimizing pharmaceutical interventions.
View details for DOI 10.1111/j.1600-065X.2011.01010.x
View details for Web of Science ID 000289468700011
View details for PubMedID 21488896
-
Th17 Cells Express Interleukin-10 Receptor and Are Controlled by Foxp3(-) and Foxp3(+) Regulatory CD4(+) T Cells in an Interleukin-10-Dependent Manner
IMMUNITY
2011; 34 (4): 554-565
Abstract
T helper 17 (Th17) cells are important for host defense against extracellular microorganisms. However, they are also implicated in autoimmune and chronic inflammatory diseases, and as such need to be tightly regulated. The mechanisms that directly control committed pathogenic Th17 cells in vivo remain unclear. We showed here that IL-17A-producing CD4+ T cells expressed interleukin-10 receptor α (IL-10Rα) in vivo. Importantly, T cell-specific blockade of IL-10 signaling led to a selective increase of IL-17A+IFN-γ⁻ (Th17) and IL-17A+IFN-γ+ (Th17+Th1) CD4+ T cells during intestinal inflammation in the small intestine. CD4+Foxp3⁻ IL-10-producing (Tr1) cells and CD4+Foxp3+ regulatory (Treg) cells were able to control Th17 and Th17+Th1 cells in an IL-10-dependent manner in vivo. Lastly, IL-10 treatment of mice with established colitis decreased Th17 and Th17+Th1 cell frequencies via direct signaling in T cells. Thus, IL-10 signaling directly suppresses Th17 and Th17+Th1 cells.
View details for DOI 10.1016/j.immuni.2011.01.020
View details for Web of Science ID 000290367300014
View details for PubMedID 21511184
-
Functional type 1 regulatory T cells develop regardless of FOXP3 mutations in patients with IPEX syndrome
EUROPEAN JOURNAL OF IMMUNOLOGY
2011; 41 (4): 1120-1131
Abstract
Mutations of forkhead box p3 (FOXP3), the master gene for naturally occurring regulatory T cells (nTregs), are responsible for the impaired function of nTregs, resulting in an autoimmune disease known as the immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. The relevance of other peripheral tolerance mechanisms, such as the presence and function of type 1 regulatory T (Tr1) cells, the major adaptive IL-10-producing Treg subset, in patients with IPEX syndrome remains to be clarified. FOXP3(mutated) Tr1-polarized cells, differentiated in vitro from CD4(+) T cells of four IPEX patients, were enriched in IL-10(+) IL-4(-) IFN-γ(+) T cells, a cytokine production profile specific for Tr1 cells, and expressed low levels of FOXP3 and high levels of Granzyme-B. IPEX Tr1 cells were hypoproliferative and suppressive, thus indicating that FOXP3 mutations did not impair their function. Furthermore, we isolated Tr1 cell clones from the peripheral blood of one FOXP3(null) patient, demonstrating that Tr1 cells are present in vivo and they can be expanded in vitro in the absence of WT FOXP3. Overall, our results (i) show that functional Tr1 cells differentiate independently of FOXP3, (ii) confirm that human Tr1 and nTregs are distinct T-cell lineages, and (iii) suggest that under favorable conditions Tr1 cells could exert regulatory functions in IPEX patients.
View details for DOI 10.1002/eji.201040909
View details for Web of Science ID 000288821000025
View details for PubMedID 21400500
-
Bone Marrow as a Source of Hematopoietic Stem Cells for Human Gene Therapy of beta-Thalassemia
HUMAN GENE THERAPY
2011; 22 (4): 507-513
Abstract
β-Thalassemia is a severe inherited anemia caused by insufficient production of β-globin chains. Allogeneic hematopoietic stem cell (HSC) transplantation is currently the only cure, and is limited by donor availability and regimen-related toxicity and mortality. Gene therapy is a promising therapeutic tool for all thalassemic patients lacking a compatible donor and potentially provides transfusion independence in the absence of transplant-related complications, such as graft rejection and graft-versus-host disease. The issue of HSC procurement is critical in this setting because of the specific features of thalassemic syndromes, which include bone marrow (BM) expansion, ineffective erythropoiesis, and splenomegaly. Little is known about the efficiency of CD34(+) cell yield from steady-state BM harvests from thalassemic patients. We have collected data on safety and cell yield from 20 pediatric patients with β-thalassemia who underwent autologous BM harvest before allogeneic HSC transplantation, and from 49 age-matched sibling donors who also underwent BM harvest. The procedure was safe, as no significant adverse events occurred. In terms of cell yield, no difference was found between patients and normal donors in the number of CD34(+) cells and total nucleated cells harvested. Most importantly, no difference was found in the proportion of myeloid and erythroid progenitors, suggesting a similar repopulating capacity. On the basis of these results, we conclude that steady-state BM can be used as a safe and efficient source of HSC for gene therapy of β-thalassemia.
View details for DOI 10.1089/hum.2010.045
View details for Web of Science ID 000289410600014
View details for PubMedID 20979441
-
HIV-1-Derived Lentiviral Vectors Directly Activate Plasmacytoid Dendritic Cells, Which in Turn Induce the Maturation of Myeloid Dendritic Cells
HUMAN GENE THERAPY
2011; 22 (2): 177-188
Abstract
Lentiviral vectors (LV) can induce type I interferon (IFN I) production from murine plasmacytoid dendritic cells (pDC), but not myeloid (my)DC. Here, we investigated whether this mechanism is conserved in human DC. MyDC and pDC were isolated from peripheral blood and transduced with increasing vector concentrations. Compared with in vitro differentiated monocyte-derived DC, the transduction efficiency of peripheral blood DC was low (ranging from <1% to 45%), with pDC showing the lowest susceptibility to LV transduction. Phenotype and function of myDC were not directly modified by LV transduction; by contrast, pDC produced significant levels of IFN-α and tumor necrosis factor-α. pDC activation was dependent on functional vector particles and was mediated by Toll-like receptor 7/9 triggering. Coculture of myDC with pDC in the presence of LV resulted in myDC activation, with CD86 up-regulation and interleukin-6 secretion. These findings demonstrate that the induction of transgene-specific immunity is triggered by an innate immune response with pDC activation and consequent myDC maturation, a response that closely resembles the one induced by functional viruses. This information is important to design strategies aimed at using LV in humans for gene therapy, where adverse immune responses must be avoided, or for cancer immunotherapy, where inducing immunity is the goal.
View details for DOI 10.1089/hum.2010.085
View details for Web of Science ID 000287447200008
View details for PubMedID 20825284
-
Integration profile of retroviral vector in gene therapy treated patients is cell-specific according to gene expression and chromatin conformation of target cell
EMBO MOLECULAR MEDICINE
2011; 3 (2): 89-101
Abstract
The analysis of genomic distribution of retroviral vectors is a powerful tool to monitor 'vector-on-host' effects in gene therapy (GT) trials but also provides crucial information about 'host-on-vector' influences based on the target cell genetic and epigenetic state. We had the unique occasion to compare the insertional profile of the same therapeutic moloney murine leukemia virus (MLV) vector in the context of the adenosine deaminase-severe combined immunodeficiency (ADA-SCID) genetic background in two GT trials based on infusions of transduced mature lymphocytes (peripheral blood lymphocytes, PBL) or a single infusion of haematopoietic stem/progenitor cells (HSC). We found that vector insertions are cell-specific according to the differential expression profile of target cells, favouring, in PBL-GT, genes involved in immune system and T-cell functions/pathways as well as T-cell DNase hypersensitive sites, differently from HSC-GT. Chromatin conformations and histone modifications influenced integration preferences but we discovered that only H3K27me3 was cell-specifically disfavoured, thus representing a key epigenetic determinant of cell-type dependent insertion distribution. Our study shows that MLV vector insertional profile is cell-specific according to the genetic/chromatin state of the target cell both in vitro and in vivo in patients several years after GT.
View details for DOI 10.1002/emmm.201000108
View details for Web of Science ID 000287781100006
View details for PubMedID 21243617
-
Methods for in vitro generation of human type 1 regulatory T cells.
Methods in molecular biology (Clifton, N.J.)
2011; 677: 31-46
Abstract
Type 1 regulatory T (Tr1) cells are adaptive regulatory T cells that are induced in the periphery upon chronic exposure to antigen (Ag) in a tolerogenic environment containing interleukin (IL)-10. Tr1 cells are Ag-specific; they produce high levels of IL-10 and TGF-β in the absence of IL-4 and suppress T-cell responses via a cytokine-dependent mechanism. During the last decade, several protocols have been developed to generate Tr1 cell lines in vitro. In this chapter, we outline protocols to generate non-Ag- and Ag-specific Tr1 cell lines and assays used to characterize Tr1 cell phenotype and functions.
View details for DOI 10.1007/978-1-60761-869-0_3
View details for PubMedID 20941601
-
Manipulating immune tolerance with micro-RNA regulated gene therapy
FRONTIERS IN MICROBIOLOGY
2011; 2
View details for DOI 10.3389/fmicb.2011.00221
View details for Web of Science ID 000208863500229
-
Manipulating Immune Tolerance with Micro-RNA Regulated Gene Therapy.
Frontiers in microbiology
2011; 2: 221-?
Abstract
The success of in vivo gene therapy greatly depends on the ability to control the immune response toward the therapeutic transgene. Over the last decade several vector-based and pharmacological approaches have been explored to control the immune-mediated clearance of transgene-expressing cells after viral delivery. One important outcome from these studies is the concept that expression of a transgene in tolerance-promoting organs, such as the liver and tolerogenic antigen-presenting cells, can help safeguard transgene-expressing cells from immune-mediated clearance. Gene therapists are now manipulating vectors to target naturally occurring tolerogenic properties of the body by: (i) incorporating tissue/cell specific promoters for targeted expression, (ii) using viral-capsid engineering to alter tropism and avoid pre-existing immunity, and (iii) regulating cell and activation dependent expression by including micro-RNA (miR) targets into expression cassettes. The combination of these three layers of vector regulation greatly enhances the targeting of tolerogenic cells and limits off-target expression of the transgene, which can lead to the induction of transgene-specific pathogenic effector T cells. In this review, we discuss the application of using miR transgene regulation to generate tolerogenic responses and speculate on possible mechanisms used by the liver to induce the transgene-specific regulatory T cells.
View details for DOI 10.3389/fmicb.2011.00221
View details for PubMedID 22144977
-
Molecular and functional characterization of allogantigen-specific anergic T cells suitable for cell therapy
HAEMATOLOGICA-THE HEMATOLOGY JOURNAL
2010; 95 (12): 2134-2143
Abstract
CD4(+) regulatory T cells are a specialized subset of T cells that actively control immune responses. Several experimental protocols have been used to expand natural regulatory T cells and to generate adaptive type 1 regulatory T cells for regulatory T-cell-based therapies.The ability of exogenous recombinant human interleukin-10 to induce alloantigen-specific anergy in T cells was investigated and compared to that of interleukin-10 derived from tolerogenic dendritic cells, in mixed lymphocyte cultures. A detailed characterization of the effector functions of the resulting anergized T cells is reported.Interleukin-10, whether exogenous or derived from tolerogenic dendritic cells, induces a population of alloantigen-specific T cells (interleukin-10-anergized T cells) containing type 1 regulatory T cells, which are anergic and actively suppress alloantigen-specific effector T cells present within the mixed population. Interleukin-10-induced anergy is transforming growth factor-β independent, and is associated with a decreased frequency of alloantigen-specific cytotoxic T lymphocyte precursors, but interleukin-10-anergized T cells are still responsive to third-party, bacterial, and viral antigens. Tolerogenic dendritic cells are more powerful than exogenous interleukin-10 in generating type 1 regulatory T-cell precursors, and are also effective in the context of HLA-matched donors.Based on these studies, we have developed an efficient and reproducible in vitro method to generate antigen-specific type 1 regulatory T-cell precursors starting from total peripheral blood cells with minimal cell manipulation and suitable for generating type 1 regulatory T cells for regulatory T-cell-based therapies.
View details for DOI 10.3324/haematol.2010.025825
View details for Web of Science ID 000285571400021
View details for PubMedID 20713457
-
Point mutants of forkhead box P3 that cause immune dysregulation, polyendocrinopathy, enteropathy, X-linked have diverse abilities to reprogram T cells into regulatory T cells
JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY
2010; 126 (6): 1242-1251
Abstract
Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) is a primary immunodeficiency with autoimmunity caused by mutations in forkhead box P3 (FOXP3), which encodes a transcription factor involved in regulatory T (Treg) cell function. The mechanistic basis for how different mutations in FOXP3 cause distinct manifestations of IPEX remains unclear.To determine whether 3 different point mutants of FOXP3 that cause severe or mild IPEX differ in their ability to reprogram conventional T cells into Treg cells.Human CD4(+) T cells were transduced with wild-type or point mutant forms of FOXP3, and changes in cell surface marker expression, cytokine production, proliferation and suppressive capacity were assessed. Ex vivo T(H)17 cells were also transduced with different forms of FOXP3 to monitor changes in IL-17 production.The forkhead mutant F373A failed to upregulate CD25 and CCR4, did not suppress cytokine production, and induced suppressive activity less effectively than wild-type FOXP3. In contrast, although the forkhead mutant R347H was also defective in upregulation of CD25, it suppressed the production of cytokines, conferred suppressive capacity on CD4(+) T cells, and suppressed IL-17 production. F324L, a mutant outside the forkhead domain associated with mild IPEX, was equivalent to wild-type FOXP3 in all aspects tested.Mutations in FOXP3 that cause IPEX do not uniformly abrogate the ability of FOXP3 to regulate transcription and drive the development of Treg cells. These data support the notion that factors in addition to functional changes in Treg cells, such as alterations in conventional T cells, are involved in the pathogenesis of IPEX.
View details for DOI 10.1016/j.jaci.2010.09.001
View details for Web of Science ID 000284947800024
View details for PubMedID 21036387
-
Granulocyte-colony stimulating factor drives the in vitro differentiation of human dendritic cells that induce anergy in naive T cells
EUROPEAN JOURNAL OF IMMUNOLOGY
2010; 40 (11): 3097-3106
Abstract
G-CSF is a modulator of T-cell and DC functions. Previous reports show that monocytes from G-CSF-treated (post-G) healthy donors differentiate into tolerogenic DC in vitro in the presence of autologous serum, containing high levels of IL-10 and IFN-α, and in turn induce type 1 Treg (Tr1) cells. However, the direct effect of G-CSF on DC differentiation was not investigated. Here, we show that monocytes differentiated in the presence of exogenous G-CSF (G-DC) remain CD14(+) CD1a(-) , but acquire a DC-like morphology, express CD83 and CD86 and low levels of the tolerogenic markers Ig-like transcript (ILT)4 and HLA-G. G-DC spontaneously produce IL-10 and, upon stimulation, low levels of IL-12. G-DC display low stimulatory capacity and induce anergy in naïve T cells, but do not confer suppressive function. Therefore, in vitro differentiation of monocyte-derived DC in the presence of G-CSF can replicate some but not all features of post-G DC. These findings indicate that the tolerogenic properties of G-CSF do not exclusively reside in its direct effect on DC, which in turn induce T-cell anergy, but also in its ability to generate a tolerogenic milieu in vivo, which is necessary for Tr1 cell induction and cannot be replicated in vitro.
View details for DOI 10.1002/eji.201040659
View details for Web of Science ID 000284059000015
View details for PubMedID 20957751
-
Role of reduced intensity conditioning in T-cell and B-cell immune reconstitution after HLA-identical bone marrow transplantation in adenosine-deaminase severe combined immunodeficiency
HAEMATOLOGICA-THE HEMATOLOGY JOURNAL
2010; 95 (10): 1778-1782
Abstract
The treatment of choice for severe combined immunodeficiency is bone marrow transplantation from an HLA-identical donor sibling without conditioning. However, this may result in low donor stem cell chimerism, leading to reduced long-term immune reconstitution. We compared engraftment, metabolic, and T-cell and B-cell immune reconstitution of HLA-identical sibling bone marrow transplantation performed in 2 severe combined immunodeficiency infants with adenosine deaminase deficiency from the same family treated with or without a reduced intensity conditioning regimen (busulfan/fludarabine). Only the patient who received conditioning showed a stable mixed chimerism in all lineages, including bone marrow myeloid and B cells. The use of conditioning resulted in higher thymus-derived naïve T cells and T-cell receptor excision circles, normalization of the T-cell repertoire, and faster and complete B-cell and metabolic reconstitution. These results suggest the utility of exploring the use of reduced intensity conditioning in bone marrow transplantation from HLA-identical donor in severe combined immunodeficiency to improve long-term immune reconstitution.
View details for DOI 10.3324/haematol.2010.025098
View details for Web of Science ID 000283275000023
View details for PubMedID 20460637
-
Differentiation of type 1 T regulatory cells (Tr1) by tolerogenic DC-10 requires the IL-10-dependent ILT4/HLA-G pathway
BLOOD
2010; 116 (6): 935-944
Abstract
Type 1 T regulatory (Tr1) cells suppress immune responses in vivo and in vitro and play a key role in maintaining tolerance to self- and non-self-antigens. Interleukin-10 (IL-10) is the crucial driving factor for Tr1 cell differentiation, but the molecular mechanisms underlying this induction remain unknown. We identified and characterized a subset of IL-10-producing human dendritic cells (DCs), termed DC-10, which are present in vivo and can be induced in vitro in the presence of IL-10. DC-10 are CD14(+), CD16(+), CD11c(+), CD11b(+), HLA-DR(+), CD83(+), CD1a(-), CD1c(-), express the Ig-like transcripts (ILTs) ILT2, ILT3, ILT4, and HLA-G antigen, display high levels of CD40 and CD86, and up-regulate CD80 after differentiation in vitro. DC-10 isolated from peripheral blood or generated in vitro are potent inducers of antigen-specific IL-10-producing Tr1 cells. Induction of Tr1 cells by DC-10 is IL-10-dependent and requires the ILT4/HLA-G signaling pathway. Our data indicate that DC-10 represents a novel subset of tolerogenic DCs, which secrete high levels of IL-10, express ILT4 and HLA-G, and have the specific function to induce Tr1 cells.
View details for DOI 10.1182/blood-2009-07-234872
View details for Web of Science ID 000280881700015
View details for PubMedID 20448110
-
Correction of beta-thalassemia major by gene transfer in haematopoietic progenitors of pediatric patients
EMBO MOLECULAR MEDICINE
2010; 2 (8): 315-328
Abstract
Beta-thalassemia is a common monogenic disorder due to mutations in the beta-globin gene and gene therapy, based on autologous transplantation of genetically corrected haematopoietic stem cells (HSCs), holds the promise to treat patients lacking a compatible bone marrow (BM) donor. We recently showed correction of murine beta-thalassemia by gene transfer in HSCs with the GLOBE lentiviral vector (LV), expressing a transcriptionally regulated human beta-globin gene. Here, we report successful correction of thalassemia major in human cells, by studying a large cohort of pediatric patients of diverse ethnic origin, carriers of different mutations and all candidates to BM transplantation. Extensive characterization of BM-derived CD34(+) cells before and following gene transfer shows the achievement of high frequency of transduction, restoration of haemoglobin A synthesis, rescue from apoptosis and correction of ineffective erythropoiesis. The procedure does not significantly affect the differentiating potential and the relative proportion of haematopoietic progenitors. Analysis of vector integrations shows preferential targeting of transcriptionally active regions, without bias for cancer-related genes. Overall, these results provide a solid rationale for a future clinical translation.
View details for DOI 10.1002/emmm.201000083
View details for Web of Science ID 000281520500005
View details for PubMedID 20665635
-
Fatal vancomycin- and linezolid-resistant Enterococcus faecium sepsis in a child undergoing allogeneic haematopoietic stem cell transplantation for beta-thalassaemia major
JOURNAL OF MEDICAL MICROBIOLOGY
2010; 59 (7): 839-842
Abstract
Recently vancomycin-resistant and sporadically linezolid-resistant Enterococcus species have been described in adults. We report what we believe to be the first case of a child with prolonged bone marrow aplasia following haematopoietic stem cell transplantation developing a fatal sepsis caused by Enterococcus faecium resistant to glycopeptides and linezolid.
View details for DOI 10.1099/jmm.0.018598-0
View details for Web of Science ID 000279838400015
View details for PubMedID 20299507
-
Escalating doses of donor lymphocytes for incipient graft rejection following SCT for thalassemia
BONE MARROW TRANSPLANTATION
2010; 45 (6): 1047-1051
Abstract
Mixed chimerism (MC) and secondary graft failure are frequent events following SCT for thalassemia. There is limited information regarding the outcome of donor lymphocyte infusion (DLI) to prevent rejection, mainly from case reports describing only successful cases. We describe a series of seven children affected by beta-thalassemia treated with escalating doses of DLI for level 2-3 MC (donor<90%) following myeloablative SCT from a matched family donor. The infusions were safe and no acute or chronic GVHD were documented; five patients experienced neutropenia and thrombocytopenia resolving spontaneously. DLI was successful in converting to full donor chimerism two patients stratified in the low-risk class (Pesaro class II). Conversely, for five high-risk patients, DLI was not effective in preventing secondary graft failure. This limited series suggests that escalating doses of DLI are safe in thalassemia patients post myeloablative therapy but efficacy may be jeopardized by rapidly growing anti-donor alloimmunity in high-risk patients. We suggest giving escalating doses of donor T cells to attempt a graft-versus-thalassemia as soon as level 2-3 MC is detected.
View details for DOI 10.1038/bmt.2009.298
View details for Web of Science ID 000278573600013
View details for PubMedID 19881553
-
Platelet transfusion refractoriness in highly immunized beta thalassemia children undergoing stem cell transplantation
PEDIATRIC TRANSPLANTATION
2010; 14 (3): 393-401
Abstract
Immune-mediated refractoriness to platelet transfusion is a major problem in patients undergoing HSCT. In a cohort of 50 pediatric patients affected by beta thalassemia coming from Middle East countries, we experienced a high incidence of refractoriness because of anti-HLA antibodies during post-HSCT aplasia. In a risk factors analysis, factors predicting a negative transfusion outcome were presence of spleen and the number of anti-HLA antibodies. We adopted a policy to select platelet donors by avoiding HLA antigens against which the patient had specific antibodies. Transfusion of dedicated units resulted in 26% refractoriness compared to 74% to random units (p < 0.0001). When dedicated transfusions were used, the presence of spleen did not influence transfusion outcome. Analyzing transfusion outcome depending on the degree of HLA match and ABO compatibility, 76% successful transfusions were obtained with HLA-matched- ABO compatible followed by 67% in HLA-1mismatch- ABO compatible or HLA-matched- ABO incompatible and by 46% in HLA-1mismatch- ABO incompatible. In conclusion, we provide evidence that the selection of platelet donors according to patient characteristics, anti-HLA antibodies and ABO matching, is successful in reducing platelet refractoriness in heavily alloimmunized thalassemia patients undergoing transplantation.
View details for DOI 10.1111/j.1399-3046.2009.01282.x
View details for Web of Science ID 000276495900021
View details for PubMedID 20070557
-
Unpredictability of Intravenous Busulfan Pharmacokinetics in Children Undergoing Hematopoietic Stem Cell Transplantation for Advanced Beta Thalassemia: Limited Toxicity with a Dose-Adjustment Policy
BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION
2010; 16 (5): 622-628
Abstract
beta-thalassemia is a major health problem worldwide, and stem cell transplantation (SCT) is the only curative option. Oral Busulfan (Bu) based conditioning is widely used in this setting. Due to the variability of Bu systemic exposure, intravenous (i.v.) Bu has been proposed as a standard of care, with no need for drug monitoring and dose adjustment. Patients with beta-thalassemia from countries with limited resources might be at higher risk of erratic Bu metabolism because of liver dysfunction, severe iron overload, and specific ethnic/genetic features. We studied Bu pharmacokinetics in 53 children with advanced beta-thalassemia from Middle Eastern countries who underwent a total of 57 matched related donor SCTs. Forty-two percent of the children required dose adjustment because they did not achieve the therapeutic window after the first dose. With a Bu dose-adjustment policy, regimen-related toxicity was limited. At a median follow-up of 564 days, the probabilities of 2-year survival, current thalassemia-free survival, rejection, and treatment-related mortality were 96%, 88%, 21%, and 4%, respectively. Conditioning with i.v. Bu and dose adjustment is feasible and well tolerated, although recurrence of thalassemia remains an unsolved problem in children with advanced disease.
View details for DOI 10.1016/j.bbmt.2009.11.024
View details for Web of Science ID 000277168200007
View details for PubMedID 19963071
-
Induction of anergic allergen-specific suppressor T cells using tolerogenic dendritic cells derived from children with allergies to house dust mites
JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY
2010; 125 (3): 727-736
Abstract
Dendritic cells (DCs) regulate the immune response to allergens in the lung; they induce either effector or regulatory T cells, which promote or suppress, respectively, the development of allergy. IL-10 is a potent immunosuppressive cytokine that induces type 1 regulatory (Tr1) T cells.To generate allergen-specific Tr1 cells in vitro from children with allergy.Monocyte-derived DCs from children with allergy to house dust mites (HDM) were generated by incubating the cells with IL-10 and pulsing them with Der p 2, a major HDM allergen, or by pulsing them with Der p 2 and incubating them with IL-10 during their last 2 days of differentiation.Der p 2-specific T-cell proliferation and T(H)2 cytokine production were significantly reduced when T cells from patients with allergy to HDM were activated with autologous Der p 2-pulsed DCs that had been differentiated or incubated with IL-10. T-cell lines generated with Der p 2-pulsed DCs that were differentiated with IL-10 were hyporesponsive to reactivation with Der p 2 and able to suppress Der p 2-specific T(H)2 effector cells.Dendritic cells differentiated in the presence of IL-10 and pulsed with allergen gave rise to a population of tolerogenic DCs that induced allergen-specific Tr1 cells. This finding represents an important step forward to the prospective clinical application of tolerogenic DCs to modulate allergen-specific T-cell responses.
View details for DOI 10.1016/j.jaci.2009.12.004
View details for Web of Science ID 000275883200032
View details for PubMedID 20153036
-
High incidence of severe cyclosporine neurotoxicity in children affected by haemoglobinopaties undergoing myeloablative haematopoietic stem cell transplantation: early diagnosis and prompt intervention ameliorates neurological outcome
ITALIAN JOURNAL OF PEDIATRICS
2010; 36
Abstract
Neurotoxicity is a recognized complication of cyclosporine A (CSA) treatment. The incidence of severe CSA-related neurological complications following hematopoietic stem cell transplantation (HSCT) is 4-11%.We describe 6 cases of CSA related neurotoxicity out of 67 matched related HSCT performed in paediatric Middle East patients affected by haemoglobinopaties (5 beta thalassemia major, 1 sickle cell disease-SCD). Conditioning regimen consisted of iv busulphan, cyclophosphamide and graft-versus-host-disease (GvHD) prophylaxis with CSA, methylprednisolone, methotrexate and ATG.All 6 patients presented prodromes such as arterial hypertension, headache, visual disturbances and vomiting, one to two days before overt CSA neurotoxicity. CSA neurotoxicity consisted of generalized seizures, signs of endocranial hypertension and visual disturbances at a median day of onset of 11 days after HSCT (range +1 to +40). Brain magnetic resonance imaging (MRI) performed in all subjects showed reversible leukoencephalopathy predominantly in the posterior regions of the brain (PRES) in 5/6 patients. EEG performed in 5/6 patients was always abnormal. Neurotoxicity was not explainable by high CSA blood levels, as all patients had CSA in the therapeutic range with a median of 178 ng/ml (range 69-250). CSA was promptly stopped and switched to tacrolimus with disappearance of clinical and radiological findings. All patients are symptoms-free at a median follow up of 882 days (range 60-1065).Our experience suggests that paediatric patients with haemoglobinopaties have a high incidence of CSA related neurological events with no correlation between serum CSA levels and neurotoxicity. Prognosis is good following CSA removal. Specific prodromes such as arterial hypertension, headache or visual disturbances occurring in the early post-transplant period should be carefully evaluated with electrophysiological and MRI-based imaging in order to intervene promptly and avoid irreversible sequels.
View details for DOI 10.1186/1824-7288-36-14
View details for Web of Science ID 000293226700001
View details for PubMedID 20181110
-
Revertant T lymphocytes in a patient with Wiskott-Aldrich syndrome: Analysis of function and distribution in lymphoid organs
JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY
2010; 125 (2): 439-448
Abstract
The Wiskott-Aldrich syndrome (WAS) is a rare genetic disease characterized by thrombocytopenia, immunodeficiency, autoimmunity, and hematologic malignancies. Secondary mutations leading to re-expression of WAS protein (WASP) are relatively frequent in patients with WAS.The tissue distribution and function of revertant cells were investigated in a novel case of WAS gene secondary mutation.A vast combination of approaches was used to characterize the second-site mutation, to investigate revertant cell function, and to track their distribution over a 18-year clinical follow-up.The WAS gene secondary mutation was a 4-nucleotide insertion, 4 nucleotides downstream of the original deletion. This somatic mutation allowed the T-cell-restricted expression of a stable, full-length WASP with a 3-amino acid change compared with the wild-type protein. WASP(+) T cells appeared early in the spleen (age 10 years) and were highly enriched in a mesenteric lymph node at a later time (age 23 years). Revertant T cells had a diversified T-cell-receptor repertoire and displayed in vitro and in vivo selective advantage. They proliferated and produced cytokines normally on T-cell-receptor stimulation. Consistently, the revertant WASP correctly localized to the immunologic synapse and to the leading edge of migrating T cells.Despite the high proportion of functional revertant T cells, the patient still has severe infections and autoimmune disorders, suggesting that re-expression of WASP in T cells is not sufficient to normalize immune functions fully in patients with WAS.
View details for DOI 10.1016/j.jaci.2009.11.034
View details for Web of Science ID 000274764000024
View details for PubMedID 20159256
-
Antigen-Specific Dependence of Tr1-Cell Therapy in Preclinical Models of Islet Transplant
DIABETES
2010; 59 (2): 433-439
Abstract
In type 1 diabetes, allogeneic pancreatic islet transplant restores insulin production, but life-threatening immunosuppression is required to avoid graft rejection. Induction of antigen (Ag)-specific tolerance by cell therapy with regulatory T-cells (Tregs) represents an attractive alternative approach but its therapeutic efficacy in islet transplant remains to be determined. Among the different subsets of CD4(+) Tregs, the T inducible regulatory type 1 (Tr1) cells can be generated from naive T-cells in the presence of interleukin-10 (IL-10) and represent one promising therapeutic choice. This study was designed to define the efficacy of Tr1-cell therapy in preclinical models of islet transplant.Non-Ag-specific polyclonal Tr1 cells and donor Ag-specific Tr1 cells were transferred, in the absence of any pharmacological treatment, in two distinct mouse models of islet transplant. The two models differed in their therapeutic stringency, based on the mean rejection time of untreated mice that underwent a transplant.Transfer of polyclonal Tr1 cells engendered graft tolerance only in the nonstringent mouse model. Conversely, cell therapy with Ag-specific Tr1 cells induced an IL-10-dependent tolerance in the stringent mouse model of islet transplant. The therapeutic advantage of Ag-specific Tr1 cells over polyclonal Tr1 cells was due to their donor Ag specificity.These results demonstrate that Tr1-cell therapy leads to tolerance in settings of islet transplant and that its therapeutic efficacy is highly dependent on the antigen specificity of these cells.
View details for DOI 10.2337/db09-1168
View details for Web of Science ID 000274435900014
View details for PubMedID 19934002
-
In vivo delivery of a microRNA-regulated transgene induces antigen-specific regulatory T cells and promotes immunologic tolerance
BLOOD
2009; 114 (25): 5152-5161
Abstract
We previously showed that incorporating target sequences for the hematopoietic-specific microRNA miR-142 into an antigen-encoding transgene prevents antigen expression in antigen-presenting cells (APCs). To determine whether this approach induces immunologic tolerance, we treated mice with a miR-142-regulated lentiviral vector encoding green fluorescent protein (GFP), and subsequently vaccinated the mice against GFP. In contrast to control mice, no anti-GFP response was observed, indicating that robust tolerance to the transgene-encoded antigen was achieved. Furthermore, injection of the miR-142-regulated vector induced a population of GFP-specific regulatory T cells. Interestingly, an anti-GFP response was observed when microRNA miR-122a was inserted into the vector and antigen expression was detargeted from hepatocytes as well as APCs. This demonstrates that, in the context of lentiviral vector-mediated gene transfer, detargeting antigen expression from professional APCs, coupled with expression in hepatocytes, can induce antigen-specific immunologic tolerance.
View details for DOI 10.1182/blood-2009-04-214569
View details for Web of Science ID 000272612100007
View details for PubMedID 19794140
-
Autoimmune diabetic patients undergoing allogeneic islet transplantation: are we ready for a regulatory T-cell therapy?
IMMUNOLOGY LETTERS
2009; 127 (1): 1-7
Abstract
Regulatory T cells (Tregs) are thought to be pivotal in controlling both autoimmune and allogeneic undesired immune responses. Recently, an extensive effort has been devoted to design clinical trials with Tregs in T cell-mediated diseases (such as autoimmune diseases or transplantation). Theoretically, this approach can be used also in patients with autoimmunity (e.g., type 1 diabetes) undergoing allogeneic transplantation (e.g., pancreatic islet transplant). However, in this latter case Tregs must control two distinct effector immune responses: a pre-existing response towards self-antigens and a de novo response induced by the newly transplanted allogeneic cells. In this review we summarize results supporting the use of Tregs in controlling either autoimmunity or allo-transplantation. We also provide our view on how Treg therapy can achieve the final goal of immunological tolerance in the extremely challenging clinical setting of type 1 diabetic subjects transplanted with allogeneic islets.
View details for DOI 10.1016/j.imlet.2009.07.007
View details for Web of Science ID 000272412700001
View details for PubMedID 19643137
-
The Tregs' world according to GARP
EUROPEAN JOURNAL OF IMMUNOLOGY
2009; 39 (12): 3296-3300
Abstract
Naturally occurring CD4+CD25(high) regulatory T cells (nTreg) are essential for maintaining tolerance. FOXP3 has been established as a molecular marker of nTreg; however, FOXP3 cannot be used as a reliable marker for bona fide human nTreg since effector T cells also up-regulate FOXP3 expression upon activation. Despite the important function of nTreg, the underlying molecular mechanisms of nTreg-mediated suppression are far from defined. Previous studies have demonstrated that the TGF-beta latency-associated peptide (LAP) is expressed on the surface of nTreg, and that immunosuppression can be mediated by membrane TGF-beta; however, it remains unknown how LAP is bound to nTreg and what is the functional significance of its selective expression on activated nTreg. The nTreg's world may now change according to GARP, an orphan toll-like receptor composed of leucine-rich repeats. In this issue of the European Journal of Immunology, a study provides further demonstration that GARP is selectively expressed only in activated human nTreg and nTreg cell clones but not in activated effector T cells, confirming GARP as a bona fide nTreg marker. In addition, GARP binds directly to LAP; yet, GARP over-expression is insufficient to induce modification of latent TGF-beta into active TGF-beta further clarifying its role in nTreg-mediated suppression.
View details for DOI 10.1002/eji.200940117
View details for Web of Science ID 000272928300006
View details for PubMedID 19904770
-
[Gene therapy in pediatrics].
Minerva pediatrica
2009; 61 (6): 775-778
View details for PubMedID 19935549
-
Role of human leukocyte antigen-G in the induction of adaptive type 1 regulatory T cells
HUMAN IMMUNOLOGY
2009; 70 (12): 966-969
Abstract
Adaptive type 1 regulatory T (Tr1) cells are suppressor cells characterized by the production of interleukin (IL)-10 in the absence of IL-4. IL-10 is essential not only for suppression of effector cells by Tr1 cells, but also for their differentiation in vitro and in vivo. However, little is known on the molecular mechanisms underneath the IL-10-mediated induction of Tr1 cells. Human Leukocyte Antigen (HLA)-G, a non-classical HLA class I molecule, has both direct inhibitory effects on natural killer cells, dendritic cells (DC), and T cells and long-term tolerogenic indirect effects by inducing regulatory T (Tr) cells. In the present review, we discuss current findings on Tr-cell induction by the different isoforms of HLA-G, focusing on the relationship among HLA-G, its ligands, and IL-10. We recently described a subset of human DC, termed DC-10, that express high levels of HLA-G and ILT4, secrete high amounts of IL-10, and induce allospecific Tr1 cells in vitro via an IL-10-dependent ILT4/HLA-G pathway. IL-10, HLA-G, and ILT4 may also be involved in Tr1-cell induction in vivo. Overall, these data demonstrate that cross-regulation between IL-10 and HLA-G may be instrumental for Tr1-cell induction and tolerance.
View details for DOI 10.1016/j.humimm.2009.07.022
View details for Web of Science ID 000272014000002
View details for PubMedID 19664675
-
Wild-type FOXP3 is selectively active in CD4(+)CD25(hi) regulatory T cells of healthy female carriers of different FOXP3 mutations
BLOOD
2009; 114 (19): 4138-4141
Abstract
Forkhead box P3 (FOXP3) is constitutively expressed by CD4(+)CD25(hi) regulatory T cells (nTregs). Mutations of FOXP3 cause a severe autoimmune syndrome known as immune dysregulation polyendocrinopathy enteropathy X-linked, in which nTregs are absent or dysfunctional. Whether FOXP3 is essential for both differentiation and function of human nTreg cells remains to be demonstrated. Because FOXP3 is an X-linked gene subject to X-chromosome inactivation (XCI), we studied 9 healthy female carriers of FOXP3 mutations to investigate the role of wild-type (WT) versus mutated FOXP3 in different cell subsets. Analysis of active WT versus mutated (mut)-FOXP3 allele distribution revealed a random pattern of XCI in peripheral blood lymphocytes and in naive and memory CD4(+)T cells, whereas nTregs expressed only the active WT-FOXP3. These data demonstrate that expression of WT-FOXP3 is indispensable for the presence of a normal nTreg compartment and suggest that FOXP3 is not necessary for effector T-cell differentiation in humans.
View details for DOI 10.1182/blood-2009-04-214593
View details for Web of Science ID 000271495500026
View details for PubMedID 19738030
-
Absence of VOD in paediatric thalassaemic HSCT recipients using defibrotide prophylaxis and intravenous Busulphan
BRITISH JOURNAL OF HAEMATOLOGY
2009; 147 (4): 554-560
Abstract
Hepatic veno-occlusive disease (VOD) is a common complication of haematopoietic stem cell transplantation (HSCT), with reported incidences of 5-40% in children. Recently, defibrotide (DF) has been successfully used as prophylaxis and treatment of VOD. This study reports data on 63 human leucocyte antigen-matched HSCT performed in 57 children affected by beta thalassemia at very high risk for developing VOD (liver fibrosis, iron overload, hepatitis C virus infections, busulphan-based conditioning, methotraexate + ciclosporine). All patients received a busulphan-based conditioning regimen, either orally (four HSCT) or intravenously (59 HSCT). All patients received oral DF (40 mg/kg per day, final dose) as VOD prophylaxis from median day -9 to median day +29. In order to overcome the lack of oral paediatric formulations, a galenic formulation was administered. DF was well tolerated. Only one patient fulfilled Seattle Criteria for VOD diagnosis. This patient had discontinued DF 6 d prior to VOD onset, due to high risk of haemorrhage. We concluded that oral defibrotide prophylaxis and i.v. busulphan safely abated VOD incidence in high-risk patients who had undergone HSCT. A galenic preparation of oral DF also permits this treatment in low-weight patients. Costs of DF prophylaxis are acceptable considering the reduced incidence of VOD.
View details for DOI 10.1111/j.1365-2141.2009.07871.x
View details for Web of Science ID 000271240900015
View details for PubMedID 19747363
-
Integration of retroviral vectors induces minor changes in the transcriptional activity of T cells from ADA-SCID patients treated with gene therapy
BLOOD
2009; 114 (17): 3546-3556
Abstract
Gene transfer into hematopoietic stem cells by gamma-retroviral vectors (RVs) is an effective treatment for inherited blood disorders, although potentially limited by the risk of insertional mutagenesis. We evaluated the genomic impact of RV integration in T lymphocytes from adenosine deaminase-deficient severe combined immunodeficiency (ADA-SCID) patients 10 to 30 months after infusion of autologous, genetically corrected CD34(+) cells. Expression profiling on ex vivo T-cell bulk population revealed no difference with respect to healthy controls. To assess the effect of vector integration on gene expression at the single-cell level, primary T-cell clones were isolated from 2 patients. T-cell clones harbored either 1 (89.8%) or 2 (10.2%) vector copies per cell and displayed partial to full correction of ADA expression, purine metabolism, and T-cell receptor-driven functions. Analysis of RV integration sites indicated a high diversity in T-cell origin, consistently with the polyclonal T-cell receptor-Vbeta repertoire. Quantitative transcript analysis of 120 genes within a 200-kb window around RV integration sites showed modest (2.8- to 5.2-fold) dysregulation of 5.8% genes in 18.6% of the T-cell clones compared with controls. Nonetheless, affected clones maintained a stable phenotype and normal in vitro functions. These results confirm that RV-mediated gene transfer for ADA-SCID is safe, and provide crucial information for the development of future gene therapy protocols. The trials described herein have been registered at http://www.clinicaltrials.gov as #NCT00598481 and #NCT00599781.
View details for DOI 10.1182/blood-2009-02-202085
View details for Web of Science ID 000271024500008
View details for PubMedID 19652199
-
ADA-deficient SCID is associated with a specific microenvironment and bone phenotype characterized by RANKL/OPG imbalance and osteoblast insufficiency
BLOOD
2009; 114 (15): 3216-3226
Abstract
Adenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations, including skeletal abnormalities. We report that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoprotegerin axis, causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro, osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore, the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy, bone marrow transplantation, or gene therapy resulted in full recovery of the altered bone parameters. Remarkably, untreated ADA-severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and children's growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling.
View details for DOI 10.1182/blood-2009-03-209221
View details for Web of Science ID 000270595700014
View details for PubMedID 19633200
-
Type 1 regulatory T cells are associated with persistent split erythroid/lymphoid chimerism after allogeneic hematopoietic stem cell transplantation for thalassemia
HAEMATOLOGICA-THE HEMATOLOGY JOURNAL
2009; 94 (10): 1415-1426
Abstract
Thalassemia major can be cured with allogeneic hematopoietic stem cell transplantation. Persistent mixed chimerism develops in around 10% of transplanted thalassemic patients, but the biological mechanisms underlying this phenomenon are poorly understood.The presence of interleukin-10-producing T cells in the peripheral blood of eight patients with persistent mixed chimerism and five with full donor chimerism was investigated. A detailed characterization was then performed, by T-cell cloning, of the effector and regulatory T-cell repertoire of one patient with persistent mixed chimerism, who developed stable split erythroid/lymphoid chimerism after a hematopoietic stem cell transplant from an HLA-matched unrelated donor.Higher levels of interleukin-10 were produced by peripheral blood mononuclear cells from patients with persistent mixed chimerism than by the same cells from patients with complete donor chimerism or normal donors. T-cell clones of both host and donor origin could be isolated from the peripheral blood of one, selected patient with persistent mixed chimerism. Together with effector T-cell clones reactive against host or donor alloantigens, regulatory T-cell clones with a cytokine secretion profile typical of type 1 regulatory cells were identified at high frequencies. Type 1 regulatory cell clones, of both donor and host origin, were able to inhibit the function of effector T cells of either donor or host origin in vitro.Overall these results suggest that interleukin-10 and type 1 regulatory cells are associated with persistent mixed chimerism and may play an important role in sustaining long-term tolerance in vivo. These data provide new insights into the mechanisms of peripheral tolerance in chimeric patients and support the use of cellular therapy with regulatory T cells following hematopoietic stem cell transplantation.
View details for DOI 10.3324/haematol.2008.003129
View details for Web of Science ID 000271092500014
View details for PubMedID 19608686
-
Characterization of New Arylsulfatase A Gene Mutations Reinforces Genotype-Phenotype Correlation in Metachromatic Leukodystrophy
HUMAN MUTATION
2009; 30 (10): E936-E945
Abstract
Metachromatic Leukodystrophy (MLD) is a rare inherited lysosomal storage disorder caused by the deficiency of Arylsulfatase A (ARSA). The disease manifests itself with a broad spectrum of clinical variants, all characterized by progressive neurodegeneration in the central and peripheral nervous systems. The correlation between mutations in the ARSA gene, residual enzymatic activity associated with the mutated alleles and patients' phenotype, which has been extensively drawn for common ARSA mutations, has recently been expanded to rare ones. In this context, functional studies on the rare allelic variances acquire particular relevance for patients' prognostic evaluation. Here we have characterized eight newly identified ARSA mutations, through lentiviral vector-based expression studies on cell lines and ARSA defective murine fibroblasts. In each case, the residual activity associated with the new mutant allele correlates well with the patient's phenotype. Therefore, our results confirm the importance of functional characterization of mutant alleles for a precise genotype-based classification and definition of prognosis in MLD patients, which is particularly relevant for pre-symptomatic diagnosis.
View details for DOI 10.1002/humu.21093
View details for Web of Science ID 000279980600004
View details for PubMedID 19606494
-
Loss of Mismatched HLA in Leukemia after Stem-Cell Transplantation.
NEW ENGLAND JOURNAL OF MEDICINE
2009; 361 (5): 478-488
Abstract
Transplantation of hematopoietic stem cells from partially matched family donors is a promising therapy for patients who have a hematologic cancer and are at high risk for relapse. The donor T-cell infusions associated with such transplantation can promote post-transplantation immune reconstitution and control residual disease.We identified 43 patients who underwent haploidentical transplantation and infusion of donor T cells for acute myeloid leukemia or myelodysplastic syndrome and conducted post-transplantation studies that included morphologic examination of bone marrow, assessment of hematopoietic chimerism with the use of short-tandem-repeat amplification, and HLA typing. The genomic rearrangements in mutant variants of leukemia were studied with the use of genomic HLA typing, microsatellite mapping, and single-nucleotide-polymorphism arrays. The post-transplantation immune responses against the original cells and the mutated leukemic cells were analyzed with the use of mixed lymphocyte cultures.In 5 of 17 patients with leukemia relapse after haploidentical transplantation and infusion of donor T cells, we identified mutant variants of the original leukemic cells. In the mutant leukemic cells, the HLA haplotype that differed from the donor's haplotype had been lost because of acquired uniparental disomy of chromosome 6p. T cells from the donor and the patient after transplantation did not recognize the mutant leukemic cells, whereas the original leukemic cells taken at the time of diagnosis were efficiently recognized and killed.After transplantation of haploidentical hematopoietic stem cells and infusion of donor T cells, leukemic cells can escape from the donor's antileukemic T cells through the loss of the mismatched HLA haplotype. This event leads to relapse.
View details for Web of Science ID 000268443900008
View details for PubMedID 19641204
-
Hematopoietic stem cell gene therapy for adenosine deaminase deficient-SCID
IMMUNOLOGIC RESEARCH
2009; 44 (1-3): 150-159
Abstract
Gene therapy is a highly attractive strategy for many types of inherited disorders of the immune system. Adenosine deaminase (ADA) deficient-severe combined immunodeficiency (SCID) has been the target of several clinical trials based on the use of hematopoietic stem/progenitor cells engineered with retroviral vectors. The introduction of a low intensity conditioning regimen has been a crucial factor in achieving stable engrafment of hematopoietic stem cells and therapeutic levels of ADA-expressing cells. Recent studies have demonstrated that gene therapy for ADA-SCID has favorable safety profile and is effective in restoring normal purine metabolism and immune functions. Stem cell gene therapy combined with appropriate conditioning regimens might be extended to other genetic disorders of the hematopoietic system.
View details for DOI 10.1007/s12026-009-8107-8
View details for Web of Science ID 000266582800016
View details for PubMedID 19224139
-
The Fate of Human Treg Cells
IMMUNITY
2009; 30 (6): 763-765
Abstract
In this issue of Immunity, Miyara et al. (2009) demonstrate that FoxP3(+) cells in human peripheral blood are heterogeneous in function, and CD45RA expression defines their different stages of differentiation.
View details for DOI 10.1016/j.immuni.2009.06.006
View details for Web of Science ID 000267270700005
View details for PubMedID 19538927
-
Recent advances in understanding the pathophysiology of Wiskott-Aldrich syndrome
BLOOD
2009; 113 (25): 6288-6295
Abstract
Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency caused by mutations in the gene encoding for WASP, a key regulator of signaling and cytoskeletal reorganization in hematopoietic cells. Mutations in WASP result in a wide spectrum of clinical manifestations ranging from the relatively mild X-linked thrombocytopenia to the classic full-blown WAS phenotype characterized by thrombocytopenia, immunodeficiency, eczema, and high susceptibility to developing tumors and autoimmune manifestations. The life expectancy of patients affected by severe WAS is reduced, unless they are successfully cured by bone marrow transplantation from related identical or matched unrelated donors. Because many patients lack a compatible bone marrow donor, the administration of WAS gene-corrected autologous hematopoietic stem cells could represent an alternative therapeutic approach. In the present review, we focus on recent progress in understanding the molecular and cellular mechanisms contributing to the pathophysiology of WAS. Although molecular and cellular studies have extensively analyzed the mechanisms leading to defects in T, B, and dendritic cells, the basis of autoimmunity and thrombocytopenia still remains poorly understood. A full understanding of these mechanisms is still needed to further implement new therapeutic strategies for this peculiar immunodeficiency.
View details for DOI 10.1182/blood-2008-12-115253
View details for Web of Science ID 000267147400009
View details for PubMedID 19351959
-
Evidence for Long-term Efficacy and Safety of Gene Therapy for Wiskott-Aldrich Syndrome in Preclinical Models
MOLECULAR THERAPY
2009; 17 (6): 1073-1082
Abstract
Wiskott-Aldrich Syndrome (WAS) is a life-threatening X-linked disease characterized by immunodeficiency, thrombocytopenia, autoimmunity, and malignancies. Gene therapy could represent a therapeutic option for patients lacking a suitable bone marrow (BM) donor. In this study, we analyzed the long-term outcome of WAS gene therapy mediated by a clinically compatible lentiviral vector (LV) in a large cohort of was(null) mice. We demonstrated stable and full donor engraftment and Wiskott-Aldrich Syndrome protein (WASP) expression in various hematopoietic lineages, up to 12 months after gene therapy. Importantly, we observed a selective advantage for T and B lymphocytes expressing transgenic WASP. T-cell receptor (TCR)-driven T-cell activation, as well as B-cell's ability to migrate in response to CXCL13, was fully restored. Safety was evaluated throughout the long-term follow-up of primary and secondary recipients of WAS gene therapy. WAS gene therapy did not affect the lifespan of treated animals. Both hematopoietic and nonhematopoietic tumors arose, but we excluded the association with gene therapy in all cases. Demonstration of long-term efficacy and safety of WAS gene therapy mediated by a clinically applicable LV is a key step toward the implementation of a gene therapy clinical trial for WAS.
View details for DOI 10.1038/mt.2009.31
View details for Web of Science ID 000266540100018
View details for PubMedID 19259069
-
The Wiskott-Aldrich syndrome protein is required for iNKT cell maturation and function
JOURNAL OF EXPERIMENTAL MEDICINE
2009; 206 (4): 735-742
Abstract
The Wiskott-Aldrich syndrome (WAS) protein (WASp) is a regulator of actin cytoskeleton in hematopoietic cells. Mutations of the WASp gene cause WAS. Although WASp is involved in various immune cell functions, its role in invariant natural killer T (iNKT) cells has never been investigated. Defects of iNKT cells could indeed contribute to several WAS features, such as recurrent infections and high tumor incidence. We found a profound reduction of circulating iNKT cells in WAS patients, directly correlating with the severity of clinical phenotype. To better characterize iNKT cell defect in the absence of WASp, we analyzed was(-/-) mice. iNKT cell numbers were significantly reduced in the thymus and periphery of was(-/-) mice as compared with wild-type controls. Moreover analysis of was(-/-) iNKT cell maturation revealed a complete arrest at the CD44(+) NK1.1(-) intermediate stage. Notably, generation of BM chimeras demonstrated a was(-/-) iNKT cell-autonomous developmental defect. was(-/-) iNKT cells were also functionally impaired, as suggested by the reduced secretion of interleukin 4 and interferon gamma upon in vivo activation. Altogether, these results demonstrate the relevance of WASp in integrating signals critical for development and functional differentiation of iNKT cells and suggest that defects in these cells may play a role in WAS pathology.
View details for DOI 10.1084/jem.20081773
View details for Web of Science ID 000266009600003
View details for PubMedID 19307326
-
Rapamycin Prevents and Breaks the Anti-CD3-Induced Tolerance in NOD Mice
DIABETES
2009; 58 (4): 875-881
Abstract
Non-Fc-binding anti-CD3-specific antibodies represent a promising therapy for preserving C-peptide production in subjects with recent-onset type 1 diabetes. However, the mechanisms by which anti-CD3 exerts its beneficial effect are still poorly understood, and it is questionable whether this therapeutic approach will prove durable with regard to its ability to impart metabolic preservation without additional actions designed to maintain immunological tolerance. We used the NOD mouse model to test whether rapamycin, a compound well-known for its immunomodulatory activity in mice and humans, could increase the therapeutic effectiveness of anti-CD3 treatment in type 1 diabetes.Rapamycin was administered to diabetic NOD mice simultaneously with anti-CD3 or to NOD mice cured by anti-CD3 therapy. The ability of this combined therapy to revert type 1 diabetes and maintain a state of long-term tolerance was monitored and compared with that of anti-CD3 therapy alone.Rapamycin inhibited the ability of anti-CD3 to revert disease without affecting the frequency/phenotype of T-cells. Rapamycin also reinstated diabetes in mice whose disease was previously reversed by anti-CD3. Withdrawal of rapamycin in these latter animals promptly restored a normoglycemic state.Our findings indicate that, when combined with anti-CD3, rapamycin exerts a detrimental effect on the disease outcome in NOD mice for as long as it is administered. These results suggest strong caution with regard to combining these treatments in type 1 diabetic patients.
View details for DOI 10.2337/db08-1432
View details for Web of Science ID 000264819700013
View details for PubMedID 19151201
-
Gene Therapy for Immunodeficiency Due to Adenosine Deaminase Deficiency.
NEW ENGLAND JOURNAL OF MEDICINE
2009; 360 (5): 447-458
Abstract
We investigated the long-term outcome of gene therapy for severe combined immunodeficiency (SCID) due to the lack of adenosine deaminase (ADA), a fatal disorder of purine metabolism and immunodeficiency.We infused autologous CD34+ bone marrow cells transduced with a retroviral vector containing the ADA gene into 10 children with SCID due to ADA deficiency who lacked an HLA-identical sibling donor, after nonmyeloablative conditioning with busulfan. Enzyme-replacement therapy was not given after infusion of the cells.All patients are alive after a median follow-up of 4.0 years (range, 1.8 to 8.0). Transduced hematopoietic stem cells have stably engrafted and differentiated into myeloid cells containing ADA (mean range at 1 year in bone marrow lineages, 3.5 to 8.9%) and lymphoid cells (mean range in peripheral blood, 52.4 to 88.0%). Eight patients do not require enzyme-replacement therapy, their blood cells continue to express ADA, and they have no signs of defective detoxification of purine metabolites. Nine patients had immune reconstitution with increases in T-cell counts (median count at 3 years, 1.07x10(9) per liter) and normalization of T-cell function. In the five patients in whom intravenous immune globulin replacement was discontinued, antigen-specific antibody responses were elicited after exposure to vaccines or viral antigens. Effective protection against infections and improvement in physical development made a normal lifestyle possible. Serious adverse events included prolonged neutropenia (in two patients), hypertension (in one), central-venous-catheter-related infections (in two), Epstein-Barr virus reactivation (in one), and autoimmune hepatitis (in one).Gene therapy, combined with reduced-intensity conditioning, is a safe and effective treatment for SCID in patients with ADA deficiency. (ClinicalTrials.gov numbers, NCT00598481 and NCT00599781.)
View details for Web of Science ID 000262812400004
View details for PubMedID 19179314
-
Ten years of gene therapy for primary immune deficiencies.
Hematology / the Education Program of the American Society of Hematology. American Society of Hematology. Education Program
2009: 682-689
Abstract
Gene therapy with hematopoietic stem cells (HSC) is an attractive therapeutic strategy for several forms of primary immunodeficiencies. Current approaches are based on ex vivo gene transfer of the therapeutic gene into autologous HSC by vector-mediated gene transfer. In the past decade, substantial progress has been achieved in the treatment of severe combined immundeficiencies (SCID)-X1, adenosine deaminase (ADA)-deficient SCID, and chronic granulomatous disease (CGD). Results of the SCID gene therapy trials have shown long-term restoration of immune competence and clinical benefit in over 30 patients. The inclusion of reduced-dose conditioning in the ADA-SCID has allowed the engraftment of multipotent gene-corrected HSC at substantial level. In the CGD trial significant engraftment and transgene expression were observed, but the therapeutic effect was transient. The occurrence of adverse events related to insertional mutagenesis in the SCID-X1 and CGD trial has highlighted the limitations of current retroviral vector technology. For future applications the risk-benefit evaluation should include the type of vector employed, the disease background and the nature of the transgene. The use of self-inactivating lentiviral vectors will provide significant advantages in terms of natural gene regulation and reduction in the potential for adverse mutagenic events. Following recent advances in preclinical studies, lentiviral vectors are now being translated into new clinical approaches, such as Wiskott-Aldrich Syndrome.
View details for DOI 10.1182/asheducation-2009.1.682
View details for PubMedID 20008254
-
Molecular Regulation of Cellular Immunity by FOXP3
FORKHEAD TRANSCRIPTION FACTORS: VITAL ELEMENTS IN BIOLOGY AND MEDICINE
2009; 665: 30-46
Abstract
The immune system is responsible for not only eliminating threats to the body, but also for protecting the body from its own immune responses that would cause harm if left unchecked. Forkhead box protein 3 (FOXP3) is a forkhead family member with an important role in the development and function of a type of CD4+ T cell called T regulatory cells that is fundamental for maintaining immune tolerance to self. This chapter reviews the structure of FOXP3 and how its role in the immune system was discovered. Studies of patients with mutations in FOXP3 who suffer from a syndrome known as IPEX (immune dysregulation, polyendocrinopathy, enteropathy, x-linked) are also discussed. Investigation into how expression of FOXP3 is regulated and how it interacts with other proteins have recently provided considerable insight into mechanisms by which the lack of this protein could cause disease. We also discuss how FOXP3 is involved in the reciprocal development ofT regulatory cells and proinflammatory T-cells that produce IL-17. A better understanding of how FOXP3 is regulated and the molecular basis for its function will ultimately contribute to the development ofT regulatory cell-based cellular therapies that could be used to restore dysregulated immune responses.
View details for Web of Science ID 000271833300003
View details for PubMedID 20429414
-
Clinical and molecular profile of a new series of patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome: inconsistent correlation between forkhead box protein 3 expression and disease severity.
journal of allergy and clinical immunology
2008; 122 (6): 1105-1112 e1
Abstract
Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is an autoimmune genetic disorder caused by mutation of the forkhead box protein 3 gene (FOXP3), a key regulator of immune tolerance.We sought to provide clinical and molecular indicators that facilitate the understanding and diagnosis of IPEX syndrome.In 14 unrelated affected male subjects who were given diagnoses of IPEX syndrome based on FOXP3 gene sequencing, we determined whether particular FOXP3 mutations affected FOXP3 protein expression and correlated the molecular and clinical data.Molecular analysis of FOXP3 in the 14 subjects revealed 13 missense and splice-site mutations, including 7 novel mutations. Enteropathy, generally associated with endocrinopathy and eczema, was reported in all patients, particularly in those carrying mutations within FOXP3 functional domains or mutations that altered protein expression. However, similar genotypes did not always result in similar phenotypes in terms of disease presentation and severity. In addition, FOXP3 protein expression did not correlate with disease severity.Severe autoimmune enteropathy, which is often associated with increased IgE levels and eosinophilia, is the most prominent early manifestation of IPEX syndrome. Nevertheless, the disease course is variable and somewhat unpredictable. Therefore genetic analysis of FOXP3 should always be performed to ensure an accurate diagnosis, and FOXP3 protein expression analysis should not be the only diagnostic tool for IPEX syndrome.
View details for DOI 10.1016/j.jaci.2008.09.027
View details for PubMedID 18951619
-
Temporal, quantitative, and functional characteristics of single-KIR-positive alloreactive natural killer cell recovery account for impaired graft-versus-leukemia activity after haploidentical hematopoietic stem cell transplantation
BLOOD
2008; 112 (8): 3488-3499
Abstract
In this study, we have characterized reconstitution of the natural killer (NK) cell repertoire after haploidentical CD34(+) selected hematopoietic stem cell transplantation (HSCT) for high-risk hematologic malignancies. Analysis focused on alloreactive single-KIR(+) NK cells, which reportedly are potent antileukemic effectors. One month after HSCT, CD56(bright)/CD56(dim) NK-cell subsets showed inverted ratio and phenotypic features. CD25 and CD117 down-regulation on CD56(bright), and NKG2A and CD62L up-regulation on CD56(dim), suggest sequential CD56(bright)-to-CD56(dim) NK-cell maturation in vivo. Consistently, the functional potential of these maturation intermediates against leukemic blasts was impaired. Mature receptor repertoire reconstitution took at least 3 months. Importantly, at this time point, supposedly alloreactive, single-KIR(+) NK cells were not yet fully functional. Frequency of these cells was highly variable, independently from predicted NK alloreactivity, and below 1% of NK cells in 3 of 6 alloreactive patients studied. In line with these observations, no clinical benefit of predicted NK alloreactivity was observed in the total cohort of 56 patients. Our findings unravel the kinetics, and limits, of NK-cell differentiation from purified haploidentical hematopoietic stem cells in vivo, and suggest that NK-cell antileukemic potential could be best exploited by infusion of mature single-KIR(+) NK cells selected from an alloreactive donor.
View details for DOI 10.1182/blood-2007-07-103325
View details for Web of Science ID 000259866100066
View details for PubMedID 18645039
-
Metachromatic leukodystrophy - mutation analysis provides further evidence of genotype-phenotype correlation
CLINICAL GENETICS
2008; 74 (4): 349-357
Abstract
Metachromatic leukodystrophy (MLD) is a rare lysosomal storage disorder resulting from the inherited deficiency of the arylsulfatase A (ARSA) enzyme. Currently, no valid therapeutic options are available for affected patients. A thorough knowledge of disease progression in its diverse clinical variants, together with the identification of reliable prognostic factors, could be instrumental in accurate patient selection for new upcoming therapeutic opportunities, such as enzyme replacement and gene therapy. The described correlation between genotype and clinical presentation proved helpful in predicting patient's prognosis, only in the minority of MLD patients harboring common mutations. Molecular characterization of a cohort of 26 MLD patients allowed us to identify 18 mutations, excluding the common 0 and R alleles, 10 of which are rare and 8 are novel. By categorizing the rare mutations, we were able to confirm a correlation between ARSA gene mutations, age at onset and patterns of disease progression, not only in those patients bearing common mutations, but also in those carrying rare mutant alleles. Moreover, in the case of absent or delayed molecular diagnosis, or of newly identified mutations, the involvement of peripheral nervous system from disease onset proved to be a sensitive prognostic marker predicting a severe progression.
View details for DOI 10.1111/j.1399-0004.2008.01058.x
View details for Web of Science ID 000259270300010
View details for PubMedID 18786133
-
Clinical improvement and normalized Th1 cytokine profile in early and long-term interferon-alpha treatment in a suspected case of hyper-IgE syndrome
PEDIATRIC ALLERGY AND IMMUNOLOGY
2008; 19 (6): 564-568
Abstract
We are reporting on a 7-months-old boy with suspected hyper-IgE syndrome, presenting with a therapy resistant severe eczema and an overall reduction of in vitro cytokine production. Interferon-alpha (IFN-alpha) treatment resulted in a marked and stable clinical improvement and normalization of in vitro T-cell cytokine production, indicating a valid therapeutic potential of IFN-alpha as immunomodulating drug.
View details for DOI 10.1111/j.1399-3038.2007.00683.x
View details for Web of Science ID 000258582000016
View details for PubMedID 18844858
-
Multiple BM harvests in pediatric donors for thalassemic siblings: safety, efficacy and ethical issues
BONE MARROW TRANSPLANTATION
2008; 42 (6): 379-384
Abstract
Allogeneic BMT represents the only chance of cure for beta-thalassemia. Occasionally, two affected individuals from the same family share a matched healthy sibling. Moreover, a high incidence of transplant rejection is still observed in Pesaro class III patients, requiring a second BMT procedure. In these settings, one option is to perform a second BM harvest from the same donor. Although BM harvest is a safe procedure in children, ethical issues concerning this invasive practice still arise. Here, we describe our series of seven pediatric, healthy donors, who donated BM more than once in favor of their beta-thalassemic HLA-identical siblings between June 2005 and January 2008. Three donors donated BM twice to two affected siblings and four donors donated twice for the same sibling following graft rejection of the first BMT. All donors tolerated the procedures well and no relevant side effects occurred. There was no significant difference between the two harvests concerning cell yield and time to engraftment. Our experience shows that for pediatric donors, a second BM donation is safe and feasible and good cellularity can be obtained. We suggest that a second harvest of a pediatric donor can be performed when a strong indication for BMT exists.
View details for DOI 10.1038/bmt.2008.177
View details for Web of Science ID 000260007400002
View details for PubMedID 18574444
-
Second hematopoietic SCT in patients with thalassemia recurrence following rejection of the first graft
BONE MARROW TRANSPLANTATION
2008; 42 (6): 397-404
Abstract
There is a substantial incidence of graft failure in patients with thalassemia after myeloablative conditioning regimens especially in class 3 patients in whom its incidence could be as high as 8-38.5%. Most patients with graft failure have recurrence of thalassemic marrow. Historically, results of second transplants for thalassemia were poor because of a high rejection rate and/or increased TRM. Sixteen patients with thalassemia recurrence following rejection of the first graft and with a median age of 9 years (range, 4-20) were given second transplants using BM (n=7) or PBSC (n=9) after preparation with a new treatment protocol. All but two patients received stem cells from the same donor. The median interval between two transplants was 28 months (range, 8-204). The sustained engraftment rate was high (94%) with only one patient having primary graft failure. The probability of overall survival, event-free survival, TRM and graft failure were 79, 79, 16 and 6%, respectively. There were three transplant-related deaths. Thirteen patients are alive with Lansky/Karnofsky score of 100. This intensified treatment protocol was well tolerated with no significant increase in toxicity. The excellent results obtained with this new preparative regimen allow us to recommend it for second transplantation for patients with thalassemia recurrence.
View details for DOI 10.1038/bmt.2008.175
View details for Web of Science ID 000260007400005
View details for PubMedID 18574445
-
Rapamycin monotherapy in patients with type 1 diabetes modifies CD4(+)CD25(+)FOXP3(+) regulatory T-Cells
DIABETES
2008; 57 (9): 2341-2347
Abstract
Rapamycin is an immunosuppressive drug currently used to prevent graft rejection in humans, which is considered permissive for tolerance induction. Rapamycin allows expansion of both murine and human naturally occurring CD4(+)CD25(+)FOXP3(+) T regulatory cells (nTregs), which are pivotal for the induction and maintenance of peripheral tolerance. Preclinical murine models have shown that rapamycin enhances nTreg proliferation and regulatory function also in vivo. Objective of this study was to assess whether rapamycin has in vivo effects on human nTregs.nTreg numbers and function were examined in a unique set of patients with type 1 diabetes who underwent rapamycin monotherapy before islet transplantation.We found that rapamycin monotherapy did not alter the frequency and functional features, namely proliferation and cytokine production, of circulating nTregs. However, nTregs isolated from type 1 diabetic patients under rapamycin treatment had an increased capability to suppress proliferation of CD4(+)CD25(-) effector T-cells compared with that before treatment.These findings demonstrate that rapamycin directly affects human nTreg function in vivo, which consists of refitting their suppressive activity, whereas it does not directly change effector T-cell function.
View details for DOI 10.2337/db08-0138
View details for Web of Science ID 000258869000011
View details for PubMedID 18559659
-
Activation of the aryl hydrocarbon receptor promotes allograft-specific tolerance through direct and dendritic cell-mediated effects on regulatory T cells
BLOOD
2008; 112 (4): 1214-1222
Abstract
VAF347 is a low-molecular-weight compound, which activates the aryl hydrocarbon receptor (AhR). Herein, we report that oral administration of a water-soluble derivative of VAF347 (VAG539) promotes long-term graft acceptance and active tolerance in Balb/c mice that receive a transplant of MHC-mismatched pancreatic islet allografts. In vivo VAG539 treatment results in increased frequency of splenic CD4(+) T cells expressing CD25 and Foxp3, markers associated with regulatory T (Tr) cells, and in vitro VAF347 treatment of splenic CD4(+) T cells improved CD4(+)CD25(+)Foxp3(+) T-cell survival. Interestingly, transfer of CD11c(+) dendritic cells (DCs), but not of CD4(+) T or CD19(+) B cells, from VAG539-treated long-term tolerant hosts into mice that recently underwent transplantation resulted in donor (C57Bl/6)-specific graft acceptance and in a significantly higher frequency of splenic CD4(+)CD25(+)Foxp3(+) Tr cells. Furthermore, the transfer of CD4(+)CD25(+) T cells from these mice into mice that recently underwent transplantation promoted graft acceptance. Similarly, cell therapy with in vitro VAF347-treated bone marrow-derived mature DCs prevented islet graft rejection, and reduced OVA-specific T-cell responses in OVA-immunized mice. Collectively, our data indicate that AhR activation induces islet allograft-specific tolerance through direct as well as DC-mediated effects on Tr-cell survival and function.
View details for DOI 10.1182/blood-2007-08-109843
View details for Web of Science ID 000258392300046
View details for PubMedID 18550851
-
CD4(+) T-regulatory cells: toward therapy for human diseases
IMMUNOLOGICAL REVIEWS
2008; 223: 391-421
Abstract
T-regulatory cells (Tregs) have a fundamental role in the establishment and maintenance of peripheral tolerance. There is now compelling evidence that deficits in the numbers and/or function of different types of Tregs can lead to autoimmunity, allergy, and graft rejection, whereas an over-abundance of Tregs can inhibit anti-tumor and anti-pathogen immunity. Experimental models in mice have demonstrated that manipulating the numbers and/or function of Tregs can decrease pathology in a wide range of contexts, including transplantation, autoimmunity, and cancer, and it is widely assumed that similar approaches will be possible in humans. Research into how Tregs can be manipulated therapeutically in humans is most advanced for two main types of CD4(+) Tregs: forkhead box protein 3 (FOXP3)(+) Tregs and interleukin-10-producing type 1 Tregs (Tr1 cells). The aim of this review is to highlight current information on the characteristics of human FOXP3(+) Tregs and Tr1 cells that make them an attractive therapeutic target. We discuss the progress and limitations that must be overcome to develop methods to enhance Tregs in vivo, expand or induce them in vitro for adoptive transfer, and/or inhibit their function in vivo. Although many technical and theoretical challenges remain, the next decade will see the first clinical trials testing whether Treg-based therapies are effective in humans.
View details for Web of Science ID 000257565200024
View details for PubMedID 18613849
-
Altered intracellular and extracellular signaling leads to impaired T-cell functions in ADA-SCID patients
BLOOD
2008; 111 (8): 4209-4219
Abstract
Mutations in the adenosine deaminase (ADA) gene are responsible for a form of severe combined immunodeficiency (SCID) caused by the lymphotoxic accumulation of ADA substrates, adenosine and 2'-deoxy-adenosine. The molecular mechanisms underlying T-cell dysfunction in humans remain to be elucidated. Here, we show that CD4(+) T cells from ADA-SCID patients have severely compromised TCR/CD28-driven proliferation and cytokine production, both at the transcriptional and protein levels. Such an impairment is associated with an intrinsically reduced ZAP-70 phosphorylation, Ca(2+) flux, and ERK1/2 signaling and to defective transcriptional events linked to CREB and NF-kappaB. Moreover, exposure to 2'-deoxy-adenosine results in a stronger inhibition of T-cell activation, mediated by the aberrant A(2A) adenosine receptor signaling engagement and PKA hyperactivation, or in a direct apoptotic effect at higher doses. Conversely, in T cells isolated from patients after gene therapy with retrovirally transduced hematopoietic stem/progenitor cells, the biochemical events after TCR triggering occur properly, leading to restored effector functions and normal sensitivity to apoptosis. Overall, our findings provide a better understanding of the pathogenesis of the immune defects associated with an altered purine metabolism and confirm that ADA gene transfer is an efficacious treatment for ADA-SCID. The trials in this study are enrolled at www.ClinicalTrials.gov as #NCT00598481 and #NCT0059978.
View details for DOI 10.1182/blood-2007-05-092429
View details for Web of Science ID 000255134700045
View details for PubMedID 18218852
-
Is FOXP3 a bona fide marker for human regulatory T cells?
EUROPEAN JOURNAL OF IMMUNOLOGY
2008; 38 (4): 925-927
Abstract
FOXP3 is a specific marker for naturally occurring regulatory T cells (nTreg). FOXP3 expression is correlated with development and function of nTreg. Recent evidence suggests that, upon activation, human effector T (Teff) cells and type 1 regulatory T (Tr1) cells can express FOXP3, albeit transiently. While the role of transient FOXP3 expression in Teff cells is poorly understood, it is becoming clear that it does not correlate with suppressor function. Furthermore, FOXP3-independent mechanisms, mediated by IL-10, contribute to the induction and suppressor functions of Tr1 cells.
View details for DOI 10.1002/eji.200838168
View details for Web of Science ID 000255355900035
View details for PubMedID 18395862
-
[Biomedical research: a need or a luxury?].
KOS
2008; 25 (264): 53-61
View details for PubMedID 20503555
-
STAT5-signaling cytokines regulate the expression of FOXP3 in CD4(+)CD25(+) regulatory T cells and CD4(+)CD25(-) effector T cells
INTERNATIONAL IMMUNOLOGY
2008; 20 (3): 421-431
Abstract
Forkhead box P3 (FOXP3) is considered a specific marker for CD4(+)CD25(+) regulatory T (Treg) cells, but increasing evidence suggests that human CD4(+)CD25(-) effector T (Teff) cells can transiently express FOXP3 upon activation. We demonstrate that the signal transducer and activator of transcription 5 (STAT5)-signaling cytokines, IL-2, IL-15 and to a lesser extent IL-7, induce FOXP3 up-regulation in vitro in activated human Teff cells. In contrast, cytokines which do not activate STAT5, such as IL-4 or transforming growth factor-beta alone, do not directly induce FOXP3 expression in activated Teff cells. Moreover, expression of a constitutively active form of STAT5a is sufficient to induce FOXP3 expression in Teff cells. Expression of FOXP3 in activated Teff cells requires both TCR-mediated activation and endogenous IL-2, but is not dependent on cell division and does not induce suppressive function. The presence of STAT5-activating cytokines is also required to maintain high FOXP3 expression and suppressive activity of Treg cells in vitro. These data indicate that activation of STAT5 sustains FOXP3 expression in both Treg and Teff cells and contribute to our understanding of how cytokines affect the expression of FOXP3.
View details for DOI 10.1093/intimm/dxn002
View details for Web of Science ID 000253747100014
View details for PubMedID 18270368
-
Development of lentiviral gene therapy for Wiskott Aldrich syndrome
EXPERT OPINION ON BIOLOGICAL THERAPY
2008; 8 (2): 181-190
Abstract
Wiskott Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency. This complex disease is characterised by microthrombocytopenia, recurrent infections, eczema and is associated with a high incidence of autoimmunity and of lymphoid malignancies. WAS is attracting growing attention not only because it highlights the rich cellular and systems biology revolving around cytoskeletal regulation but also because it is candidate for a haematopoietic stem cell gene therapy indication.As several groups are developing this novel approach, this review discusses the state of the art and challenges in clinical development of gene therapy for WAS, with particular regard to biosafety.In spite of the successes of haematopoietic gene therapy for genetic immune deficiencies, there is a need for more efficient transduction protocols and for vectors with a superior safety profile. Preclinical studies have provided reasonable expectations that haematopoietic gene therapy with a self-inactivated HIV-1-derived vector using the native gene promoter for expression of the WAS transgene will be safe and will lead to the restoration of WAS protein in the haematopoietic and immune system at levels sufficient to provide an improvement in the condition of WAS patients.Phase I/II clinical studies will soon be initiated in several European centres to assess the safety and efficacy of this lentiviral vector in WAS patients.
View details for DOI 10.1517/14712598.8.2.181
View details for Web of Science ID 000252878300005
View details for PubMedID 18194074
-
Re-establishing immune tolerance in type 1 diabetes via regulatory T cells.
Novartis Foundation symposium
2008; 292: 174-183
Abstract
Type 1 diabetes (T1D) is a disease in which tolerance to self-antigens, such as insulin, is broken leading to expansion of autoreactive T cells that attack pancreatic beta cells with consequent loss of insulin production. Regulatory T cells (Tregs) represent a specific T cell subset that plays a key role in inducing and maintaining immunological tolerance to self and non-self antigens. The naturally occurring CD4+CD25+ Tregs (nTregs) originate from the thymus, constitutively express the transcription factor FOXP3, and suppress immune responses mainly via cell-cell contact. Depletion of nTregs results in systemic autoimmune diseases in mice and, vice versa, transfer of nTregs prevents development of autoimmune diseases. Regulatory T type 1 (Tr1) cells are inducible Tregs generated in the periphery by chronic exposure to antigens in the presence of interleukin (IL)10. Tr1 cells are defined by their unique cytokine production profile (i.e. IL10++, IL5+, TGFbeta+, IL4-, IL2(low), IFNgamma(low). Tr1 cells are induced by a specialized subset of tolerogenic dendritic cells and suppress undesired immune responses mainly through production of IL10 and TGFbeta. Interestingly,Trl cells modulate responses to self-antigens such as insulin- and islet-derived peptides. In vitro expansion/induction of Tregs can be therefore envisaged as a therapeutic tool for re-establishing self-tolerance in T1D subjects.
View details for PubMedID 19203099
-
Generation of potent and stable human CD4(+) T regulatory cells by activation-independent expression of FOXP3
MOLECULAR THERAPY
2008; 16 (1): 194-202
Abstract
Therapies based on enhancing the numbers and/or function of T regulatory cells (Tregs) represent one of the most promising approaches to restoring tolerance in many immune-mediated diseases. Several groups have investigated whether human Tregs suitable for cellular therapy can be obtained by in vitro expansion, in vitro conversion of conventional T cells into Tregs, or gene transfer of the FOXP3 transcription factor. To date, however, none of these approaches has resulted in a homogeneous and stable population of cells that is as potently suppressive as ex vivo Tregs. We developed a lentivirus-based strategy to ectopically express high levels of FOXP3 that do not fluctuate with the state of T-cell activation. This method consistently results in the development of suppressive cells that are as potent as Tregs and can be propagated as a homogeneous population. Moreover, using this system, both naïve and memory CD4(+) T cells can be efficiently converted into Tregs. To date, this is the most efficient and reliable protocol for generating large numbers of suppressive CD4(+) Tregs, which can be used for further biological study and developed for antigen-specific cellular therapy applications.
View details for DOI 10.1038/sj.mt.6300341
View details for Web of Science ID 000251821000030
View details for PubMedID 17984976
-
Progress and prospects: Gene therapy clinical trials (part 2)
GENE THERAPY
2007; 14 (22): 1555-1563
Abstract
This is the second part of a review summarizing progress and prospects in gene therapy clinical research. Twenty key diseases/strategies are succinctly described and commented on by leaders in the field. This part includes clinical trials for skin diseases, neurological disorders, HIV/AIDS, ornithine transcarbamylase deficiency, alpha(1)-antitrypsin deficiency, haemophilia and cancer.
View details for DOI 10.1038/sj.gt.3303033
View details for Web of Science ID 000250714900001
View details for PubMedID 17984995
-
The role of tissue adaptation and graft size in immune tolerance
TRANSPLANT IMMUNOLOGY
2007; 18 (2): 122-125
Abstract
Understanding how immune tolerance is induced and maintained is critical for our approach to immune-related diseases. Ecoimmunity is a new theory that views the immune system-tissue interaction as a co-adapting predator-prey system. Ecoimmunity suggests that tissues adapt to the selective immune pressure during ontogeny and throughout life. Therefore, immune tolerance towards 'self' represents a symmetric balance between the propensity of the immune system to prey on 'self' cells, and the tissue's specific capacity to undergo phenotypic adaptations in order to avoid destructive immune interaction. According to this theory, we hypothesized that tissues of adult immune-deficient mice, which are not exposed to selective immune pressure, will not withstand immune activity and will therefore display higher susceptibility to graft rejection. To test this prediction, C57Bl/6 wild type female mice were rendered diabetic by streptozotocin and transplanted with syngeneic pancreatic islets isolated from either immune-deficient C57Bl/6 SCID or wild type females. Remarkably, recipients of islet grafts from immune-deficient syngeneic donors displayed significantly impaired glucose homeostasis compared to mice transplanted with islets of wild type donors (p<0.001, two way repeated measures ANOVA). The severity of this impairment was correlated with islet graft size, suggesting a capacity of transplanted islets to gradually acquire a tolerogenic phenotype. These findings support the view of graft survival that is based on 'natural selection' of tissue cells. In addition, we describe a new experimental system for molecular characterization of self-tolerance.
View details for DOI 10.1016/j.trim.2007.05.006
View details for Web of Science ID 000251629600008
View details for PubMedID 18005855
-
The immune response to lentiviral-delivered transgene is modulated in vivo by transgene-expressing antigen-presenting cells but not by CD4(+)CD25(+) regulatory T cells
BLOOD
2007; 110 (6): 1788-1796
Abstract
Systemic delivery of lentiviral vector (LV) in immunocompetent mice leads to efficient in vivo cell transduction and expression of the encoded protein under the control of the ubiquitous promoter of human cytomegalovirus (CMV). However, antitransgene immune response results in clearance of transduced cells 4 weeks after injection. T regulatory cells (Tregs), which have been demonstrated to control immune responses in vivo, were tested for their ability to suppress antitransgene response leading to stable long-term expression. Adoptive transfer of natural CD4(+)CD25(+) Tregs (nTregs) isolated from wild type (wt) mice or from transgene tolerant transgenic (tg) mice did not suppress the antitransgene immune response after LV delivery. These data demonstrate that neither increasing the endogenous pool of natural Tregs nor transferring nTregs selected in a transgene-expressing thymus can modulate the immune response and mediate sustained transgene expression. Conversely, adoptive transfer of antigen-presenting cells (APCs) isolated from transgene-tolerant tg mice efficiently reduced the immune response leading to stable LV-encoded protein expression in vivo. Reduction of CD8(+) effector T cells was observed in LV-treated mice coinjected with transgene-expressing APCs compared with control mice. These data indicate that antitransgene immune response can be modulated by transgene-expressing APCs possibly through deletion of effector T cells.
View details for DOI 10.1182/blood-200611-059873
View details for Web of Science ID 000249671700019
View details for PubMedID 17495135
-
Frequency and targeted detection of HLA-DPB1 T cell epitope disparities relevant in unrelated hematopoietic stem cell transplantation
BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION
2007; 13 (9): 1031-1040
Abstract
The majority of unrelated donor (UD) hematopoietic stem cell (HSC) transplants are performed across HLA-DP mismatches, which, if involving disparity in a host-versus-graft (HVG) direction for an alloreactive T cell epitope (TCE), have been shown by our group to be associated with poor clinical outcome in 2 cohorts of patients transplanted for hematopoietic malignancies and beta-thalassemia, respectively. Using site-directed mutagenesis of DPB1*0901, we show here that the TCE is abrogated by the presence of amino acids LFQG in positions 8-11 of the DP beta-chain. Based on this and on alloreactive T cell responsiveness, we have determined the presence or absence of the TCE for 72 DPB1 alleles reported in the ethnic groups representative of the worldwide UD registries, and predict that 67%-87% (mean 77%) of UD recipient pairs will not present a DPB1 TCE disparity in the HVG direction. We developed and validated in 112 healthy Italian blood donors an innovative approach of DPB1 epitope-specific typing (EST), based on 2 PCR reactions. Our data show that DPB1 TCE disparities may hamper the clinical success of a considerable number of transplants when DPB1 matching is not included into the donor selection criteria, and that a donor without DPB1 TCE disparities in the HVG direction can be found for the majority of patients. Moreover, the study describes the first protocol of targeted epitope-specific DPB1 donor-recipient matching for unrelated HSC transplantation. This protocol will facilitate large-scale retrospective clinical studies warranted to more precisely determine the clinical relevance of DPB1 TCE disparities in different transplant conditions.
View details for DOI 10.1016/j.bbmt.2007.05.010
View details for Web of Science ID 000249152200005
View details for PubMedID 17697965
-
Role of regulatory T cells and FOXP3 in human diseases
JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY
2007; 120 (2): 227-235
Abstract
Immune regulation and tolerance are specific functions of the immune system, meaning at prevention or limitation of effector immune responses against inner and external insults. Regulatory T (Treg) cells are crucial players in this immune balance network. Research over the last 10 years has significantly contributed to characterizing Treg cell features, their mechanisms of function, and their role in human pathologies. The discovery of FOXP3 as an essential transcription factor not only for differentiation and function of naturally occurring Treg cells but also for regulation of intracellular molecules related to effector T-cell responses has provided new insights into the pathogenesis of immune-mediated diseases. Interestingly, there is increasing evidence that the individual signature of genes relevant for immune regulation definitely influences the final outcome of an immune response.
View details for DOI 10.1016/j.jaci.2007.06.023
View details for Web of Science ID 000248654900001
View details for PubMedID 17666212
-
Regulatory T-cell immunotherapy for tolerance to self antigens and alloantigens in humans
NATURE REVIEWS IMMUNOLOGY
2007; 7 (8): 585-598
Abstract
Substantial progress in understanding the biology of regulatory T cells and their roles in health and disease has been achieved in the past 10 years. This has led to an increasing interest in the possibility of using regulatory T cells as a biological therapy to preserve and restore tolerance to self antigens and alloantigens. Immunotherapy by the adoptive transfer of regulatory T cells may have several advantages over conventional treatments. However, several hurdles have to be overcome before such a therapy can enter clinical practice. This Review summarizes our current knowledge of regulatory T cells, illustrates the ongoing regulatory T-cell-based clinical trials, analyses the strengths and pitfalls of this new therapeutic approach, and highlights the future perspectives.
View details for DOI 10.1038/nri2138
View details for Web of Science ID 000248394100011
View details for PubMedID 17653126
-
Multilineage hematopoietic reconstitution without clonal selection in ADA-SCID patients treated with stem cell gene therapy
JOURNAL OF CLINICAL INVESTIGATION
2007; 117 (8): 2233-2240
Abstract
Gene transfer into HSCs is an effective treatment for SCID, although potentially limited by the risk of insertional mutagenesis. We performed a genome-wide analysis of retroviral vector integrations in genetically corrected HSCs and their multilineage progeny before and up to 47 months after transplantation into 5 patients with adenosine deaminase-deficient SCID. Gene-dense regions, promoters, and transcriptionally active genes were preferred retroviral integrations sites (RISs) both in preinfusion transduced CD34(+) cells and in vivo after gene therapy. The occurrence of insertion sites proximal to protooncogenes or genes controlling cell growth and self renewal, including LMO2, was not associated with clonal selection or expansion in vivo. Clonal analysis of long-term repopulating cell progeny in vivo revealed highly polyclonal T cell populations and shared RISs among multiple lineages, demonstrating the engraftment of multipotent HSCs. These data have important implications for the biology of retroviral vectors, the dynamics of genetically modified HSCs, and the safety of gene therapy.
View details for DOI 10.1172/JCI31666
View details for Web of Science ID 000248478100031
View details for PubMedID 17671653
View details for PubMedCentralID PMC1934603
-
Molecular purging of multiple myeloma cells by ex-vivo culture and retroviral transduction of mobilized-blood CD34(+) cells
JOURNAL OF TRANSLATIONAL MEDICINE
2007; 5
Abstract
Tumor cell contamination of the apheresis in multiple myeloma is likely to affect disease-free and overall survival after autografting.To purge myeloma aphereses from tumor contaminants with a novel culture-based purging method.We cultured myeloma-positive CD34+ PB samples in conditions that retained multipotency of hematopoietic stem cells, but were unfavourable to survival of plasma cells. Moreover, we exploited the resistance of myeloma plasma cells to retroviral transduction by targeting the hematopoietic CD34+ cell population with a retroviral vector carrying a selectable marker (the truncated form of the human receptor for nerve growth factor, DeltaNGFR). We performed therefore a further myeloma purging step by selecting the transduced cells at the end of the culture.Overall recovery of CD34+ cells after culture was 128.5%; DeltaNGFR transduction rate was 28.8% for CD34+ cells and 0% for CD138-selected primary myeloma cells, respectively. Recovery of CD34+ cells after DeltaNGFR selection was 22.3%. By patient-specific Ig-gene rearrangements, we assessed a decrease of 0.7-1.4 logs in tumor load after the CD34+ cell selection, and up to 2.3 logs after culture and DeltaNGFR selection.We conclude that ex-vivo culture and retroviral-mediated transduction of myeloma leukaphereses provide an efficient tumor cell purging.
View details for DOI 10.1186/1479-5876-5-35
View details for Web of Science ID 000249030500001
View details for PubMedID 17626627
-
A hypomorphic R229Q Rag2 mouse mutant recapitulates human Omenn syndrome
JOURNAL OF CLINICAL INVESTIGATION
2007; 117 (5): 1260-1269
Abstract
Rag enzymes are the main players in V(D)J recombination, the process responsible for rearrangement of TCR and Ig genes. Hypomorphic Rag mutations in humans, which maintain partial V(D)J activity, cause a peculiar SCID associated with autoimmune-like manifestations, Omenn syndrome (OS). Although a deficient ability to sustain thymopoiesis and to produce a diverse T and B cell repertoire explains the increased susceptibility to severe infections, the molecular and cellular mechanisms underlying the spectrum of clinical and immunological features of OS remain poorly defined. In order to better define the molecular and cellular pathophysiology of OS, we generated a knockin murine model carrying the Rag2 R229Q mutation previously described in several patients with OS and leaky forms of SCID. These Rag2(R229Q/R229Q) mice showed oligoclonal T cells, absence of circulating B cells, and peripheral eosinophilia. In addition, activated T cells infiltrated gut and skin, causing diarrhea, alopecia, and, in some cases, severe erythrodermia. These findings were associated with reduced thymic expression of Aire and markedly reduced numbers of naturally occurring Tregs and NKT lymphocytes. In conclusion, Rag2(R229Q/R229Q) mice mimicked most symptoms of human OS; our findings support the notion that impaired immune tolerance and defective immune regulation are involved in the pathophysiology of OS.
View details for DOI 10.1172/JCI30928
View details for Web of Science ID 000246347000020
View details for PubMedID 17476358
-
In vivo administration of lentiviral vectors triggers a type I interferon response that restricts hepatocyte gene transfer and promotes vector clearance
BLOOD
2007; 109 (7): 2797-2805
Abstract
Liver gene transfer is a highly sought goal for the treatment of inherited and infectious diseases. Lentiviral vectors (LVs) have many desirable properties for hepatocyte-directed gene delivery, including the ability to integrate into nondividing cells. Unfortunately, upon systemic administration, LV transduces hepatocytes relatively inefficiently compared with nonparenchymal cells, and the duration of transgene expression is often limited by immune responses. Here, we investigated the role of innate antiviral responses in these events. We show that administration of LVs to mice triggers a rapid and transient IFNalphabeta response. This effect was dependent on functional vector particles, and in vitro challenge of antigen-presenting cells suggested that plasmacytoid dendritic cells initiated the response. Remarkably, when LVs were administered to animals that lack the capacity to respond to IFNalphabeta, there was a dramatic increase in hepatocyte transduction, and stable transgene expression was achieved. These findings indicate that, even in the setting of acute delivery of replication-defective vectors, IFNs effectively interfere with transduction in a cell-type-specific manner. Moreover, because disabling a single component of the innate/immune network was sufficient to establish persistent xenoantigen expression, our results raise the hope that the immunologic barriers to gene therapy are less insurmountable than expected.
View details for DOI 10.1182/blood-2006-10-049312
View details for Web of Science ID 000245639000029
View details for PubMedID 17170119
-
Activation-induced FOXP3 in human T effector cells does not suppress proliferation or cytokine production
INTERNATIONAL IMMUNOLOGY
2007; 19 (4): 345-354
Abstract
Forkhead box P3 (FOXP3) is currently thought to be the most specific marker for naturally occurring CD4(+)CD25(+) T regulatory cells (nTregs). In mice, expression of FoxP3 is strictly correlated with regulatory activity, whereas increasing evidence suggests that in humans, activated T effector cells (Teffs) may also express FOXP3. In order to better define the role of FOXP3 in human Teff cells, we investigated the intensity and kinetics of expression in ex vivo Teff cells, suppressed Teff cells and Teff cell lines. We found that all dividing Teff cells expressed FOXP3, but only transiently, and at levels that were significantly lower than those in suppressive nTregs. This temporary expression in Teff cells was insufficient to suppress expression of reported targets of FOXP3 repressor activity, including CD127, IL-2 and IFN-gamma, and was not correlated with induction of a nTreg phenotype. Thus expression of FOXP3 is a normal consequence of CD4(+) T cell activation and, in humans, it can no longer be used as an exclusive marker of nTregs. These data indicate that our current understanding of how FOXP3 contributes to immune tolerance in humans needs to be re-evaluated.
View details for DOI 10.1093/intimm/dxm014
View details for Web of Science ID 000245352800001
View details for PubMedID 17329235
-
Lentiviral vectors targeting WASp expression to hematopoietic cells, efficiently transduce and correct cells from WAS patients
GENE THERAPY
2007; 14 (5): 415-428
Abstract
Gene therapy has been proposed as a potential treatment for Wiskott-Aldrich syndrome (WAS), a severe primary immune deficiency characterized by multiple hematopoietic-specific cellular defects. In order to develop an optimal lentiviral gene transfer cassette for this application, we compared the performance of several internal promoters in a variety of cell lineages from human WAS patients. Vectors using endogenous promoters derived from short (0.5 kb) or long (1.6 kb) 5' flanking sequences of the WAS gene, expressed the transgene in T, B, dendritic cells as well as CD34(+) progenitor cells, but functioned poorly in non-hematopoietic cells. Defects of T-cell proliferation and interleukin-2 production, and the cytoskeletal anomalies in WAS dendritic cells were also corrected. The levels of reconstitution were comparable to those obtained following transduction with similar lentiviral vectors incorporating constitutive PGK-1, EF1-alpha promoters or the spleen focus forming virus gammaretroviral LTR. Thus, native regulatory sequences target the expression of the therapeutic WAS transgene to the hematopoietic system, as is naturally the case for WAS, and are effective for correction of multiple cellular defects. These vectors may have significant advantages for clinical application in terms of natural gene regulation, and reduction in the potential for adverse mutagenic events.
View details for DOI 10.1038/sj.gt.3302863
View details for Web of Science ID 000244274200004
View details for PubMedID 17051251
-
Current understanding of the Wiskott-Aldrich syndrome and prospects for gene therapy.
Expert review of clinical immunology
2007; 3 (2): 205-215
Abstract
Gene therapy, based on the transplantation of genetically corrected autologous hematopoietic stem cells (HSCs), has proven to be an effective therapeutic approach as an alternative to allogenic HSC transplantation for the cure of severe combined immunodeficiencies (SCID). In this article, the recent preclinical studies aiming towards gene therapy trials for the Wiskott-Aldrich syndrome (WAS), a life-threatening immunodeficiency characterized by infections, hemorrhages, autoimmune disorders and lymphomas, will be reviewed. An update of the safety and efficacy data obtained in studies performed in murine disease models and in cells from WAS patients will be presented. Based on these data and on the clinical results of the recent trials for SCID, the most critical issues regarding the implementation of a gene therapy approach for WAS will be discussed.
View details for DOI 10.1586/1744666X.3.2.205
View details for PubMedID 20477109
-
WASP regulates suppressor activity of human and murine CD4(+)CD25(+)FOXP3(+) natural regulatory T cells
JOURNAL OF EXPERIMENTAL MEDICINE
2007; 204 (2): 369-380
Abstract
A large proportion of Wiskott-Aldrich syndrome (WAS) patients develop autoimmunity and allergy. CD4(+)CD25(+)FOXP3(+) natural regulatory T (nTreg) cells play a key role in peripheral tolerance to prevent immune responses to self-antigens and allergens. Therefore, we investigated the effect of WAS protein (WASP) deficiency on the distribution and suppressor function of nTreg cells. In WAS(-/-) mice, the steady-state distribution and phenotype of nTreg cells in the thymus and spleen were normal. However, WAS(-/-) nTreg cells engrafted poorly in immunized mice, indicating perturbed homeostasis. Moreover, WAS(-/-) nTreg cells failed to proliferate and to produce transforming growth factor beta upon T cell receptor (TCR)/CD28 triggering. WASP-dependent F-actin polarization to the site of TCR triggering might not be involved in WAS(-/-) nTreg cell defects because this process was also inefficient in wild-type (WT) nTreg cells. Compared with WT nTreg cells, WAS(-/-) nTreg cells showed reduced in vitro suppressor activity on both WT and WAS(-/-) effector T cells. Similarly, peripheral nTreg cells were present at normal levels in WAS patients but failed to suppress proliferation of autologous and allogeneic CD4(+) effector T cells in vitro. Thus, WASP appears to play an important role in the activation and suppressor function of nTreg cells, and a dysfunction or incorrect localization of nTreg cells may contribute to the development of autoimmunity in WAS patients.
View details for DOI 10.1084/jem.20061334
View details for Web of Science ID 000244504100016
View details for PubMedID 17296785
-
Immunological lessons learnt from patients transplanted with fully mismatched stem cells
1st Robert-A-Good-Society Symposium
HUMANA PRESS INC. 2007: 201–9
Abstract
Fully HLA-mismatched stem cells from human fetal livers were transplanted into 17 infants and two fetuses to treat severe combined immunodeficiency disease in 1976-2000. Donor cell engraftment and immunological reconstitution were obtained in 14/19 patients, three of whom have been extensively and repeatedly studied immunologically during prolonged follow-up. T-cells were derived totally from donor cells; B-cells and antigen-presenting cells (APC) remained mainly of host origin. Due to class I and II mismatches between T-cells and all other cells (APC, B-cells, virus-infected target cells), limitations in the defense against infections in vivo and in T-cell functions in vitro (helper and cytotoxic activities) were predicted; however, these did not occur. Anti-tetanus toxoid responses (including specific antibody production) developed despite HLA disparities between T-cells and B-cells or APC in the chimeric children. Similarly, cytotoxic T-cells (of donor HLA phenotype) recognized host Epstein-Barr virus-infected target cells. Recognition of antigenic peptide by T-cells under these conditions involved presentation by host allogeneic HLA molecules and not by self HLA antigens. Tolerance to donor antigens was acquired by clonal deletion; tolerance to host antigens existed despite the presence of many host-reactive T-cells and involved clonal anergy.
View details for DOI 10.1007/s12026-007-0002-6
View details for Web of Science ID 000250372300024
View details for PubMedID 17917026
-
Isolation, expansion, and characterization of human natural and adaptive regulatory T cells.
Methods in molecular biology (Clifton, N.J.)
2007; 380: 83-105
Abstract
Regulatory T cells play a central role in controlling homeostasis, and in inducing and maintaining tolerance to both foreign and self-antigens. Several types of T cells with regulatory activity have been described both in mice and humans, and those within the CD4+ subset have been extensively studied. Among them, the best characterized are the naturally occurring CD4+CD25+ regulatory T (Treg) cells, and the adaptive type 1 regulatory T (Tr1) cells. Natural Treg cells can arise directly from the thymus, are characterized by the constitutive expression of the transcription factor Foxp3, and suppress T cell responses in a cell-cell contact mediated mechanism. On the contrary, adaptive Tr1 cells arise in the periphery upon encountering antigen in a tolerogenic environment, produce high levels of interleukin (IL)-10 and mediate suppression via IL-10. During the last decade, much effort has been placed on developing protocols to generate regulatory T-cell lines and clones, to further define the similarities and differences between various regulatory T-cell subsets. In this chapter, we will outline protocols to expand naturally occurring Treg cells, to differentiate homogeneous population of Tr1 cells in vitro, and to generate natural Treg and Tr1 cell clones and cell lines.
View details for PubMedID 17876089
-
Rapamycin promotes expansion of functional CD4(+)CD25(+)FOXP3(+) regulatory T cells of both healthy subjects and type 1 diabetic patients
JOURNAL OF IMMUNOLOGY
2006; 177 (12): 8338-8347
Abstract
CD4+CD25+FOXP3+ T regulatory cells (Tregs) are pivotal for the induction and maintenance of peripheral tolerance in both mice and humans. Rapamycin has been shown to promote tolerance in experimental models and to favor CD4+CD25+ Treg-dependent suppression. We recently reported that rapamycin allows in vitro expansion of murine CD4+CD25+FoxP3+ Tregs, which preserve their suppressive function. In the current study, we show that activation of human CD4+ T cells from healthy subjects in the presence of rapamycin leads to growth of CD4+CD25+FOXP3+ Tregs and to selective depletion of CD4+CD25- T effector cells, which are highly sensitive to the antiproliferative effect of the compound. The rapamycin-expanded Tregs suppress proliferation of both syngeneic and allogeneic CD4+ and CD8+ T cells. Interestingly, rapamycin promotes expansion of functional CD4+CD25+FOXP3+ Tregs also in type 1 diabetic patients, in whom a defect in freshly isolated CD4+CD25+ Tregs has been reported. The capacity of rapamycin to allow growth of functional CD4+CD25+FOXP3+ Tregs, but also to deplete T effector cells, can be exploited for the design of novel and safe in vitro protocols for cellular immunotherapy in T cell-mediated diseases.
View details for Web of Science ID 000243416800009
View details for PubMedID 17142730
-
Defective Th1 cytokine gene transcription in CD4(+) and CD8(+) T cells from Wiskott-Aldrich syndrome patients
JOURNAL OF IMMUNOLOGY
2006; 177 (10): 7451-7461
Abstract
Wiskott-Aldrich syndrome (WAS) protein (WASP) plays a key role in TCR-mediated activation and immunological synapse formation. However, the effects of WASP deficiency on effector functions of human CD4+ and CD8+ T cells remain to be determined. In this study, we report that TCR/CD28-driven proliferation and secretion of IL-2, IFN-gamma, and TNF-alpha are strongly reduced in CD8+ T cells from WAS patients, compared with healthy donor CD8+ T cells. Furthermore, WAS CD4+ T cells secrete low levels of IL-2 and fail to produce IFN-gamma and TNF-alpha, while the production of IL-4, IL-5, and IL-10 is only minimally affected. Defective IL-2 and IFN-gamma production persists after culture of naive WAS CD4+ T cells in Th1-polarizing conditions. The defect in Th1 cytokine production by WAS CD4+ and CD8+ T cells is also present at the transcriptional level, as shown by reduced IL-2 and IFN-gamma mRNA transcripts after TCR/CD28 triggering. The reduced transcription of Th1 cytokine genes in WAS CD4+ T cells is associated with a defective induction of T-bet mRNA and a reduction in the early nuclear recruitment of NFAT-1, while the defective activation of WAS CD8+ T cells correlates with reduced nuclear recruitment of both NFAT-1 and NFAT-2. Together, our data indicate that WASP regulates the transcriptional activation of T cells and is required specifically for Th1 cytokine production.
View details for Web of Science ID 000242009700100
View details for PubMedID 17082665
-
Ex vivo gene therapy with lentiviral vectors rescues adenosine deaminase (ADA)-deficient mice and corrects their immune and metabolic defects
BLOOD
2006; 108 (9): 2979-2988
Abstract
Adenosine deaminase (ADA) deficiency is caused by a purine metabolic dysfunction, leading to severe combined immunodeficiency (SCID) and multiple organ damage. To investigate the efficacy of ex vivo gene therapy with self-inactivating lentiviral vectors (LVs) in correcting this complex phenotype, we used an ADA(-/-) mouse model characterized by early postnatal lethality. LV-mediated ADA gene transfer into bone marrow cells combined with low-dose irradiation rescued mice from lethality and restored their growth, as did transplantation of wild-type bone marrow. Mixed chimerism with multilineage engraftment of transduced cells was detected in the long term in animals that underwent transplantation. ADA activity was normalized in lymphocytes and partially corrected in red blood cells (RBCs), resulting in full metabolic detoxification and prevention of severe pulmonary insufficiency. Moreover, gene therapy restored normal lymphoid differentiation and immune functions, including antigen-specific antibody production. Similar degrees of detoxification and immune reconstitution were obtained in mice treated early after birth or after 1 month of enzyme-replacement therapy, mimicking 2 potential applications for ADA-SCID. Overall, this study demonstrates the efficacy of LV gene transfer in correcting both the immunological and metabolic phenotypes of ADA-SCID and supports the future clinical use of this approach.
View details for DOI 10.1182/blood-2006-05-023507
View details for Web of Science ID 000241586100023
View details for PubMedID 16835374
-
Gliadin-specific type 1 regulatory T cells from the intestinal mucosa of treated celiac patients inhibit pathogenic T cells
JOURNAL OF IMMUNOLOGY
2006; 177 (6): 4178-4186
Abstract
Celiac disease (CD) results from a permanent intolerance to dietary gluten and is due to a massive T cell-mediated immune response to gliadin, the main component of gluten. In this disease, the regulation of immune responses to dietary gliadin is altered. Herein, we investigated whether IL-10 could modulate anti-gliadin immune responses and whether gliadin-specific type 1 regulatory T (Tr1) cells could be isolated from the intestinal mucosa of CD patients in remission. Short-term T cell lines were generated from jejunal biopsies, either freshly processed or cultured ex vivo with gliadin in the presence or absence of IL-10. Ex vivo stimulation of CD biopsies with gliadin in the presence of IL-10 resulted in suppression of Ag-specific proliferation and cytokine production, indicating that pathogenic T cells are susceptible to IL-10-mediated immune regulation. T cell clones generated from intestinal T cell lines were tested for gliadin specificity by cytokine production and proliferative responses. The majority of gliadin-specific T cell clones had a Th0 cytokine production profile with secretion of IL-2, IL-4, IFN-gamma, and IL-10 and proliferated in response to gliadin. Tr1 cell clones were also isolated. These Tr1 cells were anergic, restricted by DQ2 (a CD-associated HLA), and produced IL-10 and IFN-gamma, but little or no IL-2 or IL-4 upon activation with gliadin or polyclonal stimuli. Importantly, gliadin-specific Tr1 cell clones suppressed proliferation of pathogenic Th0 cells. In conclusion, dietary Ag-specific Tr1 cells are present in the human intestinal mucosa, and strategies to boost their numbers and/or function may offer new therapeutic opportunities to restore gut homeostasis.
View details for Web of Science ID 000240475300076
View details for PubMedID 16951383
-
Induction of transplantation tolerance via regulatory T cells.
Inflammation & allergy drug targets
2006; 5 (3): 157-165
Abstract
In the last two decades, graft survival has been greatly improved by the introduction of efficient immunosuppressive drugs. On the other hand, late graft loss caused by chronic rejection together with the side effects of long-term immunosuppression, remain major obstacles for successful transplantation. Operational tolerance, which is defined by the lack of acute and chronic rejection and indefinite graft survival with normal graft function in the absence of chronic immunosuppression, represents an attractive alternative. Several approaches have been explored to achieve transplantational tolerance, which is considered the "Holy Grail" of transplantation, including induction of central tolerance by establishing mixed chimerism through hematopoietic stem cell transplantation or induction of peripheral tolerance through modulation of allogeneic immune responses. Graft-specific alloreactive T cells, which largely mediate graft rejection, can be silenced through different mechanisms, including deletion, which may occur within the thymus or in the lymphoid organs; anergy, in which alloreactive T cells cannot adequately respond following restimulation with the specific antigen; and suppression, which may be mediated by direct interactions with regulatory T cells (Tregs) or by soluble factors produced by Tregs. This review attempts to summarize the most novel and successful strategies to achieve operational tolerance via induction of Tregs.
View details for PubMedID 16918479
-
Interleukin-10-secreting type 1 regulatory T cells in rodents and humans
IMMUNOLOGICAL REVIEWS
2006; 212: 28-50
Abstract
Interleukin-10 (IL-10)-secreting T regulatory type 1 (Tr1) cells are defined by their specific cytokine production profile, which includes the secretion of high levels of IL-10 and transforming growth factor-beta(TGF-beta), and by their ability to suppress antigen-specific effector T-cell responses via a cytokine-dependent mechanism. In contrast to the naturally occurring CD4+ CD25+ T regulatory cells (Tregs) that emerge directly from the thymus, Tr1 cells are induced by antigen stimulation via an IL-10-dependent process in vitro and in vivo. Specialized IL-10-producing dendritic cells, such as those in an immature state or those modulated by tolerogenic stimuli, play a key role in this process. We propose to use the term Tr1 cells for all IL-10-producing T-cell populations that are induced by IL-10 and have regulatory activity. The full biological characterization of Tr1 cells has been hampered by the difficulty in generating these cells in vitro and by the lack of specific marker molecules. However, it is clear that Tr1 cells play a key role in regulating adaptive immune responses both in mice and in humans. Further work to delineate the specific molecular signature of Tr1 cells, to determine their relationship with CD4+ CD25+ Tregs, and to elucidate their respective role in maintaining peripheral tolerance is crucial to advance our knowledge on this Treg subset. Furthermore, results from clinical protocols using Tr1 cells to modulate immune responses in vivo in autoimmunity, transplantation, and chronic inflammatory diseases will undoubtedly prove the biological relevance of these cells in immunotolerance.
View details for Web of Science ID 000239189600003
View details for PubMedID 16903904
-
Design and optimization of lentiviral vectors for transfer of GALC expression in Twitcher brain
JOURNAL OF GENE MEDICINE
2006; 8 (8): 962-971
Abstract
Demyelination in globoid cell leukodystrophy (GLD) is due to a deficiency of galactocerebrosidase (GALC) activity. Up to now, in vivo brain viral gene transfer of GALC showed modest impact on disease development in Twitcher mice, an animal model for GLD. Lentiviral vectors, which are highly efficient to transfer the expression of therapeutic genes in neurons and glial cells, have not been evaluated for direct cerebral therapy in GLD mice.Lentiviral vectors containing the untagged cDNA or the hemagglutinin (HA)-tagged cDNA for the full-length mouse GALC sequence were generated and validated in vitro. In vivo therapeutic efficacy of these vectors was evaluated by histology, biochemistry and electrophysiology after transduction of ependymal or subependymal layers in young Twitcher pups.Both GALC lentiviral vectors transduced neurons, oligodendrocytes and astrocytes with efficiencies above 75% and conferred high levels of enzyme activity. GALC accumulated in lysosomes of transduced cells and was also secreted to the extracellular medium. Conditioned GALC medium was able to correct the enzyme deficiency when added to non-transduced Twitcher glial cultures. Mice that received intraventricular injections of GALC vector showed accumulation of GALC in ependymal cells but no diffusion of the enzyme from the ependymal ventricular tree into the cerebral parenchyma. Significant expression of GALC-HA was detected in neuroglioblasts when GALC-HA lentiviral vectors were injected in the subventricular zone of Twitcher mice. Life span and motor conduction in both groups of treated Twitcher mice were not significantly ameliorated.Lentiviral vectors showed to be efficient for reconstitution of the GALC expression in Twitcher neural cells. GALC was able to accumulate in lysosomes as well as to enter the secretory pathway of lysosomal enzymes, two fundamental aspects for gene therapy of lysosomal storage diseases. Our in vivo results, while showing the capacity of lentiviral vectors to transfer expression of therapeutic GALC in the Twitcher brain, did not limit progression of disease in Twitchers and highlight the need to evaluate other routes of administration.
View details for DOI 10.1002/jgm.924
View details for Web of Science ID 000240163900003
View details for PubMedID 16732552
-
Defective regulatory and effector T cell functions in patients with FOXP3 mutations
JOURNAL OF CLINICAL INVESTIGATION
2006; 116 (6): 1713-1722
Abstract
The autoimmune disease immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) is caused by mutations in the forkhead box protein P3 (FOXP3) gene. In the mouse model of FOXP3 deficiency, the lack of CD4+ CD25+ Tregs is responsible for lethal autoimmunity, indicating that FOXP3 is required for the differentiation of this Treg subset. We show that the number and phenotype of CD4+ CD25+ T cells from IPEX patients are comparable to those of normal donors. CD4+ CD25high T cells from IPEX patients who express FOXP3 protein suppressed the in vitro proliferation of effector T cells from normal donors, when activated by "weak" TCR stimuli. In contrast, the suppressive function of CD4+ CD25high T cells from IPEX patients who do not express FOXP3 protein was profoundly impaired. Importantly, CD4+ CD25high T cells from either FOXP3+ or FOXP3- IPEX patients showed altered suppression toward autologous effector T cells. Interestingly, IL-2 and IFN-gamma production by PBMCs from IPEX patients was significantly decreased. These findings indicate that FOXP3 mutations in IPEX patients result in heterogeneous biological abnormalities, leading not necessarily to a lack of differentiation of CD4+ CD25high Tregs but rather to a dysfunction in these cells and in effector T cells.
View details for DOI 10.1172/JCI25112
View details for Web of Science ID 000237979700034
View details for PubMedID 16741580
-
Induction of tolerance in type 1 diabetes via both CD4(+) CD25(+) T regulatory cells and T regulatory type 1 cells
DIABETES
2006; 55 (6): 1571-1580
Abstract
Success in developing novel therapies to recommence self-tolerance in autoimmunity depends on the induction of T regulatory (Tr) cells. Here, we report that rapamycin combined with interleukin (IL)-10 efficiently blocks type 1 diabetes development and induces long-term immunotolerance in the absence of chronic immunosuppression in nonobese diabetic (NOD) mice. Rapamycin mediates accumulation in the pancreas of suppressive CD4(+)CD25(+)FoxP3(+) Tr cells, which prevent diabetes. IL-10 induces Tr type 1 (Tr1) cells, which reside in the spleen and prevent migration of diabetogenic T-cells to the draining lymph nodes. These two Tr cell subsets act in concert to control diabetogenic T-cells that are still present in long-term tolerant mice. Rapamycin plus IL-10 treatment, promoting distinct subsets of Tr cells, may constitute a novel and potent tolerance-inducing protocol for immune-mediated diseases.
View details for DOI 10.2337/db05-1576
View details for Web of Science ID 000238053400005
View details for PubMedID 16731819
-
Tr1 cells: From discovery to their clinical application
SEMINARS IN IMMUNOLOGY
2006; 18 (2): 120-127
Abstract
Peripheral tolerance is mediated by multiple mechanisms such as anergy and/or active suppression of effector T cells by T regulatory (Tr) cells. Among the CD4(+) Tr cells, T regulatory type 1 cells (Tr1) have been shown to down-modulate immune responses through production of the immunosuppressive cytokines IL-10 and TGF-beta. Tr1 cells maintain peripheral tolerance, control autoimmunity, and prevent allograft rejection and graft versus host disease (GvHD). Cellular therapy with ex vivo generated Tr1 cells has been proven to be effective in several preclinical models of T cell-mediated pathologies and therefore, represents a promising approach for clinical application. This review will summarize the new findings on Tr1 cells, the recent development of methods for their ex vivo expansion, and their potential clinical relevance as cellular therapy.
View details for DOI 10.1016/j.smim.2006.01.007
View details for Web of Science ID 000236041400007
View details for PubMedID 16464609
-
Efficacy of gene therapy for Wiskott-Aldrich syndrome using a WAS promoter/cDNA-containing lentiviral vector and nonlethal irradiation
HUMAN GENE THERAPY
2006; 17 (3): 303-313
Abstract
Wiskott-Aldrich syndrome (WAS) is a life-threatening X-linked primary immunodeficiency characterized by infections, hemorrhages, autoimmune disorders, and lymphomas. Transplantation of genetically corrected autologous hematopoietic stem cells (HSCs) could represent an alternative treatment to allogeneic HSC transplantation, the latter being often associated with severe complications. We used WAS-/- mice to test the efficacy of a gene therapy approach based on nonlethal irradiation followed by transplantation of WAS-/- HSCs transduced with lentiviral vectors encoding the WAS protein (WASP) from either the ubiquitous PGK promoter or the tissue- specific WAS promoter. The procedure resulted in significant levels of engraftment of WASP-expressing T cells, B cells, platelets, and myeloid cells. T cells harbored one or two vector copies and displayed partial to full correction of T cell receptor-driven interleukin-2 production and proliferation. In addition, polymerization of F-actin and localization of WASP at the site of the immunological synapse were restored. The treatment was well tolerated and no pathology was detected by systematic blood analysis and autopsy. The efficacy of WAS gene transfer into HSCs, using the WAS promoter-containing lentiviral vector, combined with nonlethal irradiation provides a strong rationale for the development of gene therapy for WAS patients.
View details for Web of Science ID 000236225800005
View details for PubMedID 16544979
-
Rapamycin and interleukin-10 treatment induces T regulatory type 1 cells that mediate antigen-specific transplantation tolerance
DIABETES
2006; 55 (1): 40-49
Abstract
Islet transplantation is a cure for type 1 diabetes, but its potential is limited by the need for constant immunosuppression. One solution to this problem is the induction of transplantation tolerance mediated by T regulatory cells. T regulatory type 1 (Tr1) cells are characterized by their production of high levels of interleukin (IL)-10, which is crucial for their differentiation and suppressive function. We investigated the effects of IL-10 administered in combination with rapamycin on the induction of Tr1 cells that could mediate a state of tolerance in diabetic mice after pancreatic islet transplantation. The efficacy of this treatment was compared with IL-10 alone and standard immunosuppression. Stable long-term tolerance that was not reversible by alloantigen rechallenge was achieved only in mice treated with rapamycin plus IL-10. Tr1 cells that produced high levels of IL-10 and suppressed T-cell proliferation were isolated from splenocytes of rapamycin plus IL-10-treated mice after treatment withdrawal. In rapamycin plus IL-10-treated mice, endogenous IL-10 mediated an active state of tolerance, as was observed when the blockade of IL-10 activity rapidly induced graft rejection >100 days after transplantation. CD4(+) T-cells from rapamycin plus IL-10-treated mice transferred antigen-specific tolerance in mice that received new transplants. Thus rapamycin plus IL-10 not only prevented allograft rejection but also induced Tr1 cells that mediated stable antigen-specific, long-term tolerance in vivo.
View details for Web of Science ID 000234349500006
View details for PubMedID 16380475
-
CD4(+) regulatory T cells: Mechanisms of induction and effector function
Conference on Immunodysregulation Diseases - From Basic Sciences to Clinic
ELSEVIER SCIENCE BV. 2005: 491–96
Abstract
The main subsets of CD4+ regulatory T (Tr) cells are the CD4+ CD25+ Tr cells and the type 1 regulatory (Tr1) cells. Both subsets are essential for the maintenance of peripheral tolerance. CD4+ CD25+ Tr cells control immune homeostasis (e.g., in autoimmunity) mainly by cell contact-dependent interactions. In contrast, Tr1 cells regulate immune responses in transplantation, allergy, and autoimmune diseases, via an IL-10- and TGF-beta-dependent mechanism. Identification of the mechanisms responsible for the induction of the different Tr subsets in vivo is a matter of intense investigation. Studies on dendritic cells (DC), performed in the last years by several groups, have significantly contributed to clarify this point. Indeed, it is now clear that the role of DCs is not only to sense danger, but also to tolerize the immune system to antigens (Ags) encountered in the absence of maturation/inflammatory stimuli. Therefore, if a naïve T cell encounters the antigen on immature DCs (iDCs), it differentiates into a Tr cell rather than an effector T cell. A better understanding of the mechanisms underlying the induction and functions of Tr cells in controlling the immune system is critical in view of a future cellular therapy to modulate immune-mediated pathologies.
View details for DOI 10.1016/j.autrev.2005.04.005
View details for Web of Science ID 000233255900003
View details for PubMedID 16214084
-
Regulatory T cells: prospective for clinical application in hematopoietic stem cell transplantation
CURRENT OPINION IN HEMATOLOGY
2005; 12 (6): 451-456
Abstract
Regulatory T cells exert a dominant effect in controlling autoimmunity and maintaining peripheral tolerance. Regulatory T cells are also involved in preventing allograft rejection and graft versus host disease. Cellular therapy with expanded regulatory T cells represents a promising approach to control T-cell mediated pathology. In this review we will summarize the efforts to design new methods for expanding regulatory T cells and exploit their regulatory function as cellular therapy for the treatment of graft versus host disease after hematopoietic stem cell transplantation.Among CD4+ T cells, the best described are the naturally occurring CD4+CD25+ regulatory T cells and type 1 regulatory T cells. Recent progress has been made in the characterization of both subsets in terms of isolation and induction, respectively. However, a clear definition of their mechanisms of action has still to be achieved.Better understanding of the mechanisms of suppression mediated by regulatory T cells might enable their use to modulate specific immune responses. Moreover, the recent development of methods allowing the ex-vivo expansion of regulatory T cells, to provide sufficient number of cells for in-vivo infusion, represents the first step toward the use of these cells as cellular therapy for the treatment of immunologic and hematological diseases.
View details for Web of Science ID 000232713100003
View details for PubMedID 16217161
-
The role of 2 FOXP3 isoforms in the generation of human CD4(+) Tregs
JOURNAL OF CLINICAL INVESTIGATION
2005; 115 (11): 3276-3284
Abstract
Little is known about the molecules that control the development and function of CD4+ CD25+ Tregs. Recently, it was shown that the transcription factor FOXP3 is necessary and sufficient for the generation of CD4+ CD25+ Tregs in mice. We investigated the capacity of FOXP3 to drive the generation of suppressive CD4+ CD25+ Tregs in humans. Surprisingly, although ectopic expression of FOXP3 in human CD4+ T cells resulted in induction of hyporesponsiveness and suppression of IL-2 production, it did not lead to acquisition of significant suppressor activity in vitro. Similarly, ectopic expression of FOXP3delta2, an isoform found in human CD4+ CD25+ Tregs that lacks exon 2, also failed to induce the development of suppressor T cells. Moreover, when FOXP3 and FOXP3delta2 were simultaneously overexpressed, although the expression of several Treg-associated cell surface markers was significantly increased, only a modest suppressive activity was induced. These data indicate that in humans, overexpression of FOXP3 alone or together with FOXP3delta2 is not an effective method to generate potent suppressor T cells in vitro and suggest that factors in addition to FOXP3 are required during the process of activation and/or differentiation for the development of bona fide Tregs.
View details for DOI 10.1172/JCI24685
View details for Web of Science ID 000233022100037
View details for PubMedID 16211090
-
Myelin deterioration in twitcher mice: Motor evoked potentials and magnetic resonance imaging as in vivo monitoring tools
JOURNAL OF NEUROSCIENCE RESEARCH
2005; 81 (4): 597-604
Abstract
We have used magnetic resonance imaging (MRI) and motor evoked potentials (MEPs) for monitoring disease progression within the CNS of the Twitcher mouse, the murine model for globoid cell leukodystrophy (GLD). GLD is a lysosomal storage disorder, resulting from galactocerebrosidase deficiency, causing central and peripheral myelin impairment, leading to death, usually during early infancy. Neuroradiological, electrophysiological, and pathological parameters of myelin maturation were evaluated in Twitcher mice between postnatal days 20 and 45. Healthy controls showed a gradual-appearance MRI T2-weighted hypointensity of the corpus callosum (CC) starting at about P30 and ending at about P37, whereas MRI of age-matched Twitcher mice showed a complete loss of the CC-related MRI signal. MEPs allowed the functional assessment of myelin maturation within corticospinal motor pathways and showed a progressive deterioration of MEPs in Twitcher mice with increased central conduction time (CCT; 5.12 +/- 0.49 msec at P27 to 6.45 +/- 1.96 msec at P32), whereas physiological CCT shortening was found in healthy controls (3.01 +/- 0.81 msec at P27 to 2.5 +/- 0.27 msec at P32). These findings were not paralleled by traditional histological stainings. Optical observation of Bielchowsky and Luxol fast blue-PAS stainings showed mild axonal/myelin deterioration of the Twitcher brain within this time frame. Our results demonstrate that serial MRI and MEP readings are sensitive evaluation tools for in vivo monitoring of dysmyelination in Twitcher mice and underscore their potential use for longitudinal evaluation of the therapeutic impact of gene and cell therapies on these animals.
View details for DOI 10.1002/jrn.20574
View details for Web of Science ID 000230945000015
View details for PubMedID 15948181
-
Rapamycin selectively expands CD4(+)CD25(+)FoxP3(+) regulatory T cells
BLOOD
2005; 105 (12): 4743-4748
Abstract
Rapamycin is an immunosuppressive compound that is currently used to prevent acute graft rejection in humans. In addition, rapamycin has been shown to allow operational tolerance in murine models. However, a direct effect of rapamycin on T regulatory (Tr) cells, which play a key role in induction and maintenance of peripheral tolerance, has not been demonstrated so far. Here, we provide new evidence that rapamycin selectively expands the murine naturally occurring CD4(+)CD25(+)FoxP3(+) Tr cells in vitro. These expanded Tr cells suppress proliferation of syngeneic T cells in vitro and prevent allograft rejection in vivo. Interestingly, rapamycin does not block activation-induced cell death and proliferation of CD4(+) T cells in vitro. Based on this new mode of action, rapamycin can be used to expand CD4(+)CD25(+)FoxP3(+) Tr cells for ex vivo cellular therapy in T-cell-mediated diseases.
View details for DOI 10.1182/blood-2004-10-3932
View details for Web of Science ID 000229757300040
View details for PubMedID 15746082
-
A proportion of patients with lymphoma may harbor mutations of the perforin gene
BLOOD
2005; 105 (11): 4424-4428
Abstract
Perforin mutations have been demonstrated in a proportion of patients diagnosed with the familial form of hemophagocytic lymphohistiocytosis (HLH). In the present study, we evaluated whether some patients with lymphoma sharing clinical characteristics with HLH might harbor mutations of the perforin gene. We analyzed 29 patients and found that 4 patients, who developed either Hodgkin or non-Hodgkin lymphoma, had biallelic mutations of the perforin gene. One of these 4 patients, a 19-year-old female with T-cell lymphoma, had a brother carrying the same mutations who developed HLH. In 2 of the 4 patients with biallelic mutations of the perforin gene, we evaluated perforin expression by flow cytometry and natural killer (NK) activity and both were found to be absent. Moreover, we documented the presence of monoallelic mutations of the perforin gene in 4 more patients. One of these 4 latter patients also carried a mutation of the Fas gene. These data indicate that perforin deficiency, either alone or in combination with other mutations of genes involved in lymphocyte survival or functional activity, may be present in patients with lymphoma. These findings suggest that perforin also plays a key role in the mechanisms of immune surveillance that prevent tumor growth and/or development.
View details for DOI 10.1182/blood-2004-04-1477
View details for Web of Science ID 000229292500045
View details for PubMedID 15728124
-
SAP controls the cytolytic activity of CD8(+) T cells against EBV-infected cells
BLOOD
2005; 105 (11): 4383-4389
Abstract
The adaptor protein SAP regulates signaling through signaling lymphocytic activation molecule (SLAM)-family receptors expressed on T and natural killer (NK) cells. In patients affected by X-linked lymphoproliferative (XLP) disease, mutations in the SH2D1A gene result in defective lytic activity. However, the mechanism by which SAP controls cytotoxic activity remains unclear. T-cell-receptor (TCR) activation of CD8(+) cytotoxic T cells (CTLs) results in down-regulation of SAP, suggesting that this protein is involved in early activation events. Here, we show that SAP-deficient CTLs from patients with XLP and hemophagocytic lymphohistiocytosis (HLH) display a specific lytic defect against autologous and allogeneic Epstein-Barr virus (EBV)-positive B cells. This defect is associated with the defective polarization of 2B4, perforin, and lipid rafts at the contact area of CTLs with EBV-positive targets. Blockade of 2B4 in normal CTLs reproduces the defects in lysis and polarization observed in SAP-deficient CTLs. Expression and regulation of the SLAM-family receptors SLAM, CD84, and 2B4, as well as the lytic effectors perforin and granzyme-B are normal in SAP-deficient CTLs. In addition, TCR stimulation leads to normal proliferation and production of interleukin 2 (IL-2), IL-4, and interferon-gamma (IFN-gamma). These results demonstrate that the SAP/2B4 pathway plays a key role in CTL lytic activity against EBV-positive targets by promoting the polarization of the lytic machinery.
View details for DOI 10.1182/blood-2004-08-3269
View details for Web of Science ID 000229292500040
View details for PubMedID 15677558
-
Human CD4+regulatory T cells and activation-induced tolerance
MICROBES AND INFECTION
2005; 7 (7-8): 1023-1032
Abstract
In the past years growing attention has been given to the role of regulatory T (Tr) cells in inducing and monitoring peripheral tolerance. Various subsets of Tr cells have been described based on their surface phenotype and cytokine production. However, presently there are no specific reliable markers for any of the Tr subsets and their classification is based predominantly upon their mode of suppression. In addition, the molecular mechanisms underlying the induction and mode of action of all Tr cell subsets remain to be elucidated. Here we review recent developments regarding human CD4+ Tr cells, their origin, phenotype, antigen specificity and mode of suppression.
View details for DOI 10.1016/j.micinf.2005.03.027
View details for Web of Science ID 000230979200009
View details for PubMedID 15893494
-
Utilizing regulatory T cells to control alloreactivity
CYTOTHERAPY
2005; 7 (2): 158-165
Abstract
Effective resetting of the immune system cannot be achieved by non-specific immunosuppression. Instead, novel strategies aim at harnessing the body's natural tolerance mechanisms to rectify an Ag-specific response without disturbing other immune functions. Fine-tuning of the balance between Ag-specific effector and regulatory T (Tr) cells is a promising strategy that requires detailed understanding of the differentiation and expansion pathways of the relevant Tr cell subsets. Here we review recent developments regarding the control of alloreactivity by induction and expansion of Tr cells. T-cell activation in the presence of tolerogenic APC and cytokines leads to the induction of Tr cells, which can mediate tolerance through cytokine-dependent and/or contact-dependent mechanisms. Better understanding of the mechanisms of immune regulation mediated by Tr cells may enable fine-tuning of specific immune responses and pave the way for novel therapeutic approaches.
View details for DOI 10.1080/14653240510018154
View details for Web of Science ID 000230106400008
View details for PubMedID 16040395
-
Beneficial autoimmunity in Type 1 diabetes mellitus
TRENDS IN IMMUNOLOGY
2005; 26 (5): 248-253
Abstract
The trigger that leads to the pathogenesis of type 1 diabetes is currently unknown. It is well established that the pathophysiology of the disease is biphasic. In the first stage, leukocytes infiltrate the pancreatic islets in a response that does not cause damage. In the second phase, which occurs only in diabetes-prone individuals and strains, autoreactive T cells acquire aggressive potential and destroy the majority of the pancreatic islets. Rodents and humans exhibit a physiological ripple of apoptotic beta-cell death shortly after birth, which induces an adaptive autoimmune response towards islet-antigens, both in diabetes-prone non-obese diabetic (NOD) mice and in mice that do not develop diabetes. Here, we propose that the early T cell-mediated autoimmune response towards islet-antigens is physiological, purposeful and beneficial.
View details for DOI 10.1016/j.it.2005.03.004
View details for Web of Science ID 000229249900005
View details for PubMedID 15866237
-
An anti-CD45RO/RB monoclonal antibody modulates T cell responses via induction of apoptosis and generation of regulatory T cells
JOURNAL OF EXPERIMENTAL MEDICINE
2005; 201 (8): 1293-1305
Abstract
The effects of a chimeric monoclonal antibody (chA6 mAb) that recognizes both the RO and RB isoforms of the transmembrane protein tyrosine phosphatase CD45 on human T cells were investigated. Chimeric A6 (chA6) mAb potently inhibited antigen-specific and polyclonal T cell responses. ChA6 mAb induced activation-independent apoptosis in CD4(+)CD45RO/RB(high) T cells but not in CD8(+) T cells. In addition, CD4(+) T cell lines specific for tetanus toxoid (TT) generated in the presence of chA6 mAb were anergic and suppressed the proliferation and interferon (IFN)-gamma production by TT-specific effector T cells by an interleukin-10-dependent mechanism, indicating that these cells were equivalent to type 1 regulatory T cells. Similarly, CD8(+) T cell lines specific for the influenza A matrix protein-derived peptide (MP.58-66) generated in the presence of chA6 mAb were anergic and suppressed IFN-gamma production by MP.58-66-specific effector CD8(+) T cells. Furthermore, chA6 mAb significantly prolonged human pancreatic islet allograft survival in nonobese diabetic/severe combined immunodeficiency mice injected with human peripheral blood lymphocytes (hu-PBL-NOD/SCID). Together, these results demonstrate that the chA6 mAb is a new immunomodulatory agent with multiple modes of action, including deletion of preexisting memory and recently activated T cells and induction of anergic CD4(+) and CD8(+) regulatory T cells.
View details for DOI 10.1084/jem.20040912
View details for Web of Science ID 000228521900011
View details for PubMedID 15837814
-
Impaired humoral immunity in X-linked lymphoproliferative disease is associated with defective IL-10 production by CD4+ T cells
JOURNAL OF CLINICAL INVESTIGATION
2005; 115 (4): 1049-1059
Abstract
X-linked lymphoproliferative disease (XLP) is an often-fatal immunodeficiency characterized by hypogammaglobulinemia, fulminant infectious mononucleosis, and/or lymphoma. The genetic lesion in XLP, SH2D1A, encodes the adaptor protein SAP (signaling lymphocytic activation molecule-associated [SLAM-associated] protein); however, the mechanism(s) by which mutations in SH2D1A causes hypogammaglobulinemia is unknown. Our analysis of 14 XLP patients revealed normal B cell development but a marked reduction in the number of memory B cells. The few memory cells detected were IgM(+), revealing deficient isotype switching in vivo. However, XLP B cells underwent proliferation and differentiation in vitro as efficiently as control B cells, which indicates that the block in differentiation in vivo is B cell extrinsic. This possibility is supported by the finding that XLP CD4(+) T cells did not efficiently differentiate into IL-10(+) effector cells or provide optimal B cell help in vitro. Importantly, the B cell help provided by SAP-deficient CD4(+) T cells was improved by provision of exogenous IL-10 or ectopic expression of SAP, which resulted in increased IL-10 production by T cells. XLP CD4(+) T cells also failed to efficiently upregulate expression of inducible costimulator (ICOS), a potent inducer of IL-10 production by CD4(+) T cells. Thus, insufficient IL-10 production may contribute to hypogammaglobulinemia in XLP. This finding suggests new strategies for treating this immunodeficiency.
View details for Web of Science ID 000228145700037
View details for PubMedID 15761493
-
Differentiation of Tr1 cells by immature dendritic cells requires IL-10 but not CD25(+)CD4(+) Tr cells
BLOOD
2005; 105 (3): 1162-1169
Abstract
Dendritic cells (DCs) are specialized antigen-presenting cells that monitor the antigenic environment and activate naive T cells. The role of DCs is not only to sense danger but also to tolerize the immune system to antigens encountered in the absence of maturation/inflammatory stimuli. Indeed, if a naive T cell encounters its antigen on immature DCs (iDCs), it may differentiate into a T-regulatory (Tr) rather than a T-effector cell. However, little is known about the mechanisms by which iDCs differentiate Tr cells. We developed a standardized and highly reproducible protocol to differentiate Tr cells by repetitive exposure of naive peripheral blood CD4(+) T cells to allogeneic iDCs. The resultant Tr cells are phenotypically and functionally identical to type 1 Tr (Tr1) cells because their generation requires production of IL-10 by iDCs, and they suppress T-cell responses through an interleukin-10 (IL-10)- and a transforming growth factor beta (TGF-beta)-dependent mechanism. In addition, Tr1 cells induced by iDCs do not require the presence of CD4(+)CD25(+) Tr cells for their generation, nor do they express high constitutive levels of CD25 or the transcription factor FoxP3. Thus, iDCs can drive the differentiation of Tr1 cells and can be used to generate large numbers of alloantigen-specific Tr1 cells for clinical use as a cellular therapy to restore peripheral tolerance.
View details for DOI 10.1182/blood-2004-03-1211
View details for Web of Science ID 000226596700045
View details for PubMedID 15479730
-
Induction of transplantation tolerance in humans using fetal cell transplants
20th International Congress of the Transplantation-Society
ELSEVIER SCIENCE INC. 2005: 65–66
Abstract
When engrafted with donor stem cells and lymphoid cells, patients develop transplantation tolerance to donor antigens. We analyzed the mechanism of tolerance induction in immunoincompetent recipients whose immunity has been reconstituted by transplantation of mismatched stem cells. Seven infants or human fetuses received fetal liver transplants as a treatment for severe combined immunodeficiency disease. After reconstitution of immunity by lymphocytes developed from donor stem cells, T-cell clones were produced and analyzed. Because donors and recipients were HLA mismatched, it was easy to demonstrate the donor origin of the T-cell clones. These clones were shown to have developed tolerance to histocompatibility antigens of the stem cell donor via a process of clonal deletion (probably as a result of contact with donor-derived macrophages and dendritic cells). They were also tolerant to histocompatibility antigens of the host but through a different mechanism: many clones recognized these antigens but had no detrimental effect on the target cells exhibiting host antigens, either in vitro or in vivo. Clonal anergy was therefore the cause of this tolerance to host determinants, resulting in a lack of graft-versus-host disease and of autoimmunity. The contact between developing T cells of donor origin and host epithelial cells within the host thymus may explain this colonal anergy. It should be noted that all patients had high serum levels of interleukin-10, which might have contributed to the persistent engraftment and tolerance.
View details for DOI 10.1016/j.transproceed.2004.12.006
View details for Web of Science ID 000228091300024
View details for PubMedID 15808548
-
Recombinant human interleukin 10 suppresses gliadin dependent T cell activation in ex vivo cultured coeliac intestinal mucosa
GUT
2005; 54 (1): 46-53
Abstract
Enteropathy in coeliac disease (CD) is sustained by a gliadin specific Th1 response. Interleukin (IL)-10 can downregulate Th1 immune responses.We investigated the ability of recombinant human (rh) IL-10 to suppress gliadin induced Th1 response.IL-10 RNA transcripts were analysed by competitive reverse transcription-polymerase chain reaction in duodenal biopsies from untreated and treated CD patients, non-coeliac enteropathies (NCE), and controls. CD biopsies were cultured with a peptic-tryptic digest of gliadin with or without rhIL-10. The proportion of CD80+ and CD25+ cells in the lamina propria, epithelial expression of Fas, intraepithelial infiltration of CD3+ cells, as well as cytokine synthesis (interferon gamma (IFN-gamma) and IL-2) were measured. Short term T cell lines (TCLs) obtained from treated CD biopsies cultured with gliadin with or without rhIL-10 were analysed by ELISPOT for gliadin specific production of IFN-gamma.In untreated CD and NCE, IL-10 RNA transcripts were significantly upregulated. In ex vivo organ cultures, rhIL-10 downregulated gliadin induced cytokine synthesis, inhibited intraepithelial migration of CD3+ cells, and reduced the proportion of lamina propria CD25+ and CD80+ cells whereas it did not interfere with epithelial Fas expression. In short term TCLs, rhIL-10 abrogated the IFN-gamma response to gliadin.rhIL-10 suppresses gliadin specific T cell activation. It may interfere with the antigen presenting capacity of lamina propria mononuclear cells as it reduces the expression of CD80. Interestingly, rhIL-10 also induces a long term hyporesponsiveness of gliadin specific mucosal T cells. These results offer new perspectives for therapeutic strategies in coeliac patients based on immune modulation by IL-10.
View details for DOI 10.1136/gut.2003.023150
View details for Web of Science ID 000225659500010
View details for PubMedID 15591503
-
Phenotypic and functional differences between human CD4(+)CD25(+) and type 1 regulatory T cells
CD4-PLUSCD25-PLUS REGULATORY T CELLS: ORIGIN, FUNCTION AND THERAPEUTIC POTENTIAL
2005; 293: 303-326
Abstract
T regulatory (Tr) cells have an essential role in the induction and maintenance of tolerance to both and foreign self-antigens. Many types of T cells with regulatory activity have been described in mice and humans, and those within the CD4+ subset have been extensively characterized. CD4+ Type-1 regulatory T (Tr1) cells produce high levels of IL-10 and mediate IL-10-dependent suppression, whereas the effects of naturally occurring CD4+CD25+ Tr cells appear to be cell-contact-dependent. Tr1 cells arise in the periphery upon encountering antigen in a tolerogenic environment. In contrast, it appears that CD4+CD25+ Tr cells can either arise directly in the thymus or be induced by antigen in the periphery. We have been interested in defining the phenotype and function of different subsets of CD4+ Tr cells present in human peripheral blood, with the ultimate aim of designing therapeutic strategies to harness their immunoregulatory effects. This review will discuss the similarities and differences between human Tr1 and naturally occurring CD4+CD25+ Tr cells, as well as evidence that indicates that they have nonoverlapping, but synergistic roles in immune homeostasis.
View details for Web of Science ID 000232051100014
View details for PubMedID 15981486
-
IL-3 or IL-7 increases ex vivo gene transfer efficiency in ADA-SCID BM CD34(+) cells while maintaining in vivo lymphoid potential
MOLECULAR THERAPY
2004; 10 (6): 1096-1108
Abstract
To improve maintenance and gene transfer of human lymphoid progenitors for clinical use in gene therapy of adenosine deaminase (ADA)-deficient SCID we investigated several gene transfer protocols using various stem cell-enriched sources. The lymphoid differentiation potential was measured by an in vitro clonal assay for B/NK cells and in the in vivo SCID-hu mouse model. Ex vivo culture with the cytokines TPO, FLT3-ligand, and SCF (T/F/S) plus IL-3 or IL-7 substantially increased the yield of transduced bone marrow (BM) CD34(+) cells purified from ADA-SCID patients or healthy donors, compared to T/F/S alone. Moreover, the use of IL-3 or IL-7 significantly improved the maintenance of in vitro B cell progenitors from ADA-SCID BM cells and allowed the efficient transduction of B and NK cell progenitors. Under these optimized conditions transduced CD34(+) cells were efficiently engrafted into SCID-hu mice and gave rise to B and T cell progeny, demonstrating the maintenance of in vivo lymphoid reconstitution capacity. The protocol based on the T/F/S + IL-3 combination was included in a gene therapy clinical trial for ADA-SCID, resulting in long-term engraftment of stem/progenitor cells. Remarkably, gene-corrected BM CD34(+) cells obtained from one patient 4 and 11 months after gene therapy were capable of repopulating the lymphoid compartment of SCID-hu hosts.
View details for DOI 10.1016/j.ymthe.2004.08.014
View details for Web of Science ID 000226001000014
View details for PubMedID 15564141
-
Lentiviral vector-mediated gene transfer in T cells from Wiskott-Aldrich syndrome patients leads to functional correction
MOLECULAR THERAPY
2004; 10 (5): 903-915
Abstract
Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with a median survival below the age of 20 due to infections, severe hemorrhage, and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical sibling donors is a resolutive treatment, but is available for a minority of patients. Transplantation of genetically corrected autologous hematopoietic stem cells or T cells could represent an alternative treatment applicable to all patients. We investigated whether WAS gene transfer with MMLV-based oncoretroviral and HIV-based lentiviral vectors could restore normal functions of patients' T cells. T cells transduced either with lentiviral vectors expressing the WAS protein (WASP) from the ubiquitous PGK promoter or the tissue-specific WASP promoter or with an oncoretroviral vector expressing WASP from the LTR, reached normal levels of WASP with correction of functional defects, including proliferation, IL-2 production, and lipid raft upregulation. Lentiviral vectors transduced T cells from WAS patients at higher rates, compared to oncoretroviral vectors, and efficiently transduced both activated and naive WAS T cells. Furthermore, a selective growth advantage of T cells corrected with the lentiviral vectors was demonstrated. The observation that lentiviral vector-mediated gene transfer results in correction of T cell defects in vitro supports their application for gene therapy in WAS patients.
View details for DOI 10.1016/j.ymthe.2004.08.008
View details for Web of Science ID 000225709400014
View details for PubMedID 15509508
-
Analysis of galactocerebrosidase activity in the mouse brain by a new histological staining method
JOURNAL OF NEUROSCIENCE RESEARCH
2004; 77 (3): 462-464
Abstract
Gene therapy of galactocerebrosidase (GALC) deficient mice (Twitcher mutants) requires a fast and sensitive assay to detect transduced cells in vitro and in vivo. We have developed a new rapid histochemical method that specifically detects GALC activity in situ in neural cells using 5-Br-3Cl-beta-galactopiranoside (X-Gal) in the presence of taurodeoxycholic and oleic acids to enhance suspension of the substrate at low pH. Using this method, we observed robust X-Gal staining in diverse neuronal populations and interfascicular oligodendrocytes in sections from normal mouse brain. In contrast, sections of Twitcher brain did not show a specific staining pattern in neurons or glial cells. The availability of this new sensitive and rapid in situ detection assay is fundamental for the follow-up of Twitcher mice under gene or cellular therapies to correct central GALC deficiency.
View details for DOI 10.1002/jnr.20169
View details for Web of Science ID 000222881300015
View details for PubMedID 15248301
-
Targeting lentiviral vector expression to hepatocytes limits transgene-specific immune response and establishes long-term expression of human antihemophilic factor IX in mice
BLOOD
2004; 103 (10): 3700-3709
Abstract
Stable gene replacement by in vivo administration of lentiviral vectors (LVs) has therapeutic potential for metabolic disorders and other systemic diseases. We studied the expression of intracellular and secreted proteins by LVs in immunocompetent mice. Liver, spleen, and bone marrow cells were efficiently transduced. However, transgene expression, driven by a ubiquitous promoter, was limited by transgene-specific cellular and humoral immune responses, leading to the clearance of transduced cells. After green fluorescent protein (GFP) gene transfer, the liver showed infiltration of CD8(+) cytotoxic T cells, and GFP-specific CD8(+) T cells were isolated from the spleen. After human factor IX (hF.IX) gene transfer, anti-hF.IX antibodies were induced. These immune responses were not detected in mice injected with heat-inactivated or genome-lacking LVs or in GFP-transgenic mice, indicating that they were specifically triggered by transgene expression in vivo. Intriguingly, selective targeting of LV expression to hepatocytes limited the immune responses to the transgenes. By this approach, high levels of hF.IX, potentially in the therapeutic range, were reached and maintained long term in immunocompetent mice, without inducing antibody formation. These results prompt further studies in relevant animal models to explore the potential of in vivo LV administration for the gene therapy of hemophilias and other liver-based diseases.
View details for DOI 10.1182/blood-2003-09-3217
View details for Web of Science ID 000221642800024
View details for PubMedID 14701690
-
Efficient gene transfer into primitive hematopoietic progenitors using a bone marrow microenvironment cell line engineered to produce retroviral vectors
HAEMATOLOGICA
2004; 89 (4): 462-470
Abstract
Effective gene transfer into human hematopoietic stem/progenitor cells is a compromise between achieving high transduction efficiency and maintaining the desired biological characteristics of the target cell. The aim of our work was to exploit the stromal microenvironment to increase gene transfer and maintenance of hematopoietic progenitors.The murine bone marrow stromal cell line MS-5, known to support primitive human progenitors, was modified into an amphotropic packaging cell, by the stable introduction of DNA coding for retroviral structural proteins, and a viral vector encoding a marker gene. The gene transfer efficiency of the recombinant virus was evaluated by flow cytometry, in vitro assays for committed (CFC) and primitive (LTC-CFC) progenitors, as well as a clonal assay for B and NK lymphoid progenitors.The new packaging cell line (NEXUS) produced equivalent levels of virus as did the established GP+Am12 system, also under serum-free conditions. On average 30% of human mobilized peripheral blood CD34(+) cells were transduced by a single exposure to NEXUS supernatant, representing a three-fold increase over GP+Am12-based technology. Gene transfer into both committed and primitive progenitors increased on average two-fold using NEXUS retroviral supernatant. Furthermore, CD34(+) CD38(low) early progenitor cells purified from umbilical cord blood were efficiently transduced with NEXUS retroviral vector and gave rise to a high frequency of marked B and NK lymphocytes.Our data show that that an established bone marrow stromal cell can be engineered to enhance the genetic modification of primitive hematopoietic and lymphoid progenitors using a clinically relevant method.
View details for Web of Science ID 000220630600010
View details for PubMedID 15075080
-
Mobilized blood CD341 cells transduced and selected with a clinically applicable protocol reconstitute lymphopoiesis in SCID-Hu mice
HUMAN GENE THERAPY
2004; 15 (3): 305-311
Abstract
We developed a clinically applicable gene transfer procedure into mobilized peripheral blood (MPB) CD34(+) hematopoietic progenitor cells, based on single viral exposure and selection of engineered cells. CD34(+) cells were transduced with a retroviral vector carrying the truncated form of the nerve growth factor receptor (Delta NGFR) marker gene, and immunoselected for Delta NGFR expression. Optimal time and procedure for viral exposure, length of culture, and transgene expression of MPB CD34(+) cells were determined using in vitro assays. The multipotent capacity of MPB CD34(+)-transduced cells was demonstrated in the SCID-hu bone/liver/thymus mouse model. Transduced Delta NGFR(+) cells retained 50% of long-term culture-colony forming cells (LTC-CFC) compared to unmanipulated CD34(+) cells. In SCID-hu mice, 52% of CD45(+) cells, 27% of CD34(+) cells, 49% of B cells, and more than 50% of T cells were derived from transplanted CD34(+)/Delta NGFR(+) cells. Furthermore, transplantation of purified transduced cells greatly reduced the competition with untransduced progenitors occurring in unselected grafts. These data demonstrate that MPB CD34(+) cells, transduced with a single viral exposure and selected by transgene expression, retain multilineage reconstitution capacity and remarkable transgene expression.
View details for Web of Science ID 000220213100008
View details for PubMedID 15018739
-
The role of cytokines (and not only) in inducing and expanding T regulatory type 1 cells
3rd Beaune Seminar in Transplant Research
LIPPINCOTT WILLIAMS & WILKINS. 2004: S16–S18
Abstract
T regulatory (Tr) type 1 cells are one of the most studied of CD4+ Tr cell subsets. They are distinct from other T-cell subsets in their cytokine production profile and in their ability to suppress immune responses both in vitro and in vivo. Human and murine Tr1 cells share many properties, but signals regarding their in vitro induction are different. Although culture of murine naive CD4+ cells in the presence of high doses of interleukin (IL)-10 is sufficient for Tr1 induction, human naive CD4+ T cells can give rise to Tr1 cells when cultured with IL-10 and interferon-alpha. Tr1 cells can also be induced in vivo, although the mere administration of IL-10 is not sufficient to generate Tr1 cells in a context of murine allogenic transplant. IL-10 needs to be combined with immunosuppressive compounds, which are permissive for tolerance induction, to generate or expand Tr1 cells in vivo and mediate antigen-specific tolerance. Whether this combined treatment can also be used in humans is still an open question.
View details for DOI 10.1097/01.TP.0000106468.96542.26
View details for Web of Science ID 000188423400007
View details for PubMedID 14726763
-
Reappraisal of in utero stem cell transplantation based on long-term results
FETAL DIAGNOSIS AND THERAPY
2004; 19 (4): 305-312
Abstract
The therapeutic field of in utero transplantation of stem cells, into human fetuses, has developed since 1988 with the hope of improved probability of engraftment and tolerance, due to immune immaturity of the host. Fifteen years later, it is possible to evaluate the results that we and others have obtained in the treatment of several fetal diseases. Seven fetal patients have been treated in Lyon: In 2 cases, pregnancy termination was induced by the in utero injection; in the 5 other cases, engraftment was obtained and repeatedly documented with presence of donor HLA antigens and/or Y chromosome in recipients. In the 2 patients with combined immunodeficiency disease, a sustained reconstitution of immunity was obtained as a result of the transplant but other complications occurred thereafter. In patients with thalassemia major, Niemann-Pick disease or hemophilia, a very partial and very transitory benefit was only obtained. Approximately 33 other patients with immunodeficiencies, hemoglobinopathies or inborn errors of metabolism have been treated worldwide, over the last 13 years, with a comparable method, using parental or fetal stem cells transplanted in utero. Successful treatment has usually been recorded in immunodeficiencies, and insufficient results have been obtained in the other cases. This form of treatment can therefore be recommended after prenatal diagnosis of combined immunodeficiency but additional research is required to improve the degree of engraftment, the lack of resistance of the host and the 'space' available for hematopoiesis in the other conditions.
View details for DOI 10.1159/000077957
View details for Web of Science ID 000221942700001
View details for PubMedID 15192288
-
IL-10-producing T regulatory type 1 cells and oral tolerance
Conference on Oral Tolerance
NEW YORK ACAD SCIENCES. 2004: 142–153
Abstract
Oral tolerance is mediated by multiple mechanisms such as anergy and/or active suppression of antigen-specific effector T cells by T regulatory (Tr) cells. Among the CD4(+) Tr cells, T regulatory type 1 cells (Tr1) have been shown to downmodulate immune responses through production of the immunosuppressive cytokines IL-10 and TGF-beta. Human Tr1 cells can be induced to differentiate in vitro by IL-10 1 IFN-alpha or after stimulation by immature dendritic cells (DCs) or DCs rendered tolerogenic by exposure to immunomodulatory compounds. Murine Tr1 cells can be induced to differentiate in vitro by activating naive CD4(+) T cells in the presence of high doses of IL-10. Several protocols for induction of oral tolerance, including oral administration of the antigen with IL-10, have been shown to induce antigen-specific Tr1 cells that suppress undesired immune responses toward self-antigens, allergens, and food antigens. Overall, these data demonstrate that IL-10-producing Tr1 cells play a central role in the induction of oral as well as systemic tolerance.
View details for DOI 10.1196/annals.1309.031
View details for Web of Science ID 000227728600017
View details for PubMedID 15681753
-
Gene therapy for adenosine deaminase deficiency.
Current opinion in allergy and clinical immunology
2003; 3 (6): 461-466
Abstract
Gene therapy for severe combined immunodeficiency due to adenosine deaminase deficiency has moved from the early trials of safety and feasibility to recent studies demonstrating efficacy and clinical benefit. This review describes the latest advances in gene therapy trials for this condition using peripheral blood lymphocytes or hematopoietic progenitors.In the first patients with severe combined immunodeficiency due to adenosine deaminase deficiency treated with peripheral blood lymphocytes, transduced T cells have been shown to persist for over 10 years, expressing transgenic adenosine deaminase, but the therapeutic effect of gene therapy remained difficult to assess because of the concomitant treatment with bovine adenosine deaminase conjugated to polyethylene-glycol (PEG-ADA). A recent report showed that discontinuation of PEG-ADA resulted in a strong selective advantage of gene corrected T cells associated with restoration of T cell functions and antibody responses to neoantigen, but incomplete correction of the metabolic defect. Follow-up studies in patients treated with engineered hematopoietic progenitors in the early trials revealed low marking levels of long-term living progenitors and limited clinical effect. Recently, an improved gene transfer protocol in bone marrow CD34+ cells combined with low-dose busulfan resulted in multilineage, stable engraftment of transduced progenitors at substantial levels, restoration of immune functions, correction of the adenosine deaminase metabolic defect, and proven clinical benefit, in the absence of PEG-ADA. Overall, no adverse effect or toxicity has been observed in patients treated with adenosine deaminase gene transfer in mature lymphocytes or hematopoietic progenitors.Gene transfer in hematopoietic stem cells combined with nonmyeloablative conditioning is efficacious and might be extended to the treatment of other inherited and acquired disorders of the hematopoietic system.
View details for PubMedID 14612670
-
IL-10 and TGF-beta induce alloreactive CD4(+)CD25(-) T cells to acquire regulatory cell function
BLOOD
2003; 101 (12): 5076-5083
Abstract
We previously reported that interleukin-10 (IL-10) and transforming growth factor (TGF)-beta treatment of primary mixed lymphocyte reaction (MLR) cultures resulted in secondary alloantigen-specific hyporesponsiveness and protection from graft-versus-host disease (GVHD) lethality. Here, we report that CD4+ T cells recovered from the IL-10- and TGF-beta-treated primary MLR cultures have immunoregulatory function. Tolerized cells significantly inhibited proliferation of naive alloreactive CD4+ T cells in a primary MLR. Inhibition of the naive alloresponse was observed with as few as 1 tolerized cell to 10 naive responder cells. Tolerized cells were able to significantly reduce GVHD lethality when injected with naive alloreactive CD4+ T cells into major histocombatibility class (MHC) II disparate recipients. Rigorous CD25 depletion of the primary MLR had no effect on generation of a regulatory capacity, suggesting that the regulatory cells likely originated from CD4+CD25- T cells. Immune suppression was mediated independently of IL-10 and TGF-beta production, as neutralizing antibodies for IL-10, IL-10R, and TGF-beta were unable to revert suppression, and IL-10- deficient CD4+ T cells were able to mediate in vitro and in vivo suppression. The generation of immunoregulatory cells from a CD4+CD25- population during tolerization with IL-10 and TGF-beta provides an additional mechanism to prevent GVHD lethality by T cells that may escape full tolerance induction.
View details for DOI 10.1182/blood-2002-09-2798
View details for Web of Science ID 000183481700072
View details for PubMedID 12609834
-
The role of interleukin 10 in the control of autoimmunity
JOURNAL OF AUTOIMMUNITY
2003; 20 (4): 269-272
View details for DOI 10.1016/S0896-8411(03)00047-7
View details for Web of Science ID 000183215600002
View details for PubMedID 12791310
-
Type 1 T regulatory cells and their relationship with CD4+CD25+ T regulatory cells.
Novartis Foundation symposium
2003; 252: 115-127
Abstract
Suppression by T regulatory (T(reg)) cells is essential for the induction of peripheral tolerance. Several types of CD4+ T(reg) cells have been described in a number of systems. Although the precise mechanisms which mediate T(reg) cells effector activity remain to be defined, it is well established that they can suppress Immune responses via cell-cell interactions and/or the production of interleukin (IL)10 and transforming growth factor (TGF)beta. Type 1 T regulatory (T(reg)) cells are defined by their ability to produce high levels of IL10 and TGFbeta, and these cytokines mediate their ability to suppress pathological immune responses in the settings of transplantation, allergy, and autoimmune diseases. T(reg) cell activity is not necessarily beneficial, and they can also suppress immune responses to antigens from tumours and pathogens. The differentiation of T(reg) cells in vivo is likely controlled by certain dendritic cells that promote IL10 production and may express tolerogenic co-stimulatory molecules. Another subset of CD4+ T(reg) cells is defined by constitutive expression of CD25. Naturally occurring human CD4+CD25+ T(reg) cells are distinct from T(reg1) cells. Suppressive CD4+CD25+ T cell clones do not synthesize IL10 but produce TGFbeta which contributes to the suppression of proliferation mediated by these cells. However, CD4+CD25+ T(reg) cells may be involved in the process inducing the differentiation of T(reg1) cells. In conclusions, many questions on the basic biology of T(reg) cells remain to be answered, but the development of therapeutic strategies designed to harness their immunoregulatory effects can already be envisaged.
View details for PubMedID 14609215
-
The role of IL-10 and TGF-beta in the differentiation and effector function of T regulatory cells
INTERNATIONAL ARCHIVES OF ALLERGY AND IMMUNOLOGY
2002; 129 (4): 263-276
Abstract
Suppression by T regulatory (Tr) cells is essential for the induction of peripheral tolerance. Many types of CD4+ Tr cells have been described in a number of systems, and although the precise mechanisms which mediate their effects remain to be defined, it is well established that they can suppress immune responses via cell-cell interactions and/or the production of interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta). Type 1 T regulatory (Tr1) cells are defined by their ability to produce high levels of IL-10 and TGF-beta, and these cytokines mediate their ability to suppress pathological immune responses in the settings of transplantation, allergy and autoimmune disease. Tr1 cell activity is not necessarily beneficial, and they can also suppress immune responses to antigens from tumours and pathogens. In vivo, the differentiation of Tr1 cells is likely controlled by certain dendritic cells which promote IL-10 production and may express tolerogenic costimulatory molecules. Another subset of CD4+ Tr cells is defined by constitutive expression of CD25, and although these CD4+CD25+ Tr cells appear to suppress via mechanisms which are largely independent of cytokines, they may actively promote the differentiation of Tr1 cells. Many questions about the basic biology of Tr1 cells remain to be answered, but the development of therapeutic strategies designed to harness their immunoregulatory effects can already be contemplated.
View details for DOI 10.1159/000067596
View details for Web of Science ID 000180558100002
View details for PubMedID 12483031
-
Human CD25(+) CD4(+) T suppressor cell clones produce transforming growth factor beta, but not interleukin 10, and are distinct from type 1 T regulatory cells
JOURNAL OF EXPERIMENTAL MEDICINE
2002; 196 (10): 1335-1346
Abstract
T regulatory (Tr) cells are essential for the induction of peripheral tolerance. Several types of Tr cells exist, including CD4(+) T cells which express CD25 constitutively and suppress immune responses via direct cell-to-cell interactions, and type 1 T regulatory (Tr1) cells, which function via secretion of interleukin (IL)-10 and transforming growth factor (TGF)-beta. The relationship between CD25(+)CD4(+) T cells and Tr1 cells remains unclear. Here, we demonstrate at the clonal level that Tr1 and CD25(+)CD4(+) T cells are two distinct subsets of regulatory cells with different cytokine production profiles. Furthermore, CD25(-)CD4(+) T cells can be rendered anergic by IL-10 and differentiated into Tr1 cells in the absence of CD25(+)CD4(+) T cells. Cloned human CD25(+)CD4(+) T cell populations are heterogeneous and only a subset of clones continues to express high levels of CD25 and is suppressive. The intensity of CD25, cytotoxic T lymphocyte antigen (CTLA)-4, and glucocorticoid-induced tumor necrosis factor (TNF) receptor expression correlates with the suppressive capacity of the T cell clones. None of the CD25(+)CD4(+) T cell clones with suppressive function produce IL-10, but all produce TGF-beta. Suppression mediated by CD25(+)CD4(+) T cell clones is partially dependent on TGF-beta, but not on constitutive high expression of CD25. Together these data indicate that naturally occurring human CD25(+)CD4(+) T cells are distinct from IL-10-producing Tr1 cells.
View details for DOI 10.1084/jem.20021139
View details for Web of Science ID 000179412600006
View details for PubMedID 12438424
-
Human insulin production and amelioration of diabetes in mice by electrotransfer-enhanced plasmid DNA gene transfer to the skeletal muscle
GENE THERAPY
2002; 9 (21): 1429-1437
Abstract
A first-line gene therapy for type 1 diabetes should be based on a safe procedure to engineer an accessible tissue for insulin release. We evaluated the ability of the skeletal muscle to release human insulin after electrotransfer (ET)-enhanced plasmid DNA injection in mice. A furin-cleavable proinsulin cDNA under the CMV or the MFG promoter was electrotransferred to immune-incompetent mice with STZ-induced severe diabetes. At 1 week, mature human insulin was detected in the serum of 17/20 mice. After an initial peak of 68.5 +/- 34.9 microU/ml, insulin was consistently detected at significant levels up to 6 weeks after gene transfer. Importantly, untreated diabetic animals died within 3 weeks after STZ, whereas treated mice survived up to 10 weeks. Fed blood glucose (BG) was reduced in correspondence with the insulin peak. Fasting BG was near-normalized when insulin levels were 12.9 +/- 5.3 (CMV group, 2 weeks) and 7.7 +/- 2.6 microU/ml (MFG group, 4 weeks), without frank hypoglycemia. These data indicate that ET-enhanced DNA injection in muscle leads to the release of biologically active insulin, with restoration of basal insulin levels, and lowering of fasting BG with increased survival in severe diabetes. Therefore the skeletal muscle can be considered as a platform for basal insulin secretion.
View details for DOI 10.1038/sj.gt.3301804
View details for Web of Science ID 000178902600003
View details for PubMedID 12378405
-
Growth and expansion of human T regulatory type 1 cells are independent from TCR activation but require exogenous cytokines
EUROPEAN JOURNAL OF IMMUNOLOGY
2002; 32 (8): 2237-2245
Abstract
Cloned T regulatory type 1 (Tr1) cells produce IL-10, TGF-beta, IFN-gamma, and very low or non-detectable levels of IL-2 and IL-4, following TCR-mediated activation. In addition, upon TCR stimulation, Tr1 cell clones up-regulate activation markers but show low proliferative responses, partially due to the suppressive effect of autocrine IL-10 and TGF-beta. Here we show that Tr1 cells have growth requirements different from those of Th1 and Th2 cells. Exogenous IL-15, and to a lesser extent IL-2, induce and support the proliferation of Tr1 cells in the absence of TCR activation. This strong cytokine response correlates with high constitutive levels of the IL-2/15Rbeta and common gamma chains expressed by Tr1 cell clones. Furthermore, suboptimal doses of IL-15, in combination with IL-2, induce a significant growth (median value: 25-fold increase in cell number) of Tr1 cell clones during a culture period of 11 days, which leads to an in vitro expansion of Tr1 cell clones comparable to that of Th1 and Th2 cell clones. Tr1 cell clones cultured in IL-15 continue to secrete immunosuppressive cytokines and to proliferate poorly upon reactivation via TCR. These findings indicate that Tr1 cells are constitutively capable of responding to cytokines and mainly to IL-15. This growth factor enables a significant in vitro expansion of Tr1 cells facilitating further biological and biochemical characterization of this unique T cell subset.
View details for Web of Science ID 000177605900016
View details for PubMedID 12209636
-
Wiskott-Aldrich syndrome protein regulates lipid raft dynamics during immunological synapse formation
IMMUNITY
2002; 17 (2): 157-166
Abstract
Immunological synapse assembly relies on the clustering of lipid rafts and is required for optimal T cell activation. We demonstrate that the Wiskott-Aldrich syndrome protein (WASP) is recruited to lipid rafts immediately after TCR and CD28 triggering and is required for the movements of lipid rafts. T cells from Wiskott-Aldrich syndrome (WAS) patients, lacking WASP, proliferate poorly after TCR/CD28 activation and have impaired capacities to cluster the lipid raft marker GM1 and to upregulate GM1 cell surface expression. T cell proliferation and lipid raft clustering are restored by retroviral transfer of the WASP gene. These results demonstrate that WASP plays a central role in the movements of lipid rafts and identify a potential mechanism underlying the T cell defect affecting WAS patients.
View details for Web of Science ID 000177669400004
View details for PubMedID 12196287
-
Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning
SCIENCE
2002; 296 (5577): 2410-2413
Abstract
Hematopoietic stem cell (HSC) gene therapy for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID) has shown limited clinical efficacy because of the small proportion of engrafted genetically corrected HSCs. We describe an improved protocol for gene transfer into HSCs associated with nonmyeloablative conditioning. This protocol was used in two patients for whom enzyme replacement therapy was not available, which allowed the effect of gene therapy alone to be evaluated. Sustained engraftment of engineered HSCs with differentiation into multiple lineages resulted in increased lymphocyte counts, improved immune functions (including antigen-specific responses), and lower toxic metabolites. Both patients are currently at home and clinically well, with normal growth and development. These results indicate the safety and efficacy of HSC gene therapy combined with nonmyeloablative conditioning for the treatment of SCID.
View details for Web of Science ID 000176467200047
View details for PubMedID 12089448
-
Immune reconstitution in ADA-SCID after PBL gene therapy and discontinuation of enzyme replacement
NATURE MEDICINE
2002; 8 (5): 423-425
View details for DOI 10.1038/nm0502-423
View details for Web of Science ID 000175336800002
View details for PubMedID 11984564
-
The puzzling world of murine T regulatory cells
MICROBES AND INFECTION
2002; 4 (5): 559-566
Abstract
T regulatory cells are essential for downregulation of undesired immune responses and prevention of autoimmune diseases, organ rejection, and graft versus host disease. This review describes the considerable progress made in the recent years in the characterization of the many subsets that constitute the puzzled world of murine T regulatory cells.
View details for Web of Science ID 000175495100006
View details for PubMedID 11959512
-
Therapeutic applications for hematopoietic stem cell gene transfer
NATURE IMMUNOLOGY
2002; 3 (4): 318-321
View details for Web of Science ID 000174623600004
View details for PubMedID 11919567
-
A novel human packaging cell line with hematopoietic supportive capacity increases gene transfer into early hematopoietic progenitors
HUMAN GENE THERAPY
2001; 12 (16): 1979-1988
Abstract
The hematopoietic stem/progenitor cell (HSPC) represents the ideal target for gene therapy of disorders of the hematopoietic system, but still faces problems related to ex vivo manipulation and gene transfer efficiency. We demonstrate that soluble factors from the human endothelial-like cell line ECV 304/T24 support the growth of human CD34(+) progenitor cells as primary human bone marrow stroma and increase the rate of gene transfer into progenitor cells up to 5-fold. ECV 304/T24 was used to generate split-function amphotropic packaging cell lines (named APEX) with the purpose of combining, in the same cells, hematopoietic support and gene transfer vehicle functions. The APEX cell lines were negative for the presence of replication-competent retroviruses and produced complement-resistant vector particles. When mobilized peripheral blood or umbilical cord blood CD34(+) cells were exposed once to APEX supernatants, the level of gene transfer was equivalent to that observed with GP + Am12, in spite of the lower titer of the APEX producers. More importantly, APEX supernatants gave rise reproducibly to a 2-fold increase in transduction of early progenitors (long-term culture-initiating cells), reaching on average 50% gene transfer. This novel packaging cell represents a significant advance in HSPC genetic modification technology, combining both a beneficial hematopoietic supportive effect and the gene transfer vector function in a human-based system.
View details for Web of Science ID 000171919200004
View details for PubMedID 11686939
-
Type 1 T regulatory cells
IMMUNOLOGICAL REVIEWS
2001; 182: 68-79
Abstract
Suppression by T regulatory (Tr) cells is essential for induction of tolerance. Many types of Tr cells have been described in a number of systems, and their biology has been the subject of intensive investigation. Although many aspects of the mechanisms by which these cells exert their effects remain to be elucidated, it is well established that Tr cells suppress immune responses via cell-to-cell interactions and/or the production of interleukin (IL)-10 and transforming growth factor (TGF)-beta. Type-1 T regulatory (Tr1) cells are defined by their ability to produce high levels of IL-10 and TGF-beta. Tr1 cells specific for a variety of antigens arise in vivo, but may also differentiate from naive CD4+ T cells in the presence of IL-10 in vitro. Tr1 cells have a low proliferative capacity, which can be overcome by IL-15. Tr1 cells suppress naive and memory T helper type 1 or 2 responses via production of IL-10 and TGF-beta. Further characterisation of Tr1 cells at the molecular level will define their mechanisms of action and clarify their relationship with other subsets of Tr cells. The use of Tr1 cells to identify novel targets for the development of new therapeutic agents, and as a cellular therapy to modulate peripheral tolerance, can be foreseen.
View details for Web of Science ID 000172329800005
View details for PubMedID 11722624
-
Human CD25(+)CD4(+) T regulatory cells suppress naive and memory T cell proliferation and can be expanded in vitro without loss of function
JOURNAL OF EXPERIMENTAL MEDICINE
2001; 193 (11): 1295-1301
Abstract
Active suppression by T regulatory (Tr) cells plays an important role in the downregulation of T cell responses to foreign and self-antigens. Mouse CD4(+) Tr cells that express CD25 possess remarkable suppressive activity in vitro and in autoimmune disease models in vivo. Thus far, the existence of a similar subset of CD25(+)CD4(+) Tr cells in humans has not been reported. Here we show that human CD25(+)CD4(+) Tr cells isolated from peripheral blood failed to proliferate and displayed reduced expression of CD40 ligand (CD40L), in response to T cell receptor-mediated polyclonal activation, but strongly upregulated cytotoxic T lymphocyte-associated antigen (CTLA)-4. Human CD25(+)CD4(+) Tr cells also did not proliferate in response to allogeneic antigen-presenting cells, but they produced interleukin (IL)-10, transforming growth factor (TGF)-beta, low levels of interferon (IFN)-gamma, and no IL-4 or IL-2. Importantly, CD25(+)CD4(+) Tr cells strongly inhibited the proliferative responses of both naive and memory CD4(+) T cells to alloantigens, but neither IL-10, TGF-beta, nor CTLA-4 seemed to be directly required for their suppressive effects. CD25(+)CD4(+) Tr cells could be expanded in vitro in the presence of IL-2 and allogeneic feeder cells and maintained their suppressive capacities. These findings that CD25(+)CD4(+) Tr cells with immunosuppressive effects can be isolated from peripheral blood and expanded in vitro without loss of function represent a major advance towards the therapeutic use of these cells in T cell-mediated diseases.
View details for Web of Science ID 000169177500008
View details for PubMedID 11390436
-
Construction of human-SCID chimeric mice.
Current protocols in immunology / edited by John E. Coligan ... [et al.]
2001; Chapter 4: Unit 4 8-?
Abstract
Until recently, testing of new therapeutic agents has relied extensively upon the use of mice and nonhuman primates for in vivo preclinical studies. Unfortunately, these animal models do not always mimic the physiological and pathophysiological processes that occur in humans. The finding that C.B-17 severe combined immunodeficiency (SCID) mice lack a competent immune system, and therefore are unable to mount effective cellular and humoral responses to foreign antigens, has led to their use as recipients for xenografts of human tissues. This unit is focused on the construction of human-SCID chimeric through the surgical implantation of human fetal hematolymphoid tissues into SCID mice (SCID-hu mice). The Basic Protocol describes the surgical implantation of human fetal thymus and liver under the kidney capsules of SCID mice (SCID-hu Thy/Liv model). Subcutaneous transplantation of human fetal bone marrow and thymus (SCID-hu Bm/Thy mice) is described in the Alternate Protocol. Additional support protocols provide procedures to analyze human lymphocyte populations in the peripheral blood and grafted organs of SCID-hu mice. The advantages and disadvantages of each protocol and potential applications are discussed in the Commentary.
View details for DOI 10.1002/0471142735.im0408s25
View details for PubMedID 18432798
-
IFN-alpha and IL-10 induce the differentiation of human type 1 T regulatory cells
JOURNAL OF IMMUNOLOGY
2001; 166 (9): 5530-5539
Abstract
CD4(+) T regulatory type 1 (Tr1) cells suppress Ag-specific immune responses in vitro and in vivo. Although IL-10 is critical for the differentiation of Tr1 cells, the effects of other cytokines on differentiation of naive T cells into Tr1 cells have not been investigated. Here we demonstrate that endogenous or exogenous IL-10 in combination with IFN-alpha, but not TGF-beta, induces naive CD4(+) T cells derived from cord blood to differentiate into Tr1 cells: IL-10(+)IFN-gamma(+)IL-2(-/low)IL-4(-). Naive CD4(+) T cells derived from peripheral blood require both exogenous IL-10 and IFN-alpha for Tr1 cell differentiation. The proliferative responses of the Tr1-containing lymphocyte populations, following activation with anti-CD3 and anti-CD28 mAbs, were reduced. Similarly, cultures containing Tr1 cells displayed reduced responses to alloantigens via a mechanism that was partially mediated by IL-10 and TGF-beta. More importantly, Tr1-containing populations strongly suppressed responses of naive T cells to alloantigens. Collectively, these results show that IFN-alpha strongly enhances IL-10-induced differentiation of functional Tr1 cells, which represents a first major step in establishing specific culture conditions to generate T regulatory cells for biological and biochemical analysis, and for cellular therapy to induce peripheral tolerance in humans.
View details for Web of Science ID 000171906500026
View details for PubMedID 11313392
-
Differentiation of T regulatory cells by immature dendritic cells
JOURNAL OF EXPERIMENTAL MEDICINE
2001; 193 (2): F5-F9
View details for Web of Science ID 000166431400002
View details for PubMedID 11208869
-
Altered T-cell receptor+CD28-mediated signaling and blocked cell cycle progression in interleukin 10 and transforming growth factor-beta-treated alloreactive T cells that do not induce graft-versus-host disease
BLOOD
2001; 97 (2): 565-571
Abstract
The induction of anergy in T cells, although widely accepted as critical for the maintenance of tolerance, is still poorly understood at the molecular level. Recent evidence demonstrates that in addition to blockade of costimulation using monoclonal antibodies (mAbs) directed against cell surface determinants, treatment of mixed lymphocyte reaction (MLR) cultures with interleukin 10 (IL-10) and transforming growth factor-beta (TGF-beta) results in induction of tolerance, rendering alloreactive murine CD4(+) T cells incapable of inducing graft-versus-host disease (GVHD) after in vivo transfer to histoincompatible recipients. The present study, using these cells prior to adoptive transfer, determined that IL-10 + TGF-beta-tolerant CD4(+) T cells exhibit an altered pattern of T-cell receptor (TCR) + CD28-mediated signaling and are incapable of progressing out of the G(1) phase of the cell cycle during stimulation with HLA class II disparate antigen-presenting cells. TGFbeta + IL-10-tolerant cells were incapable of phosphorylating TCR-zeta, or activating ZAP-70, Ras, and MAPK, similarly to T-cell tolerized by blockade of B7/CD28 and CD40/CD40L pathways. Moreover, these cells were incapable of clonal expansion due to defective synthesis of cyclin D3 and cyclin A, and defective activation of cyclin-dependent kinase (cdk)4, cdk6, and cdk2. These cells also exhibited defective down-regulation of p27(kip1) cdk inhibitor and lack of cyclin D2-cdk4 activation, Rb hyperphosphorylation, and progression to the S phase of the cell cycle. These data link anergy-specific proximal biochemical alterations and the downstream nuclear pathways that control T-cell expansion and provide a biochemical profile of IL-10 + TGF-beta-tolerant alloreactive T cells that do not induce GVHD when transferred into MHC class II disparate recipients in vivo.
View details for Web of Science ID 000166388000033
View details for PubMedID 11154238
-
The role of different subsets of T regulatory cells in controlling autoimmunity
CURRENT OPINION IN IMMUNOLOGY
2000; 12 (6): 676-683
Abstract
T regulatory cells-in addition to clonal deletion and anergy-are essential for the downregulation of T cell responses to both foreign and self antigens, and for the prevention of autoimmunity. Recent progress has been made in characterising the different subsets of T regulatory cells, the factors that drive their differentiation, and their mode of action. The resolution of these mechanisms will make it possible to use T regulatory cells therapeutically in human autoimmune diseases.
View details for Web of Science ID 000165218000011
View details for PubMedID 11102772
-
T cell receptor-dependent activation of human lymphocytes through cell surface ganglioside GT1b: implications for innate immunity
EUROPEAN JOURNAL OF IMMUNOLOGY
2000; 30 (11): 3199-3206
Abstract
Gangliosides form a component of the glycosphingolipid-rich membrane microdomains recently shown to play an important role in receptor signal transduction. Specific gangliosides also serve as receptors for binding and internalization of bacterial toxins. In the course of characterizing the basis of the native tetanus toxin (TTx) reactivity of a human gamma delta T cell clone, we observed that transfer of the TCR was required to impart TTx reactivity on a TCR-negative recipient T cell. However, the reconstitution of toxin reactivity could be achieved regardless of the antigen specificity of the TCR chains. Further analysis showed that the T cell recognition of native TTx was dependent on the presence of its ganglioside receptor, GT1b, on the T cell surface. Incorporation of exogenous GT1b into plasma membrane conferred TTx reactivity on otherwise non-reactive T cells provided these cells expressed the TCR. Finally, reconstitution of TCR-negative Jurkat T cells with a CD8-CD3zeta chain chimera demonstrated that the cytoplasmic region of the CD3zeta chain was sufficient to couple ganglioside-mediated TTx binding to T cell activation. These data reveal a novel mode of TCR-dependent reactivity to a bacterial toxin that could mobilize a large subset of T cells, thus representing a form of innate immunity. Given the possibility that endogenous ligands may bind to cell surface gangliosides, regulation of their levels and topology on the cell surface may constitute an immunoregulatory mechanism.
View details for Web of Science ID 000165253100016
View details for PubMedID 11093135
-
T-regulatory 1 cells: A novel subset of CD4(+)T cells with immunoregulatory properties
Meeting on New Trends in Immunopharmacology 1998
MOSBY-ELSEVIER. 2000: S109–S112
Abstract
Clonal deletion and clonal anergy are well established mechanisms of peripheral tolerance. A role has also been described for clonal suppression by regulatory cells in the induction of peripheral tolerance to a variety of antigens. However, it has been difficult to isolate regulatory cells and to define their mechanism of action. We have recently reported the in vitro generation and characterization of a novel subset of CD4(+) T cells that have regulatory properties and are able to suppress antigen-specific immune responses in vitro and in vivo. These T-regulatory 1 (Tr1) cells are defined by their unique profile of cytokine production and make high levels of IL-10 and TGF-beta, but no IL-4 or IL-2. The IL-10 and TGF-beta produced by these cells mediate the inhibition of primary naive T cells in vitro. There is also evidence that Tr1 cells exist in vivo, and we have documented the presence of high IL-10-producing CD4(+) T cells in patients with severe combined immunodeficiency who have received allogeneic stem-cell transplants. These findings support the notion that Tr1 cells are involved in the regulation of peripheral tolerance and that they could potentially be used as a cellular therapy to modulate immune responses in vivo.
View details for Web of Science ID 000088482600016
View details for PubMedID 10887343
-
Prognostic significance of increased IL-10 production in patients prior to allogeneic bone marrow transplantation
BONE MARROW TRANSPLANTATION
2000; 25 (3): 237-241
Abstract
IL-10 is a potent immunosuppressant which inhibits allo-antigen-specific T cell responses. In addition, IL-10 is a strong endogenous anti-inflammatory cytokine. To investigate the role of IL-10 in the induction of acute GVHD following allogeneic bone marrow transplantation (BMT) we performed a prospective study on spontaneous IL-10 production by peripheral blood mononuclear cells (PBMNC) in 84 patients admitted for allogeneic BMT. High spontaneous IL-10 production by PBMNC at the time of admission and prior to any preparative treatment correlated with a subsequent low incidence of GVHD and transplant-related mortality (8%), as compared to patients with low or intermediate IL-10 production (50%, P < 0. 01). Our data demonstrate the prognostic significance of increased IL-10 production in BMT patients and suggest a major role of IL-10 in maintaining immunobalance in the setting of allogeneic BMT. Bone Marrow Transplantation (2000) 25, 237-241.
View details for Web of Science ID 000085378100002
View details for PubMedID 10673693
-
Generation of primary antigen-specific human T- and B-cell responses in immunocompetent SCID-hu mice
NATURE MEDICINE
2000; 6 (1): 103-106
View details for Web of Science ID 000084583300045
View details for PubMedID 10613834
-
Induction of CD4(+) T cell alloantigen-specific hyporesponsiveness by IL-10 and TGF-beta(1)
JOURNAL OF IMMUNOLOGY
1999; 163 (7): 3684-3691
Abstract
Induction and maintenance of Ag-specific tolerance are pivotal for immune homeostasis, prevention of autoimmune disorders, and the goal of transplantation. Recent studies suggest that certain cytokines, notably IL-10 and TGF-beta, may play a role in down-regulating immune functions. To further examine the role of cytokines in Ag-specific hyporesponsiveness, murine CD4+ T cells were exposed ex vivo to alloantigen-bearing stimulators in the presence of exogenous IL-10 and/or TGF-beta. Primary but not secondary alloantigen proliferative responses were inhibited by IL-10 alone. However, the combined addition of IL-10 + TGF-beta markedly induced alloantigen hyporesponsiveness in both primary and secondary MLR cultures. Alloantigen-specific hyporesponsiveness was observed also under conditions in which nominal Ag responses were intact. In adoptive transfer experiments, IL-10 + TGF-beta-treated CD4+ T cells, but not T cells treated with either cytokine alone, were markedly impaired in inducing graft-vs-host disease alloresponses to MHC class II disparate recipients. These data provide the first formal evidence that IL-10 and TGF-beta have at least an additive effect in inducing alloantigen-specific tolerance, and that in vitro cytokines can be exploited to suppress CD4+ T cell-mediated Ag-specific responses in vivo.
View details for Web of Science ID 000082744900017
View details for PubMedID 10490963
-
IL-10 transgenic mice present a defect in T cell development reminiscent to SCID patients
JOURNAL OF IMMUNOLOGY
1999; 163 (3): 1420-1427
Abstract
To analyze the effect of IL-10 overexpressed by APCs as observed in some SCID patients, we have expressed the human IL-10 cDNA under the control of the murine MHC class II promoter in transgenic mice. Similar to SCID patients, these mice presented a defect in T cell maturation characterized by a rapid thymic aplasia that started after birth. The blockage in T cell maturation was strictly restricted to TCR-alpha beta T cells as the absolute number of thymic dendritic, TCR-gamma delta and NK1.1 T cells were equivalent to control littermates. Crossing IL-10 transgenic mice with TCR transgenic mice or treatment with staphylococcal enterotoxin B showed that the defect was not related to the impairment of positive or negative selection. However, repopulating of IL-10 transgenic mouse-fetal thymic organ culture with different stages of triple negative T cells isolated from control mice showed that the blockage occurred specifically at the pre-T cell stage and was reverted by treatment with blocking anti-IL-10 mAbs. These results demonstrate that IL-10 regulates T cell maturation and that dysregulation of IL-10 expression can lead to severe T cell immunodeficiency.
View details for Web of Science ID 000081640000042
View details for PubMedID 10415042
-
Administration of Flk2/Flt3 ligand induces expansion of human high-proliferative potential colony-forming cells in the SCID-hu mouse
EXPERIMENTAL HEMATOLOGY
1999; 27 (6): 1029-1037
Abstract
The effects of Flk2/Flt3 ligand (FL) administration on human hematopoiesis were investigated using SCID-hu mice transplanted with human fetal bone fragments. Treatment with recombinant human FL induced significant increases in the frequencies of the high-proliferative potential colony-forming cells and low-proliferative potential colony-forming cells in steady-state human bone marrow. FL also promoted the expansion of high-proliferative potential colony-forming cells and low-proliferative potential colony-forming cells in the human bone marrow during the recovery phase after irradiation, which was evident in increases in the frequencies as well as in the absolute numbers of colony-forming cells. Furthermore, higher percentages of CD33+ CD15- cells were found in the marrows treated with FL as compared to that of controls, indicating that FL hastened the recovery of at least some aspect of myelopoiesis after irradiation. These results indicate that FL induces the expansion of primitive hematopoietic progenitor cells in vivo and, therefore, may be useful in treating patients to promote an early hematopoietic recovery after cytoablative therapies.
View details for Web of Science ID 000080830900008
View details for PubMedID 10378892
-
High spontaneous IL-10 production in unrelated bone marrow transplant recipients is associated with fewer transplant-related complications and early deaths
BONE MARROW TRANSPLANTATION
1999; 23 (11): 1123-1129
Abstract
Interleukin 10 (IL-10) is a potent inhibitor of proliferative T cell responses toward alloantigens, and suppresses the production of pro-inflammatory cytokines which are important in cellular activation and recruitment to sites of inflammation. Because of these properties, we hypothesized that high IL-10 production in patients prior to BMT may predict a better outcome. To investigate this, peripheral blood mononuclear cells (PBMNC) were obtained from 58 recipients (11 autologous, 25 related donor (RD), and 22 unrelated donor (URD)), prior to conditioning therapy. PBMNC were cultured for 24 h in the presence and absence of lipopolysaccharide (LPS) and culture supernatants were assayed for IL-10 using an ELISA method. Spontaneously produced and LPS-stimulated IL-10 levels were correlated with the development of transplant-related complications (TRC) including grade II-IV acute GVHD, veno-occlusive disease, idiopathic pneumonia syndrome and multi-organ dysfunction syndrome, and with death before day 100. For the autologous group, there were no TRC and only one death prior to day 100; therefore, no statistical comparisons to IL-10 levels could be made. In the RD group, 36% developed one or more TRC and 24% died before day 100; however, there were no statistically significant associations between spontaneous or LPS-induced IL-10 levels. In URD patients 41% developed TRC and 55% died prior to day 100. In this group, higher levels of spontaneous IL-10 production were associated with a lower overall occurrence of TRC (P = 0.03) and early death (P = 0.04). Our data would indicate that higher levels of IL-10 production prior to URD BMT may predict fewer TRC, as well as early deaths. The hypothesis that high IL-10 production prior to BMT may decrease complications following URD BMT warrants further testing.
View details for Web of Science ID 000080621100005
View details for PubMedID 10382951
-
Ex vivo manipulations alter the reconstitution potential of mobilized human CD34(+) peripheral blood progenitors
LEUKEMIA
1999; 13 (3): 438-452
Abstract
The phenotype and functions of CD34+ cells isolated from peripheral blood (PB) of steady-state healthy volunteers (ssPB-CD34), and of patients or healthy volunteers after mobilization (mPB-CD34) were investigated. ssPB-CD34+ cells contain a lymphoid cell population that co-express T or B cell markers, while mPB-CD34+ cells lack this population. After 5-day culture, significantly higher levels of expansion in cell, CD34+ cell, and HPP-CFC numbers were induced in ssPB-CD34+ cells, as compared to mPB-CD34+ cells. Hematopoietic reconstitution potential of these ex vivo manipulated CD34+ PBPC was evaluated in SCID-hu mice. It was found that ssPB-CD34+ cells retained the potential to reconstitute human bone marrow (BM), as well as thymus implanted in SCID animals. In contrast, only very low levels of reconstitution were detected in human hematopoietic tissues injected with cultured mPB-CD34+ cells. Reconstitution was restricted to myeloid cells, and no B cell reconstitution in bone marrow, or T cell reconstitution in thymus was achieved by these cells. The loss of B cell reconstitution potential of mPB-CD34+ cells was shown to be induced in a time-dependent manner during culture. These results indicate that mPB-CD34+ cells have different phenotypic and functional properties from ssPB-CD34+ cells. This may affect the efficacy of cell and gene therapy with mobilized PBPC.
View details for Web of Science ID 000079086000016
View details for PubMedID 10086735
-
A transgenic model to analyze the immunoregulatory role of IL-10 secreted by antigen-presenting cells
JOURNAL OF IMMUNOLOGY
1999; 162 (3): 1723-1729
Abstract
IL-10 is a cytokine secreted by a wide variety of cells type that has pleiotropic stimulatory and suppressive activities on both lymphoid and myeloid cells in vitro. To analyze the consequences of high IL-10 secretion by APCs in immune responses, we produced transgenic mice expressing human IL-10 directed by the MHC class II Ea promoter. Despite alterations in the development of T and B cells, no gross abnormalities were detected in peripheral lymphocyte populations or serum Ig levels. However, when immunized using conditions that give either a Th2-type or a Th1-type response, IL-10 transgenic mice failed to mount a significant T or B cell immune response to OVA. IL-10 transgenic mice were also highly susceptible to infection with intracellular pathogens like Listeria monocytogenes or Leishmania major, in contrast to IL-10 transgenic mice, where the transgene was express in T cells. Finally, the recently described stimulatory effect of IL-10 on CD8+ T cells was confirmed by the ability of IL-10 transgenic mice to limit the growth of immunogenic tumors by a CTL-mediated mechanism. These results demonstrate, that, depending on the type of immune response, IL-10 can mediate immunosuppressive or immunostimulatory activities in vivo.
View details for Web of Science ID 000078261000064
View details for PubMedID 9973435
-
Interleukin-10 dose-dependent regulation of CD4(+) and CD8(+) T cell-mediated graft-versus-host disease
TRANSPLANTATION
1998; 66 (9): 1220-1229
Abstract
Endogenous interleukin (IL)-10 production has been associated with the lack of graft-versus-host disease (GVHD) in human recipients of MHC-disparate donor grafts. Paradoxically, we have shown that the exogenous administration of high doses (30 microg/dose) of IL-10 to murine recipients of MHC-disparate grafts accelerates GVHD lethality.The effects of IL-10 on GVHD mediated by either CD4+ or CD8+ T cells was examined in studies involving exogenous IL-10 administration or the infusion of T cells from IL-10-deficient (-/-) donor mice. The role of interferon (IFN)-gamma on IL-10-induced GVHD acceleration was studied using IFN-gamma-deficient (-/-) donor mice or neutralizing monoclonal antibody.IL-10 was found to have a dose-dependent effect on the GVHD lethality mediated by either CD4+ or CD8+ T cells. High doses of exogenous IL-10 accelerated GVHD lethality. IFN-gamma release was not responsible for the IL-10 facilitation of GVHD lethality. Paradoxically, low doses of IL-10 protected mice against GVHD lethality. The GVHD protective effect of the bioavailability of small amounts of IL-10 was confirmed by demonstrating that the infusion of T cells from IL-10 -/- donors accelerated GVHD lethality.The results suggest that IL-10 has a dose-dependent effect on the GVHD lethality mediated by CD4+ or CD8+ T cells, such that high doses accelerate lethality, while low amounts of bioavailable IL-10 are protective.
View details for Web of Science ID 000077007500018
View details for PubMedID 9825821
-
The X-linked lymphoproliferative-disease gene product SAP regulates signals induced through the co-receptor SLAM
NATURE
1998; 395 (6701): 462-469
Abstract
In addition to triggering the activation of B- or T-cell antigen receptors, the binding of a ligand to its receptor at the cell surface can sometimes determine the physiological outcome of interactions between antigen-presenting cells, T and B lymphocytes. The protein SLAM (also known as CDw150), which is present on the surface of B and T cells, forms such a receptor-ligand pair as it is a self-ligand. We now show that a T-cell-specific, SLAM-associated protein (SAP), which contains an SH2 domain and a short tall, acts as an inhibitor by blocking recruitment of the SH2-domain-containing signal-transduction molecule SHP-2 to a docking site in the SLAM cytoplasmic region. The gene encoding SAP maps to the same area of the X chromosome as the locus for X-linked lymphoproliferative disease (XLP) and we found mutations in the SAP gene in three XLP patients. Absence of the inhibitor SAP in XLP patients affects T/B-cell interactions induced by SLAM, leading to an inability to control B-cell proliferation caused by Epstein-Barr virus infections.
View details for Web of Science ID 000076212200046
View details for PubMedID 9774102
-
Transgene expression of bcl-x(L) permits anti-immunoglobulin (Ig)-induced proliferation in xid B cells
JOURNAL OF EXPERIMENTAL MEDICINE
1998; 187 (7): 1081-1091
Abstract
Mutations in the tyrosine kinase, Btk, result in a mild immunodeficiency in mice (xid). While B lymphocytes from xid mice do not proliferate to anti-immunoglobulin (Ig), we show here induction of the complete complement of cell cycle regulatory molecules, though the level of induction is about half that detected in normal B cells. Cell cycle analysis reveals that anti-Ig stimulated xid B cells enter S phase, but fail to complete the cell cycle, exhibiting a high rate of apoptosis. This correlated with a decreased ability to induce the anti-apoptosis regulatory protein, Bcl-xL. Ectopic expression of Bcl-xL in xid B cells permitted anti-Ig induced cell cycle progression demonstrating dual requirements for induction of anti-apoptotic proteins plus cell cycle regulatory proteins during antigen receptor mediated proliferation. Furthermore, our results link one of the immunodeficient traits caused by mutant Btk with the failure to properly regulate Bcl-xL.
View details for Web of Science ID 000073075900011
View details for PubMedID 9529324
-
Inhibitory and stimulatory effects of IL-10 on human CD8(+) T cells
JOURNAL OF IMMUNOLOGY
1998; 160 (7): 3188-3193
Abstract
IL-10 is a well-documented immunosuppressant that inhibits macrophage-dependent Ag presentation and CD4+ T cell proliferation in vitro. We report that IL-10 inhibits alloantigen-specific proliferative responses and induces a long lasting anergic state in human purified CD8+ T cells when added concomitantly with the Ag in the presence of APC. Moreover, the generation of allospecific cytotoxic activity is inhibited by IL-10. These effects are indirect and are mediated through inhibition of the costimulatory functions of APC. In contrast, IL-10 has no direct inhibitory effects on the proliferation of purified CD8+ T cells activated by anti-CD3 mAb and promotes the growth of activated CD8+ T cells in combination with low doses of IL-2. Taken together, these results indicate that IL-10 has differential effects on CD8+ T cells depending on their state of activation, which may explain both the enhancing and inhibitory effects observed after IL-10 treatment in different in vivo experimental models.
View details for Web of Science ID 000072700100015
View details for PubMedID 9531274
-
Successful reconstitution of human hematopoiesis in the SCID-hu mouse by genetically modified, highly enriched progenitors isolated from fetal liver
BLOOD
1997; 90 (9): 3496-3506
Abstract
Highly purified CD34++CD38-Lin- hematopoietic progenitors isolated from human fetal liver were infected with the murine retroviral vector, MFG nls-LacZ, which encodes a modified version of the Escherichia coli beta-galactosidase gene. Progenitors that were cocultured with the packaging cell line could reconstitute human bone marrow or thymus implanted in SCID-hu mice. Expression of the beta-galactosidase gene was observed in primitive and committed clonogenic progenitors, mature myeloid, B-lineage cells, and T-lineage cells for up to 4 months after injection into SCID-hu mice. Furthermore, hematopoietic reconstitution by genetically modified progenitor cells could be achieved by the injection of the cells generated from as few as 500 CD34++CD38-Lin- cells, suggesting efficient retroviral gene transfer into fetal liver progenitors.
View details for Web of Science ID A1997YD10800025
View details for PubMedID 9345033
-
A CD4(+) T-cell subset inhibits antigen-specific T-cell responses and prevents colitis
NATURE
1997; 389 (6652): 737-742
Abstract
Induction and maintenance of peripheral tolerance are important mechanisms to maintain the balance of the immune system. In addition to the deletion of T cells and their failure to respond in certain circumstances, active suppression mediated by T cells or T-cell factors has been proposed as a mechanism for maintaining peripheral tolerance. However, the inability to isolate and clone regulatory T cells involved in antigen-specific inhibition of immune responses has made it difficult to understand the mechanisms underlying such active suppression. Here we show that chronic activation of both human and murine CD4+ T cells in the presence of interleukin (IL)-10 gives rise to CD4+ T-cell clones with low proliferative capacity, producing high levels of IL-10, low levels of IL-2 and no IL-4. These antigen-specific T-cell clones suppress the proliferation of CD4+ T cells in response to antigen, and prevent colitis induced in SCID mice by pathogenic CD4+CD45RB(high) splenic T cells. Thus IL-10 drives the generation of a CD4+ T-cell subset, designated T regulatory cells 1 (Tr1), which suppresses antigen-specific immune responses and actively downregulates a pathological immune response in vivo.
View details for Web of Science ID A1997YA95900057
View details for PubMedID 9338786
-
Immunological tolerance following stem cell transplantation in human fetuses in utero
2nd Congress on Pregnancy After Transplantation
ELSEVIER SCIENCE INC. 1997: 2477–77
View details for Web of Science ID A1997XR13700076
View details for PubMedID 9270816
-
Induction of human T helper cell type 1 differentiation results in loss of IFN-gamma receptor beta-chain expression
JOURNAL OF IMMUNOLOGY
1997; 158 (12): 5627-5631
Abstract
Differential expression of cytokine receptors accounts for an important regulatory mechanism in differentiation of Th1/Th2 subsets. Here, we report that human Th0 and Th2 clones constitutively express transcripts for the IFN-gammaR beta-chain, whereas mRNA for this signaling component of the IFN-gamma receptor is absent in Th1 clones. Activation of T cell clones, however, resulted in a transient induction or enhancement of IFN-gammaR beta-chain mRNA expression in Th1 clones and Th0/Th2 clones, respectively. IL-12-mediated Th1 cell differentiation of naive CD4+, CD45RA+ cord blood T cells, which constitutively express IFN-gammaR beta-chain mRNA, resulted in a loss of expression of this cytokine receptor chain after 6 to 12 days of culture. In contrast, Th2 populations, differentiated from CD4+, CD45RA+ cord blood T cells in the presence of IL-4, continued to express high levels of IFN-gammaR beta-chain transcripts. The loss of IFN-gammaR beta-chain expression in Th1 populations was accompanied by a failure of IFN-gamma to induce the expression of the IFN-gamma-inducible gene, IFN response factor-1, whereas IFN-gamma was effective in inducing IFN response factor-1 mRNA expression in Th0 and Th2 cells. These results indicate that down-regulation of the IFN-gammaR beta-chain correlates with impaired IFN-gamma-induced signaling in Th1 cells. Finally, Th2 populations, generated in the presence of both IL-4 and IFN-gamma, expressed levels of IFN-gammaR beta-chain transcripts similar to those produced by cells differentiated in the presence of IL-4 only, demonstrating that IFN-gamma does not modulate the expression of its receptor. Together, these data indicate that human Th0/Th2 and Th1 subsets, respectively, can be distinguished based on the expression of the IFN-gammaR beta-chain.
View details for Web of Science ID A1997XE74900008
View details for PubMedID 9190910
-
Colony-forming cells expressing high levels of CD34 are the main targets for granulocyte colony-stimulating factor and macrophage colony-stimulating factor in the human fetal liver
EXPERIMENTAL HEMATOLOGY
1997; 25 (4): 277-287
Abstract
The effects of the granulocyte (G) and macrophage (M) colony-stimulating factors (CSFs) on the growth of purified subpopulations of human fetal liver progenitors were investigated. In contradiction to the characterization of these cytokines as CSFs acting late in the course of hematopoiesis, both G-CSF and M-CSF were most potent in promoting the growth of fetal liver colony-forming cells (CFCs) that express high levels of CD34 and CD38 (CD34++CD38+) and are depleted of cells expressing a panel of lineage markers (Lin-). Cultures of these cells in serum-deprived conditions generated a mean of 11.2 and 39.1 low-proliferative potential (LPP)-CFCs per 1.0 x 10(3) CD34++CD38+Lin- cells grown in G-CSF and M-CSF, respectively. Cultures of more mature progenitors, isolated based on a lower level of CD34 expression (CD34+ Lin-), generated few LPP-CFCs and 6.3 and 4.7 clusters per 1.0 x 10(3) CD34+Lin- cells in response to G-CSFs and M-CSF, respectively. G-CSF was also found to synergistically enhance colony growth by either kit-ligand (KL) or fit-3/flk-2 ligand (FL) in cultures of CD34++CD38+Lin- cells as well as the more primitive compartment of CD34++CD38-Lin- cells. Synergism between G-CSF and KL or FL was also observed in liquid cultures of CD34++CD38-Lin- cells. The effects of G-CSF on CD342++CD38-Lin- cells were further demonstrated by the ability of G-CSF to support the short-term survival of these cells in clonal cultures. In contrast, M-CSF did not affect the growth or survival of CD34++CD38-Lin- cells, a finding that was also supported by the observation that the receptor for M-CSF (CD115 or fms) was only expressed on CD34++CD38+Lin- cells. G-CSF receptor expression and flt-3/flk-2 expression were detected by flow cytometry on both the CD38- and CD38+ subpopulations of CD34++Lin- cells, but these receptors were not detected on CD34+ cells. Receptors for KL (CD117) and interleukin-3 (CD123), for which the ligands are active on a broad range of fetal liver progenitors, were detected on cells expressing both high and low levels of CD34. These data help to define the potential roles of cytokines in human fetal hematopoiesis.
View details for Web of Science ID A1997WV89500001
View details for PubMedID 9131001
-
Inflammatory reactions induced by pretransplant conditioning - An alternative target for modulation of acute GVHD and complications following allogeneic bone marrow transplantation?
LEUKEMIA & LYMPHOMA
1997; 25 (3-4): 217-224
Abstract
Intensity of pretransplant conditioning has been closely correlated with regimen related toxicity in patients receiving allogeneic bone marrow transplantation (BMT). In this review, we summarize evidence for a direct link between inflammatory reactions induced by irradiation and cytotoxic treatment and occurrence of acute graft-versus-host disease (GvHD) as well as endothelial complications: In our studies, de novo release of TNFalpha during conditioning was associated with an increased risk of severe GvHD and mortality following BMT, whereas increased spontaneous production of IL-10, an endogenous TNF-antagonist, prior to conditioning protected from these complications. Immunogenetic differences in cytokine regulation and costimulation by endotoxin proved to be important cofactors determining the extent of inflammatory cytokine release in individual patients. Pathophysiological relevance of these findings seems to be confirmed by experimental as well as first clinical trials using TNF-antibodies and related antagonists during pretransplant conditioning. Preclinical experiments suggest additional, cytokine independent inflammatory reactions induced by irradiation such as expression of ICAM-1 and endothelial cell apoptosis. Although the exact impact of these findings on pathophysiology of BMT related complications needs further clarification by future studies, conditioning related inflammation as a first crucial step in induction of GvHD and complications has to be considered when designing new protocols for preparation of patients for allogeneic BMT.
View details for Web of Science ID A1997XA30800003
View details for PubMedID 9168432
-
Phenotypic and functional evidence for the expression of CD4 by hematopoietic stem cells isolated from human fetal liver
BLOOD
1997; 89 (4): 1364-1375
Abstract
Expression of the CD4 antigen was observed on human fetal liver, fetal bone marrow (BM), and umbilical cord blood progenitors expressing high levels of CD34. Using clonal and liquid-culture assays, CD4+ CD34++ Lin- (lineage = CD3, CD8, CD10, CD14, CD15, CD16, CD19, CD20, and glycophorin A) fetal liver progenitors were found to have a greater proliferative potential than CD4- CD34++ Lin- progenitors, whereas the CD4- fraction was more enriched for erythroid progenitors. Both the CD4+ and the CD4- progenitor subpopulations also gave rise to multilineage engraftment upon transplantation into human fetal bone fragments, supportive of B-lymphoid and myeloid growth, or into human fetal thymic fragments, supportive of T-cell growth, implanted in scid/scid (SCID) mice. However, in SCID-hu mice transplanted with graded doses of donor cells ranging from 2.0 x 10(2) to 2.0 x 10(4) cells, BM reconstitution by the CD4+ fraction of CD34++ Lin- cells was more frequent than by the CD4- fraction when low numbers of cells were injected. These functional data strongly suggest that stem cells reside among CD4+ CD34++ Lin- fetal liver cells. This hypothesis was further supported by the observations that CD4+ CD34++ Lin- fetal liver cells were enriched for CDw90+ (Thy-1), CD117+ (kit), CD123+, HLA-DR+, CD7-, CD38-, CD45RA-, CD71-, CD115- (fms), and rhodamine 123(dull) cells, a phenotypic profile believed to represent fetal stem cells. Furthermore, all CD4+ CD34++ Lin- fetal liver cells also expressed CD13 and CD33.
View details for Web of Science ID A1997WG79700028
View details for PubMedID 9028960
-
Antigen-specific cytotoxic T cells mediate human fetal pancreas allograft rejection in SCID-hu mice
JOURNAL OF IMMUNOLOGY
1996; 157 (12): 5710-5720
Abstract
Human allograft rejection was studied in SCID mice transplanted with human fetal liver and thymus tissue (SCID-hu mice). These SCID-hu mice have functional, mature T cells with a polyclonal TCR repertoire. Within 12 to 36 wk after construction, SCID-hu mice were transplanted with an HLA-mismatched human fetal pancreas. In contrast to control SCID mice transplanted with pancreas alone, cellular infiltration, induction of HLA-DR on pancreatic epithelial cells, and tissue destruction of the allogenic pancreata were observed in SCID-hu mice. In addition, human insulin was not detected in the serum of SCID-hu mice in which pancreas rejection occurred. The infiltrating cells were mainly human CD3+ T lymphocytes of thymic origin, expressing the CD45RO isoform. T cell lines and CD4+ T cell clones obtained from the rejected tissues proliferated vigorously when stimulated with EBV-transformed B cell lines of pancreas donor origin. Furthermore, the majority of these CD4+ T cell clones displayed strong allospecific cytotoxicity. In addition, CD8+ T cell clones cytotoxic for EBV-transformed B cell lines of pancreas donors were isolated. Blocking experiments with anti-HLA mAbs and panel studies with HLA-matched cell lines showed that these CD4+ and CD8+ T cell clones were specific for the HLA class II and class I molecules, respectively, expressed by the pancreas donor. These data indicate that human T lymphocytes developing in SCID-hu mice are able to mount in vivo responses against allogenic organs, resulting in tissue infiltration and rejection. In addition, they show that both CD4(+)- and CD8(+)-allospecific CTL can be isolated from rejected allogenic pancreata.
View details for Web of Science ID A1996VX02600061
View details for PubMedID 8955225
-
Treatment of X-linked severe combined immunodeficiency by in utero transplantation of paternal bone marrow
NEW ENGLAND JOURNAL OF MEDICINE
1996; 335 (24): 1806-1810
View details for Web of Science ID A1996VW68300004
View details for PubMedID 8943162
-
Human T-and B-cell functions in SCID-hu mice.
Seminars in immunology
1996; 8 (4): 207-213
Abstract
SCID mice transplanted with human fetal liver and thymus (SCID-hu Thy/Liv) provide a unique in-vivo model to study human T-cell development and clonal selection mechanisms. This SCID-hu mouse model can be adapted to study the role of thymic epithelial cells, or bone marrow-derived cells in transplantation tolerance. In addition, these mice have circulating human T cells, which mediate human allograft rejection in vivo. SCID-hu mice constructed with fetal bones and thymus (SCID-hu BM/Thy) have both circulating human T and B cells, and can be used to study human B-cell development and B-cell functions. In addition, human T-B-cell interactions resulting in human lg production and the modulating effects of cytokines and cytokine receptor antagonists on this process, can be monitored. Collectively, this information indicates that the SCID-hu mouse is a powerful and versatile model to study human immune responses in vivo.
View details for PubMedID 8883143
-
Regulatory roles of the ligand for Flk2/Flt3 tyrosine kinase receptor on human hematopoiesis
STEM CELLS
1996; 14 (4): 388-395
Abstract
The biological activities of the ligand for the Flk2/Flt3 receptor tyrosine kinase (FL) on human hematopoietic cells are reviewed. In in vitro studies, FL shows relatively few effects by itself on the proliferation and differentiation of hematopoietic cells, but exhibits a potent costimulatory activity in enhancing the proliferation of progenitor cells of multiple lineages. FL promotes the growth of clonogenic myeloid progenitor cells in the presence of other cytokines known to be active on myeloid progenitors, including GM-CSF, interleukin 3 (IL-3), kit ligand (KL), M-CSF and G-CSF. In addition, FL synergizes with IL-7 in inducing the proliferation of pro-B cells, whereas FL has little effect on the growth of clonogenic erythroid progenitors. Furthermore, FL induces the in vitro expansion of the high proliferative potential colony-forming cells (HPP-CFC) and stimulates the proliferation of long-term culture-initiating cells (LTC-IC), suggesting an activity on the proliferation of putative stem cells. Thus, FL plays important roles in regulating the proliferation of hematopoietic progenitor cells and, therefore, may have therapeutic applications.
View details for Web of Science ID A1996UZ87700003
View details for PubMedID 8843540
-
Interleukin-10 induces a long-term antigen-specific anergic state in human CD4(+) T cells
JOURNAL OF EXPERIMENTAL MEDICINE
1996; 184 (1): 19-29
Abstract
Human CD4+ T cells, activated by allogeneic monocytes in a primary mixed lymphocyte reaction in the presence of exogenous interleukin (IL) 10, specifically failed to proliferate after restimulation with the same alloantigens. A comparable state of T cell unresponsiveness could be induced by activation of CD4+ T cells by cross-linked anti-CD3 monoclonal antibodies (mAbs) in the presence of exogenous IL-10. The anergic T cells failed to produce IL-2, IL-5, IL-10, interferon gamma, tumor necrosis factor alpha, and granulocyte/macrophage colony-stimulating factor. The IL-10-induced anergic state was long-lasting. T cell anergy could not be reversed after restimulation of the cells with anti-CD3 and anti-CD28 mAbs, although CD3 and CD28 expression was normal. In addition, restimulation of anergized T cells with anti-CD3 mAbs induced normal Ca2+ fluxes and resulted in increased CD3, CD28, and class II major histocompatibility complex expression, indicating that calcineurin-mediated signaling occurs in these anergic cells. However, the expression of the IL-2 receptor alpha chain was not upregulated, which may account for the failure of exogenous IL-2 to reverse the anergic state. Interestingly, anergic T cells and their nonanergic counterparts showed comparable levels of proliferation and cytokine production after activation with phorbol myristate acetate and Ca2+ ionophore, indicating that a direct activation of a protein kinase C-dependent pathway can overcome the tolerizing effect of IL-10. Taken together, these data demonstrate that IL-10 induces T cell anergy and therefore may play an important role in the induction and maintenance of antigen-specific T cell tolerance.
View details for Web of Science ID A1996UW67700003
View details for PubMedID 8691133
-
Immune functions of cord blood cells before and after transplantation.
Journal of hematotherapy
1996; 5 (2): 157-160
Abstract
There have been numerous reports of decreased acute and chronic graft-versus-host disease in patients receiving HLA-matched or HLA-disparate umbilical cord transplants. It has been proposed that this may be due to the unique properties of the neonatal immune system, which permit the development of tolerance to alloantigens. This review discusses experimental evidence contrasting the immune functions of cells derived from cord blood and those from peripheral blood.
View details for PubMedID 8723794
-
The FLK2/FLT3 ligand synergizes with interleukin-7 in promoting stromal-cell-independent expansion and differentiation of human fetal pro-B cells in vitro
BLOOD
1996; 87 (5): 1881-1890
Abstract
The effects of a novel cytokine FLK2/FLT3 ligand (FL) on human fetal bone marrow-derived CD34+CD19+ pro-B cells were analyzed in a stromal-cell-independent, serum-deprived culture system. FL, like interleukin-3 (IL-3), synergized with IL-7 in promoting pro-B cell growth, and differentiation of these cells into CD34-CD19+clgM+slgM- pre-B cells, whereas a small proportion of these cells even differentiate into more mature slgM+ B cells. In contrast, KIT ligand (KL) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were ineffective in promoting IL-7-dependent pro-B cell growth and differentiation. Maximal levels of pro-B cell expansion, generally resulting in 15- to 30-fold increases in cellularity, were obtained in cultures supplemented with optimal doses of FL + IL-7 + IL-3. The addition of mouse bone marrow stromal cells further enhanced the proliferation and differentiation of pro-B cells obtained in the presence of these three cytokines. Under these conditions, cultures could be maintained for more than 4 weeks, and in general 40- to 50-fold increases in cell numbers were observed by 3 weeks of culture. The percentages of clgM+ and slgM+ B cells increased 1.5- to 3-fold and 2-fold, respectively, suggesting that stromal cells may provide additional costimulatory signals for human B-cell growth and differentiation that are different from IL-7, IL-3, and FL. Collectively, our results indicate that FL, in contrast to KL, strongly promotes long-term expansion and differentiation of human pro-B cells in the presence of IL-7 or in combination of IL-7 and IL-3, which is a novel property of this hematopoietic growth factor.
View details for Web of Science ID A1996TY33900027
View details for PubMedID 8634436
-
The role of interleukin-10 in T-cell tolerance
International Symposium on Immune Tolerance
EDITIONS SCIENTIFIQUES ET MEDICALES ELSEVIER. 1996: 141–147
View details for Web of Science ID A1996BH33J00014
-
IL-4 INDUCES HUMAN B-CELL MATURATION AND IGE SYNTHESIS IN SCID-HU MICE - INHIBITION OF ONGOING IGE PRODUCTION BY IN-VIVO TREATMENT WITH AN IL-4/IL-13 RECEPTOR ANTAGONIST
JOURNAL OF IMMUNOLOGY
1995; 155 (9): 4162-4170
Abstract
The effect of cytokine treatment on the in vivo maturation and Ig isotype switching of human B cells was studied in a modified SCID-hu mouse model. SCID mice, subcutaneously cotransplanted with small fragments of fetal human thymus and bone (SCID-hu BM/T mice) generated all human leukocyte lineages including T and B lymphocytes, macrophages, and granulocytes. All SCID-hu BM/T mice spontaneously produced human IgM and IgG, whereas IgE and IgA were detected in 37 and 80% of the mice, respectively, indicating that productive human T-B cell interactions resulting in Ig isotype switching occur in these mice. Administration of IL-4 to SCID-hu BM/T mice enhanced human B cell maturation, as judged by the increase in the percentages of CD45+, CD19+ bone marrow B cells expressing CD20, CD23, CD40, sIgM, and sIgD. Furthermore, these cells were also functionally more mature because they spontaneously produced human IgG/IgG4 in vitro and could be induced to secrete human IgE by addition of anti-CD40 mAb alone. In contrast, B cells isolated from PBS-treated mice only produced significant Ig levels after stimulation with anti-CD40 mAb in the presence of exogenous IL-4. IL-4 administration also induced human IgE synthesis in 44% of the mice, which had no serum IgE before treatment. More importantly, ongoing human IgE synthesis in SCID-hu BM/T mice was suppressed by > 90% following administration of an IL-4 mutant protein, which acts as an IL-4 and IL-13 receptor antagonist. These results suggest that IL-4/IL-13 receptor antagonists have potential clinical utility in treating human atopic diseases associated with enhanced IgE production.
View details for Web of Science ID A1995TB46800006
View details for PubMedID 7594571
-
HLA-HAPLOIDENTICAL UMBILICAL-CORD BLOOD STEM-CELL TRANSPLANTATION IN A CHILD WITH ADVANCED LEUKEMIA - CLINICAL OUTCOME AND ANALYSIS OF HEMATOPOIETIC RECOVERY
BONE MARROW TRANSPLANTATION
1995; 16 (2): 229-240
Abstract
Growing attention has been focused on cord blood as a source of transplantable hematopoietic stem cells. However, clinical experience is rather limited. In this study we describe a child with advanced acute lymphoblastic leukemia who received an HLA-haploidentical cord blood transplant. The patient was transplanted in third complete remission after conditioning with fractionated total body irradiation, thiotepa and cyclophosphamide. Forty-one milliliters of cryopreserved umbilical cord blood, containing 0.15 x 10(8) nucleated cells/kg and 0.25 x 10(4) CFU-GM/kg, were infused. Cyclosporine and prednisone were administered for graft-versus-host disease (GVHD) prophylaxis. The patient received G-CSF from day +1 to day +35, but no improvement in granulocyte counts was observed. Therefore, administration of GM-CSF was started on day +36 to day +59, which resulted in a significant increase in white blood cells and granulocyte counts. Sustained myeloid engraftment was evidenced by a granulocyte count > 0.5 x 10(9)/l by day +41. The presence of donor-derived cells could be documented in the peripheral blood and bone marrow of the patient by cytogenetic analysis, HLA phenotyping and DNA studies. Forty-one days after transplant, clonogenic bone marrow assays showed the presence of low frequencies of primitive hematopoietic progenitor cells (BFU-E = 19/10(5) and CFU-GM = 8/10(5)). The chimerism was complete and no host-derived cells could be detected. However, the engraftment was restricted to the myeloid lineage whereas lymphoid and megakaryocytic engraftments were inadequate. The immunophenotype of the patient's peripheral blood showed the presence of T lymphocytes expressing an immature phenotype (CD2+ CD3-) at day +21.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1995RR31700005
View details for PubMedID 7581141
-
UNIQUE CYTOKINE PRODUCTION PROFILE OF ANERGIC HUMAN T-CELLS IN SCID-HU MICE AFTER STAPHYLOCOCCAL-ENTEROTOXIN-B ADMINISTRATION
JOURNAL OF IMMUNOLOGY
1995; 154 (7): 3204-3212
Abstract
Severe combined immunodeficient mice transplanted with human organs (SCID-hu mice), provide a unique in vivo model for studying human intrathymic T cell selection and development of tolerance. In vivo administration of staphylococcal enterotoxin B (SEB) to SCID-hu mice causes intrathymic clonal deletion of SEB-specific V beta+ T cells that occurs already at the immature CD4+8+ double positive stage. The expression of activation markers such as CD25, CD71, and HLA-DR was specifically increased on V beta+ T cells responding to SEB. The remaining SEB-specific human T cells that had not been deleted in vivo failed to proliferate when rechallenged with SEB in vitro. These SEB-specific T cells that were rendered anergic in vivo had a unique cytokine production profile. They failed to produce IL-2, which correlated with the lack of proliferation of these cells. In addition, they failed to produce TNF-alpha. However, the anergized T cells synthesized considerable amounts of IFN-gamma, granulocyte-macrophage CSF and IL-10 after SEB stimulation. This clonal anergy can be completely reversed in vitro by stimulating the SEB-specific cells in the presence of exogenous IL-2 or by triggering of the CD28/CTLA-4 activation pathway. Under these stimulation conditions, anergic T cells produced levels of IL-2 and TNF-alpha that were comparable to their non-anergized counterparts, whereas the levels of granulocyte-macrophage CSF, IL-10 and IFN-gamma production were even higher. Collectively, these data demonstrate that in vivo administration of SEB to SCID-hu mice leads to activation, deletion, and anergy of SEB-specific human thymocytes and that the production of IL-2 and TNF-alpha is selectively switched off in these anergic T cells.
View details for Web of Science ID A1995QN45900017
View details for PubMedID 7534791
-
DYSFUNCTIONAL CYTOKINE PRODUCTION BY HOST-REACTIVE T-CELL - CLONES ISOLATED FROM A CHIMERIC SEVERE COMBINED IMMUNODEFICIENCY PATIENT TRANSPLANTED WITH HAPLOIDENTICAL BONE-MARROW
BLOOD
1995; 85 (7): 1944-1953
Abstract
We have investigated the mechanism of tolerance in a patient with severe combined immunodeficiency (SCID) transplanted with HLA-haploidentical, T cell-depleted bone marrow cells obtained from the mother. At 4 years after transplantation, T cells, natural killer (NK) cells, and a small percentage (2%) of B cells were found to be of donor origin, whereas monocytes and the majority of B cells remained of host origin. In primary mixed lymphocyte cultures (MLC), the engrafted T cells of the donor did not proliferate in response to the host cells, whereas untransplanted donor T cells showed good proliferative responses. However, CD4+ and CD8+ T-cell clones of donor origin with specificity for class II and class I HLA determinants of the host were isolated. CD8+, host-reactive T-cell clones displayed normal cytotoxic activity after stimulation with the host cells, but proliferative responses of CD4+, host-reactive T-cell clones were considerably reduced. In addition, both CD8+ and CD4+, host-reactive T-cell clones produced very low to undetectable levels of interleukin-2 (IL-2), IL-4, IL-5, IL-10, interferon-gamma, and granulocyte-macrophage colony-stimulating factor after specific antigenic activation, which may be responsible for their nonresponsive state in vivo. Expression of the CD3 zeta subunit of the T-cell receptor (TcR) was normal, and after stimulation via CD3, Raf-1 and p42 mitogen activated protein (MAP) kinase were phosphorylated, indicating that this part of the signaling pathway after triggering of the TcR/CD3 complex is present. These results, together with our previous observation that dysfunctional, host-reactive T-cell clones can be isolated in SCID patients transplanted with fetal liver stem cells, demonstrate that lack of clonal deletion of host-reactive T cells is a general phenomenon after HLA-mismatched stem cell transplantation.
View details for Web of Science ID A1995QP61300033
View details for PubMedID 7703497
-
TRACING THE EXPRESSION OF CD7 AND OTHER ANTIGENS DURING T-CELL AND MYELOID-CELL DIFFERENTIATION IN THE HUMAN FETAL LIVER AND THYMUS
LEUKEMIA & LYMPHOMA
1995; 17 (1-2): 1-11
Abstract
During the last decade, the function/s of the cell membrane CD7 antigen have been investigated in human mature T and NK cells, showing the direct involvement of this molecule in multiple effector functions related with activation, proliferation, production of cytokines and modification of adhesion properties. The CD7 glycoprotein is not only expressed by mature lymphoid cells, but also by early hematopoietic progenitors and several types of leukemias, suggesting a role of CD7 during hematopoiesis. However, the function of CD7 in the early stages of hematopoietic development has not yet been elucidated. CD7 has been classically considered the earliest T-cell specific marker. This assumption was based on data indicating the presence of CD45+CD7+CD3-CD4-CD8- cells in the human embryonic/fetal liver at the gestational age at which the thymic rudiment is colonized by T-cell progenitors. In the present article, we review recent results obtained by several groups concerning the expression of CD7 and various other cell surface antigens by T-, B- and myeloid-cell progenitors generated in the adult bone marrow and fetal liver. In addition, we present an hypothetical model of hematopoiesis in the fetal liver and thymus.
View details for Web of Science ID A1995QJ89900001
View details for PubMedID 7539656
-
FLK-2/FLT-3 LIGAND REGULATES THE GROWTH OF EARLY MYELOID PROGENITORS ISOLATED FROM HUMAN FETAL LIVER
BLOOD
1995; 85 (4): 963-972
Abstract
The effects of the recently identified FLK-2/FLT-3 ligand (FL) on the growth of purified human fetal liver progenitors were investigated under serum-deprived culture conditions. FL alone was found to stimulate modest proliferation in short-term cultures of CD34++ CD38+ lineage (Lin)- light-density fetal liver (LDFL) cells and the more primitive CD34++ CD38- Lin- LDFL cells. However, the low levels of growth induced by FL were insufficient for colony formation in clonal cultures. Synergism between FL and either granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) or KIT ligand (KL) was observed in promoting the growth of high-proliferative potential (HPP) colony-forming cells (CF) and/or low-proliferative potential (LPP)-CFC in cultures of CD34++ CD38+ Lin- and CD34++ CD38- Lin- LDFL-cells. FL, alone or in combination with other cytokines, was not found to affect the growth of CD34+ Lin- LDFL cells, the most mature subpopulation of fetal liver progenitors investigated. The growth of the most primitive subset of progenitors studied, CD34++ CD38- Lin- LDFL cells, required the interactions of at least two cytokines, because only very low levels of growth were observed in response to either FL, GM-CSF, IL-3 or KL alone. However, the results of delayed cytokine-addition experiments suggested that individually these cytokines did promote the survival of this early population of progenitors. Although two-factor combinations of FL, KL, and GM-CSF were observed to promote the growth of early progenitors in a synergistic manner, neither of these factors was found to make fetal liver progenitors more responsive to suboptimal concentrations of a second cytokine. Only myeloid cells were recovered from liquid cultures of CD34++ CD38- Lin- LDFL cells grown in the presence of combinations of FL, KL, and GM-CSF. These results indicate that FL is part of a network of growth factors that regulate the growth and survival of early hematopoietic progenitors.
View details for Web of Science ID A1995QG99300013
View details for PubMedID 7531516
-
TRANSPLANTATION OF MISMATCHED HUMAN FETAL LIVER-CELLS - TOLERANCE INDUCTION VIA CLONAL DELETION AND CLONAL ANERGY
XVth World Congress of the Transplantation-Society
ELSEVIER SCIENCE INC. 1995: 622–24
View details for Web of Science ID A1995QJ19900252
View details for PubMedID 7879123
-
T-cell subsets and their cytokine profiles in transplantation and tolerance
Conference on Bone Marrow Transplantation in the 90s - Into the 21st-Century
NEW YORK ACAD SCIENCES. 1995: 141–148
View details for Web of Science ID A1995BF30X00013
View details for PubMedID 8597356
-
PROGRESS IN THE EX-VIVO EXPANSION OF HEMATOPOIETIC PROGENITORS
LEUKEMIA & LYMPHOMA
1994; 16 (1-2): 1-11
Abstract
In this review we describe how studies on the cytokine-stimulated growth of murine bone marrow (BM) progenitors have lead to the observations that large increases in progenitor numbers can be achieved in short-term cytokine-stimulated liquid cultures. Transplantation of these ex vivo expanded murine BM cells was shown to decrease the number of BM cells required to confer radioprotection and to increase the recovery rate of both myeloid and erythroid peripheral blood cells. The ex vivo expansion of murine BM cells does not however, markedly diminish stem cells capable of long-term hematopoietic reconstitution. Investigations on the expansion of human BM, peripheral blood, umbilical cord blood and fetal hematopoietic progenitors have demonstrated that clinically useful increases in progenitor numbers from these tissues are possible. Thus, ex vivo progenitor expansion may soon be of use in transplantation protocols to accelerate hematopoietic reconstitution and in gene therapy protocols if hematopoietic stem cells can be maintained during ex vivo culture.
View details for Web of Science ID A1994PZ96900001
View details for PubMedID 7696914
-
ANTI-SCID MOUSE REACTIVITY SHAPES THE HUMAN CD4(+) T-CELL REPERTOIRE IN HU-P8L-SCID CHIMERAS
JOURNAL OF EXPERIMENTAL MEDICINE
1994; 180 (5): 1817-1827
Abstract
Injecting human peripheral blood mononuclear cells into severe combined immunodeficient (SCID) mice results in long-term engraftment of human lymphocytes, of which > 98% are phenotypically mature, activated T cells. Here we have characterized the human T cells that populate such hu-PBL-SCID chimeras. We report that these human T cells do not mobilize Ca2+ after CD3 stimulation, i.e., their T cell receptor (TCR)-mediated signal transduction is deficient. Chimera-derived human T cells do not secrete lymphokines or undergo blastogenesis after CD3 stimulation, but proliferate in response to interleukin 2 (IL-2), defining the chimera derived human T cells as anergic. Anergy was seen in both the CD4+ and the CD8+ subpopulations. We established human T cell lines from chimeras. These T cells retained their anergic state for 1-2 mo in culture, after which they simultaneously regained the ability to mobilize Ca2+, secrete lymphokines, and to undergo blastogenesis following stimulation via the TCR. Once regaining proliferative responsiveness to CD3 stimulation, these CD4+ T cell lines displayed anti-SCID mouse reactivity and showed no specificity for recall antigens. All CD3-responsive CD4+ T cell clones obtained from such lines were SCID mouse specific, recognizing native major histocompatibility complex class II products on the murine cells. In contrast, chimera-derived human CD8+ cell lines and clones did not display detectable anti-mouse reactivity. The data show that the human T cell system in long term hu-PBL-SCID chimeras is nonfunctional due to both anergy and the limitation of the CD4+ repertoire to xenoreactive clones. The data suggest that long-term hu-PBL-SCID chimerism represents an atypical graft-versus-host reaction in which the human effector T cells become anergic in the murine environment.
View details for Web of Science ID A1994PP54300024
View details for PubMedID 7964463
-
IDENTIFICATION OF A COMMON T-NATURAL-KILLER-CELL PROGENITOR IN HUMAN FETAL THYMUS
JOURNAL OF EXPERIMENTAL MEDICINE
1994; 180 (2): 569-576
Abstract
The phenotypic similarities between natural killer (NK) and T cells have led to the hypothesis that these distinctive lymphocyte subsets may be developmentally related and thus may share a common progenitor (Lanier, L. L., H. Spits, and J. H. Phillips, 1992. Immunol. Today. 13:392; Rodewald, H.-R., P. Moingeon, J. L. Lurich, C. Dosiou, P. Lopez, and E. L. Reinherz. 1992. Cell. 69:139). In this report, we have investigated the potential of human CD34+ triple negative thymocytes ([TN] CD3-, CD4-, CD8-) to generate both T cells and NK cells in murine fetal thymic organ cultures (mFTOC) and in vitro clonogenic assays. CD34+ TN thymocytes, the majority of which express prominent cytoplasmic CD3 epsilon (cytoCD3 epsilon) protein, can be divided into high (CD34Bright) and low (CD34Dim) surface expressing populations. CD34Bright TN thymocytes were capable of differentiating into T and NK cells when transferred into mFTOC, and demonstrated high NK cell clonogenic capabilities when cultured in interleukin (IL)-2, IL-7, and stem cell factor (SCF). Likewise, CD34Bright TN thymocyte clones after 5 d in culture were capable of generating NK and T cells when transferred into mFTOC but demonstrated clonogenic NK cell differentiation capabilities when maintained in culture with IL-2. CD34Dim TN thymocytes, however, possessed only T cell differentiation capabilities in mFTOC but were not expandable in clonogenic conditions containing IL-2, IL-7, and SCF. No significant differentiation of other cell lineage was detected in either mFTOC or in clonogenic assays from CD34+ TN thymocytes. These results represent the first definitive evidence of a common T/NK cell progenitor in the human fetal thymus and delineate the point in thymocyte differentiation where T and NK cells diverge.
View details for Web of Science ID A1994NZ38500016
View details for PubMedID 7519241
-
LYMPHOID AND MYELOID DIFFERENTIATION OF FETAL LIVER CD34+ LINEAGE(-) CELLS IN HUMAN THYMIC ORGAN-CULTURE
JOURNAL OF EXPERIMENTAL MEDICINE
1994; 180 (1): 123-132
Abstract
In this article, we report that the human fetal thymus contains CD34bright cells (< 0.01% of total thymocytes) with a phenotype that resembles that of multipotent hematopoietic progenitors in the fetal bone marrow. CD34bright thymocytes were CD33-/dull and were negative for CD38, CD2, and CD5 as well as for the lineage markers CD3, CD4, and CD8 (T cells), CD19 and CD20 (B cells), CD56 (NK cells), glycophorin (erythrocytes), and CD14 (monocytes). In addition, total CD34+ lineage negative (lin-) thymocytes contained a low number of primitive myeloid progenitor cells, thus suggesting that the different hematopoietic lineages present in the thymus may be derived from primitive hematopoietic progenitor cells seeding the thymus. To investigate whether the thymus is permissive for the development of non-T cells, human fetal organ culture (FTOC) assays were performed by microinjecting sorted CD34+lin- fetal liver cells into fragments of HLA-mismatched fetal thymus. Sequential phenotypic analysis of the FTOC-derived progeny of CD34+lin- cells indicated that the differentiation into T cells was preceded by a wave of myeloid differentiation into CD14+CD11b+CD4dull cells. Donor-derived B cells (CD19+CD20+) were also generated, which produced immunoglobulins (IgG and IgM) when cultured under appropriate conditions, as well as functional CD56+CD3- NK cells, which efficiently killed K562 target cells in cytotoxicity assays. These results demonstrate that the microinjection of fetal liver hematopoietic progenitors into fetal thymic organ fragments results in multilineage differentiation in vitro.
View details for Web of Science ID A1994NR98900015
View details for PubMedID 7516402
-
EXPRESSION OF CD33, CD38, AND HLA-DR ON CD34+ HUMAN FETAL LIVER PROGENITORS WITH A HIGH PROLIFERATIVE POTENTIAL
BLOOD
1994; 83 (11): 3170-3181
Abstract
High proliferative-potential colony-forming cells (HPP-CFC) have been identified in the bone marrow of mice and adult humans, and have been characterized as a compartment of primitive progenitors possibly including stem cells. In this report we describe the human fetal liver (FL) as a source of HPP-CFC. These FL HPP-CFC develop in clonal cultures in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) within 3 to 4 weeks. The median frequency of HPP-CFC in FL tissues between 16 and 21 weeks of gestational age was 1 in 3,000 total FL cells. After 4 weeks of growth, FL HPP-CFC grew to a median colony size of 8.3 x 10(4) cells/colony. Using cell-sorting techniques FL HPP-CFC were shown to be predominantly contained in the CD34+ CD33+ CD38- fraction of FL cells. FL HPP-CFC were heterogeneous for HLA-DR expression, and no differences in proliferative capacities were observed between HLA-DR+ and HLA-DR- HPP-CFC. The CD34+ CD33-HLA-DR- CD38- population, previously suggested to contain stem cells, was observed to be very rare in the FL, representing approximately 1 in 1.7 x 10(5) light-density FL cells and containing almost no CFC. Therefore, it is possible that stem cells are contained in the CD33+ fraction of FL cells. Phenotypic characterization of CD34+ CD33+ CD38- lin -LDFL cells showed that these cells are also CD13+, predominantly Thy-1+, CD45RA-, CD45RO-, CD71-, and heterogenoeous for c-kit expression. These data suggest that FL HPP-CFC represent a heterogeneous compartment of primitive myeloid progenitors that may include stem cells.
View details for Web of Science ID A1994NN98800010
View details for PubMedID 7514903
-
LIGAND FOR FLT3 FLK2 RECEPTOR TYROSINE KINASE REGULATES GROWTH OF HEMATOPOIETIC STEM-CELLS AND IS ENCODED BY VARIANT RNAS
NATURE
1994; 368 (6472): 643-648
View details for Web of Science ID A1994NF39200063
-
IL-2 REVERSES HUMAN T-CELL UNRESPONSIVENESS INDUCED BY THYMIC EPITHELIUM IN SCID-HU MICE
JOURNAL OF IMMUNOLOGY
1994; 152 (5): 2198-2206
Abstract
In this study we investigated the mechanism responsible for the unresponsiveness of thymus-reactive T cells obtained from severe combined immunodeficient (SCID) mice constructed with human fetal liver (FL) stem cells from donor A and an allogeneic human fetal thymus (FT) from donor B (A/B SCID-hu mice). These A/B SCID-hu mice have a human thymus containing B cells, macrophages, and dendritic cells from FL donor A but thymic epithelial cells from FT donor B. The repertoire of human T cells developing in this chimeric thymus is depleted of T cells specific for the HLA Ags of the FL donor, whereas T cells reactive against the HLA Ags expressed by the FT donor are still present. However, these thymocytes failed to proliferate and expressed low levels of the activation markers CD25, CD71, and HLA-DR after stimulation with the EBV-LCL of the FT donor in primary MLRs. This unresponsiveness could be completely reversed by IL-2. Restoration of T cell responsiveness was Ag specific and a unique property of IL-2. The T cells produced very low levels of IL-2 when stimulated with the HLA Ags of FT donor B, whereas they secreted normal levels of IL-2 after activation by third party alloantigens. Low IL-2 production was also observed at the clonal level. CD4+ T cell clones from A/B SCID-hu mice, specific for the HLA Ags of B, produced significantly less IL-2 and granulocyte macrophage-CSF than control CD4+ T cell clones. However, these T cell clones synthesized normal levels of IL-2 and granulocyte macrophage-CSF after stimulation with combinations of PMA/Calo or PMA/anti-CD3 mAb. Thus, T cells that differentiate in a chimeric thymus containing allogeneic host thymic epithelium are rendered tolerant to the HLA Ags expressed by the thymic epithelial cells. This tolerance results in the presence of T cells that do not proliferate properly after Ag-specific stimulation. This lack of proliferation is primarily related to their inability to produce sufficient levels of IL-2 and can be restored by exogenous IL-2.
View details for Web of Science ID A1994NB98800014
View details for PubMedID 8133034
-
IN-VIVO CYTOKINE EXPRESSION IN THE THYMUS CD3(HIGH) HUMAN THYMOCYTES ARE ACTIVATED AND ALREADY FUNCTIONALLY DIFFERENTIATED IN HELPER AND CYTOTOXIC-CELLS
JOURNAL OF IMMUNOLOGY
1994; 152 (4): 1738-1743
Abstract
CD4 and CD8 are expressed on mutually exclusive T cell populations that can recognize peptides bound to class II and class I MHC Ags, respectively. These populations have different functions and are different in their capacity to produce cytokines. In this paper we demonstrate that this functional differentiation occurs at the CD3low- CD3high transitional stage: single positive mature CD4+ thymocytes express IL-2 mRNA in vivo, whereas CD8+ thymocytes primarily express perforin. IL-2, perforin, and IFN-gamma mRNAs were almost absent in CD4+CD8+ CD3low but were clearly detectable in CD4+CD8+ CD3high cells, indicating that these genes are induced at the CD3low- CD3high transitional stage. In contrast, IL-4 mRNA levels were highest in the precursor cells but dropped sharply at the CD3low-CD3high transitional stage. These data are consistent with and link two earlier observations, i.e., that activation occurs at the CD3low-CD3high transition and that functional differentiation in helper and cytotoxic cells is already accomplished in the single positive thymocytes. This activation may reflect positive selection and concomitant functional differentiation into helper and cytotoxic T cells.
View details for Web of Science ID A1994MY84400025
View details for PubMedID 8120382
-
HIGH-LEVELS OF INTERLEUKIN-10 PRODUCTION IN-VIVO ARE ASSOCIATED WITH TOLERANCE IN SCID PATIENTS TRANSPLANTED WITH HLA MISMATCHED HEMATOPOIETIC STEM-CELLS
JOURNAL OF EXPERIMENTAL MEDICINE
1994; 179 (2): 493-502
Abstract
Transplantation of HLA mismatched hematopoietic stem cells in patients with severe combined immunodeficiency (SCID) can result in a selective engraftment of T cells of donor origin with complete immunologic reconstitution and in vivo tolerance. The latter may occur in the absence of clonal deletion of donor T lymphocytes able to recognize the host HLA antigens. The activity of these host-reactive T cells is suppressed in vivo, since no graft-vs. -host disease is observed in these human chimeras. Here it is shown that the CD4+ host-reactive T cell clones isolated from a SCID patient transplanted with fetal liver stem cells produce unusually high quantities of interleukin 10 (IL-10) and very low amounts of IL-2 after antigen-specific stimulation in vitro. The specific proliferative responses of the host-reactive T cell clones were considerably enhanced in the presence of neutralizing concentrations of an anti-IL-10 monoclonal antibody, suggesting that high levels of endogenous IL-10 suppress the activity of these cells. These in vitro data correlate with observations made in vivo. Semi-quantitative polymerase chain reaction analysis carried out on freshly isolated peripheral blood mononuclear cells (PBMC) of the patient indicated that the levels of IL-10 messenger RNA (mRNA) expression were strongly enhanced, whereas IL-2 mRNA expression was much lower than that in PBMC of healthy donors. In vivo IL-10 mRNA expression was not only high in the T cells, but also in the non-T cell fraction, indicating that host cells also contributed to the high levels of IL-10 in vivo. Patient-derived monocytes were found to be major IL-10 producers. Although no circulating IL-10 could be detected, freshly isolated monocytes of the patient showed a reduced expression of class II HLA antigens. However, their capacity to stimulate T cells of normal donors in primary mixed lymphocyte cultures was within the normal range. Interestingly, similar high in vivo IL-10 mRNA expressions in the T and non-T cell compartment were also observed in three SCID patients transplanted with fetal liver stem cells and in four SCID patients transplanted with T cell-depleted haploidentical bone marrow stem cells. Taken together, these data indicate that high endogenous IL-10 production is a general phenomenon in SCID patients in whom allogenic stem cell transplantation results in immunologic reconstitution and induction of tolerance. Both donor T cells and host accessory cells contribute to these high levels of IL-10, which would suppress the activity of host-reactive T cell in vivo.
View details for Web of Science ID A1994MU12900013
View details for PubMedID 7905018
-
IN SEARCH OF T-CELL PROGENITORS IN THE HUMAN FETAL LIVER
RESEARCH IN IMMUNOLOGY
1994; 145 (2): 120-123
View details for Web of Science ID A1994NK25100005
View details for PubMedID 7521535
-
IMMUNE-RESPONSES BY CORD-BLOOD CELLS
BLOOD CELLS
1994; 20 (2-3): 573-586
Abstract
In the present study, the biological properties of cord blood cells were investigated. Cord blood mononuclear cells and T cells responded normally to activation by alloantigens in primary mixed leukocyte reactions (MLRs), indicating that cord blood T cells can be normally activated via their TcR and have normal proliferative capacities. In addition, they expressed normal levels of accessory molecules such as CD28 and LFA-1, which contribute to amplify their responses. In contrast, cord blood mononuclear cells, but not cord blood monocytes, had a reduced capacity to stimulate allogeneic cells in primary MLRs. In addition, cord blood monocytes express lower levels of HLA-DR and ICAM-1 compared to adult peripheral blood monocytes. Cord blood mononuclear cells were also impaired in their capacity to generate allogeneic cytotoxic activity in primary mixed leukocyte cultures (MLCs). In contrast, cord blood B cells were similar to adult B cells in their capacity to switch to immunoglobulin E producing cells when incubated with interleukin-4 (IL-4) and anti-CD40 monoclonal antibody. We also demonstrated that IL-2, IL-6, and tumor necrosis factor-alpha (TNF-alpha) production by activated cord blood mononuclear cells was comparable to that observed with peripheral blood mononuclear cells isolated from normal adult donors. In contrast, interferon-gamma (IFN-gamma) was significantly decreased, whereas IL-4 and IL-5 were absent. Granulocyte-macrophage colony-stimulating factor (GM-CSF) levels were in general higher in the supernatants of cord blood cells. Thus, cord blood immune responses differ from those of peripheral blood at several levels. Whether these differences account for a reduced capacity of transplanted cord blood cells to modulate graft vs. host disease remains to be determined.
View details for Web of Science ID A1994PZ37500047
View details for PubMedID 7749123
-
ALIVE HUMAN HLA CHIMERAS
25th Conference on Transplantation and Clinical Immunology - Rejection and Tolerance
KLUWER ACADEMIC PUBL. 1994: 400–400
View details for Web of Science ID A1994BD04M00034
-
ROLE OF IL-10 IN TRANSPLANTATION TOLERANCE
25th Conference on Transplantation and Clinical Immunology - Rejection and Tolerance
KLUWER ACADEMIC PUBL. 1994: 279–290
View details for Web of Science ID A1994BD04M00027
-
TOLERANCE TO ALLOANTIGENS AND RECOGNITION FOR ALLO+X INDUCED IN HUMANS BY FETAL STEM CELL TRANSPLANTATION
25th Conference on Transplantation and Clinical Immunology - Rejection and Tolerance
KLUWER ACADEMIC PUBL. 1994: 265–277
View details for Web of Science ID A1994BD04M00026
-
PHENOTYPIC AND FUNCTIONAL-ANALYSIS OF T-CELL PRECURSORS IN THE HUMAN FETAL LIVER AND THYMUS - CD7 EXPRESSION IN THE EARLY STAGES OF T-CELL AND MYELOID-CELL DEVELOPMENT
BLOOD
1993; 82 (11): 3401-3414
Abstract
It has been proposed that the CD7 molecule is the first antigen expressed on the membrane of cells committed to the T-cell lineage during human fetal T-cell ontogeny. To further identify the pre-T cell subpopulation that migrates to the thymus early in ontogeny, we analyzed the phenotypic and functional characteristics of the fetal liver populations separated on the basis of CD7 expression. Three populations expressing different levels of CD7 were observed: CD7bright, CD7dull, and CD7-. A CD7bright population depleted of mature T, B, and myeloid cells (lineage negative, lin-) and mostly composed of CD56+ CD34- natural killer cells did not mature into T cells in a fetal thymic organ culture (FTOC) assay and was devoid of myeloid progenitors in a clonal colony-forming cell assay. In contrast, the CD7-/dull CD34+ lin- populations were capable of differentiating into phenotypically mature T cells after injection into FTOC and contained early myeloid progenitors. Here we phenotypically compared the fetal liver CD7 populations with the most immature fetal thymic subset that differentiated in the FTOC assay, namely the triple negative (TN, CD3-CD4-CD8-) thymocytes. Fetal TN lin- expressed high levels of CD34 marker and were further subdivided by their expression of CD1 antigen, because CD1- TN thymocytes express higher levels of CD34 antigen compared with CD1+ TN cells. CD1- lin -TN thymocytes are characterized by expressing high levels of CD2, CD7, and CD34 markers and dull levels of CD5, CD10, and CD28 molecules. We could not find fetal liver pre-T cells with a phenotype equivalent to that of TN thymocytes. Our data show that CD7 does not necessarily identify T-cell precursors during fetal T-cell development and strongly support the hypothesis that the acquisition of early T-cell markers as CD2, CD28, and CD5 molecules on the cell surface of T-cell progenitors takes place intrathymically.
View details for Web of Science ID A1993MJ70800025
View details for PubMedID 7694684
-
T-LYMPHOCYTES FROM HUMAN CHIMERAS DO RECOGNIZE ANTIGEN IN THE CONTEXT OF ALLOGENEIC DETERMINANTS OF THE MAJOR HISTOCOMPATIBILITY COMPLEX
Beer-Sheva-Lyon Symposium on Immunology
ELSEVIER SCIENCE BV. 1993: 9–12
Abstract
Human stem cells from the fetal liver can be transplanted to immunodeficient patients and reconstitute their immunity by giving rise to immunocompetent T lymphocytes of donor origin. Despite full HLA mismatch between donor and host, the helper T cells and the cytotoxic T cells which develop in these chimeric patients are totally functional. They recognize the antigenic peptides presented in the context of the foreign HLA molecules of the recipient, indicating that donor stem cells have been positively selected in the host environment, probably the thymic epithelial cells. By contrast, negative selection appears to be imposed upon T cells by donor hemopoietic cells, probably macrophages or dendritic cells, migrating from the transplant to the host thymus. Clonal deletion is then responsible for tolerance to donor HLA antigens, while clonal anergy explains tolerance to host HLA antigens.
View details for Web of Science ID A1993MV05300002
View details for PubMedID 7511565
-
HUMAN IG PRODUCTION AND ISOTYPE SWITCHING IN SEVERE COMBINED IMMUNODEFICIENT-HUMAN MICE
JOURNAL OF IMMUNOLOGY
1993; 151 (1): 128-137
Abstract
Severe combined immunodeficient (SCID) mice were transplanted with different human fetal organs (SCID-hu mice), including thymus, liver, spleen, and omentum, and the serum levels of human IgM, IgG, IgE, and IgA were measured. In all SCID-hu mice significant levels (up to 590 ng/ml) of IgM were detected, irrespective of the organs transplanted. In contrast, IgG was present (up to 530 ng/ml) only when the fetal thymus was transplanted together with the fetal liver, indicating that the presence of human T cell is a prerequisite for in vivo isotypes switching by human B cells in SCID-hu mice. Additional transplantation of fetal spleen did not significantly increase IgG levels. was observed 4 months after transplantation. At that time, analysis by IEF showed that human IgG present in SCID-hu serum was at least oligoclonal. Furthermore, all IgG subclasses were represented in the human IgG pool. Human B cells were undetectable in the peripheral blood, spleen, and bone marrow of these SCID-hu mice; in contrast, B cells expressing CD19 could be isolated from the SCID-hu thymus. Considerable proportions of the CD19+ B cells coexpressed CD5, CD7, CD10, CD40, and CD2. These B cells spontaneously produced IgM and IgG in vitro and could be induced to switch to IgE-producing cells when cocultured with cloned activated CD4+ T cells in the presence of IL-4. Collectively, these data demonstrate that functionally mature B cells able to produce IgM and IgG in vivo, and IgE in vitro, are present in the SCID-hu human thymus.
View details for Web of Science ID A1993LM57900014
View details for PubMedID 8326122
-
INDUCING AND ENHANCING EFFECTS OF IL-3, IL-5, AND IL-6 AND GM-CSF ON HISTAMINE-RELEASE FROM HUMAN BASOPHILS
CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY
1993; 67 (3): 210-215
Abstract
The effects of interleukin (IL)-2, -3, -4, -5, -6, and -7 and granulocyte-macrophage colony-stimulating factor (GM-CSF) on histamine release from human basophils were evaluated. IL-3 was the only cytokine with histamine-releasing activity. This activity was observed predominantly on basophils from allergic patients (mean release +/- SEM, 33.9 +/- 9.5%; n = 12), whereas basophils from normal subjects responded less frequently to stimulation with IL-3 (mean release +/- SEM, 2.8 +/- 1.0%; n = 22). The effect of IL-3 was time and temperature dependent, since release was optimal after incubation for 120 min at 37 degrees C. When cell-bound IgE were eluted at acid pH, basophils became unresponsive to IL-3; however, IL-3-induced histamine release correlated with anti-IgE-induced histamine release in allergics, but not in normals. IL-3, IL-5, IL-6, and GM-CSF enhanced significantly anti-IgE- and FMLP-induced histamine release. In contrast, IL-2, IL-4, and IL-7 were devoid of any significant histamine-releasing or -potentiating activity. These results indicate that IL-3 can induce and IL-3, -5, and -6 and GM-CSF can enhance histamine release from human basophils, suggesting a possible role of these cytokines in the expression of allergic reactions.
View details for Web of Science ID A1993LF99500005
View details for PubMedID 7684660
-
BACTERIAL SUPERANTIGENS MEDIATE T-CELL DELETIONS IN THE MOUSE SEVERE COMBINED IMMUNODEFICIENCY HUMAN LIVER THYMUS MODEL
JOURNAL OF EXPERIMENTAL MEDICINE
1993; 177 (5): 1481-1485
Abstract
The ability to analyze T cell receptor (TCR) thymic repertoire shaping in humans by self and foreign ligands is hampered by the lack of suitable models. We recently documented that the mouse severe combined immunodeficiency (SCID)-human fetal liver/thymus model recapitulates the TCR V beta gene repertoire of human thymocytes. Here, we show that an exogenous superantigen, staphylococcal enterotoxin B, administered to such mice induces clonal deletions in both CD4+8- and CD8+4- cells involving the same human V beta clones that are selected in vitro by this toxin. This model, therefore, may allow comprehensive studies into the effects of microbial and other agents on human T cell thymic selection processes in a biologically relevant setting.
View details for Web of Science ID A1993KY84900026
View details for PubMedID 8478618
-
CHIMERISM AND TOLERANCE TO HOST AND DONOR IN SEVERE COMBINED IMMUNODEFICIENCIES TRANSPLANTED WITH FETAL LIVER STEM-CELLS
JOURNAL OF CLINICAL INVESTIGATION
1993; 91 (3): 1067-1078
Abstract
We have studied the peripheral T cell repertoire of two patients with severe combined immunodeficiency who were successfully treated with human histocompatibility leukocyte antigen (HLA)-mismatched fetal liver stem cell transplantation. The patients presented a split chimerism. T cells were of donor origin, whereas the B cells/monocytes were of the host phenotype. Interestingly, the natural killer (NK) cells in one patient were donor derived and in the other patient of host origin. The NK cells were functional but did not have antihost or donor reactivity. Despite the HLA mismatch between donor and host cells, complete tolerance was achieved in vivo, and a specific unresponsiveness of peripheral blood mononuclear cells from both patients toward the host cells was demonstrated in vitro. Nevertheless, we could isolate T cell receptor (TCR)alpha beta, CD4+ or CD8+, T cell clones specifically reacting with HLA class I and II molecules of the host. The CD4+ host-reactive T cell clones from both patients produced interleukins 2 and 5, interferon-gamma, granulocyte/macrophage colony-stimulating factor but are specifically defective in interleukin 4 production. The frequencies of CD8+ host-reactive T cells were high, and were in the same range as those observed for CD8+ alloreactive T cells. In contrast, no donor-reactive CD8+ T cells or host or donor-reactive TCR gamma delta + T cells were detected. These data indicate that, after fetal stem cell transplantation, donor-reactive, but not host-reactive cells, are deleted from the T cell repertoire. Therefore, a peripheral mechanism of suppression or clonal anergy, rather than clonal deletion, is involved in maintaining in vivo tolerance toward the host.
View details for Web of Science ID A1993KR46100040
View details for PubMedID 8450037
-
FETAL LIVER-TRANSPLANTATION - BIOLOGY AND CLINICAL-RESULTS
INTERNATIONAL SYMP ON BONE MARROW TRANSPLANTATION FROM ALTERNATIVE DONORS : BIOLOGY, CLINICAL RESULTS, REGISTRY ACTIVITIES
NATURE PUBLISHING GROUP. 1993: 119–122
Abstract
Over the last 18 years, we have developed the transplantation of fetal liver cells to treat severe immunodeficiencies, hematological disorders and inborn errors of metabolism. Post-natally, this treatment is successful in two-third of patients and it is therefore very valuable, especially when there is no perfectly matched donor for a bone marrow transplant. Since 1988 we have carried out these fetal liver transplants (FLTs) in utero, immediately after prenatal diagnosis. Engraftment and reconstitution have been obtained, and several advantages appear to be associated with in utero FLT: increased probability of graft take, ideal isolation of the patient (in the maternal uterus) and optimal environment for the differentiation of the transplanted fetal liver cells (in the fetal host).
View details for Web of Science ID A1993KK14800035
View details for PubMedID 8448534
-
INTERLEUKIN-10 INHIBITS ALLOGENEIC PROLIFERATIVE AND CYTOTOXIC T-CELL RESPONSES GENERATED IN PRIMARY MIXED LYMPHOCYTE-CULTURES
INTERNATIONAL IMMUNOLOGY
1992; 4 (12): 1389-1397
Abstract
The effects of IL-10 on the generation of alloreactivity in primary mixed lymphocyte cultures (MLCs) were investigated. IL-10 inhibited in a dose-dependent fashion the alloantigen-induced proliferative responses. The suppressive effect was maximal when IL-10 was added at the beginning of the cultures, suggesting that it acts on the early stages of T cell activation. The proliferative responses were enhanced in the presence of a neutralizing anti-IL-10 mAb, indicating that endogenously produced IL-10 suppresses proliferation in primary MLC. The inhibitory effects of IL-10 were observed irrespective of whether irradiated allogeneic peripheral blood mononuclear cells, purified monocytes or freshly isolated B cells were used as stimulator cells. The proliferation of both the CD4+ and CD8+ T cell subsets was inhibited to a similar extent. The reduced proliferative responses were only minimally restored by high concentrations of exogenous IL-2, indicating that the effects of IL-10 are not exclusively due to inhibition of IL-2 synthesis. Furthermore, the production of IL-2, interferon (IFN)-gamma, IL-6, granulocyte macrophage colony stimulating factor, and tumor necrosis factor-alpha in primary MLCs was diminished by IL-10 and enhanced in the presence of anti-IL-10 mAb. The strongest effects were observed on the production of IFN-gamma. Although IL-10 reduces the proliferative responses, the ratios of CD3+CD4+ and CD3+CD8+ T cells remained the same in IL-10 treated and control cultures, yet the percentages of activated CD3+ T cells, as judged by CD25 and HLA-DR expression, were consistently reduced.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1992KE10500006
View details for PubMedID 1286062
-
THYMIC SELECTION OF THE HUMAN T-CELL RECEPTOR V-BETA REPERTOIRE IN SCID-HU MICE
JOURNAL OF EXPERIMENTAL MEDICINE
1992; 176 (6): 1619-1624
Abstract
Implantation of pieces of human fetal liver and thymus into SCID mice results in the development of a human thymus-like organ, in which sustained lymphopoiesis is reproducibly observed. In this model, T cell development can be experimentally manipulated. To study the influence of thymic selection on the development of the human T cell repertoire, the T cell receptor (TCR) V beta gene repertoire of double-positive (CD4+CD8+) and single-positive (CD4+CD8- and CD4-CD8+) T cells was analyzed in the SCID-hu thymus using a multiprobe ribonuclease protection assay. TCR diversity in double-positive SCID-hu thymocytes was found to be comparable with that present in the thymus of the fetal liver donor, did not change with time, and was independent of the origin of the thymus donor. Thymic selection in SCID-hu thymus induces changes in V beta usage by the single-positive CD4+ or CD8+ T cells comparable with those previously reported for single-positive cells present in a normal human thymus. Finally, significant differences were observed in the V beta usage by CD4 or CD8 single-positive T cells that matured from genetically identical stem cells in different thymic environments. Collectively, these data suggest: first, that the generation of TCR diversity at the double-positive stage is determined by the genotype of the stem cells; and second, that polymorphic determinants expressed by thymic epithelium measurably influence the V beta repertoire of mature single-positive T cells.
View details for Web of Science ID A1992KA48100016
View details for PubMedID 1460421
-
IL-10 IS PRODUCED BY SUBSETS OF HUMAN CD4+ T-CELL CLONES AND PERIPHERAL-BLOOD T-CELLS
JOURNAL OF IMMUNOLOGY
1992; 149 (7): 2378-2384
Abstract
Murine IL-10 has been reported originally to be produced by the Th2 subset of CD4+ T cell clones. In this study, we demonstrate that human IL-10 is produced by Th0, Th1-, and Th2-like CD4+ T cell clones after both Ag-specific and polyclonal activation. In purified peripheral blood T cells, low, but significant, levels of IL-10 were found to be produced by the CD4+CD45RA+ population, whereas CD4+CD45RA- "memory" cells secreted 5- to 20-fold higher levels of IL-10. In addition, IL-10 was produced by activated CD8+ peripheral blood T cells. Optimal induction of IL-10 was observed after activation by specific Ag and by the combination of anti-CD3 mAb and the phorbol ester tetradecanoyl phorbol acetate, whereas the combination of calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate acetate was a poor inducer of IL-10 production. Kinetic studies indicated that IL-10 was produced relatively late as compared with other cytokines. Maximal IL-10 mRNA expression in CD4+ T cell clones and purified peripheral blood T cells was obtained after 24 h, whereas maximal IL-10 protein synthesis occurred between 24 h and 48 h after activation. No differences were observed in the kinetics of IL-10 production among Th0, Th1-, and Th2-like subsets of CD4+ T cell clones. The results indicate a regulatory role for IL-10 in later phases of the immune response.
View details for Web of Science ID A1992JP18900020
View details for PubMedID 1356125
-
INTERLEUKIN-10
CURRENT OPINION IN IMMUNOLOGY
1992; 4 (3): 314-320
Abstract
Despite the short history of interleukin-10, accumulated evidence indicates that this interleukin plays a major role in suppressing immune and inflammatory responses. Yet interleukin-10 also maintains cell viability and acts as a cofactor to promote the growth of lymphoid and myeloid cells in vitro. Here we review the present knowledge on the structure and function of interleukin-10.
View details for Web of Science ID A1992JD79700012
View details for PubMedID 1418711
-
INDUCTION OF ISOTYPE SWITCHING AND IG PRODUCTION BY CD5+ AND CD10+ HUMAN FETAL B-CELLS
JOURNAL OF IMMUNOLOGY
1992; 148 (11): 3398-3404
Abstract
In the present study the capacity of early fetal B cells to produce Ig was investigated. It is shown that B cells from fetal liver, spleen, and bone marrow (BM) can be induced to produce IgM, IgG, IgG4, and IgE, but not IgA, in response to IL-4 in the presence of anti-CD40 mAb or cloned CD4+ T cells. Even splenic B cells from a human fetus of only 12 wk of gestation produced these Ig isotypes. IFN-alpha, IFN-gamma, and transforming growth factor-beta inhibited IL-4-induced IgE production in fetal B cells, as described for mature B cells. The majority of B cells in fetal spleen expressed CD5 and CD10 and greater than 99% of B cells in fetal BM were CD10+. Highly purified CD10+, CD19+ immature B cells and CD5+, CD19+ B cells could be induced to produce Ig, including IgG4 and IgE, in similar amounts as unseparated CD19+ B cells. Virtually all CD19+ cells still expressed CD10 after 12 days of culture. However, the IgE-producing cells at the end of the culture period were found in the CD19-,CD10- cell population, suggesting differentiation of CD19+,CD10+ B cells into CD19-,CD10- plasma cells. Pre-B cells are characterized by their lack of expression of surface IgM (sIgM). Only 30 to 40% of BM B cells expressed sIgM. However, in contrast to sIgM+,CD10+,CD19+ immature B cells, sorted sIgM-,CD10+,CD19+ pre-B cells failed to differentiate into Ig-secreting cells under the present culture conditions. Addition of IL-6 to these cultures was ineffective. Taken together, these results indicate that fetal CD5+ and CD10+ B cells are mature in their capacity to be induced to Ig isotype switching in vitro as soon as they express sIgM.
View details for Web of Science ID A1992HW13600010
View details for PubMedID 1375243
-
STRATEGIES OF ANTICYTOKINE MONOCLONAL-ANTIBODY DEVELOPMENT - IMMUNOASSAY OF IL-10 AND IL-5 IN CLINICAL-SAMPLES
IMMUNOLOGICAL REVIEWS
1992; 127: 5-24
View details for Web of Science ID A1992JD81800001
View details for PubMedID 1387110
-
MEMBRANES OF ACTIVATED CD4+ T-CELLS EXPRESSING T-CELL RECEPTOR (TCR) ALPHA-BETA OR TCR GAMMA-DELTA INDUCE IGE SYNTHESIS BY HUMAN B-CELLS IN THE PRESENCE OF INTERLEUKIN-4
EUROPEAN JOURNAL OF IMMUNOLOGY
1992; 22 (5): 1133-1141
Abstract
In the present study it is demonstrated that human B cells can be induced to switch to IgE production following a contact-mediated signal provided by activated T cell receptor (TcR) gamma delta+, CD4+ and TcR alpha beta+, CD4+ T cell clones and interleukin (IL)-4. The signal provided by these T cell clones was antigen nonspecific, indicating that the TcR alpha beta/CD3 or TcR gamma delta/CD3 complexes were not involved in these T-B cell interactions. Activated TcR alpha beta+, CD8+, and TcR gamma delta+, CD4-CD8-, or resting CD4+ T cell clones were ineffective. Intact TcR alpha beta+ or TcR gamma delta+, CD4+ T cell clones could be replaced by plasma membrane-enriched fractions isolated from these activated CD4+ T cell clones. In contrast, membranes isolated from resting TcR alpha beta+, CD4+, TcR gamma delta+, CD4+ T cell clones or an Epstein-Barr virus (EBV)-transformed B cell line (EBV-LCL) failed to provide the costimulatory signal that, in addition to IL-4, is required for induction of IgE synthesis. As described for intact CD4+ T cells, CD4+ T cell membranes induced purified surface IgM+ B cells to switch to IgG4- and IgE- but not to IgA-producing cells, excluding the possibility of a preferential outgrowth of IgG4- and IgE-committed B cells. The membrane activity was inhibited by protease or heat treatment. Induction of IgE synthesis by B cells co-cultured with both TcR alpha beta+, CD4+ and TcR gamma delta+, CD4+ T cell clones and membrane preparations of these cells was blocked by anti-class II major histocompatibility complex (MHC) monoclonal antibodies (mAb), whereas various anti-CD4 mAb had differential blocking effects. Murine L cells, or EBV-LCL transfected with CD4 could not replace CD4+ T cell clones. These results indicate that, although CD4 and class II MHC antigens are required for productive CD4+ T cell clone-B cell interactions, an additional signal, provided by a membrane associated (glyco)protein that is induced by activation of both TcR alpha beta and TcR gamma delta, CD4+ T cells, is needed for induction of IgE production in the presence of IL-4.
View details for Web of Science ID A1992HV81700004
View details for PubMedID 1349531
-
HUMAN HEMATOPOIETIC-CELLS AND THYMIC EPITHELIAL-CELLS INDUCE TOLERANCE VIA DIFFERENT MECHANISMS IN THE SCID-HU MOUSE THYMUS
JOURNAL OF EXPERIMENTAL MEDICINE
1992; 175 (4): 1033-1043
Abstract
To study the role of thymic education on the development of the human T cell repertoire, SCID-hu mice were constructed with fetal liver and fetal thymus obtained from the same or two different donors. These animals were studied between 7 and 12 mo after transplantation, at which times all thymocytes and peripheral T cells were derived from stem cells of the fetal liver graft. Immunohistology of the thymus grafts demonstrated that thymic epithelial cells were of fetal thymus donor (FTD) origin. Dendritic cells and macrophages of fetal liver donor (FLD) origin were abundantly present in the medullary and cortico-medullary areas. Thymocytes of SCID-hu mice transplanted with liver and thymus of two different donors (FLDA/FTDB animals) were nonresponsive to Epstein-Barr virus-transformed B cell lines (B-LCL) established from both the FLDA and FTDB, but proliferated vigorously when stimulated with third-party allogeneic B-LCL. Mixing experiments showed that the nonresponsiveness to FTDB was not due to suppression. Limiting dilution analysis revealed that T cells reacting with the human histocompatibility leukocyte antigens (HLA) of the FLD were undetectable in the CD8+ T cell population and barely measurable in the CD4+ subset. On the other hand, CD4+ and CD8+ T cells reactive to the HLA antigens of the FTD were readily detectable. These results indicate that FLD-reactive cells were clonally deleted, whereas FTD-reactive cells were not. However, the frequencies of FTD-reactive T cells were consistently twofold lower than those of T cells specific for any third-party B-LCL. In addition, the cytotoxic activity and interleukin 2 production by FTD-specific T cells were lower compared with that of third-party-reactive T cell clones, suggesting that FTD-specific cells are anergic. These data demonstrate that T cells become tolerant to autologous and allogeneic HLA antigens expressed in the thymus via two different mechanisms: hematopoietic cells present in the thymus induce tolerance to "self"-antigens by clonal deletion, whereas thymic epithelial cells induce tolerance by clonal energy and possibly deletion of high affinity clones.
View details for Web of Science ID A1992HL01400017
View details for PubMedID 1348080
-
ALLO-RESTRICTED HELPER AND CYTOTOXIC LYMPHOCYTES-T IN A SCID PATIENT FOLLOWING INCOMPATIBLE FETAL LIVER-CELL TRANSPLANTATION
24TH INTERNATIONAL CONF ON TRANSPLANTATION AND CLINICAL IMMUNOLOGY
ELSEVIER SCIENCE PUBL B V. 1992: 338–338
View details for Web of Science ID A1992BX50M00076
-
INUTERO TRANSPLANTATION OF STEM-CELLS IN HUMANS - IMMUNOLOGICAL ASPECTS AND CLINICAL FOLLOW-UP OF PATIENTS
SYMP ON FOETAL AND NEONATAL CELL TRANSPLANTATION AND RETROVIRAL GENE THERAPY
STOCKTON PRESS. 1992: 121–126
Abstract
Four human fetuses were treated by transplantation of human fetal liver stem cells. Two of them had severe immunodeficiency disease and the two other ones had thalassemia major. Three of these in utero transplants were followed by engraftment. The three patients are now born: the first one is now very healthy thanks to the reconstitution of cell-mediated immunity associated with this transplant, and he lives normally at home; the two other ones, who have been more recently treated, have a significant improvement of their condition and they also live normally at home. This procedure, for the first time used in humans, has therefore demonstrated its feasibility and its efficacy: during early fetal development, foreign cells engraft readily and may result in cure or significant correction of a large variety of inherited diseases.
View details for Web of Science ID A1992JD06400033
View details for PubMedID 1354520
-
T-CELL REPERTOIRE AND TOLERANCE AFTER FETAL STEM-CELL TRANSPLANTATION
SYMP ON FOETAL AND NEONATAL CELL TRANSPLANTATION AND RETROVIRAL GENE THERAPY
STOCKTON PRESS. 1992: 127–128
Abstract
We studied the T cell repertoire and the mechanism of tolerance in two patients with severe combined immunodeficiency transplanted with HLA mismatched fetal liver stem cells. They are 17 and 5 years old now, healthy, and show normal immunoresponses to recall antigens. Their T cells are of donor origin, whereas monocytes and B cells remained of the host. The NK cells have different sources since in one patient they derive from the donor and in the other one from the host. Despite the HLA mismatch between donor and host cells, no acute or chronic graft-versus-host disease was observed. In vitro experiments with PBMC showed specific nonresponsiveness for the HLA antigens expressed by the host cells. However, an extensive clonal analysis showed that CD4+ and CD8+ host-reactive T cell clones recognizing class II and class I HLA molecules of the host, respectively, were present in the peripheral blood of both patients. Limiting dilution experiments indicated that the frequency of CD8+ host-reactive cells was in the same range as that observed for alloreactive T cells. In contrast, no donor reactive CD8+ T cells could be isolated. Host-reactive CD4+ and CD8+ T cell clones were normal in their capacity to produce IL-2, IFN-gamma, GM-CSF and IL-5, but they failed completely to synthesize IL-4. In addition, CD4+ T cell clones from patient RV secreted very high levels of IL-10. Interestingly, exogenous IL-10 was able to inhibit the proliferative responses of the CD4+ host-reactive T cell clones. Our data demonstrate that host-reactive cells are not deleted from the donor T cell repertoire following allogenic fetal liver stem cell transplantation. Therefore, in vivo tolerance between the host and the donor is maintained by a peripheral autoregulatory mechanism in which cytokines may play a role.
View details for Web of Science ID A1992JD06400034
View details for PubMedID 1354521
-
SCID-HU MICE AS A MODEL TO STUDY TOLERANCE AFTER FETAL STEM-CELL TRANSPLANTATION
SYMP ON FOETAL AND NEONATAL CELL TRANSPLANTATION AND RETROVIRAL GENE THERAPY
STOCKTON PRESS. 1992: 83–84
Abstract
SCID-hu mice were constructed with human fetal liver and human fetal thymus, obtained from the same or from different donors. Hematopoietic cells originating from the fetal liver migrate to the fetal thymus and give rise to medullary and cortico-medullary macrophages and dendritic cells. Thymic epithelial cells remain of thymic donor origin. The fetal liver donor derived stem cells differentiate in this environment into double positive and finally single positive CD4 or CD8 expressing T cells. The TCR V beta repertoire generated at the double positive stage is identical to that generated in the thymus of the donor. Thymic selection induces changes in V beta usage comparable to those previously reported for normal human thymus. Single-positive, functionally mature, and mainly TCR alpha beta+ T cells reach the peripheral blood compartment of the SCID-hu mice. These T cells are able of specific proliferative and cytotoxic alloresponses. No autoreactivity is observed. Single positive T cells which differentiated in the thymus of SCID-hu mice transplanted with liver and thymus of two different donors are tolerant to the HLA antigens of both donors. By limiting dilution analysis, it could be demonstrated that tolerance to the fetal liver donor is due to clonal deletion whereas tolerance to the fetal thymus donor is not. These data show that human T cell development is progressing normally in these mice and gives rise to a mature, functional and polyclonal T cell repertoire which is comparable to that observed in normal individuals.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1992JD06400021
View details for PubMedID 1354529
-
RECIPROCAL HYBRID JOINTS DEMONSTRATE SUCCESSIVE V-J REARRANGEMENTS ON THE SAME CHROMOSOME IN THE HUMAN TCR GAMMA LOCUS
INTERNATIONAL IMMUNOLOGY
1991; 3 (10): 973-982
Abstract
Novel variable (V)--joining (J) gene rearrangements are described in the human T cell receptor gamma locus, in which, on the one hand, the V3 variable gene is joined to the heptamer--nonamer recombination signals of the J1 segment and, on the other hand, the J1 segment is joined to the V3 recombination signals through head-to-head fusion. These recombination products, or hybrid joints, have been originated through an inversion of 47 kb DNA. Interestingly the inverted DNA stretch contains a normal V9-J9 rearrangement. These findings are the first direct demonstration that successive rearrangements occur, on the same chromosome, in the human T cell receptor gamma locus, and suggest that the chronology of the joining events plays a role in the ontogeny of T cells and their differentiation in gamma/delta + and alpha/beta + lineages.
View details for Web of Science ID A1991GK70300005
View details for PubMedID 1836738
-
INTERLEUKIN-10 (IL-10) AND VIRAL-IL-10 STRONGLY REDUCE ANTIGEN-SPECIFIC HUMAN T-CELL PROLIFERATION BY DIMINISHING THE ANTIGEN-PRESENTING CAPACITY OF MONOCYTES VIA DOWN-REGULATION OF CLASS-II MAJOR HISTOCOMPATIBILITY COMPLEX EXPRESSION
JOURNAL OF EXPERIMENTAL MEDICINE
1991; 174 (4): 915-924
Abstract
Interleukin 10 (IL-10) and viral IL-10 (v-IL-10) strongly reduced antigen-specific proliferation of human T cells and CD4+ T cell clones when monocytes were used as antigen-presenting cells. In contrast, IL-10 and v-IL-10 did not affect the proliferative responses to antigens presented by autologous Epstein-Barr virus-lymphoblastoid cell line (EBV-LCL). Inhibition of antigen-specific T cell responses was associated with downregulation of constitutive, as well as interferon gamma- or IL-4-induced, class II MHC expression on monocytes by IL-10 and v-IL-10, resulting in the reduction in antigen-presenting capacity of these cells. In contrast, IL-10 and v-IL-10 had no effect on class II major histocompatibility complex (MHC) expression on EBV-LCL. The reduced antigen-presenting capacity of monocytes correlated with a decreased capacity to mobilize intracellular Ca2+ in the responder T cell clones. The diminished antigen-presenting capacities of monocytes were not due to inhibitory effects of IL-10 and v-IL-10 on antigen processing, since the proliferative T cell responses to antigenic peptides, which did not require processing, were equally well inhibited. Furthermore, the inhibitory effects of IL-10 and v-IL-10 on antigen-specific proliferative T cell responses could not be neutralized by exogenous IL-2 or IL-4. Although IL-10 and v-IL-10 suppressed IL-1 alpha, IL-1 beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 production by monocytes, it was excluded that these cytokines played a role in antigen-specific T cell proliferation, since normal antigen-specific responses were observed in the presence of neutralizing anti-IL-1, -IL-6, and -TNF-alpha mAbs. Furthermore, addition of saturating concentrations of IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha to the cultures had no effect on the reduced proliferative T cell responses in the presence of IL-10, or v-IL-10. Collectively, our data indicate that IL-10 and v-IL-10 can completely prevent antigen-specific T cell proliferation by inhibition of the antigen-presenting capacity of monocytes through downregulation of class II MHC antigens on monocytes.
View details for Web of Science ID A1991GJ10900020
View details for PubMedID 1655948
-
NATURAL-KILLER-CELL CLONES CAN EFFICIENTLY PROCESS AND PRESENT PROTEIN ANTIGENS
JOURNAL OF IMMUNOLOGY
1991; 147 (3): 781-787
Abstract
NK cell clones obtained from three different donors were tested for their ability to present soluble proteins to Ag-specific T cell clones. All NK clones were CD2+CD3-CD56+, whereas the expression of CD16 varied from clone to clone. The NK cell clones were able to process and present tetanus toxoid (TT) to TT-specific T cell clones in a class II HLA restricted manner. The capacity of NK cell clones to function as APC was also observed using the house dust mite allergen Der p I and the Der p I-derived peptide Val89-Cys117. As with EBV-transformed B cell line, NK cell clones could present the peptide 3-13 derived from the 65-kDa heat shock protein of Mycobacterium leprae, but they were unable to present the whole M. leprae Ag. Freshly isolated NK cells, IL-2-activated NK cells, and NK cell lines expanded in vitro could also process and present TT. The ability of the different NK populations to act as accessory cells correlated with their levels of class II HLA expression. These data demonstrate that NK cell clones can efficiently function as APC, however they may be restricted in the types of Ag that they can process.
View details for Web of Science ID A1991FY29200006
View details for PubMedID 1861074
-
CLONAL ANALYSIS OF THE PERIPHERAL T-CELL COMPARTMENT OF THE SCID-HU MOUSE
JOURNAL OF IMMUNOLOGY
1991; 146 (12): 4173-4179
Abstract
Severe combined immunodeficiency (SCID) mice can be transplanted successfully with human fetal liver and thymus (SCID-hu mice). Precursor cells derived from the fetal liver differentiate in the thymus and migrate into the blood as mature T cells. In the present paper, the peripheral T cell compartment of such mice was studied. Peripheral WBC were activated by PHA and cultured in the presence of irradiated human feeder cells. The resultant cell population consisted exclusively of human CD1- CD2+ CD3+ CD7+ T lymphocytes; up to 4% of the T cells expressed the TCR gamma delta, whereas 95 to 100% were TCR alpha beta +. The CD4bright (42 to 66%) and CD8bright (30 to 54%) populations coexpressed variable but low levels of CD8 and CD4, respectively. The T cell cultures from the SCID-hu mice did not display reactivity towards the autologous human EBV-transformed B cell lines (B-LCL). On the other hand, these human T cells proliferated and were cytotoxic against allogeneic human B-LCL. T cell clones were established from cultured SCID-hu T cells. All T cell clones were TCR alpha beta + CD3+ CD2+; 61% of the clones were CD4+ CD8-, 27% were CD8+ CD4-, 11% were CD8+ CD4lo, and 2% were CD4+ CD8lo. None of these clones recognized the autologous B-LCL established from the fetal human donor. Fourteen of 100 T cell clones had specific alloreactivity, as tested on a panel of five B-LCL. Of these 14, two CD8+ CD4lo and two CD8+ CD4- clones were cytotoxic and did not proliferate in response to specific stimulator cells. Furthermore, two CD4+ CD8lo and eight CD4+ CD8- clones proliferated specifically in response to alloantigens. In conclusion, the peripheral human T cells of SCID-hu animals are functional and their TCR repertoire is polyclonal, alloreactive, and devoid of self-reactive cells. Therefore, the SCID-hu mouse can be a suitable model for the study of alloreactivity and allotolerance in vivo, as well as for the study of negative selection in the human thymus.
View details for Web of Science ID A1991FQ77300016
View details for PubMedID 1674954
-
LOW MW B-CELL GROWTH-FACTOR POTENTIATES HISTAMINE-RELEASE FROM HUMAN BASOPHIL LEUKOCYTES
IMMUNOLOGY
1991; 73 (2): 217-221
Abstract
The aim of the present study was to evaluate the effects of a commercial preparation of low molecular weight (MW) B-cell growth factor (BCGF), a product of activated human T cells, on histamine release from stimulated human basophils. In all the 23 cases tested, BCGF enhanced significantly the histamine release from stimulated human basophil leucocytes. After leucocyte incubation with 1% v/v BCGF, anti-IgE-induced histamine release increased from 11.9 +/- 1.9% to 31.8 +/- 3.3% (mean +/- SEM; P less than 0.001), formyl-methionine peptide-induced histamine release increased from 24.9 +/- 3.9% to 50.3 +/- 5.3% (P less than 0.001) and complement-mediated (i.e. induced by zymosan-activated human serum) histamine release increased from 10.7 +/- 1.8% to 29.2 +/- 5.2% (P less than 0.001). The enhancing effect of BCGF on histamine release was related to dose, temperature and time, since it was optimal after incubation with 10% v/v BCGF at 37 degrees for 60 min. BCGF activity persisted when the cells were washed after incubation and could also be observed when leucocyte incubation was carried out in the absence of extracellular Ca2+. It is noteworthy that basophils which showed a low response to activating agents released significant amounts of histamine after incubation with BCGF. These observations support the hypothesis that lymphocytes and lymphocyte-derived products may play a role in triggering and maintaining allergic reactions.
View details for Web of Science ID A1991FR55700015
View details for PubMedID 2071166
-
Cloning of a novel cell type from human fetal liver expressing cytoplasmic CD3 delta and epsilon but not membrane CD3.
International immunology
1991; 3 (4): 353-357
Abstract
Seventeen-week human fetal liver cells cultured with a feeder cell mixture of irradiated PBL, irradiated JY cells (an EBV-transformed B cell line) and PHA contained a subpopulation of CD3- cells in addition to a major population of T cells with the mature phenotype. After 12 days in culture, CD3- CD16- cells were sorted and cloned by limiting dilution. Two representative clones, FL121 and FL125, were expanded and characterized. They shared the phenotype of CD2+CD3-CD4-CD5-CD6+CD16-CD56+. FL121 did not express CD8 whereas FL125 expressed CD8 alpha but not CD8 beta. Both clones were found to express cytoplasmic CD3 delta and CD3 epsilon Ag while CD3- NK clones isolated from PBL were negative for them. These results indicate that FL121 and FL125 were committed to the T-cell lineage. Southern blot analysis showed that the TCR beta genes and the TCR gamma genes of these clones were in the germ-line configuration. The establishment of FL121 and FL125 may provide a novel insight into the earliest stage of T-cell development in man.
View details for PubMedID 1831653
-
CLONING OF A NOVEL CELL TYPE FROM HUMAN FETAL LIVER EXPRESSING CYTOPLASMIC CD3-DELTA AND CD3-EPSILON BUT NOT MEMBRANE CD3
INTERNATIONAL IMMUNOLOGY
1991; 3 (4): 353-357
View details for Web of Science ID A1991FK38000008
-
REGULATION OF HUMAN IGE SYNTHESIS - THE ROLE OF CD4+ AND CD8+ T-CELLS AND THE INHIBITORY EFFECTS OF INTEREFON-ALPHA
EUROPEAN RESPIRATORY JOURNAL
1991; 4: S30-S38
View details for Web of Science ID A1991FR81600005
-
Regulation of human IgE synthesis: the role of CD4+ and CD8+ T-cells and the inhibitory effects of interferon-alpha.
The European respiratory journal. Supplement
1991; 13: 31s-38s
Abstract
Interleukin-4 (IL-4) has been shown to induce immunoglobulin E (IgE) synthesis by human peripheral blood lymphocytes (PBL). Here we show that highly purified B-cells (greater than 99.5%) failed to produce IgE in the presence of IL-4 but IgE synthesis was restored by the addition of autologous CD4+ T-cells or by CD4+ T-cell clones with non-relevant specificities, derived from different donors. In contrast, autologous CD8+ T-lymphocytes or one allogeneic CD8+ T-cell clone failed to restore IgE synthesis. Moreover, autologous CD8+ T-cells suppressed IgE synthesis induced by autologous CD4+ T-cells in a dose-dependent fashion. IgE production could be induced in cultures containing as few as 20 B-cells. Collectively these data indicate that in addition to IL-4, a second signal derived from CD4+ T-cells is required to induce B-cells to switch to IgE producing cells. In a second set of experiments we showed that IFN-alpha blocked both IL-4-induced IgE synthesis by PBL of healthy donors and spontaneous IgE synthesis by PBL of allergic or atopic patients in a dose-dependent fashion. This inhibition occurred at the IgE messenger ribonucleic acid (mRNA) transcription level. The strongest inhibitory effects of interferon-alpha (IFN-alpha) were observed at the 2.2 kb productive mRNA transcript, whereas weaker inhibitory effects were observed on the 1.7 kb germline IgE mRNA transcript.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for PubMedID 1683237
-
HUMAN B-CELL CLONES CAN BE INDUCED TO PROLIFERATE AND TO SWITCH TO IGE AND IGG4 SYNTHESIS BY INTERLEUKIN-4 AND A SIGNAL PROVIDED BY ACTIVATED CD4+ T-CELL CLONES
JOURNAL OF EXPERIMENTAL MEDICINE
1991; 173 (3): 747-750
Abstract
In the present study, it is demonstrated that cloned surface IgM-positive human B cells can be induced to proliferate and to switch with high frequencies to IgG4 and IgE production after a contact-mediated signal provided by T cell clones and interleukin 4 (IL-4). This T cell signal is antigen nonspecific and is provided by activated CD4+ cells, whereas activated CD8+ or resting CD4+ T cell clones are ineffective. 15-35% of the B cell clones cultured with cloned CD4+ T cells and IL-4 produced antibodies; 35-45% of those wells in which antibodies were produced contained IgE and IgG4. In addition to B cell clones that produced IgG4 or IgE only, B cell clones producing multiple isotypes were observed. Simultaneous production of IgG4 and IgE, IgM, IgE, and IgM, or IgG4 and IgE was detected, suggesting that during clonal expansion switching might occur in successive steps from IgM to IgG4 and IgE. In addition, production of only IgM, IgG4, and IgE during clonal expansion indicates that this isotype switching is directed by the way a B cell is stimulated and that it is not a stochastic process.
View details for Web of Science ID A1991EZ66300026
View details for PubMedID 1997653
-
ISOLATION AND EXPRESSION OF HUMAN CYTOKINE SYNTHESIS INHIBITORY FACTOR CDNA CLONES - HOMOLOGY TO EPSTEIN-BARR-VIRUS OPEN READING FRAME BCRFI
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1991; 88 (4): 1172-1176
Abstract
We have demonstrated the existence of human cytokine synthesis inhibitory factor (CSIF) [interleukin 10 (IL-10)]. cDNA clones encoding human IL-10 (hIL-10) were isolated from a tetanus toxin-specific human T-cell clone. Like mouse IL-10, hIL-10 exhibits strong DNA and amino acid sequence homology to an open reading frame in the Epstein-Barr virus, BCRFI. hIL-10 and the BCRFI product inhibit cytokine synthesis by activated human peripheral blood mononuclear cells and by a mouse Th1 clone. Both hIL-10 and mouse IL-10 sustain the viability of a mouse mast cell line in culture, but BCRFI lacks comparable activity in this assay, suggesting that BCRFI may have conserved only a subset of hIL-10 activities.
View details for Web of Science ID A1991EY61700019
View details for PubMedID 1847510
-
IMMUNOLOGICAL EVALUATION OF HEMATOPOIETIC CHIMERIC RHESUS-MONKEYS
13TH INTERNATIONAL CONGRESS OF THE TRANSPLANTATION SOC
ELSEVIER SCIENCE INC. 1991: 841–43
View details for Web of Science ID A1991EV39000338
View details for PubMedID 1671308
-
In utero transplantation of hemopoietic stem cells in humans.
Transplantation proceedings
1991; 23 (1): 1706-1708
View details for PubMedID 1671181
-
INUTERO TRANSPLANTATION OF HEMATOPOIETIC STEM-CELLS IN HUMANS
TRANSPLANTATION PROCEEDINGS
1991; 23 (1): 1706-1708
View details for Web of Science ID A1991EV39100323
-
DO HUMAN TH1 AND TH2 CD4+ CLONES EXIST
RESEARCH IN IMMUNOLOGY
1991; 142 (1): 59-63
View details for Web of Science ID A1991FE79600013
View details for PubMedID 1829261
-
A SCID PATIENT RECONSTITUTED WITH HLA-INCOMPATIBLE FETAL STEM-CELLS AS A MODEL FOR STUDYING TRANSPLANTATION TOLERANCE
6TH ANNUAL SYMP ON MOLECULAR BIOLOGY OF HEMOPOIESIS - FRONTIERS IN BONE MARROW TRANSPLANTATION : FETAL HEMATOPOIESIS
SPRINGER VERLAG. 1991: 391–402
Abstract
We studied a severe combined immunodeficiency (SCID) patient who received transplantations with completely HLA-mismatched fetal liver and thymus from two different donors. The patient is now 14 years old, healthy and shows normal immunoresponses to recall antigens. His T cells are of donor origin, whereas the monocytes, B cells, and natural killer (NK) cells are of the recipient. The successful immunological reconstitution raised questions as to how T and B cells could collaborate across an HLA barrier and how tolerance was achieved. We have shown that tetanus toxin-specific T cell clones isolated from this patient recognized this antigen in the context of host and not of donor HLA-DR, indicating that those cells were educated in the host environment, presumably the thymus. Despite this, an unexpectedly high frequency of host-reactive clones was found that could recognize MHC antigens of the host. It was particularly striking that CD8+ CTL clones were obtained that recognized class I MHC antigens on the host cells. Nevertheless, the patient did not show any sign of acute or chronic graft-versus-host disease (GVHD). These data indicated that no or only incomplete clonal deletion had taken place in this patient and suggest the presence of a peripheral suppressor mechanism. Thus far, we have no indication for the existence of suppressor T cells. Inasmuch as it was found that host-reactive T cells fail to produce IL-4, which is exceptional for CD4+ T cells, we are exploring the possibility that abnormal cytokine production patterns of host-reactive T cells are associated with suppression of these cells in vivo.
View details for Web of Science ID A1991FN56900020
View details for PubMedID 1680508
-
NEW DEVELOPMENTS IN STEM-CELL TRANSPLANTATION WITH SPECIAL REFERENCE TO THE 1ST INUTERO TRANSPLANTS IN HUMANS
INTERNATIONAL COURSE ON BONE MARROW TRANSPLANTATION IN CHILDREN
STOCKTON PRESS. 1991: 92–97
Abstract
Based on the experience acquired in post-natal liver transplantation since 1974, we recently initiated pre-natal, in utero stem cell transplantation from the human fetal liver. The first two fetuses that we treated had immunodeficiencies, the third one had thalassemia major. Donors and recipients were not matched. The fetal cells were infused in the umbilical vein of the first two patients and injected intraperitoneally into the third one, under ultrasonic visualization. The first patient, born in 1988, has both engraftment of donor cells and reconstitution of cell-mediated immunity. This child, who had bare lymphocyte syndrome, has no clinical manifestation of the disease and he lives normally at home. The second child, born in 1989, has not yet developed a significant reconstitution of immunity although donor cell engraftment has been proven (Y chromosome in this female patient). The third patient has also evidence of donor cell take (Y chromosome in a female patient) but the effect on thalassemia has not yet been fully analyzed (donor hemoglobin present in small quantity). In all 3 cases, no side-effect of any kind developed in the mother nor in the fetus. Several advantages appear to be associated with in utero FLT: increased probability of graft take, ideal isolation of patient (in the uterus), optimal environment for fetal cell development (in the fetal host).
View details for Web of Science ID A1991FR13700023
View details for PubMedID 1855099
-
IL-4 INDUCED IGE SYNTHESIS AND IL-4 PRODUCTION IN NEWBORNS
RIVISTA ITALIANA DI PEDIATRIA-ITALIAN JOURNAL OF PEDIATRICS
1990; 16 (6): 683-688
View details for Web of Science ID A1990EX14300011
-
CORD BLOOD B-CELLS ARE MATURE IN THEIR CAPACITY TO SWITCH TO IGE-PRODUCING CELLS IN RESPONSE TO INTERLEUKIN-4 INVITRO
CLINICAL AND EXPERIMENTAL IMMUNOLOGY
1990; 82 (1): 114-119
Abstract
Neonatal B cells have been considered immature because of their impaired capacity to produce immunoglobulins in response to polyclonal activators in vitro. Here we demonstrate that cord blood mononuclear cells (MNC) produce normal levels of IgE in vitro when cultured in the presence of interleukin-4 (IL-4), indicating that the B cells are mature in their capacity to switch to IgE-producing cells. However, in contrast to adult peripheral blood T cells, cord blood T cells failed to produce detectable levels of IL-4 upon activation by phytohaemagglutinin (PHA) concanavalin A (Con A) or combinations of PHA and the phorbol ester TPA. Interferon-gamma (IFN-gamma) production by cord blood T cells following activation by Con A or PHA was also strongly reduced. However, high levels of IFN-gamma, significantly higher than those produced by adult T cells, were synthesized in response to combinations of PHA and TPA, indicating that IFN-gamma production by cord blood T cells is not intrinsically defective. In contrast, cord blood T cells produced levels of IL-2 that were significantly higher than those obtained by adult T cells tested in parallel. Collectively, our data indicate that the minimal levels of IgE production measured in cord blood (less than 1 U/ml) are not due to immaturity of the cord blood B cells, but may be associated with the failure of cord blood T cells to produce detectable levels of IL-4, which has been shown to be responsible for induction of IgE synthesis both in vitro and in vivo.
View details for Web of Science ID A1990EA58800021
View details for PubMedID 2119917
-
THE CAPACITY OF INTERLEUKIN-4 TO INDUCE INVITRO IGE SYNTHESIS BY B-CELLS OF PATIENTS WITH COMMON VARIABLE IMMUNODEFICIENCY
CLINICAL AND EXPERIMENTAL IMMUNOLOGY
1990; 82 (1): 120-127
Abstract
Interleukin-4 (IL-4) has been shown to induce IgE synthesis by peripheral blood mononuclear cells (PBMC) of normal donors in vitro. However, induction of PBMC of patients with common variable immunodeficiency (CVI) with IL-4 resulted in IgE production in only two out of eight cases tested. PBMC of the first patient that produced IgE in response to IL-4 also secreted normal levels of IL-4 upon activation. PBMC of the second patient secreted very low levels of IL-4 in vitro which may account for the very low serum IgE levels in this patient. Of the other six patients who had very low serum IgE levels and whose PBMC failed to produce IgE in response to IL-4 in vitro, five did not secrete IL-4 upon in vitro activation. The capacity of the T cells to produce IL-4 was intact in the sixth patient. Collectively our data indicate that PBMC of the majority of patients with CVI are defective since they failed to respond appropriately to IL-4 and they failed to produce IL-4, contributing to the view that CVI is a heterogeneous disorder in which a variety of T and B cell defects occur.
View details for Web of Science ID A1990EA58800022
View details for PubMedID 2119918
-
PRESENCE OF HOST-REACTIVE AND MHC-RESTRICTED T-CELLS IN A TRANSPLANTED SEVERE COMBINED IMMUNODEFICIENT (SCID) PATIENT SUGGEST POSITIVE SELECTION AND ABSENCE OF CLONAL DELETION
IMMUNOLOGICAL REVIEWS
1990; 116: 101-116
View details for Web of Science ID A1990EA41100006
View details for PubMedID 2227991
-
HOST-REACTIVE CD4+ AND CD8+ T-CELL CLONES ISOLATED FROM A HUMAN CHIMERA PRODUCE IL-5, IL-2, IFN-GAMMA- AND GRANULOCYTE MACROPHAGE-COLONY-STIMULATING FACTOR BUT NOT IL-4
JOURNAL OF IMMUNOLOGY
1990; 144 (3): 902-908
Abstract
In the present study, we investigated the lymphokine production patterns in a series of CD4+ and CD8+ host-reactive T cell clones isolated from PBL of a SCID patient, who was immunologically reconstituted by two allogeneic fetal liver and thymus transplantations 13 years ago. We demonstrate that these donor-derived T cell clones, specifically reacting with the MHC Ag expressed on the recipient cells, do not produce IL-4 and do not express IL-4 mRNA upon Ag or polyclonal stimulations. In contrast, CD4+ tetanus toxin-specific T cell clones isolated from the same patient and having the same HLA phenotype produced normal amounts of IL-4 upon activation. These data suggest that the failure to produce IL-4 is a specific characteristic of these host-reactive clones and is not due to a genetic defect of the transplanted cells. Furthermore, different modes of activation resulted in simultaneous production of IL-5, IL-2, IFN-gamma, granulocyte/macrophage-CSF, and transcription of the TNF-beta gene by the host-reactive clones, indicating that the lack of IL-4 production is not related to the mode of activation. The finding that some of these clones produce significant levels of IL-5 but no IL-4 indicates that the IL-4 and IL-5 genes are not always coexpressed in activated human T cells.
View details for Web of Science ID A1990CL02700019
View details for PubMedID 1967279
-
PERIPHERAL-BLOOD LYMPHOCYTES OF PATIENTS WITH COMMON VARIABLE IMMUNODEFICIENCY (CVI) PRODUCE REDUCED LEVELS OF INTERLEUKIN-4, INTERLEUKIN-2 AND INTERFERON-GAMMA, BUT PROLIFERATE NORMALLY UPON ACTIVATION BY MITOGENS
CLINICAL AND EXPERIMENTAL IMMUNOLOGY
1989; 78 (3): 334-340
Abstract
Peripheral blood lymphocytes (PBL) of 11 patients with CVI produced reduced levels of interleukin-4 (IL-4) upon activation by mitogens as compared with those secreted by PBL of healthy donors. The interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production by PBL of a series of 15 patients with CVI was also reduced. Decreased levels of IL-4 or IL-2 and IFN-gamma production were not only observed after activation by phytohaemagglutinin (PHA) at concentrations of 10 and 1 micrograms/ml, but also after activation by concanavalin A (Con A, 10 micrograms/ml). Longitudinal studies indicated that this defective lymphokine production was consistent upon testing periods up to 5 months. No correlation between reduced IL-4, IL-2 or IFN-gamma production was observed. PBL of patients that produced reduced levels of one lymphokine generally secreted normal levels of the other two lymphokines. Despite the reduced synthesis of the T cell growth factors IL-2 and IL-4, the proliferative responses of the PBL of the patients were in the normal range, which is compatible with the finding that IL-2 and IL-4 have synergistic effects on lymphocyte proliferation, particularly when one of these lymphokines is present at suboptimal concentrations. Since IL-2, IL-4 and IFN-gamma can act as B cell growth and differentiation factors, our data suggest that the reduced synthesis of these lymphokines may contribute to the deficient immunoglobulin production in patients with CVI.
View details for Web of Science ID A1989CD55300004
View details for PubMedID 2515013
-
INTERLEUKIN-4 SUPPRESSES IMMUNOGLOBULIN PRODUCTION BY PERIPHERAL-BLOOD LYMPHOCYTES OF PATIENTS WITH COMMON VARIABLE IMMUNODEFICIENCY (CVI) INDUCED BY SUPERNATANTS OF T-CELL CLONES
CLINICAL AND EXPERIMENTAL IMMUNOLOGY
1989; 78 (3): 341-347
Abstract
Supernatants of both CD4+ and CD8+ alloreactive T cell clones induced IgM, IgG and IgA synthesis by peripheral blood lymphocytes (PBL) of healthy donors in vitro. These supernatants were also tested on their capacity to induce immunoglobulin production by PBL of four patients with CVI and one patient with CVI and thymoma. A low degree of IgM, IgG and IgA production was induced in one patient with CVI. In the patient with CVI and thymoma, induction of IgG and IgA synthesis was in the normal range, whereas IgM production was reduced. In the three other patients only a low production of IgM was induced. Interestingly, pre-incubation of the PBL for 24 h with interleukin-4 (IL-4) suppressed immunoglobulin production both by PBL of the patients with CVI and healthy donors. The strongest inhibitory effects were observed on IgA synthesis. These data indicate that B cells of three patients with CVI can not be induced to switch to IgG or IgA producing cells in vitro. In contrast, B cells of the patient with CVI and thymoma were able to respond to the relevant B cell growth and differentiation factors present in the T cell clone supernatants, suggesting that the T cells of this patient may fail to produce these factors. However, the proliferative responses of the T cells to phytohaemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM), were normal in all five patients tested. In addition, the interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production by PBL of the five patients was also in the normal range. Although only a small number of patients was tested, these results support the view that defects in both regulatory T cell functions and/or intrinsic B cell defects may contribute to the pathogenesis of CVI.
View details for Web of Science ID A1989CD55300005
View details for PubMedID 2575470
-
INUTERO TRANSPLANTATION OF STEM-CELLS IN BARE LYMPHOCYTE SYNDROME
LANCET
1989; 1 (8651): 1382-1382
View details for Web of Science ID A1989AB23400020
View details for PubMedID 2567387
-
[Phenotype expression and production of IL-2 by tonsillar and blood lymphocytes in patients with tonsil pathology].
Acta otorhinolaryngologica Italica
1989; 9 (2): 149-159
Abstract
In recent years several studies have attempted to investigate immunological responses in different tonsillar pathologies, including recurrent tonsillitis and focal infections. The present study was performed on ten patients who had undergone tonsillectomy for a) simple hypertrophy, b) recurrent tonsillitis and c) recurrent tonsillitis with focal manifestations. The blood (PBMNC) and tonsillar (TMNC) lymphocytes were tested separately. A subsequent investigation was performed on the PBMNC six months after surgery. The phenotypical aspects of the different subpopulations were studied using fluorescent antiserums and monoclonal antibodies. A second field of investigation concerned the in vitro blastogenesis, which was measured under spontaneous and PHA-P induced conditions. Finally, the IL-2 production was evaluated using the induced-growth capacity of an IL-2 dependent clone of a T murine cell line. The most interesting findings are presented. The phenotypical studies confirmed some peculiar aspects of the representation of tonsillar subsets. According to the hyperactivation ratio, the proliferation data proved to be higher in the tonsil than in the blood. A greater number of positive HLA-DR and IL-2 receptor cells (both antibodies being activated cell markers) was seen in the tonsil than in the blood and this, as well as the sporadic presence of spontaneous blastogenesis, suggests the possibility of an in vivo pre-activated condition. As far as the secretion of IL-2 is concerned, when compared to the peripheral blood ratio, greater production was found in the tonsil. Finally, a different production kinetic appears to be a constant result of the present study. The performed tests were unable to demonstrate any particular differences among the three different groups of pathologies.
View details for PubMedID 2788350
-
UNMATCHED STEM-CELL TRANSPLANTATION AS A POSSIBLE ALTERNATIVE TO BONE-MARROW TRANSPLANTATION
TRANSPLANTATION PROCEEDINGS
1989; 21 (1): 3112-3113
View details for Web of Science ID A1989U152500260
View details for PubMedID 2565068
-
HYPOGAMMAGLOBULINEMIA WITH HYPER-IGM - CLINICAL AND IMMUNOLOGICAL FEATURES IN 2 PATIENTS
RIVISTA ITALIANA DI PEDIATRIA-ITALIAN JOURNAL OF PEDIATRICS
1989; 15 (1): 97-100
View details for Web of Science ID A1989T346500023
-
ANTIGEN RECOGNITION BY MHC-INCOMPATIBLE CELLS OF A HUMAN MISMATCHED CHIMERA
JOURNAL OF EXPERIMENTAL MEDICINE
1988; 168 (6): 2139-2152
Abstract
Tetanus toxin (TT)-specific T cell clones of donor origin were obtained from a patient with severe combined immunodeficiency (SCID) successfully reconstituted by transplantation of allogeneic fetal liver and thymus cells from two different donors performed 10 yr ago. A series of these clones recognized TT in the context of "allo" class II HLA determinants expressed by recipient APC. The restriction element of two T cell clones with the HLA phenotype of the first donor (HLA-DR1,8) and one T cell clone with the HLA phenotype of the second transplant (HLA-DR3,9) was HLA-DR4 of the recipient, whereas other T cell clones derived from the second transplant recognized TT in the context of HLA-DR5 of the recipient's APC. These latter T cell clones were not able to proliferate in response to TT when autologous APC were used. These data demonstrate that recipient and donor cells having different HLA phenotypes could cooperate across the allogeneic barrier and that MHC restriction of antigen (Ag) recognition is independent from the MHC genotype of the T cells but is influenced by the environment in which the T cells mature. We also isolated T cell clones that were able to recognize processed TT presented by all allogeneic EBV cell lines tested, indicating that the Ag specificity of these clones was not restricted by a particular class II MHC molecule. The Ag-specific proliferative response of one of these clones could be blocked by anti-class II MHC mAbs. These results demonstrate that in addition to Ag recognition in the context of specific class II MHC Ags, other types of Ag-specific responses may occur in this human chimera. It is not clear whether this "allo" plus Ag recognition is the result of education of transplanted fetal cells in the host thymus. Taking into consideration our previous findings indicating that alloreactive T cell clones specific for the recipient cells could be isolated in vitro from the PBL of the same patient, our data suggest that the mechanism for deletion of self-reactive clones and the generation of MHC-restricted responses are different.
View details for Web of Science ID A1988R362000015
View details for PubMedID 2462006
-
GENOMIC ORGANIZATION OF THE HUMAN T-CELL ANTIGEN-RECEPTOR ALPHA,SIGMA LOCUS
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1988; 85 (21): 8166-8170
Abstract
Two clusters of overlapping cosmid clones comprising about 100 kilobases (kb) at the human T-cell antigen-receptor alpha/delta locus were isolated from a genomic library. The structure of the germ-line V delta 1 variable gene segment was determined. V delta 1 is located 8.5 kb downstream of the V alpha 13.1 gene segment, and both V segments are arranged in the same transcriptional orientation. The V alpha 17.1 segment is located between V delta 1 and the D delta, J delta, C delta region (containing the diversity, joining, and constant gene segments). Thus, V delta and V alpha segments are interspersed along the chromosome. The germ-line organization of the D delta 2, J delta 1, and J delta 2 segments was determined. Linkage of C delta to the J alpha region was established by identification of J alpha segments within 20 kb downstream of C delta. The organization of the locus was also analyzed by field-inversion gel electrophoresis. The unrearranged V delta 1 and D delta, J delta, C delta regions are quite distant from each other, apparently separated by a minimum of 175-180 kb.
View details for Web of Science ID A1988Q834100071
View details for PubMedID 3186718
-
INTERLEUKIN-2 PRODUCTION AND INTERLEUKIN-2 RECEPTOR EXPRESSION IN CHILDREN WITH NEWLY DIAGNOSED DIABETES
CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY
1988; 49 (1): 53-62
Abstract
In the present study, we investigated the interleukin-2 (IL-2) production and the proliferative responses by peripheral blood mononuclear cells (PBMNC) of 23 children suffering from insulin-dependent diabetes mellitus (IDDM). In addition, the presence of circulating activated T lymphocytes expressing the interleukin-2 receptor (IL-2 R) and HLA-DR antigens was evaluated. The patients were tested at hospital admittance, before starting insulin treatment. Decreased IL-2 production by phytohemagglutinin (PHA)-activated PBMNC of IDDM patients was observed when compared to normal donors. In contrast, the proliferative responses of PBMNC to PHA and Con A were in the normal range. The expression of IL-2 R on patient's lymphocytes was not different from that observed in normal donors, whereas the relative and absolute number of HLA-DR+ T cells was increased. These results confirm the presence in IDDM patients of an imbalanced cellular immune response and demonstrate that the IL-2 deficiency is already present at the diagnosis and is not correlated with insulin administration.
View details for Web of Science ID A1988Q122100006
View details for PubMedID 3136960
-
AUTOREACTIVE T-CELL CLONES SPECIFIC FOR CLASS-I AND CLASS-II HLA ANTIGENS ISOLATED FROM A HUMAN CHIMERA
JOURNAL OF EXPERIMENTAL MEDICINE
1988; 167 (5): 1523-1534
Abstract
T cell clones of donor origin that specifically react with recipient cells were obtained from a SCID patient successfully reconstituted by allogeneic fetal liver and thymus transplantation performed 10 yr ago. The majority of these clones displayed both cytotoxic and proliferative responses towards PBL and an EBV-transformed B cell line derived from the patient. In addition, these T cell clones had proliferative and cytotoxic responses towards the parental PBL, EBV cell lines, and PHA blasts. Blocking studies with anti-class I and anti-class II HLA mAbs indicated that the activity of the CD4+ T cell clones was specifically directed against class II HLA antigens of the recipient. On the other hand, the cytotoxic and proliferative responses of the CD8+ T cell clones were specific for class I HLA antigens which are ubiquitously expressed on the recipient cells. Thus, the establishment of transplantation tolerance observed in this stable human chimera is not due to the elimination of host-reactive T cells from the repertoire and suggests the presence of a peripheral autoregulatory suppressor mechanism.
View details for Web of Science ID A1988N306900001
View details for PubMedID 3284961
-
FETAL TISSUE-TRANSPLANTATION, BONE-MARROW TRANSPLANTATION AND PROSPECTIVE GENE-THERAPY IN SEVERE IMMUNODEFICIENCIES AND ENZYME DEFICIENCIES
THYMUS
1987; 10 (1-2): 75-87
Abstract
The successful development of fetal tissue transplantation has resulted in therapeutical solutions for patients with a variety of diseases. Fetal liver transplants as well as bone marrow transplants, can completely cure patients with severe combined immunodeficiency disease. These transplants can also be applied to treat other types of immunodeficiency, hemopathies, and inborn errors of metabolism, in association with immunosuppressive therapy. Despite complete HLA incompatibility between transplanted stem cells and host cells, functional activities of donor-derived T-lymphocytes are not restricted. In severe forms of Di George syndrome, immunological reconstitution can be obtained by fetal thymus transplantation. It is expected that, in the near future, pure stem cell transplants and gene transplants will develop and will provide remarkable solutions for the therapy of a large number of diseases.
View details for Web of Science ID A1987K642900008
View details for PubMedID 3324405
-
COOPERATION BETWEEN MAJOR HISTOCOMPATIBILITY COMPLEX MISMATCHED MONONUCLEAR-CELLS FROM A HUMAN CHIMERA IN THE PRODUCTION OF ANTIGEN-SPECIFIC ANTIBODY
JOURNAL OF CLINICAL INVESTIGATION
1986; 77 (3): 673-680
Abstract
Fetal liver and thymus transplantation can be successfully employed for the treatment of severe combined immunodeficiency disease. In virtually all cases, donor and recipient cells are HLA mismatched. In a patient suffering from a severe combined immunodeficiency disease, full immunological reconstitution was obtained after fetal liver and thymus transplantation. HLA typing revealed that the patient's T cells were of donor origin, while the B cells and monocytes were of host origin. Despite this complete HLA mismatch, the patient was found to mount a subnormal to normal antibody response in vivo. This finding is in contrast with the concept that antigen recognition by T cells is major histocompatibility complex (MHC) restricted. To define the mechanism responsible for this in vivo antibody response, antibody production by peripheral blood mononuclear cells from the patient was tested in vitro after in vivo booster. The in vitro anti-tetanus toxoid antibody production was similar to that of the control group. In addition, specific proliferative responses to tetanus toxoid were obtained. Immunoglobulin allotype determination showed that antibodies were synthetized by host B cells. The results of the present study indicate that transplanted T lymphocytes and recipient cells cooperate despite complete HLA mismatch.
View details for Web of Science ID A1986A542900003
View details for PubMedID 3456356
-
STUDIES OF EBV-LYMPHOID CELL-INTERACTIONS IN 2 PATIENTS WITH THE X-LINKED LYMPHOPROLIFERATIVE SYNDROME - NORMAL EBV-SPECIFIC HLA-RESTRICTED CYTOTOXICITY
CLINICAL AND EXPERIMENTAL IMMUNOLOGY
1986; 63 (2): 280-289
Abstract
Two X-linked lymphoproliferative syndrome (XLP) patients with the hypogammaglobulinemia phenotype were investigated at a time remote from their primary infection with the Epstein-Barr virus (EBV). The lymphoblastoid cell lines derived from these patients expressed the phenotypic markers characteristic of normal mature B lymphocytes and produced normal levels of immunoglobulins (Ig). These observations imply that at least some of their B cells are phenotypically normal. The natural killer (NK) activity of the two patients was low. In one patient, activated lymphocyte killer (ALK) activity was inefficient. These two XLP patients expressed a normal EBV-specific, HLA-restricted cytotoxic activity. It thus appears, from the present findings and those in cases published previously (6/11 patients expressing normal EBV-specific cytotoxic activity), that the notion of poor specific T cell memory for EBV may not be as pivotal ass suggested or, alternatively, that this defect may not be common in hypogammaglobulinemic survivors.
View details for Web of Science ID A1986AYN6400003
View details for PubMedID 3009061
-
Fetal liver transplantation in immunodeficiencies and inborn errors of metabolism.
Progress in clinical and biological research
1985; 193: 299-313
View details for PubMedID 3937157
-
INTRAVENOUS IGG TREATMENT IN HYPOGAMMAGLOBULINEMIC PATIENTS
RIVISTA ITALIANA DI PEDIATRIA-ITALIAN JOURNAL OF PEDIATRICS
1984; 10 (4): 373-378
View details for Web of Science ID A1984TH71600008