My professional goal as a pediatrician specializing in immunology, has been to challenge the limits of "inexplicable" and "untreatable" diseases, and apply current scientific knowledge to understand the mechanisms of impaired cellular immune function underlying the clinical manifestations in order to develop curative treatments.

Following the completion of my pediatrics residency, I received training in molecular and cellular immunology in France (UNICET, Lyon) and the United States (DNAX Research Institute of Molecular and Cellular Biology, Palo Alto) under scientists, who critically influenced my scientific development. There, I was first exposed to the importance of integrating in depth laboratory research with clinical observations to develop a translational research approach to science. I then worked for fifteen years at the San Raffaele Scientific Institute in the Telethon Institute for Gene Therapy (HSR-TIGET), where I focused on dissecting the genetic and immunological basis of primary immunodeficiencies with autoimmune manifestations that might be treated by gene therapy.

Honors & Awards

  • Associate Professor in Pediatrics, Italian Ministry of University (01/2014)

Boards, Advisory Committees, Professional Organizations

  • Member, European Society of Immunodeficiency (ESID) (2014 - Present)
  • Member, Federal of Clinical Immunology Societies (FOCIS and CIS) (2014 - Present)
  • Associate Editor, Frontiers in Primary Immunodeficiencies (2014 - Present)
  • Reviewer, J Autoimmunity (2014 - Present)
  • Reviewer, European Journal of Immunology (2014 - Present)
  • Reviewer, Transplantation (2014 - Present)
  • Grants revisions, European Research Council (2014 - Present)
  • Grants revisions, INSERM (2014 - Present)
  • Grants revision, LSBR Foundation (2014 - Present)
  • Grants revision, Wellcome Trust (2014 - Present)
  • Grants revision, ANR-France (2014 - Present)
  • Teaching activities ad Hoc lessons, Vita-Salute University Medical School and Biotechnology, Milan (2014 - Present)
  • Teaching activities ad hoc lessons, Tor Vergata University, Rome (2014 - Present)
  • Teaching activities ad hoc lessons, Undergraduate Course at Stanford University, Stanford, Palo Alto, CA (2014 - Present)

Professional Education

  • Fellowship, University of Turin, Italy, General Pediatrics Immunology (1991)
  • MD, University of Turin, Italy, Medicine (1987)


  • Rosa Bacchetta. "United States Patent WO2007/131575 Tr1 dendritic cells, method to generate regulatory type 1 T (Tr1) cells and uses thereof", Rosa Bacchetta, Apr 1, 2007
  • Rosa Bacchetta. "United States Patent 6884410 Methods for modulating antigen-specific immune responses", Rosa Bacchetta, Apr 26, 2005
  • Rosa Bacchetta. "United States Patent 6277635 Use of Interleukin-10 produce a population of suppressor cells", Rosa Bacchetta, Aug 21, 2001

Current Research and Scholarly Interests

In the coming years, I plan to further determine the genetic and immunological basis of diseases with autoimmunity or immune dysregulation in children. I believe that much can still be learned from the in depth mechanistic studies of pediatric autoimmune diseases. Genomic analysis of the patients' samples has become possible which may provide a rapid indication of altered target molecules. I plan to implement robust functional studies to define the consequences of these genetic abnormalities and bridge them to the patient's clinical phenotype.

Understanding functional consequences of gene mutations in single case/family first and then validating the molecular and cellular defects in other patients with similar phenotypes, will anticipate and complement cellular and gene therapy strategies.

For further information please visit the Bacchetta Lab website:

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.

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  • 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

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2023-24 Courses

Stanford Advisees

Graduate and Fellowship Programs

All Publications

  • Identification of unstable regulatory and autoreactive effector T cells that are expanded in patients with FOXP3 mutations. Science translational medicine Borna, Š., Lee, E., Nideffer, J., Ramachandran, A., Wang, B., Baker, J., Mavers, M., Lakshmanan, U., Narula, M., Garrett, A. K., Schulze, J., Olek, S., Marois, L., Gernez, Y., Bhatia, M., Chong, H. J., Walter, J., Kitcharoensakkul, M., Lang, A., Cooper, M. A., Bertaina, A., Roncarolo, M. G., Meffre, E., Bacchetta, R. 2023; 15 (727): eadg6822


    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

  • IPEX Syndrome from diagnosis to cure, learning along the way. The Journal of allergy and clinical immunology Bacchetta, R., Roncarolo, M. G. 2023


    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

  • FOXP3 deficiency, from the mechanisms of the disease to curative strategies. Immunological reviews Borna, S., Meffre, E., Bacchetta, R. 2023


    FOXP3 gene is a key transcription factor driving immune tolerance and its deficiency causes immune dysregulation, polyendocrinopathy, enteropathy X-linked syndrome (IPEX), a prototypic primary immune regulatory disorder (PIRD) with defective regulatory T (Treg) cells. Although life-threatening, the increased awareness and early diagnosis have contributed to improved control of the disease. IPEX currently comprises a broad spectrum of clinical autoimmune manifestations from severe early onset organ involvement to moderate, recurrent manifestations. This review focuses on the mechanistic advancements that, since the IPEX discovery in early 2000, have informed the role of the human FOXP3+ Treg cells in controlling peripheral tolerance and shaping the overall immune landscape of IPEX patients and carrier mothers, contributing to defining new treatments.

    View details for DOI 10.1111/imr.13289

    View details for PubMedID 37994657

  • Epigenetic and Immunological Indicators of IPEX Disease in subjects with FOXP3 gene mutation. The Journal of allergy and clinical immunology Narula, M., Lakshmanan, U., Borna, S., Schulze, J. J., Holmes, T. H., Harre, N., Kirkey, M., Ramachandran, A., Tagi, V. M., Barzaghi, F., Grunebaum, E., Upton, J. E., Hong-Diep Kim, V., Wysocki, C., Dimitriades, V. R., Weinberg, K., Weinacht, K. G., Gernez, Y., Sathi, B. K., Schelotto, M., Johnson, M., Olek, S., Sachsenmaier, C., Roncarolo, M. G., Bacchetta, R. 2022


    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

  • Towards gene therapy for IPEX syndrome. European journal of immunology Borna, S., Lee, E., Sato, Y., Bacchetta, R. 2022


    Immune dysregulation polyendocrinopathy enteropathy X linked (IPEX) syndrome is an uncurable disease of the immune system, with immune dysregulation that is caused by mutations in FOXP3. Current treatment options, such as pharmacological immune suppression and allogeneic hematopoietic stem cell transplantation, have been beneficial but present limitations, and their life-long consequences are ill defined. Other similar blood monogenic diseases have been successfully treated using gene transfer in autologous patient cells, thus providing an effective and less invasive therapeutic. Development of gene therapy for patients with IPEX is particularly challenging because successful strategies must restore the complex expression profile of the transcription factor FOXP3, ensuring it is tightly regulated, and its cell subset-specific roles are maintained. This review summarizes current efforts toward achieving gene therapy to treat the immune dysregulation in IPEX patients. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1002/eji.202149210

    View details for PubMedID 35355253

  • Thymic origins of autoimmunity-lessons from inborn errors of immunity. Seminars in immunopathology Bacchetta, R., Weinberg, K. 2021


    During their intrathymic development, nascent T cells are empowered to protect against pathogens and to be operative for a life-long acceptance of self. While autoreactive effector T (Teff) cell progenitors are eliminated by clonal deletion, the intrathymic mechanisms by which thymic regulatory T cell (tTreg) progenitors maintain specificity for self-antigens but escape deletion to exert their regulatory functions are less well understood. Both tTreg and Teff development and selection result from finely coordinated interactions between their clonotypic T cell receptors (TCR) and peptide/MHC complexes expressed by antigen-presenting cells, such as thymic epithelial cells and thymic dendritic cells. tTreg function is dependent on expression of the FOXP3 transcription factor, and induction of FOXP3 gene expression by tTreg occurs during their thymic development, particularly within the thymic medulla. While initial expression of FOXP3 is downstream of TCR activation, constitutive expression is fixed by interactions with various transcription factors that are regulated by other extracellular signals like TCR and cytokines, leading to epigenetic modification of the FOXP3 gene. Most of the understanding of the molecular events underlying tTreg generation is based on studies of murine models, whereas gaining similar insight in the human system has been very challenging. In this review, we will elucidate how inborn errors of immunity illuminate the critical non-redundant roles of certain molecules during tTreg development, shedding light on how their abnormal development and function cause well-defined diseases that manifest with autoimmunity alone or are associated with states of immune deficiency and autoinflammation.

    View details for DOI 10.1007/s00281-020-00835-8

    View details for PubMedID 33532929

  • CRISPR-based gene editing enables FOXP3 gene repair in IPEX patient cells SCIENCE ADVANCES Goodwin, M., Lee, E., Lakshmanan, U., Shipp, S., Froessl, L., Barzaghi, F., Passerini, L., Narula, M., Sheikali, A., Lee, C. M., Bao, G., Bauer, C. S., Miller, H. K., Garcia-Lloret, M., Butte, M. J., Bertaina, A., Shah, A., Pavel-Dinu, M., Hendel, A., Porteus, M., Roncarolo, M. G., Bacchetta, R. 2020; 6 (19)
  • Human inborn errors of immunity: An expanding universe. Science immunology Notarangelo, L. D., Bacchetta, R. n., Casanova, J. L., Su, H. C. 2020; 5 (49)


    Molecular, cellular, and clinical studies of human inborn errors of immunity have revolutionized our understanding of their pathogenesis, considerably broadened their spectrum of immunological and clinical phenotypes, and enabled successful targeted therapeutic interventions. These studies have also been of great scientific merit, challenging a number of immunological notions initially established in inbred mice while revealing previously unrecognized mechanisms of host defense by leukocytes and other cells and of both innate and adaptive tolerance to self.

    View details for DOI 10.1126/sciimmunol.abb1662

    View details for PubMedID 32651211

  • Hematopoietic Cell Transplantation in Patients With Primary Immune Regulatory Disorders (PIRD): A Primary Immune Deficiency Treatment Consortium (PIDTC) Survey. Frontiers in immunology Chan, A. Y., Leiding, J. W., Liu, X., Logan, B. R., Burroughs, L. M., Allenspach, E. J., Skoda-Smith, S., Uzel, G., Notarangelo, L. D., Slatter, M., Gennery, A. R., Smith, A. R., Pai, S., Jordan, M. B., Marsh, R. A., Cowan, M. J., Dvorak, C. C., Craddock, J. A., Prockop, S. E., Chandrakasan, S., Kapoor, N., Buckley, R. H., Parikh, S., Chellapandian, D., Oshrine, B. R., Bednarski, J. J., Cooper, M. A., Shenoy, S., Davila Saldana, B. J., Forbes, L. R., Martinez, C., Haddad, E., Shyr, D. C., Chen, K., Sullivan, K. E., Heimall, J., Wright, N., Bhatia, M., Cuvelier, G. D., Goldman, F. D., Meyts, I., Miller, H. K., Seidel, M. G., Vander Lugt, M. T., Bacchetta, R., Weinacht, K. G., Andolina, J. R., Caywood, E., Chong, H., de la Morena, M. T., Aquino, V. M., Shereck, E., Walter, J. E., Dorsey, M. J., Seroogy, C. M., Griffith, L. M., Kohn, D. B., Puck, J. M., Pulsipher, M. A., Torgerson, T. R. 2020; 11: 239


    Primary Immune Regulatory Disorders (PIRD) are an expanding group of diseases caused by gene defects in several different immune pathways, such as regulatory T cell function. Patients with PIRD develop clinical manifestations associated with diminished and exaggerated immune responses. Management of these patients is complicated; oftentimes immunosuppressive therapies are insufficient, and patients may require hematopoietic cell transplant (HCT) for treatment. Analysis of HCT data in PIRD patients have previously focused on a single gene defect. This study surveyed transplanted patients with a phenotypic clinical picture consistent with PIRD treated in 33 Primary Immune Deficiency Treatment Consortium centers and European centers. Our data showed that PIRD patients often had immunodeficient and autoimmune features affecting multiple organ systems. Transplantation resulted in resolution of disease manifestations in more than half of the patients with an overall 5-years survival of 67%. This study, the first to encompass disorders across the PIRD spectrum, highlights the need for further research in PIRD management.

    View details for DOI 10.3389/fimmu.2020.00239

    View details for PubMedID 32153572

  • Human-engineered Treg-like cells suppress FOXP3-deficient T cells but preserve adaptive immune responses in vivo. Clinical & translational immunology Sato, Y. n., Passerini, L. n., Piening, B. D., Uyeda, M. J., Goodwin, M. n., Gregori, S. n., Snyder, M. P., Bertaina, A. n., Roncarolo, M. G., Bacchetta, R. n. 2020; 9 (11): e1214


    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

  • The autoimmune targets in IPEX are dominated by gut epithelial proteins JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY Eriksson, D., Bacchetta, R., Gunnarsson, H., Chan, A., Barzaghi, F., Ehl, S., Hallgren, A., van Gool, F., Sardh, F., Lundqvist, C., Laakso, S. M., Ronnblom, A., Ekwall, O., Makitie, O., Bensing, S., Husebye, E. S., Anderson, M., Kampe, O., Landegren, N. 2019; 144 (1): 327-+
  • Case Study: Mechanism for Increased Follicular Helper T Cell Development in Activated PI3K Delta Syndrome. Frontiers in immunology Thauland, T. J., Pellerin, L., Ohgami, R. S., Bacchetta, R., Butte, M. J. 2019; 10: 753


    Gain-of-function variants in p110δ, the catalytic subunit of phosphatidylinositol 3-kinase (PI3K) expressed in lymphocytes, cause activated PI3-kinase δ syndrome (APDS), a primary immunodeficiency that is characterized by recurrent infections, viremia, lymphadenopathy, and autoimmunity. The mechanism of autoimmunity in APDS has not been well-understood. Here, we show the profound skewing of peripheral CD4+ T cells to a T follicular helper (TFH) phenotype in a patient with APDS bearing a novel p110δ variant, Y524S. We also saw a diminishment of transient Foxp3 expression in activated T cells. Mechanistic studies revealed that both the new variant and a previously described, pathogenic variant (E81K) enhanced an interaction between intracellular Osteopontin and p85α. This interaction had been shown in mice to promote TFH differentiation. Our results demonstrate a new influence of PI3K on human T cell differentiation that is unrelated to its lipid-kinase activity and suggest that TFH should be monitored in APDS patients.

    View details for DOI 10.3389/fimmu.2019.00753

    View details for PubMedID 31031754

    View details for PubMedCentralID PMC6473200

  • Treatment with Rapamycin Can Restore Regulatory T Cell Function in IPEX Patients. The Journal of allergy and clinical immunology Passerini, L. n., Barzaghi, F. n., Curto, R. n., Sartirana, C. n., Barera, G. n., Tucci, F. n., Albarello, L. n., Mariani, A. n., AlbertoTestoni, P. n., Bazzigaluppi, E. n., Bosi, E. n., Lampasona, V. n., Neth, O. n., Zama, D. n., Hoenig, M. n., Schulz, A. n., Seidel, M. G., Rabbone, I. n., Olek, S. n., Roncarolo, M. G., Cicalese, M. P., Aiuti, A. n., Bacchetta, R. n. 2019


    Immune-dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome is a lethal disease caused by mutations in a transcription factor critical for the function of thymus-derived (t) regulatory T cells (Tregs), i.e., Forkhead-box-P3 (FOXP3), resulting in impaired Treg function and autoimmunity. At present, hematopoietic stem cell transplantation is the therapy of choice for IPEX patients. If not available, multiple immunosuppressive regimens have been used with poor disease-free survival at long-term follow-up. Rapamycin has been shown to suppress peripheral T cells while sparing Tregs expressing wild-type FOXP3, thereby proving beneficial in the clinical setting of immune-dysregulation. However, the mechanisms of immunosuppression selective to IPEX patients' Tregs are unclear.To determine the cellular and molecular basis of the clinical benefit observed under rapamycin treatment in six IPEX patients with different FOXP3 mutations.We tested phenotype and function of FOXP3-mutated Tregs from rapamycin-treated IPEX patients by flow cytometry and in vitro suppression assays, and the gene expression profile of rapamycin-conditioned Tregs by droplet-digital-PCR.Clinical and histological improvements in patients correlated with partially restored Treg function, independent of FOXP3 expression or Treg frequency. Expression of Tumor-Necrosis-Factor-Receptor-Superfamily-Member18 (TNFRSF18, GITR) and Epstein-Barr-virus-Induced3 (EBI3, an IL-35 subunit) in patients' Tregs increased during treatment as compared to that of Tregs from untreated healthy subjects. Furthermore inhibition of GITR and Ebi3 partially reverted in vitro suppression by in vivo rapamycin-conditioned Tregs.Rapamycin is able to impact on Treg suppressive function via a FOXP3-independent mechanism, thus sustaining the clinical improvement observed in IPEX patients under rapamycin treatment.

    View details for DOI 10.1016/j.jaci.2019.11.043

    View details for PubMedID 31874182

  • Tregopathies: Monogenic diseases resulting in regulatory T-cell deficiency. The Journal of allergy and clinical immunology Cepika, A., Sato, Y., Liu, J. M., Uyeda, M. J., Bacchetta, R., Roncarolo, M. G. 2018; 142 (6): 1679–95


    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

  • Epigenetic immune cell counting in human blood samples for immunodiagnostics SCIENCE TRANSLATIONAL MEDICINE Baron, U., Werner, J., Schildknecht, K., Schulze, J. J., Mulu, A., Liebert, U., Sack, U., Speckmann, C., Gossen, M., Wong, R. J., Stevenson, D. K., Babel, N., Schuermann, D., Baldinger, T., Bacchetta, R., Gruetzkau, A., Borte, S., Olek, S. 2018; 10 (452)


    Immune cell profiles provide valuable diagnostic information for hematologic and immunologic diseases. Although it is the most widely applied analytical approach, flow cytometry is limited to liquid blood. Moreover, either analysis must be performed with fresh samples or cell integrity needs to be guaranteed during storage and transport. We developed epigenetic real-time quantitative polymerase chain reaction (qPCR) assays for analysis of human leukocyte subpopulations. After method establishment, whole blood from 25 healthy donors and 97 HIV+ patients as well as dried spots from 250 healthy newborns and 24 newborns with primary immunodeficiencies were analyzed. Concordance between flow cytometric and epigenetic data for neutrophils and B, natural killer, CD3+ T, CD8+ T, CD4+ T, and FOXP3+ regulatory T cells was evaluated, demonstrating substantial equivalence between epigenetic qPCR analysis and flow cytometry. Epigenetic qPCR achieves both relative and absolute quantifications. Applied to dried blood spots, epigenetic immune cell quantification was shown to identify newborns suffering from various primary immunodeficiencies. Using epigenetic qPCR not only provides a precise means for immune cell counting in fresh-frozen blood but also extends applicability to dried blood spots. This method could expand the ability for screening immune defects and facilitates diagnostics of unobservantly collected samples, for example, in underdeveloped areas, where logistics are major barriers to screening.

    View details for PubMedID 30068569

  • Reprogramming human T cell function and specificity with non-viral genome targeting NATURE Roth, T. L., Puig-Saus, C., Yu, R., Shifrut, E., Carnevale, J., Li, P., Hiatt, J., Saco, J., Krystofinski, P., Li, H., Tobin, V., Nguyen, D. N., Lee, M. R., Putnam, A. L., Ferris, A. L., Chen, J. W., Schickel, J., Pellerin, L., Carmody, D., Alkorta-Aranburu, G., del Gaudio, D., Matsumoto, H., Morell, M., Mao, Y., Cho, M., Quadros, R. M., Gurumurthy, C. B., Smith, B., Haugwitz, M., Hughes, S. H., Weissman, J. S., Schumann, K., Esensten, J. H., May, A. P., Ashworth, A., Kupfer, G. M., Greeley, S. W., Bacchetta, R., Meffre, E., Roncarolo, M., Romberg, N., Herold, K. C., Ribas, A., Leonetti, M. D., Marson, A. 2018; 559 (7714): 405-+


    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

  • Neutralizing Anti-Cytokine Autoantibodies Against Interferon-alpha in Immunodysregulation Polyendocrinopathy Enteropathy X-Linked FRONTIERS IN IMMUNOLOGY Rosenberg, J. M., Maccari, M. E., Barzaghi, F., Allenspach, E. J., Pignata, C., Weber, G., Torgerson, T. R., Utz, P. J., Bacchetta, R. 2018; 9: 544


    Anti-cytokine autoantibodies (ACAAs) have been described in a growing number of primary immunodeficiencies with autoimmune features, including autoimmune polyendocrine syndrome type I (APS-1), a prototypical disease of defective T cell-mediated central tolerance. Whether defects in peripheral tolerance lead to similar ACAAs is unknown. Immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) is caused by mutations in FOXP3, a master regulator of T regulatory cells (Treg), and consequently results in defective T cell-mediated peripheral tolerance. Unique autoantibodies have previously been described in IPEX. To test the hypothesis that ACAAs are present in IPEX, we designed and fabricated antigen microarrays. We discovered elevated levels of IgG ACAAs against interferon-α (IFN-α) in a cohort of IPEX patients. Serum from IPEX patients blocked IFN-α signaling in vitro and blocking activity was tightly correlated with ACAA titer. To show that blocking activity was mediated by IgG and not other serum factors, we purified IgG and showed that blocking activity was contained entirely in the immunoglobulin fraction. We also screened for ACAAs against IFN-α in a second geographically distinct cohort. In these samples, ACAAs against IFN-α were elevated in a post hoc analysis. In summary, we report the discovery of ACAAs against IFN-α in IPEX, an experiment of nature demonstrating the important role of peripheral T cell tolerance.

    View details for PubMedID 29651287

  • Long-term follow-up of IPEX syndrome patients after different therapeutic strategies: An international multicenter retrospective study JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY Barzaghi, F., Hernandez, L., Neven, B., Ricci, S., Kucuk, Z., Bleesing, J. J., Nademi, Z., Slatter, M., Ulloa, E., Shcherbina, A., Roppelt, A., Worth, A., Silva, J., Aiuti, A., Murguia-Favela, L., Speckmann, C., Carneiro-Sampaio, M., Fernandes, J., Baris, S., Ozen, A., Karakoc-Aydiner, E., Kiykim, A., Schulz, A., Steinmann, S., Notarangelo, L., Gambineri, E., Lionetti, P., Shearer, W., Forbes, L. R., Martinez, C., Moshous, D., Blanche, S., Fisher, A., Ruemmele, F. M., Tissandier, C., Ouachee-Chardin, M., Rieux-Laucat, F., Cavazzana, M., Qasim, W., Lucarelli, B., Albert, M. H., Kobayashi, I., Alonso, L., De Heredia, C., Kanegane, H., Lawitschka, A., Seo, J., Gonzalez-Vicent, M., Diaz, M., Goyal, R., Sauer, M. G., Yesilipek, A., Kim, M., Yilmaz-Demirdag, Y., Bhatia, M., Khlevner, J., Padilla, E., Martino, S., Montin, D., Neth, O., Molinos-Quintana, A., Valverde-Fernandez, J., Broides, A., Pinsk, V., Ballauf, A., Haerynck, F., Bordon, V., Dhooge, C., Garcia-Lloret, M., Bredius, R. G., Kawak, K., Haddad, E., Seidel, M., Duckers, G., Pai, S., Dvorak, C. C., Ehl, S., Locatelli, F., Goldman, F., Gennery, A., Cowan, M. J., Roncarolo, M., Bacchetta, R., PIDTC, IEWP, European Soc Blood Marrow 2018; 141 (3): 1036-+


    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

  • Forkhead-Box-P3 Gene Transfer in Human CD4(+) T Conventional Cells for the Generation of Stable and Efficient Regulatory T Cells, Suitable for Immune Modulatory Therapy FRONTIERS IN IMMUNOLOGY Passerini, L., Bacchetta, R. 2017; 8: 1282


    The development of novel approaches to control immune responses to self- and allogenic tissues/organs represents an ambitious goal for the management of autoimmune diseases and in transplantation. Regulatory T cells (Tregs) are recognized as key players in the maintenance of peripheral tolerance in physiological and pathological conditions, and Treg-based cell therapies to restore tolerance in T cell-mediated disorders have been designed. However, several hurdles, including insufficient number of Tregs, their stability, and their antigen specificity, have challenged Tregs clinical applicability. In the past decade, the ability to engineer T cells has proven a powerful tool to redirect specificity and function of different cell types for specific therapeutic purposes. By using lentivirus-mediated gene transfer of the thymic-derived Treg transcription factor forkhead-box-P3 (FOXP3) in conventional CD4+ T cells, we converted effector T cells into Treg-like cells, endowed with potent in vitro and in vivo suppressive activity. The resulting CD4FOXP3 T-cell population displays stable phenotype and suppressive function. We showed that this strategy restores Treg function in T lymphocytes from patients carrying mutations in FOXP3 [immune-dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX)], in whom CD4FOXP3 T cell could be used as therapeutics to control autoimmunity. Here, we will discuss the potential advantages of using CD4FOXP3 T cells for in vivo application in inflammatory diseases, where tissue inflammation may undermine the function of natural Tregs. These findings pave the way for the use of engineered Tregs not only in IPEX syndrome but also in autoimmune disorders of different origin and in the context of stem cell and organ transplantation.

    View details for DOI 10.3389/fimmu.2017.01282

    View details for Web of Science ID 000412734100001

    View details for PubMedID 29075264

    View details for PubMedCentralID PMC5643480

  • From IPEX syndrome to FOXP3 mutation: a lesson on immune dysregulation. Annals of the New York Academy of Sciences Bacchetta, R., Barzaghi, F., Roncarolo, M. 2016


    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

  • Congenital diarrhoeal disorders: advances in this evolving web of inherited enteropathies NATURE REVIEWS GASTROENTEROLOGY & HEPATOLOGY Canani, R. B., Castaldo, G., Bacchetta, R., Martin, M. G., Goulet, O. 2015; 12 (5): 293-302


    Congenital diarrhoeal disorders (CDDs) represent an evolving web of rare chronic enteropathies, with a typical onset early in life. In many of these conditions, severe chronic diarrhoea represents the primary clinical manifestation, whereas in others diarrhoea is only a component of a more complex multi-organ or systemic disorder. Typically, within the first days of life, diarrhoea leads to a life-threatening condition highlighted by severe dehydration and serum electrolyte abnormalities. Thus, in the vast majority of cases appropriate therapy must be started immediately to prevent dehydration and long-term, sometimes severe, complications. The number of well-characterized disorders attributed to CDDs has gradually increased over the past several years, and many new genes have been identified and functionally related to CDDs, opening new diagnostic and therapeutic perspectives. Molecular analysis has changed the diagnostic scenario in CDDs, and led to a reduction in invasive and expensive procedures. Major advances have been made in terms of pathogenesis, enabling a better understanding not only of these rare conditions but also of more common diseases mechanisms.

    View details for DOI 10.1038/nrgastro.2015.44

    View details for Web of Science ID 000354070800007

    View details for PubMedID 25782092

  • Forkhead box P3: The Peacekeeper of the Immune System INTERNATIONAL REVIEWS OF IMMUNOLOGY Passerini, L., de Sio, F. R., Roncarolo, M. G., Bacchetta, R. 2014; 33 (2): 129-145


    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 Haploidentical-HSC Transplanted Patients Treated with IL-10-Anergized Donor T Cells. Frontiers in immunology Bacchetta, R., Lucarelli, B., Sartirana, C., Gregori, S., Lupo Stanghellini, M. T., Miqueu, P., Tomiuk, S., Hernandez-Fuentes, M., Gianolini, M. E., Greco, R., Bernardi, M., Zappone, E., Rossini, S., Janssen, U., Ambrosi, A., Salomoni, M., Peccatori, J., Ciceri, F., Roncarolo, M. 2014; 5: 16-?


    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 PubMedID 24550909

    View details for PubMedCentralID PMC3907718

  • Gene/Cell Therapy Approaches for Immune Dysregulation Polyendocrinopathy Enteropathy X-Linked Syndrome CURRENT GENE THERAPY Passerini, L., de Sio, F. R., Porteus, M. H., Bacchetta, R. 2014; 14 (6): 422-428


    Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome is a rare autoimmune disease due to mutations in the gene encoding for Forkhead box P3 (FOXP3), a transcription factor fundamental for the function of thymus-derived (t) regulatory T (Treg) cells. The dysfunction of Treg cells results in the development of devastating autoimmune manifestations affecting multiple organs, eventually leading to premature death in infants, if not promptly treated by hematopoietic stem cell transplantation (HSCT). Novel gene therapy strategies can be developed for IPEX syndrome as more definitive cure than allogeneic HSCT. Here we describe the therapeutic approaches, alternative to HSCT, currently under development. We described that effector T cells can be converted in regulatory T cells by LV-mediated FOXP3-gene transfer in differentiated T lymphocytes. Despite FOXP3 mutations mainly affect a highly specific T cell subset, manipulation of stem cells could be required for long-term remission of the disease. Therefore, we believe that a more comprehensive strategy should aim at correcting FOXP3-mutated stem cells. Potentials and hurdles of both strategies will be highlighted here.

    View details for Web of Science ID 000345248000002

    View details for PubMedCentralID PMC4443799

  • CD4(+) T Cells from IPEX Patients Convert into Functional and Stable Regulatory T Cells by FOXP3 Gene Transfer SCIENCE TRANSLATIONAL MEDICINE Passerini, L., Mel, E. R., Sartirana, C., Fousteri, G., Bondanza, A., Naldini, L., Roncarolo, M. G., Bacchetta, R. 2013; 5 (215)


    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

  • Autoantibodies to Harmonin and Villin Are Diagnostic Markers in Children with IPEX Syndrome PLOS ONE Lampasona, V., Passerini, L., Barzaghi, F., Lombardoni, C., Bazzigaluppi, E., Brigatti, C., Bacchetta, R., Bosi, E. 2013; 8 (11)


    Autoantibodies to enterocyte antigens harmonin (75 kDa USH1C protein) and villin (actin-binding 95 kDa protein) are associated with the Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome. In this study we evaluated the diagnostic value of harmonin and villin autoantibodies in IPEX and IPEX-like syndromes. Harmonin and villin autoantibodies were measured by a novel Luminescent-Immuno-Precipitation-System (LIPS) quantitative assay, in patients with IPEX, IPEX-like syndrome, Primary Immunodeficiencies (PID) with enteropathy, all diagnosed by sequencing of the FOXP3 gene, and in type 1 diabetes (T1D), celiac disease and healthy blood donors as control groups. Harmonin and villin autoantibodies were detected in 12 (92%) and 6 (46%) of 13 IPEX patients, and in none of the IPEX-like, PID, T1D, celiac patients, respectively. All IPEX patients, including one case with late and atypical clinical presentation, had either harmonin and/or villin autoantibodies and tested positive for enterocyte antibodies by indirect immunofluorescence. When measured in IPEX patients in remission after immunosuppressive therapy or hematopoietic stem cell transplantation, harmonin and villin autoantibodies became undetectable or persisted at low titers in all cases but one in whom harmonin autoantibodies remained constantly high. In one patient, a peak of harmonin antibodies paralleled a relapse phase of enteropathy. Our study demonstrates that harmonin and villin autoantibodies, measured by LIPS, are sensitive and specific markers of IPEX, differentiate IPEX, including atypical cases, from other early childhood disorders associated with enteropathy, and are useful for screening and clinical monitoring of affected children.

    View details for DOI 10.1371/journal.pone.0078664

    View details for Web of Science ID 000327216200039

    View details for PubMedID 24250806

  • Coexpression of CD49b and LAG-3 identifies human and mouse T regulatory type 1 cells NATURE MEDICINE Gagliani, N., Magnani, C. F., Huber, S., Gianolini, M. E., Pala, M., Licona-Limon, P., Guo, B., Herbert, D. R., Bulfone, A., Trentini, F., Di Serio, C., Bacchetta, R., Andreani, M., Brockmann, L., Gregori, S., Flavell, R. A., Roncarolo, M. 2013; 19 (6): 739-?


    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

  • Human IL2RA null mutation mediates immunodeficiency with lymphoproliferation and autoimmunity CLINICAL IMMUNOLOGY Goudy, K., Aydin, D., Barzaghi, F., Gambineri, E., Vignoli, M., Mannurita, S. C., Doglioni, C., Ponzoni, M., Cicalese, M. P., Assanelli, A., Tommasini, A., Brigida, I., Dellepiane, R. M., Martino, S., Olek, S., Aiuti, A., Ciceri, F., Roncarolo, M. G., Bacchetta, R. 2013; 146 (3): 248-261


    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

  • Accumulation of peripheral autoreactive B cells in the absence of functional human regulatory T cells BLOOD Kinnunen, T., Chamberlain, N., Morbach, H., Choi, J., Kim, S., Craft, J., Mayer, L., Cancrini, C., Passerini, L., Bacchetta, R., Ochs, H. D., Torgerson, T. R., Meffre, E. 2013; 121 (9): 1595-1603


    Regulatory T cells (Tregs) play an essential role in preventing autoimmunity. Mutations in the forkhead box protein 3 (FOXP3) gene, which encodes a transcription factor critical for Treg function, result in a severe autoimmune disorder and the production of various autoantibodies in mice and in IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) patients. However, it is unknown whether Tregs normally suppress autoreactive B cells. To investigate a role for Tregs in maintaining human B-cell tolerance, we tested the reactivity of recombinant antibodies isolated from single B cells isolated from IPEX patients. Characteristics and reactivity of antibodies expressed by new emigrant/transitional B cells from IPEX patients were similar to those from healthy donors, demonstrating that defective Treg function does not impact central B-cell tolerance. In contrast, mature naive B cells from IPEX patients often expressed autoreactive antibodies, suggesting an important role for Tregs in maintaining peripheral B-cell tolerance. T cells displayed an activated phenotype in IPEX patients, including their Treg-like cells, and showed up-regulation of CD40L, PD-1, and inducibl T-cell costimulator (ICOS), which may favor the accumulation of autoreactive mature naive B cells in these patients. Hence, our data demonstrate an essential role for Tregs in the establishment and the maintenance of peripheral B-cell tolerance in humans.

    View details for DOI 10.1182/blood-2012-09-457465

    View details for Web of Science ID 000321750300021

    View details for PubMedID 23223361

  • AML/T cell interactomics uncover correlates of patient outcomes and the key role of ICAM1 in T cell killing of AML. Leukemia Sayitoglu, E. C., Luca, B. A., Boss, A. P., Thomas, B. C., Freeborn, R. A., Uyeda, M. J., Chen, P. P., Nakauchi, Y., Waichler, C., Lacayo, N., Bacchetta, R., Majeti, R., Gentles, A. J., Cepika, A. M., Roncarolo, M. G. 2024


    T cells are important for the control of acute myeloid leukemia (AML), a common and often deadly malignancy. We observed that some AML patient samples are resistant to killing by human-engineered cytotoxic CD4+ T cells. Single-cell RNA-seq of primary AML samples and CD4+ T cells before and after their interaction uncovered transcriptional programs that correlate with AML sensitivity or resistance to CD4+ T cell killing. Resistance-associated AML programs were enriched in AML patients with poor survival, and killing-resistant AML cells did not engage T cells in vitro. Killing-sensitive AML potently activated T cells before being killed, and upregulated ICAM1, a key component of the immune synapse with T cells. Without ICAM1, killing-sensitive AML became resistant to killing by primary ex vivo-isolated CD8+ T cells in vitro, and engineered CD4+ T cells in vitro and in vivo. While AML heterogeneity implies that multiple factors may determine their sensitivity to T cell killing, these data show that ICAM1 acts as an immune trigger, allowing T cell killing, and could play a role in AML patient survival in vivo.

    View details for DOI 10.1038/s41375-024-02255-1

    View details for PubMedID 38724673

    View details for PubMedCentralID 6239928

  • A novel FOXP3 knockout-humanized mouse model for pre-clinical safety and efficacy evaluation of Treg-like cell products. Molecular therapy. Methods & clinical development Sato, Y., Nathan, A., Shipp, S., Wright, J. F., Tate, K. M., Wani, P., Roncarolo, M. G., Bacchetta, R. 2023; 31: 101150


    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

  • T-ALLO10 INFUSION AFTER A.DEPLETED-HSCT IN CHILDREN AND YOUNG ADULTS WITH HEMATOLOGIC MALIGNANCIES: IMPROVED IMMUNE RECONSTITUTION IN THE ABSENCE OF SEVERE GVHD Bertaina, A., Bacchetta, R., Shyr, D., Saini, G., Lee, J., Kristovich, K., Agarwal-Hashmi, R., Klein, O., Melsop, K., Tate, K., Barbarito, G., Oppizzi, L., Chen, P., Cepika, A., Roncarolo, M. SPRINGERNATURE. 2023: 232-234
  • Discovery of Key Transcriptional Regulators of Alloantigen-Inducible Tregs Used for Cell Therapy Cepika, A., Amaya, L., Waichler, C., Narula, M., Thomas, B. C., Chen, P. P., Mantilla, M. M., Pavel-Dinu, M., Freeborn, R., Porteus, M. H., Bacchetta, R., Mueller, F., Greenleaf, W. J., Chang, H. Y., Roncarolo, M. CELL PRESS. 2023: 370-371
  • FOXP3 TSDR Measurement Could Assist Variant Classification and Diagnosis of IPEX Syndrome. Journal of clinical immunology Wyatt, R. C., Olek, S., De Franco, E., Samans, B., Patel, K., Houghton, J., Walter, S., Schulze, J., Bacchetta, R., Hattersley, A. T., Flanagan, S. E., Johnson, M. B. 2023


    Pathogenic FOXP3 variants cause immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, a progressive autoimmune disease resulting from disruption of the regulatory T cell (Treg) compartment. Assigning pathogenicity to novel variants in FOXP3 is challenging due to the heterogeneous phenotype and variable immunological abnormalities. The number of cells with demethylation at the Treg cell-specific demethylated region (TSDR) is an independent biomarker of IPEX. We aimed to investigate if diagnosing IPEX at presentation with isolated diabetes could allow for effective monitoring of disease progression and assess whether TSDR analysis can aid FOXP3 variant classification and predict disease course. We describe a large genetically diagnosed IPEX cohort (n = 65) and 13 individuals with other monogenic autoimmunity subtypes in whom we quantified the proportion of cells with FOXP3 TSDR demethylation, normalized to the number with CD4 demethylation (%TSDR/CD4) and compare them to 29 unaffected controls. IPEX patients presenting with isolated diabetes (50/65, 77%) often later developed enteropathy (20/50, 40%) with a median interval of 23.5 weeks. %TSDR/CD4 was a good discriminator of IPEX vs. unaffected controls (ROC-AUC 0.81, median 13.6% vs. 8.5%, p < 0.0001) with higher levels of demethylation associated with more severe disease. Patients with other monogenic autoimmunity had a similar %TSDR/CD4 to controls (median 8.7%, p = 1.0). Identifying increased %TSDR/CD4 in patients with novel FOXP3 mutations presenting with isolated diabetes facilitates diagnosis and could offer an opportunity to monitor patients and begin immune modulatory treatment before onset of severe enteropathy.

    View details for DOI 10.1007/s10875-022-01428-w

    View details for PubMedID 36600150

  • Analyses of thymocyte commitment to regulatory T cell lineage in thymus of healthy subjects and patients with 22q11.2 deletion syndrome. Frontiers in immunology Borna, S., Dejene, B., Lakshmanan, U., Schulze, J., Weinberg, K., Bacchetta, R. 2023; 14: 1088059


    The Chromosome 22q11.2 deletion syndrome (22q11.2DS) results in an inborn error of immunity due to defective thymic organogenesis. Immunological abnormalities in 22q11.2DS patients are thymic hypoplasia, reduced output of T lymphocytes by the thymus, immunodeficiency and increased incidence of autoimmunity. While the precise mechanism responsible for increased incidence of autoimmunity is not completely understood, a previous study suggested a defect in regulatory T cells (Treg) cell lineage commitment during T cell development in thymus. Here, we aimed to analyze this defect in more detail. Since Treg development in human is still ill-defined, we first analyzed where Treg lineage commitment occurs. We performed systematic epigenetic analyses of the Treg specific demethylation region (TSDR) of the FOXP3 gene in sorted thymocytes at different developmental stages. We defined CD3+CD4+CD8+ FOXP3+CD25+ as the T cell developmental stage in human where TSDR demethylation first occurs. Using this knowledge, we analyzed the intrathymic defect in Treg development in 22q11.2DS patients by combination of TSDR, CD3, CD4, CD8 locus epigenetics and multicolor flow cytometry. Our data showed no significant differences in Treg cell frequencies nor in their basic phenotype. Collectively, these data suggest that although 22q11.2DS patients present with reduced thymic size and T cell output, the frequencies and the phenotype of Treg cell at each developmental stage are surprisingly well preserved.

    View details for DOI 10.3389/fimmu.2023.1088059

    View details for PubMedID 37006241

  • Rare immune diseases paving the road for genome editing-based precision medicine. Frontiers in genome editing Pavel-Dinu, M., Borna, S., Bacchetta, R. 2023; 5: 1114996


    Clustered regularly interspaced short palindromic repeats (CRISPR) genome editing platform heralds a new era of gene therapy. Innovative treatments for life-threatening monogenic diseases of the blood and immune system are transitioning from semi-random gene addition to precise modification of defective genes. As these therapies enter first-in-human clinical trials, their long-term safety and efficacy will inform the future generation of genome editing-based medicine. Here we discuss the significance of Inborn Errors of Immunity as disease prototypes for establishing and advancing precision medicine. We will review the feasibility of clustered regularly interspaced short palindromic repeats-based genome editing platforms to modify the DNA sequence of primary cells and describe two emerging genome editing approaches to treat RAG2 deficiency, a primary immunodeficiency, and FOXP3 deficiency, a primary immune regulatory disorder.

    View details for DOI 10.3389/fgeed.2023.1114996

    View details for PubMedID 36846437

  • Case report: Refractory Evans syndrome in two patients with spondyloenchondrodysplasia with immune dysregulation treated successfully with JAK1/JAK2 inhibition. Frontiers in immunology Gernez, Y., Narula, M., Cepika, A., Valdes Camacho, J., Hoyte, E. G., Mouradian, K., Glader, B., Singh, D., Sathi, B., Rao, L., Tolin, A. L., Weinberg, K. I., Lewis, D. B., Bacchetta, R., Weinacht, K. G. 2023; 14: 1328005


    Biallelic mutations in the ACP5 gene cause spondyloenchondrodysplasia with immune dysregulation (SPENCDI). SPENCDI is characterized by the phenotypic triad of skeletal dysplasia, innate and adaptive immune dysfunction, and variable neurologic findings ranging from asymptomatic brain calcifications to severe developmental delay with spasticity. Immune dysregulation in SPENCDI is often refractory to standard immunosuppressive treatments. Here, we present the cases of two patients with SPENCDI and recalcitrant autoimmune cytopenias who demonstrated a favorable clinical response to targeted JAK inhibition over a period of more than 3 years. One of the patients exhibited steadily rising IgG levels and a bone marrow biopsy revealed smoldering multiple myeloma. A review of the literature uncovered that approximately half of the SPENCDI patients reported to date exhibited increased IgG levels. Screening for multiple myeloma in SPENCDI patients with rising IgG levels should therefore be considered.

    View details for DOI 10.3389/fimmu.2023.1328005

    View details for PubMedID 38347954

  • Autoantibody discovery across monogenic, acquired, and COVID19-associated autoimmunity with scalable PhIP-Seq. eLife Vazquez, S. E., Mann, S. A., Bodansky, A., Kung, A. F., Quandt, Z., Ferre, E. M., Landegren, N., Eriksson, D., Bastard, P., Zhang, S., Liu, J., Mitchell, A., Proekt, I., Yu, D., Mandel-Brehm, C., Wang, C., Miao, B., Sowa, G., Zorn, K., Chan, A. Y., Tagi, V. M., Shimizu, C., Tremoulet, A., Lynch, K., Wilson, M. R., Kampe, O., Dobbs, K., Delmonte, O. M., Bacchetta, R., Notarangelo, L. D., Burns, J. C., Casanova, J., Lionakis, M. S., Torgerson, T. R., Anderson, M. S., DeRisi, J. L. 2022; 11


    Phage Immunoprecipitation-Sequencing (PhIP-Seq) allows for unbiased, proteome-wide autoantibody discovery across a variety of disease settings, with identification of disease-specific autoantigens providing new insight into previously poorly understood forms of immune dysregulation. Despite several successful implementations of PhIP-Seq for autoantigen discovery, including our previous work (Vazquez et al. 2020), current protocols are inherently difficult to scale to accommodate large cohorts of cases and importantly, healthy controls. Here, we develop and validate a high throughput extension of PhIP-seq in various etiologies of autoimmune and inflammatory diseases, including APS1, IPEX, RAG1/2 deficiency, Kawasaki Disease (KD), Multisystem Inflammatory Syndrome in Children (MIS-C), and finally, mild and severe forms of COVID19. We demonstrate that these scaled datasets enable machine-learning approaches that result in robust prediction of disease status, as well as the ability to detect both known and novel autoantigens, such as PDYN in APS1 patients, and intestinally expressed proteins BEST4 and BTNL8 in IPEX patients. Remarkably, BEST4 antibodies were also found in 2 patients with RAG1/2 deficiency, one of whom had very early onset IBD. Scaled PhIP-Seq examination of both MIS-C and KD demonstrated rare, overlapping antigens, including CGNL1, as well as several strongly enriched putative pneumonia-associated antigens in severe COVID19, including the endosomal protein EEA1. Together, scaled PhIP-Seq provides a valuable tool for broadly assessing both rare and common autoantigen overlap between autoimmune diseases of varying origins and etiologies.

    View details for DOI 10.7554/eLife.78550

    View details for PubMedID 36300623

  • Design of experiments as a decision tool for cell therapy manufacturing. Cytotherapy Lee, E., Shah, D., Porteus, M., Wright, J. F., Bacchetta, R. 2022


    BACKGROUND AIMS: Cell therapies are costlier to manufacture than small molecules and protein therapeutics because they require multiple manipulations and are often produced in an autologous manner. Strategies to lower the cost of goods to produce a cell therapy could make a significant impact on its total cost.METHODS: Borrowing from the field of bioprocess development, the authors took a design of experiments (DoE)-based approach to understanding the manufacture of a cell therapy product in pre-clinical development, analyzing main cost factors in the production process. The cells used for these studies were autologous CD4+ T lymphocytes gene-edited using CRISPR/Cas9 and recombinant adeno-associated virus (AAV) to restore normal FOXP3 gene expression as a prospective investigational product for patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome.RESULTS: Using gene editing efficiency as the response variable, an initial screen was conducted for other variables that could influence the editing frequency. The multiplicity of infection (MOI) of AAV and amount of single guide RNA (sgRNA) were the significant factors used for the optimization step to generate a response contour plot. Cost analysis was done for multiple points in the design space to find cost drivers that could be reduced. For the range of values tested (50 000-750 000 vg/cell AAV and 0.8-4 mug sgRNA), editing with the highest MOI and sgRNA yielded the best gene editing frequency. However, cost analysis showed the optimal solution was gene editing at 193 000 vg/cell AAV and 1.78 mug sgRNA.CONCLUSIONS: The authors used DoE to define key factors affecting the gene editing process for a potential investigational therapeutic, providing a novel and faster data-based approach to understanding factors driving complex biological processes. This approach could be applied in process development and aid in achieving more robust strategies for the manufacture of cellular therapeutics.

    View details for DOI 10.1016/j.jcyt.2022.01.009

    View details for PubMedID 35227602

  • Editorial: IPEX 2020: An Expanding Disease Spectrum and Novel Precision Therapies. Frontiers in pediatrics Bacchetta, R., Chatila, T. 2022; 10: 856920

    View details for DOI 10.3389/fped.2022.856920

    View details for PubMedID 35273932

  • Alloantigen-specific type 1 regulatory T cells suppress through CTLA-4 and PD-1 pathways and persist long-term in patients. Science translational medicine Chen, P. P., Cepika, A., Agarwal-Hashmi, R., Saini, G., Uyeda, M. J., Louis, D. M., Cieniewicz, B., Narula, M., Amaya Hernandez, L. C., Harre, N., Xu, L., Thomas, B. C., Ji, X., Shiraz, P., Tate, K. M., Margittai, D., Bhatia, N., Meyer, E., Bertaina, A., Davis, M. M., Bacchetta, R., Roncarolo, M. G. 2021; 13 (617): eabf5264


    [Figure: see text].

    View details for DOI 10.1126/scitranslmed.abf5264

    View details for PubMedID 34705520

  • 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 Uyeda, M. J., Freeborn, R. A., Cieniewicz, B., Romano, R., Chen, P. P., Liu, J. M., Thomas, B., Lee, E., Cepika, A., Bacchetta, R., Roncarolo, M. 2021
  • Co-Expression of FOXP3FL and FOXP3Δ2 Isoforms Is Required for Optimal Treg-Like Cell Phenotypes and Suppressive Function. Frontiers in immunology Sato, Y., Liu, J., Lee, E., Perriman, R., Roncarolo, M. G., Bacchetta, R. 2021; 12: 752394


    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

  • A beta T-Cell/CD19 B-Cell Depleted Haploidentical Stem Cell Transplantation: A New Platform for Curing Rare and Monogenic Disorders Bertaina, A., Bacchetta, R., Lewis, D. B., Grimm, P. C., Shah, A. J., Agarwal, R., Concepcion, W., Czechowicz, A., Bhatia, N., Lahiri, P., Weinberg, K. I., Parkman, R., Porteus, M., Roncarolo, M. ELSEVIER SCIENCE INC. 2020: S288
  • 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 Mayers, M., Schulze, J., Barbarito, G., Lakshmanan, U., Parkman, R., Weinberg, K. I., Chu, J., Agarwal, R., Roncarolo, M., Sachsenmaier, C., Bacchetta, R., Bertaina, A. ELSEVIER SCIENCE INC. 2020: S305
  • Engineered type 1 regulatory T cells designed for clinical use kill primary pediatric acute myeloid leukemia cells Haematologica Cieniewicz, B., Uyeda, M. J., Chen, P. P., Sayitoglu, E. C., Liu, J. M., Andolfi, G., Greenthal, K., Bertaina, A., Gregori, S., Bacchetta, R., Lacayo, N. J., Cepika, A., Roncarolo, M. 2020
  • 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 Cepika, A., Chen, P. P., Uyeda, M. J., Cieniewicz, B., Narula, M., Amaya, L., Louis, D. M., Xu, L., Ji, X., Bertaina, A., Agarwal-Hashmi, R., Davis, M. M., Meyer, E., Bacchetta, R., Roncarolo, M. 2020: A159–A159
  • APVO210: A Bispecific Anti-CD86-IL-10 Fusion Protein (ADAPTIR (TM)) to Induce Antigen-Specific T Regulatory Type 1 Cells FRONTIERS IN IMMUNOLOGY Pellerin, L., Chen, P., Gregori, S., Hernandez-Hoyos, G., Bacchetta, R., Roncarolo, M. 2018; 9
  • CRISPR-Based Therapy for IPEX Syndrome as a Model of Genetic Autoimmunity Goodwin, M., Lee, E., Lakshmanan, U., Shipp, S., Roncarolo, M., Porteus, M., Bacchetta, R. CELL PRESS. 2018: 95–96
  • Minimum Information about T Regulatory Cells: A Step toward Reproducibility and Standardization FRONTIERS IN IMMUNOLOGY Fuchs, A., Gliwinski, M., Grageda, N., Spiering, R., Abbas, A. K., Appel, S., Bacchetta, R., Battaglia, M., Berglund, D., Blazar, B., Bluestone, J. A., Bornhaeuser, M., ten Brinke, A., Brusko, T. M., Cools, N., Cuturi, M., Geissler, E., Giannoukakis, N., Golab, K., Hafler, D. A., van Ham, S., Hester, J., Hippen, K., Di Ianni, M., Ilic, N., Isaacs, J., Issa, F., Iwaszkiewicz-Grzes, D., Jaeckel, E., Joosten, I., Klatzmann, D., Koenen, H., van Kooten, C., Korsgren, O., Kretschmer, K., Levings, M., Marek-Trzonkowska, N., Martinez-Llordella, M., Miljkovic, D., Mills, K. G., Miranda, J. P., Piccirillo, C. A., Putnam, A. L., Ritter, T., Roncarolo, M., Sakaguchi, S., Sanchez-Ramon, S., Sawitzki, B., Sofronic-Milosavljevic, L., Sykes, M., Tang, Q., Vives-Pi, M., Waldmann, H., Witkowski, P., Wood, K. J., Gregori, S., Hilkens, C. U., Lombardi, G., Lord, P., Martinez-Caceres, E. M., Trzonkowski, P. 2018; 8: 1844


    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 Pellerin, L., Jenks, J., Chinthrajah, S., Dominguez, T., Block, W., Zhou, X., Noshirvan, A., Gregori, S., Roncarolo, M., Nadeau, K., Bacchetta, R. 2018; 141 (1): 202-+


    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 Kunicki, M. A., Hernandez, L., Davis, K. L., Bacchetta, R., Roncarolo, M. 2018; 200 (1): 336–46


    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

  • Hurdles in therapy with regulatory T cells. Science translational medicine Trzonkowski, P., Bacchetta, R., Battaglia, M., Berglund, D., Bohnenkamp, H. R., Ten brinke, A., Bushell, A., Cools, N., Geissler, E. K., Gregori, S., Marieke van Ham, S., Hilkens, C., Hutchinson, J. A., Lombardi, G., Madrigal, J. A., Marek-Trzonkowska, N., Martinez-Caceres, E. M., Roncarolo, M. G., Sanchez-Ramon, S., Saudemont, A., Sawitzki, B. 2015; 7 (304): 304ps18-?


    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

  • Hurdles in therapy with regulatory T cells. Science translational medicine Trzonkowski, P., Bacchetta, R., Battaglia, M., Berglund, D., Bohnenkamp, H. R., Ten brinke, A., Bushell, A., Cools, N., Geissler, E. K., Gregori, S., Marieke van Ham, S., Hilkens, C., Hutchinson, J. A., Lombardi, G., Madrigal, J. A., Marek-Trzonkowska, N., Martinez-Caceres, E. M., Roncarolo, M. G., Sanchez-Ramon, S., Saudemont, A., Sawitzki, B. 2015; 7 (304): 304ps18-?

    View details for DOI 10.1126/scitranslmed.aaa7721

    View details for PubMedID 26355029

  • Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nature biotechnology Hendel, A., Bak, R. O., Clark, J. T., Kennedy, A. B., Ryan, D. E., Roy, S., Steinfeld, I., Lunstad, B. D., Kaiser, R. J., Wilkens, A. B., Bacchetta, R., Tsalenko, A., Dellinger, D., Bruhn, L., Porteus, M. H. 2015; 33 (9): 985-989


    CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34(+) hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.

    View details for DOI 10.1038/nbt.3290

    View details for PubMedID 26121415

  • Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nature biotechnology Hendel, A., Bak, R. O., Clark, J. T., Kennedy, A. B., Ryan, D. E., Roy, S., Steinfeld, I., Lunstad, B. D., Kaiser, R. J., Wilkens, A. B., Bacchetta, R., Tsalenko, A., Dellinger, D., Bruhn, L., Porteus, M. H. 2015; 33 (9): 985-989

    View details for DOI 10.1038/nbt.3290

    View details for PubMedID 26121415

  • Clinical Features and Follow-Up in Patients with 22q11.2 Deletion Syndrome JOURNAL OF PEDIATRICS Cancrini, C., Puliafito, P., Digilio, M. C., Soresina, A., Martino, S., Rondelli, R., Consolini, R., Ruga, E. M., Cardinale, F., Finocchi, A., Romiti, M. L., Martire, B., Bacchetta, R., Albano, V., Carotti, A., Specchia, F., Montin, D., Cirillo, E., Cocchi, G., Trizzino, A., Bossi, G., Milanesi, O., Azzari, C., Corsello, G., Pignata, C., Aiuti, A., Pietrogrande, M. C., Marino, B., Ugazio, A. G., Plebani, A., Rossi, P. 2014; 164 (6): 1475-?


    To investigate the clinical manifestations at diagnosis and during follow-up in patients with 22q11.2 deletion syndrome to better define the natural history of the disease.A retrospective and prospective multicenter study was conducted with 228 patients in the context of the Italian Network for Primary Immunodeficiencies. Clinical diagnosis was confirmed by cytogenetic or molecular analysis.The cohort consisted of 112 males and 116 females; median age at diagnosis was 4 months (range 0 to 36 years 10 months). The diagnosis was made before 2 years of age in 71% of patients, predominantly related to the presence of heart anomalies and neonatal hypocalcemia. In patients diagnosed after 2 years of age, clinical features such as speech and language impairment, developmental delay, minor cardiac defects, recurrent infections, and facial features were the main elements leading to diagnosis. During follow-up (available for 172 patients), the frequency of autoimmune manifestations (P = .015) and speech disorders (P = .002) increased. After a median follow-up of 43 months, the survival probability was 0.92 at 15 years from diagnosis.Our data show a delay in the diagnosis of 22q11.2 deletion syndrome with noncardiac symptoms. This study provides guidelines for pediatricians and specialists for early identification of cases that can be confirmed by genetic testing, which would permit the provision of appropriate clinical management.

    View details for DOI 10.1016/j.jpeds.2014.01.056

    View details for Web of Science ID 000336503200046

    View details for PubMedID 24657119

  • Identification of STAT5A and STAT5B Target Genes in Human T Cells. PloS one Kanai, T., Seki, S., Jenks, J. A., Kohli, A., Kawli, T., Martin, D. P., Snyder, M., Bacchetta, R., Nadeau, K. C. 2014; 9 (1)

    View details for DOI 10.1371/journal.pone.0086790

    View details for PubMedID 24497979

  • Identification of STAT5A and STAT5B target genes in human T cells. PloS one Kanai, T., Seki, S., Jenks, J. A., Kohli, A., Kawli, T., Martin, D. P., Snyder, M., Bacchetta, R., Nadeau, K. C. 2014; 9 (1)


    Signal transducer and activator of transcription (STAT) comprises a family of universal transcription factors that help cells sense and respond to environmental signals. STAT5 refers to two highly related proteins, STAT5A and STAT5B, with critical function: their complete deficiency is lethal in mice; in humans, STAT5B deficiency alone leads to endocrine and immunological problems, while STAT5A deficiency has not been reported. STAT5A and STAT5B show peptide sequence similarities greater than 90%, but subtle structural differences suggest possible non-redundant roles in gene regulation. However, these roles remain unclear in humans. We applied chromatin immunoprecipitation followed by DNA sequencing using human CD4(+) T cells to detect candidate genes regulated by STAT5A and/or STAT5B, and quantitative-PCR in STAT5A or STAT5B knock-down (KD) human CD4(+) T cells to validate the findings. Our data show STAT5A and STAT5B play redundant roles in cell proliferation and apoptosis via SGK1 interaction. Interestingly, we found a novel, unique role for STAT5A in binding to genes involved in neural development and function (NDRG1, DNAJC6, and SSH2), while STAT5B appears to play a distinct role in T cell development and function via DOCK8, SNX9, FOXP3 and IL2RA binding. Our results also suggest that one or more co-activators for STAT5A and/or STAT5B may play important roles in establishing different binding abilities and gene regulation behaviors. The new identification of these genes regulated by STAT5A and/or STAT5B has major implications for understanding the pathophysiology of cancer progression, neural disorders, and immune abnormalities.

    View details for DOI 10.1371/journal.pone.0086790

    View details for PubMedID 24497979

    View details for PubMedCentralID PMC3907443

  • Tr1 cells and the counter-regulation of immunity: natural mechanisms and therapeutic applications. Current topics in microbiology and immunology Roncarolo, M. G., Gregori, S., Bacchetta, R., Battaglia, M. 2014; 380: 39-68


    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

  • IL-21 signalling via STAT3 primes human naive B cells to respond to IL-2 to enhance their differentiation into plasmablasts BLOOD Berglund, L. J., Avery, D. T., Ma, C. S., Moens, L., Deenick, E. K., Bustamante, J., Boisson-Dupuis, S., Wong, M., Adelstein, S., Arkwright, P. D., Bacchetta, R., Bezrodnik, L., Dadi, H., Roifman, C. M., Fulcher, D. A., Ziegler, J. B., Smart, J. M., Kobayashi, M., Picard, C., Durandy, A., Cook, M. C., Casanova, J., Uzel, G., Tangye, S. G. 2013; 122 (24): 3940-3950


    B-cell responses are guided by the integration of signals through the B-cell receptor (BCR), CD40, and cytokine receptors. The common γ chain (γc)-binding cytokine interleukin (IL)-21 drives humoral immune responses via STAT3-dependent induction of transcription factors required for plasma cell generation. We investigated additional mechanisms by which IL-21/STAT3 signaling modulates human B-cell responses by studying patients with STAT3 mutations. IL-21 strongly induced CD25 (IL-2Rα) in normal, but not STAT3-deficient, CD40L-stimulated naïve B cells. Chromatin immunoprecipitation confirmed IL2RA as a direct target of STAT3. IL-21-induced CD25 expression was also impaired on B cells from patients with IL2RG or IL21R mutations, confirming a requirement for intact IL-21R signaling in this process. IL-2 increased plasmablast generation and immunoglobulin secretion from normal, but not CD25-deficient, naïve B cells stimulated with CD40L/IL-21. IL-2 and IL-21 were produced by T follicular helper cells, and neutralizing both cytokines abolished the B-cell helper capacity of these cells. Our results demonstrate that IL-21, via STAT3, sensitizes B cells to the stimulatory effects of IL-2. Thus, IL-2 may play an adjunctive role in IL-21-induced B-cell differentiation. Lack of this secondary effect of IL-21 may amplify the humoral immunodeficiency in patients with mutations in STAT3, IL2RG, or IL21R due to impaired responsiveness to IL-21.

    View details for DOI 10.1182/blood-2013-06-506865

    View details for Web of Science ID 000329737600018

    View details for PubMedID 24159173

  • Combined DOCK8 and CLEC7A mutations causing immunodeficiency in 3 brothers with diarrhea, eczema, and infections JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY Dinwiddie, D. L., Kingsmore, S. F., Caracciolo, S., Rossi, G., Moratto, D., Mazza, C., Sabelli, C., Bacchetta, R., Passerini, L., Magri, C., Bell, C. J., Miller, N. A., Hateley, S. L., Saunders, C. J., Zhang, L., Schroth, G. P., Barlati, S., Badolato, R. 2013; 131 (2): 594-597

    View details for DOI 10.1016/j.jaci.2012.10.062

    View details for Web of Science ID 000314661500045

    View details for PubMedID 23374272

  • Immunodeficiency with autoimmunity: beyond the paradox. Frontiers in immunology Bacchetta, R., Notarangelo, L. D. 2013; 4: 77-?

    View details for DOI 10.3389/fimmu.2013.00077

    View details for PubMedID 23630524