Crystal L Mackall MD is the Ernest and Amelia Gallo Family Professor of Pediatrics and Internal Medicine at Stanford University. She serves as Founding Director of the Stanford Center for Cancer Cell Therapy, Associate Director of Stanford Cancer Institute, Leader of the Cancer Immunology and Immunotherapy Program and Director of the Parker Institute for Cancer Immunotherapy at Stanford. During a 27 year tenure culminating as Chief of the Pediatric Oncology Branch, NCI, and now at Stanford, she has led an internationally recognized translational research program spanning basic studies of T cell homeostasis and tumor immunology, and clinical trials of immune based therapies for cancer. Her work is credited with identifying an essential role for the thymus in human T cell regeneration and discovering IL-7 as the master regulator of T cell homeostasis. She has led numerous first-in-human and first-in-child clinical trials spanning dendritic cell vaccines, cytokines, and adoptive immunotherapy using NK cells and genetically modified T cells. Her group was among the first to demonstrate impressive activity of CD19-CAR in pediatric leukemia, recently demonstrated impressive activity of CD22-CARs for leukemia and has identified T cell exhaustion as a major feature limiting the activity of CAR T cells.. Dr. Mackall’s clinical trials are notable for the incorporation of deep biologic endpoints that further our understanding of the basis for success and failure of novel immunotherapeutics. She has published over 185 manuscripts and serves in numerous leadership positions, including co-PI on the NCI Pediatric Cancer Immunotherapy Network (U54), Leader of the NCI Pediatric Cancer Immunotherapy Trials Network, and co-Leader of the St. Baldrick’s-StandUp2Cancer Pediatric Dream Team. She is Board Certified in Pediatrics, Pediatric Hematology-Oncology and Internal Medicine.
- Pediatric Hematology-Oncology
Founding Director, Stanford Center for Cancer Cell Therapy (2017 - Present)
Director, Parker Institute for Cancer Immunotherapy at Stanford (2016 - Present)
Associate Director, Stanford Cancer Institute (2016 - Present)
Leader, Cancer Immunology and Immunotherapy Program, Stanford Cancer Institute (2016 - Present)
Director, Cancer Immunotherapy Program, Department of Pediatrics (2016 - Present)
Honors & Awards
BJ Kennedy Keynote Lecturer, Masonic Cancer Center, Minneapolis, MN (2018)
Top 10 Clinical Research Award for New CAR-T Cell Therapy for Relapsed Leukemia, Top 10 Clinical Research Award (2018)
Chair, Pediatric Cancer Working Group, American Association for Cancer Research (2017-18)
Stephen Max Memorial Lectureship, University of Maryland (2017)
Warren Stow Distinguished Lectureship, MD Anderson Cancer Center (2017)
Nitschke-Kaskake Visiting Professorship, Oklahoma City Children's Hospital (2016)
G. Burroughs Mider Lectureship, National Institutes of Health (2015)
Member, Association of American Physicians (2015)
William Hathaway Visiting Professor Award, University of Colorado (2015)
Co-Leader, St.Baldrick's/StandUp2Cancer Pediatric Dream Team (2013-present)
Alexandra Scott Lectureship in Pediatric Oncology, Children's Hospital of Philadelphia (2013)
Director's Award, National Institutes of Health (2013)
Great Teacher Lectureship, National Institutes of Health (2012)
Vineberg Lectureship, Montreal Children's Hospital (2011)
Alex Koufos Memorial Lectureship, Akron Children's Hospital (2009)
Merit Award, National Institutes of Health (2007)
Member, Best Doctors in American (2006-present)
Member, American Society for Clinical Investigation (2005)
Distinguished Alumni Award, Northeastern Ohio Universities College of Medicine (2004)
Commendation Medal, United States Public Health Service (2003)
Director's Award, National Cancer Institute (2003)
Distinguished Clinical Teacher Award, National Institutes of Health (2000)
Member, Alpha Omega Alpha, Honorary Medical Society (1984)
Boards, Advisory Committees, Professional Organizations
Executive Board, Federation of Clinical Immunology Societies (FOCIS) (2001 - 2002)
Member, Biologic Response Modifiers Advisory Committee, Food and Drug Administration (2002 - 2003)
Member, NIH Central Tenure Committee (2004 - 2008)
Education Committee, American Society of Clinical Oncology (2006 - 2009)
Member, DNA Advisory Committee, US Food and Drug Administration (2008 - 2008)
Advisory Board for Clinical Research, NIH Clinical Center (2008 - 2012)
Vice Chair and Chair, Scientific Committee on Immunology and Host Defense, American Society of Hematology (2011 - 2012)
Member, Immunology Steering Committee, American Association for Cancer Research (2013 - 2014)
Chair, Program Committee on Immunology, American Association for Cancer Research (2014 - 2015)
Chair-Elect, Pediatric Cancer Working Group, American Association for Cancer Research (2015 - 2017)
Member, Committee on Scientific Affairs, American Society of Hematology (2016 - 2017)
Chair, Pediatric Cancer Working Group, American Association for Cancer Research (2017 - Present)
Board Certification: Pediatric Hematology-Oncology, American Board of Pediatrics (1992)
Fellowship:National Cancer Institute - Center Cancer Research (1992) MD
Board Certification: Pediatrics, American Board of Pediatrics (1989)
Board Certification: Internal Medicine, American Board of Internal Medicine (1989)
Residency:Akron General Hospital (1988) OH
MD, Northeastern Ohio Universities College of Medicine, Medicine (1984)
Medical Education:Northeastern Ohio Universities (1984) OH
BS, University of Akron, Natural Sciences (1980)
Rimas Orentas, Ira Pastan, Crystal Mackall. "United States Patent 61/549,516 Anti-CD22 Chimeric Antigen Receptors, patent pending", National Cancer Institute
Rimas Orentas, Ira Pastan, Crystal Mackall, Dimiter Dimitrov. "United States Patent 61/717,960 M971 Chimeric Antigen Receptors, patent pending", National Cancer Institute
Dimiter Dimitrov, Rimas Orentas, Crystal Mackall. "United States Patent 61/805001 Anti-CD276 polypeptides, proteins and chimeric antigen receptors, patent pending", National Cancer Institute
Rimas Orentas, Dimiter Dimitrov, Crystal Mackall. "United States Patent 61/900,906 ALK Antibodies, Conjugates and Chimeric Antigen Receptors, patent pending", National Cancer Institute
Terry Fry, Haiying Qin, Crystal Mackall, Rimas Orentas. "United States Patent 62/135,442 Dual Specific Anti-CD22-Anti-CD19-Chimeric Antigen Receptors, patent pending", National Cancer Institute
Crystal Mackall, Yongzhi Cui. "United States Patent 62/216,447 Anti-CD276 Chimeric Antigen Receptors, patent pending", National Cancer Institute
Jay Berzofsky, Lee Helman, Crystal Mackall. "United States Patent 7,867,977 A Peptide Epitope and Improvement Thereof Inducting T Cell Immunity to Alveolar Rhabdomyosarcoma in HLA-B7 positive Individuals.", National Cancer Institute
Hokyung Kay Chung, Michael Z Lin, Crystal Mackall, Robbie Majzner, Louai Labanieh. "United States Patent 62/694,830 Chimeric Antigen Receptor Polypeptides and Methods of Using Same", Leland Stanford Junior University, Jul 16, 2018
Crystal L Mackall, Michelle Monje, Christopher Mount, Robbie Majzner. "United States Patent 041839 CAR T cell therapy to treat H3K27M midline gliomas", Leland Stanford Junior University, Jul 12, 2018
Tom Wandless, Rachel Lynn, Sanjay Malhotra, Evan Weber, Crystal Mackall. "United States Patent 025394 Transiently regulated CAR-T cells engineered to prevent T-cell exhaustion and improve immunotherapy", Leland Stanford Junior University, Mar 30, 2018
Tom Wandless, Rachel Lynn, Sanjay Malhotra, Evan Weber, Crystal Mackall. "United States Patent PCT/US2018/025459 Methods of Treating T Cell Exhaustion by Inhibiting Modulating T Cell Receptor Signaling", Leland Stanford Junior University, Mar 30, 2018
Rachel Lynn, Evan Weber, Crystal Mackall, Elena Sotillo. "United States Patent 62/599.299 Compositions and Methods for Inhibiting T Cell Exhaustion", Leland Stanford Junior University, Dec 15, 2017
Jay Berzofsky, Lee Helman, Crystal Mackall. "United States Patent 12/092,449 Immunogenic Peptides And Methods Of Use For Treating And Preventing Cancer", National Cancer Institute, May 2, 2008
Current Research and Scholarly Interests
We are impressed by the potency of T cell immune responses for the treatment of cancer and our work focuses on enhancing the effectiveness of T cell based immunotherapies for cancer. Our approach is to simultaneously conduct basic studies alongside clinical trials, leveraging an iterative bench-to-bedside-bench rotation to efficiently optimize clinically relevant cancer immunotherapies. Our laboratory seeks to develop novel therapies for early phase testing in clinical trials, and also conducts intensive studies on clinical samples obtained from patients treated on immunotherapy trials. We also seek to enhance fundamental understanding of human T cell biology.
We focus primarily on using genetically engineered T cells to treat cancer, with an emphasis on chimeric antigen receptors (CARs). CARs are non-natural receptors, created using synthetic biology, that endow T cells with the capacity for antigen-specific, MHC-unrestricted killing. Some clinical results using CAR based therapies have been impressive, but we believe that further progress will emerge as a result of focus on these three major areas:
1.T cell exhaustion, a state whereby continued T cell activation leads to diminished functionality, is a fundamental barrier limiting the efficacy of many cancer immunotherapies. Our laboratory is focused on using high dimensional, single cell analyses to better define human T cell exhaustion and to enhance understanding of the biological mechanisms responsible for this phenomena. We believe that enhanced understanding of T cell exhaustion will give rise to novel approaches to prevent or reverse this phenomenon in the context of cancer immunotherapy.
2.Effective immunotherapies require a therapeutic window which allows the immune cell to preferentially or exclusively attack the neoplastic cell while sparing non-neoplastic, vital tissues. Our laboratory is focused on identifying novel targets for T cell based immunotherapies and for enhancing our understanding of the basis for differential antigen recognition using CAR T cells for cancer therapy. We are also interested in using novel approaches for combinatorial recognition, both to diminish the risk for tumor escape due to loss of antigen expression, and to allow targeting of tumor antigens that pose a risk due to co-expression on healthy, vital tissues.
3.The tumor microenvironment is potently immunosuppressive and can prevent potent antigen specific immune responses from effectively mediating antitumor effects. Our laboratory focuses on enhancing understanding of the immunosuppressive tumor microenvironment and on developing novel approaches to diminish the ability of the tumor microenvironment to limit the efficacy of T cell based immunotherapies.
Anti-CD22 Chimeric Receptor T Cells in Pediatric and Young Adults With Recurrent or Refractory CD22-expressing B Cell Malignancies
Background: - One type of cancer therapy takes blood cells from a person, changes them in a lab, then gives the cells back to the person. In this study, researchers are using an anti-CD22 gene, a virus, and an immune receptor to change the cells. Objective: - To see if giving anti-CD22 Chimeric Antigen Receptor (CAR) cells to young people with certain cancers is safe and effective. Eligibility: - People ages 1-30 with a leukemia or lymphoma that has not been cured by standard therapy. Design: - Participants will be screened to ensure their cancer cells express the CD22 protein. They will also have medical history, physical exam, blood and urine tests, heart tests, scans, and x-rays. They may give spinal fluid or have bone marrow tests. - Participants may have eye and neurologic exams. - Participants will get a central venous catheter or a catheter in a large vein. - Participants will have white blood cells removed. Blood is removed through a needle in an arm. White blood cells are removed. The rest of the blood is returned by needle in the other arm. - The cells will be changed in a laboratory. - Participants will get two IV chemotherapy drugs over 4 days. Some will stay in the hospital for this. - All participants will be in the hospital to get anti-CD22 CAR cells through IV. They will stay until any bad side effects are gone. - Participants will have many blood tests. They may repeat some screening exams. - Participants will have monthly visits for 2-3 months, then every 3-6 months. They may repeat some screening exams. - Participants will have follow-up for 15 years.
Axicabtagene Ciloleucel Expanded Access Study
A multicenter, open-label expanded access protocol for the treatment of subjects with relapsed/refractory large B-cell lymphoma.
CD19/CD22 Chimeric Antigen Receptor T Cells and Chemotherapy in Treating Children or Young Adults With Recurrent or Refractory CD19 Positive B Acute Lymphoblastic Leukemia
This phase I trial studies the best dose and side effects of CD19/CD22 chimeric antigen receptor (CAR) T cells when given together with chemotherapy, and to see how well they work in treating children or young adults with CD19 positive B acute lymphoblastic leukemia that has come back or does not respond to treatment. A CAR is a genetically-engineered receptor made so that immune cells (T cells) can attack cancer cells by recognizing and responding to the CD19/CD22 proteins. These proteins are commonly found on B acute lymphoblastic leukemia. Drugs used in chemotherapy, such as fludarabine phosphate and cyclophosphamide, work in different ways to stop the growth of cancer cells, either by killing the cells, by stopping them from dividing, or by stopping them from spreading. Giving CD19/CD22-CAR T cells and chemotherapy may work better in treating children or young adults with B acute lymphoblastic leukemia.
CD19/CD22 Chimeric Antigen Receptor T Cells and Chemotherapy in Treating Patients With Recurrent or Refractory CD19 Positive Diffuse Large B-Cell Lymphoma or B Acute Lymphoblastic Leukemia
This phase I trial studies the side effects of CD19/CD22 chimeric antigen receptor (CAR) T cells when given together with chemotherapy, and to see how well they work in treating patients with CD19 positive diffuse large B-cell lymphoma or B acute lymphoblastic leukemia that has come back or does not respond to treatment. A CAR is a genetically-engineered receptor made so that immune cells (T cells) can attack cancer cells by recognizing and responding to the CD19/CD22 proteins. These proteins are commonly found on diffuse large B-cell lymphoma and B acute lymphoblastic leukemia. Drugs used in chemotherapy, such as cyclophosphamide and fludarabine phosphate, work in different ways to stop the growth of cancer cells, either by killing the cells, by stopping them from dividing, or by stopping them from spreading. Giving CD19/CD22-CAR T cells and chemotherapy may work better in treating patients with diffuse large B-cell lymphoma or B acute lymphoblastic leukemia.
Nivolumab With or Without Ipilimumab in Treating Younger Patients With Recurrent or Refractory Solid Tumors or Sarcomas
This phase I/II trial studies the side effects and best dose of nivolumab when given with or without ipilimumab to see how well they work in treating younger patients with solid tumors or sarcomas that have come back (recurrent) or do not respond to treatment (refractory). Monoclonal antibodies, such as nivolumab and ipilimumab, may block tumor growth in different ways by targeting certain cells. It is not yet known whether nivolumab works better alone or with ipilimumab in treating patients with recurrent or refractory solid tumors or sarcomas.
Screening Protocol for Tumor Antigen Expression Profiling and HLA Typing for Eligibility Determination
This screening study is intended for men and women at least 18 years of age who have advanced solid or hematologic malignancy. The study will assess a subject's human leukocyte antigen (HLA) subtype and tumor antigen expression profile. Based on the results, it will be determined if a subject is eligible to be considered for Adaptimmune sponsored clinical trials testing the safety and efficacy of genetically changed T cells targeting specific tumor antigens. No treatment intervention will occur as part of this screening study. Upon enrollment, subjects will be required to provide a blood sample for HLA subtype analysis. If the results of the analysis match the HLA-A subtypes noted in the inclusion criteria and do not express the HLA subtypes that are exclusionary for the available interventional clinical trial(s), then the subject will be required to provide either an archival tumor specimen or fresh tumor tissue biopsy. The tumor specimen will be screened at a central laboratory for the expression (protein or gene) of multiple antigens which may include, but are not limited to NY-ESO-1 and/or LAGE-1a and MAGE A10. Based upon the results of these diagnostic analyses, if eligible, subjects will be referred to an appropriate available interventional clinical trial(s) at the discretion of the Investigator. Following screening, tumor samples will be retained by Adaptimmune for the purpose of developing and validating in vitro diagnostic (IVD) assay(s) for antigen expression profiling which is required for regulatory approval of a new therapeutic product indication.
Enoblituzumab (MGA271) in Children With B7-H3-expressing Solid Tumors
This study is a Phase 1, open-label, dose escalation and cohort expansion trial designed to characterize the safety, tolerability, PK, PD, immunogenicity and preliminary antitumor activity of enoblituzumab administered IV on a weekly schedule for up to 96 doses (approximately 2 years) in children and young adults with B7-H3-expressing relapsed or refractory malignant solid tumors.
Stanford is currently not accepting patients for this trial. For more information, please contact Cancer Clinical Trials Office (CCTO), 650-498-7061.
Enrollment on the Childhood Cancer Research Network (CCRN) of the Children s Oncology Group
Background: - The Children s Oncology Group has established a research network, the Childhood Cancer Research Network (CCRN), to collect information about children with cancer and other conditions that are benign but involve abnormal cell growth in order to help doctors and scientists better understand childhood cancer. The CCRN's goal is to collect clinical information about every child diagnosed with cancer and similar conditions in the United States and Canada, to allow researchers to study patterns, characteristics, and causes of childhood cancer. The information can also help researchers study the causes of childhood cancer. To expand the CCRN, parents of children who have been diagnosed with cancer will be asked to provide information about themselves and their child for research purposes. Objectives: - To obtain informed consent from parents (and the child, when appropriate) of infants, children, adolescents, and young adults newly diagnosed with cancer to enter their names and certain information concerning their child into the Childhood Cancer Research Network. - To obtain informed consent from parents (and the child, when appropriate) of infants, children, adolescents, and young adults newly diagnosed with cancer for permission to be contacted in the future to consider participating in non-therapeutic and prevention research studies involving the parents and/or the child. Eligibility: - Parents of children who have been seen at or treated by a hospital that is a member of the Children s Oncology Group. Design: - Parents will provide permission to have personal information sent from their child s hospital to the CCRN, including the child and parents' names; child's gender, birth date, race, and ethnicity; information about the disease; and the treating institution. - Parents will also give permission for CCRN to contact the diagnostic laboratory to obtain specific information about the tumor or cancer cells. - Parents will be asked if they are willing to be contacted in the future to consider participating in CCRN research studies, and will provide contact information (name, home address, and telephone number) to be entered in the CCRN. - Parents or patients who change their minds about having information available in the CCRN can ask the treatment institution to restrict access to the identifying information. Parents or patients who refuse to have information included in the CCRN or be contacted in the future will still be able to enter clinical cancer research studies.
Stanford is currently not accepting patients for this trial. For more information, please contact Peds Hem/Onc CRAs, 650-723-5535.
Study of bb2121 in Multiple Myeloma
Study CRB-401 is a 2-part, non-randomized, open label, multi-site Phase 1 study of bb2121 in adults with relapsed/refractory multiple myeloma (MM).
Stanford is currently not accepting patients for this trial. For more information, please contact Reneth Tien, 650-723-0646.
- CAR T cell therapy: inroads to response and resistance. Nature reviews. Immunology 2019
Chimeric antigen receptor (CAR) T therapies for the treatment of hematologic malignancies: clinical perspective and significance.
Journal for immunotherapy of cancer
2018; 6 (1): 137
Chimeric Antigen Receptor (CAR) T cell therapies - adoptive T cell therapies that have been genetically engineered for a new antigen-specificity - have displayed significant success in treating patients with hematologic malignancies, leading to three recent US Food and Drug Administration approvals. Based on the promise generated from these successes, the field is rapidly evolving to include new disease indications and CAR designs, while simultaneously reviewing and optimizing toxicity and management protocols. As such, this review provides expert perspective on the significance and clinical considerations of CAR T cell therapies in order to provide timely information to clinicians about this revolutionary new therapeutic class.
View details for DOI 10.1186/s40425-018-0460-5
View details for PubMedID 30514386
Systematic Evaluation of Neurotoxicity in Children and Young Adults Undergoing CD22 Chimeric Antigen Receptor T-Cell Therapy
JOURNAL OF IMMUNOTHERAPY
2018; 41 (7): 350–58
Neurotoxicity associated with CAR-T cell therapy can be life-threatening. With rapid development of CAR-T therapies, a systematic method is needed to identify and monitor symptoms of neurotoxicity, elucidate potential etiologies, and compare toxicity across trials. This paper presents a systematic evaluation developed and used to prospectively assess neurotoxicity in our phase I anti-CD22 CAR-T-cell trial and describes the symptoms of neurotoxicity identified using this methodology. Central nervous system (CNS) studies included routine lumbar punctures performed for disease evaluation pretherapy and posttherapy and a baseline brain MRI. Brief cognitive evaluations, assessing 4 domains (attention, working memory, cognitive flexibility, and processing speed), were administered preinfusion and postinfusion. A newly developed CAR-T-specific neurological symptom checklist (NSC) was completed by caregivers at 3 designated time-points. Serial serum cytokine levels were compared with neurotoxicity symptoms and severity. The majority of the first 22 consecutively treated subjects (ages, 7-30) demonstrated stable or improved cognitive test scores following therapy and no irreversible neurotoxicity, despite CAR-T-related antileukemic response, cytokine release syndrome, and trafficking of CAR-T cells to the CSF. The NSC allowed us to document the type and timing of symptoms and explore the etiology of neurotoxicity associated with CD22 CAR-T therapy. Cytokine profiling demonstrated that more concerning symptoms of neurotoxicity, such as hallucination and disorientation, were significantly associated with higher serum cytokine levels, supporting the hypothesis of inflammation-driven neurotoxicity. Systematic assessments of neurotoxicity were feasible in acutely ill children and young adults and served to characterize and monitor the symptoms associated with CAR-T therapy. We recommend these evaluations be incorporated into future immunotherapy protocols.
View details for DOI 10.1097/CJI.0000000000000241
View details for Web of Science ID 000441913100008
View details for PubMedID 30048343
View details for PubMedCentralID PMC6086728
Screening Clinical Cell Products for Replication Competent Retrovirus: The National Gene Vector Biorepository Experience
MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT
2018; 10: 371–78
Replication-competent retrovirus (RCR) is a safety concern for individuals treated with retroviral gene therapy. RCR detection assays are used to detect RCR in manufactured vector, transduced cell products infused into research subjects, and in the research subjects after treatment. In this study, we reviewed 286 control (n = 4) and transduced cell products (n = 282) screened for RCR in the National Gene Vector Biorepository. The transduced cell samples were submitted from 14 clinical trials. All vector products were previously shown to be negative for RCR prior to use in cell transduction. After transduction, all 282 transduced cell products were negative for RCR. In addition, 241 of the clinical trial participants were also screened for RCR by analyzing peripheral blood at least 1 month after infusion, all of which were also negative for evidence of RCR infection. The majority of vector products used in the clinical trials were generated in the PG13 packaging cell line. The findings suggest that screening of the retroviral vector product generated in PG13 cell line may be sufficient and that further screening of transduced cells does not provide added value.
View details for DOI 10.1016/j.omtm.2018.08.006
View details for Web of Science ID 000451115100034
View details for PubMedID 30211249
View details for PubMedCentralID PMC6134358
Tumor Antigen Escape from CAR T-cell Therapy.
Emerging data from chimeric antigen receptor (CAR) T-cell trials in B-cell malignancies demonstrate that a common mechanism of resistance to this novel class of therapeutics is the emergence of tumors with loss or downregulation of the target antigen. Antigen loss or antigen-low escape is likely to emerge as an even greater barrier to success in solid tumors, which manifest greater heterogeneity in target antigen expression. Potential approaches to overcome this challenge include engineering CAR T cells to achieve multispecificity and to respond to lower levels of target antigen and more efficient induction of natural antitumor immune responses as a result of CAR-induced inflammation. In this article, we review the evidence to date for antigen escape and downregulation and discuss approaches currently under study to overcome these obstacles.Significance: Antigen escape and downregulation have emerged as major issues impacting the durability of CAR T-cell therapy. Here, we explore their incidence and ways to overcome these obstacles in order to improve clinical outcomes. Cancer Discov; 8(10); 1-8. ©2018 AACR.
View details for DOI 10.1158/2159-8290.CD-18-0442
View details for PubMedID 30135176
Antitumor Activity Associated with Prolonged Persistence of Adoptively Transferred NY-ESO-1(c259) T Cells in Synovial Sarcoma
2018; 8 (8): 944–57
We evaluated the safety and activity of autologous T cells expressing NY-ESO-1c259, an affinity-enhanced T-cell receptor (TCR) recognizing an HLA-A2-restricted NY-ESO-1/LAGE1a-derived peptide, in patients with metastatic synovial sarcoma (NY-ESO-1c259T cells). Confirmed antitumor responses occurred in 50% of patients (6/12) and were characterized by tumor shrinkage over several months. Circulating NY-ESO-1c259T cells were present postinfusion in all patients and persisted for at least 6 months in all responders. Most of the infused NY-ESO-1c259T cells exhibited an effector memory phenotype following ex vivo expansion, but the persisting pools comprised largely central memory and stem-cell memory subsets, which remained polyfunctional and showed no evidence of T-cell exhaustion despite persistent tumor burdens. Next-generation sequencing of endogenous TCRs in CD8+ NY-ESO-1c259T cells revealed clonal diversity without contraction over time. These data suggest that regenerative pools of NY-ESO-1c259T cells produced a continuing supply of effector cells to mediate sustained, clinically meaningful antitumor effects.Significance: Metastatic synovial sarcoma is incurable with standard therapy. We employed engineered T cells targeting NY-ESO-1, and the data suggest that robust, self-regenerating pools of CD8+ NY-ESO-1c259T cells produce a continuing supply of effector cells over several months that mediate clinically meaningful antitumor effects despite prolonged exposure to antigen. Cancer Discov; 8(8); 944-57. ©2018 AACR.See related commentary by Keung and Tawbi, p. 914This article is highlighted in the In This Issue feature, p. 899.
View details for DOI 10.1158/2159-8290.CD-17-1417
View details for Web of Science ID 000440825900023
View details for PubMedID 29891538
ANTI-GD2 CHIMERIC ANTIGEN RECEPTOR T CELLS AS A POTENT IMMUNOTHERAPY REGIMEN IN XENOGRAFT MODELS OF HISTONE 3 K27M MUTANT DIFFUSE MIDLINE GLIOMA
OXFORD UNIV PRESS INC. 2018: 56
View details for Web of Science ID 000438339000131
CHECKPOINT MOLECULE B7-H3 IS HIGHLY EXPRESSED ON ATYPICAL RHABDOID TERATOID TUMOR (ATRT) AND IS A PROMISING CANDIDATE FOR CAR T CELL THERAPY
OXFORD UNIV PRESS INC. 2018: 33
View details for Web of Science ID 000438339000025
Fludarabine and neurotoxicity in engineered T-cell therapy
2018; 25 (3): 176–91
Adoptive T-cell therapy, incorporating engineered T cell receptors (TCRs) or chimeric antigen receptors (CARs), target tumor antigens with high affinity and specificity. To increase the potency of adoptively transferred T cells, patients are conditioned with lymphodepleting chemotherapy regimens prior to adoptive T-cell transfer (ACT), and data suggest that fludarabine is an important component of an effective regimen. In a recent clinical trial using CAR-T cells engineered to target the CD19 B-cell antigen to treat acute lymphoblastic leukemia, JCAR-015 (NCT02535364), two patient deaths due to cerebral edema led to trial suspension. The lymphodepleting agent fludarabine was suggested as the causative agent, in part due to its known association with neurotoxicity and its ability to induce greater potency. In a similar CAR-T study also incorporating fludarabine in the preconditioning regimen, ZUMA-1 (NCT02348216), one patient died of cerebral edema. However, subsequent deaths in the JCAR-015 study after removal of fludarabine and improved understanding behind the mechanisms of CAR-T-related encephalopathy syndrome (CRES) indicate that fludarabine is not the primary causative agent of cerebral edema and that it can be safely incorporated into the preconditioning regimen for ACT. Since entering clinical use in the late 1980s as a chemotherapy agent, fludarabine and similar analogs have been associated with lethal neurological toxicity, yet the manifestation and timing of symptoms are distinct to those observed recently in ACT. Herein, we review the history of fludarabine development as a chemotherapeutic agent, and discuss the safety of its continued use in preconditioning regimens for ACT.
View details for DOI 10.1038/s41434-018-0019-6
View details for Web of Science ID 000437316500005
View details for PubMedID 29789639
- Programming CAR-T cells to kill cancer NATURE BIOMEDICAL ENGINEERING 2018; 2 (6): 377–91
CAR T CELLS TARGETING B7-H3, A PAN-CANCER ANTIGEN, DEMONSTRATE POTENT PRECLINICAL ACTIVITY AGAINST PEDIATRIC SOLID TUMORS AND BRAIN TUMORS
View details for Web of Science ID 000428851200005
B7-H3 CAR T CELLS MEDIATE IN VITRO AND IN VIVO ACTIVITY AGAINST NEUROBLASTOMA XENOGRAFTS
View details for Web of Science ID 000428851200460
- Engineering a designer immunotherapy Tailoring cytokine selectivity by engineering receptor-ligand pairs circumvents toxicity SCIENCE 2018; 359 (6379): 990–91
- Neurotoxicity Associated with a High-Affinity GD2 CAR-Letter. Cancer immunology research 2018; 6 (4): 494–95
Potent antitumor efficacy of anti-GD2 CAR T cells in H3-K27M+ diffuse midline gliomas.
Diffuse intrinsic pontine glioma (DIPG) and other diffuse midline gliomas (DMGs) with mutated histone H3 K27M (H3-K27M)1-5 are aggressive and universally fatal pediatric brain cancers 6 . Chimeric antigen receptor (CAR)-expressing T cells have mediated impressive clinical activity in B cell malignancies7-10, and recent results suggest benefit in central nervous system malignancies11-13. Here, we report that patient-derived H3-K27M-mutant glioma cell cultures exhibit uniform, high expression of the disialoganglioside GD2. Anti-GD2 CAR T cells incorporating a 4-1BBz costimulatory domain 14 demonstrated robust antigen-dependent cytokine generation and killing of DMG cells in vitro. In five independent patient-derived H3-K27M+ DMG orthotopic xenograft models, systemic administration of GD2-targeted CAR T cells cleared engrafted tumors except for a small number of residual GD2lo glioma cells. To date, GD2-targeted CAR T cells have been well tolerated in clinical trials15-17. Although GD2-targeted CAR T cell administration was tolerated in the majority of mice bearing orthotopic xenografts, peritumoral neuroinflammation during the acute phase of antitumor activity resulted in hydrocephalus that was lethal in a fraction of animals. Given the precarious neuroanatomical location of midline gliomas, careful monitoring and aggressive neurointensive care management will be required for human translation. With a cautious multidisciplinary clinical approach, GD2-targeted CAR T cell therapy for H3-K27M+ diffuse gliomas of pons, thalamus and spinal cord could prove transformative for these lethal childhood cancers.
View details for DOI 10.1038/s41591-018-0006-x
View details for PubMedID 29662203
Autologous lymphapheresis for the production of chimeric antigen receptor T cells.
2017; 57 (5): 1133-1141
The first step in manufacturing chimeric antigen receptor (CAR) T cells is to collect autologous CD3+ lymphocytes by apheresis. Patients, however, often have leukopenia or have other disease-related complications. We evaluated the feasibility of collecting adequate numbers of CD3+ cells, risk factors for inadequate collections, and the rate of adverse events.Apheresis lymphocyte collections from patients participating in three CAR T-cell clinical trials were reviewed. Collections were performed on the COBE Spectra by experienced nurses, with the goal of obtaining a minimum of 0.6 × 109 and a target of 2 × 109 CD3+ cells. Preapheresis peripheral blood counts, apheresis parameters, and product cell counts were analyzed.Of the 71 collections, 69 (97%) achieved the minimum and 55 (77%) achieved the target. Before apheresis, the 16 patients with yields below the target had significantly lower proportions and absolute numbers of circulating lymphocytes and CD3+ lymphocytes and higher proportions of circulating blasts and NK cells than those who achieved the target (470 × 106 lymphocytes/L vs. 1340 × 106 lymphocytes/L, p = 0.008; 349 × 106 CD3+ cells/L vs. 914 × 106 CD3+ cells/L, p = 0.001; 17.6% blasts vs. 4.55% blasts, p = 0.029). Enrichment of blasts in the product compared to the peripheral blood occurred in four patients, including the two patients whose collections did not yield the minimum number of CD3+ cells. Apheresis complications occurred in 11 patients (15%) and, with one exception, were easily managed in the apheresis clinic.In most patients undergoing CAR T-cell therapy, leukapheresis is well tolerated, and adequate numbers of CD3+ lymphocytes are collected.
View details for DOI 10.1111/trf.14003
View details for PubMedID 28236305
View details for PubMedCentralID PMC5398918
Harnessing the Immunotherapy Revolution for the Treatment of Childhood Cancers
2017; 31 (4): 476-485
Cancer immunotherapies can be classified into agents that amplify natural immune responses (e.g., checkpoint inhibitors) versus synthetic immunotherapies designed to initiate new responses (e.g., monoclonal antibodies [mAbs], chimeric antigen receptors [CARs]). Checkpoint inhibitors mediate unprecedented benefit in some adult cancers, but have not demonstrated significant activity in pediatric cancers, likely due their paucity of neoantigens. In contrast, synthetic immunotherapies such as mAbs and CAR T cells demonstrate impressive effects against childhood cancers. Intense efforts are underway to enhance the effectiveness of pediatric cancer immunotherapies through improved engineering of synthetic immunotherapies and by combining these with agents designed to amplify immune responses.
View details for DOI 10.1016/j.ccell.2017.03.002
View details for Web of Science ID 000398670600005
View details for PubMedID 28366678
Elutriated lymphocytes for manufacturing chimeric antigen receptor T cells
JOURNAL OF TRANSLATIONAL MEDICINE
Clinical trials of Chimeric Antigen Receptor (CAR) T cells manufactured from autologous peripheral blood mononuclear cell (PBMC) concentrates for the treatment of hematologic malignancies have been promising, but CAR T cell yields have been variable. This variability is due in part to the contamination of the PBMC concentrates with monocytes and granulocytes.Counter-flow elutriation allows for the closed system separation of lymphocytes from monocytes and granulocytes. We investigated the use of PBMC concentrates enriched for lymphocytes using elutriation for manufacturing 8 CD19- and 5 GD2-CAR T cell products.When compared to PBMC concentrates, lymphocyte-enriched elutriation fractions contained greater proportions of CD3+ and CD56+ cells and reduced proportions of CD14+ and CD15+ cells. All 13 CAR T cell products manufactured using the elutriated lymphocytes yielded sufficient quantities of transduced CAR T cells to meet clinical dose criteria. The GD2-CAR T cell products contained significantly more T cells and transduced T cells than the CD19-CAR T cell products. A comparison of the yields of CAR T cells produced from elutriated lymphocytes with the yields of CAR T cells previous produced from cells isolated from PBMC concentrates by anti-CD3/CD28 bead selection or by anti-CD3/CD28 bead selection plus plastic adherence found that greater quantities of GD2-CAR T cells were produced from elutriated lymphocytes, but not CD19-CAR T cells.Enrichment of PBMC concentrates for lymphocytes using elutriation increased the quantity of GD2-CAR T cells produced. These results provide further evidence that CAR T cell expansion is inhibited by monocytes and granulocytes.
View details for DOI 10.1186/s12967-017-1160-5
View details for Web of Science ID 000396942100001
View details for PubMedID 28298232
New developments in immunotherapy for pediatric solid tumors.
Current opinion in pediatrics
Building upon preclinical advances, we are uncovering immunotherapy strategies that are translating into improved outcomes in tumor subsets. Advanced pediatric solid tumors carry poor prognoses and resultant robust efforts to apply immunotherapy advances to pediatric solid tumors are in progress. Here, we discuss recent developments in the field using mAb and mAb-based therapies including checkpoint blockade and chimeric antigen receptors (CARs).The pediatric solid tumor mAb experience targeting the diganglioside, GD2, for patients with neuroblastoma has been the most compelling to date. GD2 and alternative antigen-specific mAbs are now being incorporated into antibody-drug conjugates, bispecific antibodies and CARs for treatment of solid tumors. CARs in pediatric solid tumors have not yet achieved comparative responses to the hematologic CAR experience; however, novel strategies such as bispecific targeting, intratumoral administration and improved understanding of T-cell biology may yield enhanced CAR-efficacy. Therapeutic effect using single-agent checkpoint blocking antibodies in pediatric solid tumors also remains limited to date. Combinatorial strategies continue to hold promise and the clinical effect in tumor subsets with high antigenic burden is being explored.Pediatric immunotherapy remains at early stages of translation, yet we anticipate that with advanced technology, we will achieve widespread, efficacious use of immunotherapy for pediatric solid tumors.
View details for DOI 10.1097/MOP.0000000000000564
View details for PubMedID 29189429
Assessment of programmed death-ligand 1 expression and tumor-associated immune cells in pediatric cancer tissues.
Programmed death 1 (PD-1) signaling in the tumor microenvironment dampens immune responses to cancer, and blocking this axis induces antitumor effects in several malignancies. Clinical studies of PD-1 blockade are only now being initiated in pediatric patients, and little is known regarding programmed death-ligand 1 (PD-L1) expression in common childhood cancers. The authors characterized PD-L1 expression and tumor-associated immune cells (TAICs) (lymphocytes and macrophages) in common pediatric cancers.Whole slide sections and tissue microarrays were evaluated by immunohistochemistry for PD-L1 expression and for the presence of TAICs. TAICs were also screened for PD-L1 expression.Thirty-nine of 451 evaluable tumors (9%) expressed PD-L1 in at least 1% of tumor cells. The highest frequency histotypes comprised Burkitt lymphoma (80%; 8 of 10 tumors), glioblastoma multiforme (36%; 5 of 14 tumors), and neuroblastoma (14%; 17 of 118 tumors). PD-L1 staining was associated with inferior survival among patients with neuroblastoma (P = .004). Seventy-four percent of tumors contained lymphocytes and/or macrophages. Macrophages were significantly more likely to be identified in PD-L1-positive versus PD-L1-negative tumors (P < .001).A subset of diagnostic pediatric cancers exhibit PD-L1 expression, whereas a much larger fraction demonstrates infiltration with tumor-associated lymphocytes. PD-L1 expression may be a biomarker for poor outcome in neuroblastoma. Further preclinical and clinical investigation will define the predictive nature of PD-L1 expression in childhood cancers both at diagnosis and after exposure to chemoradiotherapy. Cancer 2017. © 2017 American Cancer Society.
View details for DOI 10.1002/cncr.30724
View details for PubMedID 28608950
Tumor Antigen and Receptor Densities Regulate Efficacy of a Chimeric Antigen Receptor Targeting Anaplastic Lymphoma Kinase.
Molecular therapy : the journal of the American Society of Gene Therapy
We explored the utility of targeting anaplastic lymphoma kinase (ALK), a cell surface receptor overexpressed on pediatric solid tumors, using chimeric antigen receptor (CAR)-based immunotherapy. T cells expressing a CAR incorporating the single-chain variable fragment sequence of the ALK48 mAb linked to a 4-1BB-CD3ζ signaling domain lysed ALK-expressing tumor lines and produced interferon-gamma upon antigen stimulation but had limited anti-tumor efficacy in two xenograft models of human neuroblastoma. Further exploration demonstrated that cytokine production was highly dependent upon ALK target density and that target density of ALK on neuroblastoma cell lines was insufficient for maximal activation of CAR T cells. In addition, ALK CAR T cells demonstrated rapid and complete antigen-induced loss of receptor from the T cell surface via internalization. Using a model that simultaneously modulated antigen density and CAR expression, we demonstrated that CAR functionality is regulated by target antigen and CAR density and that low expression of either contributes to limited anti-tumor efficacy of the ALK CAR. These data suggest that stoichiometric relationships between CAR receptors and target antigens may significantly impact the anti-tumor efficacy of CAR T cells and that manipulation of these parameters could allow precise tuning of CAR T cell activity.
View details for DOI 10.1016/j.ymthe.2017.06.008
View details for PubMedID 28676342
Identification of GPC2 as an Oncoprotein and Candidate Immunotherapeutic Target in High-Risk Neuroblastoma.
2017; 32 (3): 295–309.e12
We developed an RNA-sequencing-based pipeline to discover differentially expressed cell-surface molecules in neuroblastoma that meet criteria for optimal immunotherapeutic target safety and efficacy. Here, we show that GPC2 is a strong candidate immunotherapeutic target in this childhood cancer. We demonstrate high GPC2 expression in neuroblastoma due to MYCN transcriptional activation and/or somatic gain of the GPC2 locus. We confirm GPC2 to be highly expressed on most neuroblastomas, but not detectable at appreciable levels in normal childhood tissues. In addition, we demonstrate that GPC2 is required for neuroblastoma proliferation. Finally, we develop a GPC2-directed antibody-drug conjugate that is potently cytotoxic to GPC2-expressing neuroblastoma cells. Collectively, these findings validate GPC2 as a non-mutated neuroblastoma oncoprotein and candidate immunotherapeutic target.
View details for DOI 10.1016/j.ccell.2017.08.003
View details for PubMedID 28898695
View details for PubMedCentralID PMC5600520
CNS Endothelial Cell Activation Emerges as a Driver of CAR T Cell-Associated Neurotoxicity.
2017; 7 (12): 1371–73
Central nervous system (CNS) toxicity associated with chimeric antigen receptor-based therapeutics has emerged as a significant cause of morbidity and mortality, and insights into the pathophysiology of this syndrome have been lacking. A new study provides evidence that cytokine-induced CNS endothelial cell activation leading to disruption of the blood-brain barrier plays an early and critical role in this phenomenon. These insights provide new opportunities for targeted therapeutic interventions to modulate endothelial cell activation. Cancer Discov; 7(12); 1371-3. ©2017 AACRSee related article by Gust et al., p. 1404.
View details for DOI 10.1158/2159-8290.CD-17-1084
View details for PubMedID 29208775
CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy.
Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent effects in relapsed and/or refractory pre-B cell acute lymphoblastic leukemia (B-ALL), but antigen loss is a frequent cause of resistance to CD19-targeted immunotherapy. CD22 is also expressed in most cases of B-ALL and is usually retained following CD19 loss. We report results from a phase 1 trial testing a new CD22-targeted CAR (CD22-CAR) in 21 children and adults, including 17 who were previously treated with CD19-directed immunotherapy. Dose-dependent antileukemic activity was observed, with complete remission obtained in 73% (11/15) of patients receiving ≥1 × 106 CD22-CAR T cells per kg body weight, including 5 of 5 patients with CD19dim or CD19- B-ALL. Median remission duration was 6 months. Relapses were associated with diminished CD22 site density that likely permitted CD22+ cell escape from killing by CD22-CAR T cells. These results are the first to establish the clinical activity of a CD22-CAR in B-ALL, including leukemia resistant to anti-CD19 immunotherapy, demonstrating potency against B-ALL comparable to that of CD19-CAR at biologically active doses. Our results also highlight the critical role played by antigen density in regulating CAR function.
View details for DOI 10.1038/nm.4441
View details for PubMedID 29155426
Reduction of MDSCs with All-trans Retinoic Acid Improves CAR Therapy Efficacy for Sarcomas
CANCER IMMUNOLOGY RESEARCH
2016; 4 (10): 869-880
Genetically engineered T cells expressing CD19-specific chimeric antigen receptors (CAR) have shown impressive activity against B-cell malignancies, and preliminary results suggest that T cells expressing a first-generation disialoganglioside (GD2)-specific CAR can also provide clinical benefit in patients with neuroblastoma. We sought to assess the potential of GD2-CAR therapies to treat pediatric sarcomas. We observed that 18 of 18 (100%) of osteosarcomas, 2 of 15 (13%) of rhabdomyosarcomas, and 7 of 35 (20%) of Ewing sarcomas expressed GD2. T cells engineered to express a third-generation GD2-CAR incorporating the 14g2a-scFv with the CD28, OX40, and CD3ζ signaling domains (14g2a.CD28.OX40.ζ) mediated efficient and comparable lysis of both GD2(+) sarcoma and neuroblastoma cell lines in vitro However, in xenograft models, GD2-CAR T cells had no antitumor effect against GD2(+) sarcoma, despite effectively controlling GD2(+) neuroblastoma. We observed that pediatric sarcoma xenografts, but not neuroblastoma xenografts, induced large populations of monocytic and granulocytic murine myeloid-derived suppressor cells (MDSC) that inhibited human CAR T-cell responses in vitro Treatment of sarcoma-bearing mice with all-trans retinoic acid (ATRA) largely eradicated monocytic MDSCs and diminished the suppressive capacity of granulocytic MDSCs. Combined therapy using GD2-CAR T cells plus ATRA significantly improved antitumor efficacy against sarcoma xenografts. We conclude that retinoids provide a clinically accessible class of agents capable of diminishing the suppressive effects of MDSCs, and that co-administration of retinoids may enhance the efficacy of CAR therapies targeting solid tumors. Cancer Immunol Res; 4(10); 869-80. ©2016 AACR.
View details for DOI 10.1158/2326-6066.CIR-15-0230
View details for Web of Science ID 000385632900007
View details for PubMedID 27549124
View details for PubMedCentralID PMC5050151
Induction of Immune Response after Allogeneic Wilms' Tumor 1 Dendritic Cell Vaccination and Donor Lymphocyte Infusion in Patients with Hematologic Malignancies and Post-Transplantation Relapse.
Biology of blood and marrow transplantation
Relapse of hematologic malignancies is the primary cause of treatment failure after allogeneic hematopoietic stem cell transplantation (HCT). Treatment for post-HCT relapse using donor lymphocyte infusion (DLI) has limited utility, particularly in the setting of acute leukemia, and can result in the development of graft-versus-host disease (GVHD). The Wilms' tumor 1 (WT1) gene product is a tumor-associated antigen that is expressed in acute leukemia and other hematologic malignancies, with limited expression in normal tissues. In this pilot trial, we assessed safety and feasibility of a WT1 peptide-loaded donor-derived dendritic cell (DC) vaccine given with DLI designed to enhance and direct the graft-versus-leukemia effect. Secondary objectives were to evaluate immunologic and clinical responses. A total of 5 subjects, median age 17 years (range, 9 to 19 years), with post-HCT relapse were enrolled. Disease subtypes included acute lymphoblastic leukemia (n = 3), acute myelogenous leukemia (n = 1), and Hodgkin lymphoma (n = 1). Successful vaccine production was feasible from all donors. DC vaccination and DLI were well tolerated. One recipient developed grade 1 skin GVHD not requiring systemic therapy. The most common adverse events included grade 1 reversible pain and pruritus at the vaccine injection and delayed-type hypersensitivity (DTH) skin testing sites. There were no grade 3 or higher adverse events related to the research. Immune responses consisted of ELISpot response in 3 recipients and positive DTH tests to WT1 peptide cocktail in 2 subjects. Our study provides 1 of the first attempts to apply tumor-specific vaccine therapy to the allogeneic setting. Preliminary results show the DC-based vaccination is safe and feasible after allogeneic HCT, with a suggestion that this approach can be used to sensitize the repopulated allogeneic-donor immune system to WT1. Future directions may include testing of vaccination strategies in the early post-transplantation setting for relapse prevention.
View details for DOI 10.1016/j.bbmt.2016.08.028
View details for PubMedID 27634018
Impact of Two Measures of Micrometastatic Disease on Clinical Outcomes in Patients with Newly Diagnosed Ewing Sarcoma: A Report from the Children's Oncology Group.
Clinical cancer research
2016; 22 (14): 3643-3650
Flow cytometry and RT-PCR can detect occult Ewing sarcoma cells in the blood and bone marrow. These techniques were used to evaluate the prognostic significance of micrometastatic disease in Ewing sarcoma.Newly diagnosed patients with Ewing sarcoma were enrolled on two prospective multicenter studies. In the flow cytometry cohort, patients were defined as "positive" for bone marrow micrometastatic disease if their CD99(+)/CD45(-) values were above the upper limit in 22 control patients. In the PCR cohort, RT-PCR on blood or bone marrow samples classified the patients as "positive" or "negative" for EWSR1/FLI1 translocations. The association between micrometastatic disease burden with clinical features and outcome was assessed. Coexpression of insulin-like growth factor-1 receptor (IGF-1R) on detected tumor cells was performed in a subset of flow cytometry samples.The median total bone marrow CD99(+)CD45(-) percent was 0.0012% (range 0%-1.10%) in the flow cytometry cohort, with 14 of 109 (12.8%) of Ewing sarcoma patients defined as "positive." In the PCR cohort, 19.6% (44/225) patients were "positive" for any EWSR1/FLI1 translocation in blood or bone marrow. There were no differences in baseline clinical features or event-free or overall survival between patients classified as "positive" versus "negative" by either method. CD99(+)CD45(-) cells had significantly higher IGF-1R expression compared with CD45(+) hematopoietic cells (mean geometric mean fluorescence intensity 982.7 vs. 190.9; P < 0.001).The detection of micrometastatic disease at initial diagnosis by flow cytometry or RT-PCR is not associated with outcome in newly diagnosed patients with Ewing sarcoma. Flow cytometry provides a tool to characterize occult micrometastatic tumor cells for proteins of interest. Clin Cancer Res; 1-8. ©2016 AACR.
View details for DOI 10.1158/1078-0432.CCR-15-2516
View details for PubMedID 26861456
Adjuvant Immunotherapy to Improve Outcome in High-Risk Pediatric Sarcomas.
Clinical cancer research
2016; 22 (13): 3182-3191
Patients with metastatic or relapsed pediatric sarcomas receive cytotoxic regimens that induce high remission rates associated with profound lymphocyte depletion, but ultimately few survive long term. We administered adjuvant immunotherapy to patients with metastatic and recurrent pediatric sarcomas in an effort to improve outcomes.Mononuclear cells were collected via apheresis, and tumor lysate was acquired via percutaneous biopsy at enrollment. Participants received standard antineoplastic therapy, followed by autologous lymphocytes, tumor lysate/keyhole limpet hemocyanin-pulsed dendritic cell vaccinations ± recombinant human IL7. Primary outcomes were toxicity and vaccine responses. Secondary outcomes were immune reconstitution, event-free survival, and overall survival (OS).Forty-three patients enrolled and 29 received immunotherapy. The regimen was well tolerated. Intent-to-treat analysis demonstrated 5-year OS of 51% with significant differences based upon histologic group (63% vs. 0% for Ewing/rhabdomyosarcoma vs. other sarcomas) and response to standard therapy (74% no residual disease vs. 0% residual disease). Five-year intent-to-treat OS of patients with newly diagnosed metastatic Ewing/rhabdomyosarcoma was 77%, higher than previously reported in this population and higher than observed in a similar group treated with an earlier adjuvant immunotherapy regimen (25% 5-year OS). T-cell responses to autologous tumor lysate were identified in 62% of immunotherapy recipients, and survival was higher in those patients (73% 5-year OS with vs. 37% without immune response,P= 0.017). Immune reconstitution, measured by CD4 count recovery, was significantly enhanced in subjects treated with recombinant human IL7.Adjuvant immunotherapy may improve survival in patients with metastatic pediatric sarcoma.Clin Cancer Res; 1-10. ©2016 AACR.
View details for DOI 10.1158/1078-0432.CCR-15-2550
View details for PubMedID 26823601
CD19 CAR immune pressure induces B-precursor acute lymphoblastic leukaemia lineage switch exposing inherent leukaemic plasticity
Adoptive immunotherapy using chimeric antigen receptor (CAR) expressing T cells targeting the CD19 B lineage receptor has demonstrated marked success in relapsed pre-B-cell acute lymphoblastic leukaemia (ALL). Persisting CAR-T cells generate sustained pressure against CD19 that may drive unique mechanisms of resistance. Pre-B ALL originates from a committed pre-B cell or an earlier progenitor, with potential to reprogram into other hematopoietic lineages. Here we report changes in lineage markers including myeloid conversion in patients following CD19 CAR therapy. Using murine ALL models we study the long-term effects of CD19 CAR-T cells and demonstrate partial or complete lineage switch as a consistent mechanism of CAR resistance depending on the underlying genetic oncogenic driver. Deletion of Pax5 or Ebf1 recapitulates lineage reprogramming occurring during CD19 CAR pressure. Our findings establish lineage switch as a mechanism of CAR resistance exposing inherent plasticity in genetic subtypes of pre-B-cell ALL.
View details for DOI 10.1038/ncomms12320
View details for Web of Science ID 000380538200001
View details for PubMedID 27460500
Myeloid cells in peripheral blood mononuclear cell concentrates inhibit the expansion of chimeric antigen receptor T cells
2016; 18 (7): 893-901
Autologous chimeric antigen receptor (CAR) T-cell therapies have shown promising clinical outcomes, but T-cell yields have been variable. CD19- and GD2-CAR T-cell manufacturing records were reviewed to identify sources of variability.CD19-CAR T cells were used to treat 43 patients with acute lymphocytic leukemia or lymphoma and GD2-CAR T cells to treat eight patients with osteosarcoma and three with neuroblastoma. Both types of CAR T cells were manufactured using autologous peripheral blood mononuclear cells (PBMC) concentrates and anti-CD3/CD28 beads for T-cell enrichment and simulation.A comparison of the first 6 GD2- and the first 22 CD19-CAR T-cell products manufactured revealed that GD2-CAR T-cell products contained fewer transduced cells than CD19-CAR T-cell products (147 ± 102 × 10(6) vs 1502 ± 1066 × 10(6); P = 0.0059), and their PBMC concentrates contained more monocytes (31.4 ± 12.4% vs 18.5 ± 13.7%; P = 0.019). Among the first 28 CD19-CAR T-cell products manufactured, four had poor expansion yielding less than 1 × 10(6) transduced T cells per kilogram. When PBMC concentrates from these four patients were compared with the 24 others, PBMC concentrates of poorly expanding products contained greater quantities of monocytes (39.8 ± 12.9% vs. 15.3 ± 10.8%, P = 0.0014). Among the patients whose CD19-CAR T cells expanded poorly, manufacturing for two patients was repeated using cryopreserved PBMC concentrates but incorporating a monocyte depleting plastic adherence step, and an adequate dose of CAR T cells was produced for both patients.Variability in CAR T-cell expansion is due, at least in part, to the contamination of the starting PBMC concentrates with monocytes.
View details for DOI 10.1016/j.jcyt.2016.04.003
View details for Web of Science ID 000376886900009
View details for PubMedID 27210719
Current state of pediatric sarcoma biology and opportunities for future discovery: A report from the sarcoma translational research workshop.
2016; 209 (5): 182-194
Sarcomas are a rare subgroup of pediatric cancers comprised of a variety of bone and soft-tissue tumors. While significant advances have been made in improving outcomes of patients with localized pediatric sarcomas since the addition of systemic chemotherapy to local control many decades ago, outcomes for patients with metastatic and relapsed sarcoma remain poor with few novel therapeutics identified to date. With the advent of new technologies to study cancer genomes, transcriptomes and epigenomes, our understanding of sarcoma biology has improved tremendously in a relatively short period of time. However, much remains to be accomplished in this arena especially with regard to translating all of this new knowledge to the bedside. To this end, a meeting was convened in Philadelphia, PA, on April 18, 2015 sponsored by the QuadW foundation, Children's Oncology Group and CureSearch for Children's Cancer that brought together sarcoma clinicians and scientists from North America to review the current state of pediatric sarcoma biology and ongoing/planned genomics based clinical trials in an effort to identify and bridge knowledge gaps that continue to exist at present. At the conclusion of the workshop, three key objectives that would significantly further our understanding of sarcoma were identified and a proposal was put forward to develop an all-encompassing pediatric sarcoma biology protocol that would address these specific needs. This review summarizes the proceedings of the workshop.
View details for DOI 10.1016/j.cancergen.2016.03.004
View details for PubMedID 27132463
Activation of Hematopoietic Stem/Progenitor Cells Promotes Immunosuppression Within the Pre-metastatic Niche.
2016; 76 (6): 1335-1347
Metastatic tumors have been shown to establish microenvironments in distant tissues that are permissive to disseminated tumor cells. Hematopoietic cells contribute to this microenvironment, yet the precise initiating events responsible for establishing the pre-metastatic niche remain unclear. Here, we tracked the developmental fate of hematopoietic stem and progenitor cells (HSPC) in tumor-bearing mice. We show that a distant primary tumor drives the expansion of HSPCs within the bone marrow and their mobilization to the bloodstream. Treatment of purified HSPCs cultured ex vivo with tumor-conditioned media induced their proliferation as well as their differentiation into immunosuppressive myeloid cells. We furthered tracked purified HSPCs in vivo and found they differentiated into myeloid-derived suppressor cells in early metastatic sites of tumor-bearing mice. The number of CD11b(+)Ly6g(+) cells in metastatic sites was significantly increased by HSPC mobilization and decreased if tumor-mediated mobilization was inhibited. Moreover, pharmacologic mobilization of HSPCs increased metastasis, whereas depletion of Gr1(+) cells abrogated the metastasis-promoting effects of HSPC mobilization. Finally, we detected elevated levels of HSPCs in the circulation of newly diagnosed cancer patients, which correlated with increased risk for metastatic progression. Taken together, our results highlight bone marrow activation as one of the earliest steps of the metastatic process and identify circulating HSPCs as potential clinical indicators of metastatic niche formation. Cancer Res; 76(6); 1335-47. ©2015 AACR.
View details for DOI 10.1158/0008-5472.CAN-15-0204
View details for PubMedID 26719537
Phase I Clinical Trial of Ipilimumab in Pediatric Patients with Advanced Solid Tumors
CLINICAL CANCER RESEARCH
2016; 22 (6): 1364-1370
Ipilimumab is a first-in-class immune checkpoint inhibitor approved for treatment of metastatic melanoma but not studied in children until this phase I protocol.This study examined safety, pharmacokinetics, and immunogenicity, and immune correlates of ipilimumab administered to subjects ≤21 years old with recurrent or progressive solid tumors. Dose escalation cohorts received 1, 3, 5, or 10 mg/m(2) intravenously every 3 weeks in a 3 + 3 design. Response was assessed after 6 weeks and 12 weeks, and then every 3 months. Treatment was continued until disease progression or unacceptable toxicity.Thirty-three patients received 72 doses of ipilimumab. Patients enrolled had melanoma (n = 12), sarcoma (n = 17), or other refractory solid tumors (n = 4). Immune-related adverse events included pancreatitis, pneumonitis, colitis, endocrinopathies, and transaminitis with dose-limiting toxicities observed at 5 and 10 mg/kg dose levels. Pharmacokinetics revealed a half-life of 8 to 15 days. At day 21, subjects had increased levels of cycling T cells, but no change in regulatory T-cell populations. Six subjects had confirmed stable disease for 4 to 10 cycles (melanoma, osteosarcoma, clear cell sarcoma, and synovial sarcoma).Ipilimumab was safely administered to pediatric patients using management algorithms for immune-related toxicities. The spectrum of immune-related adverse events is similar to those described in adults; however, many of the pediatric toxicities were evident after a single dose. Although no objective tumor regressions were observed with ipilimumab as a single agent, subjects with immune-related toxicities had an increased overall survival compared with those who showed no evidence of breaking tolerance. Clin Cancer Res; 22(6); 1364-70. ©2015 AACR.
View details for DOI 10.1158/1078-0432.CCR-15-0491
View details for Web of Science ID 000373358900011
View details for PubMedID 26534966
Nature reviews. Disease primers
2016; 2: 16078-?
Neuroblastoma is the most common extracranial solid tumour occurring in childhood and has a diverse clinical presentation and course depending on the tumour biology. Unique features of these neuroendocrine tumours are the early age of onset, the high frequency of metastatic disease at diagnosis and the tendency for spontaneous regression of tumours in infancy. The most malignant tumours have amplification of the MYCN oncogene (encoding a transcription factor), which is usually associated with poor survival, even in localized disease. Although transgenic mouse models have shown that MYCN overexpression can be a tumour-initiating factor, many other cooperating genes and tumour suppressor genes are still under investigation and might also have a role in tumour development. Segmental chromosome alterations are frequent in neuroblastoma and are associated with worse outcome. The rare familial neuroblastomas are usually associated with germline mutations in ALK, which is mutated in 10-15% of primary tumours, and provides a potential therapeutic target. Risk-stratified therapy has facilitated the reduction of therapy for children with low-risk and intermediate-risk disease. Advances in therapy for patients with high-risk disease include intensive induction chemotherapy and myeloablative chemotherapy, followed by the treatment of minimal residual disease using differentiation therapy and immunotherapy; these have improved 5-year overall survival to 50%. Currently, new approaches targeting the noradrenaline transporter, genetic pathways and the tumour microenvironment hold promise for further improvements in survival and long-term quality of life.
View details for DOI 10.1038/nrdp.2016.78
View details for PubMedID 27830764
Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy
2015; 5 (12): 1282-1295
The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms.CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.
View details for DOI 10.1158/2159-8290.CD-15-1020
View details for Web of Science ID 000368577500024
View details for PubMedID 26516065
4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors
2015; 21 (6): 581-590
Chimeric antigen receptors (CARs) targeting CD19 have mediated dramatic antitumor responses in hematologic malignancies, but tumor regression has rarely occurred using CARs targeting other antigens. It remains unknown whether the impressive effects of CD19 CARs relate to greater susceptibility of hematologic malignancies to CAR therapies, or superior functionality of the CD19 CAR itself. We show that tonic CAR CD3-ζ phosphorylation, triggered by antigen-independent clustering of CAR single-chain variable fragments, can induce early exhaustion of CAR T cells that limits antitumor efficacy. Such activation is present to varying degrees in all CARs studied, except the highly effective CD19 CAR. We further determine that CD28 costimulation augments, whereas 4-1BB costimulation reduces, exhaustion induced by persistent CAR signaling. Our results provide biological explanations for the antitumor effects of CD19 CARs and for the observations that CD19 CAR T cells incorporating the 4-1BB costimulatory domain are more persistent than those incorporating CD28 in clinical trials.
View details for DOI 10.1038/nm.3838
View details for Web of Science ID 000355778300014
View details for PubMedID 25939063
Thymic expression of a T-cell receptor targeting a tumor-associated antigen coexpressed in the thymus induces T-ALL
2015; 125 (19): 2958-2967
T-cell receptors (TCRs) and chimeric antigen receptors recognizing tumor-associated antigens (TAAs) can now be engineered to be expressed on a wide array of immune effectors. Engineered receptors targeting TAAs have most commonly been expressed on mature T cells, however, some have postulated that receptor expression on immune progenitors could yield T cells with enhanced potency. We generated mice (survivin-TCR-transgenic [Sur-TCR-Tg]) expressing a TCR recognizing the immunodominant epitope (Sur20-28) of murine survivin during early stages of thymopoiesis. Spontaneous T-cell acute lymphoblastic leukemia (T-ALL) occurred in 100% of Sur-TCR-Tg mice derived from 3 separate founders. The leukemias expressed the Sur-TCR and signaled in response to the Sur20-28 peptide. In preleukemic mice, we observed increased cycling of double-negative thymocytes expressing the Sur-TCR and increased nuclear translocation of nuclear factor of activated T cells, consistent with TCR signaling induced by survivin expression in the murine thymus. β2M(-/-) Sur-TCR-Tg mice, which cannot effectively present survivin peptides on class I major histocompatibility complex, had significantly diminished rates of leukemia. We conclude that TCR signaling during the early stages of thymopoiesis mediates an oncogenic signal, and therefore expression of signaling receptors on developing thymocytes with specificity for TAAs expressed in the thymus could pose a risk for neoplasia, independent of insertional mutagenesis.
View details for DOI 10.1182/blood-2014-10-609271
View details for Web of Science ID 000355687500016
View details for PubMedID 25814528
Going Back to Class I: MHC and Immunotherapies for Childhood Cancer
PEDIATRIC BLOOD & CANCER
2015; 62 (4): 571-576
After decades of unfulfilled promise, immunotherapies for cancer have reached a tipping point, with several FDA approved products now on the market and many more showing promise in both adult and pediatric clinical trials. Tumor cell expression of MHC class I has emerged as a potential determinant of the therapeutic success of many immunotherapy approaches. Here we review current knowledge regarding MHC class I expression in pediatric cancers including a discussion of prognostic significance, the opposing influence of MHC on T-cell versus NK-mediated therapies, and strategies to reverse or circumvent MHC down-regulation.
View details for DOI 10.1002/pbc.25359
View details for Web of Science ID 000349985300005
View details for PubMedID 25524394
T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial
2015; 385 (9967): 517-528
Chimeric antigen receptor (CAR) modified T cells targeting CD19 have shown activity in case series of patients with acute and chronic lymphocytic leukaemia and B-cell lymphomas, but feasibility, toxicity, and response rates of consecutively enrolled patients treated with a consistent regimen and assessed on an intention-to-treat basis have not been reported. We aimed to define feasibility, toxicity, maximum tolerated dose, response rate, and biological correlates of response in children and young adults with refractory B-cell malignancies treated with CD19-CAR T cells.This phase 1, dose-escalation trial consecutively enrolled children and young adults (aged 1-30 years) with relapsed or refractory acute lymphoblastic leukaemia or non-Hodgkin lymphoma. Autologous T cells were engineered via an 11-day manufacturing process to express a CD19-CAR incorporating an anti-CD19 single-chain variable fragment plus TCR zeta and CD28 signalling domains. All patients received fludarabine and cyclophosphamide before a single infusion of CD19-CAR T cells. Using a standard 3 + 3 design to establish the maximum tolerated dose, patients received either 1 × 10(6) CAR-transduced T cells per kg (dose 1), 3 × 10(6) CAR-transduced T cells per kg (dose 2), or the entire CAR T-cell product if sufficient numbers of cells to meet the assigned dose were not generated. After the dose-escalation phase, an expansion cohort was treated at the maximum tolerated dose. The trial is registered with ClinicalTrials.gov, number NCT01593696.Between July 2, 2012, and June 20, 2014, 21 patients (including eight who had previously undergone allogeneic haematopoietic stem-cell transplantation) were enrolled and infused with CD19-CAR T cells. 19 received the prescribed dose of CD19-CAR T cells, whereas the assigned dose concentration could not be generated for two patients (90% feasible). All patients enrolled were assessed for response. The maximum tolerated dose was defined as 1 × 10(6) CD19-CAR T cells per kg. All toxicities were fully reversible, with the most severe being grade 4 cytokine release syndrome that occurred in three (14%) of 21 patients (95% CI 3·0-36·3). The most common non-haematological grade 3 adverse events were fever (nine [43%] of 21 patients), hypokalaemia (nine [43%] of 21 patients), fever and neutropenia (eight [38%] of 21 patients), and cytokine release syndrome (three [14%) of 21 patients).CD19-CAR T cell therapy is feasible, safe, and mediates potent anti-leukaemic activity in children and young adults with chemotherapy-resistant B-precursor acute lymphoblastic leukaemia. All toxicities were reversible and prolonged B-cell aplasia did not occur.National Institutes of Health Intramural funds and St Baldrick's Foundation.
View details for DOI 10.1016/S0140-6736(14)61403-3
View details for Web of Science ID 000349213600030
View details for PubMedID 25319501
Acute GVHD in patients receiving IL-15/4-1BBL activated NK cells following T-cell-depleted stem cell transplantation
2015; 125 (5): 784-792
Natural killer (NK) cells can enhance engraftment and mediate graft-versus-leukemia following allogeneic hematopoietic stem cell transplantation (HSCT), but the potency of graft-versus-leukemia mediated by naturally reconstituting NK cells following HSCT is limited. Preclinical studies demonstrate that activation of NK cells using interleukin-15 (IL-15) plus 4-1BBL upregulates activating receptor expression and augments killing capacity. In an effort to amplify the beneficial effects of NK cells post-HSCT, we conducted a first-in-human trial of adoptive transfer of donor-derived IL-15/4-1BBL-activated NK cells (aNK-DLI) following HLA-matched, T-cell-depleted (1-2 × 10(4) T cells/kg) nonmyeloablative peripheral blood stem cell transplantation in children and young adults with ultra-high-risk solid tumors. aNK-DLI were CD3(+)-depleted, CD56(+)-selected lymphocytes, cultured for 9 to 11 days with recombinant human IL-15 plus 4-1BBL(+)IL-15Rα(+) artificial antigen-presenting cells. aNK-DLI demonstrated potent killing capacity and displayed high levels of activating receptor expression. Five of 9 transplant recipients experienced acute graft-versus-host disease (GVHD) following aNK-DLI, with grade 4 GVHD observed in 3 subjects. GVHD was more common in matched unrelated donor vs matched sibling donor recipients and was associated with higher donor CD3 chimerism. Given that the T-cell dose was below the threshold required for GVHD in this setting, we conclude that aNK-DLI contributed to the acute GVHD observed, likely by augmenting underlying T-cell alloreactivity. This trial was registered at www.clinicaltrials.gov as #NCT01287104.
View details for DOI 10.1182/blood-2014-07-592881
View details for Web of Science ID 000350814900012
View details for PubMedID 25452614
Emerging Immunotherapies for Cancer and Their Potential for Application in Pediatric Oncology.
Critical reviews in oncogenesis
2015; 20 (3-4): 315-327
After decades of basic research, immune-based therapeutics for the treatment of cancer are showing evidence of efficacy in clinical trials; several immunotherapeutics already incorporated into standard treatment regimens. Intensive research is underway to improve the efficacy of immunotherapeutics and to expand the application of immunotherapy to a wider array of cancers. The therapeutic options that comprise immunotherapy for cancer are vast and span monoclonal antibodies, tumor vaccines, adoptive cellular therapies, as well as therapies aimed at reversing immunosuppression and enhancing immune reactivity globally and/or locally within the tumor microenvironment. In pediatric cancer, monoclonal antibodies have demonstrated efficacy in hematologic malignancies, and neuroblastoma and bispecific antibodies that activate resident T cells, as well as adoptive cell therapy, have shown recent exciting results for the treatment of acute lymphoblastic leukemia in childhood. This review discusses the basic principles of tumor immunology driving clinical development of new immunotherapies, describes immunotherapeutics with demonstrated efficacy and several currently in clinical trials, and highlight agents that seem to be most promising for the treatment of pediatric cancer.
View details for PubMedID 26349422
Immune-based therapies for childhood cancer
NATURE REVIEWS CLINICAL ONCOLOGY
2014; 11 (12): 693-703
After decades of research, immunotherapies for cancer are demonstrating increasing success. These agents can amplify existent antitumour immunity or induce durable antitumour immune responses in a wide array of cancers. The spectrum of immunotherapeutics is broad, spanning monoclonal antibodies and their derivatives, tumour vaccines, and adoptive therapies using T cells and natural killer cells. Only a small number of immunotherapies have been tested in paediatric cancers, but impressive antitumour effects have already been observed. Mononclonal antibodies targeting GD2 that induce antibody-dependent cell-mediated cytotoxicity improve survival in high-risk neuroblastoma. Bi-specific monoclonal antibodies that simultaneously target CD19 and activate T cells can induce remission in acute B-cell lymphoblastic leukaemia (B-ALL) and adoptive immunotherapy using T cells genetically engineered to express chimeric antigen receptors targeting CD19 induce impressive responses in B-ALL. Efforts are underway to generate and test new immunotherapies in a wider array of paediatric cancers. Major challenges include a need to identify immunotherapy targets on the most lethal childhood cancers, to expand availability of technology-intense platforms, such as adoptive cell therapy, to optimize management of novel toxicities associated with this new class of cancer therapies and to determine how best to incorporate these therapies into standard treatment paradigms.
View details for DOI 10.1038/nrclinonc.2014.177
View details for Web of Science ID 000345566100005
View details for PubMedID 25348789
- T-Cell Immunotherapy: Looking Forward MOLECULAR THERAPY 2014; 22 (9): 1564-1574
Current concepts in the diagnosis and management of cytokine release syndrome
2014; 124 (2): 188-195
As immune-based therapies for cancer become potent, more effective, and more widely available, optimal management of their unique toxicities becomes increasingly important. Cytokine release syndrome (CRS) is a potentially life-threatening toxicity that has been observed following administration of natural and bispecific antibodies and, more recently, following adoptive T-cell therapies for cancer. CRS is associated with elevated circulating levels of several cytokines including interleukin (IL)-6 and interferon γ, and uncontrolled studies demonstrate that immunosuppression using tocilizumab, an anti-IL-6 receptor antibody, with or without corticosteroids, can reverse the syndrome. However, because early and aggressive immunosuppression could limit the efficacy of the immunotherapy, current approaches seek to limit administration of immunosuppressive therapy to patients at risk for life-threatening consequences of the syndrome. This report presents a novel system to grade the severity of CRS in individual patients and a treatment algorithm for management of CRS based on severity. The goal of our approach is to maximize the chance for therapeutic benefit from the immunotherapy while minimizing the risk for life threatening complications of CRS.
View details for DOI 10.1182/blood-2014-05-552729
View details for Web of Science ID 000342618600011
View details for PubMedID 24876563
Disruption of CXCR2-Mediated MDSC Tumor Trafficking Enhances Anti-PD1 Efficacy
SCIENCE TRANSLATIONAL MEDICINE
2014; 6 (237)
Suppression of the host's immune system plays a major role in cancer progression. Tumor signaling of programmed death 1 (PD1) on T cells and expansion of myeloid-derived suppressor cells (MDSCs) are major mechanisms of tumor immune escape. We sought to target these pathways in rhabdomyosarcoma (RMS), the most common soft tissue sarcoma of childhood. Murine RMS showed high surface expression of PD-L1, and anti-PD1 prevented tumor growth if initiated early after tumor inoculation; however, delayed anti-PD1 had limited benefit. RMS induced robust expansion of CXCR2(+)CD11b(+)Ly6G(hi) MDSCs, and CXCR2 deficiency prevented CD11b(+)Ly6G(hi) MDSC trafficking to the tumor. When tumor trafficking of MDSCs was inhibited by CXCR2 deficiency, or after anti-CXCR2 monoclonal antibody therapy, delayed anti-PD1 treatment induced significant antitumor effects. Thus, CXCR2(+)CD11b(+)Ly6G(hi) MDSCs mediate local immunosuppression, which limits the efficacy of checkpoint blockade in murine RMS. Human pediatric sarcomas also produce CXCR2 ligands, including CXCL8. Patients with metastatic pediatric sarcomas display elevated serum CXCR2 ligands, and elevated CXCL8 is associated with diminished survival in this population. We conclude that accumulation of MDSCs in the tumor bed limits the efficacy of checkpoint blockade in cancer. We also identify CXCR2 as a novel target for modulating tumor immune escape and present evidence that CXCR2(+)CD11b(+)Ly6G(hi) MDSCs are an important suppressive myeloid subset in pediatric sarcomas. These findings present a translatable strategy to improve the efficacy of checkpoint blockade by preventing trafficking of MDSCs to the tumor site.
View details for DOI 10.1126/scitranslmed.3007974
View details for Web of Science ID 000336668800007
View details for PubMedID 24848257
- Introduction to the series of reviews on "Antibody Derivatives as New Therapeutics for Hematologic Malignancies" BLOOD 2014; 123 (15): 2283-2284
Mass spectrometry in cancer biomarker research: a case for immunodepletion of abundant blood-derived proteins from clinical tissue specimens
BIOMARKERS IN MEDICINE
2014; 8 (2): 269-286
The discovery of clinically relevant cancer biomarkers using mass spectrometry (MS)-based proteomics has proven difficult, primarily because of the enormous dynamic range of blood-derived protein concentrations and the fact that the 22 most abundant blood-derived proteins constitute approximately 99% of the total plasma protein mass. Immunodepletion of clinical body fluid specimens (e.g., serum/plasma) for the removal of highly abundant proteins is a reasonable and reproducible solution. Often overlooked, clinical tissue specimens also contain a formidable amount of highly abundant blood-derived proteins present in tissue-embedded networks of blood/lymph capillaries and interstitial fluid. Hence, the dynamic range impediment to biomarker discovery remains a formidable obstacle, regardless of clinical sample type (solid tissue and/or body fluid). Thus, we optimized and applied simultaneous immunodepletion of blood-derived proteins from solid tissue and peripheral blood, using clear cell renal cell carcinoma as a model disease. Integrative analysis of data from this approach and genomic data obtained from the same type of tumor revealed concordant key pathways and protein targets germane to clear cell renal cell carcinoma. This includes the activation of the lipogenic pathway characterized by increased expression of adipophilin (PLIN2) along with 'cadherin switching', a phenomenon indicative of transcriptional reprogramming linked to renal epithelial dedifferentiation. We also applied immunodepletion of abundant blood-derived proteins to various tissue types (e.g., adipose tissue and breast tissue) showing unambiguously that the removal of abundant blood-derived proteins represents a powerful tool for the reproducible profiling of tissue proteomes. Herein, we show that the removal of abundant blood-derived proteins from solid tissue specimens is of equal importance to depletion of body fluids and recommend its routine use in the context of biological discovery and/or cancer biomarker research. Finally, this perspective presents the background, rationale and strategy for using tissue-directed high-resolution/accuracy MS-based shotgun proteomics to detect genuine tumor proteins in the peripheral blood of a patient diagnosed with nonmetastatic cancer, employing concurrent liquid chromatography-MS analysis of immunodepleted clinical tissue and blood specimens.
View details for DOI 10.2217/bmm.13.101
View details for Web of Science ID 000331212400023
View details for PubMedID 24521024
Bioinformatic description of immunotherapy targets for pediatric T-cell leukemia and the impact of normal gene sets used for comparison.
Frontiers in oncology
2014; 4: 134-?
Pediatric lymphoid leukemia has the highest cure rate of all pediatric malignancies, yet due to its prevalence, still accounts for the majority of childhood cancer deaths and requires long-term highly toxic therapy. The ability to target B-cell ALL with immunoglobulin-like binders, whether anti-CD22 antibody or anti-CD19 CAR-Ts, has impacted treatment options for some patients. The development of new ways to target B-cell antigens continues at rapid pace. T-cell ALL accounts for up to 20% of childhood leukemia but has yet to see a set of high-value immunotherapeutic targets identified. To find new targets for T-ALL immunotherapy, we employed a bioinformatic comparison to broad normal tissue arrays, hematopoietic stem cells (HSC), and mature lymphocytes, then filtered the results for transcripts encoding plasma membrane proteins. T-ALL bears a core T-cell signature and transcripts encoding TCR/CD3 components and canonical markers of T-cell development predominate, especially when comparison was made to normal tissue or HSC. However, when comparison to mature lymphocytes was also undertaken, we identified two antigens that may drive, or be associated with leukemogenesis; TALLA-1 and hedgehog interacting protein. In addition, TCR subfamilies, CD1, activation and adhesion markers, membrane-organizing molecules, and receptors linked to metabolism and inflammation were also identified. Of these, only CD52, CD37, and CD98 are currently being targeted clinically. This work provides a set of targets to be considered for future development of immunotherapies for T-ALL.
View details for DOI 10.3389/fonc.2014.00134
View details for PubMedID 24959420
T-cell adoptive immunotherapy for acute lymphoblastic leukemia
HEMATOLOGY-AMERICAN SOCIETY OF HEMATOLOGY EDUCATION PROGRAM
Substantial progress has been made in the treatment of precursor B-cell acute lymphoblastic leukemia (B-ALL), but recurrent disease remains a leading cause of death in children due to cancer and outcomes for adults with B-ALL remain poor. Recently, complete clinical responses have been observed in small numbers of patients with B-ALL treated with adoptive immunotherapy using T cells genetically engineered to express chimeric antigen receptors (CARs) targeting CD19, a cell surface molecule present in essentially all cases of B-ALL. Preclinical data suggest that CARs targeting CD22, another antigen present in the majority of B-ALL cases, are similarly potent. Several clinical studies already under way will soon more clearly define the rate of response to this novel therapy in B-ALL. Further work is needed to identify optimal platforms for CAR-based adoptive immunotherapy for leukemia, to establish guidelines for managing toxicity, and to determine whether the remissions induced by this approach can be rendered durable.
View details for Web of Science ID 000331900300050
View details for PubMedID 24319203
Simplified process for the production of anti-CD19-CAR-engineered T cells
2013; 15 (11): 1406-1415
Adoptive immunotherapy with the use of chimeric antigen receptor (CAR)-engineered T cells specific for CD19 has shown promising results for the treatment of B-cell lymphomas and leukemia. This therapy involves the transduction of autologous T cells with a viral vector and the subsequent cell expansion. We describe a new, simplified method to produce anti-CD19-CAR T cells.T cells were isolated from peripheral blood mononuclear cell (PBMC) with anti-CD3/anti-CD28 paramagnetic beads. After 2 days, the T cells were added to culture bags pre-treated with RetroNectin and loaded with the retroviral anti-CD19 CAR vector. The cells, beads and vector were incubated for 24 h, and a second transduction was then performed. No spinoculation was used. Cells were then expanded for an additional 9 days.The method was validated through the use of two PBMC products from a patient with B-cell chronic lymphoblastic leukemia and one PBMC product from a healthy subject. The two PBMC products from the patient with B-cell chronic lymphoblastic leukemia contained 11.4% and 12.9% T cells. The manufacturing process led to final products highly enriched in T cells with a mean CD3+ cell content of 98%, a mean expansion of 10.6-fold and a mean transduction efficiency of 68%. Similar results were obtained from the PBMCs of the first four patients with acute lymphoblastic leukemia treated at our institution.We developed a simplified, semi-closed system for the initial selection, activation, transduction and expansion of T cells with the use of anti-CD3/anti-CD28 beads and bags to produce autologous anti-CD19 CAR-transduced T cells to support an ongoing clinical trial.
View details for DOI 10.1016/j.jcyt.2013.06.003
View details for Web of Science ID 000325732800010
View details for PubMedID 23992830
A Pan-Inhibitor of DASH Family Enzymes Induces Immune-mediated Regression of Murine Sarcoma and Is a Potent Adjuvant to Dendritic Cell Vaccination and Adoptive T-cell Therapy
JOURNAL OF IMMUNOTHERAPY
2013; 36 (8): 400-411
Multimodality therapy consisting of surgery, chemotherapy, and radiation will fail in approximately 40% of patients with pediatric sarcomas and result in substantial long-term morbidity in those who are cured. Immunotherapeutic regimens for the treatment of solid tumors typically generate antigen-specific responses too weak to overcome considerable tumor burden and tumor suppressive mechanisms and are in need of adjuvant assistance. Previous work suggests that inhibitors of DASH (dipeptidyl peptidase IV activity and/or structural homologs) enzymes can mediate tumor regression by immune-mediated mechanisms. Herein, we demonstrate that the DASH inhibitor, ARI-4175, can induce regression and eradication of well-established solid tumors, both as a single agent and as an adjuvant to a dendritic cell (DC) vaccine and adoptive cell therapy (ACT) in mice implanted with the M3-9-M rhabdomyosarcoma cell line. Treatment with effective doses of ARI-4175 correlated with recruitment of myeloid (CD11b) cells, particularly myeloid DCs, to secondary lymphoid tissues and with reduced frequency of intratumoral monocytic (CD11bLy6-CLy6-G) myeloid-derived suppressor cells. In immunocompetent mice, combining ARI-4175 with a DC vaccine or ACT with tumor-primed T cells produced significant improvements in tumor responses against well-established M3-9-M tumors. In M3-9-M-bearing immunodeficient (Rag1) mice, ACT combined with ARI-4175 produced greater tumor responses and significantly improved survival compared with either treatment alone. These studies warrant the clinical investigation of ARI-4175 for treatment of sarcomas and other malignancies, particularly as an adjuvant to tumor vaccines and ACT.
View details for DOI 10.1097/CJI.0b013e3182a80213
View details for Web of Science ID 000324623100002
View details for PubMedID 23994886
- Q&A: Crystal Mackall, John Maris on Pediatrics CANCER DISCOVERY 2013; 3 (9): 961-961
Fibrocytes represent a novel MDSC subset circulating in patients with metastatic cancer
2013; 122 (7): 1105-1113
Fibrocytes are hematopoietic stem cell-derived fibroblast precursors that are implicated in chronic inflammation, fibrosis, and wound healing. Myeloid-derived suppressor cells (MDSCs) expand in cancer-bearing hosts and contribute to tumor immune evasion. They are typically described as CD11b⁺HLA-DR⁻ in humans. We report abnormal expansions of CD11b⁺HLA-DR⁺ myeloid cells in peripheral blood mononuclear fractions of subjects with metastatic pediatric sarcomas. Like classical fibrocytes, they display cell surface α smooth muscle actin, collagen I/V, and mediate angiogenesis. However, classical fibrocytes serve as antigen presenters and augment immune reactivity, whereas fibrocytes from cancer subjects suppressed anti-CD3-mediated T-cell proliferation, primarily via indoleamine oxidase (IDO). The degree of fibrocyte expansion observed in individual subjects directly correlated with the frequency of circulating GATA3⁺CD4⁺ cells (R = 0.80) and monocytes from healthy donors cultured with IL-4 differentiated into fibrocytes with the same phenotypic profile and immunosuppressive properties as those observed in patients with cancer. We thus describe a novel subset of cancer-induced myeloid-derived suppressor cells, which bear the phenotypic and functional hallmarks of fibrocytes but mediate immune suppression. These cells are likely expanded in response to Th2 immune deviation and may contribute to tumor progression via both immune evasion and angiogenesis.
View details for DOI 10.1182/blood-2012-08-449413
View details for Web of Science ID 000323392900009
View details for PubMedID 23757729
Highlights of the Third International Conference on Immunotherapy in Pediatric Oncology
PEDIATRIC HEMATOLOGY AND ONCOLOGY
2013; 30 (5): 349-366
The third International Conference on Immunotherapy in Pediatric Oncology was held in Frankfurt/Main, Germany, October 1-2, 2012. Major topics of the conference included (i) cellular therapies using antigen-specific and gene-modified T cells for targeting leukemia and pediatric solid tumors; (ii) overcoming hurdles and barriers with regard to immunogenicity, immune escape, and the role of tumor microenvironment; (iii) vaccine strategies and antigen presentation; (iv) haploidentical transplantation and innate immunity; (v) the role of immune cells in allogeneic transplantation; and (vi) current antibody/immunoconjugate approaches for the treatment of pediatric malignancies. During the past decade, major advances have been made in improving the efficacy of these modalities and regulatory hurdles have been taken. Nevertheless, there is still a long way to go to fully exploit the potential of immunotherapeutic strategies to improve the cure of children and adolescents with malignancies. This and future meetings will support new collaborations and insights for further translational and clinical immunotherapy studies.
View details for DOI 10.3109/08880018.2013.802106
View details for Web of Science ID 000321772400001
View details for PubMedID 23758210
Soluble IL7R alpha potentiates IL-7 bioactivity and promotes autoimmunity
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2013; 110 (19): E1761-E1770
Human soluble interleukin-7 receptor (sIL7R)α circulates in high molar excess compared with IL-7, but its biology remains unclear. We demonstrate that sIL7Rα has moderate affinity for IL-7 but does not bind thymic stromal lymphopoietin. Functionally, sIL7Rα competes with cell-associated IL-7 receptor to diminish excessive IL-7 consumption and, thus, enhances the bioactivity of IL-7 when the cytokine is limited, as it is presumed to be in vivo. IL-7 signaling in the presence of sIL7Rα also diminishes expression of CD95 and suppressor of cytokine signaling 1, both regulatory molecules. Murine models confirm diminished consumption of IL-7 in the presence of sIL7Rα and also demonstrate a potentiating effect of sIL7Rα on IL-7-mediated homeostatic expansion and experimental autoimmune encephalomyelitis exacerbation. In multiple sclerosis and several other autoimmune diseases, IL7R genotype influences susceptibility. We measured increased sIL7Rα levels, as well as increased IL-7 levels, in multiple sclerosis patients with the predisposing IL7R genotype, consistent with diminished IL-7 consumption in vivo. This work demonstrates that sIL7Rα potentiates IL-7 bioactivity and provides a basis to explain the increased risk of autoimmunity observed in individuals with genotype-induced elevations of sIL7Rα.
View details for DOI 10.1073/pnas.1222303110
View details for Web of Science ID 000319327700009
View details for PubMedID 23610432
Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia
2013; 121 (7): 1165-1174
Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3 constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL.
View details for DOI 10.1182/blood-2012-06-438002
View details for Web of Science ID 000314870700020
View details for PubMedID 23243285
- Enhancing Immune Reconstitution: From Bench to Bedside BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION 2013; 19 (1): S79-S83
- Enhancing immune reconstitution: from bench to bedside. Biology of blood and marrow transplantation 2013; 19 (1): S79-83
Phase I Trial and Pharmacokinetic Study of Lexatumumab in Pediatric Patients With Solid Tumors
JOURNAL OF CLINICAL ONCOLOGY
2012; 30 (33): 4141-4147
Lexatumumab is an agonistic, fully human monoclonal antibody against tumor necrosis factor-related apoptosis-inducing ligand receptor 2 with preclinical evidence of activity in pediatric solid tumors.This phase I dose-escalation study examined the safety, tolerability, pharmacokinetics, and immunogenicity of lexatumumab at doses up to, but not exceeding, the adult maximum-tolerated dose (3, 5, 8, and 10 mg/kg), administered once every 2 weeks to patients age≤21 years with recurrent or progressive solid tumors.Twenty-four patients received a total of 56 cycles of lexatumumab over all four planned dose levels. One patient had grade 2 pericarditis consistent with radiation recall, and one patient developed grade 3 pneumonia with hypoxia during the second cycle. Five patients experienced stable disease for three to 24 cycles. No patients experienced complete or partial response, but several showed evidence of antitumor activity, including one patient with recurrent progressive osteosarcoma who experienced resolution of clinical symptoms and positron emission tomography activity, ongoing more than 1 year off therapy. One patient with hepatoblastoma showed a dramatic biomarker response.Pediatric patients tolerate 10 mg/kg of lexatumumab administered once every 14 days, the maximum-tolerated dose identified in adults. The drug seems to mediate some clinical activity in pediatric solid tumors and may work with radiation to enhance antitumor effects.
View details for DOI 10.1200/JCO.2012.44.1055
View details for Web of Science ID 000311300000024
View details for PubMedID 23071222
IL-7 in human health and disease
SEMINARS IN IMMUNOLOGY
2012; 24 (3): 218-224
IL-7 plays many essential roles in human health and disease. Congenital deficiencies in IL-7 signaling result in profound immunodeficiency, polymorphisms in IL7Rα modulate susceptibility to autoimmune disease, and acquired somatic activating mutations in IL7Rα contribute to neoplastic transformation in B cell and T cell leukemia. In response to lymphopenia, IL-7 accumulates to supranormal levels, which alters T cell homeostasis by augmenting T cell reactivity toward self and cognate antigens. This physiologic response is now routinely exploited to improve the efficacy of adoptive cell therapies for cancer. Clinical trials of recombinant IL-7 have demonstrated safety and potent immunorestorative effects, and current studies are investigating whether rhIL-7 therapy can improve outcomes in chronic viral infection and in the context of cancer immunotherapies. Building upon the large fund of knowledge regarding the basic biology of IL-7, this review will discuss the many and varied roles of IL-7 in human health and disease.
View details for DOI 10.1016/j.smim.2012.02.005
View details for Web of Science ID 000304584300009
View details for PubMedID 22410365
The Future Is Now: Chimeric Antigen Receptors as New Targeted Therapies for Childhood Cancer
CLINICAL CANCER RESEARCH
2012; 18 (10): 2780-2790
Improved outcomes for children with cancer hinge on the development of new targeted therapies with acceptable short-term and long-term toxicity. Progress in basic, preclinical, and clinical arenas spanning cellular immunology, gene therapy, and cell-processing technologies have paved the way for clinical applications of chimeric antigen receptor-based therapies. This is a new form of targeted immunotherapy that merges the exquisite targeting specificity of monoclonal antibodies with the potent cytotoxicity, potential for expansion, and long-term persistence provided by cytotoxic T cells. Although this field is still in its infancy, clinical trials have already shown clinically significant antitumor activity in neuroblastoma, chronic lymphocytic leukemia, and B-cell lymphoma, and trials targeting a variety of other adult and pediatric malignancies are under way. Ongoing work is focused on identifying optimal tumor targets and elucidating and manipulating both cell- and host-associated factors to support expansion and persistence of the genetically engineered cells in vivo. In pediatric oncology, CD19 and GD2 are compelling antigens that have already been identified for targeting pre-B acute lymphoblastic leukemia and neuroblastoma, respectively, with this approach, but it is likely that other antigens expressed in a variety of childhood cancers will also soon be targeted using this therapy. The potential to target essentially any tumor-associated cell-surface antigen for which a monoclonal antibody can be made opens up an entirely new arena for targeted therapy of childhood cancer.
View details for DOI 10.1158/1078-0432.CCR-11-1920
View details for Web of Science ID 000304249800008
View details for PubMedID 22589486
Reduced-Intensity Allogeneic Stem Cell Transplantation in Children and Young Adults with Ultrahigh-Risk Pediatric Sarcomas
BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION
2012; 18 (5): 698-707
Some subsets of pediatric sarcoma patients have very poor survival rates. We sought to determine the feasibility and efficacy of allogeneic hematopoietic stem cell transplantation (alloHSCT) in pediatric sarcoma populations with <25% predicted overall survival (OS). Patients with ultrahigh-risk Ewing's sarcoma family of tumors (ESFT), alveolar rhabdomyosarcoma, or desmoplastic small round cell tumors received EPOCH-fludarabine induction, a cyclophosphamide/fludarabine/melphalan preparative regimen, and HLA matched related peripheral blood stem cells. Thirty patients enrolled; 7 did not undergo alloHSCT because of progressive disease with diminishing performance status during induction. All 23 alloHSCT recipients experienced rapid full-donor engraftment, with no peritransplantation mortality. Five of 23 alloHSCT recipients (22%) remain alive (OS of 30% by Kaplan-Meier analysis at 3 years), including 3 of 7 (42%) transplanted without overt disease (median survival 14.5 versus 29.0 months from alloHSCT for patients transplanted with versus without overt disease, respectively). Among the 28 patients who progressed on the study, the median survival from date of progression was 1.9 months for the 7 who did not receive a transplant compared with 11.4 months for the 21 transplanted (P = .0003). We found prolonged survival after posttransplantation progression with several patients exhibiting indolent tumor growth. We also saw several patients with enhanced antitumor effects from posttransplantation chemotherapy (objective response to pretransplantation EPOCH-F was 24% versus 67% to posttransplantation EOCH); however, this was associated with increased toxicity. This largest reported series of alloHSCT in sarcomas demonstrates that alloHSCT is safe in this population, and that patients undergoing alloHSCT without overt disease show higher survival rates than reported using standard therapies. Enhanced chemo- and radiosensitivity of tumors and normal tissues was observed posttransplantation.
View details for DOI 10.1016/j.bbmt.2011.08.020
View details for Web of Science ID 000303558500007
View details for PubMedID 21896345
Identification of cell surface proteins as potential immunotherapy targets in 12 pediatric cancers.
Frontiers in oncology
2012; 2: 194-?
Technological advances now allow us to rapidly produce CARs and other antibody-derived therapeutics targeting cell surface receptors. To maximize the potential of these new technologies, relevant extracellular targets must be identified. The Pediatric Oncology Branch of the NCI curates a freely accessible database of gene expression data for both pediatric cancers and normal tissues, through which we have defined discrete sets of over-expressed transcripts in 12 pediatric cancer subtypes as compared to normal tissues. We coupled gene expression profiles to current annotation databases (i.e., Affymetrix, Gene Ontology, Entrez Gene), in order to categorize transcripts by their sub-cellular location. In this manner we generated a list of potential immune targets expressed on the cell surface, ranked by their difference from normal tissue. Global differences from normal between each of the pediatric tumor types studied varied, indicating that some malignancies expressed transcript sets that were more highly diverged from normal tissues than others. The validity of our approach is seen by our findings for pre-B cell ALL, where targets currently in clinical trials were top-ranked hits (CD19, CD22). For some cancers, reagents already in development could potentially be applied to a new disease class, as exemplified by CD30 expression on sarcomas. Moreover, several potential new targets shared among several pediatric solid tumors are herein identified, such as MCAM (MUC18), metadherin (MTDH), and glypican-2 (GPC2). These targets have been identified at the mRNA level and are yet to be validated at the protein level. The safety of targeting these antigens has yet to be demonstrated and therefore the identified transcripts should be considered preliminary candidates for new CAR and therapeutic antibody targets. Prospective candidate targets will be evaluated by proteomic analysis including Westerns and immunohistochemistry of normal and tumor tissues.
View details for DOI 10.3389/fonc.2012.00194
View details for PubMedID 23251904
Immunotherapy targets in pediatric cancer.
Frontiers in oncology
2012; 2: 3-?
Immunotherapy for cancer has shown increasing success and there is ample evidence to expect that progress gleaned in immune targeting of adult cancers can be translated to pediatric oncology. This manuscript reviews principles that guide selection of targets for immunotherapy of cancer, emphasizing the similarities and distinctions between oncogene-inhibition targets and immune targets. It follows with a detailed review of molecules expressed by pediatric tumors that are already under study as immune targets or are good candidates for future studies of immune targeting. Distinctions are made between cell surface antigens that can be targeted in an MHC independent manner using antibodies, antibody derivatives, or chimeric antigen receptors versus intracellular antigens which must be targeted with MHC restricted T cell therapies. Among the most advanced immune targets for childhood cancer are CD19 and CD22 on hematologic malignancies, GD2 on solid tumors, and NY-ESO-1 expressed by a majority of synovial sarcomas, but several other molecules reviewed here also have properties which suggest that they too could serve as effective targets for immunotherapy of childhood cancer.
View details for DOI 10.3389/fonc.2012.00003
View details for PubMedID 22645714
Murine Rhabdomyosarcoma Is Immunogenic and Responsive to T-Cell-Based Immunotherapy
PEDIATRIC BLOOD & CANCER
2011; 57 (6): 921-929
Immunotherapies targeting cellular immunity are currently approved for treatment of melanoma, renal cell carcinoma, and prostate cancer. Studies on the immunogenicity and immune responsiveness of pediatric tumors are limited, therefore, it remains unclear to what extent T-cell-based immunotherapy holds promise for pediatric solid tumors.A new rhabdomyosarcoma cell line (M3-9-M) was derived from an embryonal rhabdomyosarcoma (ERMS) occurring in a C57BL/6 mouse transgenic for hepatocyte growth factor and heterozygous for mutated p53. Primary tumors and metastases derived from M3-9-M were studied for similarities to human ERMS, and for immunogenicity and immune responsiveness.Primary and metastatic tumors develop after orthotopic injection of M3-9-M into immunocompetent C57BL/6 mice, which mirror human ERMS with regard to histology, gene expression, and metastatic behavior. Whole cell vaccination using irradiated M3-9-M cells or M3-9-M-pulsed dendritic cells (DC)-induced tumor-specific T-cell responses that prevent tumor growth following low-dose tumor injection, and slow tumor growth following higher doses. Administration of anti-CD25 moAbs to deplete CD4(+)CD25(+)FOXP3(+) regulatory T cells prior to tumor vaccination enhanced the potency of the ERMS tumor vaccine. Adoptive immunotherapy with M3-9-M primed T cells plus DC-based vaccination resulted in complete eradication of day 10 M3-9-M derived tumors.M3-9-M derived murine ERMS is immunogenic and immunoresponsive; regulatory T cells contribute to immune evasion by murine rhabdomyosarcoma. Adoptive immunotherapy with DC vaccination can eradicate low tumor burdens. Future work will seek to identify the tumor-associated antigens that mediate protective and therapeutic immunity in this model.
View details for DOI 10.1002/pbc.23048
View details for Web of Science ID 000295257700007
View details for PubMedID 21462302
Killing of Resistant Cancer Cells with Low Bak by a Combination of an Antimesothelin Immunotoxin and a TRAIL Receptor 2 Agonist Antibody
CLINICAL CANCER RESEARCH
2011; 17 (18): 5926-5934
Many solid tumors express cell surface mesothelin making them attractive targets for antibody-based therapies of cancer. SS1P [antimesothelin(Fv)PE38] is a recombinant immunotoxin (RIT) that has potent cytotoxic activity on several cancer cell lines and clinical activity in mesothelioma patients. Pancreatic cancers express mesothelin and are known to be resistant to most chemotherapeutic agents. The goal of this study is to treat pancreatic cancer with RIT by targeting mesothelin.We measured the cytotoxic activity of an antimesothelin immunotoxin on pancreatic cancer cells. We also measured the levels of several pro- and antiapoptotic proteins, as well as the ability of TNF-related apoptosis-inducing ligand (TRAIL) or the anti-TRAIL receptor 2 agonist antibody (HGS-ETR2) to kill pancreatic cells, and the cytotoxic activity of the two agents together in cell culture and against tumors in mice.In two pancreatic cancer cell lines, immunotoxin treatment inhibited protein synthesis but did not produce significant cell death. The resistant lines had low levels of the proapoptotic protein Bak. Increasing Bak expression enhanced the sensitivity to immunotoxins, whereas Bak knockdown diminished it. We also found that combining immunotoxin with TRAIL or HGS-ETR2 caused synergistic cell death, and together triggered caspase-8 recruitment and activation, Bid cleavage and Bax activation. Combining SS1P with HGS-ETR2 also acted synergistically to decrease tumor burden in a mouse model.Our data show that low Bak can cause cancer cells to be resistant to immunotoxin treatment and that combining immunotoxin with TRAIL or a TRAIL agonist antibody can overcome resistance.
View details for DOI 10.1158/1078-0432.CCR-11-1235
View details for Web of Science ID 000294952400010
View details for PubMedID 21813632
Highlights of the Second International Conference on "Immunotherapy in Pediatric Oncology"
PEDIATRIC HEMATOLOGY AND ONCOLOGY
2011; 28 (6): 459-U21
The Second International Conference on Immunotherapy in Pediatric Oncology was held in Houston, Texas, USA, October 11-12, 2010, to discuss the progress and challenges that have occurred in cutting edge immunotherapeutic strategies currently being developed for pediatric oncology. Major topics included immune targeting of acute lymphoblastic leukemia and pediatric solid tumors, chimeric antigen receptors (CARs) for hematologic malignancies and solid tumors, enhancing graft-versus-leukemia for pediatric cancers, overcoming hurdles of immunotherapy, strategies to active the innate immune system, and moving immunotherapy beyond phase I studies. Significant progress has been made in the last 2 years both in the development of novel immunobiologics such as CARs, and in establishing survival benefits of an anti-GD2 monoclonal antibody in randomized studies. Although there is much excitement going forward, a great deal of laboratory and regulatory challenges lie ahead in improving the efficacy of each of these modalities as well as getting them to patients in a timely and cost-effective fashion. The resulting discussions will hopefully lead to new collaborations and insight for further translational and clinical studies.
View details for DOI 10.3109/08880018.2011.596615
View details for Web of Science ID 000294065900001
View details for PubMedID 21854215
Harnessing the biology of IL-7 for therapeutic application
NATURE REVIEWS IMMUNOLOGY
2011; 11 (5): 330-342
Interleukin-7 (IL-7) is required for T cell development and for maintaining and restoring homeostasis of mature T cells. IL-7 is a limiting resource under normal conditions, but it accumulates during lymphopaenia, leading to increased T cell proliferation. The administration of recombinant human IL-7 to normal or lymphopenic mice, non-human primates and humans results in widespread T cell proliferation, increased T cell numbers, modulation of peripheral T cell subsets and increased T cell receptor repertoire diversity. These effects raise the prospect that IL-7 could mediate therapeutic benefits in several clinical settings. This Review summarizes the biology of IL-7 and the results of its clinical use that are available so far to provide a perspective on the opportunities for clinical application of this cytokine.
View details for DOI 10.1038/nri2970
View details for Web of Science ID 000289774700013
View details for PubMedID 21508983
Activating Signals Dominate Inhibitory Signals in CD137L/IL-15 Activated Natural Killer Cells
JOURNAL OF IMMUNOTHERAPY
2011; 34 (2): 187-195
Natural killer (NK) cells can mediate potent antitumor effects, but factors regulating the efficiency of tumor lysis remain unclear. Studies in allogeneic stem cell transplantation highlight an important role for killer cell immunoglobulin-like receptor (KIR) mismatch in overcoming human leukocyte antigen-mediated inhibitory signals. However, other activating and inhibitory signals also modulate tumor lysis by NK cells. We used rhIL15 and artificial antigen presenting cells expressing CD137L and IL15Rα to activate and expand peripheral blood NK cells (CD137L/IL15 NK) up to 1000-fold in 3 weeks. Compared with resting NK cells, CD137L/IL15 NK cells show modest increases in KIR expression and substantial increases in NKG2D, tumor necrosis factor-related apoptosis-inducing ligand, and natural cytotoxicity receptors (NCRs: NKp30, NKp44, NKp46). Compared with resting NK cells, CD137L/IL15 NK cells mediate enhanced cytotoxicity against allogeneic and autologous tumors and KIR signaling did not substantially inhibit cytotoxicity. Rather, tumor lysis by CD137L/IL15 activated NK cells was predominantly driven by NCR signaling as blockade of NCRs dramatically diminished the lysis of a wide array of tumor targets. Furthermore, tumor lysis by CD137L/IL15 NK cells was tightly linked to NCR expression levels that peaked on day 8 to 10 after NK activation, and cytotoxicity diminished on subsequent days as NCR expression declined. We conclude that KIR mismatch is not a prerequisite for tumor killing by CD137L/IL15 NK cells and that NCR expression provides a biomarker for predicting potency of CD137L/IL15 NK cells in studies of NK cell-based immunotherapy.
View details for DOI 10.1097/CJI.0b013e31820d2a21
View details for Web of Science ID 000287186200008
View details for PubMedID 21304401
Tumor Regression in Patients With Metastatic Synovial Cell Sarcoma and Melanoma Using Genetically Engineered Lymphocytes Reactive With NY-ESO-1
JOURNAL OF CLINICAL ONCOLOGY
2011; 29 (7): 917-924
Adoptive immunotherapy using tumor-infiltrating lymphocytes represents an effective cancer treatment for patients with metastatic melanoma. The NY-ESO-1 cancer/testis antigen, which is expressed in 80% of patients with synovial cell sarcoma and approximately 25% of patients with melanoma and common epithelial tumors, represents an attractive target for immune-based therapies. The current trial was carried out to evaluate the ability of adoptively transferred autologous T cells transduced with a T-cell receptor (TCR) directed against NY-ESO-1 to mediate tumor regression in patients with metastatic melanoma and synovial cell sarcoma.A clinical trial was performed in patients with metastatic melanoma or metastatic synovial cell sarcoma refractory to all standard treatments. Patients with NY-ESO-1-positive tumors were treated with autologous TCR-transduced T cells plus 720,000 iU/kg of interleukin-2 to tolerance after preparative chemotherapy. Objective clinical responses were evaluated using Response Evaluation Criteria in Solid Tumors (RECIST).Objective clinical responses were observed in four of six patients with synovial cell sarcoma and five of 11 patients with melanoma bearing tumors expressing NY-ESO-1. Two of 11 patients with melanoma demonstrated complete regressions that persisted after 1 year. A partial response lasting 18 months was observed in one patient with synovial cell sarcoma.These observations indicate that TCR-based gene therapies directed against NY-ESO-1 represent a new and effective therapeutic approach for patients with melanoma and synovial cell sarcoma. To our knowledge, this represents the first demonstration of the successful treatment of a nonmelanoma tumor using TCR-transduced T cells.
View details for DOI 10.1200/JCO.2010.32.2537
View details for Web of Science ID 000287729900035
View details for PubMedID 21282551
- In search of targeted therapies for childhood cancer. Frontiers in oncology 2011; 1: 18-?
Novel Gamma-Chain Cytokines as Candidate Immune Modulators in Immune Therapies for Cancer
2010; 16 (4): 392-398
Cytokines that signal through the common-gamma chain are potent growth factors for T cells and natural killer cells. Interleukin (IL)-2, the gammac prototype, can mediate antitumor effects as a single agent or in the context of multimodality regimens but is limited by side effects and a propensity for expansion of regulatory T cells. IL-7, IL-15, and IL-21 each possess properties that can be exploited in the context of immunotherapy for cancer. Each has been demonstrated to mediate potent vaccine adjuvant effects in tumor models, and each can enhance the effectiveness of adoptive immunotherapies. Although the overlap among the agents is significant, IL-7 is uniquely immunorestorative and preferentially augments reactivity of naive populations, IL-15 potently augments reactivity of CD8 memory cells and natural killer cells, and IL-21 preferentially expands the inflammatory Th17 subset and may limit terminal differentiation of effector CD8 cells. Clinical trials of IL-7 and IL-21 have already been completed and, so far, demonstrate safety and biologic activity of these agents. Clinical trials of IL-15 are expected soon. Ultimately, these agents are expected to be most effective in the context of multimodal immunotherapy regimens, and careful clinical trial design will be needed to efficiently identify the proper doses, regimens, and settings in which to exploit their biologic properties for therapeutic gain.
View details for DOI 10.1097/PPO.0b013e3181eacbc4
View details for Web of Science ID 000280672100016
View details for PubMedID 20693852
NCI First International Workshop on the Biology, Prevention, and Treatment of Relapse After Allogeneic Hematopoietic Stem Cell Transplantation: Report from the Committee on the Biological Considerations of Hematological Relapse following Allogeneic Stem Cell Transplantation Unrelated to Graft-versus-Tumor Effects: State of the Science
BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION
2010; 16 (6): 709-728
Hematopoietic malignant relapse still remains the major cause of death following allogeneic hematopoietic stem cell transplantation (HSCT). Although there has been a large focus on the immunologic mechanisms responsible for the graft-versus-tumor (GVT) effect or lack thereof, there has been little attention paid to investigating the biologic basis of hematologic malignant disease relapse following allogeneic HSCT. There are a large number of factors that are responsible for the biologic resistance of hematopoietic tumors following allogeneic HSCT. We have focused on 5 major areas including clonal evolution of cancer drug resistance, cancer radiation resistance, genomic basis of leukemia resistance, cancer epigenetics, and resistant leukemia stem cells. We recommend increased funding to pursue 3 broad areas that will significantly enhance our understanding of the biologic basis of malignant relapse after allogeneic HSCT, including: (1) genomic and epigenetic alterations, (2) cancer stem cell biology, and (3) clonal cancer drug and radiation resistance.
View details for DOI 10.1016/j.bbmt.2010.03.002
View details for Web of Science ID 000278046700001
View details for PubMedID 20227509
Pharmacologic modulation of niche accessibility via tyrosine kinase inhibition enhances marrow and thymic engraftment after hematopoietic stem cell transplantation
2010; 115 (20): 4120-4129
Essential survival signals within hematopoietic stem cell (HSC) and thymic niches are mediated by receptor tyrosine kinases, which can be reversibly inhibited using clinically available drugs. We studied whether sunitinib, a multityrosine kinase inhibitor that inhibits KIT, enhances engraftment after bone marrow transplantation (BMT) in mice. Sunitinib diminished hematopoietic progenitor cell numbers, and sunitinib enhanced marrow, peripheral myeloid, and lymphoid engraftment after BMT in Rag1(-/-) mice. Sunitinib augmented HSC engraftment because recipients displayed increased myeloid and lymphoid engraftment and because sunitinib-treated recipients of purified HSCs showed enhanced engraftment of secondary hosts. However, sunitinib preferentially augmented T-cell engraftment with lesser effects on myeloid and HSC engraftment. Consistent with this, sunitinib preferentially depleted the early thymic progenitor subset in the thymus. Sunitinib did not increase engraftment in mice with deficient KIT signaling, and the pattern of more potent effects on T cell compared with HSC engraftment observed in sunitinib-treated hosts was also observed after BMT into KIT(W/Wv) mice. These results implicate KIT as a critical modulator of thymic niches. We conclude that transient, pharmacologic inhibition of KIT enhances accessibility of marrow and thymic niches, and provides a novel, noncytotoxic approach to accomplish engraftment after stem cell transplantation.
View details for DOI 10.1182/blood-2009-10-248898
View details for Web of Science ID 000277923600017
View details for PubMedID 20231424
In Vivo Role of Flt3 Ligand and Dendritic Cells in NK Cell Homeostasis
JOURNAL OF IMMUNOLOGY
2010; 184 (6): 2769-2775
IL-15 is required for NK cell development and homeostasis in vivo. Because IL-15 is presented in trans via its high-affinity IL-15Ralpha-chain to cells expressing the IL-15Rbetagamma complex, we postulated that certain IL-15-bearing cells must be required for NK cell homeostasis. Using IL-15(WT/WT) and IL-15(-/-) mice, bone marrow chimeras with normal cellularity, and a selective depletion of CD11c(hi) dendritic cells (DCs), we demonstrate that ablation of the resting CD11c(hi) DC population results in a highly significant decrease in the absolute number of mature NK cells. In contrast, administration of Flt3 ligand increases the CD11c(hi) DC population, which, when expressing IL-15, significantly expands mature NK cells via enhanced survival and proliferation. In summary, a CD11c(hi) DC population expressing IL-15 is required to maintain NK cell homeostasis under conditions of normal cellularity and also is required to mediate Flt3 ligand-induced NK cell expansion in vivo.
View details for DOI 10.4049/jimmunol.0900685
View details for Web of Science ID 000275389000005
View details for PubMedID 20142363
Immune-based therapeutics for pediatric cancer
EXPERT OPINION ON BIOLOGICAL THERAPY
2010; 10 (2): 163-178
Although most children with cancer are cured, there remain significant limitations of standard treatment, most notably chemotherapy resistance and non-specific toxicities. Novel immune-based therapies that target pediatric malignancies offer attractive adjuncts and/or alternatives to commonly employed cytotoxic regimens of chemotherapy or radiotherapy. Elucidation of the principles of tumor biology and the development of novel laboratory technologies over the last decade have led to substantial progress in bringing immunotherapies to the bedside.Current immunotherapeutic clinical trials in pediatric oncology and the science behind their development are reviewed.Most of the immune-based therapies studied to date have been well tolerated, and some have shown promise in the setting of refractory or high-risk malignancies, demonstrating that immunotherapy has the potential to overcome resistance to conventional chemotherapy.Some immune-based therapies, such as ch14.18 and MTP-PE, have already been proven effective in phase III randomized trials. Further studies are needed to optimize and integrate other therapies into standard regimens, and to test them in randomized trials for patients with childhood cancer.
View details for DOI 10.1517/14712590903431022
View details for Web of Science ID 000274499400002
View details for PubMedID 19947897
T-cell-based Therapies for Malignancy and Infection in Childhood
PEDIATRIC CLINICS OF NORTH AMERICA
2010; 57 (1): 83-96
One major advance in T-cell-based immunotherapy in the last 20 years has been the molecular definition of numerous viral and tumor antigens. Adoptive T-cell transfer has shown definite clinical benefit in the prophylaxis and treatment of viral infections that develop in pediatric patients after allogeneic transplant and in posttransplant lymphoproliferative disease associated with the Epstein-Barr virus. Developing adoptive T-cell therapies for other malignancies presents additional challenges. This article describes the recent advances in T-cell-based therapies for malignancy and infection in childhood and strategies to enhance the effector functions of T cells and optimize the cellular product, including gene modification and modulation of the host environment.
View details for DOI 10.1016/j.pcl.2009.11.002
View details for Web of Science ID 000277039300006
View details for PubMedID 20307713
Immunotherapy of childhood cancer: from biologic understanding to clinical application
CURRENT OPINION IN PEDIATRICS
2010; 22 (1): 2-11
Most children with cancer can be cured with combination regimens of chemotherapy, radiation, surgery, or all. However, standard therapies are toxic to normal tissues, cancer cells commonly develop resistance to chemotherapy, and relapsed malignancy is a leading cause of mortality in pediatrics. Elucidation of the principles of the normal immune response and tumor biology, coupled with technological developments, have led to important advances in the field of cancer immunotherapy. This review summarizes the biologic basis of cancer immunotherapy and highlights recent examples of progress in the application of novel humoral and cellular immunotherapies to children and adolescents with malignancy.Clinical trials of immunotherapy for pediatric cancer have recently been initiated. To date, most immune-based therapies have been well tolerated and some have shown clinically significant activity against specific refractory high-risk malignancies.Recent clinical trial results provide proof-of-principle that cancer immunotherapy has the capacity to overcome chemotherapy resistance without the usual toxicities associated with cytotoxic regimens. Immunotherapy holds promise in the treatment of children and adolescents with cancer and has the potential to improve both survival and quality of life.
View details for DOI 10.1097/MOP.0b013e3283350d3e
View details for Web of Science ID 000274036000002
View details for PubMedID 19952749
Phase I Study of Recombinant Human Interleukin-7 Administration in Subjects with Refractory Malignancy
CLINICAL CANCER RESEARCH
2010; 16 (2): 727-735
Interleukin-7 (IL-7) has critical and nonredundant roles in T-cell development, hematopoiesis, and postdevelopmental immune functions as a prototypic homeostatic cytokine. Based on a large body of preclinical evidence, it may have multiple therapeutic applications in immunodeficiency states, either physiologic (immunosenescence), pathologic (HIV), or iatrogenic (postchemotherapy and posthematopoietic stem cell transplant), and may have roles in immune reconstitution or enhancement of immunotherapy. We report here on the toxicity and biological activity of recombinant human IL-7 (rhIL-7) in humans.Subjects with incurable malignancy received rhIL-7 subcutaneously every other day for 2 weeks in a phase I interpatient dose escalation study (3, 10, 30, and 60 microg/kg/dose). The objectives were safety and dose-limiting toxicity determination, identification of a range of biologically active doses, and characterization of biological and, possibly, antitumor effects.Mild to moderate constitutional symptoms, reversible spleen and lymph node enlargement, and marked increase in peripheral CD3(+), CD4(+), and CD8(+) lymphocytes were seen in a dose-dependent and age-independent manner in all subjects receiving >or=10 microg/kg/dose, resulting in a rejuvenated circulating T-cell profile, resembling that seen earlier in life. In some subjects, rhIL-7 induced in the bone marrow a marked, transient polyclonal proliferation of pre-B cells showing a spectrum of maturation as well as an increase in circulating transitional B cells.This study shows the potent biological activity of rhIL-7 in humans over a well-tolerated dose range and allows further exploration of its possible therapeutic applications.
View details for DOI 10.1158/1078-0432.CCR-09-1303
View details for Web of Science ID 000278545000037
View details for PubMedID 20068111
Randomized Trial and Pharmacokinetic Study of Pegfilgrastim versus Filgrastim after Dose-Intensive Chemotherapy in Young Adults and Children with Sarcomas
CLINICAL CANCER RESEARCH
2009; 15 (23): 7361-7367
To compare the effectiveness, tolerance, and pharmacokinetics of a single dose of pegfilgrastim to daily filgrastim in children and young adults with sarcomas treated with dose-intensive combination chemotherapy.Patients were randomized to receive a single dose of 100 mcg/kg of pegfilgrastim s.c. or 5 mcg/kg/day of filgrastim s.c., daily until neutrophil recovery after two treatment cycles with vincristine, doxorubicin, and cyclophosphamide (VDC) and two cycles of etoposide and ifosfamide (IE). The duration of severe neutropenia (absolute neutrophil count, < or =500/mcL) during cycles 1 to 4 and cycle duration for all cycles were compared. Pharmacokinetics of pegfilgrastim and filgrastim and CD34+ stem cell mobilization were studied on cycle 1. Growth factor-related toxicity, transfusions, and episodes of fever and neutropenia and infections were collected for cycles 1 to 4.Thirty-four patients (median age, 20 years; range 3.8-25.8) were enrolled, and 32 completed cycles 1 to 4. The median (range) duration of absolute neutrophil count of <500/mcL was 5.5 (3-8) days for pegfilgrastim and 6 (0-9) days for filgrastim (P = 0.76) after VDC, and 1.5 (0-4) days for pegfilgrastim and 3.75 (0-6.5) days for filgrastim (P = 0.11) after IE. More episodes of febrile neutropenia and documented infections occurred on the filgrastim arm. Serum pegfilgrastim concentrations were highly variable. Pegfilgrastim apparent clearance (11 mL/h/kg) was similar to that reported in adults.A single dose per cycle of pegfilgrastim was well tolerated and may be as effective as daily filgrastim based on the duration of severe neutropenia and number of episodes of febrile neutropenia and documented infections after dose-intensive treatment with VDC and IE.
View details for DOI 10.1158/1078-0432.CCR-09-0761
View details for Web of Science ID 000272363700032
View details for PubMedID 19920107
Harnessing the physiology of lymphopenia to support adoptive immunotherapy in lymphoreplete hosts
2009; 114 (18): 3831-3840
Lymphopenia enhances the effectiveness of adoptive immunotherapy by facilitating expansion of transferred T cells but also limits the T-cell repertoire available to mediate immune responses and, in humans, is associated with chronic immune dysfunction. Previous studies concluded that lymphopenia augments adoptive immunotherapy by diminishing Tregs and increasing homeostatic cytokines. We sought to determine whether targeted therapies that replicate the physiology of lymphopenia in lymphoreplete hosts could provide a similarly supportive milieu. Pmel-1 T cells were transferred to B16-bearing lymphopenic versus lymphoreplete mice receiving alphaCD25 and/or recombinant human interleukin-7. Although CD25-based Treg depletion was inefficient because of peripheral expansion of CD4+CD25-FOXP3+ cells, outcomes were better in alphaCD25-treated lymphoreplete hosts than in lymphopenic hosts, and adoptive immunotherapy was most effective in lymphoreplete hosts receiving alphaCD25 plus recombinant human interleukin-7. Lymphopenic hosts supported increased proliferation of adoptively transferred antigen-specific T cells, but cells transferred to lymphoreplete recipients receiving targeted therapies showed superior function. Further, determinant spreading was substantial in lymphoreplete hosts but absent in lymphopenic hosts. These results demonstrate that targeted therapies delivered to mimic the "physiology of lymphopenia" enhance the efficacy of adoptive immunotherapy in lymphoreplete hosts and provide a potentially superior alternative to the induction of lymphopenia.
View details for DOI 10.1182/blood-2009-03-212134
View details for Web of Science ID 000271268100018
View details for PubMedID 19704119
- Background to hematopoietic cell transplantation, including post transplant immune recovery BONE MARROW TRANSPLANTATION 2009; 44 (8): 457-462
Beta4 integrin promotes osteosarcoma metastasis and interacts with ezrin
2009; 28 (38): 3401-3411
The development of pulmonary metastasis is the major cause of death in osteosarcoma, and its molecular basis is poorly understood. In this study, we show that beta4 integrin is highly expressed in human osteosarcoma cell lines and tumor samples. Furthermore, highly metastatic MNNG-HOS cells have increased levels of beta4 integrin. Suppression of beta4 integrin expression by shRNA and disruption of beta4 integrin function by transfection of dominant-negative beta4 integrin was sufficient to revert this highly metastatic phenotype in the MNNG-HOS model without significantly affecting primary tumor growth. These findings suggest a role for beta4 integrin expression in the metastatic phenotype in human osteosarcoma cells. In addition, we identified a previously uncharacterized interaction between beta4 integrin and ezrin, a membrane-cytoskeletal linker protein that is implicated in the metastatic behavior of osteosarcoma. The beta4 integrin-ezrin interaction appears to be critical for maintenance of beta4 integrin expression. These data begin to integrate ezrin and beta4 integrin expression into a model of action for the mechanism of osteosarcoma metastases.
View details for DOI 10.1038/onc.2009.206
View details for Web of Science ID 000270103300007
View details for PubMedID 19597468
Secondary Supratentorial Primitive Neuroectodermal Tumor Following Treatment of Childhood Osteosarcoma
PEDIATRIC BLOOD & CANCER
2009; 53 (3): 496-498
A 16-year-old Caucasian male was diagnosed with a primitive neuroectodermal tumor (PNET) 5 years following the diagnosis of nonmetastatic osteosarcoma of the left proximal humerus. The patient was initially treated with standard chemotherapy and limb salvage resection for osteosarcoma. Nine months after the completion of therapy, he developed lung metastases for which he underwent surgical resection and received additional chemotherapy. Almost 5 years after the osteosarcoma diagnosis, the patient was diagnosed with a supratentorial PNET, which represents the first known case reported in a patient with osteosarcoma.
View details for DOI 10.1002/pbc.22074
View details for Web of Science ID 000268443200043
View details for PubMedID 19434734
Modulating T-cell homeostasis with IL-7: preclinical and clinical studies
JOURNAL OF INTERNAL MEDICINE
2009; 266 (2): 141-153
Interleukin-7 (IL-7) is required for the development and survival of T cells and plays a critical role in modulating T-cell homeostasis. This review will address current understanding of IL-7 biology, review recent clinical experiences and discuss potential future clinical applications of IL-7, or IL-7 blockade, in the setting of disease.
View details for DOI 10.1111/j.1365-2796.2009.02085.x
View details for Web of Science ID 000267883100001
View details for PubMedID 19623690
Antigen loading of DCs with irradiated apoptotic tumor cells induces improved anti-tumor immunity compared to other approaches
CANCER IMMUNOLOGY IMMUNOTHERAPY
2009; 58 (8): 1257-1264
Dendritic cells (DCs) serve as central regulators of adaptive immunity by presenting antigens and providing necessary co-signals. Environmental information received by the DCs determines the co-signals delivered to the responding adaptive cells and, ultimately, the outcome of the interaction. DCs loaded with relevant antigens have been used as therapeutic cellular vaccines, but the optimal antigen loading method has not been determined. We compared different methods to load class I and class II epitopes from the male antigenic complex, HY, onto DCs for the potency of the immune response induced in vivo. Co-incubation of female DCs with HY peptides, RNA or cell lysate from HY expressing tumor induced immune responses equivalent to male DCs. In contrast, female DCs incubated with irradiated, apoptotic HY expressing tumor cells (or male B cells) generated a stronger immune response than male DCs or female DCs loaded using any of the other methods. DC loading with apoptotic tumor resulted in complete protection against high dose HY-expressing tumor challenge whereas 100% lethality was observed in groups receiving DCs that were loaded with peptides, RNA, or lysate. We conclude that signals provided to the DCs by apoptotic cells substantially augment the potency of DC vaccines.
View details for DOI 10.1007/s00262-008-0638-7
View details for Web of Science ID 000266372400008
View details for PubMedID 19139888
Bone marrow deficient in IFN-gamma signaling selectively reverses GVHD-associated immunosuppression and enhances a tumor-specific GVT effect
2009; 113 (20): 5002-5009
Vaccine-based expansion of T cells is one approach to enhance the graft-versus-tumor effect of allogeneic bone marrow transplantation (BMT), but the complex immunobiology of the allogeneic environment on responses to tumor vaccines has not been well characterized. We hypothesized that subclinical graft-versus-host disease (GVHD) impairs immunity, but modulation of gamma interferon (IFN-gamma) signaling could reverse this effect. Dendritic cell vaccines and donor lymphocyte infusions (DLIs) were incorporated into a minor histocompatibility antigen-mismatched, T cell-depleted, allogeneic BMT mouse model. Animals were then challenged with H-Y expressing tumors. CD4(+) and CD8(+) responses to H-Y were diminished in vaccinated allogeneic versus syngeneic BMT recipients with DLI doses below the threshold for clinical GVHD, especially in thymectomized hosts. IFN-gamma receptor 1-deficient (IFN-gammaR1(-/-)) T cells cannot cause GVHD but also have diminished vaccine responses. Remarkably, IFN-gammaR1(-/-) bone marrow abrogates GVHD, allowing higher DLI doses to be tolerated, but improves vaccine responses and tumor protection. We conclude that tumor vaccines administered after allogeneic BMT can augment graft-versus-tumor if GVHD is avoided and that prevention of IFN-gamma signaling on donor bone marrow is an effective approach to preventing GVHD while preserving immunocompetence.
View details for DOI 10.1182/blood-2008-11-187385
View details for Web of Science ID 000266090700027
View details for PubMedID 19258593
Highlights of the First International "Immunotherapy in Pediatric Oncology: Progress and Challenges" Meeting
JOURNAL OF PEDIATRIC HEMATOLOGY ONCOLOGY
2009; 31 (4): 227-234
The first annual conference on immunotherapy in pediatric oncology was held in Bethesda, MD, from September 9 to 10, 2008 to discuss the state-of-the-art of immunotherapeutic strategies currently being explored in pediatric oncology. Major topics included targeting cell surface receptors, understanding and improving T-cell-based therapies, augmenting innate immune strategies, and enhancing graft-versus-leukemia for pediatric malignancies. As can be seen in the summaries of the individual presentations, significant progress has been made in developing preclinical models of pediatric tumors and a variety of novel immunobiologic therapies are approaching, or already in, the clinic. Although there is much excitement about the potential utility of these agents, a great deal of challenges lie ahead in improving the efficacy of each of these modalities and getting them to patients in a timely fashion. The resulting discussions will hopefully lead to new collaborations and insight for further translational and clinical studies.
View details for Web of Science ID 000265156100001
View details for PubMedID 19346873
Interleukin 7 signaling in dendritic cells regulates the homeostatic proliferation and niche size of CD4(+) T cells
2009; 10 (2): 149-157
Interleukin 7 (IL-7) and T cell antigen receptor signals have been proposed to be the main drivers of homeostatic T cell proliferation. However, it is not known why CD4(+) T cells undergo less-efficient homeostatic proliferation than CD8(+) T cells do. Here we show that systemic IL-7 concentrations increased during lymphopenia because of diminished use of IL-7 but that IL-7 signaling on IL-7 receptor-alpha-positive (IL-7Ralpha(+)) dendritic cells (DCs) in lymphopenic settings paradoxically diminished the homeostatic proliferation of CD4(+) T cells. This effect was mediated at least in part by IL-7-mediated downregulation of the expression of major histocompatibility complex class II on IL-7Ralpha(+) DCs. Our results indicate that IL-7Ralpha(+) DCs are regulators of the peripheral CD4(+) T cell niche and that IL-7 signals in DCs prevent uncontrolled CD4(+) T cell population expansion in vivo.
View details for DOI 10.1038/ni.1695
View details for Web of Science ID 000263178500008
View details for PubMedID 19136960
Unusual Sites of Extraskeletal Metastases of Ewing Sarcoma After Allogeneic Hematopoietic Stem Cell Transplantation
JOURNAL OF PEDIATRIC HEMATOLOGY ONCOLOGY
2009; 31 (2): 142-144
Allogeneic stem cell transplantation (SCT) for solid tumors remains under investigation. We report a case of extended disease stability after a nonmyeloablative peripheral blood SCT for metastatic, refractory Ewing sarcoma. Of note, the patient developed metastatic disease to 2 unusual sites-the brain and small intestine. The allogeneic SCT environment may alter typical metastatic patterns, and may represent an ideal platform to manipulate and enhance the antitumor immune response. Further clinical trials are needed to evaluate the role for allogeneic SCT for this disease.
View details for Web of Science ID 000263135100015
View details for PubMedID 19194203
- GVHD: A Continuing Barrier to the Safety of Allogeneic Transplantation BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION 2009; 15 (1): 162-168
- Clinical implications of immune reconstitution following hematopoietic stem cell transplantation. Cancer treatment and research 2009; 144: 131-154
Current Approach to Pediatric Soft Tissue Sarcomas
2009; 14 (11): 1139-1153
The development of a new soft tissue lesion in an otherwise healthy child, adolescent, or young adult can present many challenges for pediatric or medical oncology teams. Although uncommon, the diagnosis of a soft tissue malignancy should always be considered in the differential diagnosis of persistent pain, even if no mass is palpable. The definitive diagnosis and treatment of a soft tissue mass is aided by timely scans, appropriate biopsy for anatomic and molecular pathology, and a treatment approach guided by the specific diagnosis. Because pediatric soft tissue sarcomas are rare, cooperative groups play a crucial role in defining the standard of care through retrospective series and well-designed prospective clinical trials. Enrollment of newly diagnosed patients in clinical studies should be encouraged in order to continue to improve outcomes and understanding of these rare tumors. This review focuses on the current recommendations for management of sarcomas that typically occur in the soft tissues of pediatric and young adult patients.
View details for DOI 10.1634/theoncologist.2009-0160
View details for Web of Science ID 000272166800010
View details for PubMedID 19897537
Perspective on Potential Clinical Applications of Recombinant Human Interleukin-7
CYTOKINE THERAPIES: NOVEL APPROACHES FOR CLINICAL INDICATIONS
2009; 1182: 28-38
Interleukin-7 has critical and nonredundant roles in T cell development, hematopoiesis, and postdevelopmental immune functions as a prototypic homeostatic cytokine. Based on a large body of preclinical evidence, it may have multiple therapeutic applications in immunodeficiency states, either physiologic (immuno-senescence), pathologic (HIV) or iatrogenic (postchemotherapy and posthematopoietic stem cell transplant) and may have roles in immune reconstitution or enhancement of immunotherapy. Early clinical development trials in humans show that, within a short time, rhIL-7 administration results in a marked preferential expansion of both naive and memory CD4 and CD8 T cell pools with a tendency toward enhanced CD8 expansion. As a result, lymphopenic or normal older hosts develop an expanded circulating T cell pool with a profile that resembles that seen earlier in life with increased T cell repertoire diversity. These results, along with a favorable toxicity profile, open a wide perspective of potential future clinical applications.
View details for DOI 10.1111/j.1749-6632.2009.05075.x
View details for Web of Science ID 000277730400003
View details for PubMedID 20074272
Cytokines as Adjuvants for Vaccine and Cellular Therapies for Cancer.
American journal of immunology
2009; 5 (3): 65-83
PROBLEM STATEMENT: The development of a potent vaccine that can help treat tumors resistant to conventional cytotoxic therapies remains elusive. While part of the problem may be that trials have focused on patients with bulky residual disease, the desire to maximize responses to the vaccine remains. APPROACH: The gamma(c) family of cytokines offer a unique opportunity to support the expansion and effector potential of vaccine-responding T-cells, as well as stimulate other effectors, such as natural killer (NK) cells, to become activated. RESULTS: Combining vaccines with cytokines seems logical but can bring unwanted toxicity, as has been observed with interleukin (IL)-2. In addition, the nonspecific activation or expansion of unwanted cell subsets, such as regulatory T-cells, can contribute to global immunosuppression and limit vaccine responses. The development of IL-7 and IL-21 for the clinic offers the promise of enhancing anti-tumor responses but with far less systemic toxicity and no expansion of regulatory T cells. Preclinical studies demonstrate that IL-15 could also improve T-cell, and especially NK-cell, responses as well. CONCLUSIONS/RECOMMENDATIONS: Future work should expand the use of vaccines with IL-7, IL-21 and hopefully IL-15 in high-risk patients, and consider treatment while in a state of minimal residual disease to maximize benefit. Identifying tumors that can signal through gamma(c) cytokines will also be essential so that induction of relapse will be avoided.
View details for PubMedID 20182648
Generation, Affinity Maturation, and Characterization of a Human Anti-Human NKG2D Monoclonal Antibody with Dual Antagonistic and Agonistic Activity
JOURNAL OF MOLECULAR BIOLOGY
2008; 384 (5): 1143-1156
In humans, NKG2D is an activating receptor on natural killer (NK) cells and a costimulatory receptor on certain T cells and plays a central role in mediating immune responses in autoimmune diseases, infectious diseases, and cancer. Monoclonal antibodies that antagonize or agonize immune responses mediated by human NKG2D are considered to be of broad and potent therapeutic utility. Nonetheless, monoclonal antibodies to NKG2D that are suitable for clinical investigations have not been published yet. Here, we describe the generation, affinity maturation, and characterization of a fully human monoclonal antibody to human NKG2D. Using phage display technology based on a newly generated naïve human Fab library in phage display vector pC3C followed by a tandem chain shuffling process designed for minimal deviation from natural human antibody sequences, we selected a human Fab, designated KYK-2.0, with high specificity and affinity to human NKG2D. KYK-2.0 Fab blocked the binding of the natural human NKG2D ligands MICA, MICB, and ULBP2 as potently as a commercially available mouse anti-human NKG2D monoclonal antibody in immunoglobulin G (IgG) format. Conversion of KYK-2.0 Fab to IgG1 resulted in subnanomolar avidity for human NKG2D. KYK-2.0 IgG1 was found to selectively recognize defined subpopulations of human lymphocytes known to express NKG2D, that is, the majority of human CD8+, CD16+, and CD56+ cells as well as a small fraction of human CD4+ cells. In solution, KYK-2.0 IgG1 interfered with the cytolytic activity of ex vivo expanded human NK cells. By contrast, immobilized KYK-2.0 IgG1 was found to strongly induce human NK cell activation. The dual antagonistic and agonistic activity promises a wide range of therapeutic applications for KYK-2.0 IgG1 and its derivatives.
View details for DOI 10.1016/j.jmb.2008.09.008
View details for Web of Science ID 000262016600012
View details for PubMedID 18809410
Association of Serum Interleukin-7 Levels With the Development of Acute Graft-Versus-Host Disease
JOURNAL OF CLINICAL ONCOLOGY
2008; 26 (35): 5735-5741
Morbidity from acute graft-versus-host disease (GVHD) limits the success of allogeneic hematopoietic stem-cell transplantation (HSCT) to treat malignancy. Interleukin-7 (IL-7), the principal homeostatic cytokine for T cells, is required for acute GVHD in murine models. In contrast to inflammatory cytokines (eg, IL-2, tumor necrosis factor alpha), IL-7 has not been studied extensively in the clinical transplant setting relative to its relationship with acute GVHD.We evaluated the association of serum IL-7 levels with acute GVHD in 31 patients who were uniformly treated in a prospective clinical trial with reduced-intensity allogeneic HSCT from human leukocyte antigen-identical siblings. GVHD prophylaxis consisted of cyclosporine and methotrexate. Serum IL-7 levels and lymphocyte populations were determined at enrollment, the day of transplantation before the allograft infusion, and at specified intervals through 12 months post-transplantation.As expected, IL-7 levels were inversely correlated with T-cell populations (P < .00001). Acute GVHD was significantly associated with higher IL-7 levels at day +7 (P = .01) and day +14 (P = .00003) post-transplantation as well as with the allograft CD34(+) cell dose (P = .01). IL-7 levels at day +14 also correlated with the severity of acute GVHD (P < .0001). In logistic regression models, these factors were highly sensitive (up to 86%) and specific (100%) for classifying whether patients developed acute GVHD.These data support preclinical observations that IL-7 plays a critical role in inducing acute GVHD and provide a rational basis for novel approaches to prevent and treat acute GVHD through modulation of the IL-7 pathway.
View details for DOI 10.1200/JCO.2008.17.1314
View details for Web of Science ID 000261528200014
View details for PubMedID 19001329
A pilot study of consolidative immunotherapy in patients with high-risk pediatric sarcomas
CLINICAL CANCER RESEARCH
2008; 14 (15): 4850-4858
Patients with metastatic or recurrent Ewing's sarcoma family of tumors and alveolar rhabdomyosarcoma have <25% 5-year survival in most studies. This study administered a novel immunotherapy regimen aimed at consolidating remission in these patients.Fifty-two patients with translocation positive, recurrent, or metastatic Ewing's sarcoma family of tumors or alveolar rhabdomyosarcoma underwent prechemotherapy cell harvest via apheresis for potential receipt of immunotherapy. Following completion of standard multimodal therapy, 30 patients ultimately initiated immunotherapy and were sequentially assigned to three cohorts. All cohorts received autologous T cells, influenza vaccinations, and dendritic cells pulsed with peptides derived from tumor-specific translocation breakpoints and E7, a peptide known to bind HLA-A2. Cohort 1 received moderate-dose recombinant human interleukin-2 (rhIL-2), cohort 2 received low-dose rhIL-2, and cohort 3 did not receive rhIL-2.All immunotherapy recipients generated influenza-specific immune responses, whereas immune responses to the translocation breakpoint peptides occurred in 39%, and only 25% of HLA-A2(+) patients developed E7-specific responses. Toxicity was minimal. Intention-to-treat analysis revealed a 31% 5-year overall survival for all patients apheresed (median potential follow-up 7.3 years) with a 43% 5-year overall survival for patients initiating immunotherapy.Consolidative immunotherapy is a scientifically based and clinically practical approach for integrating immunotherapy into a multimodal regimen for chemoresponsive cancer. Patients receiving immunotherapy experienced minimal toxicity and favorable survival. The robust influenza immune responses observed suggest that postchemotherapy immune incompetence will not fundamentally limit this approach. Future studies will seek to increase efficacy by using more immunogenic antigens and more potent dendritic cells.
View details for DOI 10.1158/1078-0432.CCR-07-4065
View details for Web of Science ID 000258217200026
View details for PubMedID 18676758
Administration of rhIL-7 in humans increases in vivo TCR repertoire diversity by preferential expansion of naive T cell subsets
JOURNAL OF EXPERIMENTAL MEDICINE
2008; 205 (7): 1701-1714
Interleukin-7 (IL-7) is a homeostatic cytokine for resting T cells with increasing serum and tissue levels during T cell depletion. In preclinical studies, IL-7 therapy exerts marked stimulating effects on T cell immune reconstitution in mice and primates. First-in-human clinical studies of recombinant human IL-7 (rhIL-7) provided the opportunity to investigate the effects of IL-7 therapy on lymphocytes in vivo. rhIL-7 induced in vivo T cell cycling, bcl-2 up-regulation, and a sustained increase in peripheral blood CD4(+) and CD8(+) T cells. This T cell expansion caused a significant broadening of circulating T cell receptor (TCR) repertoire diversity independent of the subjects' age as naive T cells, including recent thymic emigrants (RTEs), expanded preferentially, whereas the proportions of regulatory T (T reg) cells and senescent CD8(+) effectors diminished. The resulting composition of the circulating T cell pool more closely resembled that seen earlier in life. This profile, distinctive among cytokines under clinical development, suggests that rhIL-7 therapy could enhance and broaden immune responses, particularly in individuals with limited naive T cells and diminished TCR repertoire diversity, as occurs after physiological (age), pathological (human immunodeficiency virus), or iatrogenic (chemotherapy) lymphocyte depletion.
View details for DOI 10.1084/jem.20071681
View details for Web of Science ID 000258527000021
View details for PubMedID 18573906
Metabolic syndrome traits in long-term survivors of pediatric sarcoma
PEDIATRIC BLOOD & CANCER
2008; 50 (2): 341-346
The metabolic syndrome (MS), a cluster of central obesity, dyslipidemia, hyperglycemia, and hypertension, conveys an increased risk of type 2 diabetes and cardiovascular disease. This cross-sectional study investigated the prevalence of metabolic syndrome traits (MST) in long-term survivors of pediatric sarcoma (SARC) who received multi-modality therapy (MMT).Thirty-two SARC survivors (predominantly Ewings; median age 36.5; median age at MMT 15) underwent body composition, activity, and psychosocial analysis. Serum endocrine and inflammatory parameters and urine beta(2)-microglobulin (B2M) were evaluated. The prevalence of MST was compared to age- and gender-matched U.S. population data.SARC survivors were more likely to have two or more MST (OR 2.38 95% CI: [1.14, 5.04]). Analysis of individual MST demonstrated higher prevalence of hypertension (OR 2.61 95% CI: [1.20, 5.59]), hypertriglyceridemia (OR 3.63 95% CI: [1.75, 7.60]), and male visceral abdominal obesity (20-39 years old OR 4.63 95% CI: [0.91, 21.63], 40-59 years old OR infinity). Survivors 18-39 years old had a higher prevalence of the MS (OR 4.29 95% CI: [1.50, 11.21]), defined as three or more MST. Plasminogen activator inhibitory activity (P = 0.016) and B2M (P = 0.027) increased with increasing numbers of MST. In males, total testosterone declined (P = 0.0027) as the number of MST increased. Average (P = 0.014) and maximum (P = 0.021) activity levels decreased as the number of MST increased.After a median follow up of 17 years, adult SARC survivors of MMT had an increased prevalence of MST, especially those less than 40 years old. The development of MST in this population was associated with decreased testosterone and activity levels.
View details for DOI 10.1002/pbc.21363
View details for Web of Science ID 000252006000032
View details for PubMedID 17918262
- Thymic stromal lymphopoietin is not necessary or sufficient to mediate the thymopoietic effects of keratinocyte growth factor BLOOD 2008; 111 (2): 969-970
4-1BB is superior to CD28 costimulation for generating CD8(+) cytotoxic lymphocytes for adoptive immunotherapy
JOURNAL OF IMMUNOLOGY
2007; 179 (7): 4910-4918
Artificial APCs (aAPCs) genetically modified to express selective costimulatory molecules provide a reproducible, cost-effective, and convenient method for polyclonal and Ag-specific expansion of human T cells for adoptive immunotherapy. Among the variety of aAPCs that have been studied, acellular beads expressing anti-CD3/anti-CD28 efficiently expand CD4+ cells, but not CD8+ T cells. Cell-based aAPCs can effectively expand cytolytic CD8+ cells, but optimal costimulatory signals have not been defined. 4-1BB, a costimulatory molecule expressed by a minority of resting CD8+ T cells, is transiently up-regulated by all CD8+ T cells following activation. We compared expansion of human cytolytic CD8+ T cells using cell-based aAPCs providing costimulation via 4-1BB vs CD28. Whereas anti-CD3/anti-CD28 aAPCs mostly expand naive cells, anti-CD3/4-1BBL aAPCs preferentially expand memory cells, resulting in superior enrichment of Ag-reactive T cells which recognize previously primed Ags and efficient expansion of electronically sorted CD8+ populations reactive toward viral or self-Ags. Using HLA-A2-Fc fusion proteins linked to 4-1BBL aAPCs, 3-log expansion of Ag-specific CD8+ CTL was induced over 14 days, whereas similar Ag-specific CD8+ T cell expansion did not occur using HLA-A2-Fc/anti-CD28 aAPCs. Furthermore, when compared with cytolytic T cells expanded using CD28 costimulation, CTL expanded using 4-1BB costimulation mediate enhanced cytolytic capacity due, in part, to NKG2D up-regulation. These results demonstrate that 4-1BB costimulation is essential for expanding memory CD8+ T cells ex vivo and is superior to CD28 costimulation for generating Ag-specific products for adoptive cell therapy.
View details for Web of Science ID 000249752100070
View details for PubMedID 17878391
Therapy for metastatic ESFT: is it time to ask new questions?
Pediatric blood & cancer
2007; 49 (2): 115-116
View details for PubMedID 17474114
Immune reconstitution prevents metastatic recurrence of murine osteosarcoma
CANCER IMMUNOLOGY IMMUNOTHERAPY
2007; 56 (7): 1037-1046
Primary tumors developing in immunocompetent hosts escape immunosurveillance by acquiring immune evasive properties. This raises the prospect that metastases derived from such tumors will also evade immunity. To investigate whether immune surveillance plays a role in preventing metastases, we studied a murine model which mimics the clinical progression of osteosarcoma: primary tumor growth in the lower extremity, amputation, minimal residual disease followed by the development of overt metastases. K7M2 implants readily escaped immune surveillance since normal BALB/c mice, T cell deficient SCID and T/NK cell deficient SCID-bg mice showed no difference in the rate of growth of primary osteosarcomas. However, both SCID and SCID-bg mice had higher rates of metastases than immunocompetent mice. Similarly, immune reconstitution following transfer of naive T cells to SCID or SCID-bg mice did not impact primary tumor growth, but significantly diminished metastatic recurrence. T cells in osteosarcoma bearing mice produced IFNgamma in response to tumor and IFNgamma production by immune reconstituting T cells was required to prevent metastases. These results demonstrate an important role for T cell based immune surveillance in preventing metastases, even when metastases develop from tumors that adeptly evade immunosurveillance. The results further suggest that T cell depleting cancer therapies may eliminate beneficial immune responses and that immune reconstitution of lymphopenic cancer patients could prevent metastatic recurrence of solid tumors.
View details for DOI 10.1007/s00262-006-0257-0
View details for Web of Science ID 000246094800009
View details for PubMedID 17149595
Interferon-gamma sensitizes resistant Ewing's sarcoma cells to tumor necrosis factor apoptosis-inducing ligand-induced apoptosis by up-regulation of caspase-8 without altering chemosensitivity
AMERICAN JOURNAL OF PATHOLOGY
2007; 170 (6): 1917-1930
Ewing's sarcoma cells are highly susceptible to apoptosis via tumor necrosis factor apoptosis-inducing ligand (TRAIL). Resistance to TRAIL has been linked to deficient expression of caspase-8 in vitro. Here, we report on the status of caspase-8 expression in tumors from patients with Ewing's sarcoma, the effect of interferon-gamma on caspase-8 expression and apoptosis, and the role of caspase-8 for TRAIL- and chemotherapy-mediated apoptosis in Ewing's sarcoma. Using immunohistochemistry, we show that low expression of caspase-8 is seen in about 24% of tumors. Interferon-gamma induces expression of caspase-8 at concentrations achievable in humans and sensitizes cells to TRAIL. Transfection of wild type but not mutant caspase-8 into caspase-8-deficient Ewing's sarcoma cells restored sensitivity to TRAIL, indicating that up-regulation of caspase-8 is sufficient to restore TRAIL sensitivity. In contrast, no role for caspase-8 in chemotherapy-induced apoptosis was identified, because 1) transfection of caspase-8 or treatment with interferon-gamma did not alter the sensitivity of caspase-8-deficient cells to chemotherapeutics, 2) application of chemotherapy did not select for caspase-8-negative tumor cells in vivo, and 3) the caspase-8 status of tumors did not influence survival after chemotherapy-based protocols. In conclusion, our data provide a rationale for the inclusion of interferon-gamma in upcoming clinical trials with TRAIL.
View details for DOI 10.2353/ajpath.2007.060993
View details for Web of Science ID 000246955300012
View details for PubMedID 17525260
Thromboembolic events in children and young adults with pediatric sarcoma
JOURNAL OF CLINICAL ONCOLOGY
2007; 25 (12): 1519-1524
Adults with malignancy are at increased risk for venous thromboembolic events (TEs). However, data in children and young adults with cancer are limited.To determine the risk and clinical features of TEs in children and young adults with sarcoma, we reviewed records on 122 consecutive patients with sarcoma treated from October 1980 to July 2002.Twenty-three TEs were diagnosed in 19 of 122 (16%; 95% CI, 10% to 23%) patients. Prevalence by diagnosis was Ewing sarcoma, eight of 61 (13%); osteosarcoma, two of 20 (10%); rhabdomyosarcoma, four of 26 (15%); and other sarcomas, five of 15 (33%). TEs developed in 23% of patients with metastases at presentation versus 10% with localized disease (odds ratio, 2.59; 95% CI, 0.9 to 7.1; P < .06). Fifty-three percent of patients with thrombosis had a clot at presentation. A lupus anticoagulant was detected in four of five evaluated patients. There was a single fatality due to pulmonary embolism. Patients who were diagnosed with cancer after 1993 had a higher rate of TE (7% v 23%; P < .015). Of the 23 events, 43% were asymptomatic. Main sites of thromboses were deep veins of the extremities (10 of 23; 43%), pulmonary embolism (five of 23; 22%), and the inferior vena cava (four of 23; 17%). TEs were associated with tumor compression in eight of 23 (35%) and with venous catheters in three of 23 (13%).Thromboembolism is common in pediatric patients with sarcomas. Thromboses are detected frequently around the time of oncologic presentation, may be asymptomatic, and seem to be associated with a higher disease burden. Children and young adults with sarcoma should be monitored closely for thrombosis.
View details for DOI 10.1200/JCO.2006.06.9930
View details for Web of Science ID 000245920300012
View details for PubMedID 17442994
Potential role for IL-7 in Fas-mediated T cell apoptosis during HIV infection
JOURNAL OF IMMUNOLOGY
2007; 178 (8): 5340-5350
IL-7 promotes survival of resting T lymphocytes and induces T cell proliferation in lymphopenic conditions. As elevated IL-7 levels occur in HIV-infected individuals in addition to high Fas expression on T cells and increased sensitivity to Fas-induced apoptosis, we analyzed whether IL-7 has a regulatory role in Fas-mediated T cell apoptosis. We show that IL-7 up-regulates Fas expression on naive and memory T cells through a mechanism that involves translocation of Fas molecules from intracellular compartments to the cell membrane. IL-7 induced the association of Fas with the cytoskeletal component ezrin and a polarized Fas expression on the cell surface. The potential role of IL-7 in Fas up-regulation in vivo was verified in IL-7-treated macaques and in HIV-infected or chemotherapy treated patients by the correlation between serum IL-7 levels and Fas expression on T cells. IL-7 treatment primed T cells for Fas-induced apoptosis in vitro and serum IL-7 levels correlated with the sensitivity of T cells to Fas-induced apoptosis in HIV-infected individuals. Our data suggest an important role for IL-7 in Fas-mediated regulation of T cell homeostasis. Elevated IL-7 levels associated with lymphopenic conditions, including HIV-infection, might participate in the increased sensitivity of T cells for activation-induced apoptosis.
View details for Web of Science ID 000245605300079
View details for PubMedID 17404319
Immunologic, virologic, and neuropsychologic responses in human immunodeficiency virus-infected children receiving their first highly active antiretroviral therapy regimen
2007; 20 (1): 131-141
Our objective was to measure the early dynamics, evolution, and durability over 96 wk of immunologic responses in children receiving their first highly active antiretroviral therapy (HAART) regimen. The study was designed as a prospective, single-arm study. Twelve human immunodeficiency virus (HIV)-infected children (median age, 11.8 yr) were enrolled. All subjects received stavudine, nevirapine, and ritonavir. Serial measurements included HIV viral load, lymphocyte subsets, thymic volume by computed tomography (CT), neurocognitive testing, and brain CT. Baseline median CD4(+) T cell count was 589 cells/mm(3) , viral load was 3.9 log(10) HIV RNA copies/mL, and thymic volume was 16.3 cm(3) . Ten children had an undetectable viral load at week 48. Eight maintained an undetectable viral load at 96 wk. The median increase in absolute CD4(+) T cell count was 225 cells/mm(3) by week 48, and 307 cells/mm(3) by week 96. The median increase in naive (CD45RA(+) CD62L(+) ) CD4(+) T cells was 133 cells/mm(3) by week 48, and 147 cells/mm(3) by week 96. The median number of naive CD8(+) T cells increased from 205 to 284 cells/mm(3) by week 24; this increase was sustained to week 96. The number of B cells increased and was associated with a decrease in immunoglobulin levels. The number of natural killer cells was stable. There were no significant changes in thymic volume. Most children exhibited stable cognitive function over the course of the study. We conclude that, in this cohort of relatively immunocompetent HIV-infected children, an initial HAART regimen was associated with rapid and sustained increases in total CD4(+) T cells, in naive CD4(+) and CD8(+) T cells, and in B cells through 96 wk.
View details for DOI 10.1089/vim.2006.0079
View details for Web of Science ID 000245710300013
View details for PubMedID 17425427
Treatment late effects in long-term survivors of pediatric sarcoma
PEDIATRIC BLOOD & CANCER
2007; 48 (2): 192-199
To assess health and musculoskeletal function in survivors of pediatric sarcomas.Thirty-two individuals treated for Ewing sarcoma family of tumors (ESFT), rhabdomyosarcoma (RMS), or non-rhabdomyosarcoma soft tissue sarcomas (NR-STS) with multi-modality therapy were enrolled on this cross-sectional study. Median age at the time of therapy was 15.4 years (range 7.1-34.2), median age at the time of analysis was 37.4 years (17.5-55.4), and median duration of time elapsed from completion of therapy was 17.3 years (2.9-32.6). Participants underwent assessments of musculoskeletal functioning, cardiac function, metabolic and lipid analyses, renal and gonadal function, and psychological evaluation.This cohort of sarcoma survivors shows expected locoregional limitations in function of the area affected by sarcoma, and impaired global musculoskeletal functioning as evidenced by limited endurance and limited overall activity levels. The cohort also demonstrated substantial rates of cardiac dysfunction, elevated body fat index, hyperlipidemia, chronic psychological distress, and infertility in men (76%) and premature menopause (49%) in women.Sarcoma survivors demonstrate diminished locoregional and global musculoskeletal functioning which likely limit occupational opportunities and socioeconomic health. In addition, the combination of diminished cardiac reserve, limited activity levels, and lipid dysregulation in sarcoma survivors suggests that this population is at increased risk for cardiovascular disease, even many years following completion of sarcoma therapy. Sarcoma survivors may benefit from life long follow-up for cardiovascular disease and from occupational counseling upon completion of therapy.
View details for DOI 10.1002/pbc.20871
View details for Web of Science ID 000242875800013
View details for PubMedID 16642490
Functional outcomes and life satisfaction in long-term survivors of pediatric sarcomas
ARCHIVES OF PHYSICAL MEDICINE AND REHABILITATION
2006; 87 (12): 1611-1617
To describe the inter-relationships among impairments, performance, and disabilities in survivors of pediatric sarcoma and to identify measurements that profile survivors at risk for functional loss.Prospective, cross-sectional.Research facility.Thirty-two participants in National Cancer Institute clinical trials.Not applicable.Range of motion (ROM), strength, limb volume, grip strength, walk velocity, Assessment of Motor and Process Skills (AMPS); Human Activity Profile (HAP), Sickness Impact Profile (SIP), standard form of the Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36); and vocational attitudes and leisure satisfaction.Twenty of 30 survivors tested had moderate or severe loss of ROM; 13 of 31 tested had 90% or less of predicted walk velocity; all of whom had trunk or lower-extremity lesions. Women with decreased ROM (r=.50, P=.06) or strength (r=.74, P=.002) had slow gait velocity. Sixteen of 31 tested were more than 1 standard deviation below normal grip strength. Eighteen had increased limb volume. These 18 had low physical competence (SF-36) (r=-.70, P=.001) and high SIP scores (r=.73, P=.005). AMPS scores were lower than those of the matched normed sample (P<.001). HAP identified 15 of 30 who had moderately or severely reduced activity. Leisure satisfaction was higher in the subjects (P<.001). Eight reported cancer had negatively impacted work and 17 reported that it negatively impacted vocational plans.Survivors with lower-extremity or truncal lesions and women with decreased ROM and strength likely have slow walk velocity, low exercise tolerance, and high risk for functional loss. They should be identified using ROM, strength, limb volume, and walk time measures.
View details for DOI 10.1016/j.apmr.2006.08.341
View details for Web of Science ID 000242675000009
View details for PubMedID 17141641
Ewing sarcoma family of tumors in unusual sites: Confirmation by RT-PCR
PEDIATRIC AND DEVELOPMENTAL PATHOLOGY
2006; 9 (6): 488-495
Ewing sarcoma family tumors originating in the palate or adrenal gland are extremely rare and may cause difficulty in diagnosis. More common tumors primary to these sites need to be excluded before one arrives at the correct diagnosis. We have recently diagnosed 2 such cases. The 1st case was that of a 24-year-old woman who presented with a swelling in the right side of the hard palate. The 2nd case was diagnosed in a 28-year-old woman who presented with a mass in the right adrenal gland. In both cases, the diagnosis of Ewing sarcoma family of tumors was confirmed by immunohistochemical studies and reverse transcriptase-polymerase chain reaction (RT-PCR). The hard palate case is the 1st and the adrenal gland the 3rd case of Ewing sarcoma family of tumors arising in these sites, in which the diagnosis was confirmed by RT-PCR and/or cytogenetics. Accurate diagnosis of Ewing sarcoma family of tumors is crucial for the management of patients, and when found in such rare locations, diagnosis should be supported by immunohistochemical and/or molecular genetic studies.
View details for DOI 10.2350/06-01-0007.1
View details for Web of Science ID 000248596200009
View details for PubMedID 17163788
Dysregulation of IL-15-mediated T-cell homeostasis in TGF-beta dominant-negative receptor transgenic mice
2006; 108 (8): 2789-2795
T-cell subpopulations, defined by their expression of CD4, CD8, naive, and memory cell-surface markers, occupy distinct homeostatic compartments that are regulated primarily by cytokines. CD8+ memory T cells, as defined by CD44(hi) surface expression, are dependent on IL-15 as a positive regulator of their homeostatic maintenance. Manipulation of IL-15 signaling through gene aberration, overexpression, or receptor alterations has been shown to dramatically affect T-cell homeostasis, with overexpression leading to fatal leukemia. Here we show that TGF-beta is the critical negative regulator of murine CD8+ memory T-cell homeostasis with direct opposition to the positive effects of IL-15. This negative regulation is mediated, at least in part, by the ability of TGF-beta to modulate expression of the beta-chain of the IL-15 receptor, thus establishing a central axis between these 2 cytokines for homeostatic control of CD8+ memory T-cell populations. These data establish TGF-beta as a critical and dominant tumor-suppressor pathway opposing IL-15-mediated CD8+ T-cell expansion and potential malignant transformation.
View details for DOI 10.1182/blood-2006-05-025676
View details for Web of Science ID 000241131700048
View details for PubMedID 16788095
Persistent psychological distress in long-term survivors of pediatric sarcoma: The experience at a single institution
2006; 15 (10): 898-910
The long-term psychological impact of pediatric sarcoma is largely unknown. As part of a cross-sectional study examining the late effects of pediatric sarcoma therapy, we examined whether psychological distress or posttraumatic stress symptoms are present in an adult cohort of pediatric sarcoma survivors.Thirty-four patients participated in the study, an average of 17 years after their treatment ended, each completing the SCID module for Posttraumatic Stress Disorder, Impact of Events Scale, Brief Symptom Inventory (BSI) and a questionnaire assessing sociodemographic variables and psychosocial issues.Significant persistent psychological distress characterized this cohort of patients. Seventy-seven percent scored in the clinical range on the BSI. Twelve percent met diagnostic criteria for PTSD. Current psychological distress was associated with intrusive thoughts and avoidant behaviors, male gender, employment, difficulty readjusting to work/school after treatment, and enduring worries about health. No differences were found based on age, presence of metastatic disease or time since diagnosis.This is the first report of a clinical evaluation of psychological distress in a cohort of pediatric sarcoma survivors treated with intensive multimodal cancer therapy. The results suggest that survivors of pediatric sarcoma might be at high risk for adverse psychological outcomes. Appropriate interventions are proposed.
View details for DOI 10.1002/pon.1024
View details for Web of Science ID 000242309400006
View details for PubMedID 16402373
- Adolescents and young adults successfully restore lymphocyte homeostasis after intensive T-cell depleting therapy for cancer BRITISH JOURNAL OF HAEMATOLOGY 2006; 135 (2): 270-271
Autoimmunity during lymphopenia: A two-hit model
2006; 120 (2): 121-128
The immune system has evolved elaborate mechanisms to respond to diverse antigens while minimizing the risk for autoimmune reactivity. During lymphopenia, however, some mechanisms that normally serve to maintain host tolerance are temporarily suspended. Peripheral T cells proliferate in response to self-antigens in lymphopenic hosts, but proliferation toward these same antigens is prevented when T cell numbers are normal. This process, termed homeostatic peripheral expansion, augments peripheral T cell number and limits repertoire skewing during recovery from lymphopenia and also predisposes lymphopenic hosts to autoimmune disease. This paper reviews murine and human settings in which autoimmunity occurs in the context of lymphopenia. We propose a two-hit model, in which lymphopenia plus another insult is sufficient to induce autoimmune disease. Among the secondary insults that appear sufficient to induce autoimmunity during lymphopenia are overproduction of IL-21 as occurs in the NOD.SCID mouse, depletion of Tregs as demonstrated in murine colitis and gastritis models, and tissue inflammation as seen in HIV infected patients who develop immune reconstitution inflammatory syndrome (IRIS). Delineating critical cofactors which result in autoimmune disease during lymphopenia can provide insight into the pathophysiology of naturally occurring autoimmune diseases as well as generating testable hypothesis for inducing tumor-specific autoimmunity in lymphopenic hosts with cancer.
View details for DOI 10.1016/j.clim.2006.04.569
View details for Web of Science ID 000239545100001
View details for PubMedID 16766227
IL-7 in allogeneic transplant: Clinical promise and potential pitfalls
LEUKEMIA & LYMPHOMA
2006; 47 (7): 1222-1228
Much progress has been made in the field of allogeneic stem cell transplantation. However, one major barrier is the delay in immune recovery that can persist for months post-transplant and results in increased susceptibility to infection and relapse of malignancy. Strategies to improve immune recovery must be balanced with the potential for those therapies to exacerbate graft vs host disease. Interleukin 7 is a member of the gammac cytokine family that is required for T-cell development and maintenance of naïve T-cell populations. In addition, IL-7 plays a major role in the expansion of mature T-cells that occurs during lymphopenia and therapeutic IL-7 can enhance both quantitative and functional immune recovery following T-cell depletion. Thus, this agent holds much promise as an immunorestorative agent and as an adjuvant to vaccines or adoptive immunotherapy. Clinic trials with IL-7 are underway. Murine studies with IL-7 in the allogeneic transplant have demonstrated that the potent immune effects of this agent can also be achieved in this setting. However, these studies have indicated that the potential for IL-7 to worsen GVHD exists and that this effect may abrogate the immune benefits. Thus, careful consideration of how best to incorporate IL-7 into allogeneic trials will be needed if the full potential of this agent is to be realized.
View details for DOI 10.1080/10428190600555876
View details for Web of Science ID 000239922600007
View details for PubMedID 16923550
IL-7 administration to humans leads to expansion of CD8(+) and CD4(+) cells but a relative decrease of CD4(+) T-regulatory cells
JOURNAL OF IMMUNOTHERAPY
2006; 29 (3): 313-319
Lymphopenia is a serious consequence of HIV infection and the administration of cancer chemotherapeutic agents. Although growth factors can be administered to patients to increase circulating neutrophils, there is no effective method to stimulate CD8+ lymphocyte production in humans, in vivo. This report is the first to describe the administration of recombinant interleukin-7 to humans and demonstrates the ability of this cytokine to mediate selective increases in CD4+ and CD8+ lymphocytes along with a decrease in the percentage of CD4+ T-regulatory cells. These studies suggest an important role for interleukin-7 in the treatment of patients with lymphopenia.
View details for Web of Science ID 000237328400009
View details for PubMedID 16699374
CD1d-restricted natural killer T cells can down-regulate tumor immunosurveillance independent of interleukin-4 receptor-signal transducer and activator of transcription 6 or transforming growth factor-beta
2006; 66 (7): 3869-3875
It has been shown previously that the suppression of tumor immunosurveillance may be a mechanism by which tumors resist immune detection and elimination. In this study, we evaluated the role of the immunoregulatory natural killer T (NKT) cells in the biology of immunosurveillance of osteosarcoma. The K7M2 mouse osteosarcoma cell line was implanted orthotopically into wild-type and NKT cell-deficient CD1d knockout (KO) BALB/c mice, and mice were monitored for growth of primary tumors. Further, we examined the role of CD4(+) and/or CD8(+) cells by depleting the cells in vivo and measuring CTL activity in vitro. We also asked the role of interleukin (IL)-4 receptor alpha (IL-4Ralpha)-signal transducer and activator of transcription 6 (STAT6) signaling, including IL-13, and transforming growth factor beta (TGF-beta) by using gene-disrupted mice or treating mice with cytokine antagonists. We were surprised to find a high rate of rejection of osteosarcoma primary tumors in 88% (14 of 16) of CD1d KO mice compared with syngeneic wild-type BALB/c mice that showed rejection of tumor in <24% of mice. Further studies suggested that the rejection of tumor in CD1d KO mice was dependent on CD8(+) lymphocytes. Distinct from other murine tumor models, the negative regulation induced by CD1d-restricted NKT cells was not dependent on IL-4Ralpha-STAT6 signaling, including IL-13, or on TGF-beta. These data suggest that a novel CD1d-restricted NKT cell-mediated mechanism for tumor immunosuppression is active in the K7M2 osteosarcoma model and that NKT cells can regulate immunosurveillance through more than one pathway.
View details for DOI 10.1158/0008-5472.CAN-05-3421
View details for Web of Science ID 000236657800068
View details for PubMedID 16585215
Identification and epitope enhancement of a PAX-FKHR fusion protein breakpoint epitope in alveolar rhabdomyosarcoma cells created by a tumorigenic chromosomal translocation inducing CTL capable of lysing human tumors
2006; 66 (3): 1818-1823
Fusion proteins created by chromosomal translocations in tumors can create neoantigenic determinants at the breakpoint, which are unique to the tumor cells but shared by the vast majority of tumors of that histologic type. If the fusion protein is responsible for the malignant transformation, its expression cannot be lost by the tumor to escape immune responses against this tumor antigen. Here, we identify such a fusion protein breakpoint epitope in the PAX-FKHR fusion protein created by the t(2;13) translocation present in 80% of cases of alveolar rhabdomyosarcoma, a highly aggressive pediatric soft-tissue sarcoma. We use autologous dendritic cells pulsed with the RS10 breakpoint fusion peptide to raise a human CTL line from a normal healthy HLA-B7+ blood donor specific for this peptide. These CTLs are CD8+ (CD4-CD56-) and restricted by HLA-B7. These human peptide-specific CTL lyse human HLA-B7+ rhabdomyosarcoma tumor cells. Therefore, the fusion protein is endogenously processed to produce this natural epitope presented by HLA-B7 and thus this peptide is a bone fide human tumor antigen. We also define a substitution that increases the affinity for HLA-B7 without loss of antigenicity. This epitope-enhanced peptide may serve as a candidate cancer vaccine for HLA-B7+ patients with alveolar rhabdomyosarcoma.
View details for DOI 10.1158/0008-5472.CAN-05-2549
View details for Web of Science ID 000235095900070
View details for PubMedID 16452243
- Immune reconstitution: From stem cells to lymphocytes BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION 2006; 12 (1): 42-46
Lymphopenia and interleukin-2 therapy alter homeostasis of CD4(+) CD25(+) regulatory T cells
2005; 11 (11): 1238-1243
CD4(+)CD25(+) regulatory T (T(reg)) cells have a crucial role in maintaining immune tolerance. Mice and humans born lacking T(reg) cells develop severe autoimmune disease, and depletion of T(reg) cells in lymphopenic mice induces autoimmunity. Interleukin (IL)-2 signaling is required for thymic development, peripheral expansion and suppressive activity of T(reg) cells. Animals lacking IL-2 die of autoimmunity, which is prevented by administration of IL-2-responsive T(reg) cells. In light of the emerging evidence that one of the primary physiologic roles of IL-2 is to generate and maintain T(reg) cells, the question arises as to the effects of IL-2 therapy on them. We monitored T(reg) cells during immune reconstitution in individuals with cancer who did or did not receive IL-2 therapy. CD4(+)CD25(hi) cells underwent homeostatic peripheral expansion during immune reconstitution, and in lymphopenic individuals receiving IL-2, the T(reg) cell compartment was markedly increased. Mouse studies showed that IL-2 therapy induced expansion of existent T(reg) cells in normal hosts, and IL-2-induced T(reg) cell expansion was further augmented by lymphopenia. On a per-cell basis, T(reg) cells generated by IL-2 therapy expressed similar levels of FOXP3 and had similar potency for suppression compared to T(reg) cells present in normal hosts. These studies suggest that IL-2 and lymphopenia are primary modulators of CD4(+)CD25(+) T(reg) cell homeostasis.
View details for DOI 10.1038/nm1312
View details for Web of Science ID 000233115400037
View details for PubMedID 16227988
Cytokine signals in T-cell homeostasis
JOURNAL OF IMMUNOTHERAPY
2005; 28 (4): 289-294
Thymic production of T cells declines rapidly with age, and therefore homeostatic cycling (HC) of mature lymphocytes plays an important role in maintaining stable numbers of mature T lymphocytes bearing sufficient repertoire diversity. Following lymphocyte depletion, HC changes in quality and magnitude, resulting in homeostatic peripheral expansion (HPE), a state of widespread T-cell cycling that serves to increase T-cell number and to maintain T-cell repertoire diversity to the greatest extent possible. Recent studies delineating the requirements for HC and HPE have shown that naive CD4+ cells and naive CD8+ cells require both IL7 and TCR engagement for survival, cycling, and homeostatic expansion, whereas CD8+ memory cells are maintained and expanded by cytokine signals alone, independent of TCR engagement. While basal levels of IL15 are sufficient for HC and HPE of CD8+ memory cells, supranormal levels of IL7 will also suffice. The requirements for memory CD4+ cells remain unclear, but current models hypothesize that either IL7 or TCR triggering may be sufficient. Thus, the changes in immune physiology that are present in lymphopenic hosts can be largely accounted for by cytokine-driven signals, especially those rendered by IL7 or IL15. As the alterations in immune physiology present in lymphopenic hosts may be conducive to stronger antitumor immune responsiveness, careful delineation of the factors responsible may be expected to give rise to approaches to augment the effectiveness of current antitumor immunotherapies.
View details for Web of Science ID 000230219200002
View details for PubMedID 16000945
The many faces of IL-7: From lymphopoiesis to peripheral T cell maintenance
JOURNAL OF IMMUNOLOGY
2005; 174 (11): 6571-6576
IL-7 is well known as a lymphopoietic cytokine, but recent studies have also identified a critical role for IL-7 in peripheral T cell homeostasis. IL-7 is well poised to serve as a homeostatic cytokine because it is produced by resting stromal cells, the IL-7R is present on most T cells, and IL-7 down-regulates its own receptor. These features allow IL-7 to signal large numbers of resting T cells and to be efficiently used when supplies are limiting. Consistent with this, in normal hosts, IL-7 is required for survival of naive T cell populations, and IL-7 contributes to homeostatic cycling of naive and memory cells. In addition, lymphopenic hosts accumulate increased levels of IL-7, and the supranormal levels are largely responsible for inducing homeostatic peripheral expansion in response to lymphopenia. Thus, IL-7 plays critical and nonredundant roles in both T cell lymphopoiesis and in maintaining and restoring peripheral T cell homeostasis.
View details for Web of Science ID 000229298400007
View details for PubMedID 15905493
Adjuvant IL-7 or IL-15 overcomes immunodominance and improves survival of the CD8(+) memory cell pool
JOURNAL OF CLINICAL INVESTIGATION
2005; 115 (5): 1177-1187
Current models of T cell memory implicate a critical role for IL-7 in the effector-to-memory transition, raising the possibility that IL-7 therapy might enhance vaccine responses. IL-7 has not been studied, to our knowledge, before now for adjuvant activity. We administered recombinant human IL-7 (rhIL-7) to mice during immunization against the male antigen HY and compared these results with those obtained from mice immunized with rhIL-2 and rhIL-15. Administration of rhIL-7 or rhIL-15, but not rhIL-2, increased effector cells directed against these dominant antigens and dramatically enhanced CD8(+) effectors to subdominant antigens. The mechanisms by which the cytokines augmented effector pool generation were multifactorial and included rhIL-7-mediated costimulation and rhIL-15-mediated augmentation of the proliferative burst. The contraction phase of the antigen-specific response was exaggerated in cytokine-treated mice; however, CD8(+) memory pools in rhIL-7- or rhIL-15-treated groups demonstrated superior long-term survival resulting in quantitative advantages that remained long after the cytokines were discontinued, as demonstrated by improved survival after challenge with an HY-expressing tumor undertaken several weeks after cytokine cessation. These results confirm the adjuvant activity of rhIL-15 and demonstrate that rhIL-7 also serves as a potent vaccine adjuvant that broadens immunity by augmenting responses to subdominant antigens and improving the survival of the CD8(+) T cell memory pool.
View details for DOI 10.1172/JCI200523134
View details for Web of Science ID 000228908300018
View details for PubMedID 15841203
Adjuvant chemotherapy for the treatment of advanced pediatric nonrhabdomyosarcoma soft tissue sarcoma: the National Cancer Institute experience
PEDIATRIC BLOOD & CANCER
2005; 44 (5): 449-454
The survival of children and adolescents with advanced (unresectable or metastatic) nonrhabdomyosarcoma soft tissue sarcoma (NRSTS) is poor. In order to clarify the role of combining chemotherapy with aggressive local control using surgery and/or radiation, we reviewed our institutional experience with the treatment of advanced pediatric NRSTS.We reviewed the charts of all patients less than 21 years treated for an advanced NRSTS at the National Cancer Institute (NCI) between 1983 and 2003. Tumor pathology was confirmed and demographic, disease, and treatment data were abstracted. Survival was calculated using standard methods.Of the 25 patients who were treated over the study period, 15 had metastatic disease and 10 had unresectable or incompletely resected disease at presentation. Twenty-one patients received chemotherapy consisting of the combination of vincristine, doxorubicin, cyclophosphamide, ifosfamide, and etoposide, and the remaining 4 received regimens that included doxorubicin. Twenty patients (80%) had a complete (5/25) or partial (15/25) response after chemotherapy alone. After the combination of chemotherapy and local control, 14 patients (56%) had a complete response (CR). The estimated 5-year overall and event-free survival (EFS) for all patients was 0.50 (standard error = 0.11) and 0.34 (standard error = 0.10), respectively.The combination of chemotherapy with aggressive local control in this cohort of pediatric patients with advanced NRSTS yielded results comparable to those observed in patients with advanced sarcomas that are chemotherapy responsive. Prospective randomized trials are needed to quantify the contribution of chemotherapy and to determine the ideal regimen.
View details for Web of Science ID 000227990200005
View details for PubMedID 15547929
Age-dependent incidence, time course, and consequences of thymic renewal in adults
JOURNAL OF CLINICAL INVESTIGATION
2005; 115 (4): 930-939
Homeostatic regulation of T cells involves an ongoing balance of new T cell generation, peripheral expansion, and turnover. The recovery of T cells when this balance is disrupted provides insight into the mechanisms that govern homeostasis. In a long-term, single cohort study, we assessed the role of thymic function after autologous transplant in adults, correlating serial computed tomography imaging of thymic size with concurrent measurements of peripheral CD4(+) T cell populations. We established the age-dependent incidence, time course, and duration of thymic enlargement in adults and demonstrated that these changes were correlated with peripheral recovery of naive CD45RA(+)CD62L(+) and signal-joint TCR rearrangement excision circle-bearing CD4(+) populations with broad TCR diversity. Furthermore, we demonstrated that renewed thymopoiesis was critical for the restoration of peripheral CD4(+) T cell populations. This recovery encompassed the recovery of normal CD4(+) T cell numbers, a low ratio of effector to central memory cells, and a broad repertoire of TCR Vbeta diversity among these memory cells. These data define the timeline and consequences of renewal of adult thymopoietic activity at levels able to quantitatively restore peripheral T cell populations. They further suggest that structural thymic regrowth serves as a basis for the regeneration of peripheral T cell populations.
View details for Web of Science ID 000228145700024
View details for PubMedID 15776111
Immune reconstitution following hematopoietic progenitor cell transplantation: challenges for the future
BONE MARROW TRANSPLANTATION
2005; 35: S53-S57
Successful hematopoietic progenitor cell transplantation requires rapid and complete transfer of the donor hematopoietic and immune systems to the host. Whereas the uncontrolled transfer of a nontolerant donor immune system results in GVHD in many cases, strategies which diminish GVHD also diminish immune reconstitution. Thus, the reliable, rapid and safe transfer of immunity from donor to host remains a major challenge for the field. Advances in the understanding of the biology of immune reconstitution have elucidated that thymic-dependent immune reconstitution can restore global immunity, but is especially vulnerable to toxicities associated with transplant. Alternatively, homeostatic peripheral expansion can be exploited for targeted immunity toward pathogens and tumors, but is difficult to manipulate without exacerbating GVHD risk. New translatable strategies are needed to safely augment one or both of these pathways in the setting of allogeneic hematopoietic progenitor cell transplantation.
View details for DOI 10.1038/sj.bmt.1704848
View details for Web of Science ID 000228146700013
View details for PubMedID 15812532
Thymic function in HIV infection.
Current HIV/AIDS reports
2005; 2 (1): 24-28
Current models hold that CD4+ depletion occurs as a result of direct and indirect effects of HIV, which both kill peripheral CD4+ cells and prevent adequate regeneration. Although age-associated involution diminishes thymic reserve and HIV is clearly thymotoxic, clinical trials have nonetheless shown that large proportions of patients who sustain adequate control of viral replication with highly active antiretroviral therapy (HAART) will demonstrate some evidence for thymic-dependent immune reconstitution, which is associated with improved immune competence. Furthermore, patients with insufficient or absent immune reconstitution following HAART generally lack evidence for thymopoiesis. Current studies are focused on improving our understanding of the causes for thymic failure in HIV infection. Recent work has demonstrated that some HIV strains, especially those that are CXCR4 trophic, are more thymotoxic and may contribute to irreversible thymic damage in this population.
View details for PubMedID 16091245
Cancer therapy-induced immune modulation.
Cancer chemotherapy and biological response modifiers
2005; 22: 325-341
View details for PubMedID 16110619
Interferon gamma enhances the effectiveness of tumor necrosis factor-related apoptosis-inducing ligand receptor agonists in a xenograft model of Ewing's sarcoma
2004; 64 (22): 8349-8356
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces selective apoptosis in a variety of tumors, including most cell lines derived from Ewing's sarcoma family of tumors, an aggressive sarcoma that afflicts children and young adults. To determine the in vivo efficacy of TRAIL receptor agonists in Ewing's sarcoma family of tumors, mice with orthotopic xenografts were treated with anti-TRAIL-R2 monoclonal antibody or TRAIL/Apo2L in a model that can identify effects on both primary tumors and metastases. Administration of either agonist slowed tumor growth in 60% of animals and induced durable remissions in 11 to 19% but did not alter the incidence of metastatic disease. Response rates were not improved by concurrent doxorubicin treatment. Cells recovered from both TRAIL receptor agonist-treated and nontreated tumors were found to be resistant to TRAIL-induced death in vitro unless pretreated with interferon (IFN) gamma. This resistance coincided with a selective down-regulation of TRAIL receptor expression on tumor cells. In vivo treatment with IFNgamma increased tumor expression of TRAIL receptors and caspase 8, but did not increase the antitumor effect of TRAIL receptor agonists on primary tumors. However, IFNgamma treatment alone or in combination with a TRAIL receptor agonist significantly decreased the incidence of metastatic disease and the combination of TRAIL receptor agonist therapy with IFNgamma-mediated impressive effects on both primary tumors and metastatic disease. These data demonstrate that in vivo growth favors TRAIL resistance but that TRAIL receptor agonists are active in Ewing's sarcoma family of tumors and that the combination of TRAIL receptor agonists with IFNgamma is a potent regimen in this disease capable of controlling both primary and metastatic tumors.
View details for Web of Science ID 000225182300031
View details for PubMedID 15548704
Flt3 ligand enhances thymic-dependent and thymic-independent immune reconstitution
2004; 104 (9): 2794-2800
Despite recent progress in our understanding of the biology of T-cell homeostasis, clinically available therapies to substantially improve immune reconstitution in patients sustaining T-cell depletion are lacking. T cells are regenerated via a dynamic interplay between thymopoiesis and thymic-independent homeostatic peripheral expansion (HPE). Using athymic mice subjected to T-cell depletion, we observed that HPE is critically dependent on dendritic cells (DCs) for presentation of antigen, raising the possibility that the availability of DCs might be limiting in vivo for HPE to occur efficiently. Indeed, flt3 ligand (flt3L) treatment of athymic mice subjected to T-cell depletion (without DC depletion) substantially enhanced HPE and improved immune competence. Following bone marrow transplantation (BMT) in athymic hosts, both dendritic cells and T cells were profoundly depleted and flt3L therapy restored DC numbers and enhanced HPE. In addition, thymus-bearing BMT recipients treated with flt3L regenerated increased numbers of thymic-dependent progeny with increased numbers of T-cell receptor excision circle (TREC)-positive T cells, indicating increased thymopoiesis. Therefore, flt3L is a potent immunorestorative agent that enhances both thymic-dependent and thymic-independent pathways of T-cell regeneration.
View details for Web of Science ID 000224795700035
View details for PubMedID 15226184
Escape from immune surveillance does not result in tolerance to tumor-associated antigens
JOURNAL OF IMMUNOTHERAPY
2004; 27 (5): 329-338
Despite expression of tumor-associated or tumor-specific antigens by most tumors, evasion of protective T-cell immunity is the rule rather than the exception. Understanding whether tumor immune escape primarily represents T-cell neglect, anergy/tolerance, or quantitative limits of an existent immune response is central to developing new strategies to enhance antitumor immunity. The authors studied the immune response to MB49, a tumor that naturally expresses HY. Immune surveillance was effective following low-dose tumor inocula, since normal female mice showed a diminished incidence and slower growth rate of MB49 compared with T-cell-depleted female mice and male mice. Following high-dose tumor inoculation, females developed large, progressive tumors but continued to demonstrate immune responses to class I and class II restricted HY epitopes. The HY reactive T cells remained capable of executing HY immune responses since T cells adoptively transferred from MB49-bearing animals mediated accelerated HY skin graft rejection compared with those taken from naive mice. Thus, MB49 does not induce immune tolerance to HY but rather escapes immune surveillance largely due to quantitative limits of the immune response. Treatment of tumor-bearing animals with rhIL7 significantly increased the number of T cells responding to HY but did not alter tumor growth rate. These results demonstrate that escape from immune surveillance does not necessarily imply immune tolerance to tumor antigens and that immunotherapy need not overcome tumor-induced tolerance per se, and suggest that substantial opportunities remain in tumor-bearing hosts to amplify weak but persistent antitumor immune responses.
View details for Web of Science ID 000223559800001
View details for PubMedID 15314541
A dose effect of IL-7 on thymocyte development
2004; 104 (5): 1419-1427
To study interleukin-7 (IL-7) in early thymocyte development, we generated mice transgenic (Tg) for the IL-7 gene under control of the lck proximal promoter. Founder line TgA, with the lowest level of IL-7 overexpression, showed enhanced alphabeta T-cell development. In contrast, in the highest overexpressing founder line, TgB, alphabeta T-cell development was disturbed with a block at the earliest intrathymic precursor stage. This was due to decreased progenitor proliferation as assessed by Ki-67 staining and in vivo bromodeoxyuridine (BrdU) incorporation. Bcl-2 was up-regulated in T-cell-committed progenitors in all Tg lines, and accounted for greater numbers of double positive (DP), CD4 single positive (SP), and CD8SP thymocytes in TgA mice where, in contrast to TgB mice, thymocyte progenitor proliferation was normal. Mixed marrow chimeras using TgB(+) and congenic mice as donors, and experiments using anti-IL-7 monoclonal antibody (MAb) in vivo, confirmed the role of IL-7 protein in the observed TgB phenotype. In conclusion, at low Tg overexpression, IL-7 enhanced alphabeta T-cell development by increasing thymocyte progenitor survival, while at high overexpression IL-7 reduces their proliferation, inducing a dramatic block in DP production. These results show for the first time in vivo a dose effect of IL-7 on alphabeta T-cell development and have implications for IL-7 in the clinical setting.
View details for DOI 10.1182/blood-2004-01-0201
View details for Web of Science ID 000223544000037
View details for PubMedID 15155461
Recombinant interleukin-7 induces proliferation of naive macaque CD4(+) and CD8(+) T cells in vivo
JOURNAL OF VIROLOGY
2004; 78 (18): 9740-9749
Interleukin-7 (IL-7) regulates T-cell homeostasis, and its availability is augmented in lymphopenic hosts. Naive CD8+ T cells transferred to lymphopenic mice acquire a memory-like phenotype, raising the possibility that IL-7 is the biological mediator of this effect. Here, we provide direct evidence that IL-7 induces the acquisition of memory-cell markers not only in CD8+ T cells but also in CD4+ T-cell subsets in immune-competent Indian rhesus macaques. The increase of these memory-like populations was dependent on the dose of the cytokine, and these cells were found in the blood as well as secondary lymphoid organs. Memory-like CD4+ and CD8+ T cells acquired the ability to secrete tumor necrosis factor alpha and, to a lesser extent, gamma interferon following stimulation with a cognate antigen. The phenotypic change observed in naive T cells was promptly reversed after discontinuation of IL-7. Importantly, IL-7 induced cycling of both CD4+ and CD8+ central memory and effector memory T cells, demonstrating its contribution to the maintenance of the entire T-cell pool. Thus, IL-7 may be of benefit in the treatment of iatrogenic or virus-induced T-cell depletion.
View details for DOI 10.1128/JVI.78.18.9740-9749.2004
View details for Web of Science ID 000223701100018
View details for PubMedID 15331707
A role for thymic stromal lymphopoietin in CD4(+) T cell development
JOURNAL OF EXPERIMENTAL MEDICINE
2004; 200 (2): 159-168
Thymic stromal lymphopoietin (TSLP) signals via a receptor comprising the interleukin (IL)-7 receptor alpha chain and a distinctive subunit, TSLP receptor (TSLPR), which is most related to the common cytokine receptor gamma chain, gamma(c). We have generated TSLPR knockout (KO) mice and found that although these mice had normal lymphocyte numbers, gamma(c)/TSLPR double KO mice had a greater lymphoid defect than gamma(c) KO mice. This indicates that TSLP contributes to lymphoid development and accounts for some of the residual lymphoid development in gamma(c) KO mice and presumably in patients with X-linked severe combined immunodeficiency. Injection of TSLP into gamma(c) KO mice induced the expansion of T and B cells. Moreover, sublethally irradiated TSLPR KO mice showed weaker recovery of lymphocyte populations than wild-type (WT) littermates, even when neutralizing anti-IL-7 antibodies were injected. Interestingly, TSLP preferentially stimulated the proliferation and survival of CD4(+) single positive thymocytes and peripheral T cells in vitro. Additionally, CD4(+) T cells from TSLPR KO mice expanded less efficiently than WT CD4(+) T cells in irradiated hosts, and TSLP preferentially expanded CD4(+) T cells both in vitro and in vivo. Thus, as compared with other known cytokines, TSLP is distinctive in exhibiting a lineage preference for the expansion and survival of CD4(+) T cells.
View details for DOI 10.1084/jem.20031975
View details for Web of Science ID 000222926000004
View details for PubMedID 15263024
Pediatric large-volume leukapheresis: a single institution experience with heparin versus citrate-based anticoagulant regimens
2004; 44 (2): 229-238
Anticoagulant-associated toxicity may exert significant effects on the safety and efficacy of large-volume leukapheresis (LVL) in children, however, few studies specifically address management of this issue.Seventy-four consecutive LVL procedures (mean, 4 blood volumes processed) in children weighing less than or equal to 30 kg (minimum, 10.9 kg) were analyzed. The first 21 procedures were evaluated retrospectively; 11 used heparin alone (Group I) and 10 used heparin plus reduced-dose ACD-A (whole blood to anticoagulant ratio > or =20:1) (Group II). The next 53 procedures were evaluated prospectively and used full-dose ACD-A (whole blood to anticoagulant ratio < or =13:1), intravenous divalent cation prophylaxis and no heparin; 11 used calcium alone (Group III) followed by 42 with calcium plus magnesium (Group IV).Seventy-four LVL (56 PBPC and 18 MNC) collections were performed in 38 subjects. One donor in Group I experienced a significant groin hematoma at the site of line placement. One donor each in Groups III and IV had mild paresthesias. Despite a mean citrate infusion rate of 2.6 mg per kg per minute, mean postapheresis serum potassium and ionized magnesium and calcium concentrations in Group IV declined by only 9, 8, and 4 percent, respectively, and stable levels of these variables were maintained 24 hours later. Postapheresis PLT counts declined significantly from baseline preapheresis levels in all groups (mean, 52% decrease).Use of full-dose citrate anticoagulant with prophylactic intravenous divalent cation infusion offers an effective and safe approach to management of anticoagulant-related toxicity in children undergoing LVL.
View details for Web of Science ID 000189055900013
View details for PubMedID 14962314
Effect of imatinib mesylate on neuroblastoma tumorigenesis and vascular endothelial growth factor expression
JOURNAL OF THE NATIONAL CANCER INSTITUTE
2004; 96 (1): 46-55
Alternative treatment options are needed for advanced neuroblastoma patients because their prognosis remains poor after intensive chemotherapy. Neuroblastoma cells express platelet-derived growth factor (PDGF), stem cell factor (SCF), and vascular endothelial growth factor (VEGF) and their respective receptors, PDGFR, c-Kit, and Flk-1. We therefore evaluated the effects of imatinib mesylate (imatinib), a selective inhibitor of the tyrosine kinase activities of c-Kit and PDGFR, on the growth of neuroblastoma cells in vivo and in vitro.We tested seven human neuroblastoma cell lines for their sensitivity to imatinib. Cell viability was assessed by trypan blue dye exclusion. Apoptosis was evaluated by nuclear staining, flow cytometry, and western blotting. Protein assays included immunoprecipitation, western blotting, enzyme-linked immunosorbent assays, and immunohistochemistry. mRNA expression was assessed by northern blotting. We used a xenograft model in SCID mice (10 mice per group) to evaluate the effects of imatinib oral therapy (50 or 100 mg/kg every 12 hours for 14 days) on neuroblastoma tumor growth. All statistical tests were two-sided.All seven neuroblastoma cell lines treated with imatinib displayed concentration-dependent decreases in cell viability, which coincided with an induction of apoptosis, and with ligand-stimulated phosphorylation of c-Kit and PDGFR. The imatinib concentrations that caused 50% inhibition of growth and 50% inhibition of ligand-induced phosphorylation of these receptors were 9-13 micro M and 0.1-0.5 microM, respectively. Expression of VEGF, but not phosphorylation of Flk-1, its receptor, was reduced in neuroblastoma cells treated with imatinib at 10 microM or higher. Mice treated with imatinib at 50 mg/kg or 100 mg/kg had statistically significantly smaller tumors than control mice treated with vehicle (mean tumor volume in mice treated with imatinib at 50 mg/kg = 1546 mm3, in control mice = 2954 mm3; difference = 1408 mm3, 95% confidence interval [CI] = 657 to 2159 mm3; P<.001; mean tumor volume in mice treated with imatinib at 100 mg/kg = 463 mm3; difference = 2491 mm3, 95% CI = 1740 to 3242 mm3; P<.001).Imatinib inhibited the growth of neuroblastoma cells in vitro and in vivo. This inhibition was associated with suppression of PDGFR and c-Kit phosphorylation and inhibition of VEGF expression.
View details for DOI 10.1093/jnci/djh004
View details for Web of Science ID 000188253400011
View details for PubMedID 14709738
Tumor expression of 4-1BB ligand sustains tumor lytic T cells
CANCER BIOLOGY & THERAPY
2003; 2 (5): 579-586
Inadequate costimulation by solid tumors is generally believed to induce immune tolerance during primary tumor growth. We looked for tumor-specific immunity vs. tolerance in patients with Ewing's sarcoma. Circulating T cells from patients with progressively growing Ewing's tumors displayed MHC restricted tumor-induced proliferation and robust tumor lysis. Tumor-reactive T cells reside within the memory CD3+CD8+ subset and are CD28-/4-1BB+. Autologous Ewing's tumors expressed 4-1BBL, and tumor-induced T cell proliferation and activation required costimulation by 4-1BBL. Stimulation of PBL with anti-CD3/4-1BBL, but not anti-CD3/anti-CD28 induced tumor lytic effectors. Similarly, in a xenograft model, anti-CD3/4-1BBL expanded T cells controlled primary growth and prevented metastasis of autologous tumors while nonactivated and anti-CD3/anti-CD28 activated CD8+ cells did not. These results question prevailing models of tumor induced tolerance accompanying progressive tumor growth; rather, we show coexistence of progressive tumor growth and anti-tumor immunity, with costimulation provided by the tumor itself. They further demonstrate a potential new therapeutic role for 4-1BBL mediated costimulation in expanding tumor reactive CTLs for use in the adoptive immunotherapy of cancer.
View details for Web of Science ID 000187076300022
View details for PubMedID 14614331
Beta-platelet-derived growth factor receptor mediates motility and growth of Ewing's sarcoma cells
2003; 22 (15): 2334-2342
The Ewing's sarcoma family of tumors (ESFT) contain a translocation, t(11;22), which results in the novel oncogenic fusion protein EWS/FLI1. Platelet-derived growth factors (PDGF) and their receptors (PDGFR) are involved in the induction and proliferation of numerous solid tumors and are the potential candidates for novel targeted antitumor therapy. Since a relation was reported between PDGF-C and EWS/FLI1, we sought to characterize the PDGF signaling pathway in ESFT. Eight out of nine ESFT cell lines were found to express significant levels of beta-PDGFR. Interestingly, none of the tested cell lines expressed alpha-PDGFR, which is the receptor isotype required for PDGF-C binding. By immunohistochemical staining 47 of 52 (90.4%) archival tumor samples from patients with ESFT were positive for beta-PDGFR. ESFT cell lines were treated with PDGF-AA or PDGF-BB ligands to evaluate downstream signaling. Autophosphorylation of beta-PDGFR and tyrosine phosphorylation of PLC-gamma, PI3Kp85 and Shc were detected only in PDGF-BB-stimulated cells that express beta-PDGFR. Receptor function was further evaluated using chemotaxis assays that showed TC-32 cell migration towards PDGF-BB. A specific PDGFR kinase inhibitor AG1295 blocked beta-PDGFR activation, downstream signaling, growth in cell culture and chemotaxis of TC-32 cells. AG1295 also delayed tumor formation and prolonged survival in an ESFT animal model. We conclude that ESFT express beta-PDGFR and that this is a functional and potentially crucial signaling pathway. Therefore, beta-PDGFRs may provide a novel therapeutic target in ESFT that can be utilized to design better treatment modalities.
View details for DOI 10.1038/sj.onc.1206330
View details for Web of Science ID 000182231500011
View details for PubMedID 12700668
IL-7 therapy dramatically alters peripheral T-cell homeostasis in normal and SIV-infected nonhuman primates
2003; 101 (6): 2294-2299
Interleukin-7 (IL-7) is important for thymopoiesis in mice and humans because IL-7 receptor alpha (IL-7Ralpha) mutations result in a severe combined immunodeficiency phenotype with severe thymic hypoplasia. Recent evidence has indicated that IL-7 also plays an important role as a regulator of T-cell homeostasis. Here we report the immunologic effects of recombinant human IL-7 (rhIL-7) therapy in normal and simian immunodeficiency virus (SIV)-infected nonhuman primates. Cynomolgus monkeys receiving 10 days of rhIL-7 showed substantial, reversible increases in T-cell numbers involving a dramatic expansion of both naive and nonnaive phenotype CD4(+) and CD8(+) subsets. Although IL-7 is known to have thymopoietic effects in mice, we observed marked declines in the frequency and absolute number of T-cell receptor excision circle-positive (TREC(+)) cells in the peripheral blood and dramatic increases in the percentage of cycling T cells in the peripheral blood as measured by Ki-67 expression (baseline less than 5% to approximately 50% after 6 days of therapy) and ex vivo bromodeoxyuridine (BrdU) incorporation. Similarly, moderately CD4- depleted SIV-infected macaques treated with rhIL-7 also had significant increases in peripheral blood CD4(+) and CD8(+) T cells following rhIL-7 therapy. Thus, rhIL-7 induces dramatic alterations in peripheral T-cell homeostasis in both T-cell-replete and T-cell-depleted nonhuman primates. These results further implicate IL-7 as a promising immunorestorative agent but illustrate that a major component of its immunorestorative capacity reflects effects on mature cells. These results also raise the possibility that IL-7 therapy could be used to temporarily modulate T-cell cycling in vivo in the context of immunotherapies such as vaccination.
View details for DOI 10.1182/blood-2002-07-2297
View details for Web of Science ID 000181432600034
View details for PubMedID 12411295
Induction of caspase 8 by interferon gamma renders some neuroblastoma (NB) cells sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but reveals that a lack of membrane TR1/TR2 also contributes to TRAIL resistance in NB
2003; 63 (5): 1122-1129
The resistance of neuroblastoma (NB) cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis has been attributed to a lack of caspase 8 expression. Here we demonstrate a clinically applicable molecular targeting strategy that not only increases caspase 8 expression ex vivo in NB cell lines but also in the tumor tissues of NB patients receiving IFN-gamma treatment. We identify the functional caspase 8 promoter, which is different from the methylated region reported previously, and show promoter activity is up-regulated by IFN-gamma through a IFN-gamma activation site-containing region. IFN-gamma also induces TRAIL expression in NB cell lines. However, the IFN-gamma restoration of caspase 8 in some NB cells revealed persistent TRAIL resistance in most NB cell lines examined. This additional lesion in the TRAIL path is because of a loss of cell membrane TRAIL receptors (TR1/TR2) not only in cell lines but in most of the NB tumor tissues evaluated. Restoration of TR2 expression by transfection enhances IFN-gamma-induced TRAIL sensitivity. Furthermore, we have found that we can improve TRAIL sensitivity in NB by reconstituting caspase 8 with IFN-gamma and TR2 with chemotherapeutic agents.
View details for Web of Science ID 000181327700033
View details for PubMedID 12615731
Neonates support lymphopenia-induced proliferation
2003; 18 (1): 131-140
T cells expand without intentional antigen stimulation when transferred into adult lymphopenic environments. In this study, we show that the physiologic lymphopenic environment existing in neonatal mice also supports CD4 T cell proliferation. Strikingly, naive CD4 T cells that proliferate within neonates acquire the phenotypic and functional characteristics of memory cells. Such proliferation is inhibited by the presence of both memory and naive CD4 T cells, is enhanced by 3-day thymectomy, is independent of IL-7, and requires a class II MHC-TCR interaction and a CD28-mediated signal. CD44(bright) CD4 T cells in neonates have a wide repertoire as judged by the distribution of Vbeta expression. Thus, lymphopenia-induced T cell proliferation is a physiologic process that occurs during the early postnatal period.
View details for Web of Science ID 000180860600013
View details for PubMedID 12530982
Potential use of imatinib in Ewing's sarcoma: Evidence for in vitro and in vivo activity
JOURNAL OF THE NATIONAL CANCER INSTITUTE
2002; 94 (22): 1673-1679
Ewing's sarcoma cells express c-kit, a receptor tyrosine kinase, and its ligand, stem cell factor (SCF), creating a potential autocrine loop that may promote tumor survival. We thus examined whether the specific tyrosine kinase inhibitor imatinib mesylate (hereafter imatinib; formerly STI571) could inhibit the proliferation of Ewing's sarcoma cells in vitro and in vivo.The effect of imatinib on c-kit expression and phosphorylation in Ewing's sarcoma cells was examined by immunoblotting. The effect of imatinib on cell growth and apoptosis was examined with an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay and with a morphologic test and Annexin V staining, respectively. The effect of imatinib oral therapy (every 12 hours for 5-7 days) on primary tumor growth was assessed in Ewing's sarcoma xenografts in SCID/bg mice (5 or 10 mice per group).All Ewing's sarcoma cell lines tested were sensitive to imatinib-mediated apoptosis with a concentration inhibiting growth by 50% (IC50) of 10-12 micro M. Imatinib inhibited SCF-mediated c-kit phosphorylation (IC50 = 0.1-0.5 microM). In the xenograft model, imatinib treatment resulted in the regression or control of primary Ewing's sarcomas. After 6 days of treatment, the mean lower extremity volume including xenograft tumor was 3744 mm3 (95% confidence interval [CI] = 3050 to 4437 mm3), 1442 mm3 (95% CI = 931 to 1758 mm3), and 346 mm3 (95% CI = 131 to 622 mm3) in mice treated with carrier alone or with imatinib at 50 mg/kg or at 100 mg/kg, respectively.Imatinib interferes with growth of all Ewing's sarcoma cell lines tested in vitro and in vivo. Targeted inhibition of tyrosine kinase-dependent autocrine loops, therefore, may be a viable therapeutic strategy for Ewing's sarcoma.
View details for Web of Science ID 000179265000007
View details for PubMedID 12441322
Bax deficiency partially corrects interleukin-7 receptor alpha deficiency
2002; 17 (5): 561-573
The requirement for cytokines in hematopoiesis is partly attributable to the protection of cells from apoptosis. Since IL-7 is required for normal T cell development, we evaluated the role of Bax in vivo by generating mice deficient in both Bax and the IL-7 receptor alpha chain (IL-7R). Starting at birth, we observed complete recovery of all stages of alphabeta thymocyte development up to 4 weeks of age. However, by 12 weeks of age, thymic cellularity had reverted to that of mice deficient in IL-7R alone. The BH3 only proteins, Bad and Bim, were also part of the death pathway repressed by IL-7. Thus, in young mice, Bax emerges as an essential protein in the death pathway induced by IL-7 deficiency.
View details for Web of Science ID 000179335100003
View details for PubMedID 12433363
Interleukin-7 and immunorestoration in HIV: Beyond the thymus
JOURNAL OF HEMATOTHERAPY & STEM CELL RESEARCH
2002; 11 (5): 803-807
One of the hallmarks of untreated HIV infection is a progressive loss of effective immunity to both HIV-associated and non-HIV antigens. Combination antiretroviral therapy can frequently control viral replication, resulting in variable levels of immune reconstitution, but has not resulted in restoration of effective immunity to the virus. Understanding the limitations of immune reconstitution following highly active antiretroviral therapy (HAART) and identifying approaches to enhance immunity in this context may not only improve outcome for patients with HIV infection but could also provide insight for immune reconstitution in other conditions associated with T cell depletion.
View details for Web of Science ID 000178850000007
View details for PubMedID 12427286
Interleukin 7 worsens graft-versus-host disease
2002; 100 (7): 2642-2649
Impaired immune reconstitution has moved to the forefront of clinical problems limiting progress in allogeneic bone marrow transplantation (BMT). The identification of therapies that can enhance immune reconstitution by increasing thymopoiesis is critical to solving this problem. Interleukin 7 (IL-7) is the most potent thymopoietic cytokine identified thus far. To study the effects of IL-7 on immune reconstitution and graft-versus-host disease (GVHD) following allogeneic BMT, we administered recombinant human IL-7 (rhIL-7) in a murine parent into an F1 model. Results showed that rhIL-7 therapy lowered the "threshold" T-cell dose required to induce both clinical signs of GVHD as well as lethal GVHD. Histologic analysis of GVHD target tissues revealed that rhIL-7 increased the degree of inflammation and tissue damage observed at all T-cell doses studied, but did not change the pattern of organs affected or the histologic appearance of the GVHD within target organs. In addition, we evaluated the capacity for rhIL-7 to enhance thymopoiesis in the setting of allogeneic T cell-depleted (TCD) and T-cell-replete BMT. We observed that rhIL-7 therapy enhanced thymic function in TCD allogeneic BM transplant recipients, but not in animals that received even modest doses of T cells presumably due to thymic toxicity of the graft-versus-host reaction. Thus, caution must be exercised as IL-7 is developed clinically as an immunorestorative agent for use in the setting of allogeneic BMT. These results suggest that use of IL-7 should be limited to the setting of TCD BMT to obtain the greatest benefit on immune competence with the least toxicity.
View details for DOI 10.1182/blood-2002-04-1082
View details for Web of Science ID 000178266000047
View details for PubMedID 12239180
- Focus on sarcomas CANCER CELL 2002; 2 (3): 175-178
Treatment of metastatic osteosarcoma with the somatostatin analog OncoLar: Significant reduction of insulin-like growth factor-1 serum levels
37th Annual Meeting of the American-Society-of-Clinical-Oncology
LIPPINCOTT WILLIAMS & WILKINS. 2002: 440–46
Insulin-like growth factor-1 (IGF-1) has been implicated in the growth and/or metastasis of osteosarcoma (OS) and chondrosarcoma based on in vitro and experimental animal studies.To determine the degree of growth hormone (GH), IGF-1 axis blockade, toxicities, and antitumor effect of OncoLar (ONC) (Novartis, East Hanover, NJ, U.S.A.) in OS.A phase 1 study with ONC enrolled 21 OS patients (median age 19 y) in four cohorts: ONC 60 mg or 90 mg intramuscularly every 4 weeks with/without tamoxifen (TAM) 20 mg oral daily.There were no dose-limiting toxicities. Nineteen percent of patients had grade III drug-related toxicities including: 62% of patients showed progressive disease after two courses (8 wk). Nineteen percent received four courses. No clinical responses were observed. At weeks two and eight of therapy, IGF-1 serum levels dropped 46% ( < 0.0001, n = 21) and 53% ( = 0.003, n = 10). The difference of the area under the curve (AUC) minus baseline AUC (DeltaAUC) for arginine-stimulated GH serum levels at week two was lower than baseline ( < 0.01). At weeks two and eight, GH peak values were lower than baseline ( < 0.0001 and = 0.002, respectively).A long-acting somatostatin analog was able to lower IGF-1 levels of OS patients. IGF-BP-3 and GH were only transiently reduced. Although ONC was well tolerated, no sustained clinical responses were observed. The pathophysiology of serum versus tissue concentrations of IGF-1 as well as the interplay of IGFs, IGF-binding proteins, and other growth factors and cytokines in osteosarcoma warrants further investigation. A better understanding of these processes should lead to a more effective exploitation of these pathways for the targeted therapy of OS.
View details for Web of Science ID 000177943500006
View details for PubMedID 12218590
- Interleukin-7: from bench to clinic BLOOD 2002; 99 (11): 3892-3904
- Current concepts of thymic aging SPRINGER SEMINARS IN IMMUNOPATHOLOGY 2002; 24 (1): 7-22
Pilot trial of tumor-specific peptide vaccination and continuous infusion interleukin-2 in patients with recurrent Ewing sarcoma and alveolar rhabdomyosarcoma: An inter-institute NIH study
MEDICAL AND PEDIATRIC ONCOLOGY
2002; 38 (3): 158-164
Patients with recurrent Ewing sarcoma and alveolar rhabdomyosarcoma have poor prognoses and limited therapeutic options. We have investigated the use of peptide pulsed vaccination in an attempt to immunologically target the breakpoint region of tumor specific fusion proteins expressed in these tumors.Sixteen patients with recurrent, translocation positive, Ewing sarcoma, and alveolar rhabdomyosarcoma underwent apheresis for collection of peripheral blood mononuclear cells. Following countercurrent centrifugal elutriation, an apheresis product comprised predominantly of monocytes but containing small numbers of circulating immature dendritic cells was pulsed with peptides derived from the breakpoint region of the fusion proteins. Vaccines were administered intravenously concomitant with continuous intravenous rhIL-2 at 9 x 10(6) IU/m(2)/day.Toxicity was limited to IL-2 related effects and was generally mild. Following vaccination, all patients showed progressive disease, most in a rapid fashion following the first vaccine. One patient showed evidence of an immunologic response and another showed a mixed clinical response. Patients enrolled on this tumor vaccine trial showed significant immunosuppression and large bulky tumors.Peptide vaccination as administered in this trial did not alter the dismal clinical outcome for patients with recurrent pediatric sarcomas. Future trials of tumor vaccines in this population should target patient populations with improved immune competence and smaller tumor burdens. Furthermore, optimization of the antigen presenting cell populations may be important for inducing immune responses to peptide antigens.
View details for DOI 10.1002/mpo.1303
View details for Web of Science ID 000173972700002
View details for PubMedID 11836714
Interleukin-7: master regulator of peripheral T-cell homeostasis?
TRENDS IN IMMUNOLOGY
2001; 22 (10): 564-571
Recent evidence has implicated interleukin-7 (IL-7) as a master regulator of T-cell homeostasis, based upon its essential role in the homeostatic expansion of naive T-cell populations in response to low-affinity antigens (Ags) and its capacity to enhance dramatically the expansion of peripheral T-cell populations in response to high-affinity Ags. Furthermore, T-cell-depleted humans have a unique inverse relationship between the peripheral CD4(+) T-cell count and the level of circulating IL-7. Together, these data suggest that increased amounts of IL-7 become available following T-cell depletion, thus, enhancing the high- and low-affinity Ag-driven expansion of the population of residual, mature T cells and boosting thymic regenerative capacity, as a means to restore T-cell homeostasis.
View details for Web of Science ID 000171791100012
View details for PubMedID 11574281
Intramedullary Ewing sarcoma of the spinal cord: consequences of molecular diagnostics - Case report
JOURNAL OF NEUROSURGERY
2001; 95 (2): 270-275
Molecular biological techniques have begun to transform modern medicine. These techniques have shown promise in the pathological diagnosis of difficult or uncommon tumors. Accurate molecular diagnosis of the small round-cell tumors, for example, is especially important because divergent therapies may be required to eradicate such disparate lesions as neuroblastoma, lymphoma, rhabdomyosarcoma, central primitive neuroectodermal tumors/medulloblastoma, or Ewing sarcoma (ES). The authors present an unusual case of a primary, extraosseous ES arising from the intramedullary spinal cord, in which molecular studies were required for specific diagnosis and therapeutic guidance.
View details for Web of Science ID 000171434600021
View details for PubMedID 11599852
- What limits immune reconstitution in HIV infection? Divergent tools converge on thymic function AIDS 2001; 15 (14): 1881-1882
Immunomagnetic purging of Ewing's sarcoma from blood and bone marrow: Quantitation by real-time polymerase chain reaction
JOURNAL OF CLINICAL ONCOLOGY
2001; 19 (16): 3649-3659
A propensity for hematogenous spread with resulting contamination of autologous cell products complicates cellular therapies for Ewing's sarcoma. We used a new approach to purge artificially contaminated cellular specimens of Ewing's sarcoma and show the capacity for real-time polymerase chain reaction (PCR) to quantify the contamination level of Ewing's sarcoma in such specimens.Binding of monoclonal antibody (MoAb) 8H9 to Ewing's sarcoma cell lines and normal hematopoietic cells was studied using flow cytometry. Using real-time PCR--based amplification of t(11;22), levels of Ewing's contamination of experimental and clinical cellular products were monitored. Purging was accomplished using immunomagnetic-based depletion. Monitoring of the function of residual hematopoietic progenitors and T cells was performed using functional assays.MoAb 8H9 shows binding to Ewing's sarcoma but spares normal hematopoietic tissues. Nested real-time PCR is capable of detecting contaminating Ewing's sarcoma cells with a sensitivity of one cell in 10(6) normal cells. After 8H9-based purging, a 2- to 3-log reduction in contaminating Ewing's sarcoma was shown by real-time PCR, with purging to PCR negativity at levels of contamination of 1:10(6). Levels of contamination in clinical samples ranged from 1:10(5) to 10(6). Therefore, 8H9-based purging of clinical samples is predicted to reduce tumor cell contamination to a level below the limit of detection of PCR.These results demonstrate a new approach for purging contaminated cellular products of Ewing's sarcoma and demonstrate the capacity of real-time PCR to provide accurate quantitative estimates of circulating tumor burden in this disease.
View details for Web of Science ID 000170453500007
View details for PubMedID 11504746
Exploiting genetic alterations to design novel therapies for cancer
HEMATOLOGY-ONCOLOGY CLINICS OF NORTH AMERICA
2001; 15 (4): 657-?
In the 3 decades since the signing of the National Cancer Act, there has been tremendous progress in the elucidation of the molecular underpinnings of cancer. Molecular genetic studies have been particularly insightful, revealing genetic rearrangements, such as chromosomal translocations, which may be the seminal event leading to deregulated cell growth for many childhood and adult cancers. These findings have led to new diagnostic and prognostic tools but have been slow to be translated into new therapeutic modalities. This article reviews a variety of methods now under development to exploit genetic changes in cancer to develop specific anticancer agents using gene therapy, viral therapy, and immunotherapy approaches. As many of these strategies inevitably enter the clinic, it will be imperative for health care professionals to be familiar with these novel approaches as they help patients navigate the likely broad array of treatment options.
View details for Web of Science ID 000171555400005
View details for PubMedID 11676278
- Spreading the wealth: Antigen discovery in adult tumors can help hone the search for pediatric tumor antigens JOURNAL OF IMMUNOTHERAPY 2001; 24 (4): 281-282
- High-dose chemotherapy for rhabdomyosarcoma: Where do we go from here JOURNAL OF PEDIATRIC HEMATOLOGY ONCOLOGY 2001; 23 (5): 266-267
A potential role for interleukin-7 in T-cell homeostasis
2001; 97 (10): 2983-2990
Interleukin (IL)-7 is known to up-regulate thymopoietic pathways of T-cell regeneration. Recent work also has shown it to potently enhance thymic-independent peripheral expansion and to restore immunocompetence in athymic T-cell-depleted hosts. We hypothesized that endogenous IL-7 could contribute to the restoration of T-cell homeostasis following T-cell depletion. To analyze this, we evaluated circulating IL-7 levels and lymphocyte subsets in multiple clinical cohorts with T-cell depletion of varying etiologies. In pediatric (n = 41) and adult (n = 51) human immunodeficiency virus-infected CD4-depleted patients, there were strong inverse correlations between IL-7 levels and CD4 counts (r = -0.77, P <.0001, and r = -0.68, P <.0001). Declines in IL-7 were temporally correlated with recovery of CD4 counts. Similar patterns were observed in CD4-depleted patients receiving cancer chemotherapy (r = -0.65, P =.009). Therefore, in 2 disparate clinical scenarios involving CD4 depletion, IL-7 levels dynamically respond to changes in CD4 T-cell number, making this cytokine uniquely suited as a candidate regulator of T-cell homeostasis. Furthermore, in patients with idiopathic CD4 lymphopenia, a much weaker relationship between IL-7 levels and peripheral blood CD4 counts was observed, suggesting that an impaired IL-7 response to CD4 depletion may contribute to the impaired lymphocyte homeostasis observed in this population. In light of the known effects of IL-7 on T-cell regeneration, we postulate that increased availability of IL-7 could play a critical role in restoring T-cell homeostasis following T-cell depletion.
View details for Web of Science ID 000170301300009
View details for PubMedID 11342421
Molecular confirmation of Ewing sarcoma
JOURNAL OF PEDIATRIC HEMATOLOGY ONCOLOGY
2001; 23 (4): 221-224
To analyze retrospectively results of reverse transcription polymerase chain reaction (RT-PCR) testing and demographic information in patients with known or suspected Ewing sarcoma/primitive neuroectodermal tumor family of tumors referred to the National Cancer Institute and to describe factors influencing the determination of molecular marker status.Tumor samples from 76 patients from February 1997 to December 1999 were analyzed. In all cases, the diagnosis of this family of tumors was confirmed by histopathologic review.In 58 patients, the presence of a translocation associated with this family of tumors was confirmed using RT-PCR. Specifically, there were 45 Ewing sarcoma (EWS)-FLI type 1 translocations, four EWS-FLI type 2 translocations, five EWS-ERG translocations, and four less common EWS-FLI variants. Of patients with a confirmed translocation, four were confirmed only after nested RT-PCR techniques were used. In five patients who initially underwent needle biopsy, the diagnosis was confirmed only after open biopsy or repeat needle biopsy was undertaken. Samples from 18 patients were translocation-negative. Of these, seven samples were deemed inadequate for RT-PCR testing as a result of inappropriate tissue handling or the presence of necrotic material. Five patients were found to have a different diagnosis after complete histopathologic and molecular characterization. Six samples remained, in which adequate tissue was obtained with no evidence of a characteristic translocation.In apparently translocation-negative samples, close attention should be given to the possibility of an alternative diagnosis, the potential need for nested RT-PCR, and the possibility of an inadequate sample. Strong consideration should be given to the use of open biopsy as opposed to needle biopsy to avoid the need for repeat biopsies and the potential for inaccurate assessment of molecular marker status.
View details for Web of Science ID 000168710500008
View details for PubMedID 11846300
Sensitivity of Ewing's sarcoma to TRAIL-induced apoptosis
CELL DEATH AND DIFFERENTIATION
2001; 8 (5): 506-514
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to kill transformed cells. We have studied the expression and functionality of the TRAIL apoptotic pathway in Ewing's sarcoma. We demonstrate that tumors from patients with Ewing's sarcoma express receptors TRAIL-R1 and -R2. Using a panel of nine Ewing's sarcoma cell lines TRAIL could induce apoptosis in seven cell lines. Preincubation with interferon-gamma rendered the two resistant cell lines sensitive. TRAIL was the most potent inducer of apoptosis when compared to Fas ligand or TNF. TRAIL-mediated apoptosis could be inhibited by various caspase-inhibitors. No difference in the surface expression of TRAIL-receptors was observed between sensitive and resistant cell lines. Also, all cell lines had similar levels of expression of Flice-like inhibitory protein (FLIP) on immunoblot. However, the two resistant cell lines had only very low level expression of caspase 8 on RNA and protein level. In summary, we show that Ewing's sarcoma expresses receptors for TRAIL, and that cells are exquisitely sensitive to TRAIL-mediated apoptosis. These results may warrant clinical trials with TRAIL in Ewing's sarcoma once the safety of TRAIL for humans has been established.
View details for Web of Science ID 000168534800008
View details for PubMedID 11423911
Long-term virologic and immunologic responses in human immunodeficiency virus type 1-infected children treated with indinavir, zidovudine, and lamivudine
6th Conference on Retroviruses and Opportunistic Infections
OXFORD UNIV PRESS INC. 2001: 1116–20
Virologic and immunologic responses were examined for 33 human immunodeficiency virus (HIV)-infected children who participated for > or = 96 weeks in a phase 1/2 protocol of 16 weeks of indinavir monotherapy, followed by the addition of zidovudine and lamivudine. At week 96, a median increase of 199 CD4+ T cells/microL and a median decrease of 0.74 log(10) HIV RNA copies/mL were observed. The relationship between control of viral replication and CD4) T cell count was examined. Patients were categorized into 3 response groups on the basis of duration and extent of control of viral replication. Of 21 children with a transient decrease in virus load of > or = 0.7 log(10) HIV RNA copies/mL from baseline, 7 experienced sustained increases in CD4+, CD4+ CD45RA+, and CD4+ CD45RO+ T cell counts. CD4+ CD45RA+ (naive) T cells were the major contributor to CD4+ T cell expansion. Continued long-term immunologic benefit may be experienced by a subset of children, despite only transient virologic suppression.
View details for Web of Science ID 000167366900018
View details for PubMedID 11237839
Interleukin-7 restores immunity in athymic T-cell-depleted hosts
2001; 97 (6): 1525-1533
Thymic-deficient hosts rely primarily on antigen-driven expansion to restore the peripheral T-cell compartment following T-cell depletion (TCD). The degree to which this thymic-independent pathway can restore immune competence remains poorly understood but has important implications for a number of clinical conditions including stem cell transplantation and human immunodeficiency virus (HIV) infection. A model of HY-mediated skin graft rejection by athymic, TCD mice was used to show that restoration of naive and recall responses via peripheral expansion requires transfer of only 25 x 10(6) lymph node (LN) cells representing approximately 10% of the T-cell repertoire. Constitutive expression of bcl-2 in the expanding inocula restored recall responses to HY at a substantially lower LN cell dose (1 x 10(6)), which is normally insufficient to induce HY-mediated graft rejection in athymic hosts. Interestingly, bcl-2 had no effect on primary responses. Interleukin-7 (IL-7) potently enhanced thymic-independent peripheral expansion and led to HY graft rejection using an LN cell dose of 1 x 10(6) in both primary and recall models. The restoration of immune competence by IL-7 appeared to be mediated through a combination of programmed cell death inhibition, improved costimulation, and modulation of antigen-presenting cell (APC) function. These results show that immune competence for even stringent antigens such as HY can be restored in the absence of thymic function and identify IL-7 as a potent modulator of thymic-independent T-cell regeneration.
View details for Web of Science ID 000167406800001
View details for PubMedID 11238086
Clinical trial designs for the early clinical development of therapeutic cancer vaccines
JOURNAL OF CLINICAL ONCOLOGY
2001; 19 (6): 1848-1854
There are major differences between therapeutic tumor vaccines and chemotherapeutic agents that have important implications for the design of early clinical trials. Many vaccines are inherently safe and do not require phase I dose finding trials. Patients with advanced cancers and compromised immune systems are not good candidates for assessing either the toxicity or efficacy of therapeutic cancer vaccines. The rapid pace of development of new vaccine candidates and the variety of possible adjuvants and modifications in method of administration makes it important to use efficient designs for clinical screening and evaluation of vaccine regimens. We review the potential advantages of a wide range of clinical trial designs for the development of tumor vaccines. We address the role of immunological endpoints in early clinical trials of tumor vaccines, investigate the design implications of attempting to use disease stabilization as an end point and discuss the difficulties of reliably utilizing historical control data. Several conclusions for expediting the clinical development of effective cancer vaccines are proposed.
View details for Web of Science ID 000167652500032
View details for PubMedID 11251017
IL-7 increases both thymic-dependent and thymic-independent T-cell regeneration after bone marrow transplantation
2001; 97 (5): 1491-1497
Thymic-dependent differentiation of bone marrow (BM)-derived progenitors and thymic-independent antigen-driven peripheral expansion of mature T cells represent the 2 primary pathways for T-cell regeneration. These pathways are interregulated such that peripheral T-cell expansion is increased in thymectomized versus thymus-bearing hosts after bone marrow transplantation (BMT). This study shows that this interregulation is due to competition between progeny of these 2 pathways because depletion of thymic progeny leads to increased peripheral expansion in thymus-bearing hosts. To test the hypothesis that competition for growth factors modulates the magnitude of antigen-driven peripheral expansion during immune reconstitution in vivo, a variety of T-cell active cytokines were administered after BMT. Of the cytokines (interleukins) tested (IL-3, IL-12, IL-6, IL-2, and IL-7), IL-2 modestly increased peripheral expansion in the face of increasing numbers of thymic emigrants, whereas IL-7 potently accomplished this. This report also demonstrates that the beneficial effect of IL-7 on immune reconstitution is related to both increases in thymopoiesis as well as a direct increase in the magnitude of antigen-driven peripheral expansion. Therefore, the administration of exogenous IL-7, and to a lesser extent IL-2, abrogates the down-regulation in antigen-driven peripheral expansion that occurs in thymus-bearing hosts after BMT. These results suggest that one mechanism by which T-cell-depleted hosts may support antigen-driven T-cell expansion in vivo is via an increased availability of T-cell-active cytokines to support clonal expansion.
View details for Web of Science ID 000167117500043
View details for PubMedID 11222398
Development of a clinical-scale method for generation of dendritic cells from PBMC for use in cancer immunotherapy
2001; 3 (1): 19-29
There is growing interest in the use of dendritic cells (DCs) for treatment of malignancy and infectious disease. Our goal was to develop a clinical scale method to prepare autologous DCs for cancer clinical trials.PBMC were collected from normal donors or cancer patients by automated leukapheresis, purified by counterflow centrifugal elutriation and placed into culture in polystyrene flasks at 1 x 10(6) cells/mL for 5-7 days at 37 degrees C, with 5% CO(2), with IL-4 and GM-CSF. Conditions investigated included media formulation, supplementation with heat in activated allogeneic AB serum or autologous plasma and time to harvest (Day 5 or Day 7). DCs were evaluated for morphology, quantitative yield, viability, phenotype and function, including mixed leukocyte response and recall response to tetanus toxoid and influenza virus.DCs with a typical immature phenotype (CD14-negative, CD1a-positive, mannose receptor-positive, CD80-positive, CD83-negative) were generated most consistently in RPMI 1640 supplemented with 10% allogeneic AB serum or 10% autologous plasma. Cell yield was higher at Day 5 than Day 7, without detectable differences in phenotype or function. In pediatric sarcoma patients, autologous DCs had enhanced function compared with monocytes from which they were generated. In this patient group, starting with 8.0 +/- 3.7 x 10(8) fresh or cryopreserved autologous monocytes, DC yield was 2.1 +/- 1.0 x 10(8) cells, or 29% of the starting monocyte number.In the optimized clinical-scale method, purified peripheral monocytes are cultured for 5 days in flasks at 1 x 10(6) cells/mL in RPMI 1640, 10% allogeneic AB serum or autologous plasma, IL-4 and GM-CSF. This method avoids the use of FBS and results in immature DCs suitable for clinical trials.
View details for Web of Science ID 000168736800004
View details for PubMedID 12028840
Targeting pediatric malignancies for T cell-mediated immune responses.
Current oncology reports
2000; 2 (6): 539-546
Successful immune targeting of malignancies hinges upon the ability to activate specific T-cell populations to recognize and attack tumor but spare normal vital tissues. Investigators in the field of tumor immunology are currently utilizing at least three distinct approaches toward this goal. In the first approach, molecular targets of cytolytic T cells which spontaneously develop in tumor-bearing patients have been identified and are subsequently used as immunogens in immunotherapy trials. Whereas this approach originally focused upon the identification of tumor antigens in the immune-responsive tumors malignant melanoma and renal cell carcinoma, it surprisingly led to the identification of a variety of molecules that are now known to be expressed in other common pediatric and adult tumors. In the second approach, tumor-specific molecules (eg, mutant p53 and chromosomal translocations) that have been identified in individual tumors during the study of neoplastic transformation are used as immunogens. Because chromosomal translocations are common in pediatric tumors, such targets may be of particular interest in pediatric oncology. In the third approach, immunization with whole tumor cell components is undertaken with the assumption that the most immunogenic molecules within the tumor will dominate the immune response induced. The benefits and limitations for each approach, particularly as it pertains to the development of immunotherapy for pediatric tumors, are discussed in this article.
View details for PubMedID 11122890
Prolonged CD4 depletion after sequential autologous peripheral blood progenitor cell infusions in children and young adults
2000; 96 (2): 754-762
Administration of mobilized peripheral blood progenitor cells (PBPCs) after high-dose chemotherapy rapidly restores multilineage hematopoiesis, but the ability of such products to restore lymphocyte populations remains unclear. In this report, we evaluated immune reconstitution in a series of patients treated with sequential cycles of high-dose chemotherapy, followed by autologous PBPC infusions (median CD34(+) cell dose 7.2 x 10(6) cells/kg [range 2-29.3]). Although patients experienced rapid reconstitution of B cells and CD8(+) T cells, we observed CD4 depletion and diminished immune responsiveness in all patients for several months after completion of therapy. Mature CD4(+) T cells contained within the grafts did not appear to contribute substantially to immune reconstitution because CD4 counts did not differ between recipients of unmanipulated T-cell replete infusions versus CD34 selected, T-cell-depleted infusions. Rather, at 12 months after therapy, total CD4 count was inversely proportional to age (rho = -0.78, P =.04), but showed no relationship to CD34 cell dose (rho = -0.42, P =.26), suggesting that age-related changes within the host are largely responsible for the limited immune reconstitution observed. These results demonstrate that in the autologous setting, the infusion of large numbers of PBPCs is not sufficient to restore T-cell immune competence and emphasize that specific approaches to enhance immune reconstitution are necessary if immune-based therapy is to be used to eradicate minimal residual disease after autologous PBPC transplantation. (Blood. 2000;96:754-762)
View details for Web of Science ID 000088234800051
View details for PubMedID 10887145
Targeting tumor specific translocations in sarcomas in pediatric patients for immunotherapy
CLINICAL ORTHOPAEDICS AND RELATED RESEARCH
In an effort to develop more effective therapies for various sarcomas in pediatric patients, the authors have focused on using recurrent tumor-specific translocations as potential novel tumor antigens. In general, these translocations generate fusion transcription factors. Because cytotoxic T cell lymphocyte receptors recognize peptide fragments bound to major histocompatibility complex Class 1 molecules, it is possible that unique peptides spanning the translocation breakpoint region may be processed, bound to major histocompatibility complex Class I molecules and displayed on the tumor cell surface where they could be susceptible to cytotoxic T cell lymphocyte killing. The authors have investigated the PAX-3-FKHR fusion product seen in alveolar rhabdomyosarcoma, and the EWS-FLI-1 fusion product seen in Ewing's sarcoma. Peptides spanning these fusion regions contain potential major histocompatibility complex Class 1 and Class II binding motifs suggesting they may serve as novel T cell antigens. Preliminary mouse experiments suggest that cytotoxic T cell lymphocytes specific for the PAX-3-FKHR fusion peptide can be generated and can recognize and kill tumor cells bearing the PAX-3-FKHR fusion protein. Clinical trials are ongoing to determine whether this approach will be useful.
View details for Web of Science ID 000086437400006
View details for PubMedID 10810459
T-cell immunodeficiency following cytotoxic antineoplastic therapy: A review (Reprinted from The Oncologist, vol 4, pg 370-378, 1999)
2000; 18 (1): 10-18
Although cancer itself is immunosuppressive, cytotoxic antineoplastic therapy is the primary contributor to the clinical immunodeficiency observed in cancer patients. The immunodeficiency induced by cytotoxic antineoplastic therapy is primarily related to T-cell depletion, with CD4 depletion generally more severe than CD8 depletion. Myeloablative therapy, dose-intensive alkylating agents, purine nucleoside analogs, and corticosteroids substantially increase the risk of therapy-induced immunosuppression. Restoration of T-cell populations following cytotoxic antineoplastic therapy is a complex process. Efficient recovery of CD4+ T cell populations requires thymic-dependent pathways which undergo an age-dependent decline resulting in prolonged CD4+ T-cell depletion in adults following T-cell-depleting therapy. Total CD8+ T-cell numbers recover in both children and adults relatively quickly post-therapy; however, CD8+ subset disruptions often remain for a prolonged period. The clinical management of patients with therapy-induced T-cell depletion involves the maintenance of a high index of suspicion for opportunistic pathogens, irradiation of blood products, prophylaxis for viral infections, and reimmunization in selected clinical circumstances. Future research avenues include efforts to rapidly rebuild immunity following cytotoxic antineoplastic therapy so that immune-based therapies may be utilized immediately following cytotoxic therapy to target minimal residual neoplastic disease.
View details for Web of Science ID 000084998100002
View details for PubMedID 10661568
T-cell immunodeficiency following cytotoxic antineoplastic therapy: a review.
1999; 4 (5): 370-378
Although cancer itself is immunosuppressive, cytotoxic antineoplastic therapy is the primary contributor to the clinical immunodeficiency observed in cancer patients. The immunodeficiency induced by cytotoxic antineoplastic therapy is primarily related to T-cell depletion, with CD4 depletion generally more severe than CD8 depletion. Myeloablative therapy, dose-intensive alkylating agents, purine nucleoside analogs, and corticosteroids substantially increase the risk of therapy-induced immunosuppression. Restoration of T-cell populations following cytotoxic antineoplastic therapy is a complex process. Efficient recovery of CD4+ T cell populations requires thymic-dependent pathways which undergo an age-dependent decline resulting in prolonged CD4+ T-cell depletion in adults following T-cell-depleting therapy. Total CD8+ T-cell numbers recover in both children and adults relatively quickly post-therapy; however, CD8+ subset disruptions often remain for a prolonged period. The clinical management of patients with therapy-induced T-cell depletion involves the maintenance of a high index of suspicion for opportunistic pathogens, irradiation of blood products, prophylaxis for viral infections, and reimmunization in selected clinical circumstances. Future research avenues include efforts to rapidly rebuild immunity following cytotoxic antineoplastic therapy so that immune-based therapies may be utilized immediately following cytotoxic therapy to target minimal residual neoplastic disease.
View details for PubMedID 10551553
Simultaneous expression of fas and nonfunctional Fas ligand in Ewing's sarcoma
1998; 58 (24): 5842-5849
Fas-Fas ligand interactions play a central role in the regulation of the immune response. Fas ligand expression by tumors has been implicated in the abrogation of the host antitumor response by killing of Fas-positive effector lymphocytes. We have studied the presence and functional status of Fas and Fas ligand in Ewing's sarcoma. All Ewing's sarcoma cell lines tested expressed Fas on their surface. Three of the cell lines were readily killed after ligation of the Fas receptor. Four additional cell lines exhibited Fas-mediated apoptosis after preincubation with IFN-gamma and/or cycloheximide, whereas two cell lines were resistant to Fas-mediated killing. With regard to Fas ligand, all cell lines examined were positive for protein by immunoblot, and specificity was confirmed by reverse transcription-PCR. However, using flow cytometric analysis, Fas ligand could only be detected in Ewing's sarcoma cells after permeabilization. Furthermore, the cell lines were not capable of inducing apoptosis of Fas-sensitive Jurkat cells. In addition, Ewing's sarcoma cell lines were able to serve as stimulators for the generation of cytotoxic effector lymphocytes and were susceptible to lysis by them. Therefore, Fas ligand is expressed in Ewing's sarcoma but is not functional, suggesting that Ewing's sarcoma is a potential target for immunotherapy.
View details for Web of Science ID 000077499800035
View details for PubMedID 9865744
Thymic function in young/old chimeras: substantial thymic T cell regenerative capacity despite irreversible age-associated thymic involution
EUROPEAN JOURNAL OF IMMUNOLOGY
1998; 28 (6): 1886-1893
Age-associated thymic involution results in a diminished capacity to regenerate T cell populations, although the magnitude of this effect is unknown. In this report, thymic function was studied in aged vs. young adult mice after lethal irradiation and administration of T cell-depleted bone marrow (BM) from young mice. Abnormalities observed in aged thymi (reduced thymocyte numbers, histologic abnormalities) were not reversed by administration of young BM via bone marrow transplantation (BMT), but aged thymi displayed a normal thymocyte subset distribution and appropriately deleted MIs-reactive T cells after BMT. Aged BMT recipients regenerated significantly reduced numbers of splenic T cells compared to young recipients and showed increased peripheral expansion of thymic emigrants since a higher proportion of BM-derived T cells expressed a memory phenotype in aged vs. young BMT recipients. Because peripheral expansion of thymic emigrants could substantially increase the number of thymic progeny present in the spleen, we sought to measure thymic T cell regenerative capacity after BMT in a setting devoid of peripheral expansion. To do this, TCR-transgenic (Tg+) T cell-depleted BM was administered to aged and young recipients lacking antigen specific for the Tg+ TCR. Aged recipients regenerated approximately 50 % of the TCR Tg+ cells regenerated in young BMT recipients, providing evidence that even very aged thymi retain the capacity to regenerate significant numbers of mature T cell progeny. Therefore, thymic function is reduced with aged but it is not lost, suggesting that therapeutic approaches to enhance thymic function may be successful even in very aged hosts.
View details for Web of Science ID 000074258400017
View details for PubMedID 9645370
Molecular alterations in pediatric sarcomas: potential targets for immunotherapy.
1998; 2 (2): 77-87
Purpose/results/discussion. Recurrent chromosomal translocations are common features of many human malignancies. While such translocations often serve as diagnostic markers, molecular analysis of these breakpoint regions and the characterization of the affected genes is leading to a greater understanding of the causal role such translocations play in malignant transformation. A common theme that is emerging from the study of tumor-associated translocations is the generation of chimeric genes that, when expressed, frequently retain many of the functional properties of the wild-type genes from which they originated. Sarcomas, in particular, harbor chimeric genes that are often derived from transcription factors, suggesting that the resulting chimeric transcription factors contribute to tumorigenesis. The tumor-specific expression of the fusion proteins make them likely candidates for tumor-associated antigens (TAA) and are thus of interest in the development of new therapies. The focus of this review will be on the translocation events associated with Ewing's sarcomas/PNETs (ES), alveolar rhabdomyosarcoma (ARMS), malignant melanoma of soft parts (MMSP) (clear cell sarcoma), desmoplastic small round cell tumor (DSRCT), synovial sarcoma (SS), and liposarcoma (LS), and the potential for targeting the resulting chimeric proteins in novel immunotherapies.
View details for DOI 10.1080/13577149878037
View details for PubMedID 18521238
Thymic aging and T-cell regeneration
1997; 160: 91-102
Studies of T-cell regeneration using animal models have consistently shown the importance of the thymus for T-cell regeneration. In humans, recent studies have shown that declines in thymic T-cell regenerative capacity begins relatively early in life, resulting in a limited capacity for T-cell regeneration by young adulthood. As a result, adult humans who experience profound T-cell depletion regenerate T cells primarily via relatively inefficient thymic-independent pathways, resulting in prolonged CD4 depletion, CD4+ and CD8+ subset alterations, limited TCR repertoire diversity and a propensity for activation induced cell death. These limitations in T-cell regeneration have significant clinical implications in the setting of HIV infection and bone marrow transplantation and may also contribute to immunologic abnormalities associated with normal aging. While the mechanisms responsible for thymic aging are not well understood, current evidence suggests that changes within the thymus itself are primary, while age-related changes in marrow T-cell progenitors and inhibitory factors within the extrathymic host milieu contribute to a lesser extent. The development of therapies which can reverse thymic aging are critical for improving outcome in clinical settings of T-cell depletion, and could potentially improve immunologic function in normal aged hosts.
View details for Web of Science ID 000071351500009
View details for PubMedID 9476668
Restoration of T-cell homeostasis after T-cell depletion.
Seminars in immunology
1997; 9 (6): 339-346
T-cell homeostasis appears to be maintained throughout much of normal adult life independent of de-novo production from hematopoietic stem cells via thymopoiesis. Instead, peripheral mechanisms are generally sufficient to maintain normal T-cell number, function and adequate TCR repertoire diversity in healthy hosts. Studies of T-cell regeneration in animals, however, have shown that full restoration of T-cell homeostasis after profound T-cell depletion is primarily dependent upon thymopoiesis. In this setting, thymic-deficient hosts have prolonged reductions in total T-cell number, restricted TCR repertoire diversity, and limited immunocompetence. In humans, age-related reductions in thymic regenerative capacity as early as young adulthood result in incomplete restoration of T-cell homeostasis after T-cell depletion.
View details for PubMedID 9405262
Constraints on CD4 recovery postchemotherapy in adults: Thymic insufficiency and apoptotic decline of expanded peripheral CD4 cells
1997; 90 (9): 3789-3798
To examine the mechanisms of CD4 reconstitution in an adult population, lymphocyte repopulation was assessed following dose-intense chemotherapy in 25 breast cancer patients, ages 33 to 69 years. Chemotherapy resulted in a greater than 60% reduction in total CD4 T cells and, in particular, a greater than 90% loss of the CD45RA+ CD4 cells. CD4 recovery was protracted, achieving less than 50% of pretreatment levels after 12 to 14 months. Two facets of the CD4 recovery were notable. First, generation of CD45RA+ CD4 cells played only a minor role in the first year, suggesting that thymic production was not the main route of CD4 regeneration. Indeed, recovery of CD45RA+ CD4 cell levels remained limited in half of the patients even after 2 years. Second, expansion of the mature peripheral CD4 cells (CD45RO+) remaining after chemotherapy was the main source of early CD4 repopulation, peaking at 3 to 6 months postchemotherapy. This expansion was limited in duration, however, and was followed by a secondary decline, such that the total CD45RO+ CD4 levels at 9 to 12 months were lower than at 6 months. When stimulated by mitogens, an increased susceptibility to apoptosis was observed in postchemotherapy CD4 cells as compared with those from normal donors. The elevated expression of markers such as HLA-DR during chemotherapy and for several months postchemotherapy is consistent with the presence of an activated T-cell population. CD4 apoptotic frequency correlated with the frequency of HLA-DR expression on T cells. Thus, CD4 recovery is constrained in adults by a limited thymic regenerative capacity and by an increased susceptibility to apoptosis within the expanding peripheral CD4 population.
View details for Web of Science ID A1997YD10800059
View details for PubMedID 9345067
- Therapy-induced alterations in host defense in children receiving therapy for cancer JOURNAL OF PEDIATRIC HEMATOLOGY ONCOLOGY 1997; 19 (5): 399-417
A role for TGF beta 1 in Langerhans cell biology - Further characterization of the epidermal Langerhans cell defect in TGF beta 1 null mice
JOURNAL OF CLINICAL INVESTIGATION
1997; 100 (3): 575-581
Previous studies of TGFbeta1 null (-/-) mice indicated that the epidermis was devoid of Langerhans cells (LC) and that the LC deficiency was not secondary to the inflammation that is the dominant feature of the -/- phenotype (Borkowski, T.A., J.J. Letterio, A.G. Farr, and M.C. Udey. 1996. J. Exp. Med. 184:2417-2422). Herein, we demonstrate that dendritic cells could be expanded from the bone marrow of -/- mice and littermate controls. Bone marrow from -/- mice also gave rise to LC after transfer into lethally irradiated recipients. Thus, the LC defect in TGFbeta1 null mice does not result from an absolute deficiency in bone marrow precursors, and paracrine TGFbeta1 production is sufficient for LC development. Several approaches were used to assess the suitability of -/- skin for LC localization. A survey revealed that although a number of cytokine mRNAs were expressed de novo, mRNAs encoding proinflammatory cytokines known to mobilize LC from epidermis (IL-1 and TNFalpha) were not strikingly overrepresented in -/- skin. In addition, bone marrow-derived LC populated full-thickness TGFbeta1 null skin after engraftment onto BALB/c nu/nu recipients. Finally, the skin of transgenic mice expressing a truncated loricrin promoter-driven dominant-negative TGFbeta type II receptor contained normal numbers of LC. Because TGFbeta1 signaling in these mice is disrupted only in keratinocytes and the keratinocyte hyperproliferative component of the TGFbeta1 -/- phenotype is reproduced, these results strongly suggest that the LC defect in TGFbeta1 null mice is not due to an epidermal abnormality but reflects a requirement of murine LC (or their precursors) for TGFbeta1.
View details for Web of Science ID A1997XQ28000012
View details for PubMedID 9239404
Pathways of T-cell regeneration in mice and humans: Implications for bone marrow transplantation and immunotherapy
1997; 157: 61-72
Much of our understanding of the immunobiology of bone marrow transplantation (BMT) has come from studies in young adult mice reconstituted with T-cell-depleted bone marrow after lethal irradiation. Recent evidence indicates, however, that the applicability of conclusions drawn from this model to human BMT may be limited. While mice retain essentially normal thymic function well past sexual maturity, humans show significant age-related declines in thymic function relatively early in life. Therefore, thymic-deficient mice may provide a more accurate model for study of the immunobiology of BMT. T-cell regeneration in thymic-deficient mice occurs primarily via antigen-driven expansion of mature peripheral T cells resulting in limited immune competence due to quantitative deficiencies in T-cell number and severe restriction in the diversity of the regenerated T-cell receptor (TCR) repertoire. Similarly, immune reconstitution in adult humans after BMT is marked by quantitative T-cell deficiencies, especially in the CD4+ subset, and loss of TCR diversity. Taken together, prevailing evidence suggests that thymic function is suboptimal in most BMT recipients, and that thymic-independent pathways of T-cell regeneration are generally limited in their ability to restore host immune competence. New strategies to enhance thymic function in man after BMT would hold great therapeutic potential.
View details for Web of Science ID A1997XL05000005
View details for PubMedID 9255622
Distinctions between CD8(+) and CD4(+) T-cell regenerative pathways result in prolonged T-cell subset imbalance after intensive chemotherapy
1997; 89 (10): 3700-3707
Rapid recovery of CD4+ T cells after intensive chemotherapy is limited by an age-dependent decline in thymopoiesis. Here we sought to determine whether similar limitations exist for CD8+ T-cell regeneration. After intensive chemotherapy, CD8+ T cells had a faster effective doubling time than CD4+ T cells (median, 12.6 v 28.2 days, P < .05). Accordingly, at 3 months posttherapy, mean CD8+ T-cell number had returned to baseline, whereas mean CD4+ T-cell number was only 35% of pretherapy values (P < .05). These differences were primarily due to very rapid expansion of CD8+CD57+ and CD8+CD28- subsets. At 3 months posttherapy, there was no relationship between age and CD8+ T-cell number (R = -.02), whereas CD4+ T-cell number was inversely related to age (R = -.66) and there were no discernible differences in CD8+ recovery among patients with or without thymic enlargement, whereas CD4+ recovery was enhanced in patients with thymic enlargement after chemotherapy (P < .01). Therefore thymic-independent pathways of T-cell regeneration appear to rapidly regenerate substantial numbers of CD8+, but not CD4+ T cells, resulting in prolonged T-cell subset imbalance after T-cell depletion. These inherent distinctions between CD4+ v CD8+ T-cell regeneration may have significant implications for immunotherapeutic strategies undertaken to eradicate minimal residual neoplastic disease after cytoreductive chemotherapy.
View details for Web of Science ID A1997XD97600023
View details for PubMedID 9160675
- T-cell regeneration: All repertoires are not created equal IMMUNOLOGY TODAY 1997; 18 (5): 245-251
- Langerhans cells in the TGF beta 1 null mouse DENDRITIC CELLS IN FUNDAMENTAL AND CLINICAL IMMUNOLOGY, VOL 3 1997; 417: 307-310
Autoimmunity associated with TGF-beta 1-deficiency in mice is dependent on MHC class II antigen expression
JOURNAL OF CLINICAL INVESTIGATION
1996; 98 (9): 2109-2119
The progressive inflammatory process found in transforming growth factor beta1 (TGF-beta1)-deficient mice is associated with several manifestations of autoimmunity, including circulating antibodies to nuclear antigens, immune complex deposition, and increased expression of both class I and class II major histocompatibility complex (MHC) antigens. The contribution of MHC class II antigens to the genesis of this phenotype has been determined by crossing the TGF-beta1-null [TGF-beta1(-/-)] genotype into the MHC class II-deficient [MHC-II(-/-)] background. Mice homozygous for both the TGF-beta1 null allele and the class II null allele [TGF-beta1(-/-);MHC-II(-/-)] are without evidence of inflammatory infiltrates, circulating autoantibodies, or glomerular immune complex deposits. Instead, these animals exhibit extensive extramedullary hematopoiesis with progressive splenomegaly and adenopathy, surviving only slightly longer than TGF-beta1(-/-);MHC-II(+/+) mice. The role of CD4+ T cells, which are also absent in MHC class II-deficient mice, is directly demonstrated through the administration of anti-CD4 monoclonal antibodies in class II-positive, TGF-beta1(-/-) mice. The observed reduction in inflammation and improved survival emphasize the significance of CD4+ cells in the pathogenesis of the autoimmune process and suggest that the additional absence of class II antigens in TGF-beta1(-/-);MHC-II(-/-) mice may contribute to their extreme myeloid metaplasia. Thus, MHC class II antigens are essential for the expression of autoimmunity in TGF-beta1-deficient mice, and normally may cooperate with TGF-beta1 to regulate hematopoiesis.
View details for Web of Science ID A1996VR56300023
View details for PubMedID 8903331
Thymic-independent T cell regeneration occurs via antigen-driven expansion of peripheral T cells resulting in a repertoire that is limited in diversity and prone to skewing
JOURNAL OF IMMUNOLOGY
1996; 156 (12): 4609-4616
Thymic regenerative capacity in humans decreases with age, suggesting that thymic-independent pathways of T cell regeneration may predominate during adulthood. Using a murine bone marrow transplantation model, we present evidence that thymic-independent T cell regeneration occurs primarily via expansion of peripheral T cells and is Ag driven since significant expansion of CD4+ or CD8+ transgenic (Tg+)/TCR-bearing cells occurs only in the presence of Ag specific for the TCR. Such expansion resulted in skewing of the regenerated repertoire with 40 to 65% of the regenerated CD4+ or CD8+ T cells expressing the Tg+/TCR in thymectomized hosts after bone marrow transplantation. In experiments in which nontransgenic population are used as T cell inocula, we noted decreased CD4 expansion when Class II MHC was blocked by mAb treatment in vivo, an CD8 expansion failed to occur in Class I MHC-deficient hosts providing evidence that T cell regeneration in thymic-deficient hosts largely occurs via TCR-MHC-mediated selection of peripheral T cell populations. This process results in a T cell repertoire comprised exclusively of T cells recently activated by the antigenic milieu of the host, with negligible numbers of residual "naive" cells bearing TCRs for Ags absent at the time of expansion. These findings have important implications for approached to enhance T cell regeneration in humans and provide evidence that vaccine strategies could skew the T cell repertoire toward a specific antigenic target if administered to thymic-deficient hosts during immune reconstitution.
View details for Web of Science ID A1996UP66200012
View details for PubMedID 8648103
Early suppressive effects of chemotherapy and cytokine treatment on committed versus primitive haemopoietic progenitors in patient bone marrow
BRITISH JOURNAL OF HAEMATOLOGY
1996; 92 (3): 537-547
These studies investigated the effectiveness of in vivo administration of cytokines in ameliorating potential marrow damage induced by chemotherapy. Breast cancer patients received 5-fluorouracil, leucovorin, doxorubicin and cyclophosphamide (FLAC) followed by either GM-CSF, PIXY321, or no cytokine. Marrow was obtained before and after one or two cycles of FLAC once blood cell counts had recovered. Colony-forming units for granulocytes and macrophages (CFU-GM) were used to indicate the effect of therapy on recovery of committed progenitor cells responsible for early blood cell recovery. The frequency and number of CFU-GM in marrow obtained after FLAC + PIXY321 were significantly lower than in marrow obtained after FLAC+GM-CSF or FLAC without cytokine. CD34+ cell numbers were also reduced after FLAC + PIXY321. CFU-GM production in marrow long-term cultures (LTC) was used to assess the effect of therapy on primitive progenitors. After 5 weeks the number of CFU-GM in LTC of post-therapy marrow from all three treatment arms was < 15% of the number in pre-therapy LTC. Suppressive effects of FLAC on primitive progenitors were observed even when committed progenitors and CD34+ cells had recovered to pre-therapy levels. These results demonstrate that cytokine treatment did not ameliorate suppressive or toxic effects of FLAC on the functional integrity of the marrow.
View details for Web of Science ID A1996TY26500005
View details for PubMedID 8616014
DEMODICIDOSIS IN CHILDHOOD ACUTE LYMPHOBLASTIC-LEUKEMIA - AN OPPORTUNISTIC INFECTION OCCURRING WITH IMMUNOSUPPRESSION
JOURNAL OF PEDIATRICS
1995; 127 (5): 751-754
We report demodicidosis in 11 children with acute lymphoblastic leukemia and a mildly pruritic, erythematous papular dermatitis that developed in areas rich in sebaceous glands. Dermodex eruptions were safely and effectively treated with 5% permethrin. Proliferation of commensal parasites of the skin, Dermodex folliculorum and Dermodex brevis may be an opportunistic infection of the skin in the immunocompromised host; the expected abrogation of cell-mediated immunity secondary to lymphocyte depletion predisposes some children given chemotherapy for leukemia to mite proliferation.
View details for Web of Science ID A1995TD93100017
View details for PubMedID 7472831
AGE, THYMOPOIESIS, AND CD4+ T-LYMPHOCYTE REGENERATION AFTER INTENSIVE CHEMOTHERAPY
NEW ENGLAND JOURNAL OF MEDICINE
1995; 332 (3): 143-149
Inadequate reconstitution of CD4+ T lymphocytes is an important clinical problem complicating chemotherapy, human immunodeficiency virus infection, and bone marrow transplantation, but relatively little is known about how CD4+ T lymphocytes regenerate. There are two main possibilities: bone marrow-derived progenitors could reconstitute the lymphocyte population using a thymus-dependent pathway, or thymus-independent pathways could predominate. Previous studies have suggested that the CD45RA glycoprotein on CD4+ T lymphocytes is a marker for progeny generated by a thymus-dependent pathway.We studied 15 patients 1 to 24 years of age who had undergone intensive chemotherapy for cancer. The absolute numbers of CD4+ T lymphocytes in peripheral blood and the expression of CD45 isoforms (CD45RA and CD45RO) on these lymphocytes were studied serially during lymphocyte regeneration after the completion of therapy. Radiographic imaging of the thymus was performed concomitantly.There was an inverse relation between the patients' ages and the CD4+ T-lymphocyte counts six months after therapy was completed (r = -0.92). The CD4+ recovery correlated quantitatively with the appearance of CD45RA+CD4+ T lymphocytes in the blood (r = 0.64). There was a higher proportion of CD45RA+CD4+ T lymphocytes in patients with thymic enlargement after chemotherapy than in patients without such enlargement (two-sided P = 0.015).Thymus-dependent regeneration of CD4+ T lymphocytes occurs primarily in children, whereas even young adults have deficiencies in this pathway. Our results suggest that rapid T-cell regeneration requires residual thymic function in patients receiving high-dose chemotherapy.
View details for Web of Science ID A1995QB16000003
View details for PubMedID 7800006
TRANSFORMING GROWTH-FACTOR-BETA-1 NULL MICE - AN ANIMAL-MODEL FOR INFLAMMATORY DISORDERS
AMERICAN JOURNAL OF PATHOLOGY
1995; 146 (1): 264-275
Approximately 40% of transforming growth factor-beta 1 null (knockout) mice generated in our laboratory develop normally to term, but 60% die in utero. The animals appear normal during the first 2 weeks of life but develop a rapid wasting syndrome and die by 3 to 4 weeks of age. All of the knockout mice have a multifocal inflammatory disease in many tissues. The heart and lungs are most severely affected. Increased adhesion of leukocytes to the endothelium of pulmonary veins is the initial lesion seen at day 8 postnatally and is soon followed by perivascular cuffing as well as inflammatory infiltrates in lung parenchyma. The lesions in the heart begin as endocarditis and then progress to myocarditis and pericarditis. Within the lung, chronic inflammatory infiltrates consist of T and B lymphocytes, including plasma cells, whereas macrophages are the primary inflammatory cell type in the heart. Increased expression of major histocompatibility complex class I and II proteins is seen in pulmonary vascular endothelium as early as day 8. An immunoblastic response in mediastinal and mandibular lymph nodes and spleen is also seen. In the absence of any pathogens, this massive inflammatory disease, together with overexpression of major histocompatibility complex class I and II proteins and overproduction of immunoglobulins by lymphocytes, offers circumstantial evidence for an autoimmune etiology.
View details for Web of Science ID A1995QA90200030
View details for PubMedID 7856732
LYMPHOCYTE DEPLETION DURING TREATMENT WITH INTENSIVE CHEMOTHERAPY FOR CANCER
1994; 84 (7): 2221-2228
Recently we have observed an increased incidence of opportunistic infections in patients treated with intensive chemotherapy for cancer. Because T-cell depletion is associated with similar clinical events in human immunodeficiency virus infection and after bone marrow transplantation, we have analyzed peripheral blood lymphocyte populations in a series of patients during treatment with intensive chemotherapy for cancer. Although neutrophil, monocyte, and platelet numbers consistently recovered to greater than 50% of pretreatment values after each sequential cycle of therapy, lymphocyte numbers did not recover within the same time period. B cells decreased rapidly from a mean value of 149 +/- 46/mm3 before chemotherapy to 4 +/- 1/mm3 during chemotherapy (P = .01). CD4+ T cells decreased from a mean of 588 +/- 76/mm3 before chemotherapy to 105 +/- 28/mm3 during chemotherapy (P = .0002) and CD8+ T cells decreased from a mean of 382 +/- 41/mm3 before chemotherapy to 150 +/- 46/mm3 during chemotherapy (P = .0009). Natural killer cell numbers did not show significant declines (171 +/- 30/mm3 before, 114 +/- 24/mm3 during, P = .19). Based on the history of opportunistic complications in patients with other disorders who display similar degrees of CD4+ T-cell lymphopenia and preliminary observations in this population, immune incompetence could surface as a dose-limiting toxicity for highly dose-intensive chemotherapy regimens.
View details for Web of Science ID A1994PK12500021
View details for PubMedID 7919339
IMMUNE DYSREGULATION IN TGF-BETA-1-DEFICIENT MICE
JOURNAL OF IMMUNOLOGY
1994; 153 (5): 1936-1946
Approximately 2 wk after birth, mice having a TGF-beta 1 null mutation (TGF-beta 1(-/-)) exhibit a progressive wasting syndrome and death. Associated with this phenotype is a multifocal infiltration of lymphocytes and macrophages into target organs, especially the heart, lungs, and salivary glands. To explore the consequences of TGF-beta 1 deficiency on the immune system, lymphocyte phenotype and function were analyzed. Initially, lymphoid organ architecture seemed to be normal and, as symptoms developed, the thymus decreased in size, whereas lymph nodes were enlarged. Phenotypically, the TGF-beta 1(-/-) lymphoid cells seemed to be more differentiated in the thymus and activated in the lymph nodes, but remarkably unaffected in the spleen. Moreover, TGF-beta 1(-/-) spleen and lymph nodes displayed enhanced numbers of proliferating cells, as measured by proliferating cell nuclear Ag and/or cyclin-dependent kinase levels. Consistent with this hyperproliferative response, constitutive levels of IL-2 mRNA were elevated in the thymus and both IL-2 and IL-2R mRNA were increased in the lymph nodes. In contrast with the activation profile of TGF-beta 1(-/-) lymphoid cells in vivo, mitogen challenge of these cells in vitro revealed suppressed proliferation that was associated with a defect in inducible IL-2 mRNA expression and IL-2 secretion. Moreover, the addition of rIL-2 restored the deficient mitogen-induced proliferation. The mechanism leading to T cell anergy remains unclear; however, these data confirm the essential role for TGF-beta 1 in maintaining normal immune function.
View details for Web of Science ID A1994PD07500003
View details for PubMedID 8051399
T-CELL REGENERATION AFTER BONE-MARROW TRANSPLANTATION - DIFFERENTIAL CD45 ISOFORM EXPRESSION ON THYMIC-DERIVED VERSUS THYMIC-INDEPENDENT PROGENY
1993; 82 (8): 2585-2594
To study the source of regenerated T cells after bone marrow transplantation (BMT), lethally irradiated thymectomized and thymus-bearing C57BL/6 (Thy 1.2+) mice were injected with syngeneic T-cell depleted bone marrow (TCD BM) cells and graded numbers of congenic B6/Thy 1.1+ lymph node (LN) cells. LN cell expansion was the predominant source for T-cell regeneration in thymectomized hosts but was minimal in thymus-bearing hosts. Analysis of T-cell receptor (TCR) expression on LN progeny showed a diverse V beta repertoire. Therefore, peripheral T-cell progenitors exist within V beta families, but expansion of these progenitors after BMT is downregulated in the presence of a functional thymus. CD4+ cells derived from BM versus LN in thymus-bearing hosts displayed differential CD44 and CD45 isoform expression. BM-derived cells were primarily CD45RB+CD44lo and LN derived cells were nearly exclusively CD45RB- CD44hi. In thymectomized hosts, BM, host, and LN CD4+ progeny were CD45RB- CD44hi. We conclude that T-cell regeneration via peripheral T-cell progenitors predominates in hosts lacking thymic function and gives rise to T cells that display a "memory" phenotype. In contrast, the ability to generate sizable populations of "naive" type T cells after BMT appears limited to the prethymic progenitor pool and could serve as a marker for thymic regenerative capacity.
View details for Web of Science ID A1993MC27600041
View details for PubMedID 7691265
O-2 RESERVE OF LEFT-VENTRICLE OF ISOLATED, SALINE-PERFUSED RABBIT HEART
AMERICAN JOURNAL OF PHYSIOLOGY
1984; 247 (5): H861-H868
The heart from the pentobarbital-anesthetized rabbit was isolated, and a fluid-filled balloon was placed in the left ventricular chamber for assessment of isovolumic pressure development. The bicarbonate-buffered, physiological perfusate was aerated initially with 95% O2-5% CO2, and then progressive decreases in arterial O2 content (CaO2) were produced in a stepwise fashion by substituting N2 for O2 in the aerating gas mixture. If, for a specified set of experimental conditions, no change in ventricular function occurred after the initial decrease in CaO2, it was concluded that the heart was oxygenated adequately prior to the first CaO2 decrement. Accordingly, with perfusate flow constant at 35 ml/min, adequate oxygenation was achieved during aeration of perfusate with 95% O2 when ventricular contraction rate was 30 beats/min and temperature (T) 22 degrees C. The preparation may have been just marginally O2 sufficient when rate was 60 beats/min (T, 25 or 30 degrees C), but probably was hypoxic when contraction rate was 120 beats/min (T, 30 or 37 degrees C). Perfusion with pressure kept constant at 80 mmHg did not appear to alleviate this hypoxia when metabolism was elevated (rate, 120 beats/min; T, 37 degrees C). Thus, unless the contraction rate is very low, the addition of an O2-carrying vehicle to the perfusate appears to be necessary if O2 delivery to the isolated rabbit heart preparation is to be supramaximal.
View details for Web of Science ID A1984AAX5600024
View details for PubMedID 6496765