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T cell-based therapies like genetically modified immune cells expressing chimeric antigen receptors have shown robust anti-cancer activity in vivo, especially in patients with blood cancers. However, extending this approach to an "off-the-shelf" setting can be challenging, as allogeneic T cells carry a significant risk of graft-versus-host disease (GVHD). By contrast, allogeneic natural killer (NK) cells recognize malignant cells without the need for prior antigen exposure and have been used safely in multiple cancer settings without the risk of GVHD. However, similar to T cells, NK cell function is negatively impacted by tumor-induced transforming growth factor beta (TGF-β) secretion, which is a ubiquitous and potent immunosuppressive mechanism employed by most malignancies. Allogeneic NK cells for adoptive immunotherapy can be sourced from peripheral blood (PB) or cord blood (CB), and the authors' group and others have previously shown that ex vivo expansion and gene engineering can overcome CB-derived NK cells' functional immaturity and poor cytolytic activity, including in the presence of exogenous TGF-β.  However, a direct comparison of the effects of TGF-β-mediated immune suppression on ex vivo-expanded CB- versus PB-derived NK cell therapy products has not previously been performed. Here the authors show that PB- and CB-derived NK cells have distinctive gene signatures that can be overcome by ex vivo expansion. Additionally, exposure to exogenous TGF-β results in an upregulation of inhibitory receptors on NK cells, a novel immunosuppressive mechanism not previously described. Finally, the authors provide functional and genetic evidence that both PB- and CB-derived NK cells are equivalently susceptible to TGF-β-mediated immune suppression. The authors believe these results provide important mechanistic insights to consider when using ex vivo-expanded, TGF-β-resistant PB- or CB-derived NK cells as novel immunotherapy agents for cancer., Competing Interests: Declaration of Competing Interest CRYC is a consultant for the NK cell company Catamaran Bio. CMB is a co-founder and scientific advisory board member for Catamaran Bio. CRYC, CS and CMB have intellectual property related to the development of NK cell therapies., (Copyright © 2022 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Epstein-Barr virus (EBV) is a ubiquitous human tumor virus, which contributes to the development of lymphoproliferative disease, most notably in patients with impaired immunity. EBV-associated lymphoproliferation is characterized by expression of latent EBV proteins and ranges in severity from a relatively benign proliferative response to aggressive malignant lymphomas. The presence of EBV can also serve as a unique target for directed therapies for the treatment of EBV lymphoproliferative diseases, including T cell-based immune therapies. In this review, we describe the EBV-associated lymphoproliferative diseases and particularly focus on the therapies that target EBV., (© 2022 by The American Society of Hematology.)

Introduction :Widespread success of CD19 chimeric antigen receptor (CAR) T cells for the treatment of hematological malignancies have shifted the focus from conventional cancer treatments toward adoptive immunotherapy. There are major efforts to improve CAR constructs and to identify new target antigens. Even though the Food and Drug Administration has approved commercialization of some CD19 CART cell therapies, there are still some limitations that restrict their widespread clinical use. The manufacture of autologous products for individual patients is logistically cumbersome and expensive and allogeneic T cell products may pose an appreciable risk of graft-versus-host disease (GVHD). Areas covered :Natural killer (NK) cells are an attractive alternative for CART-based immunotherapies. They have the innate ability to detect and eliminate malignant cells and are safer in the 'off-the-shelf' setting. This review discusses the current progress within the CAR NK cell field, including the challenges, and future prospects. Gene engineered NK cells was used as the search term in PubMed and Google Scholar through to December 2020. Expert opinion :CAR NK cell therapies hold promise as an 'off-the-shelf' cell therapy for cancer. It is hoped that an enhanced understanding of their immunobiology and molecular mechanisms of action will improve their in vivo potency.

Cancer immunotherapy has dramatically changed the approach to cancer treatment. The aim of targeting the immune system to recognize and destroy cancer cells has afforded many patients the prospect of achieving deep, long-term remission and potential cures. However, many challenges remain for achieving the goal of effective immunotherapy for all cancer patients. Checkpoint inhibitors have been able to achieve long-term responses in a minority of patients, yet improving response rates with combination therapies increases the possibility of toxicity. Chimeric antigen receptor T cells have demonstrated high response rates in hematological cancers, although most patients experience relapse. In addition, some cancers are notoriously immunologically "cold" and typically are not effective targets for immunotherapy. Overcoming these obstacles will require new strategies to improve upon the efficacy of current agents, identify biomarkers to select appropriate therapies, and discover new modalities to expand the accessibility of immunotherapy to additional tumor types and patient populations., (© 2020 New York Academy of Sciences.)

Natural killer (NK) cells, the primary effector cells of the innate immune system, utilize multiple strategies to recognize tumor cells by (1) detecting the presence of activating receptor ligands, which are often upregulated in cancer; (2) targeting cells that have a loss of major histocompatibility complex (MHC); and (3) binding to antibodies that bind to tumor-specific antigens on the tumor cell surface. All these strategies have been successfully harnessed in adoptive NK cell immunotherapies targeting cancer. In this review, we review the applications of NK cell therapies across different tumor types. Similar to other forms of immunotherapy, tumor-induced immune escape and immune suppression can limit NK cell therapies' efficacy. Therefore, we also discuss how these limitations can be overcome by conferring NK cells with the ability to redirect their tumor-targeting capabilities and survive the immune-suppressive tumor microenvironment. Finally, we also discuss how future iterations can benefit from combination therapies with other immunotherapeutic agents., (Copyright © 2020 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

T-cell responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been described in recovered patients, and may be important for immunity following infection and vaccination as well as for the development of an adoptive immunotherapy for the treatment of immunocompromised individuals. In this report, we demonstrate that SARS-CoV-2-specific T cells can be expanded from convalescent donors and recognize immunodominant viral epitopes in conserved regions of membrane, spike, and nucleocapsid. Following in vitro expansion using a good manufacturing practice-compliant methodology (designed to allow the rapid translation of this novel SARS-CoV-2 T-cell therapy to the clinic), membrane, spike, and nucleocapsid peptides elicited interferon-γ production, in 27 (59%), 12 (26%), and 10 (22%) convalescent donors (respectively), as well as in 2 of 15 unexposed controls. We identified multiple polyfunctional CD4-restricted T-cell epitopes within a highly conserved region of membrane protein, which induced polyfunctional T-cell responses, which may be critical for the development of effective vaccine and T-cell therapies. Hence, our study shows that SARS-CoV-2 directed T-cell immunotherapy targeting structural proteins, most importantly membrane protein, should be feasible for the prevention or early treatment of SARS-CoV-2 infection in immunocompromised patients with blood disorders or after bone marrow transplantation to achieve antiviral control while mitigating uncontrolled inflammation.

Viral infections are common and are potentially life-threatening in patients with moderate to severe primary immunodeficiency disorders. Because T-cell immunity contributes to the control of many viral pathogens, adoptive immunotherapy with virus-specific T cells (VSTs) has been a logical and effective way of combating severe viral disease in immunocompromised patients in multiple phase 1 and 2 clinical trials. Common viral targets include cytomegalovirus, Epstein-Barr virus, and adenovirus, though recent published studies have successfully targeted additional pathogens, including HHV6, BK virus, and JC virus. Though most studies have used VSTs derived from allogenic stem cell donors, the use of banked VSTs derived from partially HLA-matched donors has shown efficacy in multicenter settings. Hence, this approach could shorten the time for patients to receive VST therapy thus improving accessibility. In this review, we discuss the usage of VSTs for patients with primary immunodeficiency disorders in clinical trials, as well as future potential targets and methods to broaden the applicability of virus-directed T-cell immunotherapy for this vulnerable patient population., (© 2020 by The American Society of Hematology.)

Introduction: Autologous T-cells transduced to express a chimeric antigen receptor (CAR) directed against CD19 elicit high response rates in relapsed or refractory (r/r) B-cell non-Hodgkin lymphoma (B-NHL). However, r/r B-NHL remissions are durable in fewer than half of recipients of second-generation CAR T-cells. Third-generation (3G) CARs employ two costimulatory domains, resulting in improved CAR T-cell efficacy in vitro and in animal models in vivo. This investigator-initiated, phase I dose escalation trial, termed ENABLE, will investigate the safety and preliminary efficacy of WZTL-002, comprising autologous T-cells expressing a 3G anti-CD19 CAR incorporating the intracellular signalling domains of CD28 and Toll-like receptor 2 (TLR2) for the treatment of r/r B-NHL., Methods and Analysis: Eligible participants will be adults with r/r B-NHL including diffuse large B-cell lymphoma and its variants, follicular lymphoma, transformed follicular lymphoma and mantle cell lymphoma. Participants must have satisfactory organ function, and lack other curative options. Autologous T-cells will be obtained by leukapheresis. Following WZTL-002 manufacture and product release, participants will receive lymphodepleting chemotherapy comprising intravenous fludarabine and cyclophosphamide. A single dose of WZTL-002 will be administered intravenously 2 days later. Targeted assessments for cytokine release syndrome and immune cell effector-associated neurotoxicity syndrome, graded by the American Society Transplantation and Cellular Therapy criteria, will be made. A modified 3+3 dose escalation scheme is planned starting at 5×10 4  CAR T-cells/kg with a maximum dose of 1×10 6  CAR T-cells/kg. The primary outcome of this trial is safety of WZTL-002. Secondary outcomes include feasibility of WZTL-002 manufacture and preliminary measures of efficacy., Ethics and Dissemination: Ethical approval for the study was granted by the New Zealand Health and Disability Ethics Committee (reference 19/STH/69) on 23 June 2019 for Protocol V.1.2. Trial results will be reported in a peer-reviewed journal, and results presented at scientific conferences or meetings., Trial Registration Number: NCT04049513., Competing Interests: Competing interests: Trial principal investigator, RW, and co-investigator, PG, are employees of the Malaghan Institute of Medical Research, a charitable research institute and study sponsor. The other co-investigators have no competing interests to declare. PL has proprietary interest in the intellectual property of the 1928T2z construct. CB is co-Founder and Scientific Advisory Board Member of Mana Therapeutics is on the Advisory Board of Cellectis, has Stock ownership in Torque Therapeutics and Neximmune and is a Board Member of Caballeta Bio., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

TCR-gamma delta (γδ) T-cells are considered important players in the graft-vs.-tumor effect following allogeneic hematopoietic cell transplantation (alloHCT) and have emerged as candidates for adoptive transfer immunotherapy in the treatment of both solid and hematological tumors. Systemic β-adrenergic receptor (β-AR) activation has been shown to mobilize TCR-γδ T-cells to the blood, potentially serving as an adjuvant for alloHCT and TCR-γδ T-cell therapy. We investigated if systemic β-AR activation, using acute dynamic exercise as an experimental model, can increase the mobilization, ex vivo expansion, and anti-tumor activity of TCR-γδ T-cells isolated from the blood of healthy humans. We also sought to investigate the β-AR subtypes involved, by administering a preferential β 1 -AR antagonist (bisoprolol) and a non-preferential β 1 + β 2 -AR antagonist (nadolol) prior to exercise as part of a randomized placebo controlled cross-over experiment. We found that exercise mobilized TCR-γδ cells to blood and augmented their ex vivo expansion by ~182% compared to resting blood when stimulated with IL-2 and ZOL for 14-days. Exercise also increased the proportion of CD56+, NKG2D+/CD62L-, CD158a/b/e+ and NKG2A- cells among the expanded TCR-γδ cells, and increased their cytotoxic activity against several tumor target cells (K562, U266, 221.AEH) in vitro by 40-60%. Blocking NKG2D on TCR-γδ cells in vitro eliminated the augmented cytotoxic effects of exercise against U266 target cells. Furthermore, administering a β 1 + β 2 -AR (nadolol), but not a β 1 -AR (bisoprolol) antagonist prior to exercise abrogated the exercise-induced enhancement in TCR-γδ T-cell mobilization and ex vivo expansion. Furthermore, nadolol completely abrogated while bisoprolol partially inhibited the exercise-induced increase in the cytotoxic activity of the expanded TCR-γδ T-cells. We conclude that acute systemic β-AR activation in healthy donors markedly augments the mobilization, ex vivo expansion, and anti-tumor activity of TCR-γδ T-cells and that some of these effects are due to β 2 -AR signaling and phenotypic shifts that promote a dominant activating signal via NKG2D. These findings highlight β-ARs as potential targets to favorably alter the composition of allogeneic peripheral blood stem cell grafts and improve the potency of TCR-γδ T-cell immune cell therapeutics., (Copyright © 2020 Baker, Bigley, Agha, Pedlar, O'Connor, Bond, Bollard, Katsanis and Simpson.)

Purpose of Review: The US Food and Drug Administration (FDA) approved two commercially available chimeric antigen receptor (CAR) T cell therapies for the treatment of relapsed B cell acute lymphoblastic leukemia (B-ALL) children and young adults less than 25 years of age and non-Hodgkin lymphoma in adults after promising results from early-phase single and multi-institutional clinical trials. In this review, we provide an overview of the practical aspects of a chimeric antigen T cell receptor (CAR-T) program development and the steps necessary for its successful implementation., Recent Findings: CAR-T therapy is a complex process and poses significant challenges as institutions prepare to deliver this therapy as a standard of care for the eligible patients. It requires a rigorous infrastructure with specific clinical, administrative, and regulatory demands. Institutions that led the clinical trials for CAR-T have adopted various approaches to integrate commercial CAR-T products into their program. Delivering commercial CAR-T cells outside the scope of clinical trials requires careful planning, allocation of resources, and utilization of existing infrastructure. Institutions may need to adapt the existing recommendations and guidelines and tailor them to meet the needs of their program and ensure appropriate financial reimbursement for this expensive but promising immunotherapy.

Viral infections are a serious cause of morbidity and mortality following haematopoietic stem cell transplantation (HSCT). Adoptive cellular therapy with virus-specific T cells (VSTs) has been successful in preventing or treating targeted viruses in prior studies, but the composition of ex vivo expanded VST and the critical cell populations that mediate antiviral activity in vivo are not well defined. We utilized deep sequencing of the T-cell receptor beta chain (TCRB) in order to classify and track VST populations in 12 patients who received VSTs following HSCT to prevent or treat viral infections. TCRB sequencing was performed on sorted VST products and patient peripheral blood mononuclear cells samples. TCRB diversity was gauged using the Shannon entropy index, and repertoire similarity determined using the Morisita-Horn index. Similarity indices reflected an early change in TCRB diversity in eight patients, and TCRB clonotypes corresponding to targeted viral epitopes expanded in eight patients. TCRB repertoire diversity increased in nine patients, and correlated with cytomegalovirus (CMV) viral load following VST infusion (P = 0·0071). These findings demonstrate that allogeneic VSTs can be tracked via TCRB sequencing, and suggests that T-cell receptor repertoire diversity may be critical for the control of CMV reactivation after HSCT., (© 2019 British Society for Haematology and John Wiley & Sons Ltd.)

Purpose: Tumor-associated antigen cytotoxic T cells (TAA-Ts) represent a new, potentially effective and nontoxic therapeutic approach for patients with relapsed or refractory solid tumors. In this first-in-human trial, we investigated the safety of administering TAA-Ts that target Wilms tumor gene 1, preferentially expressed antigen of melanoma, and survivin to patients with relapsed/refractory solid tumors., Materials and Methods: TAA-T products were generated from autologous peripheral blood and infused over three dose levels: 1, 2, and 4 × 10 7 cells/m 2 . Patients were eligible for up to eight infusions administered 4 to 7 weeks apart. We assessed dose limiting toxicity during the first 45 days after infusion. Disease response was determined within the context of a phase I trial., Results: There were no dose-limiting toxicities. Of 15 evaluable patients, 11 (73%) with stable disease or better at day 45 postinfusion were defined as responders. Six responders remain without progression at a median of 13.9 months (range, 4.1 to 19.9 months) after initial TAA-Ts. Patients who were treated at the highest dose level showed the best clinical outcomes, with a 6-month progression-free survival of 73% after TAA-T infusion compared with a 38% 6-month progression-free survival with prior therapy. Antigen spreading and a reduction in circulating tumor-associated antigens using digital droplet polymerase chain reaction was observed in patients after TAA-T infusion., Conclusion: TAA-Ts safely induced disease stabilization, prolonged time to progression, and were associated with antigen spreading and a reduction in circulating tumor-associated antigen DNA levels in patients with relapsed/refractory solid tumors without lymphodepleting chemotherapy before infusion. TAA-Ts are a promising new treatment approach for patients with solid tumors.

On August 30, 2017 the US Food and Drug Administration approved tisagenlecleucel (Kymriah; Novartis, Basel, Switzerland), a synthetic bioimmune product of anti-CD19 chimeric antigen receptor T cells (CAR-T), for the treatment of children and young adults with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL). With this new era of personalized cancer immunotherapy, multiple challenges are present, ranging from implementation of a CAR-T program to safe delivery of the drug, long-term toxicity monitoring, and disease assessments. To address these issues experts representing the American Society for Blood and Marrow Transplant, the European Society for Blood and Marrow Transplantation, the International Society of Cell and Gene Therapy, and the Foundation for the Accreditation of Cellular Therapy formed a global CAR-T task force to identify and address key questions pertinent for hematologists and transplant physicians regarding the clinical use of anti CD19 CAR-T therapy in patients with B-ALL. This article presents an initial roadmap for navigating common clinical practice scenarios that will become more prevalent now that the first commercially available CAR-T product for B-ALL has been approved., (Copyright © 2018 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Viral infections are common and can be potentially fatal in patients with primary immunodeficiency disorders (PIDDs). Because viral susceptibility stems from poor to absent T-cell function in most patients with moderate to severe forms of PIDD, adoptive immunotherapy with virus-specific T cells (VSTs) has been used to combat viral infections in the setting of hematopoietic stem cell transplantation in multiple clinical trials. Most trials to date have targeted cytomegalovirus, EBV, and adenovirus either alone or in combination, although newer trials have expanded the number of targeted pathogens. Use of banked VSTs produced from third-party donors has also been studied as a method of expanding access to this therapy. Here we review the clinical experience with VST therapy for patients with PIDDs as well as future potential targets and approaches for the use of VSTs to improve clinical outcomes for this specific patient population., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

In 2017, an autologous chimeric antigen receptor (CAR) T cell therapy indicated for children and young adults with relapsed and/or refractory CD19 + acute lymphoblastic leukaemia became the first gene therapy to be approved in the USA. This innovative form of cellular immunotherapy has been associated with remarkable response rates but is also associated with unique and often severe toxicities, which can lead to rapid cardiorespiratory and/or neurological deterioration. Multidisciplinary medical vigilance and the requisite health-care infrastructure are imperative to ensuring optimal patient outcomes, especially as these therapies transition from research protocols to standard care. Herein, authors representing the Pediatric Acute Lung Injury and Sepsis Investigators (PALISI) Network Hematopoietic Stem Cell Transplantation (HSCT) Subgroup and the MD Anderson Cancer Center CAR T Cell Therapy-Associated Toxicity (CARTOX) Program have collaborated to provide comprehensive consensus guidelines on the care of children receiving CAR T cell therapy.

Immune-based treatment strategies, such as checkpoint inhibition and chimeric antigen receptor (CAR) T cells, have started a new frontier for treatment in non-Hodgkin lymphoma (NHL). Checkpoint inhibition has been most successful in Hodgkin lymphoma, where higher expression of PD-L1 is correlated with better overall response rate. Combinations of checkpoint inhibition with various chemotherapy or biologics are in clinical trials, with initially promising results and manageable safety profiles. CAR T-cell therapies that target CD19 are a promising and attractive therapy for B-cell NHLs, with a product approved by the US Food and Drug Administration in 2017. Changes in the target, hinge, or costimulatory domain can dramatically alter the persistence and efficacy of the CAR T cells. The ZUMA trials from Kite used CD19-(CD28z) CAR T cells, whereas the TRANSCEND studies from Juno and the JULIET studies from Novartis used CD19-(4-1BBz) CARs. Despite the recent successes with CAR T-cell clinical trials, major concerns associated with this therapy include cytokine release syndrome, potential neurotoxicities, B-cell aplasia, loss of tumor antigen leading to relapse, and cost and accessibility of the treatment. Although first-generation CAR T-cell therapies have failed in solid malignancies, newer second- and third-generation CAR T cells that target antigens other than CD19 (such as mesothelin or B-cell maturation antigen) are being studied in clinical trials for treatment of lung cancer or multiple myeloma. Overall, immune-based treatment strategies have given oncologists and patients hope when there used to be none, as well as a new basket of tools yet to come with further research and development.

Purpose Transforming growth factor-β (TGF-β) production in the tumor microenvironment is a potent and ubiquitous tumor immune evasion mechanism that inhibits the expansion and function of tumor-directed responses; therefore, we conducted a clinical study to discover the effects of the forced expression of a dominant-negative TGF-β receptor type 2 (DNRII) on the safety, survival, and activity of infused tumor-directed T cells. Materials and Methods In a dose escalation study, eight patients with Epstein Barr virus-positive Hodgkin lymphoma received two to 12 doses of between 2 × 10 7 and 1.5 × 10 8 cells/m 2 of DNRII-expressing T cells with specificity for the Epstein Barr virus-derived tumor antigens, latent membrane protein (LMP)-1 and LMP-2 (DNRII-LSTs). Lymphodepleting chemotherapy was not used before infusion. Results DNRII-LSTs were resistant to otherwise inhibitory concentrations of TGF-β in vitro and retained their tumor antigen-specific activity. After infusion, the signal from transgenic T cells in peripheral blood increased up to 100-fold as measured by quantitative polymerase chain reaction for the transgene, with a corresponding increase in the frequency of functional LMP-specific T cells. Expansion was not associated with any acute or long-term toxicity. DNRII-LSTs persisted for up to ≥ 4 years. Four of the seven evaluable patients with active disease achieved clinical responses that were complete and ongoing in two patients at > 4 years, including in one patient who achieved only a partial response to unmodified tumor-directed T cells. Conclusion TGF-β-resistant tumor-specific T cells safely expand and persist in patients with Hodgkin lymphoma without lymphodepleting chemotherapy before infusion. DNRII-LSTs can induce complete responses even in patients with resistant disease. Expression of DNRII may be useful for the many other tumors that exploit this potent immune evasion mechanism.

Hematopoietic stem cell (HSC) transplantation and adoptive transfer immunotherapy are effective in treating blood cancers and posttransplant infections, but low-circulating cell numbers in patients and donors are oftentimes a limiting factor. We postulate that a single exercise bout will increase the yield of patient- and donor-derived HSCs and cytotoxic lymphocytes to improve this form of treatment for cancer patients.

Cord blood (CB) natural killer (NK) cells are promising effector cells for tumor immunotherapy but are currently limited by immune-suppressive cytokines in the tumor microenvironment, such as transforming growth factor (TGF-β). We observed that TGF-β inhibits expression of activating receptors such as NKG2D and DNAM1 and decreases killing activity against glioblastoma tumor cells through inhibition of perforin secretion. To overcome the detrimental effects of TGF-β, we engrafted a dominant negative TGF-β receptor II (DNRII) on CB-derived NK cells by retroviral transduction and evaluated their ability to kill glioblastoma cells in the presence of TGF-β. After manufacture using Good Manufacturing Practice-compliant methodologies and transduction with DNRII, CB-derived DNRII-transduced NK cells expanded to clinically relevant numbers and retained both their killing ability and their secretion of interferon-γ upon activation. More important, these cells maintained both perforin expression and NKG2D/DNMA1 expression in the presence of TGF-β allowing for recognition and killing of glioblastoma tumor cells. Hence, NK cells expressing a DNRII should have a functional advantage over unmodified NK cells in the presence of TGF-β-secreting tumors and may be an important therapeutic approach for patients with cancer., (Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Epstein Barr virus (EBV) is a human gamma herpes virus that establishes latency in B cells after primary infection. EBV generally only causes a mild, self-limiting viral illness but is also associated with several malignancies including posttransplantation lymphoproliferative disorder in the immunosuppressed host as well as Hodgkin and non-Hodgkin lymphoma in the immune competent host. The expression of EBV antigens by lymphoma has important applications as targets for adoptive T cell therapy. However, as many lymphomas only express subdominant EBV antigens that are less immunogenic, novel strategies are needed to manufacture EBV-specific T cell products specific for Latent Membrane Protein 1 (LMP1) and LMP2, which are expressed in lymphomas with type II and III latency. While several techniques for manufacturing EBV-CTLs are described in the literature, this chapter focuses on one method for generating Good Manufacturing Practice (GMP)-compliant EBV-specific T cell products that are enriched with LMP1 and LMP2.

Primary immunodeficiency disorders (PID) are a group of inborn errors of immunity with a broad range of clinical severity but often associated with recurrent and serious infections. While hematopoietic stem cell transplantation (HSCT) can be curative for some forms of PID, chronic and/or refractory viral infections remain a cause of morbidity and mortality both before and after HSCT. Although antiviral pharmacologic agents exist for many viral pathogens, these are associated with significant costs and toxicities and may not be effective for increasingly drug-resistant pathogens. Thus, the emergence of adoptive immunotherapy with virus-specific T lymphocytes (VSTs) is an attractive option for addressing the underlying impaired T cell immunity in many PID patients. VSTs have been utilized for PID patients following HSCT in many prior phase I trials, and may potentially be beneficial before HSCT in patients with chronic viral infections. We review the various methods of generating VSTs, clinical experience using VSTs for PID patients, and current limitations as well as potential ways to broaden the clinical applicability of adoptive immunotherapy for PID patients.

Although antiretroviral therapy (ART) has been successful in controlling HIV infection, it does not provide a permanent cure, requires lifelong treatment, and HIV-positive individuals are left with social concerns such as stigma. The recent application of T cells to treat cancer and viral reactivations post-transplant offers a potential strategy to control HIV infection. It is known that naturally occurring HIV-specific T cells can inhibit HIV initially, but this response is not sustained in the majority of people living with HIV. Genetically modifying T cells to target HIV, resist infection, and persist in the immunosuppressive environment found in chronically infected HIV-positive individuals might provide a therapeutic solution for HIV. This review focuses on successful preclinical studies and current clinical strategies using T-cell therapy to control HIV infection and mediate a functional cure solution., (Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background Aims: PR1 is an HLA-A2 restricted leukemia-associated antigen derived from neutrophil elastase and proteinase 3, both of which are normally stored in the azurophil granules of myeloid cells but overexpressed in myeloid leukemic cells. PR1-specific cytotoxic lymphocytes (PR1-CTLs) have activity against primary myeloid leukemia in vitro and in vivo and thus could have great potential in the setting of adoptive cellular therapy (ACT). Adult peripheral blood-derived PR1-CTLs are infrequent but preferentially lyse myeloid leukemia cells. We sought to examine PR1-CTLs in umbilical cord blood (UCB) because UCB units provide a rapidly available cell source and a lower risk of graft-versus-host disease, even in the setting of mismatched human leukocyte antigen (HLA) loci., Methods: We first determined the frequency of PR1-CTLs in HLA-A2(+) UCB units and then successfully expanded them ex vivo using repeated stimulation with PR1 peptide-pulsed antigen-presenting cells (APCs). After expansion, we assessed the PR1-CTL phenotype (naive, effector, memory) and function against PR1-expressing target cells., Results: PR1-CTLs are detected at an average frequency of 0.14% within the CD8(+) population of fresh UCB units, which is 45 times higher than in healthy adult peripheral blood. UCB PR1-CTLs are phenotypically naive, consistent with the UCB CD8(+) population as a whole. In addition, the cells can be expanded by stimulation with PR1 peptide-pulsed APCs. Expansion results in an increased frequency of PR1-CTLs, up to 4.56%, with an average 20-fold increase in total number. After expansion, UCB PR1-CTLs express markers consistent with effector memory T cells. Expanded UCB PR1-CTLs are functional in vitro as they are able to produce cytokines and lyse PR1-expressing leukemia cell lines., Conclusions: This study is the first report to show that T cells specific for a leukemia-associated antigen are found at a significantly higher frequency in UCB than adult blood. Our results also demonstrate specific cytotoxicity of expanded UCB-derived PR1-CTLs against PR1-expressing targets. Together, our data suggest that UCB PR1-CTLs could be useful to prevent or treat leukemia relapse in myeloid leukemia patients., (Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Viral infections are a leading fatal complication for patients with primary immunodeficiencies (PIDs) who require hematopoietic stem cell transplantation (HSCT). Use of virus-specific T lymphocytes (VSTs) has been successful for the treatment and prevention of viral infections after HSCT for malignant and nonmalignant conditions. Here we describe the clinical use of VSTs in patients with PIDs at 4 centers., Objective: We sought to evaluate the safety and efficacy of VSTs for treatment of viral infections in patients with PIDs., Methods: Patients with PIDs who have received VST therapy on previous or current protocols were reviewed in aggregate. Clinical information, including transplantation details, viral infections, and use of antiviral and immunosuppressive pharmacotherapy, were evaluated. Data regarding VST production, infusions, and adverse reactions were compared., Results: Thirty-six patients with 12 classes of PID diagnoses received 37 VST products before or after HSCT. Twenty-six (72%) patients had received a diagnosis of infection with cytomegalovirus, EBV, adenovirus, BK virus, and/or human herpesvirus 6. Two patients were treated before HSCT because of EBV-associated lymphoproliferative disease. Partial or complete responses against targeted viruses occurred in 81% of patients overall. Time to response varied from 2 weeks to 3 months (median, 28 days). Overall survival at 6 months after therapy was 80%. Four patients had graft-versus-host disease in the 45 days after VST infusion, which in most cases was therapy responsive., Conclusion: VSTs derived from either stem cell donors or third-party donors are likely safe and effective for the treatment of viral infections in patients with PIDs., (Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Cord blood transplantation, an alternative to traditional stem cell transplants (bone marrow or peripheral blood stem cell transplantation), is an attractive option for patients lacking suitable stem cell transplant donors. Cord blood units have also proven to be a valuable donor source for the development of cellular therapeutics. Virus-specific T cells and regulatory T cells are two cord blood-derived products that have shown promise in early-phase clinical trials to prevent and/or treat viral infections and graft-versus-host disease, respectively. We describe how current strategies that use cord blood-derived regulatory T cells and virus-specific T cells have been developed to improve outcomes for cord blood transplant recipients., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

A 12-year-old boy with refractory acute lymphoblastic leukemia received a haploidentical transplant from his mother. As prophylaxis for Epstein-Barr virus (EBV), cytomegalovirus (CMV) and adenovirus, he received ex vivo expanded virus-specific donor T cells 3.5 months after transplant. Four weeks later leukemic blasts bearing the E2A deletion, identified by fluorescent in situ hybridization (FISH), appeared transiently in the blood followed by a FISH-negative hematological remission, which was sustained until a testicular relapse 3.5 months later. Clearance of the circulating leukemic cells coincided with a marked increase in circulating virus-specific T cells. The virus-specific cytotoxic T-cell (CTL) line showed strong polyfunctional reactivity with the patient's leukemic cells but not phytohemagglutinin (PHA) blasts, suggesting that virus-specific CTL lines may have clinically significant antileukemia activity.

Purpose: Tumor cells from approximately 40% of patients with Hodgkin or non-Hodgkin lymphoma express the type II latency Epstein-Barr virus (EBV) antigens latent membrane protein 1 (LMP1) and LMP2, which represent attractive targets for immunotherapy. Because T cells specific for these antigens are present with low frequency and may be rendered anergic by the tumors that express them, we expanded LMP-cytotoxic T lymphocytes (CTLs) from patients with lymphoma using autologous dendritic cells and EBV-transformed B-lymphoblastoid cell lines transduced with an adenoviral vector expressing either LMP2 alone (n = 17) or both LMP2 and ΔLMP1 (n = 33)., Patients and Methods: These genetically modified antigen-presenting cells expanded CTLs that were enriched for specificity against type II latency LMP antigens. When infused into 50 patients with EBV-associated lymphoma, the expanded CTLs did not produce infusional toxicities., Results: Twenty-eight of 29 high-risk or multiple-relapse patients receiving LMP-CTLs as adjuvant therapy remained in remission at a median of 3.1 years after CTL infusion. None subsequently died as a result of lymphoma, but nine succumbed to complications associated with extensive prior chemoradiotherapy, including myocardial infarction and secondary malignancies. Of 21 patients with relapsed or resistant disease at the time of CTL infusion, 13 had clinical responses, including 11 complete responses. T cells specific for LMP as well as nonviral tumor-associated antigens (epitope spreading) could be detected in the peripheral blood within 2 months after CTL infusion, but this evidence for epitope spreading was seen only in patients achieving clinical responses., Conclusion: Autologous T cells directed to the LMP2 or LMP1 and LMP2 antigens can induce durable complete responses without significant toxicity. Their earlier use in the disease course may reduce delayed treatment-related mortality.

Adoptive immunotherapy-in particular, T-cell therapy-has recently emerged as a useful strategy with the potential to overcome many of the limitations of antiviral drugs for the treatment of viral complications after hematopietic stem cell transplantation. In this review, we briefly summarize the current methods for virus-specific T-cell isolation or selection and we report results from clinical trials that have used these techniques, focusing specifically on the strategies aimed to broaden the application of this technology., (Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Chronic granulomatous disease (CGD) patients are highly susceptible to invasive aspergillosis and might benefit from aspergillus-specific T cell immunotherapy, which has shown promise in treating those with known T cell defects such as haematopoietic stem cell transplant (HSCT) recipients. But whether such T cell defects contribute to increased risks for aspergillus infection in CGD is unclear. Hence, we set out to characterize the aspergillus-specific T cell response in CGD. In murine CGD models and in patients with CGD we showed that the CD4(+) T cell responses to aspergillus were unimpaired: aspergillus-specific T cell frequencies were even elevated in CGD mice (P < 0·01) and humans (P = 0·02), compared to their healthy counterparts. CD4-depleted murine models suggested that the role of T cells might be redundant because resistance to aspergillus infection was conserved in CD4(+) T cell-depleted mice, similar to wild-type animals. In contrast, mice depleted of neutrophils alone or neutrophils and CD4(+) T cells developed clinical and pathological evidence of pulmonary aspergillosis and increased mortality (P < 0·05 compared to non-depleted animals). Our findings that T cells in CGD have a robust aspergillus CD4(+) T cell response suggest that CD4(+) T cell-based immunotherapy for this disease is unlikely to be beneficial., (© 2013 British Society for Immunology.)

Post-transplant lymphoproliferative diseases (PTLD) associated with Epstein-Barr virus (EBV) infection often develop after organ and haematopoietic stem-cell transplantation. These lymphoproliferative diseases are tumours that usually express all latent EBV viral proteins, and are therefore amenable to T-cell-based immune therapies, such as donor lymphocyte infusions and the adoptive transfer of EBV-specific cytotoxic T lymphocytes. In this Review, we describe current approaches of T-cell-based therapies to treat PTLD, and describe strategies that improve the feasibility of such treatment.

Glioblastoma (GBM) is the most common primary brain cancer in adults and is virtually incurable. Recent studies have shown that cytomegalovirus (CMV) is present in majority of GBMs. To evaluate whether the CMV antigens pp65 and IE1, which are expressed in GBMs, could be targeted by CMV-specific T cells, we measured the frequency of T cells targeting pp65 and IE1 in the peripheral blood of a cohort of 11 sequentially diagnosed CMV-seropositive GBM patients, and evaluated whether it was feasible to expand autologous CMV-specific T cells for future clinical studies. All 11 CMV-seropositive GBM patients had T cells specific for pp65 and IE1 in their peripheral blood assessed by IFNγ enzyme-linked immunospot assay. However, the precursor frequency of pp65-specific T cells was decreased in comparison with healthy donors (P=0.001). We successfully reactivated and expanded CMV-specific T cells from 6 out of 6 GBM patients using antigen-presenting cells transduced with an adenoviral vector encoding pp65 and IE1. CMV-specific T-cell lines contained CD4 as well as CD8 T cells, recognized pp65 and IE1 targets and killed CMV-infected autologous GBM cells. Infusion of such CMV-specific T-cell lines may extend the benefits of T-cell therapy to patients with CMV GBMs.

Purpose: Patients with Hodgkin lymphoma (HL) relapsing after hematopoietic stem cell transplant have limited options for long-term cure. We have shown that infused cytotoxic T cells (CTL) targeting Epstein Barr virus (EBV)-derived proteins induced complete remissions in EBV(+) HL patients. A limitation of this approach is that up to 70% of relapsed HL tumors are EBV-negative. For these patients, an alternative is to target the cancer/testis antigen MAGE-A4 present in EBV antigen-negative HL tumors. Furthermore, epigenetic modification by clinically available demethylating agents can enhance MAGE-A4 expression in previously MAGE-negative tumors., Experimental Design: We explored the feasibility of combining adoptive T cell therapy with epigenetic modification of tumor antigen expression. We further characterized MAGE-A4-specific T-cell phenotype and function, and examined the effects of the epigenetic modifying drug decitabine on these T cells., Results: Cytotoxic T cells were generated specifically recognizing MAGE-A4 expressed by autologous HL targets and tumor cell lines. Decitabine-previously shown to increase tumor antigen expression in HL-did not compromise MAGE-A4-specific T-cell phenotype and function. In patients treated with decitabine, expanded MAGE-A4-specific T cells had a broader antitumor T cell repertoire, consistent with increased antigen stimulation in vivo., Conclusions: Adoptive transfer of MAGE-A4-specific T cells, combined with epigenetic modifying drugs to increase expression of the protein, may improve treatment of relapsed HL.

Patients with recurrent or refractory Epstein Barr Virus (EBV)-positive nasopharyngeal carcinoma (NPC) continue to have poor outcomes. Our earlier Phase I dose escalation clinical study of 10 NPC patients showed that infusion of EBV-specific cytotoxic T cells (EBV-CTLs) was safe and had antitumor activity. To better define the overall response rate and discover whether disease status, EBV-antigen specificity, and/or in vivo expansion of infused EBV-CTLs predicted outcome, we treated 13 additional NPC patients with EBV-CTLs in a fixed-dose, Phase II component of the study. We assessed toxicity, efficacy, specificity, and expansion of infused CTLs for all 23 recurrent/refractory NPC patients treated on this Phase I/II clinical study. At the time of CTL infusion, 8 relapsed NPC patients were in remission and 15 had active disease. No significant toxicity was observed. Of the relapsed patients treated in their second or subsequent remission, 62% (5/8) remain disease free (at 17 to 75 mo), whereas 48.7% (7/15) of those with active disease had a CR/CRu (33.3%) or PR (15.4%). In contrast to locoregional disease, metastatic disease was associated with an increased risk of disease progression (HR: 3.91, P=0.015) and decreased overall survival (HR: 5.55, P=0.022). Neither the specificity of the infused CTLs for particular EBV antigens nor their measurable in vivo expansion discernibly influenced outcome. In conclusion, treatment of patients with relapsed/refractory EBV-positive NPC with EBV-CTLs is safe and can be associated with significant, long-term clinical benefit, particularly for patients with locoregional disease.

Background Aims: The Food and Drug Administration (FDA) currently recommends at least 4 h of recipient monitoring after T cell infusions to detect early infusion reactions. Recent catastrophic reactions to 'first-in-man' biologic agents have emphasized the importance of this rule for initial studies of new products. The value of such monitoring for better established agents is less obvious., Methods: We reviewed infusion-related adverse events (AE) following administration of ex vivo-expanded T cell products (antigen-specific cytotoxic T lymphocytes, allodepleted T cells, and genetically modified T cells) on investigational new drug (IND) studies in our center., Results: From 1998 to 2008, we infused 381 T cell products to 180 recipients, enrolled on 18 studies, receiving T cells targeting malignancies or post-transplant viral infections. There were no grade 3-4 infusion reactions during initial monitoring or 24-h follow-up. Twenty-four mild (grade 1-2) AE occurred in 21 infusions either during or immediately following infusion (up to 6 h), most commonly nausea and vomiting (10/24, 41.6%), probably because of the dimethyl sulfoxide cryoprotectant, and hypotension (20.8%), attributable to diphenhydramine pre-medication. Twenty-two additional non-severe events were reported within 24 h of infusion, most commonly culture-negative fever, chills and nausea. An increased risk of adverse events was associated with age [incidence rate ratio (IRR) 0.98; 95% confidence interval (CI) 0.96-1.00, P = 0.05], while an increased risk of immediate infusion-related events was higher in patients reporting allergies (IRR 2.72, 95% CI 1.00-7.40, P = 0.05); sex, disease type and T cell source (allogeneic or autologous) had no effect on frequency of adverse events., Conclusions: Infusion of these T cell products was safe in the outpatient setting and associated with no severe reactions, so monitoring for 1 h after infusion is probably sufficient. As many of the AE were attributable to diphenhydramine premedication, a lower dose (0.25 mg/kg) should be selected.

Because of the necessary immunodepletion prior to cord blood transplantation as well as the immaturity of cord blood immune cells, recipients experience a high incidence of viral infection in addition to complications observed after hematopoietic stem cell transplantation, such as relapse and graft-versus-host disease. We describe current immunotherapeutic approaches to treating these complications, including the generation of antigen-specific T cells from cord blood, redirecting cord blood T cells using chimeric antigen receptors, and generating cord blood-derived natural killer cells and regulatory T cells.

Peripheral blood natural killer (NK) cell therapy for acute myeloid leukemia has shown promise in clinical trials after allogeneic stem cell transplantation. Cord blood (CB) is another potentially rich source of NK cells for adoptive immune therapy after stem cell transplantation. Tightly regulated receptor signaling between NK cells and susceptible tumor cells is essential for NK cell-mediated cytotoxicity. However, despite expressing normal surface activating and inhibitory NK receptors, CB-derived NK cells have poor cytolytic activity. In this study, we investigate the cellular mechanism and demonstrate that unmanipulated CB-NK cells exhibit an impaired ability to form F-actin immunologic synapses with target leukemia cells compared with peripheral blood-derived NK cells. In addition, there was reduced recruitment of the activating receptor CD2, integrin leukocyte function-associated antigen-1, and the cytolytic molecule perforin to the CB-NK synapse site. Exvivo interleukin (IL)-2 expansion of CB-NK cells enhanced lytic synapse formation including CD2 and leukocyte function-associated antigen-1 polarization and activity. Furthermore, the acquired antileukemic function of IL-2-expanded CB-NK cells was validated using a nonobese diabetic severe combined immunodeficient IL-2 receptor common γ-chain null mouse model. We believe our results provide important mechanistic insights for the potential use of IL-2-expanded CB-derived NK cells for adoptive immune therapy in leukemia.

Treatment of Epstein-Barr virus (EBV)-positive nasopharyngeal carcinoma (NPC) with EBV-specific cytotoxic T cells (EBV-specific CTL) has been promising, producing clinical responses. However, infused EBV-specific CTL did not expand in vivo, likely limiting their antitumor activity. Lymphodepleting patients with chemotherapy before T-cell transfer enhances in vivo T-cell expansion, but results in nonspecific destruction of the resident immune system and can have significant toxicity. To evaluate if monoclonal antibodies (mAbs) can produce a more selective lymphodepletion, we conducted a clinical study in which NPC patients received a pair of lymphodepleting mAbs targeted to the CD45 antigen (CD45 mAbs) before EBV-specific CTL infusion. Eight patients with recurrent NPC received CD45 mAbs followed by escalating doses of autologous EBV-specific CTL. Infusion of CD45 mAbs resulted in transient lymphopenia in all patients and an increase in interleukin-15 (IL-15) levels in 6 out 8 patients. All patients had an increase in their peripheral blood frequency of EBV-specific T cells after CTL infusion. Three patients with a persistent increase had clinical benefits including 1 complete response (> 24 months) and 2 with stable disease (for 12 and 15 months). Lymphodepleting mAbs prior CTL transfer may represent an alternative to chemotherapy to enhance expansion of infused CTL. This study is registered at (http://www.clinialtrials.gov) as NCT00608257.

Epstein-Barr virus (EBV)-associated tumors developing in immunocompetent individuals present a challenge to immunotherapy, since they lack expression of immunodominant viral antigens. However, the tumors consistently express viral proteins including LMP2, which are immunologically "weak" but may nonetheless be targets for immune T cells. We previously showed that a majority of cytotoxic T lymphocytes (CTLs) reactivated using EBV-transformed B-lymphoblastoid cells lines (LCLs) contained minor populations of LMP2-specific T cells and homed to tumor sites. However, they did not produce remissions in patients with bulky disease. We have now used gene transfer into antigen-presenting cells (APCs) to augment the expression and immunogenicity of LMP2. These modified APCs increased the frequency of LMP2-specific CTLs by up to 100-fold compared with unmodified LCL-APCs. The LMP2-specific population expanded and persisted in vivo without adverse effects. Nine of 10 patients treated in remission of high-risk disease remain in remission, and 5 of 6 patients with active relapsed disease had a tumor response, which was complete in 4 and sustained for more than 9 months. It is therefore possible to generate immune responses to weak tumor antigens by ex vivo genetic modification of APCs and the CTLs so produced can have substantial antitumor activity. This study is registered at http://www.cancer.gov/clinicaltrials (protocol IDs: BCM-H-9936, NCT00062868, NCT00070226).

Adoptive transfer of tumor-specific T-cells is an attractive strategy for the treatment of patients with refractory or relapsed Hodgkin's lymphoma. However, Hodgkin's lymphomas possess a range of tumor-evasion mechanisms, which must be overcome before the full potential of immunotherapies can be achieved. In this article, we discuss the promise of Epstein-Barr virus-specific cytotoxic T-lymphocytes, the roles of cytokines, and other strategies for overcoming the immune-evasion mechanisms in Hodgkin's lymphoma.

Adoptive T-cell therapy has definite clinical benefit in relapsed leukaemia after allogeneic transplant and in Epstein-Barr virus-associated post-transplant lymphoproliferative disease. However, the majority of tumour targets are weakly immunogenic self-antigens and success has been limited in part by inadequate persistence and expansion of transferred T cells and by tumour-evasion strategies. Adoptive immunotherapy presents the opportunity to activate, expand and genetically modify T cells outside the tolerising environment of the host and a number of strategies to optimize the cellular product, including gene modification and modulation of the host environment, in particular by lymphodepletion, have been developed.

Adenoviruses are lytic DNA viruses that are ubiquitous in human communities. In total, 51 different serotypes with varying tissue tropisms have been identified. Adenovirus infections, although frequent, are rarely fatal in immunocompetent individuals who have potent innate and adaptive immunity. But in immunosuppressed individuals, adenoviruses are a significant cause of morbidity and mortality, with limited treatment options. In particular, pediatric recipients of allogeneic hematopoietic stem cell transplantation frequently develop infections early in the posttransplantation period. Because the endogenous recovery of adenovirus-specific T cells has proven important in controlling infection, we explore the potential of adoptive T-cell immunotherapy as a therapeutic strategy. We discuss the advantages and limitations of T-cell therapy for the prophylaxis and treatment of adenovirus infection posttransplantation.

T cell therapies are increasingly used for the treatment of malignancies and viral-associated diseases. Initial studies focused on the use of unmanipulated T cell populations after allogeneic stem cell transplantation. More recently, the use of antigen-specific T cells has been explored. This chapter reviews the clinical experience with polyclonal Epstein-Barr virus (EBV)-specific cytotoxic T cells (CTL) for the treatment of EBV-associated malignancies. Strategies on how to improve the antitumor activity of EBV-specific CTL are being discussed. If effective, these strategies will have broad implications for T cell therapies for a range of human tumors with defined antigens.

Conventional treatment for nasopharyngeal carcinoma (NPC) frequently fails and is accompanied by severe long-term side effects. Since virtually all undifferentiated NPCs are associated with Epstein-Barr virus (EBV), this tumor is an attractive candidate for cellular immunotherapy targeted against tumor-associated viral antigens. We now demonstrate that EBV-specific cytotoxic T-cell (CTL) lines can readily be generated from individuals with NPC, notwithstanding the patients' prior exposure to chemotherapy/radiation. A total of 10 patients diagnosed with advanced NPC were treated with autologous CTLs. All patients tolerated the CTLs, although one developed increased swelling at the site of pre-existing disease. At 19 to 27 months after infusion, 4 patients treated in remission from locally advanced disease remain disease free. Of 6 patients with refractory disease prior to treatment, 2 had complete responses, and remain in remission over 11 to 23 months after treatment; 1 had a partial remission that persisted for 12 months; 1 has had stable disease for more than 14 months; and 2 had no response. These results demonstrate that administration of EBV-specific CTLs to patients with advanced NPC is feasible, appears to be safe, and can be associated with significant antitumor activity.

Epstein Barr virus (EBV)+ Hodgkin's disease (HD) expresses clearly identified tumor antigens derived from the virus and could, in principle, be a target for adoptive immunotherapy with viral antigen-specific T cells. However, like most tumor-associated antigens in immunocompetent hosts, these potential targets are only weakly immunogenic, consisting primarily of the latent membrane protein (LMP)1 and LMP2 antigens. Moreover, Hodgkin tumors possess a range of tumor evasion strategies. Therefore, the likely value of immunotherapy with EBV-specific cytotoxic effector cells has been questioned. We have now used a combination of gene marking, tetramer, and functional analyses to track the fate and assess the activity of EBV cytotoxic T lymphocyte (CTL) lines administered to 14 patients treated for relapsed EBV+ HD. Gene marking studies showed that infused effector cells could further expand by several logs in vivo, contribute to the memory pool (persisting up to 12 mo), and traffic to tumor sites. Tetramer and functional analyses showed that T cells reactive with the tumor-associated antigen LMP2 were present in the infused lines, expanded in peripheral blood after infusion, and also entered tumor. Viral load decreased, demonstrating the biologic activity of the infused CTLs. Clinically, EBV CTLs were well tolerated, could control type B symptoms (fever, night sweats, and weight loss), and had antitumor activity. After CTL infusion, five patients were in complete remission at up to 40 mo, two of whom had clearly measurable tumor at the time of treatment. One additional patient had a partial response, and five had stable disease. The performance and fate of these human tumor antigen-specific T cells in vivo suggests that they might be of value for the treatment of EBV+ Hodgkin lymphoma.

Viral diseases are a major cause of morbidity and mortality after hemopoietic stem cell transplantation. Because viral complications in these patients are clearly associated with the lack of recovery of virus-specific cellular immune responses, reconstitution of the host with in vitro expanded cytotoxic T lymphocytes is a potential approach to prevent and treat these diseases. Initial clinical studies of cytomegalovirus and Epstein-Barr virus in human stem cell transplant patients have shown that adoptively transferred donor-derived virus-specific T cells may restore protective immunity and control established infections. Preclinical studies are evaluating this approach for other viruses while strategies for generating T cells specific for multiple viruses to provide broader protection are being evaluated in clinical trials. The use of genetically modified T cells or the use of newer suicide genes may result in improved safety and efficacy.