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BACKGROUNDAdoptive transfer of donor-derived EBV-specific cytotoxic T-lymphocytes (EBV-CTLs) can eradicate EBV-associated lymphomas (EBV-PTLD) after transplantation of hematopoietic cell (HCT) or solid organ (SOT) but is unavailable for most patients.METHODSWe developed a third-party, allogeneic, off-the-shelf bank of 330 GMP-grade EBV-CTL lines from specifically consented healthy HCT donors. We treated 46 recipients of HCT (n = 33) or SOT (n = 13) with established EBV-PTLD, who had failed rituximab therapy, with third-party EBV-CTLs. Treatment cycles consisted of 3 weekly infusions of EBV-CTLs and 3 weeks of observation.RESULTSEBV-CTLs did not induce significant toxicities. One patient developed grade I skin graft-versus-host disease. Complete remission (CR) or sustained partial remission (PR) was achieved in 68% of HCT recipients and 54% of SOT recipients. For patients who achieved CR/PR or stable disease after cycle 1, one year overall survival was 88.9% and 81.8%, respectively. In addition, 3 of 5 recipients with POD after a first cycle who received EBV-CTLs from a different donor achieved CR or durable PR (60%) and survived longer than 1 year. Maximal responses were achieved after a median of 2 cycles.CONCLUSIONThird-party EBV-CTLs of defined HLA restriction provide safe, immediately accessible treatment for EBV-PTLD. Secondary treatment with EBV-CTLs restricted by a different HLA allele (switch therapy) can also induce remissions if initial EBV-CTLs are ineffective. These results suggest a promising potential therapy for patients with rituximab-refractory EBV-associated lymphoma after transplantation.TRIAL REGISTRATIONPhase II protocols (NCT01498484 and NCT00002663) were approved by the Institutional Review Board at Memorial Sloan Kettering Cancer Center, the FDA, and the National Marrow Donor Program.FUNDINGThis work was supported by NIH grants CA23766 and R21CA162002, the Aubrey Fund, the Claire Tow Foundation, the Major Family Foundation, the Max Cure Foundation, the Richard "Rick" J. Eisemann Pediatric Research Fund, the Banbury Foundation, the Edith Robertson Foundation, and the Larry Smead Foundation. Atara Biotherapeutics licensed the bank of third-party EBV-CTLs from Memorial Sloan Kettering Cancer Center in June 2015.

Chimeric antigen receptor (CAR) T cells have demonstrated clinical benefit in patients with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). We undertook a multicenter clinical trial to determine toxicity, feasibility, and response for this therapy. A total of 25 pediatric/young adult patients (age, 1-22.5 years) with R/R B-ALL were treated with 19-28z CAR T cells. Conditioning chemotherapy included high-dose (3 g/m2) cyclophosphamide (HD-Cy) for 17 patients and low-dose (≤1.5 g/m2) cyclophosphamide (LD-Cy) for 8 patients. Fifteen patients had pretreatment minimal residual disease (MRD; <5% blasts in bone marrow), and 10 patients had pretreatment morphologic evidence of disease (≥5% blasts in bone marrow). All toxicities were reversible, including severe cytokine release syndrome in 16% (4 of 25) and severe neurotoxicity in 28% (7 of 25) of patients. Treated patients were assessed for response, and, among the evaluable patients (n = 24), response and peak CAR T-cell expansion were superior in the HD-Cy/MRD cohorts, as compared with the LD-Cy/morphologic cohorts without an increase in toxicity. Our data support the safety of CD19-specific CAR T-cell therapy for R/R B-ALL. Our data also suggest that dose intensity of conditioning chemotherapy and minimal pretreatment disease burden have a positive impact on response without a negative effect on toxicity. This trial was registered at www.clinicaltrials.gov as #NCT01860937., (© 2019 by The American Society of Hematology.)

We have developed banks of EBV and CMV-specific T-cell lines generated from healthy seropositive third party donors and characterized them as to their HLA type, virus specificity, lack of alloreactivity, and HLA restriction. We here summarize results of studies employing these immediately accessible, broadly-applicable third party virus-specific T-cells for adoptive therapy of EBV lymphomas and CMV infections in allo-HCT recipients. We describe the characteristics contributing to their safety. We also discuss several distinctive advantages of banked third party virus-specific T-cells selected on the basis of their HLA restriction, particularly in the treatment of Rituximab-non-responsive EBV + lymphomas and drug refractory CMV infections complicating HLA non-identical transplants.

Infection, relapse, and GVHD can complicate allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although the effect of poor immune recovery on infection risk is well-established, there are limited data on the effect of immune reconstitution on relapse and survival, especially following T-cell depletion (TCD). To characterize the pattern of immune reconstitution in the first year after transplant and its effects on survival and relapse, we performed a retrospective study in 375 recipients of a myeloablative TCD allo-HSCT for hematologic malignancies. We noted that different subsets recover sequentially, CD8 + T cells first, followed by total CD4 + and naïve CD4 + T cells, indicating thymic recovery during the first year after HSCT. In the multivariate model, a fully HLA-matched donor and recovery of T-cell function, assessed by PHA response at 6 months, were the only factors independently associated with OS and EFS. In conclusion, T-cell recovery is an important predictor of outcome after TCD allo-HSCT.

Background: Even though hematopoietic stem cell transplantation can be curative in patients with severe combined immunodeficiency, there is a need for additional strategies boosting T cell immunity in individuals suffering from genetic disorders of lymphoid development. Here we show that image-guided intrathymic injection of hematopoietic stem and progenitor cells in NOD-scid IL2rγ null mice is feasible and facilitates the generation of functional T cells conferring protective immunity., Methods: Hematopoietic stem and progenitor cells were isolated from the bone marrow of healthy C57BL/6 mice (wild-type, Luciferase + , CD45.1 + ) and injected intravenously or intrathymically into both male and female, young or aged NOD-scid IL2rγ null recipients. The in vivo fate of injected cells was analyzed by bioluminescence imaging and flow cytometry of thymus- and spleen-derived T cell populations. In addition to T cell reconstitution, we evaluated mice for evidence of immune dysregulation based on diabetes development and graft-versus-host disease. T cell immunity following intrathymic injection of hematopoietic stem and progenitor cells in NOD-scid IL2rγ null mice was assessed in a B cell lymphoma model., Results: Despite the small size of the thymic remnant in NOD-scid IL2rγ null mice, we were able to accomplish precise intrathymic delivery of hematopoietic stem and progenitor cells by ultrasound-guided injection. Thymic reconstitution following intrathymic injection of healthy allogeneic hematopoietic cells was most effective in young male recipients, indicating that even in the setting of severe immunodeficiency, sex and age are important variables for thymic function. Allogeneic T cells generated in intrathymically injected NOD-scid IL2rγ null mice displayed anti-lymphoma activity in vivo, but we found no evidence for severe auto/alloreactivity in T cell-producing NOD-scid IL2rγ null mice, suggesting that immune dysregulation is not a major concern., Conclusions: Our findings suggest that intrathymic injection of donor hematopoietic stem and progenitor cells is a safe and effective strategy to establish protective T cell immunity in a mouse model of severe combined immunodeficiency.

Intracellular tumor antigens presented on the cell surface in the context of human leukocyte antigen (HLA) molecules have been targeted by T cell-based therapies, but there has been little progress in developing small-molecule drugs or antibodies directed to these antigens. Here we describe a bispecific T-cell engager (BiTE) antibody derived from a T-cell receptor (TCR)-mimic monoclonal antibody (mAb) ESK1, which binds a peptide derived from the intracellular oncoprotein WT1 presented on HLA-A*02:01. Despite the very low density of the complexes at the cell surface, ESK1-BiTE selectively activated and induced proliferation of cytolytic human T cells that killed cells from multiple leukemias and solid tumors in vitro and in mice. We also discovered that in an autologous in vitro setting, ESK1-BiTE induced a robust secondary CD8 T-cell response specific for tumor-associated antigens other than WT1. Our study provides an approach that targets tumor-specific intracellular antigens without using cell therapy and suggests that epitope spreading could contribute to the therapeutic efficacy of this BiTE.

We conducted a phase I trial of allogeneic T cells sensitized in vitro against a pool of pentadecapeptides (15-mer peptides) spanning the sequence of CMVpp65 for adoptive therapy of 17 allogeneic hematopoietic cell transplant recipients with cytomegalovirus (CMV) viremia or clinical infection persisting despite prolonged treatment with antiviral drugs. All but 3 of the patients had received T cell-depleted transplants without graft-versus-host disease (GVHD) prophylaxis with immunosuppressive drugs after transplantation. The CMVpp65-specific T cells (CMVpp65CTLs) generated were oligoclonal and specific for only 1 to 3 epitopes, presented by a limited set of HLA class I or II alleles. T cell infusions were well tolerated without toxicity or GVHD. Of 17 patients treated with transplant donor (n = 16) or third-party (n = 1) CMVpp65CTLs, 15 cleared viremia, including 3 of 5 with overt disease. In responding patients, the CMVpp65CTLs infused consistently proliferated and could be detected by T cell receptor Vβ usage in CMVpp65/HLA tetramer + populations for period of 120 days to up to 2 years after infusion. Thus, CMVpp65CTLs generated in response to synthetic 15-mer peptides of CMVpp65 are safe and can clear persistent CMV infections in the post-transplantation period., (Copyright © 2015 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Preventing unfavorable GVHD without inducing broad suppression of the immune system presents a major challenge of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We developed a novel strategy to ameliorate GVHD while preserving graft-versus-tumor (GVT) activity by small molecule-based inhibition of the NF-κB family member c-Rel. Underlying mechanisms included reduced alloactivation, defective gut homing, and impaired negative feedback on interleukin (IL)-2 production, resulting in optimal IL-2 levels, which, in the absence of competition by effector T cells, translated into expansion of regulatory T cells. c-Rel activity was dispensable for antigen-specific T-cell receptor (TCR) activation, allowing c-Rel-deficient T cells to display normal GVT activity. In addition, inhibition of c-Rel activity reduced alloactivation without compromising antigen-specific cytotoxicity of human T cells. Finally, we were able to demonstrate the feasibility and efficacy of systemic c-Rel inhibitor administration. Our findings validate c-Rel as a promising target for immunomodulatory therapy and demonstrate the feasibility and efficacy of pharmaceutical inhibition of c-Rel activity.

This study was conducted to retrospectively compare the clinical outcomes after transplantation of T cell-depleted (TCD) and unmodified allografts in patients with acute myeloid leukemia (AML) in first complete remission (CR1). Patients received TCD grafts at Memorial Sloan-Kettering Cancer Center (MSKCC, N = 115) between 2001 and 2010 using the following preparative regimens: hyperfractionated total body irradiation (HFTBI)+thiotepa+fludarabine; HFTBI+thiotepa+cyclophosphamide; or i.v. busulfan+melphalan+fludarabine. TCD was performed by 1 of 2 immunomagnetic CD34(+) cell selection methods for peripheral blood grafts or by soybean lectin agglutination followed by sheep red blood cell-rosette depletion for bone marrow grafts. No additional graft-versus-host disease (GVHD) prophylaxis was administered. Patients received unmodified grafts at M.D. Anderson Cancer Center (MDACC, N = 181) after conditioning with busulfan+fludarabine and GVHD prophylaxis with tacrolimus+mini-methotrexate. Patients with unrelated or human leukocyte antigen-mismatched donors received anti-thymocyte globulin (ATG) at both centers, with some recipients of matched related donor TCD transplants also receiving ATG, depending upon the preparative regimen. TCD graft recipients were more likely to be older, receive a mismatched transplant, and have peripheral blood used as the graft source. The incidences rates of grades 2 to 4 acute GVHD and chronic GVHD were significantly lower in the TCD graft group (5% versus 18%, and 13% versus 53%). Three-year relapse-free and overall survival rates were 58% and 57%, respectively, in recipients of TCD grafts, and 60% and 66% in recipients of unmodified grafts (P = not significant). Survival and relapse-free survival are similar after TCD and conventional transplants from related/unrelated donors in patients with AML in CR1, but TCD significantly reduces GVHD., (Copyright © 2013 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

The Wilms tumor protein (WT-1) is widely recognized as a tumor antigen that is expressed differentially by several malignancies. However, WT-1 peptides known to induce tumoricidal T cells are few. In the present study, we evaluated T-cell responses of 56 healthy donors to in vitro sensitization with autologous APCs loaded with a pool of overlapping 15-mer peptides spanning the sequence of WT-1. Thereafter, we mapped the WT-1 peptides eliciting responses in each individual, defined the immunogenic peptides, and identified their presenting HLA alleles. We report 41 previously unreported epitopes of WT-1: 5 presented by class II and 36 by class I alleles, including 10 that could be presented by more than 1 class I allele. IFNγ(+) T cells responding to 98% of the class I and 60% of the class II epitopes exhibited HLA-restricted cytotoxicity against peptide-loaded targets. T cells specific for 36 WT-1 peptides were evaluable for leukemocidal activity, of which 27 (75%) lysed WT-1(+) leukemic targets sharing their restricting HLA allele. Each epitope identified induced T-cell responses in most donors sharing the epitopes' presenting allele; these responses often exceeded responses to flanking peptides predicted to be more immunogenic. This series of immunogenic epitopes of WT-1 should prove useful for immunotherapies targeting WT-1(+) malignancies.

Eight centers participated in the Blood and Marrow Transplant Clinical Trials Network (BMT CTN) protocol 0303 to determine the effect of extensive T cell depletion (TCD) on the outcome of HLA matched sibling donor transplantation for acute myeloid leukemia. One goal of the study was to determine if TCD could be performed uniformly among study sites. TCD was achieved using the CliniMACS(®) CD34 Reagent System for CD34 enrichment. Processed grafts needed to contain ≥ 2.0 × 10(6) CD34(+)cells/kg with a target of 5.0 × 10(6) CD34(+) cells/kg and <10(5) CD3(+) T cells/kg. Up to 3 collections were allowed to achieve the minimum CD34(+) cell dose. In total, 86 products were processed for 44 patients. Differences in the starting cell products between centers were seen in regard to total nucleated cells, CD34(+) cells, and CD3(+) T cells, which could in part be ascribed to a higher dose of granulocyte-colony stimulating factor used for mobilization early in the trial. Differences between centers in processing outcomes were minimal and could be ascribed to starting cell parameters or to differences in graft analysis methods. Multivariate analysis showed that CD34(+) cell recovery (66.1% ± 20.3%) was inversely associated with the starting number of CD34(+) cells (P = .02). Median purity of the CD34 enriched fraction was 96.7% (61.5%-99.8%) with monocytes and B cells the most common impurity. All patients received the minimum CD34(+) cell dose, and 39 patients (89%) came within 10% or exceeded the target CD34(+) cell dose without exceeding the maximum T cell dose. All patients proceeded to transplantation and all achieved initial engraftment. Products processed at multiple centers using the CliniMACS System for CD34 enrichment were comparably and uniformly highly enriched for CD34(+) cells, with good CD34(+) cell recovery and very low CD3(+) T cell content., (Copyright © 2012 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

We evaluated HLA-compatible donor leukocyte infusions (DLIs) and HLA-compatible or HLA-disparate EBV-specific T cells (EBV-CTLs) in 49 hematopoietic cell transplantation recipients with biopsy-proven EBV-lymphoproliferative disease (EBV-LPD). DLIs and EBV-CTLs each induced durable complete or partial remissions in 73% and 68% of treated patients including 74% and 72% of patients surviving ≥ 8 days after infusion, respectively. Reversible acute GVHD occurred in recipients of DLIs (17%) but not EBV-CTLs. The probability of complete response was significantly lower among patients with multiorgan involvement. In responders, DLIs and EBV-CTLs regularly induced exponential increases in EBV-specific CTL precursor (EBV-CTLp) frequencies within 7-14 days, with subsequent clearance of EBV viremia and resolution of disease. In nonresponders, EBV-CTLps did not increase and EBV viremia persisted. Treatment failures were correlated with impaired T-cell recognition of tumor targets. Either donor-derived EBV-CTLs that had been sensitized with autologous BLCLs transformed by EBV strain B95.8 could not lyse spontaneous donor-derived EBV-transformed BLCLs expanded from the patient's blood or biopsied tumor or they failed to lyse their targets because they were selectively restricted by HLA alleles not shared by the EBV-LPD. Therefore, either unselected DLIs or EBV-specific CTLs can eradicate both untreated and Rituxan-resistant lymphomatous EBV-LPD, with failures ascribable to impaired T-cell recognition of tumor-associated viral antigens or their presenting HLA alleles.

Adoptive transfer of in vivo generated antigen-specific donor-derived T-cells is increasingly recognized as an effective approach for the treatment or prevention of EBV lymphomas and cytomegalovirus infections complicating allogeneic hematopoietic cell transplants. This review examines evidence from preclinical experiments and initial clinical trials to critically assess both the potential and current limitations of adoptive transfer of donor T-cells sensitized to selected minor alloantigens of the host or to peptide epitopes of proteins, differentially expressed by clonogenic leukemia cells, such as the Wilms tumor protein, WT-1, as a strategy to treat or prevent recurrence of leukemia in the post-transplant period., (Copyright 2010 Elsevier Ltd. All rights reserved.)

From 1985 to 2004, 49 patients with advanced myelodysplastic syndromes (MDS) (> or =5% blasts) or acute myeloid leukemia (AML) transformed from MDS underwent T cell depleted bone marrow or peripheral blood hematopoietic stem cell transplantation (HSCT) from HLA-identical siblings following conditioning with a myeloablative regimen that included total body irradiation (44 patients) or busulfan (5 patients). Thirty-six patients received chemotherapy (3 low dose and 33 induction doses) before conditioning, and 13 patients did not receive any chemotherapy. Prior to transplantation, 22 of the 36 treated patients were in hematologic remission; 4 were in a second refractory cytopenia phase (26 responders); 8 had failed to achieve remission; and 2 of the responders had progression or relapse of their MDS (10 failures). No post-transplantation pharmacologic prophylaxis for graft-versus-host disease (GVHD) was given. The median age was 48 yrs (range 13-61). Forty-five of the 49 patients engrafted; 2 had primary graft failure; and 2 died before engraftment. Only 3 patients developed acute GVHD (aGVHD) (grades I and III) and 1 chronic GVHD (cGVHD). At 3 yrs post-transplantation, the overall survival (OS) was 54% in the responders; 31% in the untreated group; and 0% in the failure group (P=.0004). The disease free survival (DFS) was 50%, 15% and 0% in each group respectively (P=.0008). In multivariate analysis, disease status before cytoreduction remained highly correlated with DFS (P<.001). The cumulative incidence (CI) of relapse at 2-yrs post-transplantation for the responders was 23%; for the untreated group was 38%; and for the failures was 50%. The CI of non-relapse mortality at 2-yrs post-transplantation, for the responders was 23%; for the untreated group was 38%; and for the failures was 40%. All survivors achieved a Karnofsky Performance Status (KPS) of > or =90. These results indicate that patients with advanced MDS who achieve and remain in remission or a second refractory cytopenia phase with chemotherapy before conditioning can achieve successful long-term remissions following a myeloablative T cell depleted allogeneic HSCT.

Sequential imaging of genetically marked effector cells after adoptive transfer in vivo has greatly enhanced analyses of their biodistribution, growth, and activity both in animal models and in clinical trials of cellular immunotherapy. However, the immunogenicity of cells expressing xenogeneic reporter constructs limits their survival and clinical utility. To address this limitation, we have evaluated a human norepinephrine transporter (hNET) permitting imaging of transduced cells in vivo with a previously approved clinical grade radiolabeled probe, metaiodobenzylguanidine (MIBG). The hNET gene cDNA was cloned from the SK-N-SH cell line and inserted into a bicistronic retroviral vector also encoding green fluorescent protein. Following transfection, human EBV-specific T lymphocytes seemed fully functional in vitro and also selectively accumulated [(123)I]MIBG. In nonobese diabetic/severe combined immunodeficient mice bearing human EBV lymphoma xenografts, as few as 10(4) transduced T cells injected into the tumors could be imaged by single-photon emission computed tomography (SPECT) or positron emission tomography (PET) after i.v. infusion of [(123)I]MIBG or [(124)I]MIBG, respectively. When hNET(+) EBV-specific T cells were infused i.v., their migration and specific accumulation in EBV(+) tumors expressing their restricting HLA allele could be imaged by SPECT or PET over 28 days. Image intensity was closely correlated with the number of T cells accumulated in targeted tumors. The use of two reporter probes (MIBG and 2'-deoxy-2'-fluoro-beta-d-arabinofuranosyl-5-iodouracil) permitted independent contemporaneous tracking of two distinct EBV-specific T-cell subpopulations expressing different reporter genes (hNET-CD4(+) T cells and HSV-TK-CD8(+) T cells) in the same animal using three-dimensional nuclear modalities (SPECT and PET). The hNET-based system described may thus have significant potential as a nonimmunogenic reporter for extended repeated quantitative in vivo imaging of transduced cells in man.

Allogeneic marrow and cytokine-mobilized peripheral blood stem cells adequately depleted of T cells prevent acute and chronic forms of graft versus host disease in HLA-matched and non-identical hosts without any posttransplant immunosuppressive prophylaxis. Current cytoreductive regimens secure consistent durable engraftment, and full donor chimerism. The risk of relapse following such transplants in patients with AML and ALL has been low, and not different from that recorded following unmodified transplants. However, in HLA-disparate hosts the risk of infections caused by EBV, CMV, and certain fungi are increased. To address this limitation, others and we are exploring adoptive immunotherapies with in vitro generated, pathogen-specific T cells. Early clinical trials already indicate the potential of such T cells to treat and prevent life threatening diseases caused by these pathogens, particularly in recipients of T cell depleted grafts who do not require ongoing treatment with immunosuppressive agents, and therefore provide a permissive environment for the expansion and persistence of the T cells following adoptive transfer. New more predictable strategies are under development, which should allow such therapies to be broadly applicable.

Purpose: Donor T cells have been shown to be reactive against and effective in adoptive immunotherapy of Epstein-Barr virus (EBV) lymphomas which develop in some leukemia patients post marrow transplantation. These T cells may be genetically modified by incorporation of a replication-incompetent viral vector (NIT) encoding both an inactive mutant nerve growth factor receptor (LNGFR), as an immunoselectable surface marker, and a herpes simplex virus thymidine kinase (HSV-TK), rendering the cells sensitive to ganciclovir. The current studies are based on the selective HSV-TK-catalyzed trapping (phosphorylation) of the thymidine analog [(131)I]-2'-fluoro-2'-deoxy-1-beta-D-arabinofuransyl-5-iodo-uracil (FIAU) as a means of stably labeling such T cells for in vivo trafficking (including tumor targeting) studies. Because of the radiosensitivity of lymphocytes and the potentially high absorbed dose to the nucleus from intracellular (131)I (even at tracer levels), the nucleus absorbed dose (D ( n )) and dose-dependent immune functionality were evaluated for NIT(+) T cells labeled ex vivo in [(131)I]FIAU-containing medium., Methods: Based on in vitro kinetic studies of [(131)I]FIAU uptake by NIT(+) T cells, D ( n ) was calculated using an adaptation of the MIRD formalism and the recently published MIRD cellular S factors. Immune cytotoxicity of [(131)I]FIAU-labeled cells was assayed against (51)Cr-labeled target cells [B-lymphoblastoid cells (BLCLs)] in a standard 4-h release assay., Results and Conclusion: At median nuclear absorbed doses up to 830 cGy, a (51)Cr-release assay against BLCLs showed no loss of immune cytotoxicity, thus demonstrating the functional integrity of genetically transduced, tumor-reactive T cells labeled at this dose level for in vivo cell trafficking and tumor targeting studies.

A HLA-DR1 transgenic mouse (NOD/scid-DR1) was derived by breeding the existing B10.M/J-[Tg]DR1 mouse with the NOD/scid mouse. The intention was to enhance engraftment of human T cells by providing human class II elements in the tissues. Thymus and spleen fragments from adult NOD/scid-DR1 mice were transplanted under the syngeneic kidney capsules, followed by injection of human cord blood mononuclear cells (CBMNC) into transplanted tissues. FACS analyses showed that human T and B cells were consistently detected in the peripheral blood and spleen, of the chimeric mice. An average of 20% of human cells was found in the spleen and the engrafted thymus/spleen tissues. Furthermore, human cells from these tissues could proliferate with anti-human CD3 antibody and these mice could generate humoral and cellular responses to allogeneic human cells. Cytokines, such as IL-10, GMCSF, IFN-gamma, and TNF-alpha were also detected in the supernatants of the cultured human cells from the chimeric mice, when they were stimulated with allogeneic cells. Therefore, a novel mouse model with functional circulating human T and B cells was established that would facilitate the exploration of vaccine, the disease processes of autoimmunity, HIV infection, and human cancer.

The Wilms tumor protein (WT1) is overexpressed in most acute and chronic leukemias. To develop a practicable, clinically applicable approach for generation of WT1-specific T cells and to comparatively evaluate the immunogenicity of WT1 in normal individuals, we sensitized T cells from 13 HLA-A0201+ and 5 HLA-A2402+ donors with autologous EBV-transformed B cells or cytokine-activated monocytes, loaded with the HLA-A0201-binding WT1 peptides (126-134)RMFPNAPYL or (187-195)SLGEQQYSV or a newly identified HLA-A2402-binding WT1 peptide (301-310)RVPGVAPTL. WT1-specific T cells were regularly generated from each donor. T cells sensitized with peptide-loaded EBV-transformed B cells generated higher numbers of WT1-specific T cells than peptide-loaded cytokine-activated monocytes. Contrary to expectations, the frequencies of WT1 peptide-specific T cells were equivalent to those generated against individual highly immunogenic HLA-A0201-binding EBV peptides. Each of these T-cell lines specifically killed WT1+ leukemias and solid tumors in an HLA-restricted manner but did not lyse autologous or HLA-matched normal CD34+ hematopoietic progenitor cells or reduce their yield of colony-forming unit-granulocyte-macrophage (CFU-GM), burst-forming unit erythroid (BFU-E), or mixed colonies (CFU-mix). Furthermore, WT1 peptide-specific T cells after adoptive transfer into nonobese diabetic-severe combined immunodeficient mice bearing subcutaneous xenografts of WT1+ and WT1- HLA-A0201+ leukemias preferentially accumulated in and induced regressions of WT1+ leukemias that expressed the restricting HLA allele. Such cells are clinically applicable and may prove useful for adoptive cell therapy of WT1+ malignant diseases in humans.

Background: Prolonged immunodeficiency after allogeneic bone marrow transplantation (allo BMT) results in significant morbidity and mortality from infection. Previous studies in murine syngeneic BMT models have demonstrated that posttransplantation insulin-like growth factor (IGF)-I administration could enhance immune reconstitution., Methods: To analyze the effects of IGF-I on immune reconstitution and graft-versus-host disease (GVHD) after allo BMT, we used murine models for MHC-matched and -mismatched allo BMT. Young (3-month-old) recipient mice received 4 mg/kg per day of human IGF-I from days 14 to 28 by continuous subcutaneous administration., Results: IGF-I administration resulted in increased thymic precursor populations (triple negative-2 and triple negative-3) as determined on day 28 but had no effect on overall thymic cellularity. In the periphery, the numbers of donor-derived splenic CD3+ T cells were increased and these cells had an improved proliferative response to mitogen stimulation. IGF-I treatment also significantly increased the numbers of pro-, pre-, and mature B cells and myeloid cell populations in the spleens of allo BMT recipients on day 28. The administration of IGF-I in combination with interleukin 7 had a remarkable additive effect on B-cell, but not on T-cell, lymphopoiesis. Finally, we tested the effects of IGF-I administration on the development of GVHD in three different MHC-matched and -mismatched models and found no changes in GVHD morbidity and mortality., Conclusion: IGF-I administration can enhance lymphoid and myeloid reconstitution after allo BMT without aggravating GVHD.

New technologies are needed to characterize the migration, survival, and function of antigen-specific T cells in vivo. Here, we demonstrate that Epstein-Barr virus (EBV)--specific T cells transduced with vectors encoding herpes simplex virus-1 thymidine kinase (HSV-TK) selectively accumulate radiolabeled 2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil (FIAU). After adoptive transfer, HSV-TK+ T cells labeled in vitro or in vivo with [131I]FIAU or [124I]FIAU can be noninvasively tracked in SCID mice bearing human tumor xenografts by serial images obtained by scintigraphy or positron emission tomography (PET), respectively. These T cells selectively accumulate in EBV+ tumors expressing the T cells' restricting HLA allele but not in EBV- or HLA-mismatched tumors. The concentrations of transduced T cells detected in tumors and tissues are closely correlated with the concentrations of label retained at each site. Radiolabeled transduced T cells retain their capacity to eliminate targeted tumors selectively. This technique for imaging the migration of ex vivo-transduced antigen-specific T cells in vivo is informative, nontoxic, and potentially applicable to humans.

Successful allogeneic hematopoietic stem cell transplantation (HSCT) requires reconstitution of normal T-cell immunity. Recipient thymic activity, biologic features of the allograft, and preparative regimens all contribute to immune reconstitution. We evaluated circulating T-cell phenotypes and T-cell receptor rearrangement excision circles (TRECs) in 331 blood samples from 158 patients who had undergone allogeneic HSCTs. All patients had received myeloablative conditioning regimens and were full donor chimeras in remission. Younger patients exhibited more rapid recovery and higher TRECs (P =.02). Recipients of T-cell-depleted allografts initially had lower TRECs than unmodified allograft recipients (P <.01), but the difference abated beyond 9 months. TREC level disparities did not achieve significance among adults with respect to type of allograft. Measurable, albeit low, TREC values correlated strongly with severe opportunistic infections (P <.01). This finding was most notable during the first 6 months after transplantation, when patients are at greatest risk but before cytofluorography can detect circulating CD45RA(+) T cells. Low TRECs also correlated strongly with extensive chronic graft-versus-host disease (P <.01). Recipients of all ages of either unmodified or T-cell-depleted allografts therefore actively generate new T cells. This generation is most notable among adult recipients of T-cell-depleted allografts, most of whom had also received antithymocyte globulin for rejection prophylaxis. Low TREC values are significantly associated with morbidity and mortality after transplantation. T-cell neogenesis, appropriate to age but delayed in adult recipients of T-cell-depleted allografts, justifies interventions to hasten this process and to stimulate desirable cellular immune responses.

Techniques for the quantitation of virus-specific and alloantigen-reactive T cells vary in their measurement of clinically relevant T-cell effector populations, their sensitivity and quantitative accuracy, and the time required to obtain measurable results. We compared frequencies of Epstein-Barr virus (EBV)-specific and major alloantigen-reactive T cells as measured by flow cytometric analysis of responding T cells producing intracellular interferon-gamma (IFN-gamma) and by limiting-dilution analysis (LDA) of cytotoxic T-cell precursors (CTLp) at sequential time points during the generation of EBV-specific T-cell lines. The expansion of EBV-specific T lymphocytes and the depletion of alloreactive T cells in cultures of T cells sensitized with autologous EBV-transformed targets followed similar kinetics when measured by either method. Frequencies of EBV- specific T cells generating intracellular IFN-gamma exceeded by 25- to 90-fold the frequencies of responding CTLp at each stage of expansion, whereas the frequencies of alloreactive T cells generating intracellular IFN-gamma exceeded by 30- to 220-fold those detected by LDA. The assay that quantitated T cells producing IFN-gamma yielded more reproducible and precise results than LDA. Furthermore, frequencies detected by the enumeration of T cells responding to immunodominant EBNA 3a and EBNA 3c peptides by IFN-gamma production or their capacity to bind peptide-HLA tetramers were strikingly similar and represented significant fractions of T cells generating IFN-gamma in response to autologous EBV B lymphoblastoid cell line. Functional analysis of responding viable T cells, fractionated on the basis of their secretion of IFN-gamma, demonstrated that EBV-specific and alloantigen cytotoxic T cells were predominantly or exclusively detected in the CD8(+)IFN-gamma(+) fraction of T cells. Strikingly, the CD4(+)IFN-gamma(+) cell fractions were not cytotoxic against EBV-transformed or allogeneic targets.

PurposeTo describe ocular outcomes in eyes with cytomegalovirus (CMV) retinitis treated with adoptive immunotherapy using systemic administration of CMV-specific cytotoxic Tlymphocytes (CMV-specific CTLs).DesignRetrospective cohort study.ParticipantsPatients with active CMV retinitis evaluated at a tertiary care academic center.MethodsTreatment of CMV retinitis with standard-of-care therapy (systemic or intravitreal antivirals) or CMV-specific CTLs (with or without concurrent standard-of-care therapies).Main outcome measuresThe electronic medical record was reviewed to determine baseline characteristics, treatment course, and ocular outcomes, including best-corrected visual acuity (BCVA), treatments administered (CMV-specific CTLs, systemic antivirals, intravitreal antivirals), resolution of CMV retinitis, any occurrence of immune recovery uveitis, cystoid macular edema, retinal detachment, or a combination thereof.ResultsSeven patients (3 of whom had bilateral disease [n = 10 eyes]) were treated with CMV-specific CTLs, whereas 20 patients (6 of whom had bilateral disease [n = 26 eyes]) received standard-of-care treatment. Indications for CMV-specific CTL therapy included persistent or progressive CMV retinitis (71.4% of patients); CMV UL54 or UL97 antiviral resistance mutations (42.9%); side effects or toxicity from antiviral agents (57.1%); patient intolerance to longstanding, frequent antiviral therapy for persistent retinitis (28.6%); or a combination thereof. Two patients (28.6%; 4 eyes [40%]) received CMV-specific CTL therapy without concurrent systemic or intravitreal antiviral therapy for active CMV retinitis, whereas 5 patients (71.4%; 6 eyes [60%]) continued to receive concurrent antiviral therapies. Resolution of CMV retinitis was achieved in 9 eyes (90%) treated with CMV-specific CTLs, with BCVA stabilizing (4 eyes [40%]) or improving (4 eyes [40%]) in 80% of eyes over an average follow-up of 33.4 months. Rates of immune recovery uveitis, new-onset cystoid macular edema, and retinal detachment were 0%, 10% (1 eye), and 20% (2 eyes), respectively. These outcomes compared favorably with a nonrandomized cohort of eyes treated with standard-of-care therapy alone, despite potentially worse baseline characteristics.ConclusionsCMV-specific CTL therapy may represent a novel monotherapy or adjunctive therapy, or both, for CMV retinitis, especially in eyes that are resistant, refractory, or intolerant of standard-of-care antiviral therapies. More generally, adoptive cell transfer and adoptive immunotherapy may have a role in refractory CMV retinitis. Larger prospective, randomized trials are necessary.

To define the tolerability and outcome of allogeneic hematopoietic stem cell transplant (allo-HSCT) following CAR T-cell therapy, we retrospectively reviewed pediatric/young adult patients with relapsed/refractory B-ALL who underwent this treatment. Fifteen patients (median age 13 years; range 1-20 years) with a median potential follow-up of 39 months demonstrated 24-month cumulative incidence of relapse, cumulative incidence of TRM, and OS of 16% (95% CI: 0-37%), 20% (95% CI: 0-40%), and 80% (95% CI: 60-100%), respectively. Severe toxicity following CAR T cells did not impact OS (p = 0.27), while greater time from CAR T cells to allo-HSCT (>80 days) was associated with a decrease in OS. In comparing CD34-selected T-cell depleted (TCD; n = 9) vs unmodified (n = 6) allo-HSCT, the cumulative incidence of relapse, TRM, and OS at 24 months was 22% (95% CI: 0-49%) vs 0% (p = 0.14), 0% vs 50% [95% CI: 10-90%] (p = 0.02) and 100% vs 50% [95% CI: 10-90%] (p = 0.02). In this small cohort of patients, CAR T cells followed by a CD34-selected TCD allo-HSCT appears to result in less TRM and favorable OS when compared with unmodified allo-HSCT. There was no evidence that disease control was impacted by the type of consolidative allo-HSCT, which demonstrates the feasibility of this approach.

Cytomegalovirus (CMV) retinitis is a potentially blinding infection that affects immunocompromised patients who are unable to generate a T-cell response against the organism. Infusion of CMV-specific leukocytes has been shown to be effective in patients with systemic CMV infection, especially those resistant to standard therapies. The authors report a case of a patient with CMV viremia with progressive retinitis in whom infusion of third-party donor-derived CMV pp65-specific T cells alone prompted resolution of CMV retinitis. This case suggests a potential role for CMV-specific leukocyte infusion in the treatment of CMV retinitis, especially in cases resistant or refractory to antiviral therapies.

BackgroundThe Primary Immune Deficiency Treatment Consortium was formed to analyze the results of hematopoietic-cell transplantation in children with severe combined immunodeficiency (SCID) and other primary immunodeficiencies. Factors associated with a good transplantation outcome need to be identified in order to design safer and more effective curative therapy, particularly for children with SCID diagnosed at birth.MethodsWe collected data retrospectively from 240 infants with SCID who had received transplants at 25 centers during a 10-year period (2000 through 2009).ResultsSurvival at 5 years, freedom from immunoglobulin substitution, and CD3+ T-cell and IgA recovery were more likely among recipients of grafts from matched sibling donors than among recipients of grafts from alternative donors. However, the survival rate was high regardless of donor type among infants who received transplants at 3.5 months of age or younger (94%) and among older infants without prior infection (90%) or with infection that had resolved (82%). Among actively infected infants without a matched sibling donor, survival was best among recipients of haploidentical T-cell-depleted transplants in the absence of any pretransplantation conditioning. Among survivors, reduced-intensity or myeloablative pretransplantation conditioning was associated with an increased likelihood of a CD3+ T-cell count of more than 1000 per cubic millimeter, freedom from immunoglobulin substitution, and IgA recovery but did not significantly affect CD4+ T-cell recovery or recovery of phytohemagglutinin-induced T-cell proliferation. The genetic subtype of SCID affected the quality of CD3+ T-cell recovery but not survival.ConclusionsTransplants from donors other than matched siblings were associated with excellent survival among infants with SCID identified before the onset of infection. All available graft sources are expected to lead to excellent survival among asymptomatic infants. (Funded by the National Institute of Allergy and Infectious Diseases and others.).