Introduction: Approved Chimeric Antigen Receptor (CAR) T-cell therapies in refractory or relapsed B-cell Non-Hodgkin lymphoma (r/r B-NHL) target CD19; however, their efficacy is limited to about half of the patients treated. We thus developed a CAR targeting CD20 (MB-CART20.1) consisting of CAR transduced CD4 /CD8 enriched T-cells, derived from an autologous leukapheresis. Here, we report on the first part of a phase I first-in-human clinical trial that evaluates the safety of MB-CART20.1 in patients with r/r B-NHL. Methods: Patients with r/r B-NHL with no curative treatment option were eligible for this phase I multi-center, open label, dose escalation trial of MB-CART20.1 (EudraCT 2017-000121-12). The primary objective was to determine safety of MB-CART20.1. Secondary objectives included response to treatment, phenotype and persistence of MB-CART20.1. The primary endpoint was determination of the maximum tolerated dose (MTD), defined as the highest dose level at which < 33% of patients experience Dose Limiting Toxicities (DLTs) until day 28 post infusion (follow-up safety period). Patient leukapheresis products were enriched for CD4/CD8 T-cells, transduced with the vector and expanded using the CliniMACS Prodigy System.. Apheresis and the MB-CART20.1 were fresh products with a turnaround time of 14 days. Patients underwent lymphodepletion with fludarabine (30 mg/m2) and cyclophosphamide (300 mg/m2), each from day -5 to day -3. Dose escalation was planned with 3 + 3 patients at Dose Level (DL) 0 (1x105 CAR T-cells/kg BW), followed by DL1 (1x106 CAR T-cells/kg BW ) and DL2 (3x106 CAR T-cells/kg BW) with 6 + 3 patients, each. Results: In total 10 patients were treated. 9 patients were included in this analysis. 1 patient was treated with an out of specification product, these data are not included. 7 patients were enrolled at DL0 and 2 at DL1. 8 of 9 patients completed the follow-up safety period. At DL0, 3 of 7 patients experienced CRS (grade I) starting 1 to 10 days post infusion. One CRS case was managed with tocilizumab. This patient further developed CAR T-cell related encephalopathy syndrome (CRES; grade II) and bone marrow aplasia (grade III). On day 17 post infusion the patient died because of a septic shock. The Safety Monitoring Board thus recommended to extend DL0 with 3 additional patients. In this extension cohort, no further neurotoxicity or any DLT occurred. 1 of 7 patients achieved complete response (CR), remained in remission until month 12 and then entered long-term follow-up. 5 patients progressed at 4 weeks (n=2), 12 weeks (n=2) and 6 months (n=1) post infusion, respectively. Analysis of expansion for the 3 patients reaching at least partial response (PR) within 12 weeks (responders) showed a notable increase of the maximum cell concentration (Cmax, values between 3,1 to 104,7 cells/µl), as well as an enhanced area under the curve from day 0 to 28 (AUC D0-28, between 28,5 and 1273,0 days/cells/µl). This increase of values was not observed in the 3 non-responders. Time to Cmax is similar between responders and non-responders (mean 16 days). At DL1, 2 of 2 patients experienced CRS shortly post infusion (grade I and II respectively). Tocilizumab was used in one patient. Neither DLT nor ICANS occurred. Short-lived Grade III and IV neutropenia was observed in both patients. Both patients completed the active part of the trial (12 months post infusion) with CR, entered the long-term follow-up and remained in CR until data cut-off. Analysis of expansion showed an increase of Cmax with 198,6 and 79,8 cells/µl, respectively and AUC D0-28 with 2495,0 and 610,1 days/cells/µl. Time to Cmax is 14 days for both patients. The sponsor stopped the trial early due to the COVID pandemic with relevant impact on enrollment and study conduct. In addition there are no current plans for further development of this vector. Conclusions: No DLT was observed in this first trial of a CD20 targeting CAR T-cell product in r/r B-NHL patients; however, early termination of the trial does not allow conclusions on a MTD. In total 3 patients achieved a CR and remained in remission for at least one year. The two patients treated at the higher DL, achieved a CR indicating a dose-response correlation. The results strongly support further evaluation of MB-CART20.1 in r/r B-cell NHL.

Recently, a rare type of relapse was reported upon treating a B cell acute lymphoblastic leukemia (B-ALL) patient with antiCD19 chimeric antigen receptor (CAR)-T cells caused by unintentional transduction of residual malignant B cells (CAR-B cells). We show that anti-CD19 and anti-CD20 CARs are presented on the surface of lentiviral vectors (LVs), inducing specific binding to the respective antigen. Binding of anti-CD19 CAR-encoding LVs containing supernatant was reduced by CD19-specific blocking antibodies in a dose-dependent manner, and binding was absent for unspecific LV containing supernatant. This suggests that LVs bind via displayed CAR molecules to CAR antigen-expressing cells. The relevance for CAR-T cell manufacturing was evaluated when PBMCs and B-ALL malignant B cells were mixed and transduced with anti-CD19 or anti-CD20 CAR-displaying LVs in clinically relevant doses to mimic transduction conditions of unpurified patient leukapheresis samples. Malignant B cells were transduced at higher levels with LVs displaying anti-CD19 CARs compared to LVs displaying non-binding control constructs. Stability of gene transfer was confirmed by applying a potent LV inhibitor and long-term cultures for 10 days. Our findings provide a potential explanation for the emergence of CAR-B cells pointing to safer manufacturing procedures with reduced risk of this rare type of relapse in the future.

Abstract Introduction This phase I first-in-human clinical study assesses the safety and preliminary efficacy of a CD19-directed, CAR (4-1BBz) gene-modified, autologous T-cell immunotherapy (MB-CART19.1) intended for use in pediatric and adult patients with relapsed or refractory B-cell acute lymphoblastic leukemia (ALL) and Non-Hodgkin lymphoma (NHL). The study also evaluates the feasibility of a hybrid manufacturing model, combining central and academic manufacturing capabilities with central QP oversight. Methods MB-CART19.1 is evaluated in a Phase I (EudraCT 2017-002848-32) multi-center, open label, dose escalation trial enrolling 33 to 48 patients in three disease cohorts, defined by disease biology and age. Pediatric (1-17 years) and adult patients are eligible if diagnosed with relapsed/refractory (r/r) CD19-expressing B-cell ALL or B-cell high-grade and low-grade (adults) NHL, including chronic lymphocytic leukemia (CLL). Enrollment is still ongoing. The starting material, a fresh patient leukapheresis product, is enriched for CD4/CD8 T-cells, transduced with a lentiviral vector (LV) and expanded using the CliniMACS Prodigy System allowing a high degree of control and consistency of the manufacturing process in both a central and decentralized facilities. MB-CART19.1 is presented as fresh cellular dispersion for single infusion and undergoes central release. Subjects undergo lymphodepletion with fludarabine (25 mg/m 2 daily for 3 days) and cyclophosphamide (1000 mg/m 2 on day -3). Dose escalation includes 3 dose levels (DL) 5x10 5 (DL I), 1x10 6 (DL II), 3x10 6 (DL III) CAR T cells/kg BW, respectively, and a safety dose level 0. The primary objective is to determine the recommended dose of MB-CART19.1. Secondary objectives are preliminary efficacy parameters evaluation of as well as CART persistence. Results Disease cohort I: pediatric ALL and aggressive NHL, 1-17 years. Up to the data lock point for interim analysis (DLP, 02 June 2021), 9 pediatric ALL patients were treated, 6 at DL I and 3 at DL II. All 9 patients completed the 28 days safety follow-up. At DL I, 5 of 6 patients experienced CRS (4 grade I-II, 1 grade III,) starting 5 to 7 days after IMP infusion. Two CRS cases were managed with tocilizumab and resolved within 1-2 weeks. 1 patient developed signs of neurotoxicity (grade IV seizure) concurrently with grade II CRS, which was effectively managed and fully resolved within 48 hours. The event was evaluated as DLT and led to the extension of the dose group from 3 to 6 patients. No further neurotoxicities occurred. Four of 6 patients treated at DL I finished the active part of the trial (12 months after administration of IMP) in CR-MRD and entered the long term follow-up. Two patients had CD19-negative relapses 4 and 10 months post MB-CART19.1 infusion. At DL II, 1 patient completed the 6 months follow up in ongoing CR, and 2 patients relapsed. Disease cohort II adult ALL: Up to the DLP, 4 adult ALL patients were treated at DL I. 1 patient died due to progression of disease on day 20 after the IMP infusion. All 4 adult patients experienced a grade I or II CRS all cases were reversible within 1-2 weeks , 1 patient received tocilizumab One patient developed neurologic symptoms (grade III visual impairment and grade III muscle weakness right-sided) with onset 41 and 72 days after administration of MB-CART19.1, respectively. 2 of the 3 patients who completed the safety follow-up finished the active part of the trial and entered the long-term follow-up, both in molecular CR up to Month 6. Disease cohort III adult NHL/CLL: 4 patients were enrolled with the starting dose of 1x10 6 CAR+ cells/kg (DL II). 1 patient experienced grade III CRS and was treated with tocilizumab. 3 patients completed the 28 days safety follow up. One patient with CLL achieved a CR, which is maintained at 6 months. Another CLL patient was in PR at the assessment visit Day 28. Data from the 2 other patients, 1 with MCL and 1 with DLBCL were in PR at month 3. Later data is not yet available. Conclusions 18 of 19 patients completed the follow-up safety period of 28 days defined as observation period for dose limiting toxicity (DLT). One DLT was observed as well as 3 grade III CRS events and 1 grade III neurological event. Early efficacy results are very encouraging. Longer follow-up will establish whether treatment results in durable responses. The hybrid manufacturing model provides flexibility and a timely delivery of the fresh drug product to the patients Disclosures Hanssens: Miltenyi Biomedicine GmbH: Current Employment. Stelljes: MSD: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Celgene/BMS: Consultancy, Speakers Bureau; Kite/Gilead: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Medac: Speakers Bureau; Pfizer: Consultancy, Research Funding, Speakers Bureau. Bethge: Novartis: Consultancy, Honoraria, Speakers Bureau; Kite-Gilead: Consultancy, Honoraria, Speakers Bureau; Miltenyi Biotec: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau. Yakushina: Miltenyi Biomedicine GmbH: Current Employment. Holtkamp: Miltenyi Biomedicine GmbH: Current Employment. Assenmacher: Miltenyi Biotec: Current Employment. Jurk: Miltenyi Biotec: Current Employment. Rauser: Miltenyi Biomedicine GmbH: Current Employment. Schneider: Employee of Lentigen Technology, a Miltenyi Biotec Company: Current Employment. Rossig: AdBoards by Amgen: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; BMS and Celgene: Honoraria.

Hematopoietic stem cell transplantation (HSCT) from haploidentical donors is a viable option for patients lacking HLA-matched donors. Here we report the results of a prospective multicenter phase I/II trial of transplantation of TCRαβ and CD19-depleted peripheral blood stem cells from haploidentical family donors after a reduced-intensity conditioning with fludarabine, thiotepa, and melphalan. Thirty pediatric and 30 adult patients with acute leukemia ( n = 43), myelodysplastic or myeloproliferative syndrome ( n = 6), multiple myeloma ( n = 1), solid tumors ( n = 6), and non-malignant disorders ( n = 4) were enrolled. TCR αβ/CD19-depleted grafts prepared decentrally at six manufacturing sites contained a median of 12.1 × 10 6 CD34 + cells/kg and 14.2 × 10 3 TCRαβ + T-cells/kg. None of the patients developed grade lll/IV acute graft-versus-host disease (GVHD) and only six patients (10%) had grade II acute GVHD. With a median follow-up of 733 days 36/60 patients are alive. The cumulative incidence of non-relapse mortality at day 100, 1 and 2 years after HSCT was 5%, 15%, and 17% for all patients, respectively. Estimated probabilities of overall and disease-free survival at 2 years were 63% and 50%, respectively. Based on these promising results in a high-risk patient cohort, haploidentical HSCT using TCRαβ/CD19-depleted grafts represents a viable treatment option.

Background Use of cell-based medicinal products (CBMPs) represents a state-of-the-art approach for reducing general immunosuppression in organ transplantation. We tested multiple regulatory CBMPs in kidney transplant trials to establish the safety of regulatory CBMPs when combined with reduced immunosuppressive treatment. Methods The ONE Study consisted of seven investigator-led, single-arm trials done internationally at eight hospitals in France, Germany, Italy, the UK, and the USA (60 week follow-up). Included patients were living-donor kidney transplant recipients aged 18 years and older. The reference group trial (RGT) was a standard-of-care group given basiliximab, tapered steroids, mycophenolate mofetil, and tacrolimus. Six non-randomised phase 1/2A cell therapy group (CTG) trials were pooled and analysed, in which patients received one of six CBMPs containing regulatory T cells, dendritic cells, or macrophages; patient selection and immunosuppression mirrored the RGT, except basiliximab induction was substituted with CBMPs and mycophenolate mofetil tapering was allowed. None of the trials were randomised and none of the individuals involved were masked. The primary endpoint was biopsy-confirmed acute rejection (BCAR) within 60 weeks after transplantation; adverse event coding was centralised. The RTG and CTG trials are registered with ClinicalTrials.gov, NCT01656135, NCT02252055, NCT02085629, NCT02244801, NCT02371434, NCT02129881, and NCT02091232. Findings The seven trials took place between Dec 11, 2012, and Nov 14, 2018. Of 782 patients assessed for eligibility, 130 (17%) patients were enrolled and 104 were treated and included in the analysis. The 66 patients who were treated in the RGT were 73% male and had a median age of 47 years. The 38 patients who were treated across six CTG trials were 71% male and had a median age of 45 years. Standard-of-care immunosuppression in the recipients in the RGT resulted in a 12% BCAR rate (expected range 3.2-18.0). The overall BCAR rate for the six parallel CTG trials was 16%. 15 (40%) patients given CBMPs were successfully weaned from mycophenolate mofetil and maintained on tacrolimus monotherapy. Combined adverse event data and BCAR episodes from all six CTG trials revealed no safety concerns when compared with the RGT. Fewer episodes of infections were registered in CTG trials versus the RGT. Interpretation Regulatory cell therapy is achievable and safe in living-donor kidney transplant recipients, and is associated with fewer infectious complications, but similar rejection rates in the first year. Therefore, immune cell therapy is a potentially useful therapeutic approach in recipients of kidney transplant to minimise the burden of general immunosuppression. Copyright (C) 2020 Elsevier Ltd. All rights reserved.

Abstract Abstract: Here we report for the first time on long term follow-up data of a cohort of 60 patients who received TCRαβ and CD19 depleted peripheral blood stem cell grafts from haploidentical family donors within a prospective, multi-center, single-arm, phase I/II clinical study (EudraCT No.: 2011-005562-38). As planned, 30 pediatric and 30 adult patients were enrolled in this trial: All patients received a reduced-intensity conditioning regimen consisting of fludarabine (160 mg/m2), thiotepa (10 mg/kg), melphalan (140 mg/m2) and either antithymocyte globulin (Grafalon, 15 or 30 mg/kg, N=53) or 7 Gy total nodal irradiation (N=7). MMF (40 mg/kg/day) was administered as single-agent GVHD prophylaxis until Day 30. Results: Sixty patients with a median age of 18.5 years (range 1-63) were treated. Twenty-five patients had AML, 17 ALL, 6 MDS/MPS and 1 each had multiple myeloma and acute undifferentiated leukemia. Six patients had solid tumors (soft tissue sarcomas and neuroblastomas) and 4 non-malignant disorders (SCID, Wiskott-Aldrich syndrome, lysosomal storage disorder and sickle cell anemia). Of the 56 patients with malignant disease, 33 were transplanted in complete (CR), 11 in partial (PR) and 12 in non-remission (NR). Twenty of the 56 patients with malignant diseases received a 2nd or 3rd transplantation within this protocol. In total, 88 depletion procedures were performed with the CliniMACS plus System (Miltenyi Biotec, Germany) at 7 GMP laboratories and resulted in a median T and B cell log depletion of 4.7 (range 3.6-5.3) and 3.4 (range: 2.3-4.5), respectively. The median number of infused CD34+ cells and TCRαβ T cells was 12.4 × 106 /kg BW (range 4.0 - 54.9) and 1.4× 104 /kg BW (range 0.06-6.4), respectively. Engraftment was rapid with a median of 13 (range 9-41) and 15 (11-38) days to reach ANC >500 cell/µl and PLT > 20,000 cells/µl. Nine patients rejected the graft. Eight of them were successfully re-transplanted and 1 patient died. One patient received stem cell boosts from the original donor due to poor graft function. On day 100, peripheral T cell chimerism was completely donor-type in 44 of 47 evaluable patients, and mixed in 3. None of the patients developed grade III/IV aGVHD and only 6 patients (10%) had grade II aGVHD. Of 47 evaluable patients 4 had severe cGVHD (9%), and 6 (13%) and 5 (11%) had moderate and mild cGVHD, respectively. CMV reactivation was seen in 25 (42%) mainly adult patients, and only 1 (2%) patient developed disease. Twenty-one (35%) patients had ADV reactivation and 7 children and 1 adult (13%) developed disease. Only 1 case of EBV disease (encephalitis) occurred (2%). A median of 221 (range 8-1230) CD3+ cells/µl was reached on day 100. The median numbers of CD3/CD4+ and CD3/CD8+ cells at 1 year post transplant were 316 (range 1-1173) and 308 (range 0-2203) /µl. As of July 15 2018, 57 patients have completed the 2 year follow-up, died or discontinued the study resulting in a median follow-up of 706 days (range 18-800). 37 patients (62%) are alive and 23 (38%) died. Relapse was the major cause of death (N=12) followed by ADV infection (N=3), ARDS (N=3) and 1 case each of cardiac arrest, multi organ failure, sepsis due to graft failure and demyelinating neuropathy. Cause of death was not reported in 1 patient. Eight of the 20 patients who received the 2nd or 3rd transplantation are alive; 1 discontinued the study prematurely. Of the 23 patients transplanted in PR or NR, 13 are alive. The Kaplan-Meier estimated probabilities of overall survival, disease-free survival (DFS) were 62% and 53%, respectively. Cumulative incidences of relapse and NRM at 2 years were 34% and 20%, respectively. For those patients with leukemia receiving a first SCT in CR, the overall survival, DFS and relapse rate were 75%, 64% and 20%, respectively. Conclusion: The transplantation of TCRαβ and CD19 depleted haploidentical hematopoietic stem cell grafts was safe and feasible. Decentralized production using the CliniMACS System was feasible and reliably resulted in grafts containing sufficient numbers of stem cells with only minimal numbers of co-infused TCRαβ T cells. None of the patients developed grade III-IV aGVHD and incidence of cGVHD was acceptable. Given the heterogeneous patient cohort with respect to age, disease, remission status and number of previous transplants, the outcome of patients after 2 years follow-up is promising. Disclosures Lang: Miltenyi Biotec: Patents & Royalties, Research Funding. Handgretinger:Miltenyi Biotec: Patents & Royalties: Co-patent holder of TcR alpha/beta depletion technologies, Research Funding. Meisel:Amgen: Consultancy. Mielke:KIADIS Pharma: Speakers Bureau; Miltenyi Biotec: Speakers Bureau; DGHO: Speakers Bureau; EHA: Speakers Bureau; Celgene: Speakers Bureau. Niederwieser:Novartis: Research Funding; Miltenyi: Speakers Bureau. Bader:Neovii: Research Funding; Medac: Patents & Royalties, Research Funding; Riemser: Research Funding; Cellgene: Consultancy; Novartis: Consultancy, Speakers Bureau. Kuball:Gadeta (www.gadeta.nl): Consultancy, Equity Ownership, Patents & Royalties: on gd T cells and receptors and isolation strategies, Research Funding; Miltenyi Biotec: Research Funding; Novartis: Research Funding. Bonig:Miltenyi Biotec GmbH: Honoraria, Research Funding. Karitzky:Miltenyi Biotec GmbH: Employment. Holtkamp:Miltenyi Biotec GmbH: Employment. Malchow:Miltenyi Biotec GmbH: Employment. Siewert:Miltenyi Biotec GmbH: Employment. Biedermann:Miltenyi Biotec GmbH: Employment. Bethge:Neovii GmbH: Honoraria, Research Funding; Miltenyi Biotec GmbH: Consultancy, Honoraria, Research Funding.

Since cellular, immune and gene therapy are evolving and promising medical fields in research and daily routine, immune monitoring has become an essential tool for determining treatment-induced effects on immune cells. Flow cytometry offers the opportunity to monitor immunological parameters in clinical trials on low amounts of cellular material (e.g. whole blood) with a reasonableamount of time. However, reproducibility of flow cytometry-based immune monitoring is a matter of high importance, especially when multiple sites are involved. Here we present results of the semi-annual inter-site comparisons conducted over 3 years as a quality assurance measure of flow cytometry-based immune monitoring for the TCRab-Haplo2010 trial. To assess the reproducibility of the immune monitoring in different flow cytometry core labs, blood from a healthy donor was centrally collected and distributed to the participating labs. Samples were assayed within 25 to 30 hours after blood collection by four operators using four different MACSQuant Analyzer devices at three different sites. Immune cell subsets, which are relevant to evaluate immune reconstitution after haploidentical hematopoietic stem cell transplantation with TCRαβ-CD19 depleted stem cell grafts (T cells, T helper cells, cytotoxic T cells, B cells, NK cells, TCRαβ+ and TCRγδ+ T cells) showed an average coefficient of variation (CV) of 6.28 % (±3.89 %) for percentage of subsets among CD45+cells and of 9.13 % (±5.48 %) for cells/µl (two platform approach). These inter-site comparisons over a period of 3 years demonstrated low variability of immune monitoring results and thereby confirm reliability and comparability of patient immune monitoring data obtained in the TCRab-Haplo2010 flow cytometry core labs.

We report the first prospective, multi-center, open-label, single-arm phase I/II clinical trial that assesses the safety and feasibility of stem cell transplantation with TCRalpha/beta and CD19-depleted haploidentical grafts generated with the CliniMACS plus System (Miltenyi Biotec, Germany) in combination with a reduced-intensity conditioning in pediatric patients suffering from various malignant and non-malignant diseases (www.clinicaltrialsregister.org; 2011-005562-38). All patients received single agent MMF as short-term GVHD prophylaxis (40mg/kg/day for 30 days). The speed of immune reconstitution was measured in two core labs using standardized methods and the MACSQuant flow cytometry device (Miltenyi Biotec, Germany).

We report the first prospective, multi-center, single-arm phase I/II study that assesses the safety and feasibility of haploidentical transplantation with TCRalpha/beta and CD19+ cells depleted peripheral blood stem cell grafts using the CliniMACS plus System (Miltenyi Biotec, Germany) in combination with a reduced-intensity conditioning (RIC) (www.clinicaltrialsregister.org; 2011-005562-38). The grafts were processed in 7 GMP laboratories and immune cell assays were performed in two core labs using standardized methods and the MACSQuant flow cytometry device (Miltenyi Biotec, Germany). The RIC regimen consisted of 15 or 30 mg ATG (Fresenius/Grafalon) or 7 Gy total nodal irradiation, 160 mg/m2 fludarabine, 10 mg/kg thiotepa, and 140 mg/m2 melphalan. MMF (40mg/kg/day) was used for GVHD prophylaxis until Day 30. The Day 100 data of the 30 pediatric patients have been presented at ASH2016; this report presents the data of the 30 adult patients with at least 100 days follow-up alive.

Several single center experiences have shown favorable outcomes using in-vitro depletion of T cell receptor (TCR)-alpha/beta cells and B cells.

A fundamental research objective in allogeneic transplantation is the selective depletion of alloreactive T cell populations that are responsible for GVHD while preserving mediators of anti-tumor immunity and immune reconstitution. Alloreactive T cells may be identified by their expression of activation markers, such as CD25, following stimulation by recipient antigen presenting cells. However, CD25 expression is also present on regulatory T cells, which suppress alloreactivity through the expression of inhibitory ligands such as CTLA-4 and GITR. In an effort to develop an optimal strategy to segregate these populations, we have examined the effect of conditions of allogeneic stimulation on the balance of alloreactive and regulatory T cell populations. Stimulation of lymphocytes with irradiated peripheral blood mononuclear cells results in a gradual increase in the percentage of CD4/CD25+ cells over 7 days of culture. Mean percentage of CD4/CD25 at day 3 and 6 was 3% and 11%, respectively. On day 3 of stimulation, these cells demonstrate a characteristic activated phenotype with low intensity of CD25 expression and absence of GITR and CTLA-4 expression. Following isolation by magnetic bead separation, CD25+ cells demonstrated high levels of proliferation in response to restimulation with allogeneic PBMC. In contrast, minimal proliferation was observed following stimulation of the CD25- fraction. On day 6 of culture increased intensity of CD25 expression was observed in a fraction of the stimulated population. A majority of this population now expressed GITR and CTLA-4, consistent with a regulatory phenotype. Lymphocytes stimulated with mature DC demonstrated a markedly different pattern. A marked rise in CD4+/CD25+ cells with high levels of CD25 expression was noted early in the culture period; Mean CD4+/CD25+ cells on day 3 of culture were 9%. These cells were uniformly positive for CTLA-4 and GITR. A further increase in the percentage of cells demonstrating a regulatory phenotype was noted on day 6 of culture. We have demonstrated that the balance of activated and regulatory cells is determined by the nature of the antigen presenting cell and the duration of T cell stimulation. Based on these findings, we plan to conduct a clinical trial in which patients will undergo allogeneic transplantation following the selective removal of alloreactive T cell populations.