GVT Effect Research Articles

Donor NK Cell Education By KIR3DL1 and HLA-B Associates with Reduced Relapse in Unrelated Bone Marrow Transplantation for Adult T-Cell Leukemia

Background: Adult T-cell leukemia/lymphoma (ATL) derives from human T-cell leukemia virus-1 (HTLV-1)-infected T cells, and its prognosis is still dismal. Whereas allogeneic stem cell transplant (allo-HSCT) may lead to long-term remission, 1-year relapse rate is still high at 35-47% (Muranushi et al, Bone Marrow Transplant. 2021). As for NK cell modulation against ATL, polymorphisms of killer-cell immunoglobulin-like receptors (KIRs) are of interest. NK cell inhibition by donor KIR/patient HLA interaction and NK cell education status have been reported to affect the prognosis of allo-HSCT, but no consensus has been reached. Furthermore, the effects of entire KIR haplotypes have not been extensively studied. Aim: Using high-resolution genotyping of all 15 functional KIR genes based on long-range PCR and long-read sequencer, correlations between KIR/HLA polymorphisms and relapses in unrelated bone marrow transplantation (UR-BMT) for ATL were evaluated. Methods: DNAs and clinical data of 264 transplants performed between 2007 and 2016 were obtained. Alleles of 15 KIRs, HLA-A, B, C, DRB1, DPB1 and DQB1 were genotyped for patients and donors. Prognostic factors for relapse were evaluated using the Fine-Gray proportional-hazards models. Results: Donor KIR3DL1 and patient HLA-B combination predictive of strong interaction (n=74) showed a trend for a lower relapse rate than weak or non-interactive combinations (n=145) (HR, 0.64; p=0.100), raising the beneficial impact of donor NK cell education at KIR3DL1/HLA-B interaction. Comprehensive analysis focusing on amino acids of donors' KIRs/HLAs specified combinations of HLA-B isoleucine 80 (80I)/ KIR3DL2 aspartic acid 42 and HLA-B threonine 80 (80T)/ KIR3DL2 glutamic acid 42 (n=76) associated with decreased relapse (HR, 0.35; 95%CI, 0.19-0.65; p=0.00087). On the other hand, combinations of HLA-B glutamic acid 152 (152E)/ KIR2DL4 cysteine 30 and HLA-B valine 152 (152V)/ KIR2DL4 tyrosine 30 (n=27) were associated with increased relapse (HR, 11.79; 95%CI, 1.6-86.58; p=0.015). Given that KIR3DL2/ KIR2DL4 do not bind HLA-B and are closely linked to KIR3DL1/S1 in KIR haplotypes, specific KIR3DL1/S1 alleles were further explored by subgroup analyses. As a result, combinations of HLA-B 80I/ KIR3DL1*015& KIR3DL2*002 and HLA-B 80T/ KIR3DL1∗001, ∗005, ∗007, ∗020, ∗038, ∗097 (n=60) were defined as the group at low risk for relapse (adjusted HR, 0.21; 95%CI, 0.09-0.48; p=0.00026) (Figure 1A), and combinations of HLA-B 152E/ KIR3DS1 and HLA-B 152V/ KIR3DL1∗001, ∗007, ∗015, ∗020, ∗029, ∗038 (n=176) were defined as the group at high-risk for relapse (adjusted HR, 5.31; 95%CI, 1.72-16.39; p=0.0037) (Figure 1B). The cases in the low-risk group and/or not in the high-risk group (n=84) had a lower relapse rate (adjusted HR, 0.20; 95%CI, 0.10-0.40; p=0.0000059) (Figure 1C) and a higher overall survival (adjusted HR, 0.66; 95%CI, 0.46-0.95; p=0.0265389) (Figure 1D) than the remaining cases (n=130). In spite of reduced relapse rate, the former showed a lower rate of grades II-IV acute GVHD (HR 0.65; p=0.049). Finally, CD107a degranulation of NK cells was measured following coincubation of HLA-null K562 cells and PBMCs from healthy donors with or without genotypes of the low-risk group. KIR3DL1(+) NK cells showed increased CD107a degranulation compared with KIR3DL1(−) NK cells in healthy donors with the low-risk group genotype (p=0.031), but this difference was not apparent in healthy donors without the low-risk group genotype (p=0.28) (Figure 2). Conclusions: Allelic genotyping of KIRs enabled to specify donors associated with reduced relapses. Donor NK cell education status at HLA-B/KIR3DL1 interaction might confer enhanced GVT effects and better prognosis in UR-BMT against ATL.

TCF-1 acts as a positive regulator in alloactivated T cells, and TCF-1 deficiency reduces GVHD yet maintains GVT responses

Abstract Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for hematological malignancies. Mature donor T cells in this graft help kill residual malignant cells (graft-versus-tumor or GVT effect). However, donor T cells also damage healthy host tissue, causing graft-versus-host disease (GVHD) in about 30–70% of allo-HSCT patients. These alloactivated T cells proliferate, migrate to target organs (liver, skin, and small intestine), and produce cytokines during GVHD. Our work focuses on modulating T cell signaling to separate GVHD from GVT, as GVHD is the main clinical barrier to widespread use of allo-HSCT. T Cell Factor-1 (TCF-1) is critical for T cell development and activation, but its role in alloactivated and mature T cells is unclear. Using a mouse model of MHC-mismatched allo-HSCT leading to GVHD, we examined the role of TCF-1 in this context. Loss of TCF-1 specifically in T cells led to reduced severity and persistence of GVHD, and reduced host tissue damage from both CD4 and CD8 T cells, yet maintained GVT effects. Cytokine production, proliferation, and survival of donor T cells, as well as donor T cell phenotype, were all affected by loss of TCF-1. Therefore, TCF-1 controls mature T cell phenotype and function during allo-HSCT, and is indispensable for GVHD damage. TCF-1 is dispensable for GVT effects, because loss of this factor does not disrupt antitumor immunity. Thus, loss of TCF-1 separates detrimental GVHD damage from beneficial GVT effects. This work will help to improve outcomes for allo-HSCT patients, who have poor survival due to GVHD and increased risk of deadly infections. This research also enhances our basic understanding of how alloactivated mature T cells are regulated during allo-HSCT.

CD4+FOXP3+ Regulatory T Cell Therapies in HLA Haploidentical Hematopoietic Transplantation.

Since their discovery CD4+FOXP3+ regulatory T cells (Tregs) represented a promising tool to induce tolerance in allogeneic hematopoietic cell transplantation. Preclinical models proved that adoptive transfer of Tregs or the use of compounds that can favor their function in vivo are effective for prevention and treatment of graft-vs.-host disease (GvHD). Following these findings, Treg-based therapies have been employed in clinical trials. Adoptive immunotherapy with Tregs effectively prevents GvHD induced by alloreactive T cells in the setting of one HLA haplotype mismatched hematopoietic transplantation. The absence of post transplant pharmacologic immunosuppression unleashes T-cell mediated graft-vs.-tumor (GvT) effect, which results in an unprecedented, almost complete control of leukemia relapse in this setting. In the present review, we will report preclinical studies and clinical trials that demonstrate Treg ability to promote donor engraftment, protect from GvHD and improve GvT effect. We will also discuss new strategies to further enhance in vivo efficacy of Treg-based therapies.

Bromodomain and Extraterminal (BET) Domain Inhibition with PLX51107 and PLX2853 Improves Survival and Decreases Acute GVHD Severity in Murine Models

Introduction: Acute Graft-versus-Host Disease (aGVHD) affects 30-70% of all allogeneic stem cell transplant (alloSCT) recipients, contributing to high non-relapse mortality. aGVHD is due to donor T cell cytotoxic inflammatory effects stimulated by exposure to foreign host antigens. Epigenetic modulation by BET inhibition boasts anti-inflammatory effects. BET proteins are a class of epigenetic "reader molecules," functioning as key mediators of transcription. BET proteins regulate inflammatory gene expression and are targetable. BET inhibition decreases NF-kB dependent cytokine expression and Th1/Th17 differentiation without affecting regulatory T cell differentiation. BET inhibition disrupts BRD4 interaction with the acetylated component of NF-kB, RelA. Therefore, we hypothesize that BET inhibition is a feasible and effective strategy to mitigate T cell mediated aGVHD inflammation. In this study, we have used PLX51107 and PLX2853 - novel BET inhibitors developed by Plexxikon Inc that possess a unique binding mode and high bioavailability ideal for preclinical evaluation. PLX2853 (second generation) was designed to increase potency and improve tolerability. Materials and Methods: aGVHD allogeneic mouse model: Lethally irradiated (1200 cGy) B6D2F1 recipients received T cell depleted bone marrow cells (TCD-BM, 10x106) and CD45.1+ B6 splenocytes (15x106) intravenously via tail vein injection. Recipients were treated by oral gavage, 3x weekly with PLX51107 (10 mg/kg), PLX2853 (4 mg/kg) or vehicle. We evaluated two dosing schedules - starting at day +1 or day +7 after transplant in the PLX51107 studies and day +1 only after infusion of allogeneic CD45.1+ B6 splenocytes in the PLX2853 studies. Survival and clinical aGVHD scores were assessed. Xenogeneic mouse model: NSG mice were conditioned with 50 cGy X-ray irradiation and injected with 15-20x106 human PBMCs and treated with PLX2853 (4 mg/kg) or vehicle starting day +1. Survival was assessed. Graft-versus-Tumor model: In vivo: Lethally irradiated F1 mice were intravenously injected with firefly luciferase-transduced murine mastocytoma P815 cells on day 0 with B6 TCD-BM ± allogeneic B6 splenocytes (n=8 per cohort). Recipients were treated with PLX2853 (4 mg/kg) or vehicle (oral gavage, 3x weekly) starting at day +1 post-transplant. Survival was assessed. In vitro: Murine CD45.1 B6 CD8+ T cells were stimulated in vitro with PMA/Ionomycin ± PLX2853 (10nM) for 5 days and then degranulation was analyzed in response to P815 tumor challenge to evaluate CTL capacity via flow cytometry of intracellular CD107a expression. Study designs are illustrated in the Figure (Panel A). Results: PLX51107 demonstrates potent biological activity and improves survival in a murine model of aGVHD. Administration of PLX51107 dramatically improved survival of recipient mice (B) and reduced aGVHD clinical scores (C). PLX2853 significantly improves survival in multiple mouse models of aGVHD. Our in-vitro data show that PLX2853 (IC50 ~10nM) is more potent than PLX51107 (IC50 ~500nM). Therefore, we validated the biological activity and efficacy of PLX2853 in mouse models of aGVHD. PLX2853 significantly prolonged survival of allogeneic transplanted recipient mice (D) and resulted in reduced clinical scores (E). Recipient mice in the xenogeneic mouse model showed improved survival with PLX2853 treatment (F). PLX2853 maintains Graft Versus Tumor response in vivo and does not abrogate CD8+ cytotoxic T lymphocyte (CTL) responses in vitro. BET inhibition retained beneficial GVT effects as seen by improvement in survival comparable to vehicle group (G). PLX2853 treated CD8+ T cells showed comparable degranulation to control as measured by CD107a mobilization (H, I). These results suggest that BET inhibition does not abrogate CD8+ CTL capacity, correlating to retention of GVT effects observed in vivo. Conclusions: PLX51107 was well tolerated at both day +1 and +7 initiation, demonstrating the future feasibility of BET inhibition as a prophylactic or therapeutic strategy for aGVHD. PLX2853 given at lower doses demonstrated similar improvements in survival and GVHD scoring, consistent with increased potency. Preliminary in vivo and in vitro studies of PLX2853 on GVT and on CD8+ CTLs show that PLX2853 retains GVT effects. Additional mechanistic in vivo and in vitro studies are ongoing. Figure Disclosures Powell: Plexxikon Inc.: Employment. Bollag:Plexxikon Inc.: Employment.

«Graft-Versus-Host» Disease after Haploidentical Hematopoietic Stem Cell Transplantation from Relative Donors in Children with Cancer, Prophylaxis Regimens and Correlation with «Graft-Versus-Tumor» Effect: a Retrospective Cohort Study

Background. «Graft-versus-host» disease (GVHD) is a major complication of allogeneic Hematopoietic Stem Cell Transplantation (HSCT), the main cause of non-relapse mortality. The actual problems of alternative donors HSCTs are GVHD prophylaxis and its distinction from «graft-versus-tumor» (GVT) effect. Objective. Assessment of incidence and severity of GVHD in children with very high-risk cancer after non-T-depleted haplo-HSCT; searching some correlation between controlled chronic GVHD (chGVHD) and GVT effect after HSCT, compare cyclosporine (CSA) and tacrolimus based GVHD prophylaxis. Methods. We provided the GVHD assessment in 74 recipients of haplo-HSCs from relatives since 2001 till 2018 in retrospective cohort study. Effect of chGVHD on the HSCT results was assessed in 23 patients with acute myeloid leukemia (AML) and 15 patients with solid tumors. We analyzed the incidence of severe acute and chronic GVHD in patients who received CSA or tacrolimus-based prophylaxis. Results. The incidence of severe aGHVD in our study was 15%, chGVHD — 12%. Switch CSA to tacrolimus resulted in reduction of severe aGVHD from 21% to 9% and full elimination of severe extensive chGVHD. Controlled chGVHD after haplo-HSCT was associated with relapse reduction from 48.7% to 30% and improvement of overall survival (OS) from 46.5% to 80.8% in patients with AML. We haven’t seen any clinically significant antitumor effect of graft in patients with solid tumors. Conclusion. In our study the incidence of severe GVHD corresponds to world data after HSCTs from alternative donors, it went down after switch from CSA to tacrolimus. We found some correlation between controlled chGVHD development and HSCT results improvement in patients with AML. The GVT effect of haplo-graft in solid tumors is almost absent.

The role of checkpoint blockade after allogeneic stem cell transplantation in diseases other than Hodgkin's Lymphoma.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment option for many malignant high-risk hematological diseases. The Graft-vs.-Tumor (GvT) effect is the major hallmark of this treatment approach. However, disease relapse remains a major limitation. Boosting the GvT effect by checkpoint inhibitors (CI) is an attractive option in this desperate situation although potentially triggering Graft-vs.-Host Disease (GvHD). Early reports in patients with Hodgkin's lymphoma support the idea that CI therapy after HSCT is feasible and effective. We have retrospectively analyzed CI therapy for treatment of disease recurrence after allo-HSCT other than Hodgkin's lymphoma including 21 patients from eight German transplant centers. The median follow-up was 59 days. The overall response rate (ORR) was 43%. Patients receiving donor lymphocyte infusion (DLI) in combination with CI had superior response (ORR 80%). Severe acute GvHD grade III-IV and moderate to severe chronic GvHD were observed in 29% of all patients. Taken together, CI therapy in relapsed patients after HSCT, especially in combination with DLI, is effective but induces severe GvHD in a considerable proportion of patients. Thus, prospective trials or EBMT registry-based validation of different dosing and application schedules including immunosuppressive regimens in those patients are urgently needed.

The Nrf2 activator dimethyl fumarate inhibits acute graft-versus-host disease with retention of graft-versus-tumor effects

Abstract Dimethyl fumarate (DMF), an activator of Nrf2, has been shown to have anti-inflammatory and immunomodulatory properties without significant immunosuppression. we therefore hypothesized that DMF could be a new candidate drug for the treatment of aGVHD with retention of GVT effect. Lethally irradiated BALB/c mice transplanted with B6 bone marrow plus splenocytes were administered DMF or vehicle solution by gavage daily from day -3 to day 3. DMF treatment inhibited aGVHD, accompanied by less weight loss and lower clinical score. DMF is capable of significantly inhibiting proliferation of alloreactive T cells in a dose-dependent manner in mixed lymphocyte reaction assay. Furthermore, the percentages of CD69+T cells and IFN-γ expressing T cells, were significantly increased in aGVHD target organs from DMF recipients. In addition, DMF treatment markedly increased the frequencies of Treg cells in target organs. Depletion of CD25+ cells in DMF recipients aggravated aGVHD mortality compared with IgG control recipients. Moreover, Treg polarizing assay suggested that DMF could promote Treg cell differentiation in vitro in a dose-dependent manner. In addition, No protective effects of DMF were observed in mice transplanted with BM and challenged with leukemia cells. However, DMF treatment significantly prolonged the survival in tumor-bearing mice that received an allogeneic BMT with donor splenocytes. we also observed that donor T cells recovered from DMF-treated recipients and controls showed comparable cytolytic activity against leukemia cells. In conclusion, our findings demonstrated that activation of Nrf2 with DMF may be of significant use in preventing aGVHD and preserving GVT responses.

A Composite Event-Free Survival End Point to Evaluate Outcome after Allogeneic Transplantation for Severe Aplastic Anemia: A Study on Behalf of the Francophone Society of Bone Marrow Transplantation and Cellular Therapy (SFGM-TC)

Background: Optimization of transplantation modalities (Graft versus host disease (GVHD) prophylaxis, conditioning regimens, high level HLA typing and supportive care) has improved overall survival (OS) of patients (pts) with idiopathic severe aplastic anemia (SAA) undergoing matched related (MRD) or 10/10 unrelated donor (MUD) allogeneic transplantation (HSCT). OS is nowadays of more than 80% and thus may not be sufficient anymore to accurately assess outcome. New composite endpoints including surrogate markers of quality of life, such as GVHD, may fit better our current practice.GVHD and relapse free survival (GRFS) have recently been reported in the context of hematological malignancies, as a surrogate marker of quality of survival (Holtan et al. blood 2014). However, this end point was not evaluated in the setting of SAA, with no need of GVT effect, but more concern regarding engraftment. Thus, we adapted from Holtan et al. a composite GRFS with the aim to provide more accurate evaluation of outcome after HSCT for SAA.Methods: We analyzed adult pts undergoing first HSCT for SAA from a MRD or 10/10 MUD between 2004 and 2013 (Clinical data obtained throughProMISe [Project Manager Internet Server], an internet-based system shared by SFGM-TC centers). Informed consent was obtained in accordance with the Declaration of Helsinki.Relevant events for GRFS were death, graft failure/loss, grade 3 to 4 acute GVHD and severe chronic GVHD, according to which one occurred first. We analyzed GRFS according to donor type. Moreover, prediction by dynamiclandmarkingwas used for continual reassessment of GRFS within the next 2 years following subsequent landmark times from 0 to 3 years after HSCT.Results: 188 pts (MRD, n=142; MUD n=46) with a median age of 30 years (18-67) were analyzed. After a median follow-up of 52 months (3-159), 2-year GRFS were 75% and 54% in pts receiving MRD and MUD HSCT, respectively (p=0.006, Figure 1A). In Causes of GRFS failure in MRD vs. MUD were grade 3-4 acute GVHD (5% vs 20%, p=0.002), extensive chronic GVHD (3% vs 4%, p=0.902), graft failure/loss (10% vs 4%, p=0.236) and death before the previous events (7% vs 18%, p=0.057). The use of peripheral blood graft (PBSC) was associated with lower 2-GRFS compared to bone marrow (51% vs 74%, p=0.004).Multivariate Cox model showed that MUD (p<0.001), age (continuous variable, p=0.001) were associated with worse GRFS, with a significant interaction between these both variables (interaction p-value: 0.008, Figure 1B). PBSC as graft source (p=0.096) and positive donor and/or recipient CMVsero-status (p=0.095) tended to decrease GRFS. Although the pts in the MUD group initially have worse overall GRFS, prediction by dynamiclandmarkingshowed that after 6 months post HSCT, they recovered the same GRFS as those transplanted with MRD. Indeed, the probabilities to stay event-free within 2 years following the 6-month landmark time was 89% and 86% in the MRD and MUD groups, respectively (Figure 2).Conclusion: GRFS after MRD HSCT is very good, with 75% of pts who did not present any events, supporting upfront HSCT for young patients with available MRD. In contrast, the higher incidence of acute GVHD (20% vs. 5%) after MUD significantly decreased GRFS compared to MRD. However, MUD pts surviving the early 6 months without may reach similar GRFS than pts transplanted with MRD. Taken together, these results suggest that although overall GRFS is good with low incidence of late events, early events such as acute GVHD remains a major cause of GRFS failure. This supports that the early post HSCT period remains a critical phase for the future good quality survival, underlining the importance of graft source choice as well as the need of GVHD prophylaxis improvement. [Display omitted] [Display omitted] DisclosuresPeffault de Latour:Amgen: Research Funding; Alexion: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Reversal of Low Donor Chimerism Following Hematopoietic Cell Transplantation Using Pentostatin and Donor Lymphocyte Infusion

Objectives: Low donor chimerism after hematopoietic cell transplantation (HCT) is associated with increased risk of post transplant relapse. Our group has shown that donor lymphocyte infusion (DLI) was ineffective for converting <40% donor CD3 chimerism to full donor chimerism. If successful, however, donor CD3 chimerism >90% elicited GVT effects (Blood 2004;103:790). To facilitate effectiveness of DLI for patients (pts) with low or declining donor CD3chimerism, a prospective multicenter trial assessing the safety and efficacy of addingpentostatin was developed.Patients and Methods: Pts at risk for rejection after HCTs, defined as low (<50%) but persistent (>5%) donor CD3 chimerism and with stable/in remission disease, were included. Pts were excluded if they had evidence of relapse/progression, ongoing grades II-IV acute (a)GVHD, or extensive chronic (ec)GVHD. Between 2003 and 2014, 36 pts were treated on the study; 35 pts received a total of 41 DLIs following a dose of pentostatin, and 1 pt received pentostatin only. Diagnoses included AML (n=13), NHL (n=4), CLL (n=7), CML (n=2), MDS (n=6), MPD (n=2), MM (n=1), NHL and MDS (n=1). Pts received nonmyeloablative (n= 35) or ablative (n=1) conditioning, followed by PBSCs from HLA-matched related (n=17) or unrelated (n=17) donors, or 1-allele mismatched unrelated donor (n=2). Median age at HCT was 58 (34-72) years. DLI was given at a median of 96 (54-339) days after HCT. A dose of pentostatin was given 2 days before infusing 1x107 CD3 cells/kg (n=26), 2x107 CD3 cells/kg (n=1), or 3x107 CD3 cells/kg (n=14). Per protocol, prophylactic immunosuppression (IS) with cyclosporine and MMF was given after DLI to the last 6 pts on study.Results: Median donor CD3chimerism beforepentostatin/DLI was 28 (5-47)%. Efficacy, defined by increases in donor CD3chimerism>10% maintained to day 56 post-DLI, was seen in 16 pts (44.4%) with a median CD3 donorchimerism of 64 (48-100)%. There was a trend for better efficacy among the 21 pts who received first treatment within 100 days after transplant (57%) compared to the 15 pts who received first treatment more than 100 days after HCT (27%) (p=0.07). Six pts received 2nd treatment ofpentostatin/DLI; among them 1 responded. Fifteen pts (12 responders) developedaGVHD after DLI [grade I (n=2), II (n=9), III (n=3), IV (n=1)] at a median of 10 (0-83) days after DLI. One pt developed skinGVHD, grade II, afterpentostatin administration and DLI was aborted. Of the 16 pts who developedaGVHD afterpentostatin/DLI, 3 had a prior history of GVHD after transplant. Thirteen pts developedcGVHD, ofwhom 10 developedecGVHD at a median of 112 (13-347) days after DLI. One pt developedecGVHD with no prioraGVHD. Among the 6 pts who received prophylaxis IS after DLI 1 pt developedaGVHD grade II, and none developedcGVHD. However, only 2 of those 6 pts responded. Seventeen pts developed grade 4cytopenia after DLI; 9 pts developed both neutropenia and thrombocytopenia, 4 pts developed neutropenia, and 4 pts developed thrombocytopenia. Among the 13 patients who developed grade 4 neutropenia, 9 (69%) were non-responders. Median time between DLI and lowest ANC count was 22 (2-100) days, and neutropenia lasted for median of 7 (1-52) days. Twelve pts received platelet transfusions started at median of 33 (1-98) days after DLI. Nineteen pts relapsed at a median of 91 (26-777) days after DLI. Among the 20 non-responders 12 relapsed (60%), compared to 7 of 16 responders (44%) (HR 0.64 (0.3-1.6), p=0.35). Ten of the 17 pts (59%) who developedcytopenia relapsed, 6 ofwhom (60%) were non-responders. Median number of significant infections was 2 (0-10) per pt. Twenty-eight pts died at a median of 522 (67-2028) days after DLI. Causes of death were relapse (n=21), respiratory failure/infections (n=4), grade IV GVHD (n=2), or other causes (n=1). Five of the 16 responders (31%) are alive, all in CR. Six of the 20 non-responders (30%) are alive, only 3 in CR (Figure 1).Summary:Pentostatin/DLI increased donor CD3chimerism in pts after HCT if was given within 100 days after transplant, and it was well tolerated. Response was associated with GVHD. Delayed treatment after HCT and prophylactic IS after DLI were associated with decreased efficacy. There was a trend for higher relapse rate among the non-responders and pts who developed cytopenia, but the differences were not statistically significant. [Display omitted] DisclosuresPulsipher:Novartis: Consultancy, Other: Advisory Board, Steering Committee for Phase II Study; Jazz Pharmaceutical: Consultancy, Other: Advisory Board; Chimerix: Consultancy, Other: Advisory Board ; Medac: Other: Travel support for a study group.

Murine Donor 1928z CAR T Cells Exert Potent Graft-Versus-Lymphoma Activity without Graft-Versus-Host-Disease

Chimeric antigen receptors (CARs) promote tumor cell target recognition of adoptively transferred T cellsand clinical studies have shown that autologous T cells transduced with CARs targeting CD19, expressed on normal B cells and several B cell malignancies, are effective against CD19+ malignancies. The potential of donor-derived CD19-CAR T cells to treat relapse after allogeneic hematopoietic stem cell transplantation (alloHSCT) has been relatively unexplored due to concerns for graft-versus-host disease (GVHD) and other toxicities. However, recent clinical trials of donor-derived CD19-CAR T cells in allo-HSCT have noted a surprisingly low incidence of GVHD. We studied the GVHD and anti-tumor potential of donor-derived mouse CD19-specific CAR 1928z (m1928z) T cells in preclinical allo-HSCT and lymphoma models.We constructed a panel of retroviral vectors targeting mouse CD19 (Figure 1A) to test various aspects of CAR stimulation and co-stimulation and generated T cells expressing these constructs. We then used these constructs in mouse allo-HSCT models comprising BALB/c recipients and B6 donors (Figure 1B).A20 lymphoma-inoculated BALB/c recipients of B6 BM treated with donor B6 m1928z T cells had significantly higher tumor-free survival and improved survival compared to m19delta treated recipients (Figure 1C, P < 0.0001). Adoptive transfer of B6 m1928z T cells caused significantly less GVHD compared to B6 m19delta T cells (P < 0.001) in BALB/c recipients of B6 BM confirming that m1928z T cells cause less GVHD. We explored the requirement of CAR signaling for attenuated GVHD using hum1928z (P < 0.001) (anti-human CD19-CAR, non reactive in mice) T cells and noted significantly worse survival and GVHD scores in mice as compared to with m1928z indicating that CAR signaling was necessary for this protection (Figure 1D). We also examined the importance of co-stimulation and noted increased GVHD mediated by m19z T cells (P < 0.05) (CAR with no co-stimulation) (Figure 1E) and m19BBz (P < 0.01) (CAR with 4-1BB co-stimulation) (Figure 1F) indicating that CD28 co-stimulation was also vital for attenuated GVHD. To study the effect of concurrent TCR and CAR signaling in vitro, we used T cells engineered to co-express 1928z CAR and OT-1 TCR in a cold-target inhibition killing assay and noted that activation of the T cells through 1928z can impair lytic activity mediated by TCR. To elucidate the consequence of concurrent TCR and CAR activation, we studied the TCR signaling pathway and found increased phosphorylated PKCa, pERK1/2, pS6, pSTAT1, pSTAT3, and pSTAT5 in donor m1928z T cells compared to m19delta T cells, indicating a hyperactivated state. In order to further study the effect of concurrent stimulation of alloreactive TCR and m1928z CAR, we utilized B6 ABM-RAG1 knockout (ABM) T cells, which express monoclonal TCR specific to I-Abm12 expressed on B6.C-H2bm12 (BM12) mice. In accordance with our previous findings, we observed significantly less GVHD in allo-HSCT BM12 recipients of ABM m1928z T cells versus m19delta T cells (P < 0.0001). We performed global transcriptional analysis of ABM m1928z T cells and m19delta harvested from adoptively transferred BM12 recipients of B6 BM and differential upregulation of Bim, Bid, FasL and Apaf1 in m1928z T cells. Phenotypic analysis of these cells also showed increased PD-1 and Fas expression, consistent with post-stimulatory programmed cell deathIn conclusion, we show that donor m1928z T cells mediate strong GVT effects with decreased GVHD. Following adoptive transfer, alloreactive m1928z T cells (but not m19BBz) undergo progressive loss of effector function and proliferative potential, and eventual deletion, which significantly decreases graft-versus-host activity. The non-alloreactive m1928z T cells present in bulk donor T cell populations retain significant anti-lymphoma activity. Our findings support the administration of donor 1928z T cells to prevent relapse of CD19+ malignancies after allo-HSCT and provide mechanistic insight into recent clinical trial results demonstrating that allogeneic CD19-specific 1928z CAR T cells promote anti-lymphoma activity while causing minimal GVHD. [Display omitted] DisclosuresDavila:Adaptive Biotechnologies: Membership on an entity’s Board of Directors or advisory committees; Celyad: Consultancy; Precision Biosciences: Membership on an entity’s Board of Directors or advisory committees; Kite Pharma: Membership on an entity’s Board of Directors or advisory committees; Servier: Membership on an entity’s Board of Directors or advisory committees. Sadelain:Juno Therapeutics: Consultancy, Equity Ownership, Patents & Royalties.

CTLA-4 polymorphisms and haplotype correlate with survival in ALL after allogeneic stem cell transplantation from related HLA-haplotype-mismatched donor.

BackgroundAllogeneic hematopoietic stem cell transplantation (allo-HSCT) has been established as an effective treatment for patients with hematological malignancies. Disease relapse remains a major cause of transplant failure. T cell homeostasis is critical to determine the potency of the GVT effect. Recent studies have shown the association of the CTLA-4 polymorphisms with the outcome after HLA-identical sibling allogeneic HSCT.MethodsIn this study, we focused on four CTLA-4 polymorphisms, and analyzed the impact of donor genotypes and haplotypes on the conditions of 152 acute leukemia patients (ALL 83) after related HLA-haplotype- mismatched transplantation. The four SNP genotypes (−1661, −318, CT60 and +49) were determined by TaqMan SNP genotyping assays.ResultsALL recipients of donors with +49 GG showed significantly lower OS (67.7 vs. 90.3 %, P = 0.015) than those with GA+AA. Multivariate analyses showed that +49 GG was an independent risk factor for OS (HR: 0.306, 95 % CI 0.111–0.842, P = 0.022) .23 ALL patients receiving mDLI showed significantly lower OS with +49 GG donor than those with GA+AA (30.0 vs. 83.1 %, P = 0.003). The haplotype analysis revealed only three haplotypes in the donor population −1661/−318/CT60/+49 i.e., ACGG, ACAA and GTGA, the frequencies were 64.1, 19.4 and 16.5 %, respectively. Donors with and without the ACGG/ACGG haplotype had the same effect on transplant outcomes as those with +49 GG and +49 GA+AA.ConclusionIn summary, the CTLA-4 +49 GG and the haplotype ACGG/ACGG reduced the overall survival in ALL after allo-HSCT from the related HLA-haplotype-mismatched donor, knowledge of the CTLA-4 polymorphism and haplotype may provide useful information for donor selection and individual application of immunosuppressive agents and immunotherapy.

CTLA-4 Polymorphisms and Haplotype Correlate with Survival and aGVHD after Allogeneic Stem Cell Transplantation from Related HLA-Haplotype-Mismatched Donor

Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been established as an effective treatment for patients with hematological malignancies. Disease relapse remains a major cause of transplant failure.T cell homeostasis is critical to determine the potency of the GVT effect. Cytotoxic T lymphocyte antigen-4 (CTLA-4 or CD152) is a T cell activation negative regulator. Recent studies have shown the association of the CTLA-4 polymorphisms with the outcome after HLA-identical sibling allogeneic HSCT.Patients and Methods: In this study, we focused on four CTLA-4 polymorphisms, and analyzed the impact of donor genotypes and haplotypes on the conditions of 154 acute leukemia patients after related HLA-haplotype-mismatched transplantation. The four SNP genotypes (-1661, -318, CT60 and +49) were determined by TaqMan SNP genotyping assays.Results: Recipients of donors with +49 GG showed significantly lower OS (69.1% vs. 85.6%, P=0.024) and higher incidence of III-IV aGVHD (10.0% vs. 2.1%, P=0.032) than those with GA + AA(Fig.1,Fig.2). Multivariate analyses showed that +49GG was an independent risk factor for OS (HR:0.457,95%CI=0.227-0.920,P=0.028). Patients receiving mDLI showed significantly lower OS with +49 GG donor than those with AG+AA (P=0.011).The haplotype analysis revealed only three haplotypes in the donor population -1661/-318/CT60/+49 i.e.,ACGG,ACAA and GTGA,the frequencies were 64.3%, 19.5%, and 16.2%, respectively.Donors with and without the ACGG/ACGG haplotype had the same effect on transplant outcome as those with +49 GG and +49 AG+AA.Conclusion: The CTLA-4 +49 GG and the haplotype ACGG/ACGG reduced the overall survival and increased the aGVHD after allo-HSCT from the related HLA-haplotype-mismatched donor,knowledge of the CTLA-4 polymorphism and haplotype may provide useful information for donor selection and individual application of immunosuppressive agents and immunotherapy.CONFLICT OF INTERESTThe authors declare no conflict of interest. DisclosuresNo relevant conflicts of interest to declare.

Allogeneic Stem Cell Transplant Can Produce Long-Term Remission, &amp; GVT Effect Exists in Myeloma

Allogeneic Stem Cell Transplant Can Produce Long-Term Remission, &amp; GVT Effect Exists in Myeloma

Non-mitogenic form of acidic fibroblast growth factor protects against graft-versus-host disease without accelerating leukemia.

Acid fibroblast growth factor (aFGF) has been shown to prevent epithelial damage under various conditions, suggesting its potential to inhibit GVHD. However, because aFGF receptors are expressed on tumor cells, it may possibly offset the graft-vs.-tumor (GVT) effects of allogeneic bone marrow transplantation (allo-BMT). Here, we addressed these questions in a B6→B6D2F1 allo-BMT model. Although aFGF administration attenuated GVHD in non-leukemic recipients, aFGF treatment markedly accelerated death in mice that received recipient-type tumor (P815) cells along with allo- or syngeneic-BMT. Similar protection against GVHD was achieved by administration of a non-mitogenic form of aFGF (naFGF). Importantly, GVT effects were fully preserved in naFGF-treated recipients. Furthermore, aFGF, but not naFGF, significantly enhanced P815 cell proliferation both in vitro and in vivo. Our data indicate that the tumor-promoting, but not GVHD-protecting, effect of aFGF largely depends on its mitogenic activity, and suggest that naFGF may provide a safer approach to inhibiting GVHD in patients with malignancies.

Granzyme B plays a differential role in CD4+ T cell subsets versus CD8+ T cells in mediating the graft-versus-tumor effect and graft-versus-host disease (TRAN3P.888)

Abstract After allogeneic hematopoietic cell transplantation, donor-derived T cells can attack host tumor cells, producing a favorable graft-vs.-tumor (GVT) effect. However, graft-vs.-host disease (GVHD) may develop when donor T cells attack normal host tissue. We previously reported that while granzyme B (GzmB) is required for CD8+ T cells to cause GVHD, GzmB-mediated damage of CD8+ T cells impairs the GVT effect. In this study, we examined the role of GzmB in GVT and GVHD mediated by CD4+ T cell subsets, using an established A20 tumor model and bioluminescence imaging to monitor tumor growth in BALB/c hosts. We found that CD4+CD25+ regulatory T cells (Tregs) derived from C57BL/6 donors significantly suppressed GVT activity mediated by both CD8+ T cells and CD4+CD25- effector T cells (Teffs). However, GzmB-/- Tregs were as equally suppressive as WT Tregs, consistent with our previous report that GzmB is not required for Treg-mediated suppression of GVHD. Nonetheless, GzmB-/- Teffs were less effective in controlling tumor growth than WT Teffs; but surprisingly, GzmB-/- Teffs caused more severe GVHD than WT Teffs. Our data reveal that GzmB is essential for CD4+CD25- Teff-mediated optimal GVT effect but diminishes the ability of Teffs to cause GVHD, which contradicts its role in CD8+ T cells where GzmB is essential to cause GVHD but impairs GVT effect. These differential roles suggest that targeting GzmB in selected T cell subsets may provide a strategy to separate GVT from GVHD.

Sensitivity of hematological malignancies to graft-versus-host effects: an EBMT megafile analysis.

After allogeneic stem cell transplantation, graft-versus-host disease (GvHD) occurs through recognition of histocompatibility mismatches by donor T lymphocytes. The same mechanism operates in eliminating malignant cells (the graft-versus-tumor or GvT effect). We hypothesized that comparing the correlation between GvHD and relapse might provide a surrogate marker for the susceptibility of diseases to allo-immune effects. We studied 48 111 first allogeneic transplants performed between 1998 and 2007. In chronic myeloid leukemia (CML), the relapse risk declined clearly and proportionally to severity of acute and chronic GvHD. Acute lymphoblastic leukemia and BCR-ABL-negative myeloproliferative neoplasias were comparably sensitive to GvHD as CML, whereas myelodysplastic syndromes and lymphoproliferative disorders showed intermediate sensitivity. GvHD was only associated with modest reductions in relapse risk in acute myeloid leukemia (AML) and plasma cell disorders (PCDs). Except for PCD, hazard rates for relapse decreased to almost 0 at 48 months of follow-up in all diseases. These data confirm observations of potent GvT effects associated with GvHD. The strength of the GvHD/GvT correlation differs significantly between hematological malignancies. The parallel drop of relapse rates in different diseases despite differences in GvHD/GvT ratios suggests that GvT effects might operate in the absence of GvHD, particularly in AML.

A 2 Step Approach To Myeloablative Haploidentical Hematopoietic Stem Cell Transplantation (HSCT): Low Non-Relapse Related Mortality (NRM) and High Overall Survival (OS) Rates Confirmed In Second Generation Trial

Over 7 years ago, we developed a 2 step myeloablative approach to haploidentical HSCT in which patients, after conditioning with 12 Gy of total body irradiation (TBI days -9 to -6), receive a fixed dose of 2 x 108/kg of donor T cells (DLI-step 1 of HSCT) immediately after the last fraction of TBI. This large dose of haploidentical lymphocytes results in high fever, and in some cases, diarrhea and rash, developing on days-5 and -4. Cyclophosphamide (CY) 60 mg/kg, given on days -3 and -2 for T cell tolerization, results in complete resolution of this “alloreaction.” Tacrolimus and MMF are begun on day-1, followed by a CD 34 selected donor product infused on day 0 (Step 2 of HSCT). The separation of the lymphoid and myeloid portions of the graft (1) avoids the exposure of allogeneic stem cells to CY, (2) avoids the polarization of T cells to a TH2 phenotype because the donor T cells are collected prior to the initiation of G-CSF, (3) allows the administration of a fixed dose of T cells establishing a consistent platform from which to compare outcomes, and (4) provides a method to deliver higher doses of T cells. Recent data suggests that T cell doses > 1.1 x108/kg are associated with superior GVT effects as compared to lower T cell doses (Guo et al. JCO, 2012;30:4084 and Colvin et al. BBMT, 2009;15:421). To date, over 180 patients have undergone 2 step haploidentical myeloablative and reduced intensity HSCT at our institution. In the initial myeloablative haploidentical trial (2006-2009), twenty-seven patients underwent treatment using this approach. Immune reconstitution was brisk with median CD3/4 and CD3/8 counts at day +28 of 33.6 and 28.7 cells/ul respectively Cumulative incidences of grades III-IV graft versus host disease (GVHD), NRM, and relapse-related mortality were 7.4%, 22.2%, and 29.8% respectively. OS for the 12 patients without disease at HSCT was 75% and 48% for the whole cohort with 51-79 (median 63) months of follow-up.A 2nd generation myeloablative 2 step trial specific to patients without morphologic evidence of disease at the time of their haploidentical HSCT completed accrual in 2013. Twenty-eight patients with AML (15), ph+ ALL (4), B cell ALL (4), T cell ALL (2), MDS (1), mantle cell NHL (1), and hepatosplenic NHL (1), were treated and are 3-32 (median 14) months post HSCT. All patients engrafted and no patients died of GVHD or infection. Median CD3/4 and CD3/8 counts at 28 days were 74 and 47 cells/ul respectively. The only death related to treatment occurred in a patient who suffered a subdural hematoma after an LP performed for fever. Four patients have relapsed (14%), 3 of whom have died of their disease. The probability of OS of the patients treated on the current trial is 85% at 15 months. The combination of the most recently treated 28 patients with the 12 patients treated on the initial trial who were in morphologic CR at the time of HSCT, has resulted in a probable OS rate of 78% with 3 to 79 (median 18 months of follow-up).For patients without morphologic evidence of their disease at the time of HSCT, the 2 step myeloablative approach to haploidentical HSCT has been associated with robust immune reconstitution resulting in low rates of infectious death. There have been no deaths from GVHD, and very low rates of treatment-related mortality. OS rates of the good risk patients treated on this approach are very high due to this low degree of NRM. The results of this 2nd generation trial have confirmed that the 2 step myeloablative approach to haploidentical HSCT is safe and efficacious and produces outcomes that are comparable to those reported by CIBMTR (2000-2010) for recipients of matched related or other alternative donor grafts. This approach has become our alternative HSCT strategy of choice allowing virtually every patient to be treated with HSCT rapidly at our institution. Disclosures:Kasner: Roche: Research Funding.

Pre-Emptive T-Rapa Cell DLI for Therapy of High-Risk Lymphoma After Low-Intensity Allogeneic HCT

AbstractAbstract 471Lymphoma progression remains an obstacle after allogeneic HCT, particularly after non-myeloablative conditioning in patients with high-risk histology (non-indolent; Kahl et al; Blood, 2007) and chemotherapy refractory disease (Bishop et al; Cancer, 2010). In murine models, rapamycin-resistant T cells favorably modulated the balance between GVHD, graft rejection, and GVT effects. To translate this, we conducted a multi-center phase II trial (NCT0074490) of T-Rapa cells infused as a pre-emptive DLI after low-intensity allogeneic HCT; here, we report overall outcome of patients with high-risk lymphoma diagnoses, many of whom were chemotherapy refractory. T-Rapa cells were manufactured by ex vivo culture of donor CD4+ T cells using CD3/CD28 co-stimulation and IL-4, IL-2, and rapamycin for 12-days (T-Rapa12) or 6-days (T-Rapa6), and administered (2.5 × 107 cells/kg) at d14 post-HCT. Both populations of T-Rapa cells expressed a mixed Th2/Th1 phenotype with minimal T-Reg content. Patients (n=42) received outpatient-intensity chemotherapy (typically, EPOCH-FR) until CD4 count was < 200 cells/μl, and then received an HLA-matched sibling mobilized allograft and GVHD prophylaxis of cyclosporine plus short-course sirolimus (to d14 post-HCT); conditioning consisted of fludarabine (120 mg/m2) and cyclophosphamide (1200 mg/m2). Table I details the high-risk diagnoses, pre-treatment history (median of 4 prior regimens), remission status at time of HCT (7/42 [17%] in CR), and presence of chemotherapy refractory disease (stable or progressive disease to prior therapy: 23/42 pts, 55%). T-Rapa cell infusion was relatively safe, with no engraftment syndrome or d100 TRM; incidences of grade II-IV acute, late acute, and classical chronic GVHD were 17%, 34%, and 36%, respectively. Initial mixed donor/host chimerism converted to predominate donor chimerism after T-Rapa cell DLI (Table I). Overall median survival probability at 24 months post-HCT is 85.7% for patients with chemotherapy sensitive disease vs. 39.1% for patients with chemotherapy refractory disease (Fig. 1; p=0.0008). All deaths were due to progressive disease except for 2 infection-related deaths at days 162 and 359 post-HCT; all surviving patients are in CR. Survival was not statistically significantly influenced by DLI type (T-Rapa12 vs. T-Rapa6) or histology-type (NHL vs. HD). Pre-emptive DLI using donor T-Rapa cells after low-intensity conditioning is safe and very effective in patients with high-risk lymphoma diagnoses and chemotherapy sensitive disease; for such patients, future randomized trials should compare low-intensity T-Rapa cell therapy to other transplantation regimens. For patients with high-risk lymphoma diagnoses and chemotherapy-refractory disease, we are evaluating a modification to the current platform that incorporates high-dose sirolimus therapy.Table IPatient CharacteristicsChimerism ResultscCD3CD15Number of Patientsn = 42Day 14 post-HCT57 (12-97)46 (8-94)Age (median, range)44 (23-68)Day 28 post-HCT77 (37-100)76 (29-98)Sex (male/female)(26/16)# of Prior Therapies (mean, range)4 (1-6)Day 100 post-HCT89 (51-100)93 (35-100)Histologyan=26 total (62%)Survival Resultsd24 Mo. Surv. Prob.NHL Categoryn=12ChemoSens/T-R1278.6% (n=7)DLBCLn=5ChemoSens/T-R685.7% (n=12)DLBCL-trn=2ChemoRefr/T-R1241.7% (n=11)DLBCL-EBVn=3ChemoRefr/T-R636.4% (n=12)PlasmacytoidDCn=3All NHL57.4% (n=26)T Celln=16 total (38%)All HD/Grey Zone68.8% (n=16)HD Categoryn=12HDn=4Grey ZoneChemo. Responseb23/42 (55%)Refractory (SD/PD)19/42 (45%)Sensitive (PR/CR)Complete Remission7/42 (17%)At Time of HCTDLBCL, diffuse large B cell lymphoma; DLBCL-tr, transformed; DLBCL-EBV, Epstein Barr Virus related; Plasmacytoid DC, dendritic cell; HD, Hodgkins Disease; SD, stable disease; PD, progressive disease; PR/CR is partial or complete response.aGrey Zone Lymphoma and Hodgkins Disease were pooled for statistical analyses.bChemotherapyresponse, as determined after last regimen prior to study entry.cChimerism determined by VNTR-PCR at days 14, 28, and 100 post-HCT (mean and range of values shown); CD3, T cell chimerism; CD15, myeloid cell chimerism.dSurvival by Kaplan-Meier analysis; 24 month median survival probability values shown; T-R12 and T-R6 indicates recipient of T-Rapa cells manufactured for 12 days vs. 6 days. [Display omitted] Disclosures:Levine:TxCell: Consultancy, Membership on an entity’s Board of Directors or advisory committees; University of Pennsylvania: financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight, financial interest due to intellectual property and patents in the field of cell and gene therapy. Conflict of interest is managed in accordance with University of Pennsylvania policy and oversight Patents & Royalties. June:Novartis: Research Funding, institution owned patents have been licensed by Novartis, institution owned patents have been licensed by Novartis Patents & Royalties. Mato:Celgene, Milennium, Genentech, Seattle Genetics: Speakers Bureau.

Cross-Presentation by Host CD8α+dendritic Cells Augments Graft-Versus-Tumor Effect without Aggravating Graft-Versus-Host Disease After Allogeneic BMT

Abstract 310Host hematopoietic derived antigen presenting cells (APCs) play an important role in the induction of GVHD and GVT. While all of the host hematopoietic derived APCs express and directly present allo-antigens that drive GVHD and GVT responses, the tumor specific antigens (TSA) expressed only on the tumor cells but not on the host professional APCs. TSAs will therefore need to be cross-presented on host hematopoietic derived APCs to generate optimal tumor antigen specific GVT response. We therefore reasoned that absence of cross-presentation by host hematopoietic derived professional APCs will not affect allo-antigen driven GVHD severity but may reduce GVT driven by TSAs. Because cross-presentation of antigens is mediated primarily by CD8α+dendritic cell (DCs) subset, we hypothesized that that cross-presentation by host CD8α+DCs will be critical for optimal GVT without aggravating GVHD. We utilized wild type (WT) B6 and B6 Batf3−/− animals (that lack the CD8α+ DCs) as recipients in well characterized MHC matched, minor mismatched C3H.SW “type=”#_x0000_t75”>B6 model of acute GVHD. WT and Batf3−/− animals received 10 Gy and were transplanted with 5 × 105 purified splenic CD8+T cells and 5 × 106 BM from either syngeneic B6 or allogeneic C3H.SW donors. Consistent with the hypothesis, the WT and the Batf3−/− animals showed similar survival and clinical severity of GVHD (P = NS). The increase in mortality was associated with a significant increase in the histopathological damage of the GVHD target organs (liver and intestine).Next, to determine the effect of host CD8α DCs on GVT responses, WT and Batf3−/−mice were conditioned and transplanted as above with syngeneic B6 or the C3H.sw donors along with 2×104MBL-2 tumor cells (which syngeneic to the host and express TSAs in addition to the allo-antigens that can be recognized by the donor T cells). All of the allogeneic WT and Batf3−/− mice developed GVHD. However, while none (0%) of the WT B6 animals died with tumor, 80% of the Batf3 KO mice had tumors at death (P<0.01, see Table). When the tumor dose was increased over two folds, 40% of the allogeneic WT animals died with tumor while 100% of the Batf3 KO animals died with tumors (P<0.05, see Table). Similar results were obtained with different tumor (EL-4) and GVHD (BALB/c→B6) models ruling tumor and strain dependent artifacts.Because toll-like receptor 3 (TLR-3) is primarily, but not exclusively, expressed on CD8α+DC subsets and has also been implicated in cross-presentation, to further confirm the relevance of cross-presentation by host APC subsets in mediating GVT, we generated [WT-B6→B6Ly5.2] and [TLR3−/−B6→B6Ly5.2] chimeras and utilized them as allo-recipients along with or without 5×105 MBL-2 tumor cells. All of the allogeneic [WT-B6→B6Ly5.2] and [TLR3−/−B6→B6Ly5.2] animals demonstrated similar GVHD. However, while only 50% of allogeneic [WT-B6→B6 Ly5.2] animals died with tumor, 100% of allogeneic [TLR3−/−→B6Ly5.2] animals died with tumor (P<0.05). To further confirm the specificity of TLR-3 and to determine whether GVT effect can be augmented by enhancing cross-presentation, we treated the chimeras with either poly I:C (which is a TLR-3 specific ligand and shown to promote cross-presentation) at 50μg/mice (day 0 and 1) or diluent control along with the MBL2-tumors after allo-BMT. Treatment with poly I:C reduced tumor induced mortality in the allogeneic [WT-B6→B6 Ly5.2] animals (50% vs. 8%, P<0.01, see Table), but did not alter tumor induced death in the [TLR3−/−→B6 Ly5.2] animals (100% vs. 93%, P=NS). Collectively, these data demonstrate, for the first time to our knowledge, that modulating antigen presentation on host APC (CD8α+ DC) subsets can augment GVT without aggravating GVHD.DonorRecipient (n)Tumor (dose)Mortality from relapseB6B6 WTMBL-2 (0.2×105)100%C3H.swB6 WTMBL-2 (0.2×105)0%**C3H.swBatf3−/−MBL-2 (0.2×105)80%C3H.swB6 WTMBL-2 (0.5×105)40%*C3H.swBatf3−/−MBL-2 (0.5×105)100%C3H.sw[B6 WTàB6Ly5.2]MBL-2 (0.5×105)50%**C3H.sw[TLR3−/−à B6Ly5.2]MBL-2 (0.5×105)100%C3H.sw[B6 WTàB6Ly5.2] +PolyI:CMBL-2 (0.5×105)8%***C3H.sw[TLR3−/−àB6Ly5.2] +PolyI:CMBL-2 (0.5×105)93%*P<0.05,**P<0.01,***p<0.001 Disclosures:No relevant conflicts of interest to declare.

Over-Expression of TRAIL on Donor T Cells Enhances GVT and Suppresses Gvhd Via Elimination of Alloreactive T Cells and Host APC

Abstract 817Strategies to suppress GVHD are often associated with broader suppression of the immune system leading to a compromised GVT effect. Using experimental models, we have demonstrated a novel strategy to enhance GVT effects and explicitly suppress GVHD using genetically engineered T lineage cells over-expressing TNF-Related Apoptosis Inducing Ligand (TRAIL). TRAIL can induce apoptotic signals through death receptor (DR) 4 and 5 molecules (only DR5 in mice) expressed on target cells. Expression of DR5 is higher on certain tumors and can be enhanced on others using small molecules rendering them susceptible to TRAIL mediated killing. TRAIL is therefore an attractive candidate for genetic engineering of donor T cells to enhance their GVT potential. We evaluated the effect of TRAIL over-expression (TRAIL+) in donor T cells (mature and precursor) on GVHD and GVT. Mature T cells derived from donor B6 splenocytes were transduced with a lentiviral TRAIL expression vector. The transduced TRAIL+ T cells were adoptively transferred on day 0 into lethally irradiated CBF1 recipients of T cell depleted allografts and LB27.4 tumor (B6 ^ CBF1+LB27.4) to assess their GVHD and GVT activity. TRAIL+ T cells displayed significantly enhanced antitumor immunity compared to T cells transduced with a control vector against LB27.4 tumor cell lines in vitro and upon transfer into tumor bearing allo-BMT recipients (p<0.01, 100% survival in TRAIL+ T cell group) (Fig 1A, also shown at the annual meeting last year). Precursor (pre)T cells have the benefit of regenerating the T cell compartment without causing GVHD and being available for “off the shelf” use. We generated TRAIL+ preT cells from transduced B6 hematopoietic stem cells and expanded them using the OP9-DL1 co-culture system. Adoptive transfer of B6 TRAIL+ preT cells into syngeneic-transplanted BALB/c mice could reconstitute the T cell compartment with TRAIL-expressing T cells and caused enhanced antitumor activity (p<0.05) compared to mock (GFP)-transduced controls.Interestingly, in addition to enhanced GVT, the recipients treated with TRAIL+ T cells had significantly less GVHD lethality and morbidity (Fig1B). This was observed across multiple GVHD models (B6 ^ CBF1, B6^ BALB/c and B10.BR^ B6). To explore the factors contributing to TRAIL-mediated suppression of GVHD, we used animals deficient in DR5 (DR5ko) in our models of GVHD. We found that GVHD suppressive effects of TRAIL were lost when hosts were DR5ko or when DR5ko TRAIL+ T cells were adoptively transferred indicating that TRAIL+ T cells suppress GVHD by targeting both host and donor compartments. We observed a higher DR5 expression in host MHC-IIhi antigen presenting cells (APC) following total body irradiation, suggesting that TRAIL+ donor T cells could potently eliminate host APC, resulting in less GVHD. Further, on transferring wild type T cells into irradiated hosts, we found that alloreactive CD25+ T cells had a significantly higher DR5 expression compared to CD25− T cells. This indicates that TRAIL+ T cells can specifically target the alloreactive CD25+ T cells in order to suppress GVHD.Collectively, our data demonstrate that donor T cells genetically engineered to express TRAIL can enhance GVT effects and suppress the development of lethal GVHD in recipients of allo-HSCT. Our data suggests that his suppression of GVHD is mediated by the elimination the alloreactive donor T cells and the elimination of GVHD-promoting residual APC. Furthermore, we demonstrated that allogeneic ex vivo generated preT cells expressing TRAIL could mediate a strong protection against tumor challenge in syngeneic HSCT recipients. TRAIL over-expression thus represents a potential off the shelf approach to enhancing GVT in both allogeneic and autologous transplantation. Despite elimination of alloreactive donor T cells, TRAIL+ T cells demonstrated enhanced GVT by directly targeting DR5+ tumors in the absence of alloreactivity. [Display omitted] Disclosures:No relevant conflicts of interest to declare.