Abstract: Background: Mantle cell lymphoma (MCL) accounts for approximately 5-7% of non-Hodgkin lymphomas (NHL). Bruton's tyrosine kinase (BTK) inhibitors, eg ibrutinib, are indicated for treatment of adults with MCL who have received ≥1 prior therapy. Primary and acquired resistance to ibrutinib is common and linked with poor outcomes, and remains an unmet medical need. Parsaclisib, a potent, highly-selective, next-generation PI3Kδ inhibitor, demonstrated clinical activity in patients (pts) with relapsed or refractory (R/R) NHL. We report preliminary results for parsaclisib monotherapy in a cohort of pts with R/R MCL who were previously treated with ibrutinib in the open-label, phase 2 study CITADEL-205 (NCT03235544). Methods: Pts must be ≥18 years of age with pathologically confirmed MCL, R/R disease to the most recent treatment, documented cyclin D1 overexpression or t(11;14) translocation, have Eastern Cooperative Oncology Group performance status (ECOG PS) ≤2, and received 1 to 3 prior systemic treatments (including ibrutinib). Prior treatment with PI3Ki was prohibited. Pts were allocated to receive parsaclisib 20 mg once daily (QD) for 8 weeks followed by either 20 mg once weekly (weekly-dosing group [WG]) or 2.5 mg QD (daily-dosing group [DG] ). Prophylaxis for Pneumocystis jirovecii pneumonia (PJP) was required. Objective response rate (ORR) was the primary endpoint; complete response rate (CRR), duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety and tolerability were secondary endpoints. All radiology-based endpoints were based on independent review. Results: From October 2017 to January 17, 2020 (data cut-off), 47 pts were treated (WG, n = 12; DG, n = 35). Enrollment is ongoing. At cut-off, 70% of pts had discontinued treatment, most commonly due to progressive disease (62%). Median exposure (range) was 2.2 (0.1-18.0) months. Median age was 70 years and 79% of pts were men. Median time since initial diagnosis was 4.7 years. Most pts (85%) had ECOG PS ≤1 and 51% had high-risk MCL International Prognostic Index. The median number of prior systemic therapies was 3; 38% of pts had prior hematopoietic stem cell transplant, and 38% were refractory to most recent systemic therapy. At data cut-off, 46 pts were evaluable for efficacy, including 12 in WG and 34 in DG (Table). Median follow-up (range) for this population was 10.2 months (1.5-25.9) overall and 7.6 months (1.5-25.9) for DG. ORR (95% confidence interval [CI]) and CRR were 28.3% (16.0-43.5) and 2.2%, respectively in all evaluable pts, and 35.3% (19.7-53.5) and 2.9%, respectively in DG. Median time to complete or partial response was 7.6 weeks. Median DOR (95% CI) was 7.3 months (0.2-not estimable) among all responders and 3.7 months (0.2-7.3) for DG. Median PFS (95% CI) was 3.65 months (1.9-3.9) overall and 3.65 months (1.9-5.5) for DG. Among 47 safety-evaluable pts, most common treatment-emergent adverse events (TEAEs) occurring in & gt;10% of pts were anemia (19.1%), diarrhea (19.1%), neutropenia (14.9%), asthenia and cough (12.8% each), decreased appetite, dyspnea, fatigue, pyrexia and rash (10.6% each). Most common grade ≥3 TEAEs reported in ≥5% of pts included anemia (12.8%), neutropenia (10.6%), thrombocytopenia and rash (6.4% each). TEAEs leading to dose interruption or reduction occurred in 31.9% and 2.1% of pts, respectively. TEAEs leading to treatment discontinuation occurred in 2 (4.3%) pts (diarrhea and colitis). Serious TEAEs reported in ≥2 pts were diarrhea, dyspnea, peripheral swelling and pneumonia (4.3% each). Two pts experienced fatal TEAEs (one pt had general physical health deterioration and respiratory tract infection, deemed not related to treatment; one pt had pneumonia, neutropenia, and septic shock, deemed related to treatment and disease progression). New or worsening grade ≥3 laboratory test values of clinical interest occurring in ≥5% of pts included decreased neutrophils (14.9%), platelets (10.6%), and hemoglobin (8.5%); there were no grade ≥3 increases in alanine/aspartate aminotransferase. Conclusion: Preliminary efficacy data indicate that parsaclisib monotherapy is clinically active in this difficult-to-treat patient population. Treatment with parsaclisib had an acceptable safety profile and was generally well tolerated. Updated study results will be presented. Disclosures Zinzani: EUSA Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kyowa Kirin: Consultancy, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Immune Design: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Sandoz: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Portola: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celltrion: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Verastem: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Immune Design: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kirin Kyowa: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Therapeutics, Inc.: Honoraria, Speakers Bureau; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Eusapharma: Consultancy, Speakers Bureau. Delwail:Amgen: Consultancy. Paneesha:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Gilead: Honoraria; Janssen: Honoraria; AbbVie: Honoraria. Rule:AstraZeneca: Consultancy; Celgene: Consultancy; Celltrion: Consultancy; Janssen Oncology: Consultancy, Research Funding, Speakers Bureau; Roche Pharma AG: Consultancy, Research Funding. Martin Garcia-Sancho:Roche, Celgene, Janssen, Servier, Gilead: Honoraria; Celgene, Eusa Pharma, Gilead, iQuone, Kyowa Kirin, Roche, Morphosys: Consultancy. Salles:Amgen: Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Gilead Sciences: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Servier: Consultancy, Honoraria; Acerta Pharma: Consultancy; Kite Pharma: Consultancy; Merck: Consultancy, Research Funding; Novimmune: Consultancy; Pfizer: Consultancy; Sanofi: Other. Sancho:Sandoz: Consultancy; Celltrion: Consultancy; Roche: Consultancy, Honoraria; Takeda: Honoraria; Novartis: Consultancy, Honoraria; Kern-Pharma: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Bristol-Myers Squibb: Honoraria. Zheng:Incyte: Current Employment, Current equity holder in publicly-traded company. DeMarini:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Jiang:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Mehta:Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Gelgene/BMS: Research Funding; Affimed: Research Funding; Merck: Research Funding; Kite/Gilead: Research Funding; Juno Parmaceuticals/BMS: Research Funding; Innate Pharmaceuticals: Research Funding; Oncotartis: Research Funding; Roche-Genentech: Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; fortyseven Inc/Gilead: Research Funding; Takeda: Research Funding.

Abstract: Background: Understanding genetic predisposition to cancer is of paramount importance to tailor therapeutic strategies. Several defects in immune checkpoint regulators have been discovered in patients with EBV-induced lymphoma (e.g. CD27, PRKCD, RASGRP1, MAGT1, SH2D1A, ITK). Here, we describe the clinical and immune phenotype of 2 unrelated patients from consanguineous families presenting with a primary immune deficiency (PID) and EBV-associated lymphoproliferation due to biallelic mutations in CD137 (TNFRSF9/4-1BB). Methods: One patient of Turkish origin (P1) and Palestinian origin (P2), respectively were evaluated. Genetic analysis using whole exome sequencing was conducted. Immunological and biochemical assays were performed on primary patient material. Results: Clinical findings included recurrent sinopulmonary and herpes virus infections from childhood on. One patient suffered from auto-immunity (AIHA and auto-immune thrombocytopenia). Abnormal immunoglobulin levels were documented (IgG 413-1670, IgM 105-714, IgA 49-68 mg/dL). Both patients developed EBV-associated lymphoproliferative disorders: P1 developed Burkitt lymphoma and was treated according to NHL-BFM2000 regimen in combination with rituximab. He is currently in remission. P2 had monoclonal EBV-positive lymphoproliferation and was successfully treated with immunosuppressive therapy (e.g. cellcept, glucocorticoids). To shed light on the underlying genetic etiology, we performed whole exome sequencing. A large homozygous deletion in CD137 (c.1_545+1716del) was identified for P1, while P2 harbored a homozygous missense mutation (c.C452T, p.Thr151Met). Impaired anti-CD3 T cell lymphocyte activation and proliferation were observed, amenable to correction upon addition of anti-CD28 monoclonal antibodies. In an attempt to provide definitive proof that the CD137 gene variant causes the activation defect of T-cells, we designed a genetic rescue experiment in patient T cells. Upon retrovirus-mediated recombinant expression of WT CD137, proliferation and activation defects in T cells were restored. Conclusions: In sum, we here show that a genetic defect in the immune checkpoint molecule CD137 causes a new primary immunodeficiency disorder with susceptibility to EBV-induced lymphomagenesis. Disclosures No relevant conflicts of interest to declare.

Abstract: The existence of circulating endothelial progenitor cells (EPC) in adult humans is under intensive investigation due to its potential clinical application. In the present study we have attempted to identify the EPC with colony forming capacity and compare their characteristics with those of the monocytic-macrophage lineage. 42 healthy donors were analysed (7 Bone Marrow, 20 steady-state Peripheral Blood (PB), 4 apheresis products from mobilised PB and 9 buffy-coat products). Median age was 38 years and M/F ratio was 19/23. EPC were obtained by culturing MNC in IMDM with VEGF and beta-FGF. At day 7 the colonies were counted and flow cytometry and immunohistochemistry studies were carried out. Sequential studies were performed on days +21, +28 and +35 of culture with vascular growth factors. Monocytic cells were obtained by adhesion to plastic surface or CD14+ cell selection using magnetic microbeads (Miltenyi, Biotech). Then, monocytic cells were cultured using the same conditions as EPC until day +21 or alternatively with VEGF, beta-FGF and IGF. Von Willebrand gene expression was analysed by RT-PCR and the formation of vascular structures was analyzed by Matrigel assays on both cell sources, EPC and monocytic cells. The mean number of EPC colonies at day 7 was significantly higher in BM (813±695) than in steady-state PB (21.2±2.5), while mobilized PB displayed intermediate values (272±274). By contrast, using the same medium as EPC, monocytic cells did not form colonies at day 7, but cord-like structures could be seen in 7 out 9 cases. However, when the cells were cultured by adding IGF to the medium, a greater number of colonies could be observed. By immunohistochemistry colonies were positive for CD45, CD31 and lysozyme but negative for vWf. Flow cytometry analysis showed that colonies were positive for CD4, CD13, CD14, CD31, CD33, CD45, lysozyme and VE-cadherin, weak positive for CD15 and CD105, and did not express CD16, CD34, CD133 or KDR. This phenotypic profile remained unchanged at all time-points analysed. The immunophenotype of cultured monocytes at day +21 was identical to that of pre-cultured monocytes and both were similar to those obtained in EPC, even for the “specific” markers lysozyme and VE-cadherin. PCR analysis showed small amounts of vWF transcripts in EPC as well as in monocytes but the expression increased after culture with VEGF. Finally, when matrigel assays were carried out, we observed that monocytes formed cord- and tubular-like structures to a higher extent than EPC. Our results in both cell subtypes suggest that the so “called” EPC belongs to the monocytic cell lineage and both populations express some “specific” endothelial antigens such as CD31 or VE-cadherin, as well as monocytic markers such as lysozyme.

Abstract: TNFRSF13B encodes transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), a B cell– specific tumor necrosis factor (TNF) receptor superfamily member. Both biallelic and monoallelic TNFRSF13B mutations were identified in patients with common variable immunodeficiency disorders. The genetic complexity and variable clinical presentation of TACI deficiency prompted us to evaluate the genetic, immunologic, and clinical condition in 50 individuals with TNFRSF13B alterations, following screening of 564 unrelated patients with hypogammaglobulinemia. We identified 13 new sequence variants. The most frequent TNFRSF13B variants (C104R and A181E; n = 39; 6.9%) were also present in a heterozygous state in 2% of 675 controls. All patients with biallelic mutations had hypogammaglobulinemia and nearly all showed impaired binding to a proliferation-inducing ligand (APRIL). However, the majority (n = 41; 82%) of the pa-tients carried monoallelic changes in TNFRSF13B. Presence of a heterozygous mutation was associated with antibody deficiency (P 〈 .001, relative risk 3.6). Heterozygosity for the most common mutation, C104R, was associated with disease (P 〈 .001, relative risk 4.2). Furthermore, heterozygosity for C104R was associated with low numbers of IgD−CD27+ B cells (P = .019), benign lymphoproliferation (P 〈 .001), and autoimmune complications (P = .001). These associations indicate that C104R heterozygosity increases the risk for common variable immunodeficiency disorders and influences clinical presentation.

Abstract: Somekh and colleagues identify CD137, a member of the tumor necrosis factor superfamily, as a novel cause of immunodeficiency associated with a risk of autoimmunity and lymphoid malignancy.

Abstract: Although the treatment paradigm for chronic lymphocytic leukemia (CLL) is rapidly changing, the disease remains incurable, except with allogeneic bone marrow transplantation, and resistance, relapsed disease, and partial responses persist as significant challenges. Recent studies have uncovered roles for epigenetic modification in the regulation of mechanisms contributing to malignant progression of CLL B cells. However, the extent to which epigenetic modifiers can be targeted for therapeutic benefit in CLL patients remains poorly explored. We report for the first time that expression of epigenetic modifier histone deacetylase 6 (HDAC6) is upregulated in CLL patient samples, cell lines, and euTCL1 transgenic mouse models compared with HDAC6 in normal controls. Genetic silencing of HDAC6 conferred survival benefit in euTCL1 mice. Administration of isoform-specific HDAC6 inhibitor ACY738 in the euTCL1 aging and adoptive transfer models deterred proliferation of CLL B cells, delayed disease onset via disruption of B-cell receptor signaling, and sensitized CLL B cells to apoptosis. Furthermore, coadministration of ACY738 and ibrutinib displayed synergistic cell kill against CLL cell lines and improved overall survival compared with either single agent in vivo. These results demonstrate for the first time the therapeutic efficacy of selective HDAC6 inhibition in preclinical CLL models and suggest a rationale for the clinical development of HDAC6 inhibitors for CLL treatment, either alone or in combination with Bruton tyrosine kinase inhibition.

Abstract: In recent years, the deeper understanding of the molecular alterations that occurs in MCL has provided the foundation for the development of new targeted therapeutic approaches. Histone deacetylases (HDACs), represent ideal targets due to their dual role not only in supporting lymphoma survival and resistance to apoptosis1,2, but also in immunologic escape and autocrine cytokine signaling which maintains the microenvironmental niche and facilitates drug resistance3,4. Recently, we have identified two members of the HDAC family, HDAC6 and HDAC3, which play integral roles in regulation of 1) survival/apoptosis, 2) microenvironment-mediated drug resistance, and 3) immunogenicity of MCL cells. Our studies to date have shown the following: First, HDAC6 and HDAC3 are overexpressed in MCL cell lines and primary cells from MCL patients. This expression is further up-regulated following adhesion of MCL cells to stromal cells, consequently increasing survival and acquisition of a drug-resistance phenotype. Second, genetic or pharmacologic inhibition of HDAC6 with isotype-selective inhibitor(s) induced cell cycle arrest, apoptosis and overcome stroma-mediated drug resistance. Of note, MCL cells lacking HDAC6 are more immunogenic and able of inducing stronger antitumor responses. This increased immunogenicity is due to a novel mechanism involving HDAC6 regulation of the immunosuppressive STAT3 signaling pathway and PD-L1 expression. Strikingly, mice lacking HDAC6 (germ-line HDAC6KO) displayed significantly reduced tumor growth kinetics when compared to the wild-type controls. Third, Genetic or pharmacologic disruption of HDAC3 resulted in decreased cell proliferation, increased apoptosis and reversal of the drug-resistance phenotype of MCL cells. This is due to HDAC3-mediated regulation of a group of small non-coding tumor suppressor microRNAs (miR-15a/16-1, miR-26 and miR29). The importance of HDAC3 as a promising therapeutic target in MCL is further highlighted by our recent findings using the HDAC3-selective inhibitor RGFP966 in human MCL cells, Four,strategies targeting both, HDAC6 and HDAC3 with selective inhibitors represent a promising strategy in MCL specially when these novel agents are combined with the BTK inhibitor Ibrutinib. Although inhibition of HDACs are extensively known for their ability to induce cell cycle arrest and apoptosis of malignant cells, their immuneregulatory effects upon tumor cell and/or immune cells are yet to be elucidated. In this study we present data that can convincingly provide a proper framework for combinatorial therapy using HDAC inhibitors in MCL. 1. Gupta M, et alLeukemia 2012; 26(6): 1356-64. 2. Kawamata N, et alBlood 2007; 110(7): 2667-73. 3. Lwin T, et alLeukemia 2007; 21(7): 1521-31. 4. Meads MB, et alNat Rev Cancer 2009; 9(9): 665-74. Disclosures Shah: Seattle Genetics, Inc.: Research Funding; NCCN: Consultancy; Celgene: Consultancy, Speakers Bureau; SWOG: Consultancy; Pharmacyclics: Consultancy; Janssen: Consultancy. Quayle:Acetylon Pharmaceuticals Inc.: Employment. Jones:Acetylon Pharmaceuticals Inc.: Employment.

Abstract: Co-inhibitory receptors and ligands such as PD-1, PD-L1, TIM3 and LAG3 are commonly over expressed in malignancies resulting in immune-surveillance evasion and generation of a tumorigenic microenvironment. Targeting of these molecules has become a new therapeutic strategy demonstrating promising and favorable clinical outcomes in a variety of human cancers. Although, inhibitory antibody therapy has been successful for many patients, other subsets of patients receive no clinical benefit. This is potentially due to the activation of redundant untargeted co-inhibitory pathways. In CLL, an immunosuppressive phenotype enables the malignant B-cell to evade immune detection and obtain a vast array of immune regulatory mechanisms, causing T-cell dysfunction and ultimately immunosuppression. In fact CD8+ effector and terminal memory subsets are skewed and over-express PD-1, LAG3 and TIM3 in a Eu-TCL1 CLL mice model. Epigenetic changes triggered by proteins such as histone deacetylases are gaining special attention particularly because of their active role in the regulation of pathogenesis and immune-related pathways in CLL; however the exact mechanism of action is yet to be elucidated. Recently, we have identified one member of the HDAC family, HDAC6 which plays a role in the regulation of immunogenicity in CLL. Utilizing CLL murine models, CLL patient samples, and CLL cell lines we have found the following: First, HDAC6 is overexpressed in human CLL Second, using selective HDAC6 inhibitor (ricolinostat) we have been able to modulate the expression of co-inhibitory molecules in CLL. Indeed, treatment of MEC1 cells with varying doses of ricolinostat for 48hrs resulted in decreased expression of PD-L1 Interestingly in primary B-cells isolated from CLL patient samples; PD-1 expression was decreased when exposed to various concentrations of ricolinostat for 48hrs. Third, silencing of HDAC6 in the Eu-TCL1 mice (Eu-TCL1/HDAC6KO mouse model) results in 1) significantly lower levels of PD-L1 expression and 2) a restoration of the T-cell CD4:CD8 ratio, from 8:10 (Eu-TCL1) to 1:1 (Eu-TCL1/HDAC6KO). Although the overall absolute count of T-cells was observed to be higher than the wild-type, the memory compartment was returned to mostly naive T-cells, doubling in numbers from 4% of total lymphocytes to 9%. Consequently, a total reduction in surface expression of co-inhibitory molecules was observed on all T-cell memory subsets, but with substantial reduction in the CD8+ effector and terminal memory phenotype. In conclusion, selective inhibition of HDAC6 in both B and T-cell compartment of CLL results in the reduction of co-inhibitory molecules, which in combination with subsequent therapeutic regimen could provide a successful immunotherapeutic strategy for this disease. Disclosures Quayle: Acetylon Pharmaceuticals: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals Inc.: Employment.

Abstract: Although thymic graft-versus-host-disease (tGVHD) has been recognized as an important contributor to impaired T cell reconstitution, limited T cell repertoire and increased infection risk in patients with GVHD, the molecular basis of interactions between donor alloreactive T cells, donor bone marrow (BM)-derived thymocytes, and host hematopoietic and non-hematopoietic thymic stromal cells in GVHD has not been well-defined. Here we analyzed the role of molecules relevant for T cell trafficking, cytolytic function, and co-stimulation and co-inhibition of alloreactive T cells in tGVHD. We first demonstrated that thymic output (as measured by RAG2+ splenic recent thymic emigrants) as well as the thymic cellularity (especially of CD4+CD8+ thymocytes) were inversely proportional to numbers of mature donor T cells infused with the allograft, suggesting that tGVHD severity was inversely associated with thymic function. We then studied the migration of alloreactive donor T cells in vivo with bioluminescence imaging (BLI) and found that luciferase-expressing donor T cells infiltrated the thymus within one week after allogeneic bone marrow transplantation (BMT) (Fig. 1). Upon adoptive transfer of CFSE-labeled donor T cells we noted that thymus-infiltrating alloreactive donor T cells were largely fast-proliferating (CFSElo) and highly activated (CD25+ CD44+). We analyzed the importance of T cell trafficking molecules for tGVHD using mice deficient for certain trafficking molecules, and assessed tGVHD by loss of BM-derived CD4+CD8+ thymocytes. We found that CCR9, b7 integrin subunit, and PSGL-1 were all partially required for tGVHD, while L-selectin and aE integrin subunit may be dispensable (Fig. 2A). Similarly, we examined the role of T cell cytolytic pathways for tGVHD, and found that FasL and TRAIL were required for tGVHD, but that perforin and TNF were dispensable (Fig. 2B). Finally, we assessed the role of various T cell co-stimulatory and co-inhibitory molecules for tGVHD, and found that CEACAM1, OX40 and CTLA4 were required, while GITR was partially required and ICOS was dispensable (Fig. 2C). Upon further analysis of donor BM-derived thymocytes, we observed that Bcl-2 expression in donor BM-derived thymocytes was decreased in recipients with GVHD vs. those without GVHD, which suggests that survival of thymocytes is decreased during tGVHD. Hollander and others have previously demonstrated in non-irradiated GVH reaction models that host non-hematopoietic thymic stroma may be an important target for donor alloreactive T cells. We assessed the expression of the death receptors Fas and DR5 in thymic stroma from normal and irradiated (850 cGy) BALB/c mice. We observed that in particular, MHC class II-negative stroma (endothelial cells and fibroblasts), as well as a population of MHC class II-intermediate stroma (epithelial cells) upregulated the expression of both Fas and DR5 after irradiation. Our study defines the specific pathways for cytolysis, trafficking and immune modulation involved in tGVHD and suggests selective therapeutic targets to attenuate tGVHD and improve post-transplant T-cell reconstitution in patients with GVHD. Fig 1. BLI demonstrate a distinct distribution pattern for alloreactive donor T cells in allogeneic BMT recipients, Allogeneic Balb/c recipients show a strong signal on day 4 post-transparent after transfer of 10×108 luc+ splenocytes as measured by total body photon emission. Ex vivo imaging confirms the infiltration of luc+ splenocytes to the thymus. Fig 1. BLI demonstrate a distinct distribution pattern for alloreactive donor T cells in allogeneic BMT recipients, Allogeneic Balb/c recipients show a strong signal on day 4 post-transparent after transfer of 10×108 luc+ splenocytes as measured by total body photon emission. Ex vivo imaging confirms the infiltration of luc+ splenocytes to the thymus. Fig 2. We assessed the role of molecules relevant for T cell trafficking (A), cytolytic function (B), and co-stimulation, co-inhibition (C). Irradiated BALB/c mice received 5×106 T cell depleted C57BL/6 bone marrow + 0.25×106 purified splenic T cells. Absolute numbers of donor-BM-derived CD4+CD8+ thymocytes are shown. Black bars indicate means. p-values were calculated vs. recipients of WT T cells(*p 〈 0.05, **p 〈 0.01) Fig 2. We assessed the role of molecules relevant for T cell trafficking (A), cytolytic function (B), and co-stimulation, co-inhibition (C). Irradiated BALB/c mice received 5×106 T cell depleted C57BL/6 bone marrow + 0.25×106 purified splenic T cells. Absolute numbers of donor-BM-derived CD4+CD8+ thymocytes are shown. Black bars indicate means. . / p-values were calculated vs. recipients of WT T cells(*p 〈 0.05, **p 〈 0.01)