ALBERT

Description: We have previously reported on the efficacy of the JAK1/2 inhibitor ruxolitinib in corticosteroid-refractory (SR) acute (a) and chronic (c) graft-versus-host disease (GVHD) in 95 patients (pts) (Leukemia 2015;29(10):2062-8). To assess long-term follow-up results, we collected data from the same pts treated in 19 centers in Europe and the US. Pts were classified as SR-aGVHD (n=54, all grade III or IV) or SR-cGvHD (n=41, all moderate or severe). Median numbers of pre-ruxolitinib GVHD treatment lines were 3 (1-7) and 3 (1-10) for SR-aGVHD and SR-cGvHD, respectively. The median follow-up was 19 and 24 months for aGVHD and cGVHD, respectively. The 1-year overall survival (OS) from was 62.4% (CI: 49.4%-75.4%) and 92.7% (CI: 84.7%-100%) for SR-aGVHD and SR-cGvHD, respectively. The estimated median OS (50% death) was 18 months for aGVHD and not reached for cGVHD patients. The median duration of ruxolitinib treatment was 5 and 10 months for patients with SR-aGVHD and SR-cGVHD, respectively reflecting the different biology of the diseases. At follow-up, 22/54 (41%) of SR-aGVHD patients and 10/41 (24%) of SR-cGVHD patients have an ongoing response and are free of any immunosuppression. GVHD relapse or progression after achieved PR/CR was observed in 14/45 (31%) and 13/36 (36%) patients with SR-aGVHD and SR-cGVHD, respectively. Response to re-treatment with Ruxolitinib or any immunosupressive therapy was seen in 11/14 (78%) and 11/13 (86%) patients with SR-aGVHD and SR-cGVHD, respectively. Cytopenia (any grade) and CMV-reactivation were observed during ruxolitinib-treatment in both SR-aGVHD (30/54, 55.6% and 18/54, 33.3%) and SR-cGVHD (7/41, 17.1% and 6/41, 14.6%) patients. These findings extend our previous report by showing that patients with SR-aGVHD and SR-cGVHD may benefit long-term from ruxolitinib treatment with an OS that is relatively high for steroid-refractory GVHD. GVHD-relapse or GVHD-progression rates were moderate and more than 75% of the relapse/progression patients responded to re-treatment with ruxolitinib or other immunosuppression. Disclosures Meyer: Stanford University: Patents & Royalties. Marks:Pfizer: Honoraria. Lübbert:Ratiopharm: Other: Study drug valproic acid; Celgene: Other: Travel Funding; Janssen-Cilag: Other: Travel Funding, Research Funding. Scheid:Novartis: Other: funding outside this work; Celgene: Other: funding outside this work; Janssen: Other: funding outside this work. Kobbe:Celgene: Honoraria, Other: travel support, Research Funding; Jansen: Honoraria, Other: travel support. Negrin:Stanford University: Patents & Royalties. Brune:Meda Pharma: Consultancy. Mielke:JAZZ Pharma: Speakers Bureau; Novartis: Consultancy; MSD: Consultancy, Other: Travel grants; Gilead: Other: Travel grants; Celgene: Other: Travel grants, Speakers Bureau. Kuball:Gadeta B.V,: Membership on an entity's Board of Directors or advisory committees. Kröger:Sanofi: Honoraria, Research Funding; Neovii: Honoraria, Research Funding; Riemser: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Peschel:MophoSys: Honoraria. von Bubnoff:BMS: Honoraria; Amgen: Honoraria; Novartis: Honoraria, Research Funding.

Description: Background: Allogeneic hematopoietic cell transplantation is a potentially curative therapy for patients with hematological malignancies. However a fraction of patients will develop corticosteroid-refractory (SR) acute (a) and chronic (c) graft-versus-host disease (GVHD) which both cause a high mortality and impaired quality of life. Pre-clinical evidence indicates the potent anti-inflammatory properties of the JAK1/2 inhibitor ruxolitinib by modification of T cells and dendritic cells. Methods: In this retrospective analysis, 19 stem cell transplant centers in Europe and the United States reported clinical outcome data from 95 patients who had received ruxolitinib as salvage-therapy for SR-GVHD. Patients were classified as having SR-aGVHD (n=54, all grade III or IV) or SR-cGvHD (n=41, all moderate or severe). The median number of previous GVHD-therapies was 3 for both SR-aGVHD (1-7) and SR-cGvHD (1-10). The median follow-up times were 26.5 (3-106) for SR-aGVHD and 22.4 (3-135) weeks for SR-cGVHD-patients. Results: The ORR was 81.5% (44/54) in SR-aGVHD including 25 CRs (46.3%), while for SR-cGVHD the ORR was 85.4% (35/41). The median time to response was 1.5 (1-11) and 3 (1-25) weeks after initiation of ruxolitinib treatment in SR-aGVHD and SR-cGVHD, respectively. Of those patients responding to ruxolitinib, the rate of GVHD-relapse was 6.8% (3/44) and 5.7% (2/35) for SR-aGVHD and SR-cGVHD, respectively. The 6-month-survival was 79% (67.3%-90.7%,95% CI) and 97.4% (92.3%-100%,95% CI) for SR-aGVHD and SR-cGVHD, respectively. Cytopenia and CMV reactivation were observed during ruxolitinib-treatment in both SR-aGVHD (30/54, 55.6% and 18/54, 33.3%) and SR-cGVHD (7/41, 17.1% and 6/41, 14.6%) patients. Relapse of the underlying malignancy occurred in 9.3% (5/54) and 2.4% (1/41) of the patients with SR-aGVHD or SR-cGVHD, respectively. Conclusion: Ruxolitinib constitutes a promising new treatment option for SR-aGVHD and SR-cGVHD. Its activity in SR-aGVHD and SR-cGVHD should be validated in a prospective trials in both, SR-aGvHD and cGvHD. Disclosures Bertz: GILEAD Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Scheid:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bug:TEVA Oncology, Astellas: Other: Travel Grant; NordMedica, Boehringer Ingelheim, Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene, Novartis: Research Funding.

Description: Chronic graft-versus-host disease (cGVHD) remains to be a major cause of mortality and morbidity after allogeneic hematopoietic cell transplantation (allo-HCT). cGVHD is characterized as autoimmune-like fibrosis and antibody production, mediated by pathogenic T and B cells. Through producing pro-inflammatory cytokines, CD4 T cells are the driving force of cGVHD. Donor B cells augment the pathogenesis of cGVHD not only by acting as antigen-presenting cells (APCs) and promoting CD4 T-cell expansion and survival, but also by producing autoantibodies. microRNA (miR)-17-92 has been shown to regulate T-cell immunity including allogeneic, anti-viral, and anti-tumor responses. Recently, miR-17-92 was found to act together with Bcl-6 to promote the differentiation of Follicular help T (Tfh) cells. Furthermore, B-cell deficiency of miR-17-92 impairs IgG2c production. Since Tfh differentiation and antibody production are required for the development of cGVHD, we hypothesize that miR-17-92 contributes to the pathogenesis of cGVHD by promoting pathogenic T- and B-cell responses. By using Cre-loxp system, we generated B6 mice with conditional deficiency of miR-17-92 in T cells (CD4cre), B cells (CD19cre), or both (CD4CD19cre). aGVHD to cGVHD transition model (B6 to BALB/c) was utilized to test the effects of individual and combinational deficiency of miR-17-92 in T and/or B cells in the development of cGVHD. BALB/c mice were lethally irradiated and transferred with splenocytes plus BM cells derived from CD4cre, CD19cre or CD4CD19cre miR-17-92flox/flox B6 mice. WT B6 (Cre- miR-17-92flox/flox) mice were used as control donors. A significantly reduction of GVHD mortality was observed only in the recipients with CD4CD19cre grafts, but not with CD4cre or CD19cre grafts. Deficiency of miR-17-92 in donor T or B cells indeed improved the clinical manifestation of cGVHD, but the deficiency in both T and B cells showed further improvement, indicating the additive role of miR-17-92 in T and B cells in the pathogenesis of cGVHD. Mechanistically, deficiency of miR-17-92 in T cells resulted in the reduction of Tfh generation (Fig. A), germinal center (GC) B-cell and plasma cell differentiation, and the expression of MHC-II and CD86 on donor B cells in recipient spleens. Furthermore, deficiency of miR-17-92 in B cells significantly reduced the levels of total IgG and IgG2c in recipient serum (Fig. A). These data suggest that miR-17-92 contributes to both T- and B-cell differentiation and function, which is required for the development of cGVHD. To extend our findings, we used a bronchiolitis obliterans cGVHD model (B6 to B10.BR). Recipient mice were pre-conditioned and received either BM alone from WT or CD19cre B6 mice, or BM plus purified T cells from WT or CD4cre B6 mice. Deficiency of miR-17-92 in T cells or BM-derived B cells resulted in significant improvement in pulmonary functions in recipient mice, as demonstrated by a decrease in resistance and elastance and an increase in compliance (Fig. B). Consistently, we found that miR-17-92 promoted Tfh and GC B-cell differentiation (Fig. B), while inhibiting differentiation of T follicular regulatory cells in recipient spleens 60 days after allo-HCT. For translational purpose, we tested whether inhibition of miR-17-92 could ameliorate cGVHD using locked nucleic acid (LNA) antagomirs specific for miR-17 or miR-19, key members in this microRNA cluster. In a SLE cGVHD model (DBA2 to BALB/c), administration of anti-miR-17, but not anti-miR-19, significantly suppressed the incidence of proteinuria and the severity of clinical manifestation by inhibiting donor splenocyte expansion, expression of costimulatory molecules on donor B cells, and differentiation of GC B cells and plasma cells (Fig. C). In addition, systemic delivery of anti-miR-17 significantly improved skin cGVHD by restraining IL-17 producing CD4 T-cell infiltration in skin-draining lymph nodes in a scleroderma-cGVHD model (B10.D2 to BALB/c). Taken together, the current work reveals that miR-17-92 is required for T- and B-cell differentiation and function, and thus for the development of cGVHD. Furthermore, pharmacological inhibition of miR-17 represents a potential therapeutic strategy for the control of cGVHD after allo-HCT. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Description: To study transcriptional pathways and develop targeted therapeutics for cGVHD, we studied the effects of emerging therapeutic agents targeting GCs. BCL6 is a master regulatory transcription factor, which facilitates GC development in a cooperative manner with emerging chromatin-associated factors, namely the EZH2 lysine methyltransferase and the BRD4 epigenetic reader protein. Using a murine multi-organ system cGVHD model induced by low T cell numbers, we have shown that cGVHD is dependent on pathogenic antibodies and the GC reaction. To determine whether strategies that disrupt GC integrity could be used to treat cGVHD we targeted Bcl6 using a direct-acting ligand (79-6), EZH2 using three structurally-distinct inhibitors (JQ-E, UNC1999 and DZNep), and a first-in-class BRD4 inhibitor (JQ1). In T cells, Bcl6 is selectively expressed in T follicular helper and regulatory cells. Using donor T cells from a Bcl6-mCherry reporter, we observed a 10-fold increase in % of CD4+/mCherry+ splenic T cells in cGVHD vs bone marrow (BM) only controls on d56. The small-molecule Bcl6 inhibitor 79-6 was synthesized and used to treat mice with active cGVHD from d28-56. Treatment reversed cGVHD-induced bronchiolitis obliterans (BO) measured by d56 pulmonary function tests (PFTs). This correlated with a 2-fold decrease in GC B cells and decrease in lung collagen (Fig 1 C). EZH2 catalyzes the methylation of lysine 27 on histone 3 (H3K4me3) silencing genes to a transcriptionally repressive state. EZH2 is massively upregulated during the GC reaction, and prevents GC B cell terminal differentiation, allowing affinity maturation to occur. Furthermore, naive T cells express low EZH2 levels, EZH2 is rapidly upregulated upon allostimulation.To determine whether EZH2 expression in donor BM or splenic T cells was critical for cGVHD, recipients were given wild type (WT) vs EZH2 floxed, Cg1-Cre (for B cell specific EZH2 deletion) BM + WT T cells, WT BM + EZH2 floxed, CD4-Cre vs WT T cells or WT BM + WT T cells. EZH2 deletion in either BM-derived B cells or splenic T cells completely prevented cGVHD (Fig 1 A) with a decrease in d56 splenic GC frequency (Fig 1 B). We compared 3 drugs that reduce H3K4me3: two pyridinone inhibitors, which inhibit SAM-dependent methylation (JQ-E and UNC1999) and DZNep, which destabilizes EZH2 complexes. At established doses given d28-56, DZNep and UNC1999 were ineffective or toxic, respectively. In contrast, JQ-E fully reversed cGVHD lung dysfunction and fibrosis around the bronchioles was significantly decreased (Fig 1 C). JQ1 is a first in class epigenetic reader that recognizes histone modifications and has been shown to reduce B cell lymphomas via inhibiting super-enhancer-associated transcripts and to treat cardiac failure-induced fibrosis. JQ1 given to cGVHD mice d28-56 significantly inhibited BO as assessed by PFTs and collagen deposition (Fig 1 D). Taken together, these data demonstrate for the first time the critical role of Bcl6 and targeting of histones that affect the transcriptional repressive states via EZH2 and a BET bromodomain epigenetic reader. These data provide a strong foundation for clinical trials of inhibitors that directly or epigenetic modifiers and readers that indirectly target Bcl6. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Key Points miR-17-92 mediates the progression of scleroderma and bronchiolitis obliterans in cGVHD by enhancing T- and B-cell responses. Pharmacologically blocking the activity of miR-17-92 with an anti–miR-17 antagomir effectively alleviates cGVHD.

Description: Graft versus host disease (GvHD) is the most common complication of hematopoietic stem cell transplant (HCT). However, our understanding of the molecular pathways that cause this disease remains incomplete, leading to inadequate targeted strategies for prevention and treatment. To address this, we determined the gene expression profile of non-human primate (NHP) T cells during active and partially controlled acute GvHD (aGvHD), in order to accomplish two goals: 1) uncover important genetic drivers of aGvHD and 2) identify novel, targetable pathways for optimal aGvHD prevention. Utilizing microarray technology, we measured the gene expression profiles of flow cytometrically purified CD3+ T cells from NHP recipients of MHC partially-matched HCT in three treatment cohorts resulting in increasing degrees of survival: 1) no immunoprophylaxis (No Rx, MST = 7.5); 2) sirolimus monotherapy (MST = 17) tacrolimus-methotrexate (Tac-Mtx) dual prophylaxis (MST = 49). Arrays were performed on T cells purified on Day +14 post-transplant (unless terminal analysis occurred earlier due to severe disease). This comparison allowed us to determine the impact of both mTOR and calcineurin inhibition on the molecular pathways dysregulated during GvHD, and to determine which genes and pathways remained dysregulated despite prophylaxis. Pathways identified by this strategy may contain new therapeutic targets unaffected by current immunoprophylactic approaches. We found that the transcriptional profile of donor T cells from HCT recipients with unprophylaxed GvHD was characterized by significant perturbation of pathways regulating T cell proliferation, effector function and cytokine synthesis (Figure 1a). By identifying pathways unaffected by sirolimus or tac-mtx therapy (Figure 1b), we discovered multiple potentially druggable targets not previously implicated in the pathophysiology of aGvHD. These targets prominently included the hedgehog and the aurora kinase A pathways. Utilizing a murine aGvHD model, we demonstrated that pharmacologic inhibition of these pathways could mitigate disease and improve survival (Figure 2a,b). These data provide the first identification of the T cell transcriptome of primate acute GvHD and the hedgehog and aurora kinase A pathways as novel potential targets for prevention of this disease. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Chronic graft-versus-host disease (cGVHD) remains a major cause of morbidity and mortality following allogeneic hematopoietic cell transplantation (allo-HCT) with limited success of current therapeutic options. We have shown that Notch inhibition in donor T cells or systemic neutralization of the Notch ligands Delta-like1 and Delta-like4 (Dll1/4) prevents acute GVHD in multiple mouse allo-HCT models (Zhang et al., Blood 2011; Sandy et al., J Immunol 2013; Tran et al., JCI 2013). However, the role of Notch signaling in cGVHD remains unknown. To address this question, we used genetic and antibody-mediated strategies of Notch inhibition in two established and pathophysiologically distinct mouse models of cGVHD characterized by dominance of sclerodermatous changes (Scl) or non-Scl multi-organ system disease that includes bronchiolitis obliterans (BO), respectively. In the B10.D2→BALB/c MHC-matched, minor antigen mismatched model of Scl-cGVHD, transgenic expression of the pan-Notch inhibitor DNMAML in T cells induced high level protection from cGVHD as assessed using clinical and pathological scoring criteria. Systemic antibody-mediated blockade of Dll1/4 Notch ligands prevented cGVHD when given early after allo-HCT, with dominant effects of Dll4 inhibition alone. However, Dll4 blockade provided no therapeutic benefits if delayed by 48 hours after allo-HCT or when targeting fully established Scl-cGVHD, suggesting that Dll4-mediated Notch signals are critical in this model when delivered to T cells early after allo-HCT. To understand the impact of Notch inhibition during priming of alloantigen-specific T cells, we studied immunodominant Vβ3+CD4+ T cells that expand in response to a host superantigen encoded by Mtv6 in BALB/c recipients. Both control and Notch-inhibited Vβ3+CD4+ T cells upregulated activation markers and expanded to comprise 〉80% of donor T cells in lymphoid and target organs by day 6. However, Notch inhibition markedly decreased IFN-γ, TNF-α, and IL-17 production in these cells, while expanding Vβ3+CD4+FoxP3+ regulatory T cells. Thus, Notch inhibition preserved in vivo T cell proliferation and expansion, but tilted the balance in favor of alloantigen-specific regulatory T cells over highly inflammatory effector T cells. In the B6→B10.BR MHC-mismatched model of allo-HCT, characterized by prominent germinal center (GC) responses and BO-cGVHD, genetic pan-Notch inhibition in T cells with DNMAML blocked GC formation and provided long-lasting high level protection from BO, as assessed using pulmonary function tests to measure resistance, elastance and compliance, as well as pathological examination (day 56 after allo-HCT). Flow cytometric evaluation showed that follicular helper T cells (CD4+PD-1hiCXCR5+) and cells with a GC B cell phenotype (CD19+GL7+CD95hi) were markedly decreased in numbers. Genetic deletion of Notch1 or Notch2 in donor T cells also prevented the immunological manifestations of cGVHD. Antibody-mediated blockade of the Notch ligands Dll1, Dll4, or both in the peri-transplant period protected recipients from BO-cGVHD. In contrast to our observations in Scl-cGVHD, delayed Dll1/4 blockade starting at day 28 after allo-HCT preserved pulmonary function, decreased GC formation and alloantibody deposition in target tissues, and ameliorated BO-cGVHD. Thus, Dll1/4 inhibition could provide therapeutic benefits even in established BO-cGVHD. Altogether, we identified a key role for early Notch signals in alloantigen-specific T cells that define subsequent cGVHD pathogenesis. Interference with Notch signaling early after allo-HCT provided long-lasting protection from cGVHD. In addition, our observations in the multi-organ system BO-cGVHD model suggest a therapeutic potential for delayed inhibition of Dll1 or Dll4 Notch ligands in an active disease setting driven by alloantibody formation. Our preclinical data suggest that Notch signaling should be explored as a novel druggable target to prevent or treat different forms of cGVHD. Disclosures Yan: Genentech, Inc.: Employment, Equity Ownership. Siebel:Genentech Inc.: Employment, Equity Ownership.

Description: Activated Protein C (APC), a trypsin-like plasma serine protease, exerts multiple beneficial pharmacological activities, and an APC variant with reduced anticoagulant activity, namely 3A-APC (Lys191-193Ala), is in phase 2 clinical trials for ischemic stroke. Although APC's beneficial effects on endothelial, epithelial, and neuronal cells have been well described, little is known about APC's effects on the immune system. Here we study APC's immunosuppressive actions using a multi-organ system murine chronic graft versus host disease (cGVHD) model with bronchiolitis obliterans (BO) and fibrosis. To determine whether APC has in vivo immune suppressive activity, mice were conditioned with high dose cyclophosphamide and total body irradiation followed by MHC-disparate bone marrow and splenocytes. Recombinant murine APC (6 µg/dose) or vehicle was administered intraperitoneally once daily from d28-56. Three forms of APC were studied: wild type (WT)-APC and two APC variants with greatly reduced anticoagulant activity but normal cell signaling activity, 3A-APC and 5A-APC (Lys191-193Ala plus Arg229-230Ala). Pulmonary function tests (compliance, resistance and elastance) indicative of cGVHD BO were markedly improved by WT-APC and both anti-coagulant defective mutants, 3A-APC and 5A-APC. Additionally, WT-APC and the variants, 3A-APC and 5A-APC, significantly reduced pulmonary fibrosis. The lack of difference in efficacy between WT and the signaling selective variants suggests APC-induced signaling, not anticoagulant activity, is primarily responsible for reduction of pulmonary pathology in cGVHD. APC treatment also significantly reduced splenic germinal center (GC) size as well as Tfollicular helper cell (CD4+CXCR5+PD1hi) and Th17 frequencies (Figure), keys for GC formation and subsequent fibrosis. Together, these data indicated that APC-induced signaling is primarily responsible for reduction of pulmonary pathology in cGVHD. Previous work has documented APC's antithrombotic, anti-inflammatory, anti-apoptotic, endothelial barrier stabilizing, and regenerative activities. Although receptors on multiple cell types have been implicated for APC's cell signaling activities, in the current major paradigm, endothelial cell protein C receptor (EPCR)-bound APC cleaves protease activated receptor (PAR) 1 at Arg46 to initiate arrestin-dependent biased signaling. In contrast, thrombin's canonical cleavage at Arg41 in PAR1 induces G-protein-dependent pro-inflammatory signaling. To determine if APC's benefits for cGVHD might require PAR1 cleavage at Arg41 or Arg46, we used ES cells and homologous recombination to generate C57BL/6 strains carrying the PAR1 mutation of either Arg41Gln (R41Q) or Arg46Gln (R46Q). Then donor T cells were prepared from each genetically altered strain carrying WT-PAR1, QQ41-PAR1, or QQ46-PAR1, and they were used to induce cGVHD. 5A-APC reduced cGVHD for transplanted T cells carrying WT-PAR1 and QQ41-PAR1, but it was completely ineffective for transplanted QQ46-PAR1 T cells, proving Arg46 in PAR1 in donor T cells is essential for 5A-APC's benefits. Since APC's cleavage at Arg46 is key for triggering PAR1 biased signaling, we infer that APC's benefits for cGVHD require, at least in part, direct biased activation of PAR1 on donor T cells and that this signaling precludes sufficient IL17 generation and GC formation to cause cGVHD. In summary, daily therapy using recombinant APC was highly effective in treating ongoing cGVHD in a BO model. Studies using signaling-selective APC mutants and donor T cells obtained from novel PAR1-genetically modified mice suggest that the mechanisms responsible for cGVHD suppression involve APC-induced signaling dependent on PAR1 cleavage at Arg46 on donor T cells, and not APC's anticoagulant activity. Such signaling resulted in suppression of Tfollicular helper cells and Th17. Given the safety and efficacy record of 3A-APC in phase 1 clinical studies, these data strongly support the consideration of 3A-APC or 5A-APC for treatment of cGVHD with BO manifestations that have been refractory to other therapies. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Description: Programmed death 1 (PD-1) and its ligands, PD-L1 and PD-L2, play an important role in the maintenance of peripheral tolerance. PD-1 is an inhibitory receptor that attenuates TCR signaling. Its expression is inducible on T-cells, B-cells, NKT-cells, and activated monoytes. Interactions between PD-1 and its ligands deliver inhibitory signals that regulate T-cell activation, tolerance, and immune-mediated tissue damage.A blocking anti-PD-1 mAb given at the time of transplant markedly accelerated acute GVHD lethality in preclinical models via an interferon-gamma dependent mechanism. Both PD-L1 and PD-L2 expression were upregulated in the spleen, liver, colon, and ileum of GVHD mice. PD-L2 expression was limited to hematopoietic cells, but hematopoietic and endothelial cells expressed PD-L1. PD-1/PD-L1, but not PD-1/PD-L2, blockade markedly accelerated GVHD-induced lethality. PD-L1-deficient hosts exhibit rapid mortality associated with increased gut T-cell homing and loss of intestinal epithelial integrity, increased donor T-cell proliferation, activation, Th1 cytokine production, and reduced apoptosis. Bioenergetics profile analysis of proliferating alloreactive donor T-cells demonstrated increased aerobic glycolysis and oxidative phosphorylation, hyperpolarized mitochondrial membrane potential, increased superoxide production, and increased expression of a glucose transporter. During acute GVHD, PD-L1 was up-regulated on donor T-cells. Surprisingly, GVHD-induced lethality was significantly reduced in recipients of donor T cells devoid of PD-L1 and associated with reduced PD-L1-/- donor T-cell infiltration into lymphoid organs and gut, a retention of intestinal epithelial integrity, and a lower production of inflammatory cytokines. During GVHD, PD-L1-/- donor T cells showed increased apoptosis and reduced proliferation, as well as reduced glycolysis, glutaminolysis, and fatty acid metabolism. A role for PD-L1 in glucose-mediated acetyl-CoA production was seen, highlighting the important of glucose as an important carbon source in in alloreactive T cells undergoing clonal expansion. Further data support the hypothesis that the PD-1/PD-L1 pathway regulates T-T interaction. Together our studies indicate that PD-L1 expression that is upregulated on alloreactive donor T cells increases their survival and alters their metabolic pathway utilization in GVHD mice. In contrast to acute GVHD models, we have found that PD-1 pathway blockade can reduce chronic GVHD in a mouse model of multi-organ system disease in which one prominent component is bronchiolitis obliterans. This may occur via effects on T follicular regulatory or germinal center B cells. In summary, we have identified distinct consequences of PD-1/PD-L1 engagement in preclinical acute and chronic GVHD models: PD-1/PD-L1 interactions restrain acute GVHD but increase chronic GVHD. These findings illustrate the important but complex regulatory features of this pathway on a wide array of cell types. Our finding suggests PD-1 pathway modulation may provide unique opportunities for altering immune regulation post-transplant. Disclosures Milone: Novartis: Patents & Royalties, Research Funding. Sharpe:Costim Pharmaceuticals: Patents & Royalties.

Description: Allogeneic hematopoietic stem cell transplantation (aHSCT) is hampered by chronic graft-versus-host disease (cGVHD), which results in multi-organ fibrosis and loss of function. In particular, bronchiolitis obliterans (BO) and scleroderma resulting from fibrotic bronchiolar and cutaneous response, respectively, are two devastating outcomes for cGVHD patients. Fibrotic manifestations often are considered irreversible and progressive. Therefore, new therapies targeting fibrosis are urgently needed. Pirfenidone (5-methyl-1-phenyl-2- (1H)-pyridone) exhibits a well-documented anti-inflammatory and anti-fibrosis function in multiple pre-clinical models and is the first and only FDA-approved drug for idiopathic pulmonary fibrosis. For this study, Pirfenidone was synthesized as a crystalline solid and found to be pure both by melting point and NMR spectroscopy. We evaluated Pirfenidone's anti-fibrosis function in 2 pathophysiologically distinct cGVHD murine models: 1. a major mismatched multi-organ system model (C57BL/6 to B10.BR) that induces BO as a result of a cGVHD-induced germinal center (GC) reaction, antibody deposition and fibrosis in the lung; and 2. a minor antigen mismatched model (B10.D2 to BALB/c) in which severe scleroderma is the major disease manifestation. In the BO model, pulmonary function loss in cGVHD mice (as reflected by increased resistance, elastance and decreased compliance of the lung) was restored by Pirfenidone treatment (400mg/kg) during both the early (day28-56) (Fig A, representative of 3 experiments with 5-8 mice per group) and late stages (day56-84) of the disease. Pathologic changes in the lung, such as collagen deposition and narrowing of bronchioles, were significantly reduced by Pirfenidone. The size and frequency of GCs in the spleen, and the frequency of GC B cells (Fig B, representative of 2 experiments with 5-8 mice per group) and T follicular helper cells were all significantly reduced in Pirfenidone- treated groups. To determine whether GCs were directly affected by Pirfenidone, we evaluated Pirfenidone in C57BL/6 mice immunized with sheep red blood cells (SRBC) to induce GCs. Interestingly, Pirfenidone did not reduce the SRBC-induced GC reaction (Fig C) (comparable frequencies of splenic GC B cells, T follicular helper cells and serum IgG levels were seen between Pirfenidone and vehicle-treated groups). These results suggested that Pirfenidone suppresses the GC reaction through a cGVHD-specific mechanism, rather than through immune regulation. Mechanistically, Pirfenidone administration attenuated the sequestration of pro-fibrogenic F4/80+ macrophages (Fig D, representative of 2 experiments) and TGF-β (Fig E, representative of 2 experiments) production within the lung. These results have led us to elucidate a potential mechanism of cGVHD: antibody deposition in the lung results in the activation of macrophages and TGF-β that drive fibrotic change and tissue damage, resulting in the exposure of auto- and allo- antigens to the immune system that support and sustain pathologic GC reactions. In the B10.D2 to BALB/c sclerodermatous cGVHD model, Pirfenidone treatment (400mg/kg, day21-55) improved clinical signs of scleroderma and reduced macrophage infiltration in the skin (Fig F). In summary, this is the first study evaluating a commercially available anti-fibrosis drug on pathologically distinct pre-clinical cGVHD models. Our data suggests Prifenidone reversed cGVHD in the BO model and, to a lesser extent, in the scleroderma model. Thus, Pirfenidone is a novel therapeutic agent for treating cGVHD patients with fibrosis that have been typically refractory to therapies. A. Resistance of lungs was measured on day56 of transplantation; Elastance and compliance correlated with resistance but were not shown here. B. Flow cytometry analysis of GC B cells of no cGVHD vs cGVHD mice treated with Pirfenidone or vehicle; C. Flow cytometry analysis of GC B cells from SRBC-immunized mice treated with Pirfenidone or vehicle; D and E. Macrophage F4/80 and TGF-β quantification of day56 lungs of no cGVHD vs cGVHD mice treated as indicated; F. Skin GVHD scores were recorded on indicated dates of irradiated BALB/c mice transplanted with B10.D2 donor BM alone or with T cells and treated as indicated. Unpaired student T test was used for statistical analysis. ****:P