ALBERT

Description: High spatial and temporal resolution remotely sensed data is of great significance for the extraction of land use/cover information and the quantitative inversion of biophysical parameters. However, due to the limitation of sensor performance and the influence of rain cloud weather, it is difficult to obtain remote sensing images with both high spatial and temporal resolution. The spatiotemporal fusion model is a crucial method to solve this problem. The spatial and temporal adaptive reflectivity fusion model (STARFM) and its improved models are the most widely used spatiotemporal adaptive fusion models. However, the existing spatiotemporal adaptive reflectivity fusion model and its improved models have great uncertainty in selecting neighboring similar pixels, especially in spatially heterogeneous areas. Therefore, it is difficult to effectively search and determine neighboring spectrally similar pixels in STARFM-like models, resulting in a decrease of imagery fusion accuracy. In this research, we modify the procedure of neighboring similar pixel selection of ESTARFM method and propose an improved ESTARFM method (I-ESTARFM). Based on the land cover endmember types and its fraction values obtained by spectral mixing analysis, the neighboring similar pixels can be effectively selected. The experimental results indicate that the I-ESTARFM method selects neighboring spectrally similar pixels more accurately than STARFM and ESTARFM models. Compared with the STARFM and ESTARFM, the correlation coefficients of the image fused by the I-ESTARFM with that of the actual image are increased and the mean square error is decreased, especially in spatially heterogeneous areas. The uncertainty of spectral similar neighborhood pixel selection is reduced and the precision of spatial-temporal fusion is improved.

Description: Histone methylation is thought to be important for regulating Ag-driven T-cell responses. However, little is known about the effect of modulating histone methylation on inflammatory T-cell responses. We demonstrate that in vivo administration of the histone methylation inhibitor 3-deazaneplanocin A (DZNep) arrests ongoing GVHD in mice after allogeneic BM transplantation. DZNep caused selective apoptosis in alloantigen-activated T cells mediating host tissue injury. This effect was associated with the ability of DZNep to selectively reduce trimethylation of histone H3 lysine 27, deplete the histone methyltransferase Ezh2 specific to trimethylation of histone H3 lysine 27, and activate proapoptotic gene Bim repressed by Ezh2 in antigenic-activated T cells. In contrast, DZNep did not affect the survival of alloantigen-unresponsive T cells in vivo and naive T cells stimulated by IL-2 or IL-7 in vitro. Importantly, inhibition of histone methylation by DZNep treatment in vivo preserved the antileukemia activity of donor T cells and did not impair the recovery of hematopoiesis and lymphocytes, leading to significantly improved survival of recipients after allogeneic BM transplantation. Our findings indicate that modulation of histone methylation may have significant implications in the development of novel approaches to treat ongoing GVHD and other T cell–mediated inflammatory disorders in a broad context.

Description: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective therapy for many hematological malignancies, however, it remains to be limited by morbidity and mortality related to graft-versus-host disease (GVHD). During GVHD, donor T cells are activated by host antigen-presenting cells (APCs) and differentiate into effector cells that mediate host tissue damage. Development of alloreactive effector T cells requires orchestrated expression of myriad genes initiated by host APCs. However, little is known about the key epigenetic factor that controls this process. Histone methylation, which is catalyzed by histone methyltransferanses (HMT), has been correlated with genes encoding effector cytokines and transcription factors critical for effector differentiation. We recently demonstrated that inhibition of histone methylation using 3-Deazaneplanocin A (DZNep) arrested ongoing GVHD through induction of apoptosis in alloreactive T cells. However, since DZNep reduced multiple histone methylation marks, including trimethylation of histone H3 at lysine 4 (H3K4me3), H3K27me3, H3K36me3 and H4K20me3, the key HMT that regulates the transcription program important for allogeneic T cell responses remains unclear. Using genetic approaches and mouse GVHD models, we identify that the HMT Ezh2, which specifically catalyzes H3K27me3 and acts primarily as a gene silencer, plays a central role in regulating allogeneic T cell proliferation, differentiation, and function during the GVHD process. Transfer of donor T cells derived from Ezh2 conditional knockout C57BL/6 (B6) mice did not induce GVHD in lethally irradiated allogeneic BALB/C mice, with all recipients surviving free of disease. In contrast, wild-type (WT) donor B6 T cells caused uniformly lethal GVHD in these recipients. We found that loss of Ezh2 selectively impaired alloantigen-specific T cell responses. Three days after allo-HSCT, both Ezh2-deficient and WT T cells were similarly activated to proliferate in vivo. However, 7 days after transplantation, there was a significant reduction in the number of highly proliferative Ezh2-deficient T cells compared to WT T cells, leading to a dramatic reduction of infiltrating Ezh2-deficient T cells in GVHD target organs. Interestingly, conditionally deleting Ezh2 did not affect proliferation and survival of B6 T cells that were transferred into lethally irradiated syngeneic B6/SJL mice by 7 days after transplantation. Thus, Ezh2 is critical for the continual proliferation of alloantigen-responding T cells during later stages of GVHD induction. In contrast, Ezh2 is dispensable for both the initial activation and proliferation of T cells upon alloantigen-priming and homeostatic expansion of T cells in response to lymphopenia. Previous studies suggest that IFN-g-producing alloreactive effector cells cause damage to the liver and gastrointestinal tract. We found that loss of Ezh2 led to selective reduction in frequency of IFN-γ-producing effector cells 7 days after allo-HSCT. This effect was accompanied with decreased expression of transcription factors TBX21 and STAT4, both of which are crucial for effector differentiation. These data suggest that in addition to regulating the transcription program critical for producing sufficient numbers of alloreactive effector T cells needed to mediate GVHD, Ezh2 is also important for orchestrating genes required to promote IFN-γ production in effector cells. Importantly, we found that Ezh2 ablation retained anti-leukemia activity in alloreactive T cells, leading to improved overall survival of transplant recipients. Collectively, these findings identify Ezh2 as a key epigenetic regulator that controls allogeneic T cell responses and GVHD. We propose that pharmacological modulation of Ezh2 using specific inhibitors should be investigated as a novel therapeutic strategy after allo-HSCT. Disclosures: No relevant conflicts of interest to declare.

Description: Host antigen-presenting cells (APCs) are critical for inducing a potent graft-versus-leukemia (GVL) response after allogeneic hematopoietic stem-cell transplantation (allo-HSCT). In this setting, host APCs activate donor T cells to become effector T cells that recognize and react to antigens in malignant cells. However, alloreactive T cells also mediate graft-versus-host disease (GVHD), which causes significant morbidity and mortality after allo-HSCT. Many studies suggest that if alloreactive T cells have reduced capacity to expand in local tissues, they will be unable to trigger severe GVHD. Thus, it is possible that host APC induction of qualitative changes in donor T cells can potentially modify their anti-host toxicities while retaining the GVL effect. Here we report the establishment of a cellular programming approach that reduces the GVHD toxicity of donor T cells using host dendritic cells (DCs) that express high levels of Dll4 (named Dll4hi DCs). We have previously identified inflammatory Dll4hi DCs. They occurred in HSCT mice early during GVHD induction and had a greater ability than Dll4-negative DCs to induce IFN-γ and IL-17 in alloantigen-activated T cells. However, only approximately 0.03 X 105 Dll4hi DCs were recovered from one HSCT mouse. To provide adequate numbers of Dll4hi DCs for therapeutic translation, we developed a novel culture system capable of producing large number of Dll4hi DCs (about 100.0 X 105) from the bone marrow (BM) of one mouse using Flt3L and the TLR agonists lipopolysaccharide (LPS) and R848, which activate TLR4 and TLR7/8, respectively. Dll4hi DCs showed significantly different phenotype as compared to conventional DCs derived from GM-CSF-stimulated BM cells (named GM-DCs), as evidenced by expressing higher levels of Dll4, Ifnb, Il4, Il6 and Ido, and producing lower levels of iNOS and arginase I. When cultured with C57BL/6 (B6) mouse CD4+ T cells (H2b) at a T cell: DC ratio of 4:1 for 5 days, BALB/c mouse Dll4hi DCs (H2d) induced 3- to 5-fold more in frequency of alloreactive effector T cells producing high levels of IFN-γ and IL-17 compared to GM-DCs. Following transfer, allogeneic Dll4hi DC-induced CD4+ T cells were unable to mediate severe GVHD in BALB/c recipients, with all of them surviving 60 days after allo-HSCT. In contrast, both unstimulated B6 CD4+ T cells and allogeneic GM-DC-induced B6 CD4+ T cells caused lethal GVHD in all BALB/c recipients, indicating that GM-DCs could not be used for reducing the GVHD toxicity of donor CD4+ T cells. Mechanistic analysis showed that Dll4hi DC-induced CD4+ T cell recipients showed 2- to 6-fold less donor CD4+ T cells in the spleen, liver, and intestine 12 days after transplantation compared to unstimulated CD4+ T cell recipients. This reduction of Dll4hi DC-induced CD4+ T cells was associated with markedly increased apoptosis in recipient mice. IFN-γ production by Dll4hi DC-induced CD4+ T cells was essential for their anti-GVHD effects. Absence of T cell IFN-γ led to improved survival and expansion of Dll4hi DC-induced CD4+ T cells in transplant recipients and caused lethal GVHD. Finally, we demonstrated that Dll4hi DC-induced alloreactive T cells had acquired the ability to kill A20 leukemic cells in BALB/c recipients and control growth of P815 mastocytoma cells in the second model of BDF1 recipients, leading to significantly improved survival of mice receiving allo-HSCT. Furthermore, in the third mouse model of GVHD directed against minor histocompatibility antigens, B6 Dll4hi DC-induced C3H.SW CD8+ T cells produced high levels of IFN-γ, had reduced capacity to mediate GVHD in B6 recipients, but preserved GVL activity against C1498 myeloid leukemic cells. In summary, our findings demonstrate that in vitro Dll4hi DC programming represents a novel and effective platform to reduce toxicities of donor T cells. This strategy has several potential advantages compared to current and developing methods for the modification of donor T cells to reduce GVHD, including a relatively short period of culture, no requirement for T cell subset selection and no need of viral transduction. Importantly, this method may lead to new strategies that can produce large amount of leukemic cell-reactive donor T cells with decrease capability of causing severe GVHD. Disclosures No relevant conflicts of interest to declare.

Description: Graft-versus-host disease (GVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). GVHD involves complex interactions of immune cells, induction of host-reactive donor effector T cells, and donor T cell-mediated injury to normal tissues. Epigenetic changes have been implicated in T cell-mediated GVHD. We previously described that genetic deletion of Ezh2, which catalyzes trimethylation of histone H3 at lysine 27 (H3K27me3), reduced GVHD in mice but preserved graft-versus-leukemia (GVL) responses. Several selective inhibitors of Ezh2 have been recently discovered (e.g. GSK126, UNC1999 and EPZ6438), which specifically reduce the levels of H3K27me3 but not EZH2 protein. Unexpectedly, our preliminary studies showed that administration of GSK126 failed to prevent GVHD in mice. This stands in contrast to our findings that genetic deletion of T cell Ezh2 leads to GVHD inhibition, and suggest that Ezh2 may regulate GVHD through a mechanism independent of H3K27me3. Identifying an optimal method to target T cell Ezh2 for controlling GVHD remains an unmet need. Using experimental mouse models, we demonstrate that functional heat shock protein (Hsp)90 is critical for maintaining Ezh2 protein stability and function in activated T cells. Pharmacological inhibition of Hsp90 destablizes Ezh2 protein in alloreactive T cells, reduces GVHD but preserves GVL effects in mice. To determinethe molecule(s) that is critical for maintaining Ezh2 protein stablility in T cells, we performed mass spectrum (MS) analysis and identified 25 Ezh2-interacting proteins that showed higher intensities than others in T cell receptor (TCR)-activated CD8+ T cells. Among them, we found a group of proteins associated with protein folding and degradation, including Hsp90. Hsp90 is a molecular chaperone required for the stability and function of several key signaling intermediates (e.g., AKT, Raf1 and ERK1/2). Using reciprocal co-immunoprecipitation assay, we confirmed that Ezh2 and Hsp90 directly interacted with each other in TCR-activated CD8+ T cells. Pharmacological inhibition of Hsp90 using its specific inhibitor AUY922, which is currently in phase II clinical trials for cancer therapy, effectively reduced Ezh2 protein without decreasing H3K27me3 24 hours after treatment. This effect was accompanied by decreased proliferation and survival of TCR-activated T cells in vitro. Retroviral overexpression of Ezh2 in T cells markedly improved their proliferation in the presence of AUY922, suggesting that reducing Ezh2 by Hsp90 inhibition is an important mechanism that reduces proliferation and survival of activated CD8+ T cells. Building on these observations, we examined the impact of inhibiting Hsp90 on GVHD by administering AUY922 to B6 mice receiving MHC-identical minor histocompatibility antigen-mismatched C3H.SW mouse CD8+ T cells and T cell-depleted bone marrow (BM). While about 80% of control B6 recipients died from severe GVHD, 80% of AUY922-treated B6 recipients survived without clinical signs of severe GVHD by 84 days after transplantation. In vivo AUY922 administration reduced the survival and expansion of alloreactive T cells, and decreased the fequency of alloreactive T effector cells producing IFN-g and TNF-a. To rule out the model-specific effect of AUY922, we used a haplo-identical B6 into BDF1 mouse model of GVHD. Using CFSE-labeled donor T cells, we first validated that in vivo administration of AUY922 to unirradiated BDF1 mice receiving parent B6 T cells selectively reduced the expansion of alloantigen-reactive donor T cells, but did not impair the expansion and survival of donor T cells that did not respond to alloantigens. In lethally irradiated BDF1 mice receiving B6 T cells and BM, AUY922 administration reduces lethal GVHD, with approximately 50% of them surviving long-time. Importantly, AUY922 treatment preserved GVL activity of donor T cells, leading to significantly improved survival of BDF1 recipients challenged with A20 leukemic cells (Fig.1). Taken together, our findings identified a previously unrecognized molecular mechanism by which Ezh2 and Hsp90 are integrated to regulate alloreactive T cell responses and GVHD. Targeting the Ezh2-Hsp90 complex using AUY922 represents a novel and clinically relevant approach to reduce GVHD while preserving GVL effects, thereby improving the efficacy of allo-HSCT. Disclosures No relevant conflicts of interest to declare.

Description: Key Points Ezh2 requires Hsp90 to maintain Ezh2 protein stability and function in alloreactive T cells. Pharmacological inhibition of Hsp90 destabilizes Ezh2 protein in alloreactive T cells and reduces GVHD but preserves graft-versus-leukemia effects.

Description: Abstract 337 Graft-versus-host disease (GVHD) remains a major barrier to the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Host antigen-presenting cells (APCs) are known to be essential for presenting alloantigens to activate donor T cells to become effector cells mediating GVHD after allo-HSCT. However, APCs are heterogeneous populations. The identity of APC subset(s) that directs effector differentiation of alloantigen-activated T cells and by which mechanism this effect may be achieved remain largely unknown. The Notch signaling pathway controls cell proliferation, differentiation and survival. Upon interaction with Notch ligands of the δ-like family (Dll1, Dll3 and Dll4) and Jagged family (J1, J2), Notch receptors (Notch 1, 2, 3, and 4) are cleaved by γ-secretase and translocate into the nucleus to modify gene transcription. We have recently demonstrated that activation of Notch receptors in donor T cells is critical to the production of alloreactive effector T cells producing multiple inflammatory cytokines (e.g., IFN-γ, TNF-α and IL-17) during GVH reaction (Blood 2011). Building on these findings, we hypothesized that: 1) Notch ligand(s) derived from APCs may be important for directing effector differentiation of alloantigen-activated T cells, and 2) the expression of Notch ligand(s) may differentiate the capability of APCs to prime GVH responses. Using mouse models of GVHD, here we report the identification of previously uncharacterized Dll4-positive (Dll4+) inflammatory plasmacytoid dendritic cells (i-pDCs) and their roles in eliciting allogeneic T-cell responses. Host-derived Dll4+ i-pDCs occurred in the spleen of allo-HSCT recipients one day after transplantation, peaked by three days and declined by seven days. In contrast, host-derived inflammatory conventional DCs (i-cDCs) were Dll4-negative (Dll4−) and rapidly diminished by three days after transplantation. Notably, donor-derived DCs which occurred seven days after HSCT did not express Dll4. In vitro mixed lymphocyte-reaction (MLR) assay showed that these host-derived Dll4+ i-pDCs induced approximately 2.5-fold and 7-fold more IFN-γ- and IL-17-producing effector T cells than Dll4− i-cDCs, respectively. Addition of neutralizing antibody specific to Dll4 to the MLR cultures markedly reduced the production of IFN-γ and IL-17 in donor T cells stimulated by host Dll4+ i-pDCs, but had minimal impact on donor T cells cultured in the presence of Dll4− i-cDCs. These results suggest that Dll4+ i-pDCs may play important roles in directing effector differentiation of alloantigen-activated T cells. Further characterization of biological properties of Dll4+ i-pDCs revealed that as compared to unstimulated host pDCs at steady state conditions, Dll4+ i-pDCs expressed higher levels of antigen-presenting and costimulatory molecules, upregulated other Notch ligands (e.g.,J1 and J2) on their surface and produced more Ifnb and Il23. Notably, Dll4+ i-pDCs were mainly located in the spleen and intestine of mice receiving allogeneic HSCT. In vivo administration of Dll4 antibody reduced donor alloreactive effector T cell producing IFN-γ, IL-17 and TNF-α in GVHD target organs (in particular of the intestine), leading to reduction of GVHD and significantly improved survival of mice after allogeneic HSCT. Furthermore, adoptive transfer of in vitro generated Dll4+ i-pDCs caused severe GVHD in MHC-II-deficient mice (in which host DCs are incapable to elicit GVHD). Our findings identify that Dll4+ i-pDCs may represent a previously uncharacterized inflammatory APC population developed during GVH reaction. These Dll4+ i-pDCs and their-derived Dll4 are critical for directing differentiation of alloreactive effector T cells and may be beneficial therapeutic targets for modulating GVHD. Disclosures: No relevant conflicts of interest to declare.

Description: Whether tumor-reactive T cells can infiltrate into the tumor to execute effector function is essential for controlling tumor growth. CD103 is an integrin protein (αE) that binds integrin β7 to form the heterodimeric integrin complex αEβ7. CD103 is important for T cell retention in peripheral tissues by interacting with E-cadherin and a promising prognosis biomarker for assessment of tumor-reactive T cells infiltrating in the tumor from various types of cancer, such as lung cancer, ovarian cancer and cervical cancers. However, CD103 is not expressed on the surface of circulating peripheral blood T cells that are genetically modified to express a chimeric antigen receptor (CAR) for adoptive T cell therapy. Whether CD103 expression on the surface of tumor-reactive CAR T cells is functionally important for their anti-tumor activity has not been previously determined. Using a preclinical model of human lymphoma expressing E-cadherin, we demonstrate that engineering of CD19-specific human CAR T cells with CD103 significantly improves their therapeutic effects on eliminating pre-established human lymphoma in immune deficient NSG mice (NOD.scid.Il2Rγcnull). We synthesized a codon optimized CD19-specific CAR containing 4-1BB and CD3zeta intracellular signaling domains (named CD19-BBz-CAR), cloned it into lentiviral vector and infected human T cells. As expected, the resultant human CD19-BBz-CAR T cells possessed potent capacity to cure human B cell leukemia in NSG mice that had been intravenously inoculated with Raji leukemic/lymphoma cells. Notably, while approximately 10% of non-CAR T cells produced high levels of CD103 from these NSG mice, CD19-BBz-CAR T cells failed to upregulate CD103, suggesting that the expression of CD19-BBz-CAR inhibits the induction of CD103 in vivo. Ex vivo assay confirmed that CD19-BBz-CAR caused dose-dependent decrease of CD103 expression in human T cells cultured in the presence of TGF-β1. This effect was mediated by the expression of costimulatory molecule 41BB, which is known essential for sustaining CD19-BBz-CAR T cells in vivo. To circumvent the repression effect of 41BB on induction of CD103, we incorporated the gene encoding integrin αE into the CAR structure to generate CD103-CD19-BBz-CAR T cells. Intriguingly, as compared to conventional CD19-BBz-CAR T cells, CD103-CD19-BBz-CAR T cells expressed high levels of CD62L and CD45RA, which resemble less differentiated T cells, produced higher levels of IL-2, which is crucial for promoting T cell expansion and function, and underwent greater expansion in cultures. Upon adoptive transfer into NSG mice that had subcutaneous human Raji lymphoma, CD103-engineering of CD19-BBz-CAR T cells dramatically decreased the distal metastasis of lymphoma, increased the infiltration of CAR T cells into the solid lymphoma, and improved the in vivo persistence of tumor-reactive CAR T cells. As a result, transfer of CD103-CD19-BBz-CAR T cells significantly increased overall survival rate of lymphoma mice compared to conventional CD19-BBz-CAR T cells (40% versus 10%, p

Description: Graft-versus-host disease (GVHD) remains a major barrier for the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We have identified the central role of the histone methyltransferase Ezh2 in regulating allogeneic T-cell expansion, differentiation and function. Conditional loss of Ezh2 in donor T cells inhibits GVHD in mice due to the inability of alloreactive T cells to persist. However, the molecular mechanism by which Ezh2 deficiency causes alloreactive T cell death remains unknown. Here we demonstrate that genetic deletion of Stromal Interaction Molecule (Stim) 1, a dynamic endoplasmic reticulum Ca2+ sensor and regulator of Ca2+ signaling, rescues antigen-activated Ezh2-null (Ezh2-/-) T cells, leading to restored persistence of alloreactive effector T cells in mice and severe GVHD. Using RNA-sequencing analysis, we found Ezh2-deficiency led to the upregulation of multiple genes (e.g., Ifng, Prf1, Ccl5, Ccl4, Upp1 and Spp1) known to be regulated by Ca2+ signals through calcineurin (CN), the primary target of the immunosuppressant cyclosporine A (CsA). This reverse correlation between Ezh2 inhibition and CsA-treatment for gene expression suggests that Ezh2 may antagonize Ca2+ signaling in activated T cells. Calcium signaling assays revealed higher cytosolic Ca2+ uptake and more frequent Ca2+ oscillations in Ezh2-/- T cells. Moreover, Ezh2-/- T cells exhibited significantly increased polarization of Stim1 and Orai1 in the cellular membrane. These data reveal an unexpected role of Ezh2 as a negative regulator of Ca2+ entry, thereby serving as a 'brake' for Ca2+ signaling. Using the C57BL/6 (B6) into Balb/c mouse GVHD model, we found significantly fewer Ezh2-/- or Stim1-/- IFN-g-secreting effector T cells compared to the WT counterparts on day 8 or 14 post-transplantation. In contrast, deleting Stim1 from Ezh2-/- donor T cells rescued the cells in the spleen and liver, producing even more donor T cells and IFN-g-secreting effector T cells compared to WT T cells and inducing severe GVHD. We further examined the cell autonomous effect of Stim1 deletion on the rescue of Ezh2-/- T cells by mixing WT T cells (B6/SJL, CD45.1) with Ezh2- and/or Stim1- conditional knockout T cells (i.e., Ezh2-/-, Stim1-/- or Ezh2-/- x Stim1-/- B6 T cells (CD45.2)) at a ratio of 1:1 before transferring into the Balb/c mice. While loss of either Ezh2 or Stim1 led to lower frequency of IFN-g+IL-2+ effector T cells, combined deletion of both genes restored the frequency and number of IFN-g+IL-2+ effector T cells to that of WT T cells. Thus, Stim1-mediated Ca2+ signals are crucial for mediating cell death in alloantigen-driven Ezh2-/- effector T cells. To further determine whether the inhibition of CN-NFAT contributes to the rescue, we treated T cell receptor (TCR)-activated Ezh2-/- T cells with CsA or the calcium release-activated channel specific inhibitor BTP2, respectively, in vitro. While BTP2 dramatically improved the survival of IFN-g-producing effector T cells, CsA did not, suggesting the involvement of CN-NFAT-independent pathways. Ca2+ overload is known to impair mitochondrial function and cause massive cell death. As compared to TCR-activated WT T cells, activated Ezh2-/- T cells displayed significantly less ATP, lower mitochondrial membrane potential, enlarged mitochondrial mass, and decreased capacity to upregulate oxidative phosphorylation. Stim1 deletion largely reversed the metabolic defect in Ezh2-/- T cells, indicating the critical role of mitochondrial metabolism in rescuing these T cells. Considered together, our findings identify the remarkable coordination between Ezh2- and Stim1-regulated effector T cell persistence. As such, these investigations may lead to new approaches to inhibit GVHD, with broad implications to defining fundamental mechanisms of T cell differentiation for control of adaptive immunity, such as tumor immunity and autoimmunity. Disclosures Reshef: Incyte: Consultancy; Takeda Pharmaceuticals: Consultancy; Pfizer: Consultancy; Kite Pharma: Consultancy; Atara Biotherapeutics: Consultancy; Bristol-Myers Squibb: Consultancy.

Description: Abstract 820 Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment option for patients with hematological malignancies. However, its success is limited by life-threatening graft-versus-host disease (GVHD). Novel approaches are needed to control GVHD. Recent studies have shown the importance of histone methylation in regulating the expression of genes associated with effector T cell differentiation and proliferation. Using several mouse models of allo-HSCT, we report that in vivo administration of the histone methylation inhibitor 3-Deazaneplanocin A (DZNep) arrested ongoing GVHD while preserving graft-versus-leukemia activity (GVL). To assess the therapeutic effect of pharmacologic modulation of histone methylation on GVHD, we administered DZNep to BALB/c mice receiving major histocompatibility-mismatched C57BL/6 mouse T cells 7 days after transplantation, in which GVHD had been fully established. Notably, injection of 12 doses of DZNep controlled the disease in these recipients, with approximately 80% of them surviving long-term without significant clinical signs of GVHD. We found that in vivo administration of DZNep caused selective apoptosis in alloantigen-activated T cells, but did not impair the generation of effector T cells that produced inflammatory cytokines (e.g., TNF-α, IFN-γ and IL-17) and cytotoxic molecules (e.g., granzyme B and Fas ligand). As a result, alloreactive T cells retained potent GVL activity, leading to improved overall survival of the recipients challenged by leukemic cells. These data suggest that DZNep-mediated inhibition of GVHD may be accounted for by reduced number of alloreactive effector T cells. In vitro culture assays showed that DZNep treatment induced apoptosis in T cells activated by anti-CD3/CD28 antibodies but not in naive T cells stimulated by IL-2 or IL-7. This effect was associated with DZNep's ability to selectively reduce trimethylation of histone H3 lysine 27 (H3K27), deplete the histone methyltranferase Ezh2 that specifically catalyzes trimethylation of H3K27, and activate Ezh2-repressed pro-apoptotic gene Bim. Inactivation of Bim partially protected alloreactive T cells from DZNep-mediated apoptosis. Importantly, unlike DNA methylation inhibitors, inhibition of histone methylation by DZNep had no toxicities to hematopoietic cells or impairment on the reconstitution of hematopoiesis and thymopoiesis. Our findings indicate that modulation of histone methylation may have significant implications in the development of novel approaches to treat established GVHD and other T cell-mediated inflammatory disorders in a broad context. Disclosures: No relevant conflicts of interest to declare.