519 We have previously shown that the pan-HDAC inhibitors LAQ824 and LBH589 inhibit IL-10 production in APCs, rendering these cells more inflammatory and capable of effectively priming naïve antigen-specific CD4+ T-cells and restoring the responsiveness of tolerant T-cells1. These findings led us to explore which HDAC(s) might be involved in the regulation of IL-10 gene transcription and be the putative target(s) of HDI-mediated IL-10 inhibition. To answer these questions we subjected the macrophage cell line RAW264.7 to shRNA screening using specific shRNAs to knockdown each known HDAC. We found that among all the HDACs, knocking down HDAC6 (HDAC6KD) was associated with a significant decrease in IL-10 mRNA and protein in response to LPS stimulation. Furthermore, HDAC6KD clones display an enhanced expression of the co-stimulatory molecule B7.2. Functionally, HDAC6KD cells were better activators of anti-HA (hemagglutinin-influenza) transgenic CD4+ T cells, leading to significantly enhanced production of IL-2 and IFN-g in response to cognate antigen. More importantly, anti-HA CD4+ anergic T cells isolated from animals bearing HA-expressing A20 B-cell lymphoma regained their ability to produce IL-2 and IFN-g when cultured in vitro with HDAC6KD cells. These results have been confirmed in APCs isolated from HDAC6 knock-out mice and in wild type APCs treated in vitro with isotype-selective HDAC6 inhibitors. Given that HDACs do not bind to DNA and they need to interact with transcription factors to regulate gene expression, we investigate next which transcription factor(s) HDAC6 might be associated with, to regulate IL-10 transcriptional activity. One likely candidate was Stat3, a well-known transcriptional activator of IL-10 gene expression that we have previously shown to play a central role in tolerance induction by APCs2. By co-immunoprecipitation studies we found that HDAC6 indeed interacts physically with Stat3. Of note, knocking down HDAC6 in APCs resulted in absence of Stat3 phosphorylation and decreased recruitment of Stat3 to the IL-10 gene promoter which might explain the inability of HDAC6KD cells to produce IL-10. The additional findings that IL-10 production by HDAC6KD cells was restored when these cells were transfected with a constitutively active mutant version of Stat3 (Stat3c) provides additional support for the important role of HDAC6 upon Stat3 activation. Further confirmation for a concerted regulatory mechanism involving HDAC6 and Stat3 in IL-10 gene regulation was provided by studies using CPA-7, a specific Stat3 inhibitor that disrupts Stat3 recruitment and binding to gene promoters. As expected, a complete abrogation of Stat3 recruitment to the IL-10 gene promoter was observed in CPA-7 treated APCs. Interestingly, such an effect was accompanied by a parallel decrease in HDAC6 recruitment to the IL-10 promoter and inhibition of IL-10 gene transcriptional activity. Taken together, we have shown for the first time that HDAC6 interacts physically with Stat3 and is required for its phosphorylation. Since Stat3 phosphorylation is absolutely necessary for activation of Stat3 target genes, HDAC6 inhibition is an enticing molecular approach to disrupt the Stat3/IL-10 axis and overcome tolerogenic mechanisms in APCs. Disclosures: No relevant conflicts of interest to declare.

359FN2 T-cells are an essential component of immune mediated tumor rejection. Adoptive transfer of T-cells has resulted in durable antitumor responses in some patients with hematologic malignancies. Further improvement in the efficacy of this treatment modality will require a better understanding of the regulatory checkpoint(s) limiting T-cell expansion and/or reactivity in vivo. Our group has been especially interested in the epigenetic regulatory mechanisms affecting T-cells, particularly those involving histone deacetylases (HDACs). HDACs are enzymes recruited to gene promoters where they regulate transcription through histone modifications. The role of HDACs in cell biology, initially limited to their effects upon histones, now encompasses more complex regulatory functions that are dependent on their tissue expression, subcellular compartment distribution and the stage of cellular differentiation. For instance, HDAC11, the most recently discovered member of the HDAC family, has been found to be predominantly expressed in the brain, kidney, testis as well as in T-cells. Recently, HDAC11 knock out (KO) mice have been generated by targeted deletion of floxed exon 3 of the HDAC11 gene. Although analysis of the T-cell compartment in these mice revealed no significant differences in the absolute number and/or percent of T-cells in lymphoid organs as compared to wildtype mice, striking functional differences were observed in HDAC11KO T cells. First, in response to in vitro stimulation with anti-CD3 plus anti-CD28 antibodies, HDAC11KO T-cells display an enhanced proliferation, produce significantly higher levels of the pro-inflammatory cytokines IL-2, IFN-gamma and TNF-alpha and are less susceptible to Treg-mediated suppression. Studies performed in HDAC11KO T-cells expressing a transgenic receptor (TCR) specific for ovalbumin (OTII;HDAC11KO mice) confirm that these T-cells are hyperreactive in an antigen-specific manner. In vivo, HDAC11KO T cells induced significantly more severe graft-versus-host disease (GVHD) than wildtype T-cells after allogeneic bone marrow transplantation. Enhanced GVHD mediated by HDAC11KO T cells was associated with increased levels of T-cell expansion and secreted Th1-cytokines such as IFN-gamma. Further demonstration of the intrinsic potency of HDAC11KO T-cells was provided by the finding that when the number of adoptively transferred T-cells was titrated down such that wildtype T-cells no longer induced GVHD, T-cells from HDAC11KO still potently did so. HDAC11KO T-cells are also endowed with a stronger antitumor effect given the prolonged survival observed in mice challenged with C1498 leukemic cells. Mechanistically, using ChIP analysis, we have found that HDAC11 is recruited to the T-bet promoter, which may explain, at least in part, the Th1 phenotype displayed by T-cells in the absence of the repressive effect of HDAC11. Taken together, we have unveiled for the first time that HDAC11 is a regulatory checkpoint in T-cells and represents a novel molecular target to improve the efficacy of T-cell adoptive immunotherapy for the treatment of hematologic malignancies. Disclosures: No relevant conflicts of interest to declare.

1363 The role of HDACs in cell biology, initially limited to their effects upon histones, encompasses now more complex regulatory functions that are dependent on their tissue expression, cellular compartment distribution and the stage of cellular differentiation. Not surprisingly, HDACs have been shown to play important roles in normal B-cell biology and, aberrant expression of these proteins has been found in some B-cell malignancies1. However, the role of specific HDACs in regulation of pro-survival and cell-cycling genes in MCL and CLL still remains poorly understood. We therefore evaluated by RT-PCR the mRNA expression of specific HDACs in MCL and CLL cell lines and in primary cells from patients with these B-cell malignancies. Our analysis revealed a unique and opposing expression of HDAC10 and HDAC11 in these malignant B-cells. While HDAC11 over-expression was frequently found in MCL and CLL cells, in particular in patients with aggressive disease, an almost complete abrogation of HDAC10 was observed in malignant B-cells as compared to normal B-cell controls. These findings led us to explore the biological consequences of manipulating HDAC11 and HDAC10 in MCL and CLL cells. First, knocking-down HDAC11 (HDAC11KD) using lentiviral shRNA resulted in downregulation of cyclin D1, Cdkn1a (p21) and bcl-2. Furthermore, HDAC11KD MCL or CLL cells displayed a slower cell proliferation relative to non-target shRNA control cells. Cell cycle analysis revealed that HDAC11KD clones are arrested in G1. Conversely, over-expression of HDAC11 in the MCL cell line Z138c or in the CLL cell line MEC1 resulted in enhanced cell survival and increased proliferative capacity. Mechanistically, we have recently found that HDAC11 over-expression is associated with increased phosphorylation of STAT3, a known survival pathway in malignant B-cells. Second, HDAC10 belongs to the class II HDAC family and its biological functions remain largely unknown. Similar to our results in aggressive MCL and CLL, a decreased HDAC10 expression has been reported in patients with aggressive solid tumors2, suggesting that loss of HDAC10 expression might confer a survival advantage to malignant cells. Indeed, over-expression of HDAC10 in Z138c and MEC1 cells resulted in a rapid induction of cell death in vitro with only 5% of cells being alive at 48 hours. Our results highlight the need for a better understanding of the expression/function of specific HDACs in MCL and CLL biology. The findings of opposing roles for HDAC11 and HDAC10 in influencing cell survival and proliferation might explain the limited efficacy of pan-HDAC inhibitors (with their indiscriminate inhibition of multiple HDACs) in these B-cell malignancies, and provide support for the development of isotype-selective inhibitors targeting HDAC11. Disclosures: Chen-Kiang: Pfizer, Inc.: Research Funding.

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