A Novel Role for Histone Deacetylase 6 (HDAC6) in the Regulation of IL-10 and Immune Tolerance Mediated by Antigen-Presenting Cells (APCs).

Abstract

Abstract 1360Poster Board I-382There is now an undisputed recognition that bone marrow (BM) derived APCs can induce T-cell activation as well as T-cell tolerance to tumor antigens. The inflammatory status of the APC at the time of tumor antigen presentation, rather than its phenotype, has been proposed as explanation for the induction of such divergent T-cell outcomes. The molecular basis by which the APC regulate this critical decision remain however to be fully elucidated.Recently, we have focused our efforts to mechanistically understand the regulation of inflammatory/anti-inflammatory genes in their natural setting, the chromatin substrate, and how changes at this level could influence the overall inflammatory status of the APC. In particular, we have studied the consequences of chromatin modification by deacetylation of histone tails (mediated by histone deacetylases) upon expression of IL-10, an immunosuppressive cytokine that plays a central role in tolerance induction. By utilizing a reporter gene carrying the IL10 promoter fused to a luciferase gene, and plasmids encoding Flag-tagged versions of specific HDACs, we found that among all the HDACs evaluated, overexpression of HDAC6 in the APC resulted in transcriptional activation of IL-10 gene expression. Conversely, knockdown of HDAC6 in APCs using shRNA specific for murine HDAC6 resulted in abrogation of IL-10 gene transcription in response to LPS, as compared to APCs transduced with nontarget control (NT). Similar results were found when APCs were treated with the hydroxamate-based selective HDAC6 inhibitors, compound 3 (ST-3-06) and compound 7 (ST-292). Treatment of APCs with either compound resulted in a dose-dependent inhibition of IL-10 production in response to LPS. This effect was specific for IL-10, since no inhibition of other cytokines was observed in HDAC6 inhibitor-treated cells. Next, we evaluated the antigen-presenting capabilities of APCs in which HDAC6 was either knocked down or pharmacologically inhibited. In vitro culture of these APCs with naïve CD4+ T cells specific for a MHC class II restricted epitope of influenza hemagglutinin (HA) in the presence of cognate HA peptide resulted in enhanced activation of antigen-specific T cells since they produce higher levels of IL-2 and IFN-g ƒnrelative to clonotypic T cells encountering antigen on control APCs. Importantly, APCs lacking HDAC6 were capable of restoring the responsiveness of tolerized CD4+ T cells isolated from tumor-bearing mice.Our results suggest a role for HDAC6 in positively regulating IL-10 gene transcription in APCs, an effect that it is opposite to the recently described role of HDAC11 as a negative regulator IL-10 gene transcription1. To address whether a potential crosstalk between these two HDACs could represent a novel mechanism to tightly regulate IL-10 gene expression, we first performed confocal studies that revealed that HDAC6 and HDAC11 indeed colocalize in the cytoplasm. Coimmunoprecipitation confirmed that HDAC6 and HDAC11 interact. Furthermore, by using Flag-tagged HDAC6 wild type (1-1215) or Flag-tagged HDAC6 mutants lacking the C-terminus domain we demonstrated that the C-terminus portion of HDAC6 is required for its interaction with HDAC11.Taken together, we have demonstrated for the first time that HDAC6 regulates IL-10 gene expression, an effect that influences the overall inflammatory status of APC and determines antigen-specific T-cell responses. Importantly, inhibition of HDAC6 in APC with specific HDAC inhibitors represents a novel therapeutic approach to tip the balance towards immune activation rather than immune tolerance, a critical decision with significant implications for cancer immunotherapy.1Villagra et al. Nature Immunology, 10:92-100, 2009 DisclosuresPinilla:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding; exelixis: Research Funding.