ALBERT

Description: Thousands of transcripts and proteins confer function and discriminate cell types in the body. Using high-parameter technologies, we can now measure many of these markers at once, and multiple platforms are now capable of analysis on a cell-by-cell basis. Three high-parameter single-cell technologies have particular potential for discovering new biomarkers, revealing disease mechanisms, and increasing our fundamental understanding of cell biology. We review these three platforms (high-parameter flow cytometry, mass cytometry, and a new class of technologies called integrated molecular cytometry platforms) in this article. We describe the underlying hardware and instrumentation, the reagents involved, and the limitations and advantages of each platform. We also highlight the emerging field of high-parameter single-cell data analysis, providing an accessible overview of the data analysis process and choice of tools.

Description: The role of histone deacetylases (HDACs) as epigenetic regulators of immune function is becoming increasingly clear. Recently, the role of specific HDACs in orchestrating T-cell maturation, survival and function has begun to emerge, giving rationale to selective therapy to direct immune responses in different disease settings, including cancer. In particular, HDAC6 has recently been characterized as a negative regulator of regulatory T-cell suppressive activity (de Zoeten, Molecular and Cellular Biology, 2011). Here we report an expanded, novel role of HDAC6 in regulating T-cell survival and activation. First, the relative expression of the eleven classic HDACs was evaluated in resting and activated T-cells from mouse and human samples. It was found that the majority of HDACs decrease in expression following activation, including HDAC6. Next, in a HDAC6KO mouse model, it was found that T-cells lacking HDAC6 had skewed survival when compared to wild-type murine T-cells. This difference seems to be the result of an increased CD4+ T-cells population in the lymph nodes, with a concomitant decrease in viable CD8+ T-cells. To determine whether this population skewing was the consequence of defects in HDAC6KO mice T-cell development, wild-type murine T-cells were treated with an isotype-selective HDAC6 inhibitor. The results seen in HDAC6KO T-cells were recapitulated when wild-type T-cells were activated and treated with HDAC6 specific inhibitors, indicating a role of HDAC6 outside of thymic development in promoting CD4+ T-cell survival at the expense of CD8+ T-cells. Interestingly, it was found that activated CD4+ T-cells displayed decreased expression of the apoptosis signaling receptor FAS after HDAC6 inhibition while no differences were observed in CD8+ T-cells under the same conditions. In addition to these results implicating HDAC6 in regulating T-cell survival, expression of surface markers was altered in both CD8+ and CD4+ T-cells, including enhanced expression of the activation molecule CD69 in stimulated T-cells treated with an isotype-selective HDAC6 inhibitor. Finally, in vivo studies in tumor-bearing HDAC6KO mice revealed a significantly delayed in tumor progression. Similar results were observed in lymphoma-bearing mice treated with HDAC6 specific inhibitors. Taken together, this data shows that HDACs are dynamic in expression with regards to T-cell activation state. More specifically, we have unveiled hereto-unexplored roles of HDAC6 in regulating T-cell survival and function, pointing at this specific HDAC as an appealing target to harness T-cell immunity in hematologic malignancies. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4728 HDAC11 is the newest member of the HDAC family. The physiological role of this HDAC was mainly unknown until the discovery by our group that HDAC11 regulates IL-10 gene expression in immune cells in-vitro1. To better elucidate the role of HDAC11 in lineage differentiation and hematopoiesis, we have utilized an HDAC11 promoter-driven eGFP reporter transgenic mice (TgHDAC11-eGFP) which allow us to “visualize” dynamic changes in HDAC11 gene expression/transcriptional activity in immune cell compartments in vivo. Thus far, our data indicates that in hematopoietic stem cells (CD34+/Lin−), transcriptional activation of HDAC11, indicated by eGFP expression appears to be absent. Also, no eGFP expression is seen in the common lymphoid progenitors (CLP-CD34+/CD127+/CD117low/Lin−) and/or the common myeloid progenitors (CMP-CD34+/CD127−/CD117high/Lin−). In the T-cell compartment, transcriptional activation of HDAC11 increases from CD4−/CD8− T-cells to CD4+/CD8+ T-cells to single positive CD4+ and CD8+ T-cells. The expression of eGFP then decreases from naive to effector memory, but then increases again at terminal effector memory. Expression of eGFP, in the bone marrow moderately increase transitioning from Pro-B-cells (CD3−/CD200+/CD19low/CD43high), Pre-B-cells (CD3−/CD200+/CD19int/CD43int), and Immature (CD3−/CD200+/CD19high/CD43low) respectively. Interestingly eGFP expression doubles in the B-1 (CD3−/CD19+/CD200low/−) stage of differentiation in the periphery. Remarkably, eGFP expression appears to be at its highest in the plasma cell compartment of the bone marrow. A second murine model also available to us, HDAC11 knockout mice (HDAC11KO) were also utilize to confirm these findings. When compared to wild-type mice, HDAC11KO mice have increased B-1 B-cells and decreased plasma cells. In the myeloid compartment, using TgHDAC11-eGFP mice, expression of HDAC11 transcript in myeloblasts (CD34+/CD45dim/CD117+/Lin-) appears to be absent. However the expression increases to 50% in the promyelocytes (Side Scatter high/CD45dim/+/CD34−/CD117+) and to 98% in the granulocytes specifically Neutrophils (Side scatter high/CD45dim+/CD34−/CD117−/CD14−/Ly6Gbright+). Strikingly, monocytes (dendritic cells and macrophages) showed no expression of eGFP. Taken together, HDAC11 appears to be essential for proper B-cells and T-cell differentiation. It also seems to play a critical role in differentiation of granulocytes and monocytes. Therefore it is plausible that HDAC11 might function as a regulator of hematopoietic differentiation and expansion in vivo. A better understanding of this previously unknown role of HDAC11 in hematopoiesis might lead to targeted epigenetic therapies in hematological malignancies to influence the appropriate differentiation of these cells, and possibly augmenting the efficacy of immunotherapeutic approaches against malignancies. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points T cells from HDAC11KO mice have increased effector functions and mediate more rapid and potent GVHD. HDAC11 associates with the Eomes and Tbet gene promoter regions in resting cells and disassociates upon activation.

Description: Abstract 840 T-cells are an essential component of immune mediated tumor rejection. Adoptive transfer of T-cells results in a durable anti-tumor response in some patients with hematological malignancies. To further improve the efficacy of T-cell adoptive transfers, a better understanding of the regulatory checkpoints of these cells is needed. Here we show that HDAC11 is a negative regulator of CD8+ T-cell function, thus representing a potential target in adoptive immunotherapy. HDACs are a group of enzymes initially known for their role in deacetylating histones, thereby condensing chromatin structure and repressing gene expression. The known roles of HDACs as epigenetic regulators have recently expanded to include more complex regulatory functions including interactions with non-histone targets. HDAC11 is the most recently identified member of the HDAC family, and is highly expressed in brain, testis and T-cells. Recently, our group reported HDAC11 as a regulator of IL-10 production in antigen presenting cells. To determine the role of HDAC11 in T-cell biology, T-cells from HDAC11 knock out (HDAC11KO) mice were compared to wild-type T-cells in number, function and phenotype. HDAC11KO T-cells had no differences in absolute number or percentages of CD4+ or CD8+ lymphocytes. However CD8+ T-cells were hyper-proliferative upon CD3/CD28 stimulation and produced significantly higher levels of the pro-inflammatory, Tc1 cytokines IL-2, INF-γ, and TNF-α. However, no significant increases in the production of the Tc2 cytokines IL-4, IL-6 or IL-10 were seen. Further investigation of phenotypic differences also revealed that HDAC11KO mice have a larger percentage of central memory CD8+ T-cells. Additionally, HDAC11KO CD8+ T-cells express higher levels of the transcription factor Eomes, a known contributor to central memory cell formation as well as a controller of granzyme B and perforin production in CD8+ T-cells. This Tc1 and central memory-like phenotype translated to delayed tumor progression and survival in vivo in C1498 AML bearing mice treated with adoptively transferred HDAC11KO T-cells, as compared with wild type T-cells. Collectively, we have demonstrated HDAC11 as a negative regulator of CD8+ T-cell function, and a novel potential target to augment the efficacy of adoptive T-cell tumor immunotherapy. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1660 Recently, we have found that HDAC6 is overexpressed in MCL cell lines and in primary human MCL cells. Knocking-down HDAC6 in MCL cells with a shRNA lentiviral system resulted in cell cycle arrest and apoptosis induction. Interestingly, MCL cells lacking HDAC6 displayed a significantly decreased STAT3 phosphorylation and abrogation of IL-10 gene transcriptional activity. ACY1215 is a novel, selective, orally bioavailable HDAC6 inhibitor. Treatment of MCL cell lines with this agent resulted in decreased cell viability and proliferation. In addition, ACY1215 inhibits IL-10 production in a dose dependent manner. Bruton tyrosine kinase (BTK) is a member of Tec family of kinases with a very distinct role in B-cell antigen receptor (BCR) signaling. The selective BTK-inhibitor PCI-32765 has shown promising pre-clinical and clinical activity in MCL. In addition to their direct anti-lymphoma effects, disruption of BTK also induces positive immunological changes such as inhibition of the immunosuppressive STAT3/IL-10 signaling pathway1. The above observations led us to determine whether the direct antitumor effects and the immunological properties of ACY1215 and PCI-32765 could be potentiated when these agents are used in combination. First, the viability of MCL cells was decreased when they were treated in vitro with either PCI-32765 or ACY1215. However, combination of these two agents resulted in a 3-fold increase in apoptosis induction, pointing to a synergistic effect of BTK and HDAC6 inhibition in MCL. The additional findings that this approach can increase the immunogenicity of MCL cells and anti-MCL immune responses has provided the proper framework for combining the selective HDAC6 inhibitor ACY1215 with BTK inhibition as a novel therapeutic strategy in MCL. Disclosures: Chen-Kiang: Bristol Myers Squibb: Consultancy; Pfizer: Research Funding. Jones:Acetylon Pharmaceuticals, Inc: Employment.

Description: Abstract 829 APCs are critical in T-cell activation and in the induction of T-cell tolerance. Epigenetic modifications of specific genes in the APC play a key role in this process, and among them, histone deacetylases (HDACs) have emerged as key participants. HDAC6 is a 131 KDa protein with preferential cytoplasmic localization where it regulates the acetylation of proteins involved in cytoskeleton, cell-cell interaction and cell migration. Emerging evidence also implicates HDAC6 in regulation of immune responses, in particular at the level of the APC/T cell immune synapse1 and in the suppressive function of regulatory T-cells2. Expanding upon these immunoregulatory properties, here we show for the first time that HDAC6 physically interacts with STAT3, a transcriptional activator of IL-10 gene expression. By co-immunoprecipitation studies and confocal studies we found that HDAC6 co-localize with STAT3 in the cytoplasm and nuclei of macrophages. Furthermore, by using several HDAC6 and STAT3 mutants we have identified that the aminoacids 503–840 of HDAC6 and the STAT3 domain comprising aminoacids 465–585 are required for this interaction. Functionally, knocking down HDAC6 in a macrophage cell line (RAW264.7-HDAC6KD) resulted in inhibition of STAT3 phosphorylation, decreased recruitment of STAT3 to the IL-10 gene promoter and abrogation of IL-10 production by these cells in response to either LPS or IL-10. Similar results were observed in dendritic cells (DCs) or macrophages isolated from HDAC6 knock-out (KO) mice. Furthermore, HDAC6KD clones or APCs from HDAC6 KO mice displayed an enhanced expression of the co-stimulatory molecule B7.2 and are better activators of antigen-specific CD4+ T-cell responses in vitro. More importantly, these APCs are able of restoring the responsiveness of anergic T-cells from lymphoma-bearing mice. Pharmacologic inhibition of HDAC6 in APCs with Tubastatin A, an isotype-selective HDAC6 inhibitor, yielded similar enhancement of APC and T-cell function in vitro. Further support for HDAC6 as an appealing target in cancer immunotherapy has been recently provided by the significant delay in tumor growth observed in either HDAC6 KO mice or in wild type mice treated with Tubastatin A. In summary, we have shown for the first time that HDAC6 interacts physically with STAT3 and such an interaction is necessary for STAT3 phosphorylation and IL-10 gene expression in APCs. Disrupting the HDAC6/STAT3/IL-10 axis in APCs with selective HDAC6 inhibitors represents a novel approach to overcome tolerogenic pathways in these cells and tip the balance towards effective antitumor immune responses. Disclosures: No relevant conflicts of interest to declare.