Choe, Justin

Abstract Introduction Generation of T cells requires dynamic epigenetic alteration of histones to control gene expression at each developmental stage. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) facilitate addition or removal of various acyl groups from lysine residues, including acetylation (KAc) and crotonylation (KCr). The Class I deacetylases HDAC1, HDAC2, and HDAC3 have been shown to have 3 functions: adaptor, decrotonylase, and deacetylase activity. The relative importance of each function in T cells is not known. Genetic knockout of HDAC3 in mice using CD2icre results in a severe block in T cell development and spontaneous development of severe colitis. Our lab determined that HDAC3 is required for three different aspects of this process: CD4+CD8 + (DP) cell survival, positive selection, and CD4+ lineage commitment. We generated a novel mouse model with CD2icre-mediated deletion of endogenous HDAC3 and linked re-expression of HDAC3 “VRPP” mutant mice, which maintained adaptor function but lacks enzymatic activity. Strikingly, these mice do not develop spontaneous colitis even up to one year of age. Methods We used flow cytometry to assess T cell development and maturation in the thymus, spleen, mLN, and colon of HDAC3 cKO vs. HDAC3 VRPP mice. CUT&RUN was utilized to determine differences in H3K9Ac, H3K27Ac, and H3K18Cr across CD8 and CD4 lineage genes. H&E was utilized to assess structural changes in thymus and colon. Results Similar to CD2icre HDAC3 cKO mice, HDAC3 VRPP mice have a block in positive selection due to failure to down regulate RORγt and decreased DP cell survival due to increased expression of P2RX7. Although fewer T cells are produced in HDAC3 VRPP mice, CD4+ lineage commitment is restored, likely mitigating the development of spontaneous colitis. Conclusion Our results demonstrate that HDAC3 enzymatic activity is necessary for several aspects of T cell development, while HDAC3 adaptor function is sufficient to restore CD4+ lineage commitment. Funding Source NIAID RO1 AI150100, T32 AI170478 Topic Categories Hematopoiesis and Immune System Development (HEM)

Abstract Description Generation of T cells requires dynamic epigenetic alteration of histones to control gene expression at each developmental stage. The Class I deacetylases HDAC1, HDAC2, and HDAC3 have been shown to have 3 functions: adaptor, decrotonylase, and deacetylase activity. The relative importance of each function in T cells is not known. CD2icre HDAC3 cKO mice develop severe colitis and demonstrate that HDAC3 is required during thymocyte development for positive selection, DP survival, and CD4 lineage commitment. Recently, a mutation was identified in HDAC3 that eradicated deacetylase activity (“VRPP”). We generated a novel mouse model with Cre-mediated deletion of endogenous HDAC3 and linked re-expression of HDAC3 VRPP, which maintained adaptor function but lacks both deacetylase and decrotonylase enzymatic activity. Similar to CD2icre HDAC3 cKO mice, HDAC3 VRPP mice have a block in positive selection due to failure to down regulate RORγt and decreased DP cell survival due to increased expression of P2RX7, demonstrating that HDAC3 enzymatic activity is required for these functions. However, when the positive selection defect is bypassed in HDAC3 VRPP mice, CD4+ lineage commitment is restored. Strikingly, this mouse does not develop spontaneous colitis even up to one year of age, although T cell development is still impaired. Thus, there are different roles for HDAC3 enzymatic vs. adaptor function during T cell development which are made evident in HDAC3 VRPP mice. Funding Sources NIAID RO1 AI150100; T32 AI170478; Mayo Clinic Graduate School of Biomedical Sciences Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Type 1 diabetes is characterized by the autoimmune destruction of pancreatic β cells, resulting in permanent loss of glucose homeostasis. Islet transplantation is a promising potential cure that remains hindered by immune rejection. We previously showed that ST8Sia6 expression on tumors reduced immune surveillance and hypothesized that this sialyltransferase could protect β cells from autoimmune destruction. Here, we demonstrate that ectopic expression of ST8Sia6 in β cells of female nonobese diabetic mice (NOD βST) decreased the spontaneous incidence of diabetes by 90% and preserved β cell mass. NOD βST mice had comparable insulitis at 8 weeks of age that did not progress over time compared with littermate controls. β Cell–autoreactive B and T cells were present in NOD βST mice, indicating a peripheral rather than central mechanism of immune tolerance. The islets of NOD βST mice displayed a dampened type 1 immune response and reduced IL-12p35 expression in dendritic cells compared with those of littermate controls. The peripheral protection persisted even after removal of ST8Sia6 expression at 20 weeks of age, indicating that transient expression was sufficient for establishment of tolerance. These results demonstrate that ST8Sia6 protects β cells from immune-mediated attack and rejection, highlighting its therapeutic potential for autoimmune disorders.

Abstract In this study, we leveraged immunomodulatory Siglec-sialic acid interactions to protect beta-cells against autoimmune rejection in Type 1 Diabetes (T1D). Our lab previously demonstrated that the sialyltransferase ST8Sia6 generates ligands that bind the Siglec-E receptor expressed on murine islet-resident macrophages. Here, mice were engineered to express ST8Sia6 in beta cells (βST mice), backcrossed to the non-obese diabetic (NOD) mouse model of T1D, and followed for disease. 60% of female littermates spontaneously developed disease by 300 days, compared to only 6% in female βST mice. Histologic analysis of pancreas sections from non-diabetic βST and littermate mice suggested a similar degree of immune infiltration in early life, but drastically reduced insulitis only in βST mice at later timepoints. This was not due to an absence of autoreactive T cells, as an anti-PD-L1 challenge resulted in similarly rapid induction of disease in both βST and littermate mice. Our genetic model allows for temporal control of ST8Sia6 expression in βST mice using the tet-off system. Treating our mice with doxycycline at 20 weeks of age resulted in persistent protection compared to age matched littermates, while the same treatment at 8 weeks of age led to similar incidence of diabetes between βST mice and littermate controls. Flow cytometric analysis of islet infiltrating immune cells from euglycemic mice at 14 weeks of age demonstrated reductions in short-lived effector CD8 T cells and increased thymically derived Tregs in βST mice compared to littermate controls. These results highlight the therapeutic potential of sialyltransferase mediated immune regulation and present a potential path toward improved curative approaches for T1D. Supported by grants from NIH (5R21AI135858-02, 1F30DK127564)

Abstract In this study, we harnessed immunomodulatory Siglec-sialic acid interactions to mitigate autoimmune rejection of beta cells in Type 1 Diabetes (T1D). Mice engineered to express the sialyltransferase ST8Sia6 in beta cells (βST mice) were backcrossed to the non-obese diabetic mouse model of T1D and followed for disease; 60% of female littermates spontaneously developed diabetes by 300 days, compared to minimal occurrence (5%) in female βST mice. Histologic analysis of pancreas sections from non-diabetic βST and littermate mice suggested a similar degree of immune infiltration at 4 and 8 weeks of age, but the severity of insulitis was drastically reduced in βST mice beyond 20 weeks of age as compared to littermates. IHC for various immune populations suggested no difference in composition of insulitis at 8 weeks between βST and littermate mice. ELISA for serum anti-insulin antibodies demonstrated no difference in concentration between female βST and littermate mice. After female euglycemic βST and littermate mice at 16 weeks of age were challenged with anti-PD-L1 monoclonal antibodies, both groups resulted in similarly rapid induction of disease, suggesting that ST8Sia6 dependent protection occurs peripherally. Shut-off of ST8Sia6 expression in euglycemic βST mice at 20 weeks of age did not abrogate protection from disease, supporting persistent immune tolerance beyond this time. Flow analysis of islet infiltrating immune cells at 300 days confirmed reduced number of infiltrating immune cells in βST mice. Thus, induced expression of ST8Sia6 has been shown to mitigate autoimmune attack in the NOD mouse and presents a novel path forward for improving the success of curative islet transplant into T1D patients. Supported by funding from the Mayo Clinic (Mayo Clinic Center for Biomedical Discovery, Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic MSTP) and the NIH (5R21AI135858-02, 1F30DK127564). Supported by funding from the Mayo Clinic (Mayo Clinic Center for Biomedical Discovery, Mayo Clinic Graduate School of Biomedical Sciences, Mayo Clinic MSTP) and the NIH (5R21AI135858-02, 1F30DK127564).

Abstract Cell surface hypersialylation is used by malignant cells as a survival mechanism, leveraging interactions with Siglec receptors to dampen the immune response. In this study, we aimed to harness this mechanism to prevent autoimmune rejection of beta cells in the context of type 1 diabetes. Mice engineered to overexpress the sialyltransferase ST8Sia6 in beta cell (BST) were generated and crossed to the non-obese diabetic (NOD) mouse for 10 generations. The blood glucose of these mice compared to non-overexpressing littermates was assessed twice weekly to determine diabetes incidence, defined as 3 consecutive blood glucose readings above 250 mg/dL. Analysis of female BST mice aged 15 weeks or greater showed no incidence of overt diabetes, while littermates developed diabetes. BST males aged 15 weeks or greater also had no disease incidence, while littermate controls also became diabetic. An insulin tolerance test demonstrated no significant difference between BST mice and littermates, though the BST had significantly higher blood glucose during a glucose challenge, possibly representing insulin hypersecretion by littermate islets in a stressed pre-diabetic state. Histologic analysis of young BST and littermate pancreata showed no differences, with similar levels of insulitis at 8 weeks. Thus, the overexpression of ST8Sia6 has been shown to mitigate autoimmune attack in the NOD model and presents a novel path forward for prevention of islet transplant rejection.