Shapiro, Michael J.

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)

Abstract Description Inflammatory bowel disease (IBD) affects between 1-3 million people in the United States, and patients present with symptoms including diarrhea, rectal bleeding, and fatigue. ST8Sia6 is a sialic acid transferase that adds terminal a2,8 disialic acids to O-linked glycoproteins, and a SNP in ST8Sia6 is associated with Crohn’s Disease. We have found that ST8Sia6 knockout (KO) and heterozygous (het) mice exhibit a 30-fold increase in small bowel immune infiltrate, along with an increased proportion of Th1 and pathogenic Th17 CD4 cells. These mice also exhibit activated gut epithelia, increased inflammatory cytokines, and increased innate cell activation, indicative of gut inflammation. Both ST8Sia6 KO and het mice exhibit increased weight loss and decreased survival when challenged with low dose dextran sodium sulfate-induced colitis. CellChat pathway analysis demonstrates enhanced TNF and IL-1 pathway activation in ST8Sia6 KO small bowel epithelium as compared to WT mice. In addition, ST8Sia6 KO small bowel epithelium produces significantly more CCL5, which may drive immune cell migration. These data demonstrate a role for ST8Sia6 in regulating gut immune homeostasis. As TNF blockade is a first line therapy for IBD patients, elucidating the role of ST8Sia6 in regulating this pathway is critical for a mechanistic understanding of IBD pathogenesis, and this may help to improve immunotherapy options for the disease. Funding Sources NIH R01CA243545 & NIH 5T32DK124190 Topic Categories Basic Autoimmunity (BA)

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.

Total body irradiation for bone marrow transplantation (BMT) conditioning is commonly used in immunologic research. Historically, cesium-137 (137Cs) irradiators were predominantly used as a radiation source for these studies. However, 137Cs has a moderate half-life (30 years), is highly water soluble, and poses significant environmental and radiological safety concerns. As a result, the Department of Energy is promoting the replacement of 137Cs irradiation systems with orthovoltage x-ray cabinet systems, which pose no such concerns. Because the physical characteristics of x-ray and 137Cs photon beams have slightly different energy deposition characteristics, the relatively biological effectiveness (RBE) for x-rays is slightly higher than 137Cs in cell survival studies. This study aims to compare the RBE of orthovoltage and 137Cs irradiators with regards to BMT efficiency. Total body irradiation was performed on C57BL6 mice using 137Cs or 320 kVp x-rays across six dose levels to establish a RBE for lethality. Cohorts of five mice were irradiated using split-dose radiation delivered four hours apart. Daily observations and body weights were captured over a 30-day period. Mice with poor body condition scores or weight loss ≥20% were euthanized. With x-rays, 50% lethality was observed ∼750 cGy while 137Cs required ∼850 cGy, which results in an RBE of 750 cGy/850 cGy = 0.88. Bone marrow ablation and subsequent engraftment efficacy was compared in a parallel study. Cohorts of five C57BL6 CD45.1 recipient mice received total body irradiation with an established single dose 1000 cGy 137Cs regimen or a range of x-ray doses (910-1030 cGy single-dose) and then were infused with CD45.2 donor bone marrow. Ten weeks after BMT, bone marrow, spleen, thymus, and peripheral blood were analyzed via multi-color flow cytometry and the percentage of CD45.1 and CD45.2-positive cells were analyzed to determine marrow engraftment efficiency across various immune compartments. A clear dose-response relationship with bone marrow ablation and engraftment was observed in the stem cell and progenitor compartments. LSK (Lineage- Sca-1+ c-Kit+) cells and other hematopoietic progenitors in the bone marrow were efficiently engrafted (>99%) except for the lowest dose of 910 cGy (around 98%). Similarly, in the spleen, B cells, myeloid, megakaryocyte/erythrocyte, and granulocyte/macrophage lineages were also efficiently engrafted similar to that of 137Cs. Developing thymocytes were efficiently replaced by all radiation regimens, while replacement of peripheral T cell populations varied but were similar to traditional 137Cs at the highest doses. Taken together, these data demonstrate comparable BMT efficiencies with orthovoltage irradiation after appropriate dose-optimization. Citation Format: Danielle M. Burgenske, Brett L. Carlson, Matthew J. Rajcula, Michael J. Shapiro, Virginia M. Shapiro, Jann N. Sarkaria. An in vivo comparison of 137Cs and orthovoltage irradiation for murine bone marrow transplantation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4684.

Abstract Inflammatory bowel disease (IBD) including ulcerative colitis (UC) and Crohn’s disease (CD) affects between 1–3 million people in the United States and costs an estimated $11–28 billion in national healthcare costs. Patients present with symptoms including diarrhea, rectal bleeding, and fatigue. Characterization of molecules that regulate IBD is critical in understanding pathogenesis. ST8Sia6 is a sialic acid transferase that adds terminal a2,8 disialic acids to cell surface glycoproteins, and we have found that ST8Sia6 knockout (KO) mice exhibit increased spontaneous small bowel (SB) lamina propria CD4 TH1 polarization, increased inflammatory cytokine production, decreased IgA class switching of SB plasma cells, and alterations of the SB innate lymphoid cells, indicative of gut inflammation. Moreover, adult ST8Sia6 KO mice have shortened small and large bowels, likely due to baseline inflammation. These mice do not exhibit overt baseline symptoms, but ST8Sia6 KO mice exhibit increased weight loss and decreased survival when challenged with dextran sodium sulfate (DSS)-induced colitis, which was also enhanced when co-housed with wildtype (WT) mice. DSS-challenged ST8Sia6 KO mice exhibit increased histological damage and immune infiltration of the gut as compared to WT controls. Accelerated DSS-induced colitis is also observed when ST8Sia6-deficient bone marrow is transplanted into WT recipients, indicating that the effect is mediated specifically by immune cells. This data demonstrates a role for ST8Sia6 in regulating the host immune response to IBD, and further elucidating this role will aid in developing a mechanistic understanding of IBD pathogenesis and in understanding potential immunotherapies for the disease. R01CA243545, 5T32DK124190-03

Abstract Histone acetyltransferases (HATs) and histone deacetylases (HDACs) facilitate addition or removal of various acyl groups from lysine residues, including acetylation (KAc) and crotonylation (KCr). Recently, mutations in p300 and CBP were identified that abrogate their function as an acetyltransferase but not the ability to mediate crotonylation. These mutants can activate transcription independent of their ability to acetylate histones, demonstrating that crotonylation can also lead to transcriptional activation. The Class I deacetylases HDAC1, HDAC2, and HDAC3 have been shown to have both decrotonylase and deacetylase activity, but the relative importance of each function in T cells is not known. Genetic knockout of HDAC3 using CD2 icre mice results in a severe block in T cell development and spontaneous development of severe colitis. Examination of HDAC3 cKO mice has shown an aberrant ratio of Treg:Th17 cells within the gut, driving inflammation and loss of mucosal barrier integrity. Our lab has also determined that HDAC3 is required for at least three aspects of T cell development: CD4+ CD8+ (DP) cell survival, positive selection, and CD4+ lineage development. Recently, a mutation was identified in either HDAC1 or HDAC3 that eradicated deacetylase activity but retained decrotonylase activity (“VRPP”). Through this mutation we were able to generate a novel mouse model with Cre-mediated deletion of endogenous HDAC3 and linked re-expression of HDAC3 VRPP. Strikingly, this mouse does not develop colitis, although T cell development is still impaired. Thus, HDAC3 decrotonylase activity alone is sufficient to impede the IBD phenotype. Support by grants from National Institute of Allergy and Infectious Diseases (R01 AI150100)

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 Glycans decorate most proteins and lipids on the cell surface, and glycoproteins serve a critical role in cell-cell communication. The terminal modification of many glycans is a sialic acid. There are twenty different mammalian sialyltransferases, each with a unique specificity. Recently, we demonstrated that the sialyltransferase ST8Sia6, which adds a terminal alpha-2,8 disialic acid to a preexisting sialic acid on cell surface proteins, generates ligands for the inhibitory receptor Siglec-E. In patients, ST8Sia6 overexpression is associated with worse outcome. We have previously shown that ST8sia6 overexpression in a mouse tumor model accelerates tumor growth, dependent upon Siglec-E expression by immune cells. To understand the basis for ST8Sia6 function in tumors, proteomic analysis of murine tumor lines was performed to identify proteins modified by ST8Sia6. Here, we demonstrate that ST8Sia6 sialylates CD44. Siglecs vary significantly between species, with 5 non-conserved Siglecs in mice compared to 10 non-conserved Siglecs in humans. The closest ortholog to Siglec-E in mice is Siglec-7 in humans. We also demonstrate that ST8Sia6 sialylates CD44, generating ligands for Siglec-7. Thus, ST8sia6 has a conserved role in both mice and humans in modifying glycans on CD44 to generate ligands for Siglecs. As CD44 expression on tumors is associated with increased metastasis, we predict that ST8Sia6 may also have a role in this process which is under investigation. Supported by grants from 2T32AI007425-23 to DJF and NIH Glycobiology Common Fund supplement to R01CA243545-01A1 to VSS.

Abstract Glycans decorate almost all proteins and lipids on the cell surface, and they play a critical role in cell-cell interactions. Terminal sialic acid addition to glycans helps the immune system distinguish between “self” and “non-self.” The enzyme ST8sia6 adds alpha-2,8 disialic acids to cell surface receptors, and this sialic acid addition promotes binding to the inhibitory receptor Siglec-E. We have previously shown ST8sia6 overexpression in tumors accelerates tumor growth in a Siglec-E dependent manner. Proteomic analysis in murine tumor lines identified CD44 as a target for ST8sia6. CD44 expression on tumors is associated with metastasis and chemoresistance. Since humans and mice have different sialic acid structures, we examined whether ST8sia6 makes CD44 a ligand to the ortholog of Siglec-E in human cells, Siglec-7. When HEK293T cells overexpress ST8sia6 and CD44 together, Siglec-7 binding was significantly increased compared to ST8sia6 or CD44 overexpression alone. Thus, ST8sia6 has a conserved role from mouse to human in decorating CD44 with sialic acid. Our study proposes that ST8sia6 sialylation of CD44 could suppress immune responses and enables cancer progression in human. Supported by grant: 2T32AI007425-23 to DJF and R01 CA243545-01S1 to V.S.S.

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).