Shapiro, Virginia Smith

Abstract Description During the humoral immune response, B cells undergo rapid metabolic reprogramming with a high demand for nutrients, which are vital to sustain the formation of the germinal centers (GCs). Rag-GTPases sense amino acid availability to modulate the mechanistic target of rapamycin complex 1 (mTORC1) pathway and suppress transcription factor EB (TFEB) and transcription factor enhancer 3 (TFE3). However, how Rag-GTPases coordinate amino acid sensing, mTORC1 activation, and TFEB/TFE3 activity in humoral immunity remains undefined. Here, we show that B cell-intrinsic Rag-GTPases are critical for the development and activation of B cells. RagA/RagB deficient B cells fail to form GCs, produce antibodies, and generate plasmablasts in both T-dependent (TD) and T-independent (TI) humoral immune responses. Deletion of RagA/RagB in GC B cells leads to abnormal dark zone (DZ) to light zone (LZ) ratio and reduced affinity maturation. Mechanistically, the Rag-GTPase complex constrains TFEB/TFE3 activity to prevent mitophagy dysregulation and maintain mitochondrial fitness in B cells, which are independent of canonical mTORC1 activation. TFEB/TFE3 deletion restores B cell development, GC formation in Peyer’s patches and TI humoral immunity, but not TD humoral immunity in the absence of Rag-GTPases. Collectively, our data establish Rag-GTPase-TFEB/TFE3 pathway as an mTORC1 independent mechanism to coordinating nutrient sensing and mitochondrial metabolism in B cells. Funding Sources National Institute of Allergy and Infectious Diseases (NIAID) - AI162678 National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) - AR077518 Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

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 During the humoral immune response, B cells undergo rapid metabolic reprogramming with a high demand for nutrients, which are vital to sustain the formation of the germinal centers (GCs). Rag-GTPases sense amino acid availability to modulate the mechanistic target of rapamycin complex 1 (mTORC1) pathway and suppress transcription factor EB (TFEB) and transcription factor enhancer 3 (TFE3), members of the microphthalmia (MiT/TFE) family of HLH-leucine zipper transcription factors. However, how Rag-GTPases coordinate amino acid sensing, mTORC1 activation, and TFEB/TFE3 activity in humoral immunity remains undefined. Here, we show that B cell-intrinsic Rag-GTPases are critical for the development and activation of B cells. RagA/RagB deficient B cells fail to form GCs, produce antibodies, and generate plasmablasts in both T-dependent (TD) and T-independent (TI) humoral immune responses. Deletion of RagA/RagB in GC B cells leads to abnormal dark zone (DZ) to light zone (LZ) ratio and reduced affinity maturation. Mechanistically, the Rag-GTPase complex constrains TFEB/TFE3 activity to prevent mitophagy dysregulation and maintain mitochondrial fitness in B cells, which are independent of canonical mTORC1 activation. TFEB/TFE3 deletion restores B cell development, GC formation in Peyer’s patches and TI humoral immunity, but not TD humoral immunity in the absence of Rag-GTPases. Collectively, our data establish Rag-GTPase-TFEB/TFE3 axis as an mTORC1 independent mechanism to coordinating nutrient sensing and mitochondrial metabolism in B cells.

Abstract At the terminal position of many glycan chains are unique sugars known as sialic acids. Sialic acids bind sialic acid immunoglobin-like lectins (Siglecs). Our lab is interested in the role of CD22 (Siglec-2), which binds to α2,6-linked sialic acid generated by ST6GalI. CD22 can associate with itself or other molecules on the same cells in a “cis” configuration or bind in “trans” with ligands on other cells. CD22 contains four ITIMs within its cytoplasmic tail and is primarily known to function as an inhibitory co-receptor of the B cell receptor (BCR). CD22-ligand interactions restrain CD22 and BCR association which dampens BCR signaling. While CD22 is usually characterized as a B cell specific protein, we found that CD22 is highly expressed in hybrid macrophages (CD11b+ CD11c+ F4/80+) and expression decreases with activation, implying that CD22 may modulate myeloid cell responses. Using novel macrophage and dendritic cell-specific CD22 cKO mouse models, we will explore how CD22 impacts myeloid cell activation. We propose that CD22 impacts myeloid cell function and modulating this interaction may improve immune responses.

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 The lifetime risk for colorectal cancer in the United States is approximately 4%. Individuals with Inflammatory Bowel Disease, including Ulcerative Colitis and Crohn’s Disease, have a substantially increased risk of developing colorectal cancer. The mechanisms for accelerated tumorigenesis due to enhanced inflammation are not fully characterized. Siglecs (sialic acid immunoglobulin lectin-like proteins) are a family of inhibitory receptors that are negative regulators of the immune response. Siglec-E is an inhibitory receptor that is expressed by innate immune cells, including monocytes, macrophages, neutrophils and dendritic cells. Interestingly, mice deficient in Siglec-E have accelerated development of tumors and reduced survival in a spontaneous mouse model of colorectal cancer (TS4-cre LSL-KRasG12D APClox468/wt). While tumors develop at approximately six months in mice with Siglec-E, tumors develop at approximately two months in Siglec-E knockout mice. Initial results indicate that Siglec-E knockout mice also have accelerated gut inflammation using the DSS colitis model as compared to WT mice. Current studies are examining inflammation that develops during tumorigenesis in Siglec-E knockout mice. Center for Biomedical Discovery NIH R01 CA243545-01A1 to VSS Mayo Clinic Graduate School of Biomedical Sciences Deans fellowship: Initiative for maximizing student development (IMSD) R25 GM055252-26 Doyon Foundation

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.