Citations

Abstract Introduction The lifetime risk for colorectal cancer (CRC) 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. Siglecs (sialic acid immunoglobulin lectin-like proteins) are a family of receptors that regulates the immune response. Siglec-E is a mouse inhibitory receptor that is primarily expressed by innate cells. While others have demonstrated that Siglec-E KO mice reject tumors faster in multiple tumor models, we have found that colorectal cancer is accelerated in Siglec-E KO mice. Here we aim to investigate the role of Siglec-E in immune homeostasis, inflammation and colorectal cancer development. Methods We used two models of colorectal cancer to assess the role of Siglec-E in tumorigenesis: TS4-cre LSL KRASG12D APClox468/wt (TAR mice) and the DSS/AOM model of colitis-associated cancer. Colons were prepared by separating lamina propria (LP) and epithelial (EP) fractions for flow cytometry. 2cm sections were taken from the middle of colons to generate lysates for ELISA. DSS was administered to WT and Siglec-E KO mice to assess the susceptibility to intestinal inflammation. H&E and IHC for PCNA were performed on colonic tissue from experiments. Results We have found that in both the spontaneous and DSS induced models of CRC, Siglec-E KO mice grow more tumors than WT mice. Siglec-E KO mice have an exquisite susceptibility to DSS induced inflammation and reduced number and proportion of neutrophils after treatment. Lastly, we have found that Siglec-E KO mice at baseline have increased VEGF-A in the colon. Conclusion Siglec-E has a critical role in regulating immune homeostasis in the colon. Siglec-E functions as a brake for intestinal inflammation which protects from the development of colorectal cancer. Funding Source This research was funded by NIH grant CA243545-01A1 (VSS), NIH T32 AI170478 (VSS/BM), and and grant R25 GM055252-26 Deans fellowship: Initiative for maximizing student development (IMSD). Topic Categories Cellular Adhesion, Migration, and Inflammation (CAM)

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 Introduction The mitochondrial transporter ABCB7 is essential for exporting iron-sulfur (Fe-S) clusters, a function known to be critical in B cells. This study was conducted to investigate the broader, previously unknown roles of ABCB7 in regulating the overall lymphocyte development and homeostasis. Methods The study utilized conditional knockout (cKO) mouse models (CD2-iCre and Mb1-Cre) to create a specific deficiency of ABCB7 in certain immune populations. To determine if the observed effects were due to ABCB7 deletion in myeloid cells, two further models (CD2i-Cre ABCB7 cKO/C/EBPα and CD2-iCre LSL TMT ABCB7 cKO) were developed. The effects of blocking the cytokine G-CSF were also examined to understand its role in the observed myelopoiesis. Results ABCB7 deficiency was found to dramatically impair erythropoiesis while simultaneously driving emergency myelopoiesis. We confirmed that the myeloid expansion was not a direct result of ABCB7 loss in myeloid cells. A significant autoimmune phenotype also developed, characterized by hyperactivated T cells and a sharp decrease in peripheral regulatory T cells (Tregs). This Treg instability was linked to decreased thymic expression of key stability genes (like CD28 and PD-1) and reduced O-GlcNAc modification in T cells, a process crucial for maintaining Treg lineage. Conclusion The findings establish ABCB7 as a pivotal regulator of hematopoietic balance, linking mitochondrial function to both myeloid expansion and Treg stability. Funding Source T32AI170478 Topic Categories Hematopoiesis and Immune System Development (HEM)

Abstract Description Interactions between ST6GAL1-produced sialic acids and Siglec-2 (CD22) result in immune inactivation. CD22 is a well-characterized regulator of B cell function. We demonstrate that conventional type-2 dendritic cells (cDC2s) and hybrid macrophages express CD22 across the spleen and tumor microenvironment. To understand the function of CD22 on antigen-presenting cells, we generated novel CD22-floxed mice and crossed them with a) Zbtb46-Cre ones (those lacking CD22 on cDC2s and hybrid macrophages) as well as b) CX3CR1-Cre mice (without CD22 on hybrid macrophages nor microglia). From the former cross, we generated subcutaneous flank tumor and colitis-associated colorectal cancer models. CD22 expression on myeloid cells did not impact anti-tumor responses when tumors expressed low CD22 ligand levels. From here, we assess the impact of tumor cell CD22 ligand overexpression on tumor responses. Also, to evaluate CD8 T cell response to CD22, we challenged mice with Theiler’s murine encephalomyelitis virus (TMEV), modeling multiple sclerosis via CD8 T cell hyperactivation. CD22 did not mediate CD8 activation nor infiltration in brains of the latter cross. Thus, CD22 may be functionally redundant across myeloid and B cells. Topic Categories Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Abstract Description 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 the development of tumors and reduced survival in a spontaneous mouse model of colorectal cancer (TS4-cre LSL-KRasG12D APClox468/wt). Siglec-E knockout mice also have accelerated gut inflammation using the DSS colitis model as compared to WT mice. Using the DSS/AOM model of colitis-associated colorectal cancer, we found that Siglec-E KO mice develop more tumors as compared to wild-type controls. Thus, Siglec-E is an important regulator of gut homeostasis, and the loss of Siglec-E accelerates colon cancer in two murine models. Funding Sources Mayo Clinic Center for Biomedical Discovery NIH R01 CA243545-01A1 to VSS T32 AI170478 Mayo Clinic Graduate School of Biomedical Sciences Deans fellowship: Initiative for maximizing student development (IMSD) R25 GM055252-26 Doyon Foundation Topic Categories Mucosal and Regional Immunology (MUC)

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)

Mucin-1 (MUC1) is a highly relevant antigen for cancer vaccination due to its overexpression and hypo-glycosylation in a high percentage of carcinomas. To enhance the immune response to MUC1, our group has developed C3-liposomes that encapsulate the MUC1 antigen along with immunostimulatory compounds for direct delivery to antigen-presenting cells (APCs). C3-liposomes bind complement C3, which interacts with C3-receptors on APCs, resulting in liposomal uptake and the delivery of tumor antigens to APCs in a manner that mimics pathogenic uptake. In this study, MUC1 and Toll-like receptor (TLR) agonists were encapsulated in C3-liposomes to provoke an immune response in transgenic mice tolerant to MUC1. The immune response to the C3-bound MUC1 liposomal vaccine was assessed by ELISA, ELISpot, and flow cytometry. Co-administering TLR 7/8 agonists with MUC1 encapsulated in C3-liposomes resulted in a significant antibody response compared to non-encapsulated MUC1. This antibody response was significantly higher in females than in males. The co-encapsulation of three TLR agonists with MUC1 in C3-liposomes significantly increased antibody responses and eliminated sex-based differences. Furthermore, this immunization strategy resulted in a significantly increased T cell-response compared to other treatment groups. In conclusion, the co-delivery of MUC1 and TLR agonists via C3-liposomes greatly enhances the immune response to MUC1, highlighting its potential for antigen-specific cancer immunotherapy.

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 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 6 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.