Zastrow, Alexi

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 Checkpoint blockade revolutionized cancer therapy. However, only a small proportion of cancers respond to checkpoint blockade. B7-H3 is a member of the B7 superfamily, which includes both co-inhibitory and co-stimulatory receptors. B7-H3 is upregulated by many tumors which is generally associated with a worse prognosis. However, the exact mechanism by which B7-H3 suppresses the immune response remains elusive. Methods We transfected human HEK293T cells and assessed the binding to recombinant Siglecs via flow cytometry. We generated monoclonal antibody F7 against human B7-H3 and tested its blocking function using B7-H3 transfected HEK293T cells. We examined the extent of degranulation and NK cell-mediated killing on B7-H3 expressing HEK293T cells in vitro, as well as the CART19 cells mediated killing in relapse model of B7-H3 expressing CD19+ leukemia Nalm6 cells NSG mice. Results We demonstrated that sialylated B7-H3 binds to the inhibitory receptor Siglec-9 on immune cells but not to the related protein Siglec-7. A high affinity monoclonal antibody (mAb) F7 against B7-H3 blocks Siglec-9 binding. Functionally, F7 enhances NK cell killing of B7-H3 expressing target cells in vitro. F7 also dramatically enhances CART19 mediated killing of B7-H3 expressing Nalm6 cells in a relapse model in NSG mice in vivo. Conclusion We have now shown for the first time that B7-H3 binds to the inhibitory receptor Siglec-9 and generated a novel monoclonal antibody F7 that target a unique sequence in B7-H3 as compared to other monoclonal antibodies being examined for clinical efficacy. F7 can promote NK cell killing of B7-H3 expressing target cells in vitro and synergize with CART19 to enhance killing of B7-H3 expressing tumor cells in vivo. As B7-H3 is overexpressed in many human cancers, this work is critical toward understanding how B7-H3 mediates immune evasion through Siglec-9 for enhancing anti-tumor immunity. Funding Source R01CA243545-01A1 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

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 B7-H3 is a member of the B7 superfamily, which includes co-inhibitory receptors such as B7-H1/PD-L1 and co-stimulatory receptors such as B7-1/CD80 and B7-2/CD86. B7-H3 is upregulated by almost all types of tumors and is associated with worse prognosis. However, the exact mechanism by which B7-H3 suppresses anti-tumor immune response remains to be discovered as its ligand has yet to be identified. Sialic acid modifications are also increased in many cancers and are similarly associated with poor outcome. Here, we demonstrate for the first time that sialylated B7-H3 binds to the immune inhibitory receptor Siglec-9. Siglec-9 is expressed on many immune cells, including NK cells and T cells. We have shown that B7-H3 expression on target cells reduces killing by Siglec-9 expressing NK cells in vitro. We then hypothesized that antibodies that block B7-H3 binding to Siglec-9 will be an effective intervention to boost anti-tumor immunity. We have generated a high affinity anti-B7-H3 monoclonal antibody, F7, which blocks B7-H3 binding to Siglec-9. F7 functions to enhance NK cell killing of B7-H3 expressing target cells in vitro. F7 also dramatically enhances CART19 mediated killing of B7-H3 expressing Nalm6 cells in a relapse model in NSG mice in vivo. In summary, our work has demonstrated that B7-H3 binds Siglec-9, and blockade of such interaction can enhance anti-tumor immunity. Funding Sources • Mayo Clinic Center for Biomedical Discovery • NIH Glycobiology Common Fund supplement to R01CA243545-01A1 to VSS Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

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 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 Glioblastoma multiforme (GBM) is a deadly CNS malignancy with an average survival of ~12–18 months post diagnosis. The unique immune environment of the brain and the immunosuppressive features of GBM complicate the use of immunotherapies against these tumors. We aimed to promote the efficacy of αPD-1 against the GL261 murine model of GBM by supplementing this therapy with an engineered IL-2 cytokine. IL-2 therapy has been used clinically for metastatic melanoma and renal cancer, but its short half-life in circulation necessitated high doses to keep the drug concentration sufficiently high. Fusing IL-2 to the mouse serum albumin molecule (MSA-IL-2), extends the cytokine’s half-life. Neither αPD-1 nor MSA-IL-2 are effective monotherapies for GL261 tumors, but the combination provides durable tumor clearance and prevents subsequent rechallenge. Most strikingly, this combination therapy cleared established GL261 tumors even in mice incapable of MHC class I restricted antigen presentation. Instead, therapeutic efficacy was abrogated by the depletion of CD4 T cells. Our combination therapy also reversed multiple key features of GBM-associated peripheral immune suppression, including depressed CD4 T cell counts and dampened MHC class II expression. Our combination therapy’s ability to overcome these immune derangements associated with glioma growth and to function independent of MHC class I restricted antigen presentation enhance the appeal of this therapy for future translational adaptation. This work was supported by the National Institutes of Health (R01 NS103212) and the Mayo Clinic-Koch Institute Cancer Solutions Team Grant