Shapiro, M.

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)

Abstract Checkpoint blockade has improved patient outcome through reinvigorating T cells against cancer. Despite this success, many patients don’t respond, leaving them with few options. Tumors utilize many immunosuppressive mechanisms, including altering the function of innate immune cells. Cancer cells can promote differentiation of macrophages into tolerogenic “M2-like” macrophages, which support tumor growth. Macrophage differentiation by cancer cells is influenced by many receptors, including Siglec-E. Siglec-E binds to α2,8-linked disialic acids, generated by the enzyme ST8Sia6, on O-linked glycoproteins, and functions to inhibit activation of innate immune cells. In some tumor types, higher expression of ST8Sia6 correlates with poorer survival. Therefore, ST8Sia6 overexpressing tumors may engage Siglec-E, leading to immune suppression and accelerating tumor growth. Mice injected with ST8Sia6 overexpressing MC38 and B16-F10 tumors had increased tumor burden, reduced survival and increased M2 macrophage polarization, which suppresses the immune response. This acceleration of tumor growth was abrogated when injected into Siglec-E knockout mice. Thus, the primary function of ST8Sia6 in tumors is an extrinsic alteration of the immune response, rather than an intrinsic effect on cell growth. Similarly, in a spontaneous colon cancer model, ST8Sia6 overexpression in a murine colon cancer model accelerated the development of tumors, decreasing survival to 2.5 months instead of 6 months. The overexpression of ST8Sia6 leads to increased Arg-1 expression by colonic tumor macrophages. Therefore, ST8Sia6 expression in tumors inhibits the immune response to promote tumor growth in a Siglec-E dependent manner.

Abstract Inflammatory bowel disorders (IBD), including Crohn’s disease and colitis, have a prevalence between 10–20%. IBD patients experience a reduced quality of life due to symptoms such as diarrhea, abdominal cramps, anal bleeding. The exact etiology of IBD is incompletely understood, but the immune response plays a critical role. Innate immune cells integrate the sum of stimulatory and inhibitory signals to determine overall activation. Solidifying the roles of pertinent proteins and their interactions is critical in mapping the immune response and understanding the implications of clinical treatment of IBD. ST8Sia6 is a sialic acid transferase which adds terminal a2,8 disialic acids to cell surface glycoproteins, and we have found that ST8Sia6 generates ligands for Siglec-2 and Siglec-E on innate immune cells, including colonic macrophages and neutrophils. These siglecs are inhibitory sialic acid-binding membrane receptors expressed on several immune cell subsets. Restraining ST8Sia6 should therefore enhance the immune response by reducing siglec-mediated inhibition. To examine this, our lab has generated a novel ST8Sia6 knockout (KO) mouse. At baseline these mice are healthy, but we have found that ST8Sia6 KO mice challenged with dextran sodium sulfate (DSS)-induced colitis lose weight and succumb to colonic inflammation significantly faster than their wildtype (WT) counterparts, particularly at low doses. ST8Sia6 KO mice exhibit increased histological damage and immune infiltration of the colon and small bowel as compared to WT controls. This data demonstrates a role for ST8Sia6 in regulating the hose immune response to inflammatory bowel disorders.

Abstract Iron-sulfur (Fe-S) clusters are cofactors essential for the activity of numerous enzymes including DNA polymerases, helicases, and glycosylases. They are synthesized in the mitochondria as Fe-S intermediates and are exported to the cytoplasm for maturation by the mitochondrial transporter ABCB7. Here, we demonstrate that ABCB7 is required for bone marrow B cell development, proliferation, and class switch recombination, but is dispensable for peripheral B cell homeostasis in mice. Conditional deletion of ABCB7 using Mb1-cre resulted in a severe block in bone marrow B cell development at the pro-B cell stage. The loss of ABCB7 did not alter expression of transcription factors required for B cell specification or commitment. While increased intracellular iron was observed in ABCB7-deficient pro-B cells, this did not lead to increased cellular or mitochondrial reactive oxygen species, ferroptosis, or apoptosis. Interestingly, loss of ABCB7 led to replication-induced DNA damage in pro-B cells, independent of VDJ recombination, and these cells had evidence of slowed DNA replication. Interestingly, ABCB7 appeared to be dispensable for splenic B cell homeostasis, as CD23-cre ABCB7 cKO mice had normal splenic B cell populations. However, stimulated ABCB7-deficient splenic B cells from CD23-cre ABCB7 cKO mice had a striking loss of proliferation and a defect in class switching. Thus, ABCB7 is essential for early B cell development, proliferation, and class switch recombination. Supported by 1R21 AI157328-01 to V.S.S., T32AI007425 to M.J.L.

Abstract Inflammatory bowel disorders (IBD) including ulcerative colitis (UC) and Crohn’s disease (CD) affect between 1–3 million people in the United States with prevalence on the rise. Patients present with symptoms including diarrhea, abdominal cramps, and weight loss. Many factors are implicated in pathogenesis, including host immune response. Identification 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 lamina propria CD4 TH1 polarization, increased inflammatory cytokine production, and decreased IgA class switching of Peyer’s patch B cells, indicative of inflammation. Additionally, adult ST8Sia6 KO mice have shortened small bowels, likely due to baseline inflammation. These mice do not exhibit obvious 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 and after antibiotic treatment. These DSS-challenged ST8Sia6 KO mice exhibit increased histological damage and immune infiltration of the gut as compared to WT controls. The accelerated DSS-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 inflammatory bowel disorders, and further elucidating this role will aid in developing a mechanistic understanding of IBD pathogenesis. Supported by grants from NIH R01 CA243545

Abstract Checkpoint blockade has improved patient outcome through reinvigorating T cells against cancer. Despite this success, many patients don’t respond, leaving them with few options. Tumors utilize many immunosuppressive mechanisms, including altering the function of innate immune cells. Cancer cells can promote differentiation of macrophages into tolerogenic “M2-like” macrophages, which support tumor growth. Macrophage differentiation by cancer cells is influenced by many receptors, including Siglec-E. Siglec-E binds to α2,8-linked disialic acids, generated by the enzyme ST8Sia6, on O-linked glycoproteins, and functions to inhibit activation of innate immune cells. Cancer patients with high expression of ST8Sia6 exhibit reduced survival. Therefore, ST8Sia6 overexpressing tumors may engage Siglec-E, leading to immune suppression and accelerating tumor growth. Mice injected with ST8Sia6 overexpressing MC38 and B16-F10 tumors had increased tumor burden, reduced survival and an increase M2 macrophage polarization. The acceleration in tumor growth was abrogated when injected into Siglec-E knockout mice. Thus, the primary function of ST8Sia6 in tumors is an extrinsic alteration of the immune response, rather than an intrinsic effect on cell growth. Similarly, in a spontaneous colon cancer model, ST8Sia6 overexpression in a murine colon cancer model accelerated the development of tumors, decreasing survival to 2.5 months instead of 6 months. The overexpression of ST8Sia6 on tumors leads to increased Arg-1 expression by CD11b+ CD11c+Ly6C−Ly6G− cells, which suppresses the immune response. Therefore, ST8Sia6 expression in tumors inhibits the immune response to promote tumor growth in a Siglec-E dependent manner.