Belmonte, Paul

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 ABCB7 exports iron-sulfur (Fe-S) clusters from the mitochondria into the cytoplasm. Conditional deletion of ABCB7 revealed that it is required for peripheral T cell homeostasis. CD4-cre ABCB7 cKO mice have normal thymic development, but a severe reduction in peripheral T cell numbers. A majority of the peripheral T cells in the ABCB7 cKO mice were recent thymic emigrants (RTEs), indicating a loss of more mature T cells in the periphery. T cell maturation markers were found to be normal and stimulation of ABCB7 cKO RTEs revealed normal levels of TNFα production. These data indicate that the loss of peripheral T cells in CD4-cre ABCB7 cKO mice was not due to a defect in development or maturation but a defect in peripheral homeostasis. ABCB7-deficient T cells did not have evidence of elevated apoptosis. We demonstrated iron accumulation was occurring in ABCB7-deficient peripheral T cells using the metal-specific dye Phen Green SK. Additionally, lipid peroxides levels were elevated in the ABCB7 cKO T cells. Lipid peroxides are a hallmark of ferroptosis, an iron-dependent form of regulated cell death. Expression of GPX4, the master regulator of ferroptosis, was normal in ABCB7 cKO T cells. In addition, levels of glutathione, which counteracts lipid peroxides were found to be normal, indicating the ferroptosis protection pathway was intact and that the iron overload was causing ferroptosis. Interestingly, treatment with the iron chelator deferoxamine did not rescue peripheral T cells in the CD4-cre ABCB7 cKO mice. Vitamin E, a potent antioxidant, also failed to rescue peripheral T cell homeostasis. Thus, ABCB7 is required to maintain iron homeostasis and its loss leads to peripheral T cell ferroptosis.

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).

Abstract Inhibitory receptors have a critical role in the regulation of immunity. Siglecs are a family of primarily inhibitory receptors expressed by immune cells, which recognize specific sialic acid modifications on cell surface glycans. Sialyltransferases are the enzymes that catalyzing the addition of sialic acids onto glycoproteins and glycolipids. 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. Many tumors have increased sialic acid incorporation. Overexpression of the sialyltransferase ST8Sia6 on tumors led to altered macrophage polarization and increased tumor growth through engagement of ligands generated by ST8Sia6 with Siglec-E. Here, we examine the role of ST8Sia6 on immune cells in regulating anti-tumor immunity. The loss of ST8Sia6 in tumor bearing mice reduced tumor growth, increased survival, and altered the immune response as compared to ST8Sia6 sufficient tumor bearing mice. Using MC38, B16, and B16-OVA flank tumor mouse models, tumor growth was decreased in ST8Sia6 KO mice as compared to C57Bl/6 mice. Within the tumor microenvironment of tumors present in ST8Sia6 KO mice compared to tumors present in C57Bl/6 mice, macrophages and dendritic cells altered their phenotype towards enhanced immune activation. Tregs from tumors in ST8Sia6 KO mice also displayed an increase in RORγt and a reduction in TNFRII, suggesting a possible shift to a less suppressive Treg. These alterations in the immune response to tumors were due to the loss of ST8Sia6 in macrophages and dendritic cells and not T-cells. Thus, ST8Sia6 generates ligands for Siglecs that dampen anti-tumor immunity. Supported by grants from NIH (R01CA243545, 2T32AI007425) and Mayo Clinic Center of Biomedical Discovery

Abstract Cell surface hypersialylation is used by malignant cells as a survival mechanism, leveraging interactions with Siglec receptors to dampen the immune response. In this study, we aimed to harness this mechanism to prevent autoimmune rejection of beta cells in the context of type 1 diabetes. Mice engineered to overexpress the sialyltransferase ST8Sia6 in beta cell (BST) were generated and crossed to the non-obese diabetic (NOD) mouse for 10 generations. The blood glucose of these mice compared to non-overexpressing littermates was assessed twice weekly to determine diabetes incidence, defined as 3 consecutive blood glucose readings above 250 mg/dL. Analysis of female BST mice aged 15 weeks or greater showed no incidence of overt diabetes, while littermates developed diabetes. BST males aged 15 weeks or greater also had no disease incidence, while littermate controls also became diabetic. An insulin tolerance test demonstrated no significant difference between BST mice and littermates, though the BST had significantly higher blood glucose during a glucose challenge, possibly representing insulin hypersecretion by littermate islets in a stressed pre-diabetic state. Histologic analysis of young BST and littermate pancreata showed no differences, with similar levels of insulitis at 8 weeks. Thus, the overexpression of ST8Sia6 has been shown to mitigate autoimmune attack in the NOD model and presents a novel path forward for prevention of islet transplant rejection.

Abstract ABCB7 exports iron-sulfur (Fe-S) cluster intermediates from the mitochondria to the cytoplasm. Loss of ABCB7 results in mitochondrial iron accumulation in some cell types. We found that ABCB7 is required for peripheral T cell homeostasis, as CD4-cre ABCB7 cKO have normal thymic T cell development but a severe reduction in peripheral T cells, with a majority of peripheral T cells being recent thymic emigrants. The metal-specific dye Phen Green SK indicated iron accumulation was occurring in the absence of ABCB7. We hypothesized that ABCB7-deficient T cells are eliminated by ferroptosis, an iron-dependent form of regulated cell death that is marked by generation of lipid peroxides. Both Bodipy-C11 and Liperfluo, dyes specific for lipid peroxides, were elevated in ABCB7 cKO T cells. Interestingly, expression of GPX4, the master regulator of ferroptosis, was found to be normal in ABCB7-deficient T cells. A high vitamin E diet, which can reduce ferroptosis in GPX4-deficient T cells, did not restore peripheral T cell numbers in CD4-cre ABCB7 cKO mice. Additionally, treatment with the iron chelator deferoxamine did not restore peripheral T cell numbers. Thus, more than one mechanism may be involved in regulating T cell survival in the absence of ABCB7.

Abstract Type 1 diabetes is an autoimmune disease that destroys insulin-producing beta cells in the pancreatic islets. Although exogenous insulin replacement therapy is lifesaving, precise blood glucose management remains a significant challenge. Experimental islet transplantation restores normoglycemia, but the lack of available islets and graft rejection present major barriers to widespread adoption as standard therapy. The islet resident macrophage (IRM) is in close contact with beta cells, efficiently presents islet antigen to autoreactive T cells, and is critical to disease occurrence. We have demonstrated that murine IRMs express high levels of Siglec-E, an immune-regulatory receptor that preferentially binds α2,8-disialic acids in cis or in trans. When islets were exposed to streptozocin (STZ)-induced stress in culture, beta cells upregulated ligands for Siglec-E. To determine the biological significance of this, we generated a mouse model where ST8Sia6, the sialyltransferase that produces α2,8-disialic acids and thus Siglec-E ligands, is constitutively overexpressed in beta cells. This increased trans Siglec-E binding affinity to the beta cell surface, and mitigated hyperglycemia in the multiple low-dose STZ model of diabetes. Significant reduction in blood sugar was evident at 14 days post-initial injection of STZ, coinciding with T cell infiltration of islets. T cells from ST8Sia6+ islets were less activated when compared to littermates. This work reveals a novel and important role for Siglec-E in autoimmune diabetes and provides insight into how rational targeting of this immune receptor may instruct islet-residing antigen presenting cells to provide local tolerance signals in the setting of islet transplantation.

Abstract Inhibitory receptors have a critical role in the regulation of immunity. Siglecs are a family of primarily inhibitory receptors expressed by immune cells that recognize specific sialic acid modifications on cell surface glycans. Many tumors have increased sialic acid incorporation. Overexpression of the sialyltransferase ST8Sia6 on tumors led to altered immune responses and increased tumor growth. In this study, we examined the role of ST8Sia6 on immune cells in regulating antitumor immunity. ST8Sia6 knockout mice had an enhanced immune response to tumors. The loss of ST8Sia6 promoted an enhanced intratumoral activation of macrophages and dendritic cells, including upregulation of CD40. Intratumoral regulatory T cells exhibited a more inflammatory phenotype in ST8Sia6 knockout mice. Using adoptive transfer studies, the change in regulatory T cell phenotype was not cell intrinsic and depended on the loss of ST8Sia6 expression in APCs. Thus, ST8Sia6 generates ligands for Siglecs that dampen antitumor immunity.

Abstract The immune system contains a series of checks and balances that maintain tolerance and prevent autoimmunity. Sialic acid-binding Ig-type lectins (Siglecs) are cell surface receptors found on immune cells and inhibit inflammation by recruiting protein tyrosine phosphatases to ITIMs. Islet-resident macrophages express Siglec-E, and Siglec-E expression decreases on islet-resident macrophages as insulitis progresses in the NOD mouse. The sialyltransferase ST8Sia6 generates α-2,8-disialic acids that are ligands for Siglec-E in vivo. We hypothesized that engaging Siglec-E through ST8Sia6-generated ligands may inhibit the development of immune-mediated diabetes. Constitutive overexpression of ST8Sia6 in pancreatic β cells mitigated hyperglycemia in the multiple low-dose streptozotocin model of diabetes, demonstrating that engagement of this immune receptor facilitates tolerance in the setting of inflammation and autoimmune disease.

Abstract Recent thymic emigrants that fail postpositive selection maturation are targeted by complement proteins. T cells likely acquire complement resistance during maturation in the thymus, a complement-privileged organ. To test this, thymocytes and fresh serum were separately obtained and incubated together in vitro to assess complement deposition. Complement binding decreased with development and maturation. Complement binding decreased from the double-positive thymocyte to the single-positive stage, and within single-positive thymocytes, complement binding gradually decreased with increasing intrathymic maturation. Binding of the central complement protein C3 to wild-type immature thymocytes required the lectin but not the classical pathway. Specifically, MBL2 but not MBL1 was required, demonstrating a unique function for MBL2. Previous studies demonstrated that the loss of NKAP, a transcriptional regulator of T cell maturation, caused peripheral T cell lymphopenia and enhanced complement susceptibility. To determine whether complement causes NKAP-deficient T cell disappearance, both the lectin and classical pathways were genetically ablated. This blocked C3 deposition on NKAP-deficient T cells but failed to restore normal cellularity, indicating that complement contributes to clearance but is not the primary cause of peripheral T cell lymphopenia. Rather, the accumulation of lipid peroxides in NKAP-deficient T cells was observed. Lipid peroxidation is a salient feature of ferroptosis, an iron-dependent nonapoptotic cell death. Thus, wild-type thymocytes naturally acquire the ability to protect themselves from complement targeting by MBL2 with maturation. However, NKAP-deficient immature peripheral T cells remain scarce in complement-deficient mice likely due to ferroptosis.