Model Of Autoimmune Diabetes Research Articles

RIPK1 is dispensable for cell death regulation in β-cells during hyperglycemia

ObjectiveReceptor-interacting protein kinase 1 (RIPK1) orchestrates the decision between cell survival and cell death in response to tumor necrosis factor (TNF) and other cytokines. Whereas the scaffolding function of RIPK1 is crucial to prevent TNF-induced apoptosis and necroptosis, its kinase activity is required for necroptosis and partially for apoptosis. Although TNF is a proinflammatory cytokine associated with β-cell loss in diabetes, the mechanism by which TNF induces β-cell demise remains unclear. MethodsHere, we dissected the contribution of RIPK1 scaffold versus kinase functions to β-cell death regulation using mice lacking RIPK1 specifically in β-cells (Ripk1β-KO mice) or expressing a kinase-dead version of RIPK1 (Ripk1D138N mice), respectively. These mice were challenged with streptozotocin, a model of autoimmune diabetes. Moreover, Ripk1β-KO mice were further challenged with a high-fat diet to induce hyperglycemia. For mechanistic studies, pancreatic islets were subjected to various killing and sensitising agents. ResultsInhibition of RIPK1 kinase activity (Ripk1D138N mice) did not affect the onset and progression of hyperglycemia in a type 1 diabetes model. Moreover, the absence of RIPK1 expression in β-cells did not affect normoglycemia under basal conditions or hyperglycemia under diabetic challenges. Ex vivo, primary pancreatic islets are not sensitised to TNF-induced apoptosis and necroptosis in the absence of RIPK1. Intriguingly, we found that pancreatic islets display high levels of the antiapoptotic cellular FLICE-inhibitory protein (cFLIP) and low levels of apoptosis (Caspase-8) and necroptosis (RIPK3) components. Cycloheximide treatment, which led to a reduction in cFLIP levels, rendered primary islets sensitive to TNF-induced cell death which was fully blocked by caspase inhibition. ConclusionsUnlike in many other cell types (e.g., epithelial, and immune), RIPK1 is not required for cell death regulation in β-cells under physiological conditions or diabetic challenges. Moreover, in vivo and in vitro evidence suggest that pancreatic β-cells do not undergo necroptosis but mainly caspase-dependent death in response to TNF. Last, our results show that β-cells have a distinct mode of regulation of TNF-cytotoxicity that is independent of RIPK1 and that may be highly dependent on cFLIP.

Controlling autoimmune diabetes onset by targeting Protease-Activated Receptor 2

BackgroundType 1 diabetes (T1D) is a challenging autoimmune disease, characterized by an immune system assault on insulin-producing β-cells. As insulin facilitates glucose absorption into cells and tissues, β-cell deficiency leads to elevated blood glucose levels on one hand and target-tissues starvation on the other. Despite efforts to halt β-cell destruction and stimulate recovery, success has been limited. Our recent investigations identified Protease-Activated Receptor 2 (Par2) as a promising target in the battle against autoimmunity. We discovered that Par2 activation’s effects depend on its initial activation site: exacerbating the disease within the immune system but fostering regeneration in affected tissue. MethodsWe utilized tissue-specific Par2 knockout mice strains with targeted Par2 mutations in β-cells, lymphocytes, and the eye retina (as a control) in the NOD autoimmune diabetes model, examining T1D onset and β-cell survival. ResultsWe discovered that Par2 expression within the immune system accelerates autoimmune processes, while its presence in β-cells offers protection against β-cell destruction and T1D onset. This suggests a dual-strategy treatment for T1D: inhibiting Par2 in the immune system while activating it in β-cells, offering a promising strategy for T1D. ConclusionsThis study highlights Par2's potential as a drug target for autoimmune diseases, particularly T1D. Our results pave the way for precision medicine approaches in treating autoimmune conditions through targeted Par2 modulation.

Abstract 6740: Next-generation LNPs induce effective anti-tumor T cell responses

Abstract mRNA immunotherapies have gained interest for the treatment of tumors from various origins, however, their ability to provide long -term durable tumor control has been underwhelming. Here we report that novel mRNA lipid nanoparticle (LNP) vaccinations induce robust expansion of antigen-specific T cells, although this does not always correlate with their ability to infiltrate and kill solid tumors. Using a portfolio of novel ionizable lipids and a robust in vivo model that reliably identifies LNPs with prominent anti-tissue T cell activity we have identified a putative LNP candidate that provides long term tumor control in a murine syngeneic tumor model. We utilized the model antigen glycoprotein (Gp), from the lymphocytic choriomeningitis virus (LCMV), and produced mRNA encoding the immunodominant epitopes gp3333-41, gp6161-80 and gp276276-286 (3GP-mRNA). This mRNA was encapsulated in various novel LNP formulations and administered i.m. into naïve C57Bl/6 mice to track the induction of a robust CD8+ T cell response identified using tetramers to gp33 and gp276. Successful candidate LNPs were then tested in the RIPgp model of autoimmune diabetes. RIPgp transgenic mice express LCMV-Gp in the pancreatic β cells and offer a robust assay for the ability to induce antigen-specific T cells capable of breaking self-tolerance and infiltrating and killing target tissue. Utilizing the RIPgp model to screen LNP candidates we identified a lead candidate, C2-5A, that both induced a robust expansion of antigen-specific T cells and tissue destruction. When utilized in a colon adenocarcinoma model expressing the LCMV-Gp antigen (MC38-Gp), therapeutic dosing of LNP C2-5A as a monotherapy lead to long term tumor clearance in 40% of the treated animals. We believe this screening platform allows the selection of LNP candidates capable of breaking self-tolerance, and one such candidate C2-5A provides a potent vehicle and adjuvant for mRNA based cancer vaccines. Citation Format: Douglas G. Millar, Matthew J. Gold, Kirsten Olsen, Yu Wu, Yury Karpov, Robert Nechanitzky, Haritha Menon, Rajesh Krishnan, Robert Georgantas, Pamela Ohashi, Natalia Martin-Orozco. Next-generation LNPs induce effective anti-tumor T cell responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6740.

Dissecting the Roles of the Nuclear and Mitochondrial Genomes in a Mouse Model of Autoimmune Diabetes.

Mitochondria have been previously implicated in diabetes, but the specific genetic factors remain unclear. To better understand the contributions of mitochondrial genes in nuclear DNA (nDNA) versus mtDNA, we created mitochondrial-nuclear exchange (MNX) mice carrying nDNA from a diabetic strain and mtDNA from nondiabetic mice. Long-term tracking of MNX mice showed occasional large shifts in heteroplasmy consistent with an mtDNA bottleneck in the germ line. In addition, the MNX mice showed improved survival and delayed incidence of diabetes relative to the unaltered diabetic mice, which appeared to be linked to the activity of respiratory complex I.

Immunomodulation by subcutaneously injected methacrylic acid-based hydrogels and tolerogenic dendritic cells in a mouse model of autoimmune diabetes

Type 1 diabetes is an autoimmune disease associated with the destruction of insulin-producing β cells. Immunotherapies are being developed to mitigate autoimmune diabetes. One promising option is the delivery of tolerogenic dendritic cells (DCs) primed with specific β-cell-associated autoantigens. These DCs can combat autoreactive cells and promote expansion of β-cell-specific regulatory immune cells, including Tregs. Tolerogenic DCs are typically injected systemically (or near target lymph nodes) in suspension, precluding control over the microenvironment surrounding tolerogenic DC interactions with the host. In this study we show that degradable, synthetic methacrylic acid (MAA)-based hydrogels are an inherently immunomodulating delivery vehicle that enhances tolerogenic DC therapy in the context of autoimmune diabetes. MAA hydrogels were found to affect the local recruitment and activation state of macrophages, DCs, T cells and other cells. Delivering tolerogenic DCs in the MAA hydrogel improved the local host response (e.g., fewer cytotoxic T cells) and enhanced peripheral Treg expansion. Non obese diabetic (NOD) mice treated with tolerogenic DCs subcutaneously injected in MAA hydrogels showed a delay in onset of autoimmune diabetes compared to control vehicles. Our findings further demonstrate the usefulness of MAA-based hydrogels as platforms for regenerative medicine in the context of type 1 diabetes.

295-OR: Mechanisms of Antigen-Specific Immunotherapy following Tolerance Induction to a CD4 Hybrid Insulin Peptide Epitope and the Impact on CD8 T Cell Dysfunction

Type 1 diabetes is the culmination of T cell mediated autoimmunity resulting in the destruction of insulin-producing beta cells. Islet-derived hybrid insulin peptides (HIPs), recognized by autoreactive CD4 T cells, are likely dominant T cell epitopes in the NOD mouse model of autoimmune diabetes. The 2.5HIP is formed by the fusion of a fragment of insulin C-peptide with a natural cleavage product of chromogranin A, generating a neoepitope. When the 2.5HIP was delivered as an immunotherapy on tolerance inducing PLG-nanoparticles, islet grafts functioned longer in transplanted, diabetic NOD mice. Tolerance induction to this single neoepitope induced dysfunctional cytokine production in autoreactive CD4 and CD8 T cells specific for other known islet antigens. Here we used both prediabetic NOD mice and diabetic transplant recipients to examine mechanisms of peripheral tolerance induction to a HIP. Using the Tiger IL10-GFP reporter mouse, we observed a substantial increase in the proportion of (CD25+) Treg and (CD25-) Tr1 2.5HIP tetramer+ CD4 T cells that expressed IL10 in the islets, spleen, and draining lymph node of 2.5HIP nanoparticle treated mice. Autoreactive, IGRP tetramer+ CD8 T cells displayed a trafficking defect and accumulated in secondary lymphoid tissue with a concomitant reduction within islets. The proportion of cytolytic, CX3CR1+ IGRP tetramer+ CD8 T cells that infiltrated islets was also reduced. IL10 generated by both Treg and Tr1 2.5HIP tet+ T cells may directly suppress IGRP tetramer+ CD8 T cells or suppress an intermediate cell type such as a dendritic cell, causing CD8 T cell dysfunction. Examining how antigen-specific immunotherapies suppress multiple autoreactive T cells specificities may help inform future therapies without the need for immunosuppression. Disclosure J.E.Dilisio: None. R.L.Baker: None. K.M.Haskins: None. Funding National Institutes of Health (5T32DK120520-03, 2R01DK081166-11); JDRF (2-SRA-2020-907-S-B)

Suppression of IGRP-specific CD8 T cells following induction of tolerance to a Hybrid Insulin Peptide CD4 neoepitope

Abstract An autoimmune-mediated death of insulin-producing beta cells, orchestrated by effector CD4 and CD8 T cells that recognize islet antigens, results in Type 1 Diabetes. A dominant CD4 islet autoantigen in the NOD mouse model of autoimmune diabetes is a neoepitope termed the 2.5 Hybrid Insulin Peptide (2.5HIP). By delivering the 2.5HIP as an antigen-specific immunotherapy on tolerogenic PLG-nanoparticles, islet grafts in diabetic NOD mice survived longer and cytokine production in autoreactive CD4 and CD8 T cells was suppressed, including the well-studied IGRP tetramer+ (tet+) CD8 T cells. Using both diabetic transplant recipients and prediabetic NOD mice treated with 2.5HIP nanoparticles, we examined mechanisms of peripheral tolerance induction to a dominant CD4 neoepitope and the impact on the function of IGRP CD8 T cells. Following induction of tolerance to the 2.5HIP, there was an increase in dysfunctional surface marker expression (PD1+ TIM3+) on effector 2.5HIP tet+ CD4 T cells in the spleen of treated mice. Along with a decrease in effector function, we observed an increase in the fraction of both Treg and Tr1 2.5HIP tet+ T cells expressing IL10 in the islets, spleen, and draining lymph nodes of tolerized mice. Concurrently, IGRP tet+ CD8 T cells accumulated in the draining lymph nodes and were less able to traffic and infiltrate islets. Of the IGRP tet+ cells that entered islets, there were fewer cytolytic CX 3CR1 +effector cells in tolerized mice. The robust functional increase in both Treg and Tr1 2.5HIP tet+ T cells may provide a potential mechanism for the accumulation of IGRP tet+ CD8 T cells in the draining lymph node and explain their inefficient trafficking into, and function within, islets following tolerance induction. NIH - T32 5T32DK120520-03, R01 2R01DK081166-11) JDRF 2-SRA-2020-907-S-B

Umbilical cord mesenchymal stromal cells transplantation delays the onset of hyperglycemia in the RIP-B7.1 mouse model of experimental autoimmune diabetes through multiple immunosuppressive and anti-inflammatory responses.

Type 1 diabetes mellitus (T1DM) is an autoimmune disorder specifically targeting pancreatic islet beta cells. Despite many efforts focused on identifying new therapies able to counteract this autoimmune attack and/or stimulate beta cells regeneration, TD1M remains without effective clinical treatments providing no clear advantages over the conventional treatment with insulin. We previously postulated that both the inflammatory and immune responses and beta cell survival/regeneration must be simultaneously targeted to blunt the progression of disease. Umbilical cord-derived mesenchymal stromal cells (UC-MSC) exhibit anti-inflammatory, trophic, immunomodulatory and regenerative properties and have shown some beneficial yet controversial effects in clinical trials for T1DM. In order to clarify conflicting results, we herein dissected the cellular and molecular events derived from UC-MSC intraperitoneal administration (i.p.) in the RIP-B7.1 mouse model of experimental autoimmune diabetes. Intraperitoneal (i.p.) transplantation of heterologous mouse UC-MSC delayed the onset of diabetes in RIP-B7.1 mice. Importantly, UC-MSC i. p. transplantation led to a strong peritoneal recruitment of myeloid-derived suppressor cells (MDSC) followed by multiple T-, B- and myeloid cells immunosuppressive responses in peritoneal fluid cells, spleen, pancreatic lymph nodes and the pancreas, which displayed significantly reduced insulitis and pancreatic infiltration of T and B Cells and pro-inflammatory macrophages. Altogether, these results suggest that UC-MSC i. p. transplantation can block or delay the development of hyperglycemia through suppression of inflammation and the immune attack.

Effect of Acute IFN-α Exposure on β-cell Survival and Function in the Absence of Functional Autophagy

Background and Hypothesis: It has been demonstrated that the protective degradative process of autophagy is defective in the context of human T1D. Additionally, a type I interferon-inducible transcriptional signature has been detected in blood cells of children genetically at risk for T1D prior to the appearance of autoantibodies. Preliminary data demonstrate that autophagy can be stimulated by IFN-α in β-cells of non-diabetic mice, but this response is defective in β-cells of nonobese diabetic (NOD) mice, a mouse model for autoimmune diabetes. We hypothesize that impaired autophagy and exposure to IFN-α will lead to reduced β-cell survival and impaired function.Methods:Bafilomycin A, a lysosomal inhibitor, was used to inhibit autophagy in INS-1 832/13 cells and pancreatic human islets. We then utilized western blot analysis, Caspase-Glo® 3/7 Assay, and Insulin HTRF Assay to assess β-cell survival and function in response to IFN-α exposure.Results: Western blot analysis was used to monitor inhibition of autophagy and stimulation of IFN-α pathway in INS-1 832/13 cells (n=4) and pancreatic human islets (n=1). An increase in LC3II/LC3I ratio was observed after treatment with Baf A, indicating accumulation of autophagosomes. Phosphorylation of STAT2 was observed in IFN-α treated cells, indicating stimulation of the IFN-α pathway. Impaired autophagy and IFN-α exposure did not induce apoptosis in INS-1 832/13 cells (n=2). Following Baf A and IFN-α treatment, glucose stimulated insulin secretion was measured. Glucose stimulated insulin secretion is decreased in INS-1 832/13 cells following Baf A and IFN-α treatment (n=2).Conclusions: These data suggest that inhibition of autophagy followed by an acute treatment with IFN-α does not induce significant cell death, but modestly impairs β-cell function. Future studies will determine the effects of prolonged IFN-α exposure on β-cell survival and function as well as investigate other potential mechanisms of cell death.

Activation-induced cytidine deaminase expression by thymic B cells promotes T-cell tolerance and limits autoimmunity

Elimination of self-reactive Tcells in the thymus is critical to establish T-cell tolerance. A growing body of evidence suggests a role for thymic B cells in the elimination of self-reactive thymocytes. To specifically address the role of thymic B cells in central tolerance, we investigated the phenotype of thymic Bcells in various mouse strains, including non-obese diabetic (NOD) mice, a model of autoimmune diabetes. We noted that isotype switching of NOD thymic B cells is reduced as compared to other, autoimmune-resistant, mouse strains. To determine the impact of B cell isotype switching on thymocyte selection and tolerance, we generated NOD.AID-/- mice. Diabetes incidence was enhanced in these mice. Moreover, we observed reduced clonal deletion and a resulting increase in self-reactive CD4+ Tcells in NOD.AID-/- mice relative to NOD controls. Together, this study reveals that AID expression in thymic Bcells contributes to T-cell tolerance.

Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice.

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease is the transplantation of stem cell-derived beta cells. Genetic modifications, however, may be necessary to protect the transplanted cells from persistent autoimmunity. Diabetic mouse models are a useful tool for the preliminary evaluation of strategies to protect transplanted cells from autoimmune attack. Described here is a minimally invasive method for transplanting and imaging cell grafts in an adoptive transfer model of diabetes in mice. In this protocol, cells from the murine pancreatic beta cell line NIT-1 expressing the firefly luciferase transgene luc2 are transplanted subcutaneously into immunodeficient non-obese diabetic (NOD)-severe combined immunodeficient (scid) mice. These mice are simultaneously injected intravenously with splenocytes from spontaneously diabetic NOD mice to transfer autoimmunity. The grafts are imaged at regular intervals via non-invasive bioluminescent imaging to monitor the cell survival. The survival of mutant cells is compared to that of control cells transplanted into the same mouse.

Compound A attenuates proinflammatory cytokine-induced endoplasmic reticulum stress in beta cells and displays beneficial therapeutic effects in a mouse model of autoimmune diabetes.

Type 1 diabetes (T1D) is characterized by an immune-mediated progressive destruction of the insulin-producing β-cells. Proinflammatory cytokines trigger endoplasmic reticulum (ER) stress and subsequent insulin secretory deficiency in cultured β-cells, mimicking the islet microenvironment in T1D. β-cells undergo physiologic ER stress due to the high rate of insulin production and secretion under stimulated conditions. Severe and uncompensated ER stress in β-cells is induced by several pathological mechanisms before onset and during T1D. We previously described that the small drug Compound A (CpdA), a selective glucocorticoid receptor (GR/NR3C1, nuclear receptor subfamily 3, group C, member 1) ligand with demonstrated inflammation-suppressive activity in vivo, is an effective modulator of effector T and dendritic cells and of macrophages, yet, in a GR-independent manner. Here, we focus on CpdA's therapeutic potential in T1D cellular and animal models. We demonstrate that CpdA improves the unfolded protein response (UPR) by attenuating ER stress and favoring the survival and function of β-cells exposed to an environment of proinflammatory cytokines. CpdA administration to NODscid mice adoptively transferred with diabetogenic splenocytes (from diabetic NOD mice) led to a delay of disease onset and reduction of diabetes incidence. Histological analysis of the pancreas showed a reduction in islet leukocyte infiltration (insulitis) and preservation of insulin expression in CpdA-treated normoglycemic mice in comparison with control group. These new findings together with our previous reports justify further studies on the administration of this small molecule as a novel therapeutic strategy with dual targets (effector immune and β-cells) during autoimmune diabetes.

Costimulation blockade in combination with IL-2 permits regulatory T cell sparing immunomodulation that inhibits autoimmunity

Blockade of CD28 costimulation with CTLA-4-Ig/Abatacept is used to dampen effector T cell responses in autoimmune and transplantation settings. However, a significant drawback of this approach is impaired regulatory T cell homeostasis that requires CD28 signaling. Therefore, strategies that restrict the effects of costimulation blockade to effector T cells would be advantageous. Here we probe the relative roles of CD28 and IL-2 in maintaining Treg. We find provision of IL-2 counteracts the regulatory T cell loss induced by costimulation blockade while minimally affecting the conventional T cell compartment. These data suggest that combining costimulation blockade with IL-2 treatment may selectively impair effector T cell responses while maintaining regulatory T cells. Using a mouse model of autoimmune diabetes, we show combined therapy supports regulatory T cell homeostasis and protects from disease. These findings are recapitulated in humanised mice using clinically relevant reagents and provide an exemplar for rational use of a second immunotherapy to offset known limitations of the first.

Proteomic identification of MHC class I-associated peptidome derived from non-obese diabetic mouse thymus and pancreas

The peptides repertoire presented to CD8+ T cells by major histocompatibility complex (MHC) class I molecules is referred to as the MHC I-associated peptidome (MIP), which regulates thymus development, peripheral survival and function during lifetime of CD8+ T cells. Type 1 diabetes (T1D) is an organ-specific autoimmune disease caused by pancreatic β cells destruction mediated primarily by autoreactive CD8+ T cells. Non-obese diabetic (NOD) mouse is an important animal model of T1D. Here, we deeply analyzed the MIP derived from NOD mice thymus and pancreas, and demonstrated that the thymus MIP source proteins partially shared with the MIP source proteins derived from NOD mice pancreas and β cell line. One H-2Kd restricted peptide SLC35B126–34 which was shared by MIP derived from both NOD mice pancreatic tissues and islet β-cell line, but absent in MIP from NOD thymus tissues, showed ability to stimulate IFN-γ secretion and proliferation of NOD mice splenic CD8+ T cells. The global view of the MHC I-associated self-peptides repertoire in the thymus and pancreas of NOD mice may serve as a biological reference to identify potential autoantigens targeted by autoreactive CD8+ T cells in T1D. Data are available via ProteomeXchange with identifier PXD031966. SignificanceThe peptides repertoire presented to CD8+ T cells by major histocompatibility complex (MHC) class I molecules is referred to as the MHC I-associated peptidome (MIP). The MIP presented by thymic antigen presenting cells (APCs) is crucial for shaping CD8+ T cell repertoire and self-tolerance, while the MIP presented by peripheral tissues and organs is not only involved in maintaining periphery CD8+ T cell survival and homeostasis, but also mediates immune surveillance and autoimmune responses of CD8+ T cells under pathological conditions. Type 1 diabetes (T1D) is an organ-specific autoimmune disease caused by the destruction of pancreatic β cells, mediated primarily by autoreactive CD8+ T cells. Non-obese diabetic (NOD) mouse is one of important animal models of spontaneous autoimmune diabetes that shares several key features with human T1D. The global view of the MHC I-associated self-peptides repertoire in the thymus and pancreas of NOD mice may serve as a good biological reference to identify potential autoantigens targeted by autoreactive CD8+ T cells in T1D. It has great significance for further clarifying the immune recognition and effect mechanism of autoreactive CD8+ T cells in the pathogenesis of T1D, and then developing antigen-specific immune intervention strategies.

1443-P: Real-Time In Vivo Analysis of Beta-Cell Autophagy in Autoimmune Diabetes

Type 1 Diabetes (T1D) is an autoimmune disease caused by a combination of genetic and environmental factors. The onset of disease is associated with autoimmune attack against pancreatic beta-cells leading to a reduction of their number. While a role for the beta-cell in these events is widely hypothesized, a complete understanding of disease pathogenesis is still lacking. Our group recently found that beta-cell autophagy is defective in the context of human T1D as well as in the non-obese diabetic (NOD) mouse model of autoimmune diabetes. Importantly, our data suggested that defective autophagy is present prior to the onset of diabetes but stopped short of definitively demonstrating pre-existing defects. Therefore, here we evaluated autophagic flux in the pancreatic beta-cells of live mice using a fluorescent biosensor coupled to intravital microscopy. A custom beta-cell-selective autophagy biosensor (AAV8-INS-mCherry-EGFP-LC3B) was intraperitoneally (IP) injected into 7-week-old mice of three strains: wild type C57Bl/6J, NOD and NOD scid gamma (NSG) mice. Three weeks after biosensor injections, mice were placed under inhaled isoflurane anesthesia, then externalized pancreata were imaged using a LEICA SP8 DIVE two-photon microscope mounted with a 40x/1.1NA water objective. To evaluate autophagic flux, we assessed fluorescent signal over time in response to IP infusion of either saline or chloroquine, an autophagy inhibitor. Autophagic flux was assessed by quantifying the rate of change of the ph-sensitive GFP and the ph-insensitive RFP, as well as signal colocalization. In wild type mice, we observed clear evidence of autophagic flux over time that was altered in the presence of chloroquine. However, in contrast, in pre-diabetic NOD mice, islet autophagic flux did not respond effectively to chloroquine treatment. Collectively, these data support the conclusion that autophagy defects precede hyperglycemia and suggest a potential role for these defects in beta cell demise during T1D pathogenesis. Disclosure O.Melnyk: None. C.Muralidharan: None. M.M.Martinez irizarry: None. J.Crowder: None. A.K.Linnemann: Consultant; Janssen Research & Development, LLC. Funding National Institutes of Health (R01DK124380) . Startup funds to AKL from the Herman B Wells Center for Pediatric Research at Indiana University School of Medicine

Induction of tolerance to a CD4 T cell hybrid insulin peptide epitope prolongs islet graft survival and suppresses autoreactive CD8 T cells

Abstract Autoreactive T cells are thought to drive autoimmune diabetes by recognizing beta cell-derived peptide antigens. We previously discovered that hybrid insulin peptides (HIPs) are potent neoantigen peptide ligands for a subset of autoreactive CD4 T cells in both the NOD mouse model and human type 1 diabetes patients. Inducing tolerance to prominent T cell autoantigens through antigen-specific immunotherapy could prevent disease onset or recurrence after islet transplantation without the need for broad immunosuppression. Here we show that tolerogenic nanoparticle (NP) delivery of the 2.5HIP, a dominant CD4 T cell HIP epitope in the NOD mouse, can prolong islet graft survival in transplanted diabetic NOD mice. Tolerance induction to the 2.5HIP not only suppressed 2.5HIP tetramer+ CD4 T cells but also autoreactive CD4 and CD8 T cells specific for different islet antigens. 2.5HIP NP treatment induced a dysfunctional state in graft-infiltrating T cells characterized by increased expression of anergic markers on CD4 T cells and reduced inflammatory cytokine production in 2.5HIP tetramer+ CD4 T cells as well as IGRP (islet specific glucose-6-phosphatase catalytic subunit-related protein) tetramer+ CD8 T cells. In conclusion, we demonstrate that antigen-specific immunotherapy aimed at inducing tolerance to a single CD4 T cell HIP epitope can prolong islet graft survival and suppress autoreactive CD8 T cells in the NOD mouse model of autoimmune diabetes. Supported by grants from NIH (R01 DK122566, R01 DK081166, T32 DK120520) and JDRF (2-SRA-2018-566-S-B)

Glucagon-receptor-antagonism-mediated β-cell regeneration as an effective anti-diabetic therapy.

Type 1 diabetes mellitus (T1D) is a chronic disease with potentially severe complications, and β-cell deficiency underlies this disease. Despite active research, no therapy to date has been able to induce β-cell regeneration in humans. Here, we discover the β-cell regenerative effects of glucagon receptor antibody (anti-GcgR). Treatment with anti-GcgR in mouse models of β-cell deficiency leads to reversal of hyperglycemia, increase in plasma insulin levels, and restoration of β-cell mass. We demonstrate that both β-cell proliferation and α- to β-cell transdifferentiation contribute to anti-GcgR-induced β-cell regeneration. Interestingly, anti-GcgR-induced α-cell hyperplasia can be uncoupled from β-cell regeneration after antibody clearance from the body. Importantly, we are able to show that anti-GcgR-induced β-cell regeneration is also observed in non-human primates. Furthermore, anti-GcgR and anti-CD3 combination therapy reverses diabetes and increases β-cell mass in a mouse model of autoimmune diabetes.

D-mannose administration improves autoimmune hepatitis by upregulating regulatory T cells

A recent study revealed that d-mannose suppressed immunopathology in mouse models of autoimmune diabetes and airway inflammation and increased the proportion of regulatory T cells (Tregs) in mice. We investigated the effect of d-mannose on liver injury in murine autoimmune hepatitis (AIH) models induced by concanavalin A (ConA) and α-galactosylceramide (GalCer). Mouse models of AIH were created by intraperitoneal injection of GalCer or intravenous injection of ConA. Drinking water was supplemented with d-mannose and biochemically and pathologically examined over time. The administration of d-mannose to AIH model mice significantly reduced liver injury and reduced inflammatory cytokine expression. In addition, Tregs among splenocytes and intrahepatic lymphocytes were significantly increased by the administration of d-mannose. These results indicate that treatment with d-mannose reduced the inflammatory response in the liver and suppressed liver damage by increasing Tregs.

Dynamic Number and Function of IL-10-Producing Regulatory B Cells in the Immune Microenvironment at Distinct Stages of Type 1 Diabetes.

The critical role of IL-10-producing B cells (B10 cells) with a unique CD1dhiCD5+ phenotype in suppressing autoimmune responses and relieving inflammation has been demonstrated in several models of autoimmune diseases. However, the regulatory role of B10 cells in T cell-mediated autoimmune responses during the natural history of type 1 diabetes is unclear. In this study, we used the NOD mouse model of autoimmune diabetes to clarify the changes and potential mechanisms of B10 cells for disease. Compared with B10 cells present in the 4-wk-old normoglycemic NOD mice, the frequency of B10 cells was increased in the insulitis and diabetic NOD mice, with the highest proportion in the insulitis NOD mice. The changes in the relative number of B10 cells were most pronounced in the pancreas-draining lymph nodes. The pathogenic T cells, including Th1 and Th17 cells, remarkably increased. The assays in vitro showed that B10 cells in the NOD mice did not inhibit the proliferation of CD4+CD25- T cells. They also had no regulatory effect on IFN-γ and IL-4 secretion or on Foxp3 expression of T cells. B10 cells suppressed T cell-mediated autoimmune responses via an IL-10-dependent pathway. In contrast, B10 cells in the NOD mice exhibited a significant reduction in IL-10 production. In summary, a defect in the number and function of B10 cells may participate in the development and progression of type 1 diabetes.

Rational construction of controllable autoimmune diabetes model depicting clinical features.

Through animal models, particularly non-obesity diabetes model (NOD), pathological understandings of human autoimmune diabetes have been gained. However, features of those mouse models and the human disease are not sufficiently analogous; it is therefore not unexpected that interventions based on the mouse data fail at an alarming rate in clinical settings. An improvised model that maximally resembles the real pathological course is highly desirable. Here we devised a 'double-hit' strategy, pancreas was first hit by chemical damage (streptozotocin, STZ) to unleash auto-antigens, then hit second time by transient immune-inflammation (regulatory T cell depletion). Comparing to NOD model, this strategy not only induced classical diabetic symptoms, but also depicted the crucial pathogenic features absent in conventional models, such as CD8+ T cell dominant infiltrates, strong ketoacidosis and epitope-specific T cell responses. In addition, this model allowed synchronized control of disease onset, permitting more refined temporal analysis of disease progression. We believe that this model would yield research outcomes with clinically relevant prediction power unattainable previously.