NOD Mouse Model Research Articles

Inhibition of CD226 co-stimulation suppresses diabetes development in the NOD mouse by augmenting regulatory T cells and diminishing effector T cell function.

Immunotherapeutics targeting T cells are crucial for inhibiting autoimmune disease progression proximal to disease onset in type 1 diabetes. There is an outstanding need to augment the durability and effectiveness of T cell targeting therapies by directly restraining proinflammatory T cell subsets, while simultaneously augmenting regulatory T cell (Treg) activity. Here, we present a novel strategy for preventing diabetes incidence in the NOD mouse model using a blocking monoclonal antibody targeting the type 1 diabetes risk-associated T cell co-stimulatory receptor, CD226. Female NOD mice were treated with anti-CD226 at 7-8 weeks of age and then monitored for diabetes incidence and therapeutic mechanism of action. Compared with isotype-treated controls, anti-CD226-treated NOD mice showed reduced insulitis severity (0.84-fold, p=0.0002) at 12 weeks and decreased disease incidence (HR 0.41, p=0.015) at 30 weeks. Flow cytometric analysis performed 5 weeks post treatment demonstrated reduced proliferation of conventional CD4+ T cells (0.87-fold, p=0.030) and CD8+ (0.78-fold, p=0.0018) effector memory T cells in spleens of anti-CD226-treated mice. Phenotyping of pancreatic Tregs revealed increased CD25 expression (2.05-fold, p=0.0073) and signal transducer and activator of transcription 5 (STAT5) phosphorylation (1.39-fold, p=0.0007) following anti-CD226, with splenic Tregs displaying augmented suppression of CD4+ responder T cells (Tresps) (1.49-fold, p=0.0008, 1:2 Treg:Tresp) in vitro. Anti-CD226-treated mice exhibited reduced frequencies of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-reactive CD8+ T cells in the pancreas, using both ex vivo tetramer staining (0.50-fold, p=0.0317) and single-cell T cell receptor sequencing (0.61-fold, p=0.022) approaches. 51Cr-release assays demonstrated reduced cell-mediated lysis of beta cells (0.61-fold, p<0.0001, 1:1 effector:target) by anti-CD226-treated autoreactive cytotoxic T lymphocytes. CD226 blockade reduces T cell cytotoxicity and improves Treg function, representing a targeted and rational approach for restoring immune regulation in type 1 diabetes.

Efficacy of BAFF inhibition and B-cell depletion in non-obese diabetic mice as a spontaneous model for Sjögren’s disease

IntroductionThe therapeutic interest of targeting B-cell activating factor (BAFF) in Sjögren’s disease (SjD) can be suspected from the results of two phase II clinical trials but has not been evaluated...

Regulatory T Cell Insufficiency in Autoimmune Diabetes Is Driven by Selective Loss of Neuropilin-1 on Intraislet Regulatory T Cells.

Approaches to reverse or limit regulatory T cell (Treg) insufficiency are of great interest for development of immunotherapeutic treatments for autoimmune patients, including type 1 diabetes. Treg insufficiency is heavily implicated in the progression of autoimmune diabetes in the NOD mouse model and is characterized by defects in Treg numbers, development, and/or function. Utilizing a Treg-centric screen, we show that intraislet Tregs have a uniquely dysfunctional phenotype, hallmarked by an almost complete lack of neuropilin-1 (Nrp1), a cell surface receptor required to maintain Treg stability. Intraislet Nrp1- Tregs exhibit hallmark features of fragility, including reduced suppressive capacity, decreased CD73 and Helios, and increased Rorγt and Tbet. Intraislet Nrp1- Tregs also exhibit decreased Foxp3 expression on a per cell basis, suggesting that Nrp1 may also be required for long-term Treg stability. Mechanistically, Treg-restricted augmentation of Nrp1 expression limited the onset of autoimmune diabetes in NOD mice suggesting that Nrp1 critically impacts intraislet Treg function. Transcriptional analysis showed that Nrp1 restoration led to an increase in markers and pathways of TCR signaling, survival, and suppression, and when Nrp1 protein expression is examined by cellular indexing of transcriptomes and epitopes by sequencing, significant differences were observed between Nrp1+ and Nrp1- Tregs in all tissues, particularly in markers of Treg fragility. This translated into substantive differences between Nrp1+ and Nrp1- Tregs that afforded the former with a competitive advantage in the islets. Taken together, these data suggest that maintenance of Nrp1 expression and signaling on Tregs limits diabetes onset and may serve as a strategy to combat Treg insufficiency in autoimmune disease.

Inhibition of CD226 Co-Stimulation Suppresses Diabetes Development in the NOD Mouse by Augmenting Tregs and Diminishing Effector T Cell Function.

Immunotherapeutics targeting T cells are crucial for inhibiting autoimmune disease progression proximal to disease onset in type 1 diabetes. A growing number of T cell-directed therapeutics have demonstrated partial therapeutic efficacy, with anti-CD3 (α-CD3) representing the only regulatory agency-approved drug capable of slowing disease progression through a mechanism involving the induction of partial T cell exhaustion. There is an outstanding need to augment the durability and effectiveness of T cell targeting by directly restraining proinflammatory T helper type 1 (Th1) and type 1 cytotoxic CD8+ T cell (Tc1) subsets, while simultaneously augmenting regulatory T cell (Treg) activity. Here, we present a novel strategy for reducing diabetes incidence in the NOD mouse model using a blocking monoclonal antibody targeting the type 1 diabetes-risk associated T cell co-stimulatory receptor, CD226. Female NOD mice were treated with anti-CD226 between 7-8 weeks of age and then monitored for diabetes incidence and therapeutic mechanism of action. Compared to isotype-treated controls, anti-CD226 treated NOD mice showed reduced insulitis severity at 12 weeks and decreased disease incidence at 30 weeks. Flow cytometric analysis performed five weeks post-treatment demonstrated reduced proliferation of CD4+ and CD8+ effector memory T cells in spleens of anti-CD226 treated mice. Phenotyping of pancreatic Tregs revealed increased CD25 expression and STAT5 phosphorylation following anti-CD226, with splenic Tregs displaying augmented suppression of CD4+ T cell responders in vitro. Anti-CD226 treated mice exhibited reduced frequencies of islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP)-reactive CD8+ T cells in the pancreas, using both ex vivo tetramer staining and single-cell T cell receptor sequencing (scTCR-seq) approaches. 51Cr-release assays demonstrated reduced cell-mediated lysis of beta-cells by anti-CD226-treated autoreactive cytotoxic T lymphocytes. CD226 blockade reduces T cell cytotoxicity and improves Treg function, representing a targeted and rational approach for restoring immune regulation in type 1 diabetes.

Cracking the type 1 diabetes code: Genes, microbes, immunity, and the early life environment.

Type 1 diabetes (T1D) results from a complex interplay of genetic predisposition, immunological dysregulation, and environmental triggers, that culminate in the destruction of insulin-secreting pancreatic β cells. This review provides a comprehensive examination of the multiple factors underpinning T1D pathogenesis, to elucidate key mechanisms and potential therapeutic targets. Beginning with an exploration of genetic risk factors, we dissect the roles of human leukocyte antigen (HLA) haplotypes and non-HLA gene variants associated with T1D susceptibility. Mechanistic insights gleaned from the NOD mouse model provide valuable parallels to the human disease, particularly immunological intricacies underlying β cell-directed autoimmunity. Immunological drivers of T1D pathogenesis are examined, highlighting the pivotal contributions of both effector and regulatory T cells and the multiple functions of B cells and autoantibodies in β-cell destruction. Furthermore, the impact of environmental risk factors, notably modulation of host immune development by the intestinal microbiome, is examined. Lastly, the review probes human longitudinal studies, unveiling the dynamic interplay between mucosal immunity, systemic antimicrobial antibody responses, and the trajectories of T1D development. Insights garnered from these interconnected factors pave the way for targeted interventions and the identification of biomarkers to enhance T1D management and prevention strategies.

Unraveling the Role of CD137 in Treg-Mediated Suppression of Type 1 Diabetes: Insights from Treg-Specific CD137 Knockout Mice

Abstract CD137, encoded by Tnfrsf9, acts as a costimulatory molecule on activated T cells. Conversely, soluble CD137 exerts immunosuppression by binding to CD137L on T cells, inhibiting activation. In the NOD mouse model of Type 1 Diabetes (T1D), CD137 expression in Foxp3+ regulatory CD4 T cells (Tregs) inhibits T1D, but the underlying mechanism remains elusive. To investigate the role of CD137 in Treg-mediated T1D suppression, we generated Treg-specific CD137 knockout mice (NOD.Treg-Tnfrsf9-/-). While no systemic inflammation was observed, these mice developed accelerated T1D compared to wildtype control. Reduced serum-soluble CD137 in these mice indicated Tregs are its main producers. scRNA-seq revealed phenotypic alterations of Tregs isolated from pancreatic islets of NOD.Treg-Tnfrsf9-/- mice, showing lower activation and clonal expansion than wildtype counterparts. Reduced Treg activity in these mice was associated with increased frequencies of islet-activated CD8 T cell subsets and their clonal expansion. MHC class I tetramer staining revealed a higher frequency of β-cell antigen IGRP206-214 specific CD8 T cells in NOD.Treg-Tnfrsf9-/- mice. No discernible differences in the initial expansion and activation of β-cell autoreactive CD8 T cells in pancreatic lymph nodes were observed. Our results suggest CD137 expression in Tregs is critical for restraining ongoing autoimmune inflammation in islets of NOD mice but dispensable for controlling autoreactive T cells in lymphoid tissues.

Stat5b/Ezh2 axis governs high PD-L1 expressing tolerogenic dendritic cell subset in autoimmune diabetes

Dendritic cells (DCs) are specialized antigen-presenting cells that play an important role in inducing and maintaining immune tolerance. The altered distribution and/or function of DCs contributes to defective tolerance in autoimmune diseases such as type 1 diabetes (T1D). In human T1D and in NOD mouse models, DCs share some defects and are often described as less tolerogenic and excessively immunogenic. In the NOD mouse model, the autoimmune response is associated with a defect in the Stat5b signaling pathway. We have reported that expressing a constitutively active form of Stat5b in DCs of transgenic NOD mice (NOD.Stat5b-CA), re-established their tolerogenic function, restored autoimmune tolerance and conferred protection from diabetes. However, the role and molecular mechanisms of Stat5b signaling in regulating splenic conventional DCs tolerogenic signature remained unclear. In this study, we reported that, compared to immunogenic splenic DCs of NOD, splenic DCs of NOD.Stat5b-CA mice exhibited a tolerogenic profile marked by elevated PD-L1 and PD-L2 expression, reduced pro-inflammatory cytokine production, increased frequency of the cDC2 subset and decreased frequency of the cDC1 subset. This tolerogenic profile was associated with increased Ezh2 and IRF4 but decreased IRF8 expression. We also found an upregulation of PD-L1 in the cDC1 subset and high PD-L1 and PD-L2 expression in cDC2 of NOD.Stat5b-CA mice. Mechanistically, we demonstrated that Ezh2 plays an important role in the maintenance of high PD-L1 expression in cDC1 and cDC2 subsets and that Ezh2 inhibition resulted in PD-L1 but not PD-L2 downregulation which was more drastic in the cDC2 subset. Additionally, Ezh2 inhibition severely reduced the cDC2 subset and increased the cDC1 subset and Stat5b-CA.DC pro-inflammatory cytokine production. Together our data suggest that the Stat5b-Ezh2 axis is critical for the maintenance of tolerogenic high PD-L1-expressing cDC2 and autoimmune tolerance in NOD.Stat5b-CA mice.

Analysis of the Bile Acid Composition in a Fibroblast Growth Factor 19-Expressing Liver-Humanized Mouse Model and Its Use for CYP3A4-Mediated Drug-Drug Interaction Studies.

Numerous biomedical applications have been described for liver-humanized mouse models, such as in drug metabolism or drug-drug interaction (DDI) studies. However, the strong enlargement of the bile acid (BA) pool due to lack of recognition of murine intestine-derived fibroblast growth factor-15 by human hepatocytes and a resulting upregulation in the rate-controlling enzyme for BA synthesis, cytochrome P450 (CYP) 7A1, may pose a challenge in interpreting the results obtained from such mice. To address this challenge, the human fibroblast growth factor-19 (FGF19) gene was inserted into the Fah-/- , Rag2-/- , Il2rg-/- NOD (FRGN) mouse model, allowing repopulation with human hepatocytes capable of responding to FGF19. While a decrease in CYP7A1 expression in human hepatocytes from humanized FRGN19 mice (huFRGN19) and a concomitant reduction in BA production was previously shown, a detailed analysis of the BA pool in these animals has not been elucidated. Furthermore, there are sparse data on the use of this model to assess potential clinical DDI. In the present work, the change in BA composition in huFRGN19 compared with huFRGN control animals was systematically evaluated, and the ability of the model to recapitulate a clinically described CYP3A4-mediated DDI was assessed. In addition to a massive reduction in the total amount of BA, FGF19 expression in huFRGN19 mice resulted in significant changes in the profile of various primary, secondary, and sulfated BAs in serum and feces. Moreover, as observed clinically, administration of the pregnane X receptor agonist rifampicin reduced the oral exposure of the CYP3A4 substrate triazolam. SIGNIFICANCE STATEMENT: Transgenic expression of FGF19 normalizes the unphysiologically high level of bile acids in a chimeric liver-humanized mouse model and leads to massive changes in bile acid composition. These adaptations could overcome one of the potential impediments in the use of these mouse models for drug-drug interaction studies.

831-P: Allogeneic Neo-Islets (NIs), an Immune-Privileged Cellular Therapy for T1DM, Is Safe and Effective in Spontaneously Diabetic Pet Dogs, Reducing Insulin Need by up to 50% over Three Years

The purpose of this INAD pilot study was to assess the safety, feasibility and efficacy of NIs in significantly reducing or eliminating a T1DM dog's need for insulin and/or preventing, halting or reversing DM-related damage to a dog's other organs. NIs (3-D organoids made of Mesenchymal Stromal Cells (MSCs) and cultured, partially dedifferentiated islet cells) have been shown preclinically (NOD mouse model) to eliminate the need for exogenous insulin when introduced i.p. Their MSC component provides immune isolation, allowing their allogeneic use without antirejection agents. Hypothesis: Allogeneically administered NIs would safely, durably, and significantly reduce or eliminate the need for exogenous insulin in spontaneously T1DM dogs, as was found in experimental mice. 6 (4 M, 2 F) spontaneously DM, pet dogs; age 1-12 years were treated under INAD 012-776 and followed 3 years. 2×10e5 NIs/kg bw were introduced under ultrasound guidance to metabolically controlled (blood glucose, triglycerides, thyroid, adrenal functions), sedated animals. Dogs were maintained on insulin as needed, and were followed clinically (CBCs, Chemistry panels, Urinalyses, physical examination, imaging, endocrine function) by the PIs and the dogs’ regular veterinarians. Results: Over 3 years, daily insulin needs, blood and urine glucose and fructosamine fell significantly in 5 of 6 dogs; renal function, body weights, CBCs, chemistries were unchanged. Urinary protein decreased; no hypoglycemic events, and immune response to NIs were observed; no AEs or SAEs were attributed to NIs. Redosing and dosing strategies are being tested to try to eliminate exogenous insulin need. Allogeneic NIs are safe and durably effective, and has significant translational relevance. Even partial effectiveness protects from end-organ damage and preserves renal function. A successful Pre-IND meeting for NI use in humans has been held. Disclosure A.Gooch: Board Member; SymbioCellTech, LLC, Employee; SymbioCellTech, LLC, Stock/Shareholder; SymbioCellTech, LLC. C.Westenfelder: Board Member; SymbioCellTech, LLC., Employee; SymbioCellTech, LLC., Research Support; SymbioCellTech, LLC., Stock/Shareholder; SymbioCellTech, LLC.

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)

1653-P: Brown Adipocyte–Secreted Factors for Treating Type 1 Diabetes Mellitus

Hyperglycemia during type 1 Diabetes (T1D) is driven not only by a lack of insulin secretion due to autoimmune destruction of the pancreatic β-cells, but also by hypersecretion of glucagon from pancreatic α-cells. Insulin therapy can replace the lack of insulin secretion, but there is no effective therapeutic tool to counter the hyperglucagonemia. Brown adipose tissue (BAT) has gained attention for treating metabolic diseases due to its role as an endocrine organ and its effect on whole-body energy metabolism. Subcutaneous transplantation of embryonic BAT into T1D mouse models reverses hyperglycemia, independent of insulin but accompanied by reduced plasma glucagon levels, which suggests that lowering plasma glucagon plays a central role in restoring euglycemia. However, the molecular mechanism(s) underlying this action remain unclear. Towards discovering these mechanisms, we have examined the effects of secretions from immortalized brown adipocytes (imBAT) on pancreatic α-cells and mouse models of T1D. We show that the large molecule fraction (&amp;gt;100kDa) of imBAT-secreted factors suppresses glucagon secretion from both mouse and human pancreatic α-cells. Live cell fluorescence imaging shows that this fraction inhibits both intracellular Ca2+ and cAMP activities, and these inhibitions are ameliorated by blocking the insulin receptor, suggesting that imBAT-secreted factors act, at least in part, through these receptors. Injecting this imBAT-secreted fraction into the NOD mouse model reverses hyperglycemia (242 ± 47.4 to 146 ± 24.7mg/dL, n=8), suppresses hyperglucagonemia (77.6 ± 55.4 to 10.7 ± 4.75pg/mL, n=8), improves glucose tolerance (n=3), and increases thermogenic activity and differentiation of endogenous BAT (n=7). Our data provide insights into the mechanisms of long-term recovery of euglycemia by BAT transplants, and further fractionation experiments to isolate active factors are ongoing. Disclosure J.Lee: None. D.W.Piston: None. Funding The Leona M. and Harry B. Helmsley Charitable Trust (2305-06050); National Institutes of Health (R01DK123301, P30DK020579)

Gut microbiota combined with fecal metabolomics reveals the effects of FuFang Runzaoling on the microbial and metabolic profiles in NOD mouse model of Sjögren’s syndrome

ObjectiveSjögren’s syndrome (SS) is an inflammatory autoimmune disease characterized by high levels of chronic lymphocyte infiltration. Differences and dysfunction in the gut microbiota and metabolites may be closely related to the pathogenesis of SS. The purpose of this study was to reveal the relationship between the gut microbiota and metabolome in NOD mice as a model of SS and the role of FuFang Runzaoling (FRZ), which is a clinically effective in treating SS.MethodsNOD mice were gavaged with FRZ for 10 weeks. The ingested volume of drinking water, submandibular gland index, pathologic changes of the submandibular glands, and serum cytokines interleukin (IL)-6, IL-10, IL-17 A, and tumor necrosis factor-alpha (TNF-α) were determined. The roles of FRZ on gut microbiota and fecal metabolites were explored by 16 S rRNA gene sequencing and liquid chromatography-mass spectrometry (LC-MC), respectively. The correlation between them was determined by Pearson correlation analysis.ResultsCompared with the model group, the drinking water volume of NOD mice treated with FRZ increased and the submandibular gland index decreased. FRZ effectively ameliorated lymphocyte infiltration in the small submandibular glands in mice. Serum levels of IL-6, TNF-α, and IL-17 A decreased, and IL-10 increased. The Firmicutes/Bacteroidetes ratio in the FRZ treatment group was higher. FRZ significantly downregulated the relative abundance of the family Bacteroidaceae and genus Bacteroides, and significantly upregulated the relative abundance of genus Lachnospiraceae_UCG-001. Orthogonal projections to latent structures discriminant analysis (OPLS-DA) revealed the significant change in fecal metabolites after FRZ treatment. Based on criteria of OPLS-DA variable influence on projection > 1, P < 0.05, and fragmentation score > 50, a total of 109 metabolites in the FRZ-H group were differentially regulated (47 downregulated and 62 upregulated) compared to their expressions in the model group. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed enriched metabolic of sphingolipid metabolism, retrograde endocannabinoid signaling, GABAergic synapse, necroptosis, arginine biosynthesis, and metabolism of histidine, alanine, aspartate, and glutamate. Correlation analysis between gut microbiota and fecal metabolites suggested that the enriched bacteria were related to many key metabolites.ConclusionsTaken together, we found FRZ could reduce the inflammatory responses in NOD mice by regulating the gut microbiota, fecal metabolites, and their correlation to emerge a therapeutic effect on mice with SS. This will lay the foundation for the further studies and applications of FRZ, and the use of gut microbiotas as drug targets to treat SS.Graphical

Inflammation versus regulation: how interferon-gamma contributes to type 1 diabetes pathogenesis.

Type 1 diabetes is an autoimmune disease with onset from early childhood. The insulin-producing pancreatic beta cells are destroyed by CD8+ cytotoxic T cells. The disease is challenging to study mechanistically in humans because it is not possible to biopsy the pancreatic islets and the disease is most active prior to the time of clinical diagnosis. The NOD mouse model, with many similarities to, but also some significant differences from human diabetes, provides an opportunity, in a single in-bred genotype, to explore pathogenic mechanisms in molecular detail. The pleiotropic cytokine IFN-γ is believed to contribute to pathogenesis of type 1 diabetes. Evidence of IFN-γ signaling in the islets, including activation of the JAK-STAT pathway and upregulation of MHC class I, are hallmarks of the disease. IFN-γ has a proinflammatory role that is important for homing of autoreactive T cells into islets and direct recognition of beta cells by CD8+ T cells. We recently showed that IFN-γ also controls proliferation of autoreactive T cells. Therefore, inhibition of IFN-γ does not prevent type 1 diabetes and is unlikely to be a good therapeutic target. In this manuscript we review the contrasting roles of IFN-γ in driving inflammation and regulating the number of antigen specific CD8+ T cells in type 1 diabetes. We also discuss the potential to use JAK inhibitors as therapy for type 1 diabetes, to inhibit both cytokine-mediated inflammation and proliferation of T cells.

Tuning peptide specificity for T cell tolerance in Type 1 diabetes

Abstract Type 1 Diabetes (T1D) is a classical T-cell mediated autoimmune disease, and fundamental data have implicated insulin as the dominant autoantigen in T1D disease. In the NOD mouse model of T1D, notable studies have found that mice lacking native insulin expression but with an altered insulin sequence to maintain blood glucose levels are protected from insulitis and diabetes. Growing evidence also indicates how the insulin peptide is bound within MHC Class II (peptide register) is essential in determining the strength of interactions and recognition of autoreactive T-cells. We have uncovered a unique peptide binding characterized by the dominant insulin epitope InsB:9–23. The majority of InsB:9–23-specific CD4 T-cells in the periphery recognize insulin bound in the unusual register 3, and knocking in this single amino acid variation into just one copy of the insulin gene in NOD mice, confers protection against T1D. In addition, these mice were protected from T1D, even when treated with checkpoint inhibitor blockade, unlike their wild-type littermates. Introducing this superagonist epitope into the NOD mouse has allowed us to address several key questions surrounding pathways of peptide generation, presentation by MHC molecules, and recognition by auto-reactive pathogenic T cells. In addition, we have been able to model how central and peripheral tolerance mechanisms can be altered when major epitopes in the Insulin gene are mutated, allowing us to explore means of dominant tolerance to understand the potential for translation into treatments for T1D. Supported by grants from NIH (R01 DK133443-01A)

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

Correction of T-Cell Repertoire and Autoimmune Diabetes in NOD Mice by Non-myeloablative T-Cell Depleted Allogeneic HSCT.

The induction of partial tolerance toward pancreatic autoantigens in the treatment of type 1 diabetes mellitus (T1DM) can be attained by autologous hematopoietic stem cell transplantation (HSCT). However, most patients treated by autologous HSCT eventually relapse. Furthermore, allogeneic HSCT which could potentially provide a durable non-autoimmune T-cell receptor (TCR) repertoire is associated with a substantial risk for transplant-related mortality. We have previously demonstrated an effective approach for attaining engraftment without graft versus host disease (GVHD) of allogeneic T-cell depleted HSCT, following non-myeloablative conditioning, using donor-derived anti-3rd party central memory CD8 veto T cells (Tcm). In the present study, we investigated the ability of this relatively safe transplant modality to eliminate autoimmune T-cell clones in the NOD mouse model which spontaneously develop T1DM. Our results demonstrate that using this approach, marked durable chimerism is attained, without any transplant-related mortality, and with a very high rate of diabetes prevention. TCR sequencing of transplanted mice showed profound changes in the T-cell repertoire and decrease in the prevalence of specific autoimmune T-cell clones directed against pancreatic antigens. This approach could be considered as strategy to treat people destined to develop T1DM but with residual beta cell function, or as a platform for prevention of beta cell destruction after transplantation of allogenic beta cells.

Heterogeneity of Islet-Infiltrating IL-21+ CD4 T Cells in a Mouse Model of Type 1 Diabetes.

IL-21 is essential for type 1 diabetes (T1D) development in the NOD mouse model. IL-21-expressing CD4 T cells are present in pancreatic islets where they contribute to T1D progression. However, little is known about their phenotype and differentiation states. To fill this gap, we generated, to our knowledge, a novel IL-21 reporter NOD strain to further characterize IL-21+ CD4 T cells in T1D. IL-21+ CD4 T cells accumulate in pancreatic islets and recognize β cell Ags. Single-cell RNA sequencing revealed that CD4 T effector cells in islets actively express IL-21 and they are highly diabetogenic despite expressing multiple inhibitory molecules, including PD-1 and LAG3. Islet IL-21+ CD4 T cells segregate into four phenotypically and transcriptionally distinct differentiation states, that is, less differentiated early effectors, T follicular helper (Tfh)-like cells, and two Th1 subsets. Trajectory analysis predicts that early effectors differentiate into both Tfh-like and terminal Th1 cells. We further demonstrated that intrinsic IL-27 signaling controls the differentiation of islet IL-21+ CD4 T cells, contributing to their helper function. Collectively, our study reveals the heterogeneity of islet-infiltrating IL-21+ CD4 T cells and indicates that both Tfh-like and Th1 subsets produce IL-21 throughout their differentiation process, highlighting the important sources of IL-21 in T1D pathogenesis.

Inhibition of PI3K/C/EBPβ axis in tolerogenic bone marrow-derived dendritic cells of NOD mice promotes Th17 differentiation and diabetes development

Dendritic cells (DCs) are key regulators of the adaptive immune response. Tolerogenic dendritic cells play a crucial role in inducing and maintaining immune tolerance in autoimmune diseases such as type 1 diabetes in humans as well as in the NOD mouse model. We previously reported that bone marrow-derived DCs (BM.DCs) from NOD mice, generated with a low dose of GM-CSF (GM/DCs), induce Treg differentiation and are able to protect NOD mice from diabetes. We had also found that the p38 MAPK/C/EBPβ axis is involved in regulating the phenotype, as well as the production of IL-10 and IL-12p70, by tolerogenic GM/DCs. Here, we report that the inhibition of the PI3K signaling switched the cytokine profile of GM/DCs toward Th17-promoting cytokines without affecting their phenotype. PI3K inhibition abrogated the production of IL-10 by GM/DCs, whereas it enhanced their production of IL-23 and TGFβ. Inhibition of PI3K signaling in tolerogenic GM/DCs also induced naive CD4+ T cells differentiation toward Th17 cells. Mechanistically, PI3K inhibition increased the DNA-binding activity of C/EBPβ through a GSK3-dependent pathway, which is important to maintain the semimature phenotype of tolerogenic GM/DCs. Furthermore, analysis of C/EBPβ−/− GM/DCs demonstrated that C/EBPβ is required for IL-23 production. Of physiological relevance, the level of protection from diabetes following transfusion of GM/DCs into young NOD mice was significantly reduced when NOD mice were transfused with GM/DCs pretreated with a PI3K inhibitor. Our data suggest that PI3K/C/EBPβ signaling is important in controlling tolerogenic function of GM/DCs by limiting their Th17-promoting cytokines.

Immune cells and their inflammatory mediators modify β cells and cause checkpoint inhibitor-induced diabetes.

Checkpoint inhibitors (CPIs) targeting programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) have revolutionized cancer treatment but can trigger autoimmune complications, including CPI-induced diabetes mellitus (CPI-DM), which occurs preferentially with PD-1 blockade. We found evidence of pancreatic inflammation in patients with CPI-DM with shrinkage of pancreases, increased pancreatic enzymes, and in a case from a patient who died with CPI-DM, peri-islet lymphocytic infiltration. In the NOD mouse model, anti–PD-L1 but not anti–CTLA-4 induced diabetes rapidly. RNA sequencing revealed that cytolytic IFN-γ+CD8+ T cells infiltrated islets with anti–PD-L1. Changes in β cells were predominantly driven by IFN-γ and TNF-α and included induction of a potentially novel β cell population with transcriptional changes suggesting dedifferentiation. IFN-γ increased checkpoint ligand expression and activated apoptosis pathways in human β cells in vitro. Treatment with anti–IFN-γ and anti–TNF-α prevented CPI-DM in anti–PD-L1–treated NOD mice. CPIs targeting the PD-1/PD-L1 pathway resulted in transcriptional changes in β cells and immune infiltrates that may lead to the development of diabetes. Inhibition of inflammatory cytokines can prevent CPI-DM, suggesting a strategy for clinical application to prevent this complication.

1473-P: Combination Therapy with Dual-Function Peptide S1K and Insulin Reduces Blood Glucose Levels in Mouse Models of Type 1 Diabetes through Beta-Cell Regeneration and Immunosuppression

β cell regeneration is necessary to cure patients with type 1 diabetes (T1D) whose β cells are destroyed by immune cells. Although insulin therapy can control blood glucose levels, it must be administered throughout life and occurs several side effects such as hypoglycemia. New β cell can be generated through neogenesis, β cell replication or transdifferentiation from other cells in diabetic animals. In addition to β cell regeneration, immunosuppression is another important mechanism to prevent β cell destruction, but currently any drugs haven’t been approved as immunosuppressants for T1D treatments yet. A novel 9-mer peptide, Shiloah 1000 (S1K) , has dual functions of β cell regeneration and immunosuppression. In streptozotocin-induced hyperglycemic mouse model, the number of mice recovered to normoglycemia was increased by the combined treatment of S1K and insulin compared to S1K or insulin alone. In addition, the combined treatment more increased the number of β cells than S1K or insulin alone. Interestingly, insulin/glucagon double-positive cells were increased by both combined and S1K alone treatments. The increase in the number of mice recovered to normoglycemia is considered to be due to β cell regeneration through α- to β-cell transdifferentiation by S1K. In the NOD mouse model, the combined treatment of S1K and insulin suppressed hyperglycemia more than insulin alone. In addition, the serum C-peptide levels were increased by both combined and S1K treatments, but not by insulin treatment. The increase in the serum C-peptide level suggest that β cells remain undestroyed by immunosuppression. Taken together, our data show that S1K, as a drug candidate for T1D treatment in combination with insulin reduces blood glucose levels in the T1D mouse models through β cell regeneration and immunosuppression, suggesting a potential role in preventing and curing T1D. Disclosure Y. Lee: Employee; Ensolbiosciences. H. Kim: Employee; Ensolbiosciences. J. Kim: Employee; Ensol Bioscience. Y. Jang: Employee; Ensolbiosciences.