Islets Of Nonobese Diabetic Mice Research Articles

Arachidonic acid alleviates autoimmune diabetes in NOD mice

BackgroundArachidonic acid (AA) is considered to link nutrient metabolism, to inflammation and immunity, suggesting it may have a role in autoimmune diseases. Our previous study suggests that DPP-4 inhibitors (DPP-4i) might regulate AA – relative signaling in type 1 diabetes. AimsTo examine the effect of AA on autoimmune diabetes and its cross-talk with DPP-4i in The Non-Obese Diabetic (NOD) mice. MethodsThe NOD mice were divided randomly and equally into three groups: AA group, AA plus DPP-4i group and control group. The incidence of diabetes, blood glucose, insulitis and cytokine profiles were monitored. At the end of the experiment, pancreatic tissues were stained by H&E. Serum cytokine profiles were examined using a Mesco Scale Discovery multiplexed-assay kit. ResultsEven though AA or AA plus DPP-4i treatment has no effect on incidence of diabetes and weight, AA treatment reduces blood glucose, preserves islet morphology and alleviates inflammatory cell infiltration into pancreatic islets in NOD mice, accompanying with increased serum levels of IL-10, IL-1 β, IL-6, IL-5, KC/GRO and TNF-α and decreased serum levels of IL-2. ConclusionWe observed that AA treatment alleviates autoimmune diabetes in NOD mice by reducing hyperglycemia, alleviating insulitis and improving cytokine profiles. DPP-4i might alleviate the effect of AA by cross-talk. We provide evidence of AA treatment to alleviate type 1 diabetes in NOD mice, which may provide a novel therapeutic option for type 1 diabetes.

Delayed graft rejection in autoimmune islet transplantation via biomaterial immunotherapy

The induction of operational immune tolerance is a major goal in beta-cell replacement strategies for the treatment of type 1 diabetes. Our group previously reported long-term efficacy via biomaterial-mediated programmed death ligand 1 (PD-L1) immunotherapy in islet allografts in nonautoimmune models. In this study, we evaluated autoimmune recurrence and allograft rejection during islet transplantation in spontaneous nonobese diabetic (NOD) mice. Graft survival and metabolic function were significantly prolonged over 60 days in recipients of syngeneic islets receiving the biomaterial-delivered immunotherapy, but not in control animals. The biomaterial-mediated PD-L1 immunotherapy resulted in delayed allograft rejection in diabetic NOD mice compared with controls. Discrimination between responders and nonresponders was attributed to the enriched presence of CD206+ program death 1+ macrophages and exhausted signatures in the cytotoxic T cell compartment in the local graft microenvironment. Notably, draining lymph nodes had similar remodeling in innate and adaptive immune cell populations. This work establishes that our biomaterial platform for PD-L1 delivery can modulate immune responses to transplanted islets in diabetic NOD mice and, thus, can provide a platform for the development of immunologic strategies to curb the allo- and autoimmune processes in beta-cell transplant recipients.

WDFY4 deficiency in NOD mice ameliorates autoimmune diabetes and insulitis

The events that initiate autoimmune diabetes in nonobese diabetic (NOD) mice remain poorly understood. CD4+ and CD8+ T cells are both required to develop disease, but their relative roles in initiating disease are unclear. To test whether CD4+ T cell infiltration into islets requires damage to β cells induced by autoreactive CD8+ T cells, we inactivated Wdfy4 in nonobese diabetic (NOD) mice (NOD.Wdfy4-/--) using CRISPR/Cas9 targeting to eliminate cross-presentation by type 1 conventional dendritic cells (cDC1s). Similar to C57BL/6 Wdfy4-/- mice, cDC1 in NOD.Wdfy4-/- mice are unable to cross-present cell-associated antigens to prime CD8+ T cells, while cDC1 from heterozygous NOD.Wdfy4+/- mice cross-present normally. Further, NOD.Wdfy4-/- mice fail to develop diabetes while heterozygous NOD.Wdfy4+/- mice develop diabetes similarly to wild-type NOD mice. NOD.Wdfy4-/- mice remain capable of processing and presentingmajor histocompatibility complex class II (MHC-II)-restricted autoantigens and can activate β cell-specific CD4+ T cells in lymph nodes. However, disease in these mice does not progress beyond peri-islet inflammation. These results indicate that the priming of autoreactive CD8+ T cells in NOD mice requires cross-presentation by cDC1. Further, autoreactive CD8+ T cells appear to be required not only to develop diabetes, but to recruit autoreactive CD4+ T cells into islets of NOD mice, perhaps in response to progressive β cell damage.

Autoreactive CD8 T cells in NOD mice exhibit phenotypic heterogeneity but restricted TCR gene usage.

Type 1 diabetes (T1D) is an autoimmune disorder defined by CD8 T cell-mediated destruction of pancreatic β cells. We have previously shown that diabetogenic CD8 T cells in the islets of non-obese diabetic mice are phenotypically heterogeneous, but clonal heterogeneity remains relatively unexplored. Here, we use paired single-cell RNA and T-cell receptor sequencing (scRNA-seq and scTCR-seq) to characterize autoreactive CD8 T cells from the islets and spleens of non-obese diabetic mice. scTCR-seq demonstrates that CD8 T cells targeting the immunodominant β-cell epitope IGRP206-214 exhibit restricted TCR gene usage. scRNA-seq identifies six clusters of autoreactive CD8 T cells in the islets and six in the spleen, including memory and exhausted cells. Clonal overlap between IGRP206-214-reactive CD8 T cells in the islets and spleen suggests these cells may circulate between the islets and periphery. Finally, we identify correlations between TCR genes and T-cell clonal expansion and effector fate. Collectively, our work demonstrates that IGRP206-214-specific CD8 T cells are phenotypically heterogeneous but clonally restricted, raising the possibility of selectively targeting these TCR structures for therapeutic benefit.

NADPH oxidase-derived superoxide promote autoreactive T cell infiltration into islets of prediabetic NOD mice.

Abstract Proinflammatory cytokines, reactive oxygen species (ROS), and T cells contribute to β-cell destruction in Type 1 diabetes (T1D). NADPH oxidase (NOX) generate superoxide, a precursor for a variety of ROS, regulate cell survival, differentiation, signaling, and autoimmune responses in T1D. Previously, we demonstrated that NOX-derived superoxide increase chemokine synthesis and skew macrophages and T cells towards an inflammatory response in T1D. However, it is unclear if NOX is required for diabetogenic T cell trafficking to islets. Using non-obese diabetic (NOD) mice deficient in NOX-derived superoxide (NOD.Ncf1m1J), we tested the hypothesis that loss of NOX-derived superoxide delays T1D by impairing autoreactive T infiltration of islets. Female NOD.Ncf1m1J mice (n=20) exhibit a significant delay in spontaneous T1D, as only 20% become diabetic by 40-weeks of age compared to 90% NOD mice (n=23). Immunophenotyping of 12-week-old NOD.Ncf1m1J mice revealed fewer pancreatic CCR2+CD4+ (1.9-fold) and CCR5+ CD4+ T cells (2.1-fold) compared to NOD. NOD.Ncf1m1J islets also had reduced total CD4+ (1.8-fold), CD25+ CD4+(2.0-fold), and effector CD4+ T cells (2.2-fold) compared to NOD. Using tetramer staining to detect autoreactive T cells, we observed a decrease in NOD.Ncf1m1J islet-infiltrating CD4+ T cells specific for insulin (Ins) (2.6-fold), Ins-chromogranin A hybrid peptide (HIP) (3.0-fold), and Ins-islet amyloid polypeptide HIP (2.6-fold) compared to NOD. Our data provides evidence that loss of NOX-derived superoxide can impair autoreactive T cell activation and islet-infiltration as a potential mechanism to delay T1D. Future studies will determine if NOX regulates islet chemokine synthesis to induce T cell migration. Supported by R01 DK126456 R01 DK127497 T32 GM109780 T32 AI007051 F31 DK130551

Inflammatory status of the pancreas in NOD mice that do not develop overt diabetes.

Type 1 diabetes (T1D) is an autoimmune disease in which immune cells target the pancreatic islets and destroy the β-cells, resulting in hyperglycemia and decreased plasmatic insulin levels. The non-obese diabetic (NOD) mouse is the most used animal model for studying diabetes because it spontaneously develops T1D and shares similarities with the human disease. A hallmark feature of this model is the appearance of insulitis, defined as an inflammatory cell infiltration of the pancreatic islets. However, a small percentage of NOD mice do not develop overt diabetes even after 28–35 weeks of age. Thus, we questioned the status of the pancreatic islets in these non-diabetic NOD mice, with particular focus on islet inflammation and plasmatic insulin levels, in comparison to pre-diabetic (11 weeks old) and new-onset diabetic mice. Diabetes progression was evaluated by assessing blood glucose and pancreas histology. The inflammatory score was determined on Hematoxylin–Eosin (HE)-stained sections of pancreas. Plasma insulin was detected by enzyme-linked immunosorbent assay (ELISA). The results showed that inflammation increased in an age-dependent manner in all mice, irrespective of their diabetic status. Mostly affected within the analyzed groups were the 28 weeks old non-diabetic NOD mice, in which insulin production was reduced and inversely correlated with the inflammatory status. We conclude that in NOD mice, pancreatic inflammation progresses independently of diabetes onset and clinical signs of disease. Most likely, the NOD females that do not develop overt diabetes preserve a small mass of functional β-cells, which is able to provide the physiological insulin levels and avoid diabetes onset.

Syntaxin 4 Enrichment in β-Cells Prevents Conversion to Autoimmune Diabetes in Non-Obese Diabetic (NOD) Mice.

Syntaxin 4 (STX4), a plasma membrane-localized SNARE protein, regulates human islet β-cell insulin secretion and preservation of β-cell mass. We found that human type 1 diabetes (T1D) and NOD mouse islets show reduced β-cell STX4 expression, consistent with decreased STX4 expression, as a potential driver of T1D phenotypes. To test this hypothesis, we generated inducible β-cell-specific STX4-expressing NOD mice (NOD-iβSTX4). Of NOD-iβSTX4 mice, 73% had sustained normoglycemia vs. <20% of control NOD (NOD-Ctrl) mice by 25 weeks of age. At 12 weeks of age, before diabetes conversion, NOD-iβSTX4 mice demonstrated superior whole-body glucose tolerance and β-cell glucose responsiveness than NOD-Ctrl mice. Higher β-cell mass and reduced β-cell apoptosis were also detected in NOD-iβSTX4 pancreata compared with pancreata of NOD-Ctrl mice. Single-cell RNA sequencing revealed that islets from NOD-iβSTX4 had markedly reduced interferon-γ signaling and tumor necrosis factor-α signaling via nuclear factor-κB in islet β-cells, including reduced expression of the chemokine CCL5; CD4+ regulatory T cells were also enriched in NOD-iβSTX4 islets. These results provide a deeper mechanistic understanding of STX4 function in β-cell protection and warrant further investigation of STX4 enrichment as a strategy to reverse or prevent T1D in humans or protect β-cell grafts.

Pancreatic beta cell autophagy is impaired in type 1 diabetes

Aims/hypothesisPancreatic beta cells are subjected to exogenous damaging factors such as proinflammatory cytokines or excess glucose that can cause accumulation of damage-inducing reactive oxygen species during the pathogenesis of diabetes. We and others have shown that beta cell autophagy can reduce reactive oxygen species to protect against apoptosis. While impaired islet autophagy has been demonstrated in human type 2 diabetes, it is unknown if islet autophagy is perturbed in the pathogenesis of type 1 diabetes. We hypothesised that beta cell autophagy is dysfunctional in type 1 diabetes, and that there is a progressive loss during early diabetes development.MethodsPancreases were collected from chloroquine-injected and non-injected non-obese diabetes-resistant (NOR) and non-obese diabetic (NOD) mice. Age- and BMI-matched pancreas tissue sections from human organ donors (N = 34) were obtained from the Network for Pancreatic Organ Donors with Diabetes (nPOD). Tissue sections were stained with antibodies against proinsulin or insulin (beta cell markers), microtubule-associated protein 1 light chain 3 A/B (LC3A/B; autophagosome marker), lysosomal-associated membrane protein 1 (LAMP1; lysosome marker) and p62 (autophagy adaptor). Images collected on a scanning laser confocal microscope were analysed with CellProfiler and ImageJ. Secondary lysosomes and telolysosomes were assessed in electron micrographs of human pancreatic tissue sections (n = 12), and energy dispersive x-ray analysis was performed to assess distribution of elements (n = 5).ResultsWe observed increased autophagosome numbers in islets of diabetic NOD mice (p = 0.008) and increased p62 in islets of both non-diabetic and diabetic NOD mice (p < 0.001) vs NOR mice. There was also a reduction in LC3–LAMP1 colocalisation in islets of diabetic NOD mice compared with both non-diabetic NOD (p < 0.001) and NOR mice (p < 0.001). Chloroquine elicited accumulation of autophagosomes in the islets of NOR (p = 0.003) and non-diabetic NOD mice (p < 0.001), but not in islets of diabetic NOD mice; and stimulated accumulation of p62 in NOR (p < 0.001), but not in NOD mice. We observed reduced LC3–LAMP1 colocalisation (p < 0.001) in residual beta cells of human donors with type 1 diabetes vs non-diabetic participants. We also observed reduced colocalisation of proinsulin with LAMP1 in donors with type 1 diabetes (p < 0.001). Electron microscopy also revealed accumulation of telolysosomes with nitrogen-dense rings in beta cells of autoantibody-positive donors (p = 0.002).Conclusions/interpretationWe provide evidence of islet macroautophagy/crinophagy impairment in human type 1 diabetes. We also document accumulation of telolysosomes with peripheral nitrogen in beta cells of autoantibody-positive donors, demonstrating altered lysosome content that may be associated with lysosome dysfunction before clinical hyperglycaemia. Similar macroautophagy impairments are present in the NOD mouse model of type 1 diabetes.Graphical abstract

Pancreatic Alpha-Cells Contribute Together With Beta-Cells to CXCL10 Expression in Type 1 Diabetes.

C-X-C Motif Chemokine Ligand 10 (CXCL10) is a pro-inflammatory chemokine specifically recognized by the ligand receptor CXCR3 which is mostly expressed in T-lymphocytes. Although CXCL10 expression and secretion have been widely associated to pancreatic islets both in non-obese diabetic (NOD) mice and in human type 1 diabetic (T1D) donors, the specific expression pattern among pancreatic endocrine cell subtypes has not been clarified yet. Therefore, the purpose of this study was to shed light on the pancreatic islet expression of CXCL10 in NOD, in C57Bl/6J and in NOD-SCID mice as well as in human T1D pancreata from new-onset T1D patients (DiViD study) compared to non-diabetic multiorgan donors from the INNODIA European Network for Pancreatic Organ Donors with Diabetes (EUnPOD). CXCL10 was expressed in pancreatic islets of normoglycaemic and new-onset diabetic NOD mice but not in C57Bl/6J and NOD-SCID mice. CXCL10 expression was increased in pancreatic islets of new-onset diabetic NOD mice compared to normoglycaemic NOD mice. In NOD mice, CXCL10 colocalized both with insulin and glucagon. Interestingly, CXCL10-glucagon colocalization rate was significantly increased in diabetic vs. normoglycaemic NOD mouse islets, indicating an increased expression of CXCL10 also in alpha-cells. CXCL10 was expressed in pancreatic islets of T1D patients but not in non-diabetic donors. The analysis of the expression pattern of CXCL10 in human T1D pancreata from DiViD study, revealed an increased colocalization rate with glucagon compared to insulin. Of note, CXCL10 was also expressed in alpha-cells residing in insulin-deficient islets (IDI), suggesting that CXCL10 expression in alpha cells is not driven by residual beta-cells and therefore may represent an independent phenomenon. In conclusion, we show that in T1D CXCL10 is expressed by alpha-cells both in NOD mice and in T1D patients, thus pointing to an additional novel role for alpha-cells in T1D pathogenesis and progression.

Neutrophil elastase triggers the development of autoimmune diabetes by exacerbating innate immune responses in pancreatic islets of non-obese diabetic mice.

Type 1 diabetes is an autoimmune disease resulted from self-destruction of insulin-producing pancreatic β cells. However, the pathological pathways that trigger the autoimmune destruction remain poorly understood. Clinical studies have demonstrated close associations of neutrophils and neutrophil elastase (NE) with β-cell autoimmunity in patients with Type 1 diabetes. The present study aims to investigate the impact of NE inhibition on development of autoimmune diabetes in NOD mice. NE pharmacological inhibitor (sivelestat) or biological inhibitor (elafin) was supplemented into NOD mice to evaluate their effects on islet inflammation and diabetogenesis. The impact of NE inhibition on innate and adaptive immune cells was measured with flow cytometry and immunohistochemistry. A significant but transient increase in neutrophil infiltration accompanied with elevated NE activity was observed in the neonatal period of NOD mice. Treatment of NOD mice with sivelestat or elafin at the early age led to a marked reduction in spontaneous development of insulitis and autoimmune diabetes. Mechanistically, inhibition of NE significantly attenuated infiltration of macrophages and islet inflammation, thus ameliorating cytotoxic T cell-mediated autoimmune attack of pancreatic β cells. In vitro studies showed that NE directly induced inflammatory responses in both min6 β cells and RAW264.7 macrophages, and promoted macrophage migration. These findings support an important role of NE in triggering the onset and progression of β-cell autoimmunity, and suggest that pharmacological inhibition of NE may represent a promising therapeutic strategy for treatment of autoimmune diabetes.

2115-P: Beta-Cell Autophagy Is Reduced in Type 1 Diabetes

Autophagy is a dynamic recycling mechanism that maintains beta cell homeostasis, whereas the related process of crinophagy also plays a specific role in insulin granule regulation. Impairment of autophagy and crinophagy have been implicated in beta cell death and dysfunction in the context of type 2 diabetes; however, there is currently no literature describing beta cell autophagy in type 1 diabetes pathogenesis. Therefore, we set out to determine if beta cell autophagy and crinophagy are impaired in the context of type 1 diabetes. We utilized immunofluorescent staining of LC3 (autophagosome marker), Lamp1 (lysosome marker), and proinsulin to analyze pancreatic islet autophagy and crinophagy in the NOD (non-obese diabetic) mouse model of type 1 diabetes as well as in human pancreas tissue obtained from the network for Pancreatic Organ donors with Diabetes (nPOD). We observed evidence of reduced macroautophagy in the residual proinsulin-positive islet cells of human donors with type 1 diabetescompared to controls, as measured by significantly reduced autophagosome:lysosome colocalization (p=0.034). Similarly, there was a trend towards reduced autophagosome:lysosome colocalization in islets of diabetic NOD mice compared to nondiabetic NOR mice (p=0.08). We also observed significantly reduced colocalization of proinsulin with lysosomes in the proinsulin positive cells from human donors with type 1 diabetes in comparison to both controls without diabetes (p=0.0014) and autoantibody positive donors (p= 0.0278), as well as in diabetic NOD vs. nondiabetic NOR mice (p=0.0121), suggesting that crinophagy is also reduced as a function of type 1 diabetes. Collectively, we provide evidence that macroautophagy and crinophagy are reduced in the context of type 1 diabetes. Considering these data in the context of what is known regarding the cell-protective effects of islet autophagy, we suggest targeting beta cell autophagy pathways as an approach to reduce apoptosis in individuals at risk for type 1 diabetes development. Disclosure C. Muralidharan: None. M. Marasco: Employee; Spouse/Partner; Eli Lilly and Company. J. Crowder: Employee; Spouse/Partner; Eli Lilly and Company. A.M. Conteh: None. A.K. Linnemann: None.

Endogenous retrovirus Gag antigen and its gene variants are unique autoantigens expressed in the pancreatic islets of non-obese diabetic mice

Endogenous retrovirus (ERV) are remnants of ancient retroviruses that have been incorporated into the genome and evidence suggests that they may play a role in the etiology of T1D. We previously identified a murine leukemia retrovirus-like ERV whose Env and Gag antigens are involved in autoimmune responses in non-obese diabetic (NOD) mice. In this study, we show that the Gag antigen is present in the islet stromal cells. Although Gag gene transcripts were present, Gag protein was not detected in diabetes-resistant mice. Cloning and sequencing analysis of individual Gag genes revealed that NOD islets express Gag gene variants with complete open-reading frames (ORFs), in contrast to the diabetes-resistant mice, whose islet Gag gene transcripts are mostly non-ORFs. Importantly, the ORFs obtained from the NOD islets are extremely heterogenous, coding for various mutants that are absence in the genome. We further show that Gag antigens are stimulatory for autoreactive T cells and identified one islet-expressing Gag variant that contains an altered peptide ligand capable of inducing IFN-gamma release by the T cells. The data highlight a unique retrovirus-like factor in the islets of the NOD mouse strain, which may participate in key events triggering autoimmunity and T1D.

IL-21 regulates SOCS1 expression in autoreactive CD8+ T cells but is not required for acquisition of CTL activity in the islets of non-obese diabetic mice

In type 1 diabetes, maturation of activated autoreactive CD8+ T cells to fully armed effector cytotoxic T lymphocytes (CTL) occurs within the islet. At present the signals required for the maturation process are poorly defined. Cytokines could potentially provide the necessary “third signal” required to generate fully mature CTL capable of killing insulin-producing β-cells. To determine whether autoreactive CTL within islets respond to cytokines we generated non-obese diabetic (NOD) mice with a reporter for cytokine signalling. These mice express a reporter gene, hCD4, under the control of the endogenous regulatory elements for suppressor of cytokine signalling (SOCS)1, which is itself regulated by pro-inflammatory cytokines. In NOD mice, the hCD4 reporter was expressed in infiltrated islets and the expression level was positively correlated with the frequency of infiltrating CD45+ cells. SOCS1 reporter expression was induced in transferred β-cell-specific CD8+ 8.3T cells upon migration from pancreatic draining lymph nodes into islets. To determine which cytokines induced SOCS1 promoter activity in islets, we examined hCD4 reporter expression and CTL maturation in the absence of the cytokine receptors IFNAR1 or IL-21R. We show that IFNAR1 deficiency does not confer protection from diabetes in 8.3 TCR transgenic mice, nor is IFNAR1 signalling required for SOCS1 reporter upregulation or CTL maturation in islets. In contrast, IL-21R-deficient 8.3 mice have reduced diabetes incidence and reduced SOCS1 reporter activity in islet CTLs. However IL-21R deficiency did not affect islet CD8+ T cell proliferation or expression of granzyme B or IFNγ. Together these data indicate that autoreactive CD8+ T cells respond to IL-21 and not type I IFNs in the islets of NOD mice, but neither IFNAR1 nor IL-21R are required for islet intrinsic CTL maturation.

A Novel Liposome Formulation Carrying Both an Insulin Peptide and a Ligand for Invariant Natural Killer T Cells Induces Accumulation of Regulatory T Cells to Islets in Nonobese Diabetic Mice.

Type 1 diabetes (T1D) is an autoimmune disease caused by the destruction of pancreatic β cells by autoantigen-reactive diabetogenic cells. Antigen-specific therapies using islet autoantigens for restoring immune tolerance have emerged as promising approaches for the treatment of T1D but have been unsuccessful in humans. Herein, we report that RGI-3100-iB, a novel liposomal formulation carrying both α-galactosylceramide (α-GalCer), which is a representative ligand for invariant natural killer T (iNKT) cells, and insulin B chain 9–23 peptide, which is an epitope for CD4+ T cells, could induce the accumulation of regulatory T cells (Tregs) in islets in a peptide-dependent manner, followed by the remarkable prevention of diabetes onset in nonobese diabetic (NOD) mice. While multiple administrations of a monotherapy using either α-GalCer or insulin B peptide in a liposomal formulation was confirmed to delay/prevent T1D in NOD mice, RGI-3100-iB synergistically enhanced the prevention effect of each monotherapy and alleviated insulitis in NOD mice. Immunopathological analysis showed that Foxp3+ Tregs accumulated in the islets in RGI-3100-iB-treated mice. Cotransfer of diabetogenic T cells and splenocytes of NOD mice treated with RGI-3100-iB, but not liposomal α-GalCer encapsulating an unrelated peptide, to NOD-SCID mice resulted in the prevention of diabetes and elevation of Foxp3 mRNA expression in the islets. These data indicate that the migration of insulin B-peptide-specific Tregs to islet of NOD mice that are involved in the suppression of pathogenic T cells related to diabetes onset and progression could be enhanced by the administration of liposomes containing α-GalCer and insulin B peptide.

Peptides of endogenous retrovirus Gag gene variants are not agonistic ligands for autoreactive T cells in non-obese diabetic mice

Abstract Endogenous retroviruses (ERV) have been isolated from the pancreatic islets of non-obese diabetic (NOD) mice. We previously demonstrated that a murine leukemia retrovirus–like ERV Gag protein was secreted together with exosomes by an insulinoma and by primary islet mesenchymal stem cells. Here, we further demonstrated that Gag is an effective antigen to stimulate IFN-gamma production by autoreactive T cells using recombinant Gag protein or virus-like particles (VLPs) expressing the Gag gene. However, candidate peptides of this Gag protein were not effective in stimulating the T cells. Upon analyses of ERV Gag genes expressed in the islets of prediabetic NOD mice, we were surprised to find multiple Gag gene variants with complete open-reading frames. These results suggested the expression of different endogenous proviruses, or alternatively, the detection of point mutations incurred during viral replication. A new batch of altered peptides was synthesized reflecting the variations among the Gag gene variants, and a few of them induced detectable amount of cytokine secretion from the T cells. But, the levels of IFN-gamma induced by the peptides were much lower than that induced by the Gag protein and the VLPs, suggesting a low affinity activation. This weak T-cell-stimulating activity of the Gag peptides was further confirmed by testing diabetogenic T-cell clones, as well as by using MHC tetramers loaded with candidate peptides. The data suggest that ERV Gag or its gene variants may trigger autoimmunity via a pathway that could be independent from direct presentation of viral peptides to autoreactive T cells.

CD11c+ Cells Are Gatekeepers for Lymphocyte Trafficking to Infiltrated Islets During Type 1 Diabetes.

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease that affects more than 19 million people with incidence increasing rapidly worldwide. For T cells to effectively drive T1D, they must first traffic to the islets and extravasate through the islet vasculature. Understanding the cues that lead to T cell entry into inflamed islets is important because diagnosed T1D patients already have established immune infiltration of their islets. Here we show that CD11c+ cells are a key mediator of T cell trafficking to infiltrated islets in non-obese diabetic (NOD) mice. Using intravital 2-photon islet imaging we show that T cell extravasation into the islets is an extended process, with T cells arresting in the islet vasculature in close proximity to perivascular CD11c+ cells. Antigen is not required for T cell trafficking to infiltrated islets, but T cell chemokine receptor signaling is necessary. Using RNAseq, we show that islet CD11c+ cells express over 20 different chemokines that bind chemokine receptors expressed on islet T cells. One highly expressed chemokine-receptor pair is CXCL16-CXCR6. However, NOD. CXCR6−/− mice progressed normally to T1D and CXCR6 deficient T cells trafficked normally to the islets. Even with CXCR3 and CXCR6 dual deficiency, T cells trafficked to infiltrated islets. These data reinforce that chemokine receptor signaling is highly redundant for T cell trafficking to inflamed islets. Importantly, depletion of CD11c+ cells strongly inhibited T cell trafficking to infiltrated islets of NOD mice. We suggest that targeted depletion of CD11c+ cells associated with the islet vasculature may yield a therapeutic target to inhibit T cell trafficking to inflamed islets to prevent progression of T1D.

Interferon-gamma drives programmed death-ligand 1 expression on islet \u03b2 cells to limit T cell function during autoimmune diabetes

Type 1 diabetes is caused by autoreactive T cell-mediated β cell destruction. Even though co-inhibitory receptor programmed death-1 (PD-1) restrains autoimmunity, the expression and regulation of its cognate ligands on β cell remains unknown. Here, we interrogated β cell-intrinsic programmed death ligand-1 (PD-L1) expression in mouse and human islets. We measured a significant increase in the level of PD-L1 surface expression and the frequency of PD-L1+ β cells as non-obese diabetic (NOD) mice aged and developed diabetes. Increased β cell PD-L1 expression was dependent on T cell infiltration, as β cells from Rag1-deficient mice lacked PD-L1. Using Rag1-deficient NOD mouse islets, we determined that IFN-γ promotes β cell PD-L1 expression. We performed analogous experiments using human samples, and found a significant increase in β cell PD-L1 expression in type 1 diabetic samples compared to type 2 diabetic, autoantibody positive, and non-diabetic samples. Among type 1 diabetic samples, β cell PD-L1 expression correlated with insulitis. In vitro experiments with human islets from non-diabetic individuals showed that IFN-γ promoted β cell PD-L1 expression. These results suggest that insulin-producing β cells respond to pancreatic inflammation and IFN-γ production by upregulating PD-L1 expression to limit self-reactive T cells.

Revealing the specificity of regulatory T cells in murine autoimmune diabetes

Regulatory T cells (Tregs) control organ-specific autoimmunity in a tissue antigen-specific manner, yet little is known about their specificity in a natural repertoire. In this study, we used the nonobese diabetic (NOD) mouse model of autoimmune diabetes to investigate the antigen specificity of Tregs present in the inflamed tissue, the islets of Langerhans. Compared with Tregs present in spleen and lymph node, Tregs in the islets showed evidence of antigen stimulation that correlated with higher proliferation and expression of activation markers CD103, ICOS, and TIGIT. T cell receptor (TCR) repertoire profiling demonstrated that islet Treg clonotypes are expanded in the islets, suggesting localized antigen-driven expansion in inflamed islets. To determine their specificity, we captured TCRαβ pairs from islet Tregs using single-cell TCR sequencing and found direct evidence that some of these TCRs were specific for islet-derived antigens including insulin B:9-23 and proinsulin. Consistently, insulin B:9-23 tetramers readily detected insulin-specific Tregs in the islets of NOD mice. Lastly, islet Tregs from prediabetic NOD mice were effective at preventing diabetes in Treg-deficient NOD.CD28-/- recipients. These results provide a glimpse into the specificities of Tregs in a natural repertoire that are crucial for opposing the progression of autoimmune diabetes.

Direct Analysis of Insulin-Specific T Cells Provides New Insights.

Type 1 diabetes (T1D) is an autoimmune disease in which destruction of insulin-producing β-cells leads to dependence on exogenous insulin (1). T1D progression involves progressive loss of tolerance to β-cell antigens, but this has been challenging to observe in human disease. Loss of self-tolerance is most evident through development of islet cell antibodies, among which insulin antibodies typically appear first (2). Given strong genetic association of T1D with HLA-DQ8, DQ8-restricted CD4+ T cells activated by self-peptides are thought to play a crucial role in disease. Key evidence from the nonobese diabetic (NOD) mouse model supports a primary role for recognition of an epitope within the insulin B chain amino acids 9–23 (InsB9–23) peptide in disease initiation. The majority of CD4+ T cells isolated from pancreatic islets of NOD mice were shown to respond to this peptide (3). Furthermore, expression of an insulin transgene with a substitution within this peptide abrogated diabetes development (4). Efforts to directly visualize and study insulin-specific T cells in human subjects have been hampered by the biochemical instability of the HLA-DQ8 protein (5). However, recent reports highlight the development of HLA-DQ8 tetramer reagents and their application to study T1D, affirming that InsB11–23 peptide is analogously presented by IAg7 and HLA-DQ8 (6,7). In addition, two recent reports documented the presence …

KLRG1 expression identifies short-lived Foxp3+ Treg effector cells with functional plasticity in islets of NOD mice

A progressive waning in Foxp3+ regulatory T (Treg) cell function provokes autoimmunity in the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D), a cellular defect rescued by prophylactic IL-2 therapy. We showed that most islet-infiltrating Treg cells express inducible T-cell co-stimulator (ICOS) in pre-diabetic NOD mice, and that ICOS+ Treg cells display enhanced fitness and suppressive function in situ. Moreover, T1D progression is associated with decreased expansion and suppressive activity of ICOS+Foxp3+ Treg cells, in islets, an observation consistent with the exacerbated T1D seen in NOD.BDC2.5 mice in which the ICOS pathway is abrogated. Here, we show that a large proportion of islet-resident Treg cells express the KLRG1 marker of terminally differentiation, in contrast to islet-infiltrating ICOS− Treg or Teff cells. We hypothesized that KLRG1 expression designates a subpopulation of ICOS+ Treg cells in islets that progressively loses function, and contributes to the immune dysregulation observed at T1D onset. Indeed, KLRG1-expressing ICOS+ Treg cells are prone to apoptosis, and have an impaired proliferative capacity and suppressive function in vitro and in vivo. T1D protective low-dose IL-2 treatment in vivo could not rescue the loss of KLRG1-expressing Treg cells in situ. While the global pool of Foxp3+ Treg cells displays some degree of functional plasticity in vivo, the KLRG1+ ICOS+ Treg cell subset is particularly susceptible to lose Foxp3 expression and reprogram into Th1- or Th17-like effector T (Teff) cells in the pancreas microenvironment. Overall, KLRG1 expression delineates a subpopulation of dysfunctional Treg cells during T1D progression in autoantigen-specific TCR transgenic NOD mice.