Altering ß Cell Antigen Exposure to Exhausted CD8+ T Cells Prevents Autoimmune Diabetes in Mice.

Chronic destruction of insulin-producing pancreatic ß cells by T cells results in autoimmune diabetes. Similar to other chronic T cell-mediated pathologies, a role for T cell exhaustion has been identified in diabetes in humans and NOD mice. The development and differentiation of exhausted T cells depends on exposure to Ag. In this study, we manipulated ß cell Ag presentation to target exhausted autoreactive T cells by inhibiting IFN-γ-mediated MHC class I upregulation or by ectopically expressing the ß cell Ag IGRP under the MHC class II promotor in the NOD8.3 model. Islet PD-1+TIM3+CD8+ (terminally exhausted [TEX]) cells were primary producers of islet granzyme B and CD107a, suggestive of cells that have entered the exhaustion program yet maintained cytotoxic capacity. Loss of IFN-γ-mediated ß cell MHC class I upregulation correlated with a significant reduction in islet TEX cells and diabetes protection in NOD8.3 mice. In NOD.TII/8.3 mice with IGRP expression induced in APCs, IGRP-reactive T cells remained exposed to high levels of IGRP in the islets and periphery. Consequently, functionally exhausted TEX cells, with reduced granzyme B expression, were significantly increased in these mice and this correlated with diabetes protection. These results indicate that intermediate Ag exposure in wild-type NOD8.3 islets allows T cells to enter the exhaustion program without becoming functionally exhausted. Moreover, Ag exposure can be manipulated to target this key cytotoxic population either by limiting the generation of cytotoxic TIM3+ cells or by driving their functional exhaustion, with both resulting in diabetes protection.

TIGIT acts as an immune checkpoint upon inhibition of PD1 signaling in autoimmune diabetes.

Introduction: Chronic activation of self-reactive T cells with beta cell antigens results in the upregulation of immune checkpoint molecules that keep self-reactive T cells under control and delay beta cell destruction in autoimmune diabetes. Inhibiting PD1/PD-L1 signaling results in autoimmune diabetes in mice and humans with pre-existing autoimmunity against beta cells. However, it is not known if other immune checkpoint molecules, such as TIGIT, can also negatively regulate self-reactive T cells. TIGIT negatively regulates the CD226 costimulatory pathway, T-cell receptor (TCR) signaling, and hence T-cell function. Methods: The phenotype and function of TIGIT expressing islet infiltrating T cells was studied in non-obese diabetic (NOD) mice using flow cytometry and single cell RNA sequencing. To determine if TIGIT restrains self-reactive T cells, we used a TIGIT blocking antibody alone or in combination with anti-PDL1 antibody. Results: We show that TIGIT is highly expressed on activated islet infiltrating T cells in NOD mice. We identified a subset of stem-like memory CD8+ T cells expressing multiple immune checkpoints including TIGIT, PD1 and the transcription factor EOMES, which is linked to dysfunctional CD8+ T cells. A known ligand for TIGIT, CD155 was expressed on beta cells and islet infiltrating dendritic cells. However, despite TIGIT and its ligand being expressed, islet infiltrating PD1+TIGIT+CD8+ T cells were functional. Inhibiting TIGIT in NOD mice did not result in exacerbated autoimmune diabetes while inhibiting PD1-PDL1 resulted in rapid autoimmune diabetes, indicating that TIGIT does not restrain islet infiltrating T cells in autoimmune diabetes to the same degree as PD1. Partial inhibition of PD1-PDL1 in combination with TIGIT inhibition resulted in rapid diabetes in NOD mice. Discussion: These results suggest that TIGIT and PD1 act in synergy as immune checkpoints when PD1 signaling is partially impaired. Beta cell specific stem-like memory T cells retain their functionality despite expressing multiple immune checkpoints and TIGIT is below PD1 in the hierarchy of immune checkpoints in autoimmune diabetes.

Extraislet expression of islet antigen boosts T cell exhaustion to partially prevent autoimmune diabetes.

Persistent antigen exposure results in the differentiation of functionally impaired, also termed exhausted, T cells which are maintained by a distinct population of precursors of exhausted T (TPEX) cells. T cell exhaustion is well studied in the context of chronic viral infections and cancer, but it is unclear whether and how antigen-driven T cell exhaustion controls progression of autoimmune diabetes and whether this process can be harnessed to prevent diabetes. Using nonobese diabetic (NOD) mice, we show that some CD8+ T cells specific for the islet antigen, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) displayed terminal exhaustion characteristics within pancreatic islets but were maintained in the TPEX cell state in peripheral lymphoid organs (PLO). More IGRP-specific T cells resided in the PLO than in islets. To examine the impact of extraislet antigen exposure on T cell exhaustion in diabetes, we generated transgenic NOD mice with inducible IGRP expression in peripheral antigen-presenting cells. Antigen exposure in the extraislet environment induced severely exhausted IGRP-specific T cells with reduced ability to produce interferon (IFN)γ, which protected these mice from diabetes. Our data demonstrate that T cell exhaustion induced by delivery of antigen can be harnessed to prevent autoimmune diabetes.

Transcriptional reprogramming from innate immune functions to a pro-thrombotic signature by monocytes in COVID-19.

Although alterations in myeloid cells have been observed in COVID-19, the specific underlying mechanisms are not completely understood. Here, we examine the function of classical CD14+ monocytes in patients with mild and moderate COVID-19 during the acute phase of infection and in healthy individuals. Monocytes from COVID-19 patients display altered expression of cell surface receptors and a dysfunctional metabolic profile that distinguish them from healthy monocytes. Secondary pathogen sensing ex vivo leads to defects in pro-inflammatory cytokine and type-I IFN production in moderate COVID-19 cases, together with defects in glycolysis. COVID-19 monocytes switch their gene expression profile from canonical innate immune to pro-thrombotic signatures and are functionally pro-thrombotic, both at baseline and following ex vivo stimulation with SARS-CoV-2. Transcriptionally, COVID-19 monocytes are characterized by enrichment of pathways involved in hemostasis, immunothrombosis, platelet aggregation and other accessory pathways to platelet activation and clot formation. These results identify a potential mechanism by which monocyte dysfunction may contribute to COVID-19 pathology.

Interferons limit autoantigen-specific CD8+ T-cell expansion in the non-obese diabetic mouse.

Interferon gamma (IFNγ) is a proinflammatory cytokine implicated in autoimmune diseases. However, deficiency or neutralization of IFNγ is ineffective in reducing disease. We characterize islet antigen-specific T cells in non-obese diabetic (NOD) mice lacking all three IFN receptor genes. Diabetes is minimally affected, but at 125 days of age, antigen-specific CD8+ T cells, quantified using major histocompatibility complex class I tetramers, are present in 10-fold greater numbers in Ifngr-mutant NOD mice. T cells from Ifngr-mutant mice have increased proliferative responses to interleukin-2 (IL-2). They also have reduced phosphorylated STAT1 and its target gene, suppressor of cytokine signaling 1 (SOCS-1). IFNγ controls the expansion of antigen-specific CD8+ T cells by mechanisms which include increased SOCS-1 expression that regulates IL-2 signaling. The expanded CD8+ T cells are likely to contribute to normal diabetes progression despite reduced inflammation in Ifngr-mutant mice.

Antigen-Specific Regulatory T Cell Therapy in Autoimmune Diseases and Transplantation.

Regulatory T (Treg) cells are a heterogenous population of immunosuppressive T cells whose therapeutic potential for the treatment of autoimmune diseases and graft rejection is currently being explored. While clinical trial results thus far support the safety and efficacy of adoptive therapies using polyclonal Treg cells, some studies suggest that antigen-specific Treg cells are more potent in regulating and improving immune tolerance in a disease-specific manner. Hence, several approaches to generate and/or expand antigen-specific Treg cells in vitro or in vivo are currently under investigation. However, antigen-specific Treg cell therapies face additional challenges that require further consideration, including the identification of disease-relevant antigens as well as the in vivo stability and migratory behavior of Treg cells following transfer. In this review, we discuss these approaches and the potential limitations and describe prospective strategies to enhance the efficacy of antigen-specific Treg cell treatments in autoimmunity and transplantation.

Tolerance to Proinsulin-1 Reduces Autoimmune Diabetes in NOD Mice.

T-cell responses to insulin and its precursor proinsulin are central to islet autoimmunity in humans and non-obese diabetic (NOD) mice that spontaneously develop autoimmune diabetes. Mice have two proinsulin genes proinsulin -1 and 2 that are differentially expressed, with predominant proinsulin-2 expression in the thymus and proinsulin-1 in islet beta-cells. In contrast to proinsulin-2, proinsulin-1 knockout NOD mice are protected from autoimmune diabetes. This indicates that proinsulin-1 epitopes in beta-cells maybe preferentially targeted by autoreactive T cells. To study the contribution of proinsulin-1 reactive T cells in autoimmune diabetes, we generated transgenic NOD mice with tetracycline-regulated expression of proinsulin-1 in antigen presenting cells (TIP-1 mice) with an aim to induce immune tolerance. TIP-1 mice displayed a significantly reduced incidence of spontaneous diabetes, which was associated with reduced severity of insulitis and insulin autoantibody development. Antigen experienced proinsulin specific T cells were significantly reduced in in TIP-1 mice indicating immune tolerance. Moreover, T cells from TIP-1 mice expressing proinsulin-1 transferred diabetes at a significantly reduced frequency. However, proinsulin-1 expression in APCs had minimal impact on the immune responses to the downstream antigen islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) and did not prevent diabetes in NOD 8.3 mice with a pre-existing repertoire of IGRP reactive T cells. Thus, boosting immune tolerance to proinsulin-1 partially prevents islet-autoimmunity. This study further extends the previously established role of proinsulin-1 epitopes in autoimmune diabetes in NOD mice.

Harnessing CD8+ T-cell exhaustion to treat type 1 diabetes.

Although immune interventions have shown great promise in type 1 diabetes mellitus (T1D) clinical trials, none are yet in routine clinical use or able to achieve insulin independence in patients. In addition to this, the principles of T1D treatment remain essentially unchanged since the isolation of insulin, almost a century ago. T1D is characterized by insulin deficiency as a result of destruction of insulin-producing beta cells mediated by autoreactive T cells. Therapies that target beta-cell antigen-specific T cells are needed to prevent T1D. CD8+ T-cell exhaustion is an emerging area of research in chronic infection, cancer immunotherapy, and more recently, autoimmunity. Recent data suggest that exhausted T-cell populations are associated with improved markers of T1D. T-cell exhaustion is both characterized and mediated by inhibitory receptors. This review aims to identify which inhibitory receptors may prove useful to induce T-cell exhaustion to treat T1D and identify limitations and gaps in the current literature.

Deficiency of the innate immune adaptor STING promotes autoreactive T cell expansion in NOD mice.

AIMS/HYPOTHESIS: Stimulator of IFN genes (STING) is a central hub for cytosolic nucleic acid sensing and its activation results in upregulation of type I IFN production in innate immune cells. A type I IFN gene signature seen before the onset of type 1 diabetes has been suggested as a driver of disease initiation both in humans and in the NOD mouse model. A possible source of type I IFN is through activation of the STING pathway. Recent studies suggest that STING also has antiproliferative and proapoptotic functions in T cells that are independent of IFN. To investigate whether STING is involved in autoimmune diabetes, we examined the impact of genetic deletion of STING in NOD mice. METHODS: CRISPR/Cas9 gene editing was used to generate STING-deficient NOD mice. Quantitative real-time PCR was used to assess the level of type I IFN-regulated genes in islets from wild-type and STING-deficient NOD mice. The number of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206-214-specific CD8+ T cells was determined by magnetic bead-based MHC tetramer enrichment and flow cytometry. The incidence of spontaneous diabetes and diabetes after adoptive transfer of T cells was determined. RESULTS: STING deficiency partially attenuated the type I IFN gene signature in islets but did not suppress insulitis. STING-deficient NOD mice accumulated an increased number of IGRP206-214-specific CD8+ T cells (2878 ± 642 cells in NOD.STING-/- mice and 728.8 ± 196 cells in wild-type NOD mice) in peripheral lymphoid tissue, associated with a higher incidence of spontaneous diabetes (95.5% in NOD.STING-/- mice and 86.2% in wild-type NOD mice). Splenocytes from STING-deficient mice rapidly induced diabetes after adoptive transfer into irradiated NOD recipients (median survival 75 days for NOD recipients of NOD.STING-/- mouse splenocytes and 121 days for NOD recipients of NOD mouse splenocytes). CONCLUSIONS/INTERPRETATION: Data suggest that sensing of endogenous nucleic acids through the STING pathway may be partially responsible for the type I IFN gene signature but not autoimmunity in NOD mice. Our results show that the STING pathway may play an unexpected intrinsic role in suppressing the number of diabetogenic T cells.

Loss of BIM increases mitochondrial oxygen consumption and lipid oxidation, reduces adiposity and improves insulin sensitivity in mice.

BCL-2 proteins are known to engage each other to determine the fate of a cell after a death stimulus. However, their evolutionary conservation and the many other reported binding partners suggest an additional function not directly linked to apoptosis regulation. To identify such a function, we studied mice lacking the BH3-only protein BIM. BIM-/- cells had a higher mitochondrial oxygen consumption rate that was associated with higher mitochondrial complex IV activity. The consequences of increased oxygen consumption in BIM-/- mice were significantly lower body weights, reduced adiposity and lower hepatic lipid content. Consistent with reduced adiposity, BIM-/- mice had lower fasting blood glucose, improved insulin sensitivity and hepatic insulin signalling. Lipid oxidation was increased in BIM-/- mice, suggesting a mechanism for their metabolic phenotype. Our data suggest a role for BIM in regulating mitochondrial bioenergetics and metabolism and support the idea that regulation of metabolism and cell death are connected.

Perinatal tolerance to proinsulin is sufficient to prevent autoimmune diabetes.

High-affinity self-reactive thymocytes are purged in the thymus, and residual self-reactive T cells, which are detectable in healthy subjects, are controlled by peripheral tolerance mechanisms. Breakdown in these mechanisms results in autoimmune disease, but antigen-specific therapy to augment natural mechanisms can prevent this. We aimed to determine when antigen-specific therapy is most effective. Islet autoantigens, proinsulin (PI), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) were expressed in the antigen-presenting cells (APCs) of autoimmune diabetes-prone nonobese diabetic (NOD) mice in a temporally controlled manner. PI expression from gestation until weaning was sufficient to completely protect NOD mice from diabetes, insulitis, and development of insulin autoantibodies. Insulin-specific T cells were significantly diminished, were naive, and did not express IFN-γ when challenged. This long-lasting effect from a brief period of treatment suggests that autoreactive T cells are not produced subsequently. We tracked IGRP206-214-specific CD8+ T cells in NOD mice expressing IGRP in APCs. When IGRP was expressed only until weaning, IGRP206-214-specific CD8+ T cells were not detected later in life. Thus, anti-islet autoimmunity is determined during early life, and autoreactive T cells are not generated in later life. Bolstering tolerance to islet antigens in the perinatal period is sufficient to impart lasting protection from diabetes.

Analysis of antigen specific T cells in diabetes - Lessons from pre-clinical studies and early clinical trials.

Antigen-specific immune tolerance promises to provide safe and effective therapies to prevent type 1 diabetes (T1D). Antigen-specific therapy requires two components: well-defined, clinically relevant autoantigens; and safe approaches to inducing tolerance in T cells specific for these antigens. Proinsulin is a critical autoantigen in both NOD mice, based on knockout mouse studies and induction of immune tolerance to proinsulin preventing disease whereas most antigens cannot, and also in human T1D based on proinsulin-specific T cells being found in the islets of affected individuals and the early appearance of insulin autoantibodies. Effective antigen-specific therapies that prevent T1D in humans have not yet been developed although doubt remains about the best molecular form of the antigen, the dose and the route of administration. Preclinical studies suggest that antigen specific therapy is most useful when administered before onset of autoimmunity but this time-window has not been tested in humans until the recent "pre-point" study. There may be a 'window of opportunity' during the neonatal period when 'vaccine' like administration of proinsulin for a short period may be sufficient to prevent diabetes. After the onset of autoimmunity, naive antigen-specific T cells have differentiated into antigen-experienced memory cells and the immune responses have spread to multiple antigens. Induction of tolerance at this stage becomes more difficult although recent studies have suggested generation of antigen-specific TR1 cells can inhibit memory T cells. Preclinical studies are required to identify additional 'help' that is required to induce tolerance to memory T cells and develop protocols for effective therapy in individuals with established autoimmunity.

p53-upregulated-modulator-of-apoptosis (PUMA) deficiency affects food intake but does not impact on body weight or glucose homeostasis in diet-induced obesity.

BCL-2 proteins have been implicated in the control of glucose homeostasis and metabolism in different cell types. Thus, the aim of this study was to determine the role of the pro-apoptotic BH3-only protein, p53-upregulated-modulator-of-apoptosis (PUMA), in metabolic changes mediated by diet-induced obesity, using PUMA deficient mice. At 10 weeks of age, knockout and wild type mice either continued consuming a low fat chow diet (6% fat), or were fed with a high fat diet (23% fat) for 14-17 weeks. We measured body composition, glucose and insulin tolerance, insulin response in peripheral tissues, energy expenditure, oxygen consumption, and respiratory exchange ratio in vivo. All these parameters were indistinguishable between wild type and knockout mice on chow diet and were modified equally by diet-induced obesity. Interestingly, we observed decreased food intake and ambulatory capacity of PUMA knockout mice on high fat diet. This was associated with increased adipocyte size and fasted leptin concentration in the blood. Our findings suggest that although PUMA is dispensable for glucose homeostasis in lean and obese mice, it can affect leptin levels and food intake during obesity.

BIM Deficiency Protects NOD Mice From Diabetes by Diverting Thymocytes to Regulatory T Cells.

Because regulatory T-cell (Treg) development can be induced by the same agonist self-antigens that induce negative selection, perturbation of apoptosis will affect both negative selection and Treg development. But how the processes of thymocyte deletion versus Treg differentiation bifurcate and their relative importance for tolerance have not been studied in spontaneous organ-specific autoimmune disease. We addressed these questions by removing a critical mediator of thymocyte deletion, BIM, in the NOD mouse model of autoimmune diabetes. Despite substantial defects in the deletion of autoreactive thymocytes, BIM-deficient NOD (NODBim(-/-)) mice developed less insulitis and were protected from diabetes. BIM deficiency did not impair effector T-cell function; however, NODBim(-/-) mice had increased numbers of Tregs, including those specific for proinsulin, in the thymus and peripheral lymphoid tissues. Increased levels of Nur77, CD5, GITR, and phosphorylated IκB-α in thymocytes from NODBim(-/-) mice suggest that autoreactive cells receiving strong T-cell receptor signals that would normally delete them escape apoptosis and are diverted into the Treg pathway. Paradoxically, in the NOD model, reduced thymic deletion ameliorates autoimmune diabetes by increasing Tregs. Thus, modulating apoptosis may be one of the ways to increase antigen-specific Tregs and prevent autoimmune disease.

Using pancreas tissue slices for in situ studies of islet of Langerhans and acinar cell biology.

Studies on the cellular function of the pancreas are typically performed in vitro on its isolated functional units, the endocrine islets of Langerhans and the exocrine acini. However, these approaches are hampered by preparation-induced changes of cell physiology and the lack of an intact surrounding. We present here a detailed protocol for the preparation of pancreas tissue slices. This procedure is less damaging to the tissue and faster than alternative approaches, and it enables the in situ study of pancreatic endocrine and exocrine cell physiology in a conserved environment. Pancreas tissue slices facilitate the investigation of cellular mechanisms underlying the function, pathology and interaction of the endocrine and exocrine components of the pancreas. We provide examples for several experimental applications of pancreas tissue slices to study various aspects of pancreas cell biology. Furthermore, we describe the preparation of human and porcine pancreas tissue slices for the validation and translation of research findings obtained in the mouse model. Preparation of pancreas tissue slices according to the protocol described here takes less than 45 min from tissue preparation to receipt of the first slices.

Mouse pancreas tissue slice culture facilitates long-term studies of exocrine and endocrine cell physiology in situ.

Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.