Prerak Trivedi

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.

Granzyme B Is Dispensable in the Development of Diabetes in Non-Obese Diabetic Mice

Pancreatic beta cell destruction in type 1 diabetes is mediated by cytotoxic CD8+ T lymphoctyes (CTL). Granzyme B is an effector molecule used by CTL to kill target cells. We previously showed that granzyme B-deficient allogeneic CTL inefficiently killed pancreatic islets in vitro. We generated granzyme B-deficient non-obese diabetic (NOD) mice to test whether granzyme B is an important effector molecule in spontaneous type 1 diabetes. Granzyme B-deficient islet antigen-specific CD8+ T cells had impaired homing into islets of young mice. Insulitis was reduced in granzyme B-deficient mice at 70 days of age (insulitis score 0.043±0.019 in granzyme B-deficient versus 0.139±0.034 in wild-type NOD mice p<0.05), but was similar to wild-type at 100 and 150 days of age. We observed a reduced frequency of CD3+CD8+ T cells in the islets and peripheral lymphoid tissues of granzyme B-deficient mice (p<0.005 and p<0.0001 respectively), but there was no difference in cell proportions in the thymus. Antigen-specific CTL developed normally in granzyme B-deficient mice, and were able to kill NOD islet target cells as efficiently as wild-type CTL in vitro. The incidence of spontaneous diabetes in granzyme B-deficient mice was the same as wild-type NOD mice. We observed a delayed onset of diabetes in granzyme B-deficient CD8-dependent NOD8.3 mice (median onset 102.5 days in granzyme B-deficient versus 57.50 days in wild-type NOD8.3 mice), which may be due to the delayed onset of insulitis or inefficient priming at an earlier age in this accelerated model of diabetes. Our data indicate that granzyme B is dispensable for beta cell destruction in type 1 diabetes, but is required for efficient early activation of CTL.

Repurposed JAK1/JAK2 Inhibitor Reverses Established Autoimmune Insulitis in NOD Mice

Recent advances in immunotherapeutics have not yet changed the routine management of autoimmune type 1 diabetes. There is an opportunity to repurpose therapeutics used to treat other diseases to treat type 1 diabetes, especially when there is evidence for overlapping mechanisms. Janus kinase (JAK) 1/JAK2 inhibitors are in development or clinical use for indications including rheumatoid arthritis. There is good evidence for activation of the JAK1/JAK2 and signal transducer and activator of transcription (STAT) 1 pathway in human type 1 diabetes and in mouse models, especially in β-cells. We tested the hypothesis that using these drugs to block the JAK-STAT pathway would prevent autoimmune diabetes. The JAK1/JAK2 inhibitor AZD1480 blocked the effect of cytokines on mouse and human β-cells by inhibiting MHC class I upregulation. This prevented the direct interaction between CD8+ T cells and β-cells, and reduced immune cell infiltration into islets. NOD mice treated with AZD1480 were protected from autoimmune diabetes, and diabetes was reversed in newly diagnosed NOD mice. This provides mechanistic groundwork for repurposing clinically approved JAK1/JAK2 inhibitors for type 1 diabetes.

Perforin facilitates beta cell killing and regulates autoreactive CD8+ T-cell responses to antigen in mouse models of type 1 diabetes.

In type 1 diabetes, cytotoxic CD8+ T lymphocytes (CTLs) directly interact with pancreatic beta cells through major histocompatibility complex class I. An immune synapse facilitates delivery of cytotoxic granules, comprised mainly of granzymes and perforin. Perforin deficiency protects the majority of non‐obese diabetic (NOD) mice from autoimmune diabetes. Intriguingly perforin deficiency does not prevent diabetes in CD8+ T‐cell receptor transgenic NOD8.3 mice. We therefore investigated the importance of perforin‐dependent killing via CTL‐beta cell contact in autoimmune diabetes. Perforin‐deficient CTL from NOD mice or from NOD8.3 mice were significantly less efficient at adoptive transfer of autoimmune diabetes into NODRag1−/− mice, confirming that perforin is essential to facilitate beta cell destruction. However, increasing the number of transferred in vitro‐activated perforin‐deficient 8.3 T cells reversed the phenotype and resulted in diabetes. Perforin‐deficient NOD8.3 T cells were present in increased proportion in islets, and proliferated more in response to antigen in vivo indicating that perforin may regulate the activation of CTLs, possibly by controlling cytokine production. This was confirmed when we examined the requirement for direct interaction between beta cells and CD8+ T cells in NOD8.3 mice, in which beta cells specifically lack major histocompatibility complex (MHC) class I through conditional deletion of β2‐microglobulin. Although diabetes was significantly reduced, 40% of these mice developed diabetes, indicating that NOD8.3 T cells can kill beta cells in the absence of direct interaction. Our data indicate that although perforin delivery is the main mechanism that CTL use to destroy beta cells, they can employ alternative mechanisms to induce diabetes in a perforin‐independent manner.

Measuring death of pancreatic beta cells in response to stress and cytotoxic T cells.

Apoptosis of pancreatic beta cells is a feature of type 1 and type 2 diabetes, although by different effector mechanisms. In type 1 diabetes, beta cells are the targets of cytotoxic CD8(+) T cells that kill by releasing the contents of their cytotoxic granules into the immunological synapse with the target beta cell. In type 2 diabetes, the mechanisms of beta cell apoptosis are less clear, but believed to be due to cellular stresses including endoplasmic reticulum stress and oxidative stress induced by chronic exposure to high concentrations of glucose, lipids, inflammatory cytokines, or islet amyloid polypeptide. Measuring apoptosis in primary islets can be more difficult than in a beta cell line because islets exist as a cluster of cells and it is often difficult to obtain sufficient cells for any particular type of assay. Here, we describe two different methods for measuring islet cell apoptosis. The first method is the measurement of DNA fragmentation, a hallmark of apoptosis, of islets that have been cultured with reagents that induce stress. The second method is the measurement of islet lysis by activated cytotoxic T cells. We describe methods using mouse islets, but these can easily be adapted for human islets.

Granzyme A-deficiency breaks immune tolerance and promotes Autoimmune Diabetes Through a Type I Interferon-Dependent Pathway

Granzyme A is a protease implicated in the degradation of intracellular DNA. Nucleotide complexes are known triggers of systemic autoimmunity, but a role in organ-specific autoimmune disease has not been demonstrated. To investigate whether such a mechanism could be an endogenous trigger for autoimmunity, we examined the impact of granzyme A deficiency in the NOD mouse model of autoimmune diabetes. Granzyme A deficiency resulted in an increased incidence in diabetes associated with accumulation of ssDNA in immune cells and induction of an interferon response in pancreatic islets. Central tolerance to proinsulin in transgenic NOD mice was broken on a granzyme A–deficient background. We have identified a novel endogenous trigger for autoimmune diabetes and an in vivo role for granzyme A in maintaining immune tolerance.

Prevention of Islet Inflammatory Stress with JAK1/JAK2 Inhibitors

Introduction There is an opportunity to repurpose therapeutics from other diseases to autoimmunity and transplantation, especially when there is evidence for overlapping mechanisms. Inflammation is one of the main causes of islet damage and loss during islet isolation and transplantation, and autoimmune diabetes is associated with a pro-inflammatory environment around pancreatic islets. The JAK-STAT pathway is activated by inflammatory cytokines and is important in T cells and pancreatic beta cells, the target of T cell-mediated destruction. We tested the hypothesis that blocking the response to cytokines that activate the JAK-STAT pathway would prevent islet inflammation, using ruxolitinib, a drug that selectively inhibits JAK1/JAK2. Materials and Methods Isolated human or mouse islets were cultured with pro-inflammatory cytokines (IFN&ggr;, TNF&agr; and IL-1&bgr;) to induce an inflammatory gene signature, which was measured by quantitative real-time PCR. The JAK1/JAK2 inhibitor ruxolitinib was used to prevent islet inflammation and cell death, measured by flow cytometry. Results Ruxolitinib successfully blocked IFN&ggr;-induced phosphorylation of STAT1 and IFN&ggr;-induced MHC class-I upregulation in mouse and human islets. Islet cell death induced by IFN&ggr;, TNF&agr; and IL-1&bgr; was completely prevented by JAK inhibitors. Expression of CXCL10, NOS2 and IL6 was upregulated by cytokines, and this was inhibited by ruxolitinib. Ruxolitinib also prevented cytokine-induced LDHA expression and restored PDX1 expression in isolated islets. Mouse islets were protected from CTL-mediated killing in vitro by JAK1/JAK2 inhibitors. Furthermore, NOD mice treated with JAK1/JAK2 inhibitors were protected from autoimmune diabetes, and diabetes was reversed in newly diagnosed NOD mice. Discussion The transplantation of islets for the treatment of type 1 diabetes is currently limited by the loss of functional islet mass throughout the procedures of isolation and infusion at least in part due to inflammatory stress. This study investigated the potential for the JAK1/JAK2 inhibitor ruxolitinib to prevent these effects. Our results showing that ruxolitinib can prevent the effects of pro-inflammatory cytokines on islet gene expression and cell death suggest that it is a promising strategy to prevent these effects during islet transplantation. Conclusion JAK1/JAK2 inhibitors are clinically approved for other indications including rheumatoid arthritis. Our work provides mechanistic groundwork for repurposing these drugs for islet transplantation. The National Health and Medical Research Council of Australia GNT1037321.

Differential regulation of pro-inflammatory cytokine signalling by protein tyrosine phosphatases in pancreatic β-cells.

Type 1 diabetes (T1D) is characterized by the destruction of insulin-producing β-cells by immune cells in the pancreas. Pro-inflammatory including TNF-α, IFN-γ and IL-1β are released in the islet during the autoimmune assault and signal in β-cells through phosphorylation cascades, resulting in pro-apoptotic gene expression and eventually β-cell death. Protein tyrosine phosphatases (PTPs) are a family of enzymes that regulate phosphorylative signalling and are associated with the development of T1D. Here, we observed expression of PTPN6 and PTPN1 in human islets and islets from non-obese diabetic (NOD) mice. To clarify the role of these PTPs in β-cells/islets, we took advantage of CRISPR/Cas9 technology and pharmacological approaches to inactivate both proteins. We identify PTPN6 as a negative regulator of TNF-α-induced β-cell death, through JNK-dependent BCL-2 protein degradation. In contrast, PTPN1 acts as a positive regulator of IFN-γ-induced STAT1-dependent gene expression, which enhanced autoimmune destruction of β-cells. Importantly, PTPN1 inactivation by pharmacological modulation protects β-cells and primary mouse islets from cytokine-mediated cell death. Thus, our data point to a non-redundant effect of PTP regulation of cytokine signalling in β-cells in autoimmune diabetes.