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Type 1 diabetes is a complex disease characterized by the lack of endogenous insulin secreted from the pancreatic β-cells. Although β-cell targeted autoimmune processes and β-cell dysfunction are known to occur in type 1 diabetes, a complete understanding of the cell-to-cell interactions that support pancreatic function is still lacking. To characterize the pancreatic endocrine compartment, we studied pancreata from healthy adult donors and investigated a single cell surface adhesion molecule, desmoglein-2 (DSG2). Genetically-modified mice lackingDsg2were examined for islet cell mass, insulin production, responses to glucose, susceptibility to a streptozotocin-induced mouse model of hyperglycaemia, and ability to cure diabetes in a syngeneic transplantation model. Herein, we have identified DSG2 as a previously unrecognized adhesion molecule that supports β-cells. Furthermore, we reveal thatDSG2is within the top 10 percent of all genes expressed by human pancreatic islets and is expressed by the insulin-producing β-cells but not the somatostatin-producing δ-cells. In aDsg2loss-of-function mice (Dsg2lo/lo), we observed a significant reduction in the number of pancreatic islets and islet size, and consequently, there was less total insulin content per islet cluster.Dsg2lo/lomice also exhibited a reduction in blood vessel barrier integrity, an increased incidence of streptozotocin-induced diabetes, and islets isolated fromDsg2lo/lomice were more susceptible to cytokine-induced β-cell apoptosis. Following transplantation into diabetic mice, islets isolated fromDsg2lo/lomice were less effective than their wildtype counterparts at curing diabetes. In vitro assays using the Beta-TC-6 murine β-cell line suggest that DSG2 supports the actin cytoskeleton as well as the release of cytokines and chemokines. Taken together, our study suggests that DSG2 is an under-appreciated regulator of β-cell function in pancreatic islets and that a better understanding of this adhesion molecule may provide new opportunities to combat 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.

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. 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. 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). 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.

Immune checkpoint inhibitors have achieved clinical success in cancer treatment, but this treatment causes immune-related adverse events, including type 1 diabetes (T1D). Our aim was to test whether a JAK1/JAK2 inhibitor, effective at treating spontaneous autoimmune diabetes in nonobese diabetic (NOD) mice, can prevent diabetes secondary to PD-L1 blockade.Anti-PD-L1 antibody was injected into NOD mice to induce diabetes, and JAK1/JAK2 inhibitor LN3103801 was administered by oral gavage to prevent diabetes. Flow cytometry was used to study T cells and beta cells. Mesothelioma cells were inoculated into BALB/c mice to induce a transplantable tumour model.Anti-PD-L1-induced diabetes was associated with increased immune cell infiltration in the islets and upregulated MHC class I on islet cells. Anti-PD-L1 administration significantly increased islet T cell proliferation and islet-specific CD8A JAK1/JAK2 inhibitor can prevent and reverse anti-PD-L1-induced diabetes by blocking IFNγ and γc cytokine activities. Our study provides preclinical validation of JAK1/JAK2 inhibitor use in checkpoint inhibitor-induced diabetes.

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.

Aims Aim of this study is to report severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection, responsible for coronavirus disease 2019 (COVID‐19), as a possible cause for type 1 diabetes by providing an illustrative clinical case of a man aged 45 years presenting with antibody‐negative diabetic ketoacidosis post‐recovery from COVID‐19 pneumonia and to explore the potential for SARS‐CoV‐2 to adhere to human islet cells. Methods Explanted human islet cells from three independent solid organ donors were incubated with the SARS‐CoV‐2 spike protein receptor biding domain (RBD) fused to a green fluorescent protein (GFP) or a control‐GFP, with differential adherence established by flow cytometry. Results Flow cytometry revealed dose‐dependent specific binding of RBD‐GFP to islet cells when compared to control‐GFP. Conclusions Although a causal basis remains to be established, our case and in vitro data highlight a potential mechanism by which SARS‐CoV‐2 infection may result in antibody‐negative type 1 diabetes.

Cytokines that signal through the JAK-STAT pathway, such as interferon-γ (IFN-γ) and common γ chain cytokines, contribute to the destruction of insulin-secreting β cells by CD8+ T cells in type 1 diabetes (T1D). We previously showed that JAK1/JAK2 inhibitors reversed autoimmune insulitis in non-obese diabetic (NOD) mice and also blocked IFN-γ mediated MHC class I upregulation on β cells. Blocking interferons on their own does not prevent diabetes in knockout NOD mice, so we tested whether JAK inhibitor action on signaling downstream of common γ chain cytokines, including IL-2, IL-7 IL-15, and IL-21, may also affect the progression of diabetes in NOD mice. Common γ chain cytokines activate JAK1 and JAK3 to regulate T cell proliferation. We used a JAK1-selective inhibitor, ABT 317, to better understand the specific role of JAK1 signaling in autoimmune diabetes. ABT 317 reduced IL-21, IL-2, IL-15 and IL-7 signaling in T cells and IFN-γ signaling in β cells, but ABT 317 did not affect GM-CSF signaling in granulocytes. When given in vivo to NOD mice, ABT 317 reduced CD8+ T cell proliferation as well as the number of KLRG+ effector and CD44hiCD62Llo effector memory CD8+ T cells in spleen. ABT 317 also prevented MHC class I upregulation on β cells. Newly diagnosed diabetes was reversed in 94% NOD mice treated twice daily with ABT 317 while still on treatment at 40 days and 44% remained normoglycemic after a further 60 days from discontinuing the drug. Our results indicate that ABT 317 blocks common γ chain cytokines in lymphocytes and interferons in lymphocytes and β cells and are thus more effective against diabetes pathogenesis than IFN-γ receptor deficiency alone. Our studies suggest use of this class of drug for the treatment of type 1 diabetes.

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.

During immune cell activation, serine-derived lipids such as phosphatidylserine and sphingolipids contribute to the formation of protein signaling complexes within the plasma membrane. Altering lipid composition in the cell membrane can subsequently affect immune cell function and the development of autoimmune disease. Serine incorporator 1 (SERINC1) is a putative carrier protein that facilitates synthesis of serine-derived lipids. To determine if SERINC1 has a role in immune cell function and the development of autoimmunity, we characterized a mouse strain in which a retroviral insertion abolishes expression of the Serinc1 transcript. Expression analyses indicated that the Serinc1 transcript is readily detectable and expressed at relatively high levels in wildtype macrophages and lymphocytes. The ablation of Serinc1 expression in these immune cells, however, did not significantly alter serine-derived lipid composition or affect macrophage function and lymphocyte proliferation. Analyses of Serinc1-deficient mice also indicated that systemic ablation of Serinc1 expression did not affect viability, fertility or autoimmune disease susceptibility. These results suggest that Serinc1 is dispensable for certain immune cell functions and does not contribute to previously reported links between lipid composition in immune cells and autoimmunity.

Type 1, or autoimmune, diabetes is caused by the T-cell mediated destruction of the insulin-producing pancreatic beta cells. Non-obese diabetic (NOD) mice spontaneously develop autoimmune diabetes akin to human type 1 diabetes. For this reason, the NOD mouse has been the preeminent murine model for human type 1 diabetes research for several decades. However, humanized mouse models are highly sought after because they offer both the experimental tractability of a mouse model and the clinical relevance of human-based research. Autoimmune T-cell responses against insulin, and its precursor proinsulin, play central roles in the autoimmune responses against pancreatic beta cells in both humans and NOD mice. As a first step towards developing a murine model of the human autoimmune response against pancreatic beta cells we set out to replace the murine insulin 1 gene (Ins1) with the human insulin gene (INS) using CRISPR/Cas9. Here we describe a NOD mouse strain that expresses human insulin in place of murine insulin 1, referred to as HuPI. HuPI mice express human insulin, and C-peptide, in their serum and pancreata and have normal glucose tolerance. Compared with wild type NOD mice, the incidence of diabetes is much lower in HuPI mice. Only 15-20% of HuPI mice developed diabetes after 300 days, compared to more than 60% of unmodified NOD mice. Immune-cell infiltration into the pancreatic islets of HuPI mice was not detectable at 100 days but was clearly evident by 300 days. This work highlights the feasibility of using CRISPR/Cas9 to create mouse models of human diseases that express proteins pivotal to the human disease. Furthermore, it reveals that even subtle changes in proinsulin protect NOD mice from diabetes.

CD8+ T cells play a central role in beta-cell destruction in type 1 diabetes. CD8+ T cells use two main effector pathways to kill target cells, perforin plus granzymes and FAS ligand (FASL). We and others have established that in non-obese diabetic (NOD) mice, perforin is the dominant effector molecule by which autoreactive CD8+ T cells kill beta cells. However, blocking FASL pharmacologically was shown to protect NOD mice from diabetes, indicating that FASL may have some role. FASL can engage with its receptor FAS on target cells either as membrane bound or soluble FASL. It has been shown that membrane-bound FASL is required to stimulate FAS-induced apoptosis in target cells, whereas excessive soluble FASL can induce NF-κB-dependent gene expression and inflammation. Because islet inflammation is a feature of autoimmune diabetes, we tested whether soluble FASL could be important in disease pathogenesis independent of its cell death function. We generated NOD mice deficient in soluble FASL, while maintaining expression of membrane-bound FASL due to a mutation in the FASL sequence required for cleavage by metalloproteinase. NOD mice lacking soluble FASL had normal numbers of lymphocytes in their spleen and thymus. Soluble FASL deficient NOD mice had similar islet inflammation as wild-type NOD mice and were not protected from diabetes. Our data indicate that soluble FASL is not required in development of autoimmune diabetes.

Diabetogenic T cells infiltrate the pancreatic islets by transmigrating across the microcapillaries residing close to, or within, the pancreatic islets. Deficiency in IFNγ signaling prevents efficient migration of T cells into the pancreatic islets, but the IFNγ-regulated molecules that mediate this are uncertain. Homing of autoreactive T cells into target tissues may require antigen specificity through presentation of cognate antigen by MHC expressed on the vascular endothelium. We investigated the hypothesis that IFNγ promotes the migration of islet antigen-specific CD4+ T cells by upregulating MHC class II on islet endothelial cells (IEC), thereby providing an antigen-specific signal for islet infiltration. Upon IFNγ stimulation, MHC class II, which is not constitutively expressed on IEC, was induced. IFNγ-dependent upregulation of MHC class II was detected in IEC isolated from prediabetic NOD mice at the earliest stages of insulitis, before other markers of inflammation were present. Using a CD4+ T cell-mediated adoptive transfer model of autoimmune diabetes we observed that even though diabetes does not develop in recipient mice lacking IFNγ receptors, mice with MHC class II-deficient IEC were not protected from disease. Thus, IFNγ-regulated molecules, but not MHC class II or antigen presentation by IECs is required for the early migration of antigen-specific CD4+ T cells into the pancreatic islets.

Despite the benefits of islet and pancreas allotransplantation, their widespread use in type 1 diabetes is limited because of the paucity of suitable donors. Porcine xenotransplantation offers an alternative, and advances in genetic modification of pigs have opened up new potential for its clinical use. This review outlines the barriers to successful islet xenotransplantation, and genetic modifications that have been tested to overcome these.Islets from pigs lacking α1,3-galactosyltransferase, to prevent hyperacute rejection, are now used as a background strain for further genetic modifications. The instant blood-mediated inflammatory reaction is overcome by expressing complement regulators including CD46, CD55 and CD59. Prevention of immune-mediated rejection mediated by T cells, macrophages and natural killer cells remains a challenge. The use of immunosuppressive antibodies, such as anti-CD154 or anti-CD2, can be protective, and may be useful if they are produced by the islets themselves.The field of xenotransplantation has benefited enormously from the development of new genetic modification strategies. With the possibility of multiple genetic modifications in the same animal, and a detailed knowledge of the mechanism of xenograft rejection, the challenge now is to develop islets that provide long-term graft survival without systemic immunosuppression.

Nonobese diabetic (NOD) mice spontaneously develop lacrimal and salivary gland autoimmunity similar to human Sj&ouml, gren syndrome. In both humans and NOD mice, the early immune response that drives T-cell infiltration into lacrimal and salivary glands is poorly understood. In NOD mice, lacrimal gland autoimmunity spontaneously occurs only in males with testosterone playing a role in promoting lacrimal gland inflammation, while female lacrimal glands are protected by regulatory T cells (Tregs). The mechanisms of this male-specific lacrimal gland autoimmunity are not known. Here, we studied the effects of Treg depletion in hormone-manipulated NOD mice and lacrimal gland gene expression to determine early signals required for lacrimal gland inflammation. While Treg-depletion was not sufficient to drive dacryoadenitis in castrated male NOD mice, chemokines (Cxcl9, Ccl19) and other potentially disease-relevant genes (Epsti1, Ubd) were upregulated in male lacrimal glands. Expression of Cxcl9 and Ccl19, in particular, remained significantly upregulated in the lacrimal glands of lymphocyte-deficient NOD-severe combined immunodeficiency (SCID) mice and their expression was modulated by type I interferon signaling. Notably, Ifnar1-deficient NOD mice did not develop dacryoadenitis. Together these data identify disease-relevant genes upregulated in the context of male-specific dacryoadenitis and demonstrate a requisite role for type I interferon signaling in lacrimal gland autoimmunity in NOD mice.

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells in pancreatic islets are progressively destroyed. Clinical trials of immunotherapies in recently diagnosed T1D patients have only transiently and partially impacted the disease course, suggesting that other approaches are required. Our previous studies have demonstrated that heparan sulfate (HS), a glycosaminoglycan conventionally expressed in extracellular matrix, is present at high levels inside normal mouse beta cells. Intracellular HS was shown to be critical for beta cell survival and protection from oxidative damage. T1D development in Non-Obese Diabetic (NOD) mice correlated with loss of islet HS and was prevented by inhibiting HS degradation by the endoglycosidase, heparanase. In this study we investigated the distribution of HS and heparan sulfate proteoglycan (HSPG) core proteins in normal human islets, a role for HS in human beta cell viability and the clinical relevance of intra-islet HS and HSPG levels, compared to insulin, in human T1D. In normal human islets, HS (identified by 10E4 mAb) co-localized with insulin but not glucagon and correlated with the HSPG core proteins for collagen type XVIII (Col18) and syndecan-1 (Sdc1). Insulin-positive islets of T1D pancreases showed significant loss of HS, Col18 and Sdc1 and heparanase was strongly expressed by islet-infiltrating leukocytes. Human beta cells cultured with HS mimetics showed significantly improved survival and protection against hydrogen peroxide-induced death, suggesting that loss of HS could contribute to beta cell death in T1D. We conclude that HS depletion in beta cells, possibly due to heparanase produced by insulitis leukocytes, may function as an important mechanism in the pathogenesis of human T1D. Our findings raise the possibility that intervention therapy with dual activity HS replacers/heparanase inhibitors could help to protect the residual beta cell mass in patients recently diagnosed with T1D.

Type 1 diabetes results from destruction of pancreatic beta cells by autoreactive T cells. Both CD4(+) and CD8(+) T cells have been shown to mediate beta-cell killing. While CD8(+) T cells can directly recognize MHC class I on beta cells, the interaction between CD4(+) T cells and beta cells remains unclear. Genetic association studies have strongly implicated HLA-DQ alleles in human type 1 diabetes. Here we studied MHC class II expression on beta cells in nonobese diabetic mice that were induced to develop diabetes by diabetogenic CD4(+) T cells with T-cell receptors that recognize beta-cell antigens. Acute infiltration of CD4(+) T cells in islets occurred with rapid onset of diabetes. Beta cells from islets with immune infiltration expressed MHC class II mRNA and protein. Exposure of beta cells to IFN-γ increased MHC class II gene expression, and blocking IFN-γ signaling in beta cells inhibited MHC class II upregulation. IFN-γ also increased HLA-DR expression in human islets. MHC class II(+) beta cells stimulated the proliferation of beta-cell-specific CD4(+) T cells. Our study indicates that MHC class II molecules may play an important role in beta-cell interaction with CD4(+) T cells in the development of type 1 diabetes.

Type 1 diabetes results from T cell-mediated destruction of pancreatic beta cells. The mechanisms of beta cell destruction in vivo, however, remain unclear. We aimed to test the relative roles of the main cell death pathways: apoptosis, necrosis and necroptosis, in beta cell death in the development of CD4(+) T cell-mediated autoimmune diabetes.We altered expression levels of critical cell death proteins in mouse islets and tested their ability to survive CD4(+) T cell-mediated attack using an in vivo graft model.Loss of the B cell leukaemia/lymphoma 2 (BCL-2) homology domain 3-only proteins BIM, PUMA or BID did not protect beta cells from this death. Overexpression of the anti-apoptotic protein BCL-2 or combined deficiency of the pro-apoptotic multi-BCL2 homology domain proteins BAX and BAK also failed to prevent beta cell destruction. Furthermore, loss of function of the death receptor Fas or its essential downstream signalling molecule Fas-associated death domain (FADD) in islets was also not protective. Using electron microscopy we observed that dying beta cells showed features of necrosis. However, islets deficient in receptor-interacting serine/threonine protein kinase 3 (RIPK3), a critical initiator of necroptosis, were still normally susceptible to CD4(+) T cell-mediated destruction. Remarkably, simultaneous inhibition of apoptosis and necroptosis by combining loss of RIPK3 and overexpression of BCL-2 in islets did not protect them against immune attack either.Collectively, our data indicate that beta cells die by necrosis in autoimmune diabetes and that the programmed cell death pathways apoptosis and necroptosis are both dispensable for this process.

CD8+ T cells are critical in human type 1 diabetes and in the NOD mouse. In this study, we elucidated the natural history of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific CD8+ T cells in NOD diabetes using MHC-tetramer technology. IGRP206–214-specific T cells in the peripheral lymphoid tissue increased with age, and their numbers correlated with insulitis progression. IGRP206–214-specific T cells in the peripheral lymphoid tissue expressed markers of chronic Ag stimulation, and their numbers were stable after diagnosis of diabetes, consistent with their memory phenotype. IGRP206–214-specific T cells in NOD mice expand, acquire the phenotype of effector-memory T cells in the islets, and emigrate to the peripheral lymphoid tissue. Our observations suggest that enumeration of effector-memory T cells of multiple autoantigen specificities in the periphery of type 1 diabetic subjects could be a reliable reporter for progression of islet pathology.

Whilst initial rates of insulin independence following islet transplantation are encouraging, long-term function using the Edmonton Protocol remains a concern. The aim of this single-arm, multicenter study was to evaluate an immunosuppressive protocol of initial antithymocyte globulin (ATG), tacrolimus and mycophenolate mofetil (MMF) followed by switching to sirolimus and MMF. Islets were cultured for 24 h prior to transplantation. The primary end-point was an HbA1c of7% and cessation of severe hypoglycemia. Seventeen recipients were followed for ≥ 12 months. Nine islet preparations were transported interstate for transplantation. Similar outcomes were achieved at all three centers. Fourteen of the 17 (82%) recipients achieved the primary end-point. Nine (53%) recipients achieved insulin independence for a median of 26 months (range 7-39 months) and 6 (35%) remain insulin independent. All recipients were C-peptide positive for at least 3 months. All subjects with unstimulated C-peptide0.2 nmol/L had cessation of severe hypoglycemia. Nine of the 17 recipients tolerated switching from tacrolimus to sirolimus with similar graft outcomes. There was a small but significant reduction in renal function in the first 12 months. The combination of islet culture, ATG, tacrolimus and MMF is a viable alternative for islet transplantation.

Pancreatic beta-cell apoptosis is an important feature of islets in type 2 diabetes. Apoptosis can occur through two major pathways, the extrinsic or death receptor mediated pathway, and the intrinsic or Bcl-2-regulated pathway. Hyperglycaemia, hyperlipidaemia and islet amyloid poly-peptide (IAPP) represent important possible causes of increased beta-cell apoptosis. Hyperglycaemia induces islet-cell apoptosis by the intrinsic pathway involving molecules of the Bcl-2 family. High concentrations of palmitate also activate intrinsic apoptosis in islets cells. IAPP oligomers can induce apoptosis by both intrinsic and extrinsic pathways. IL-1b produced through NLRP3 inflammasome activation can also induce islet cell death. Activation of the NLRP3 inflammasome may not be important for glucose or palmitate induced apoptosis in islets but may be important for IAPP mediated cell death. Endoplasmic reticulum (ER) and oxidative stress have been observed in beta cells in type 2 diabetes, and these could be the link between upstream metabolic abnormalities and downstream apoptotic machinery.

Type 1 diabetes is caused by immune-mediated loss of pancreatic beta cells. It has been proposed that inflammatory cytokines play a role in killing beta cells. Expression of interleukin (IL)-1 and tumor necrosis factor (TNF-α) has been detected in islets from patients with type 1 diabetes, and these cytokines can induce beta cell death in vitro. We produced nonobese diabetic (NOD) mice lacking receptors for these cytokines. Islets from mice lacking IL-1RI or TNFR1 were killed when transplanted into wild-type NOD mice, suggesting that cytokine action on beta cells is not required for killing. Mice lacking TNFR1 did not develop diabetes, and mice lacking IL-1R had delayed onset of diabetes, indicating a role for these cytokines in disease development. TNFR1-deficient mice had an increased number of CD4(+) CD25(+) FoxP3(+) regulatory T cells with enhanced suppressive capacity. IL-1 was produced at higher levels in NOD mice and resulted in dilution of suppressor function of CD4(+) CD25(+) FoxP3(+) regulatory T cells. Our data suggest that blocking inflammatory cytokines may increase the capacity of the immune system to suppress type 1 diabetes through regulatory T cells.

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.

Rotavirus infection is associated with childhood progression to type 1 diabetes. Infection by monkey rotavirus RRV accelerates diabetes onset in non-obese diabetic (NOD) mice, which relates to regional lymph node infection and a T helper 1-specific immune response. When stimulated ex vivo with RRV, plasmacytoid dendritic cells (pDCs) from naïve NOD mice secrete type I interferon, which induces the activation of bystander lymphocytes, including islet-autoreactive T cells. This is our proposed mechanism for diabetes acceleration by rotaviruses. Here we demonstrate bystander lymphocyte activation in RRV-infected NOD mice, which showed pDC activation and strong upregulation of interferon-dependent gene expression, particularly within lymph nodes. The requirement for type I interferon signalling was analysed using NOD mice lacking a functional type I interferon receptor (NOD.IFNAR1−/− mice). Compared with NOD mice, NOD.IFNAR1−/− mice showed 8-fold higher RRV titers in lymph nodes and 3-fold higher titers of total RRV antibody in serum. However, RRV-infected NOD.IFNAR1−/− mice exhibited delayed pDC and lymphocyte activation, no T helper 1 bias in RRV-specific antibodies and unaltered diabetes onset when compared with uninfected controls. Thus, the type I interferon signalling induced by RRV infection is required for bystander lymphocyte activation and accelerated type 1 diabetes onset in genetically susceptible mice.

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.

Animal models have provided significant mechanistic insights into both physiology and disease. The NOD mouse, first identified and bred in Japan in the 1970s, is no exception. Studies in these mice have contributed to basic knowledge of the immune system, including costimulation, regulatory T cells, and MHC structure and function. NOD mice have also been informative about mechanisms that result in autoimmune diabetes, although it is unclear how these specifically contribute to disease pathogenesis in an individual human patient (1). Predisposing MHC genes, along with autoimmune reactions to insulin and its precursor proinsulin, are important in both mouse and human disease (2–6). Less clear is the value of animal models as part of the preclinical development of new treatments for disease. This has been particularly questioned not only for neurological diseases but also for type 1 diabetes (7). This uncertainty stems from many factors: the idea that inbred strains are a genetically homogeneous “n of 1” experiment compared with outbred humans; the many well-documented differences in the immune systems of the two species; and, most relevant to the article by Gill et al. (8), issues around the reliability, robustness, and reproducibility of data produced in trials of therapy in mice. These concerns have undermined the value of mouse studies and led to the notion that “mice lie,” and it is at least in part suboptimal design and execution of studies that have led to outcomes that cannot be translated to humans. To address these concerns, a consortium was formed by JDRF …

In the context of islet transplantation, experimental models show that induction of islet intrinsic NF-κB-dependent proinflammatory genes can contribute to islet graft rejection. Isolation of human islets triggers activation of the NF-κB and mitogen-activated kinase (MAPK) stress response pathways. However, the downstream NF-κB target genes induced in human islets during the isolation process are poorly described. Therefore, in this study, using microarray, bioinformatic, and RTqPCR approaches, we determined the pattern of genes expressed by a set of 14 human islet preparations. We found that isolated human islets express a panel of genes reminiscent of cells undergoing a marked NF-κB-dependent proinflammatory response. Expressed genes included matrix metallopeptidase 1 (MMP1) and fibronectin 1 (FN1), factors involved in tissue remodeling, adhesion, and cell migration; inflammatory cytokines IL-1β and IL-8; genes regulating cell survival including A20 and ATF3; and notably high expression of a set of chemokines that would favor neutrophil and monocyte recruitment including CXCL2, CCL2, CXCL12, CXCL1, CXCL6, and CCL28. Of note, the inflammatory profile of isolated human islets was maintained after transplantation into RAG-/- recipients. Thus, human islets can provide a reservoir of NF-κB-dependent inflammatory factors that have the potential to contribute to the anti-islet-graft immune response. To test this hypothesis, we extracted rodent islets under optimal conditions, forced activation of NF-κB, and transplanted them into allogenic recipients. These NF-κB activated islets not only expressed the same chemokine profile observed in human islets but also struggled to maintain normoglycemia posttransplantation. Further, NF-κB-activated islets were rejected with a faster tempo as compared to non-NF-κB-activated rodent islets. Thus, isolated human islets can make cell autonomous contributions to the ensuing allograft response by elaborating inflammatory factors that contribute to their own demise. These data highlight the potential importance of islet intrinsic proinflammatory responses as targets for therapeutic intervention.

Infiltration of pancreatic islets by immune cells, termed insulitis, increases progressively once it begins and leads to clinical type 1 diabetes. But even after diagnosis some islets remain unaffected and infiltration is patchy rather than uniform. Traffic of autoreactive T cells into the pancreas is likely to contribute to insulitis progression but it could also depend on T-cell proliferation within islets. This study utilizes transgenic NOD mice to assess the relative contributions of these two mechanisms. Progression of insulitis in NOD8.3 TCR transgenic mice was mildly reduced by inhibition of T-cell migration with the drug FTY720. In FTY720-treated mice, reduced beta cell MHC class I expression prevented progression of insulitis both within affected islets and to previously unaffected islets. CTL proliferation was significantly reduced in islets with reduced or absent beta cell expression of MHC class I protein. This indicates that intra-islet proliferation, apparently dependent on beta cell antigen presentation, in addition to recruitment, is a significant factor in progression of insulitis.

Pancreatic islets, transplanted into recipients with type 1 diabetes, are exposed to allogenic and auto-immune T-cell responses. We set out to develop an assay to measure these responses using PBMC. Our approach was to prepare spleen extract from the islet donors (allo-antigen) and islet extracts (auto-antigen). To our surprise, we found that spleen extracts potently inhibited the proliferation of human T cells driven by antigen (tetanus toxoid) and mitogen (anti-CD3 mAb, OKT3), whereas extracts prepared from pancreatic islets from the same donor did not suppress T-cell proliferation. Suppression mediated by spleen extracts was unaffected by blocking mAbs against the IL-10R, transforming growth factor-β or CD152 (CTLA-4). It was also unaffected by denaturing the spleen extracts by heating, exposing to reducing agents or protease digestion. Because deceased organ donors are commonly given the immunosuppressive glucocorticoid methylprednisolone prior to death, we hypothesized that suppression was due to residual methylprednisolone in the spleen extracts. Methylprednisolone could be detected by mass spectrometry in spleen extracts at concentrations that suppress T-cell proliferation. Finally, the glucocorticoid receptor antagonist mifepristone completely reversed the suppression caused by the spleen extracts. We conclude that extracts of human spleen, but not islets, from deceased organ donors contain sufficient residual methylprednisolone to suppress the proliferation of T-cells in vitro.

Recent advances in our understanding of the pathogenesis of type 1 diabetes have occurred in all steps of the disease. This review outlines the pathogenic mechanisms utilized by the immune system to mediate destruction of the pancreatic beta-cells. The autoimmune response against beta-cells appears to begin in the pancreatic lymph node where T cells, which have escaped negative selection in the thymus, first meet beta-cell antigens presented by dendritic cells. Proinsulin is an important antigen in early diabetes. T cells migrate to the islets via the circulation and establish insulitis initially around the islets. T cells within insulitis are specific for islet antigens rather than bystanders. Pathogenic CD4+ T cells may recognize peptides from proinsulin which are produced locally within the islet. CD8+ T cells differentiate into effector T cells in islets and then kill beta-cells, primarily via the perforin-granzyme pathway. Cytokines do not appear to be important cytotoxic molecules in vivo. Maturation of the immune response within the islet is now understood to contribute to diabetes, and highlights the islet as both driver and target of the disease. The majority of our knowledge of these pathogenic processes is derived from the NOD mouse model, although some processes are mirrored in the human disease. However, more work is required to translate the data from the NOD mouse to our understanding of human diabetes pathogenesis. New technology, especially MHC tetramers and modern imaging, will enhance our understanding of the pathogenic mechanisms.

Prevention of autoimmunity requires the elimination of self-reactive T cells during their development in the thymus and maturation in the periphery. Transgenic NOD mice that overexpress islet-specific glucose 6 phosphatase catalytic subunit–related protein (IGRP) in antigen-presenting cells (NOD-IGRP mice) have no IGRP-specific T cells. To study the relative contribution of central and peripheral tolerance mechanisms to deletion of antigen-specific T cells, we crossed NOD-IGRP mice to highly diabetogenic IGRP206–214 T-cell receptor transgenic mice (NOD8.3 mice) and studied the frequency and function of IGRP-specific T cells in the thymus and periphery. Peripheral tolerance was extremely efficient and completely protected NOD-IGRP/NOD8.3 mice from diabetes. Peripheral tolerance was characterized by activation of T cells in peripheral lymphoid tissue where IGRP was expressed followed by activation-induced cell death. Thymectomy showed that thymic output of IGRP-specific transgenic T cells compensated for peripheral deletion to maintain peripheral T-cell numbers. Central tolerance was undetectable until 10 weeks and complete by 15 weeks. These in vivo data indicate that peripheral tolerance alone can protect NOD8.3 mice from autoimmune diabetes and that profound changes in T-cell repertoire can follow subtle changes in thymic antigen presentation.

Diabetes mellitus are complex, multi-organ metabolic pathologies characterized by hyperglycemia. Emerging evidence shows that the highly conserved and potent JAK/STAT signaling pathway is required for normal homeostasis, and, when dysregulated, contributes to the development of obesity and diabetes. In this review, we analyze the role of JAK/STAT activation in the brain, liver, muscle, fat and pancreas, and how this affects the course of the disease. We also consider the therapeutic implications of targeting the JAK/STAT pathway in treatment of obesity and diabetes.

TNF has been implicated in the pathogenesis of type 1 diabetes. When administered early in life, TNF accelerates and increases diabetes in NOD mice. However, when administered late, TNF decreases diabetes incidence and delays onset. TNFR1-deficient NOD mice were fully protected from diabetes and only showed mild peri-insulitis. To further dissect how TNFR1 deficiency affects type 1 diabetes, these mice were crossed to β cell-specific, highly diabetogenic TCR transgenic I-Ag7–restricted NOD4.1 mice and Kd-restricted NOD8.3 mice. TNFR1-deficient NOD4.1 and NOD8.3 mice were protected from diabetes and had significantly less insulitis compared with wild type NOD4.1 and NOD8.3 controls. Diabetic NOD4.1 mice rejected TNFR1-deficient islet grafts as efficiently as control islets, confirming that TNFR1 signaling is not directly required for β cell destruction. Flow cytometric analysis showed a significant increase in the number of CD4+CD25+Foxp3+ T regulatory cells in TNFR1-deficient mice. TNFR1-deficient T regulatory cells were functionally better at suppressing effector cells than were wild type T regulatory cells both in vitro and in vivo. This study suggests that blocking TNF signaling may be beneficial in increasing the function of T regulatory cells and suppression of type 1 diabetes.

Type 1 diabetes is an autoimmune disease in which pancreatic beta cells are destroyed by autoreactive T cells. It is a common pediatric disease with increasing incidence. Islet transplantation may be a therapeutic option, however, the current limitations of this procedure mean that for most sufferers of type 1 diabetes there is no cure. The transcription factor NF-κB has been widely studied for its role in development of type 1 diabetes. Recent data have shown that NF-κB is required for activation of autoreactive T cells, and its hyperactivity in monocytes and dendritic cells results in altered cytokine secretion and antigen presentation, which ultimately contributes to the initiation of type 1 diabetes. NF-κB is also activated by a number of proinflammatory cytokines to regulate both the survival and death of beta cells. The critical role of NF-κB in type 1 diabetes renders it a promising pharmaceutical target in the intervention of this disease and further understanding of the NF- κB pathway will have an important implication on the development of novel and safe therapeutic strategies.

CD8(+) T cells kill pancreatic β-cells in a cell-cell contact-dependent mechanism in the non-obese diabetic mouse. CD4(+) T lymphocytes are also able to kill pancreatic β-cells, but they do not directly contact β-cells and may use another cell type as the actual cytotoxic cell. Natural killer (NK) cells could have this role but it is uncertain whether they are cytotoxic towards β-cells. Therefore, the requirement for NK cells in β-cell destruction in the CD4-dependent T-cell antigen receptor transgenic NOD4.1 mice was examined. NK cells failed to kill β-cells in vitro, even in the absence of major histocompatibility complex class I. We observed only 9.7±1.1% of islet infiltrating NK cells from NOD4.1 mice expressing the degranulation marker CD107a. Diabetogenic CD4(+) T cells transferred disease to NODscid.IL2Rγ(-/-) mice lacking NK cells, indicating that NK cells do not contribute to β-cell death in vitro or in vivo. However, depletion of NK cells reduced diabetes incidence in NOD4.1 mice, suggesting that NK cells may help to maintain the right environment for cytotoxicity of effector cells.

OBJECTIVE The effectiveness of tolerizing immunotherapeutic strategies, such as anti-CD40L or dendritic cells (DCs), is greater when administered to young nonobese diabetic (NOD) mice than at peak insulitis. RelBlo DCs, generated in the presence of an nuclear factor-κB inhibitor, induce T-regulatory (Treg) cells and suppress inflammation in a model of rheumatoid arthritis. Interleukin (IL)-1β is overexpressed in humans and mice at risk of type 1 diabetes, dysregulates Treg cells, and accelerates diabetes in NOD mice. We investigated the relationship between IL-1β production and the response to RelBlo DCs in the prediabetic period. RESEARCH DESIGN AND METHODS We injected RelBlo DCs subcutaneously into 4- or 14-week-old NOD mice and tracked the incidence of diabetes and effect on Treg cell function. We measured the expression of proinflammatory cytokines by stimulated splenocytes and unstimulated islets from mice of different ages and strains and proliferative and cytokine responses of T effectors to Treg in vitro. RESULTS Tolerizing RelBlo DCs significantly inhibited diabetes progression when administered to 4-week-old but not 14-week-old mice. IL-1β production by NOD splenocytes and mRNA expression by islets increased from 6 to 16 weeks of age when major histocompatibility complex (MHC)-restricted islet antigen presentation to autoreactive T-cells occurred. IL-1 reduced the capacity of Treg cells to suppress effector cells and promoted their conversion to Th17 cells. RelBlo DCs exacerbated the IL-1–dependent decline in Treg function and promoted Th17 conversion. CONCLUSIONS IL-1β, generated by islet-autoreactive cells in MHC-susceptible mice, accelerates diabetes by differentiating Th17 at the expense of Treg. Tolerizing DC therapies can regulate islet autoantigen priming and prevent diabetes, but progression past the IL-1β/IL-17 checkpoint signals the need for other strategies.

Apoptosis of beta cells is a feature of both type 1 and type 2 diabetes as well as loss of islets after transplantation. In type 1 diabetes, beta cells are destroyed by immunological mechanisms. In type 2 diabetes abnormal levels of metabolic factors contribute to beta cell failure and subsequent apoptosis. Loss of beta cells after islet transplantation is due to many factors including the stress associated with islet isolation, primary graft non-function and allogeneic graft rejection. Irrespective of the exact mediators, highly conserved intracellular pathways of apoptosis are triggered. This review will outline the molecular mediators of beta cell apoptosis and the intracellular pathways activated.

CD4(+) T cells can actively kill beta-cells in type I diabetes as well as help CD8(+) T cells become cytolytic. Cytokines have the potential to kill beta-cells, or upregulate Fas on beta-cells, and increase their susceptibility to FasL. We investigated the direct effects of cytokines on beta-cells in perforin-deficient non-obese diabetic (NOD) mice and NOD4.1 TCR transgenic mice, two models in which CD8(+) T cells play a less dominant role. Inhibiting the effects of cytokines by the overexpression of suppressor of cytokine signalling-1 (SOCS1) in beta-cells did not reduce diabetes or insulitis in perforin-deficient NOD, NOD4.1 or interleukin (IL)-1 receptor-deficient NOD4.1 mice. SOCS1 overexpression prevented Fas upregulation on NOD4.1 beta-cells, but did not prevent islet destruction because SOCS1 transgenic islets were killed when grafted into NOD4.1.scid mice. Likewise, Fas-deficient NOD.lpr islets were destroyed in NOD4.1 mice. Although blocking the effects of interferon (IFN)gamma on beta-cells did not affect diabetes in NOD4.1 mice, global deficiency of IFNgammaR2 reduced diabetes and insulitis, suggesting that IFNgamma is involved in CD4(+) T-cell activation or migration. Our data show that beta-cells under attack by CD4(+) T cells are not destroyed by the effects of cytokines including IFNgamma and IL-1 or Fas-dependent cytotoxicity.

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.

Type 1 diabetes (T1D) is characterized by immune responses against several autoantigens expressed in pancreatic beta cells. T cells specific for proinsulin and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) can induce T1D in NOD mice. However, whether immune responses to multiple autoantigens are caused by spreading from one to another or whether they develop independently of each other is unknown. As cytotoxic T cells specific for IGRP were not detected in transgenic NOD mice tolerant to proinsulin, we determined that immune responses against proinsulin are necessary for IGRP-specific T cells to develop. On the other hand, transgenic overexpression of IGRP resulted in loss of intra-islet IGRP-specific T cells but did not protect NOD mice from insulitis or T1D, providing direct evidence that the response against IGRP is downstream of the response to proinsulin. Our results suggest that pathogenic proinsulin-specific immunity in NOD mice subsequently spreads to other antigens such as IGRP.

CD8(+) cytotoxic T lymphocytes (CTL) can rapidly kill beta-cells and therefore contribute to the development of type 1 diabetes (T1D). CTL-mediated beta-cell killing can occur via perforin-mediated lysis, Fas-Fas-L interaction, and the secretion of TNF-alpha or IFN-gamma. The secretion of IFN-gamma can contribute to beta-cell death directly by eliciting nitric oxide production, and indirectly by upregulating MHC class I and 'unmasking' beta-cells for recognition by CTL. Earlier studies in the RIP-LCMV mouse model of diabetes showed that disruption of beta-cell IFN-gamma signaling alone abolished the direct detrimental effects of IFN-gamma, but not MHC class I upregulation. Suppressor of cytokine signaling-1 (SOCS-1) represses several crucial cytokine signaling pathways simultaneously, among them IFN-gamma and IL-1-beta. We therefore evaluated the protective capacity of islet cell SOCS-1 expression in the CD8(+) mediated RIP-LCMV diabetes model. Clinical disease was prevented in over 90% of the mice. Not only absence of MHC-I and Fas upregulation, but also resistance to cytokine-induced killing of beta-cells and a complete lack of CXCL-10 (IP10) production in islets led to a lack of islet infiltration and impaired activation of autoaggressive CD4(+) and CD8(+) T-cells in these mice. Thus, SOCS expression renders beta-cells resistant to CTL attack in a mouse model of T1D.

Direct interaction between auto-reactive CTL and specific peptide-MHC class I complexes on pancreatic beta cells is critical in mediating beta cell destruction in type I diabetes. We used mice with genetic modifications in three major pathways used by CTL, perforin, Fas and pro-inflammatory cytokines to assess the relative contribution of these mechanisms to beta cell death. In vitro-activated ovalbumin (OVA)-specific CTL, from OT-I TCR-transgenic mice, specifically killed transgenic beta cells expressing OVA (from RIP-mOVA mice) in a 16-h cytotoxicity assay. Perforin-deficient CTL had a reduced ability to kill OVA-expressing islets in vitro (22.1 +/- 3.8%) compared with wild-type CTL (71.4 +/- 4.6%). Fas-deficient islets were only slightly protected from wild-type CTL but were completely protected from the residual killing observed with perforin-deficient CTL. Residual cytotoxicity in perforin-deficient CTL was also prevented by overexpression of SOCS-1, which blocks multiple cytokine signaling pathways. It was also prevented by pre-incubation with anti-tumor necrosis factor-alpha (anti-TNFalpha) antibody or by blocking IFNgamma responsiveness through expressing a dominant negative IFNgamma receptor. Perforin-deficient CTL produced IFNgamma and TNFalpha that was shown to directly induce islet Fas expression during the assays. This suggests that Fas-deficiency, SOCS-1 overexpression and blockade of IFNgamma and TNFalpha all protect beta cells from residual cytotoxicity of perforin-deficient CTL by blocking Fas upregulation. These findings indicate that wild-type CTL destroy antigen-expressing islets via a perforin-dependent mechanism. However, in the absence of perforin, the Fas/FasL pathway provides an alternative mechanism dependent on islet cell Fas upregulation by cytokines IFNgamma and TNFalpha.

In the NOD mouse model of type 1 diabetes (T1D), genetically identical mice in the same environment develop diabetes at different rates. Similar heterogeneity in the rate of progression to T1D exists in humans, but the underlying mechanisms are unclear. Here, we aimed to discover peripheral blood (PB) genes in NOD mice predicting insulitis severity and rate of progression to diabetes. We then wished to use these genes to mine existing databases to identify drugs effective in diabetes. In a longitudinal study, we analyzed gene expression in PB samples from NOD.CD45.2 mice at 10 weeks of age, then scored pancreatic insulitis at 14 weeks or determined age of diabetes onset. In a multilinear regression model, Tnf and Tgfb mRNA expression in PB predicted insulitis score (R (2)=0.56, P=0.01). Expression of these genes did not predict age of diabetes onset. However, by expression-profiling PB genes in 10-week-old NOD.CD45.2 mice, we found a signature of upregulated genes that predicted delayed or no diabetes. Major associated pathways included chromatin organization, cellular protein location and regulation of nitrogen compounds and RNA. In a clinical cohort, three of these genes were differentially expressed between first-degree relatives, T1D patients and controls. Bioinformatic analysis of differentially expressed genes in NOD.CD45.2 PB identified drugs that are predicted to delay or prevent diabetes. Of these drugs, 11 overlapped with drugs predicted to induce a human 'non-progressor' expression profile. These data demonstrate that disease heterogeneity in diabetes-prone mice can be exploited to mine novel clinical T1D biomarkers and drug targets.

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.

Pro-inflammatory cytokines have been implicated in the death of pancreatic beta cells leading to type 1 diabetes. NIT-1 cells are an insulinoma cell line derived from mice expressing the SV40 large T antigen. These cells are a useful tool in analysis of beta cell death. NIT-1 cells are highly susceptible to caspase-dependent apoptosis induced by TNF-alpha alone. Primary islets are not susceptible to cell death induced by TNF-alpha alone; however, they are killed by TNF-alpha and IFN-gamma in a nitric oxide-dependent manner. We examined signal transduction in NIT-1 cells in response to cytokines to determine the mechanism for TNF-alpha-induced apoptosis. We found that NIT-1 cells are defective in the activation of nuclear factor-kappaB (NFkappaB) as a result of functionally deficient RelA activity, because overexpression of RelA protected NIT-1 cells from apoptosis. TNF-alpha also did not induce phosphorylation of c-Jun N-terminal kinase in NIT-1 cells. Together, these defects prevent expression of anti-apoptotic genes in NIT-1 cells and make them susceptible to TNF-alpha. To determine whether similar defects in primary beta cells would induce the same effect, we examined TNF-alpha-induced apoptosis in islets isolated from mice deficient in NFkappaB p50. These islets were as susceptible as wild-type islets to TNF-alpha and IFN-gamma-induced cell death. In contrast to wild-type islets, cell death was not prevented by inhibition of nitric oxide in p50-deficient islets. Blocking NFkappaB has been proposed as a mechanism for protection of beta cells from cytokine-induced cell death in vivo. Our results suggest that this would make beta cells equally or more sensitive to cytokines.

Type 1 diabetes (T1D) develops when insulin-secreting β-cells, found in the pancreatic islets of Langerhans, are destroyed by infiltrating T cells. How human T cells recognize β-cell-derived antigens remains unclear. Genetic studies have shown that HLA and insulin alleles are the most strongly associated with risk of T1D. These long-standing observations implicate CD4+ T-cell responses against (pro)insulin in the pathogenesis of T1D. To dissect the autoimmune T-cell response against human β-cells, we isolated and characterized 53 CD4+ T-cell clones from within the residual pancreatic islets of a deceased organ donor who had T1D. These 53 clones expressed 47 unique clonotypes, 8 of which encoded proinsulin-specific T-cell receptors. On an individual clone basis, 14 of 53 CD4+ T-cell clones (26%) recognized 6 distinct but overlapping epitopes in the C-peptide of proinsulin. These clones recognized C-peptide epitopes presented by HLA-DQ8 and, notably, HLA-DQ8 transdimers that form in HLA-DQ2/-DQ8 heterozygous individuals. Responses to these epitopes were detected in the peripheral blood mononuclear cells of some people with recent-onset T1D but not in HLA-matched control subjects. Hence, proinsulin-specific, HLA-DQ8, and HLA-DQ8-transdimer–restricted CD4+ T cells are strongly implicated in the autoimmune pathogenesis of human T1D.

Apoptosis of pancreatic beta cells is a feature of type 2 diabetes and its prevention may have therapeutic benefit. High glucose concentrations induce apoptosis of islet cells, and this requires the proapoptotic Bcl-2 homology domain 3 (BH3)-only proteins Bim and Puma. We studied the stress pathways induced by glucotoxicity in beta cells that result in apoptosis. High concentrations of glucose or ribose increased expression of the transcription factor CHOP (C/EBP homologous protein) but not endoplasmic reticulum (ER) chaperones, indicating activation of proapoptotic ER stress signaling. Inhibition of ER stress prevented ribose-induced upregulation of Chop and Puma mRNA, and partially protected islets from glucotoxicity. Loss of Bim or Puma partially protected islets from the canonical ER stressor thapsigargin. The antioxidant N-acetyl-cysteine also partially protected islets from glucotoxicity. Islets deficient in both Bim and Puma, but not Bim or Puma alone, were significantly protected from killing induced by the mitochondrial reactive oxygen species donor rotenone. Our data demonstrate that high concentrations of glucose induce ER and oxidative stress, which causes cell death mediated by Bim and Puma. We observed significantly higher Bim and Puma mRNA in islets of human donors with type 2 diabetes. This indicates that inhibition of Bim and Puma, or their inducers, may prevent beta-cell destruction in type 2 diabetes., info:eu-repo/semantics/published

CD8(+) T cells are prominent in autoimmune diabetes of both humans and non-obese diabetic (NOD) mice. For example, CD8(+) T cells against islet-specific glucose 6-phosphatase catalytic subunit-related protein (IGRP) can be detected readily in older NOD mice. It has been suggested that the enumeration of islet-specific CD8(+) T cells in the peripheral blood may be a predictive biomarker for autoimmune type 1 diabetes (T1D). Here, we determined the natural history of the functional endogenous IGRP(206-214)-specific cytotoxic T lymphocytes (CTLs) in NOD mice with regard to age (3- to 15-week-old pre-diabetic mice and diabetic mice) and sex. We demonstrated that in vivo IGRP(206-214)-specific CTLs significantly increased after 12 weeks of age and in vivo cytotoxicity in female NOD mice was significantly higher than in male NOD mice. To determine the in vivo IGRP(206-214)-specific CTL frequency without killing the mice, we performed splenectomies on a cohort of mice after injecting IGRP(206-214)-coated targets and then followed their diabetes progression. We found that CTL frequency correlated with future of disease onset. Thus, our data support that IGRP(206-214)-specific CTLs may be a potent biomarker for T1D.

Autoimmune diabetes is caused by selective loss of insulin-producing pancreatic beta-cells. The main factors directly implicated in beta-cell death are autoreactive, cytotoxic (islet-antigen specific) T-lymphocytes (CTL), and inflammatory cytokines. In this study, we have used an antigen-specific model of virally induced autoimmune diabetes to demonstrate that even high numbers of autoreactive CTL are unable to lyse beta-cells by perforin unless major histocompatibility complex class I is upregulated on islets. This requires the presence of inflammatory cytokines induced by viral infection of the exocrine pancreas but not of the beta-cells. Unexpectedly, we found that the resulting perforin-mediated killing of beta-cells by autoreactive CTL is not sufficient to lead to clinically overt diabetes in vivo, and it is not an absolute prerequisite for the development of insulitis, as shown by studies in perforin-deficient transgenic mice. In turn, destruction of beta-cells also requires a direct effect of gamma-interferon (IFN-gamma), which is likely to be in synergy with other cytokines, as shown in double transgenic mice that express a mutated IFN-gamma receptor on their beta-cells in addition to the viral (target) antigen and do not develop diabetes. Thus, destruction of most beta-cells occurs as cytokine-mediated death and requires IFN-gama in addition to perforin. Understanding these kinetics could be of high conceptual importance for the design of suitable interventions in prediabetic individuals at risk to develop type 1 diabetes.