Jonathan Chee

TNF Receptor 1 Deficiency Increases Regulatory T Cell Function in Nonobese Diabetic mice

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.

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.

Pathogenic mechanisms in type 1 diabetes: the islet is both target and driver of disease.

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.

Effector-Memory T Cells Develop in Islets and Report Islet Pathology in Type 1 Diabetes

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.

Proinflammatory cytokines contribute to development and function of regulatory T cells in type 1 diabetes

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.

Complete Diabetes Protection Despite Delayed Thymic Tolerance in NOD8.3 TCR Transgenic Mice Due to Antigen-Induced Extrathymic Deletion of T Cells

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.

Expression of Pro- and Antiapoptotic Molecules of the Bcl-2 Family in Human Islets Postisolation

Human islets are subjected to a number of stresses before and during their isolation that may influence their survival and engraftment after transplantation. Apoptosis is likely to be activated in response to these stresses. Apoptosis due to intrinsic stresses is regulated by pro- and antiapoptotic members of the Bcl-2 family. While the role of the Bcl-2 family in apoptosis of rodent islets is becoming increasingly understood, little is known about which of these molecules are expressed or required for apoptosis of human islets. This study investigated the expression of the Bcl-2 family of molecules in isolated human islets. RNA and protein lysates were extracted from human islets immediately postisolation. At the same time, standard quality control assays including viability staining and β-cell content were performed on each islet preparation. Microarrays, RT-PCR, and Western blotting were performed on islet RNA and protein. The prosurvival molecules Bcl-xl and Mcl-1, but not Bcl-2, were highly expressed. The multidomain proapoptotic effector molecule Bax was expressed at higher levels than Bak. Proapoptotic BH3-only molecules were expressed at low levels, with Bid being the most abundant. The proapoptotic molecules BNIP3, BNIP3L, and Beclin-1 were all highly expressed, indicating exposure of islets to oxygen and nutrient deprivation during isolation. Our data provide a comprehensive analysis of expression levels of pro- and antiapoptotic Bcl-2 family members in isolated human islets. Knowledge of which molecules are expressed will guide future research to understand the apoptotic pathways activated during isolation or after transplantation. This is crucial for the design of methods to achieve improved transplantation outcomes.

Functional cytotoxic T lymphocytes against IGRP206-214 predict diabetes in the non-obese diabetic mouse.

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 IGRP206–214‐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 IGRP206–214‐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 IGRP206–214‐specific CTL frequency without killing the mice, we performed splenectomies on a cohort of mice after injecting IGRP206–214‐coated targets and then followed their diabetes progression. We found that CTL frequency correlated with future of disease onset. Thus, our data support that IGRP206–214‐specific CTLs may be a potent biomarker for T1D.

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.

Transient Treg depletion enhances therapeutic anti‐cancer vaccination

Regulatory T cells (Treg) play an important role in suppressing anti‐ immunity and their depletion has been linked to improved outcomes. To better understand the role of Treg in limiting the efficacy of anti‐cancer immunity, we used a Diphtheria toxin (DTX) transgenic mouse model to specifically target and deplete Treg.