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ItemInduction of antigen-specific tolerance and development of autoreactive T cells in an experimental model of autoimmune diabetesJhala, Gaurang ( 2016)Immune responses to proinsulin initiate anti-islet autoimmunity in non-obese diabetic (NOD) mice and possibly in humans. This results in autoimmune destruction of insulin secreting beta cells leading to type 1 diabetes (T1D). Therapies that bolster immune tolerance to islet antigens are highly desirable, however such approaches have failed to prevent clinical T1D. The major aim of this thesis was to determine a stage of life when antigen-specific tolerance is most effective in preventing anti-islet immune responses. Chapter 2 describes generation and validation of transgenic NOD mice engineered to express islet antigens proinsulin (TIP mice) and IGRP (TII mice) in the antigen presenting cells (APCs) in a tetracycline dependent manner. MHC class II IEα promoter in combination with tet-OFF transactivator induced robust, doxycycline dependent and APC specific expression of proinsulin and IGRP in TIP and TII mice respectively. TIP mice expressing proinsulin did not develop insulitis and were protected from cyclophosphamide-induced diabetes, suggesting that proinsulin expression in TIP mice was sufficient to induce functional antigen-specific tolerance. In chapter 3, we examined the impact of antigen-specific tolerance on the development of autoreactive T cells and spontaneous diabetes by expressing islet antigens proinsulin and IGRP in the APCs during defined periods of life in TIP and TII mice. Our results indicate that tolerance to proinsulin in early life until the weaning period is sufficient to prevent diabetes development in TIP mice. The protection from diabetes was not due to dominant tolerance, but mainly due to a significant reduction in the insulin reactive T cells. Although insulin reactive T cells were not completely absent, the residual autoreactive T cells lacked pathogenic potential. By tracking IGRP reactive T cells in TII mice we demonstrate that IGRP T cells also emerge during early life. These data suggest that early life is a vulnerable period for escape of islet reactive T cells, and that boosting immune tolerance to islet antigens during this time imparts durable protection from islet autoimmunity. Immune tolerance to proinsulin-2 imparts robust protection from autoimmune diabetes in the NOD mice. Whether dampening immune responses to proinsulin-1 would influence diabetes development in NOD mice remains to be investigated. Chapter 4 describes the generation of transgenic NOD mice that express proinsulin-1 in the APCs (TIP-1 mice) in a tetracycline dependent manner. 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 number in TIP-1 mice indicating immune tolerance. Although immune response to downstream antigen IGRP was reduced in TIP-1 mice, tolerance to proinsulin-1 was unable to prevent diabetes in NOD 8.3 mice with a pre-existing repertoire of IGRP reactive T cells. Thus, despite being highly conserved to proinsulin-2, tolerance to proinsulin-1 only partially prevents islet-autoimmunity in NOD mice, which suggests an ongoing residual immune response to proinsulin-2 epitopes in TIP-1 mice.
ItemThe development and regulation of islet-specific T cells in an experimental model of autoimmune diabetesChee, Hui En Jonathan ( 2014)Type 1 diabetes (T1D) is an autoimmune disease. T cells specific for β-cell antigens such as proinsulin and islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP) are important in mediating the disease. The aims of this thesis were to study the development and regulation of T cells in the development of autoimmune diabetes in the non-obese diabetic (NOD) mouse model. Chapter 2 describes the development of IGRP-specific CD8+ T cells in autoimmune diabetes. IGRP-specific T cells in the mouse were tracked using a sensitive MHC-tetramer based magnetic enrichment. There was an increase in the number of IGRP-specific T cells in the peripheral blood and lymphoid tissue as mice age, and the increase correlated with insulitis progression. These cells had an effector-memory phenotype, which was only acquired in the inflammatory environment of the islets, and not the draining lymph nodes. Islet-specific T cells could also migrate from islets into the periphery. In the development of autoimmune diabetes, important changes to IGRP-specific T cells during the pathogenesis of diabetes occur not in the draining lymph nodes but in the islets, where they expand and differentiate into effector-memory T cells, and emigrate to the periphery, where they can report progression of islet pathology. Tumour Necrosis Factor (TNF) is an inflammatory cytokine that has been implicated in the pathogenesis of autoimmune diabetes. In chapter 3, we investigate the effects of TNF-TNFR1 signalling deficiency on the development of autoimmune diabetes, by using a NOD mouse deficient in TNF receptor 1 (TNFR1). TNFR1-/- islets grafted onto kidney capsule of diabetic mice were destroyed, showing that TNFR1 deficiency on β-cell did not confer protection against immune destruction. The specific effects of TNFR1 deficiency on the immune system of NOD mice were also examined. Adoptively transferred β-cell specific T cells proliferated normally in the pancreatic lymph nodes, but failed to migrate into the pancreas of TNFR1-/- recipient mice. Notably, analysis of immune cell subsets by flow cytometry showed an increased percentage of CD4+CD25+Foxp3+ T regulatory cells in TNFR1 deficient mice. Depletion of CD4+CD25+ regulatory T cells using GK1.5 CD4 depleting mAb restored diabetes in NOD8.3/TNFR1-/- mice. These results suggest that blockade of TNF signalling suppresses diabetes by increasing regulatory functions of the immune system. T cell responses to insulin (INS) are crucial in development of T1D. Chapter 4 of the thesis examines insulin-specific T cells in NOD mice, and in NOD mice tolerant to proinsulin II (NODPI), which do not develop diabetes or insulitis. There was no significant difference in the absolute number of insulin-specific CD8+ T-cells in NOD and NODPI mice. INS-specific CD8+ T-cells in NOD mice expanded significantly more in response to stimulation by peptide compared to NODPI. In vivo cytotoxic activity in NODPI was reduced compared to NOD. The absolute number of INS-specific CD4+ T-cells in NOD and NODPI mice was similar. The proportion of regulatory INS-specific CD4+ T cells that were Foxp3+ was also similar. INS-specific CD4+ T cells in the NOD and NODPI were tested on whether they could help CD8+ T-cells mediate diabetes. NODRAG1-/-/8.3 developed diabetes rapidly after NOD CD4+ T-cells were transferred (median=32days). 7/8 of recipients did not develop diabetes when NODPI CD4+ T-cells were transferred. In a NOD mouse tolerant to proinsulin, insulin-specific CD4+ and CD8+ T-cells were detected, suggesting that the main mechanism of tolerance is not deletion. It is more likely that these cells could have impaired function.