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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.