Medicine (St Vincent's) - Theses
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ItemMechanisms and inhibition of CD4+ T cell migration in pre-clinical and humanised mouse models of type 1 diabetes( 2017)Type 1 diabetes (T1D) is an autoimmune disease that develops when the insulin-secreting beta cells in the pancreas are destroyed. This destruction is primarily mediated by T cells, of which CD4+ T cells play a central role by controlling immune responses via the production of cytokines. The aim of this thesis was to investigate mechanisms by which CD4+ T cells migrate to the pancreatic islets and kill beta cells, to test a therapeutic method of inhibiting this process, and to develop a mouse model capable of analysing the pathogenicity of human-islet infiltrating CD4+ T cell clones. Deficiency of IFNγ receptor has been reported to prevent the adoptive transfer of CD4+ T cell mediated diabetes. In chapter three, I confirmed these findings and investigate 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 a cognate antigen signal for diapedesis across the microvessels into the islets. IFNγ treatment of islets led to MHC class II expression on IECs and high MHC class II was detected on IECs in the early stages of insulitis. However, bone marrow chimera experiments revealed MHC class II on IECs is not required for the transfer of CD4+ T cell mediated diabetes. This work rules out antigen presentation by IECs as a putative mechanism for the homing of antigen-specific CD4+ T cells into the pancreatic islets. Cytokines that signal through the JAK-STAT pathway play a role in CD4+ T cell-dependent diabetes. In chapter four, I tested whether JAK1/2 inhibition prevents CD4+ T cell mediated diabetes to determine whether this could be a viable method of blocking cytokine signalling, and pathogenic T-cell responses, in a clinical setting. AZD1480, a JAK1/2 inhibitor, delayed diabetes induced by adoptive transfer of highly diabetogenic CD4+ BDC2.5 T cells. AZD1480 slowed the development of insulitis, and decreased the absolute numbers of leukocytes, including CD4+ T cells, in the islets and pancreatic lymph nodes (PLN), especially reflected in reduced effector-memory T cells. Combined with the recent success of our laboratory in using JAK1/2 inhibitors to abrogate CD8+ T cell mediated diabetes and induce disease reversal, we envision that JAK inhibitors could be trialled in patients at risk of developing type 1 diabetes. Autoimmunity to proinsulin is crucial in the development of diabetes in mice. However, it is unclear whether T cell responses to proinsulin are required for type 1 diabetes in humans. In chapter five, I surveyed the characteristics required to accurately model, in a humanised mouse, CD4+ T-cell responses to proinsulin seen in the pancreatic islets of a deceased organ donor who suffered from T1D. Three components were determined to be necessary: human proinsulin, HLA-DQ8 and chimeric T-cell receptors containing the human TCR variable regions that encode for proinsulin recognition. This led to the generation of a human proinsulin knockin mouse, NOD.HuPI. In this mouse, the murine Ins1 gene was ‘replaced’ with human INS. In addition, chimeric TCR constructs for several of the proinsulin-specific T-cell clones were generated and tested. These constructs are now ready for use to create TCR transgenic or retrogenic mice. This humanised mouse model will serve as a vehicle to determine which proinsulin-responding clone(s) induce diabetes pathogenesis. This information is vital for designing antigen-specific therapies to prevent and treat T1D in the future.