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.
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ItemInduction of antigen-specific tolerance and development of autoreactive T cells in an experimental model of autoimmune diabetes( 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.
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ItemTargeting CD8+ T cells to protect beta cells in type 1 diabetes( 2016)Type 1 diabetes results from destruction of pancreatic beta cells by autoreactive T cells. CD8+ T cells play central role in beta cell destruction. The T cell receptor on CD8+ T cells engages with peptide-MHC class I molecules present on beta cells, and deliver cytotoxic molecules though the immunological synapse. Inhibiting the interaction between CD8+ T cells and beta cells, or blocking cytotoxic pathways could prevent beta cell destruction and hence type 1 diabetes. In this thesis I have used novel small molecule inhibitors to block recognition and killing of beta cells by CD8+ T cells. To achieve this goal in an antigen specific manner for future immunotherapy, I have also investigated the antigens recognized by islet-infiltrating CD8+ T cells from type 1 diabetic donors. In chapter 2, I investigated role of perforin as the major killing mechanism used by CD8+ T cells to kill beta cells. I confirmed that perforin is essential to facilitate beta cell destruction in vivo. In addition, perforin-deficient beta cell antigen-specific CD8+ T cells from NOD8.3 mice were activated more in response to antigen, indicating that perforin may regulate the activation of cytotoxic T lymphocytes. There are currently no therapies available that directly target cytotoxic CD8+ T cells. In chapter 3, I have tested the use of novel small molecule perforin inhibitors for prevention of beta cell death in autoimmune diabetes. Perforin inhibitors protected beta cells from CD8+ T cell killing in vitro and blocked antigen specific CD8+ T cell mediated killing of target cells in vivo. These studies pave the way for testing perforin inhibitors in mouse models of diabetes. Blocking the interaction between CD8+ T cells and beta cells holds promise for prevention of beta cell death, In chapter 4, I showed that small molecule JAK1/JAK2 inhibitors successfully blocked the interaction between beta cells and CD8+ T cells and protected beta cells from CD8+ T cell mediated killing in vitro. When used in mice JAK1/JAK2 inhibitors reduced migration of T cells to islets and prevented cytokine mediated MHC class I upregulation on beta cells, even at later stages of autoimmune diabetes in mice. These inhibitors significantly protected mice from development of autoimmune diabetes. In chapter 5, human islet-infiltrating CD8+ T cell clones from organ donors who died with type 1 diabetes were used to discover beta cell antigens. COS-7 cells co-transfected with donor specific HLA class I alleles and plasmids encoding beta cell antigens were used as antigen presenting cells. While this method worked well to identify the antigen specificity of a CD8+ T cell clone for which the antigen was already known, none of the 24 islet-infiltrating clones tested recognized any of the beta cell antigen and donor specific HLA class I encoding plasmids. This thesis shows that the use of small molecule inhibitors may be effective in protecting protect beta cells from CD8+ T cells in type 1 diabetes. Identifying beta cell antigens recognized by CD8+ T cells will help to develop therapies where these inhibitors can be used in combination with antigen-specific therapy.
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ItemThe development and regulation of islet-specific T cells in an experimental model of autoimmune diabetes( 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.