Medicine (St Vincent's) - Theses
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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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ItemCharacterization of T cell populations in human skin( 2016)Human skin serves as a primary barrier to pathogenic and environmental assault. As part of the frontlines of the skin immune system, normal skin contains a vast number of T cells. With advances in immunofluorescent staining, confocal microscopy and fluorescence activated cell sorting, enumeration and characterization of these cutaneous T cells can now be performed with improved accuracy. Furthermore, several subsets of T cells, including mucosal-associated invariant T (MAIT) cells and resident memory T (TRM) cells, have only recently been described and their exact distribution in normal human skin remains to be elucidated. The relationship of T cells to hair follicles, ubiquitous appendages in human skin, also requires further investigation. This dissertation presents a comprehensive assessment of the distribution and composition of major T cell subsets in human skin at steady state. The gene expression profiles of skin and blood T cells were compared to determine a transcriptional signature for cutaneous T cells. We also conducted a preliminary investigation of the novel T cell subsets MAIT cells and TRM, exploring their presence in normal and diseased skin. Finally, we used a targeted laser capture microdissection approach to study chemokine expression in healthy hair follicles and in the autoimmune hair loss condition alopecia areata. Our findings reconfirm the strikingly anisotropic arrangement of T cells in normal human skin, with preferences for superficial perivascular and perifollicular localization. A resident cutaneous population of MAIT cells was found, with increased frequency in the blistering skin condition dermatitis herpetiformis. Surprisingly, a strong disparity was observed between the transcriptional profiles of skin-tropic T cells isolated from normal blood and skin. As the skin-derived T cells showed significant enrichment for genes from the TRM transcriptional core signature, we hypothesized that the skin may contain additional as-yet undefined TRM or TRM-like populations. Analysis of alopecia areata hair follicles revealed the presence of perifollicular T cells of a TRM phenotype that persisted long after active hair loss had ceased. Gene expression studies of laser microdissected follicles further highlighted transcriptional abnormalities in alopecia areata follicles that spanned the natural history of the disease. The results from this dissertation form a foundation for further study of conventional and emerging T cell subsets in skin and hold implications for the pathogenesis of the dermatological diseases dermatitis herpetiformis and alopecia areata.
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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.