Medicine (St Vincent's) - Theses
Permanent URI for this collection
Search Results
1 - 2 of 2
-
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.
-
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.