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ItemThe development and regulation of islet-specific T cells in an experimental model of autoimmune diabetesChee, Hui En Jonathan ( 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.
ItemCharacterisation of susceptibility to Listeria monocytogenes infection in the non-obese diabetic (NOD) mouseWANG, NANCY ( 2012)The immune system has evolved the ability to prevent infection by a wide range of pathogens while maintaining tolerance to self-tissues. Due to the strong selective pressure imposed by microbial pathogens, susceptibility to infection can be modulated by a large number of genetic loci. It is postulated that, at least in some cases, allelic variants for particular loci confer increased resistance to pathogens while simultaneously increasing the risk of developing autoimmune diseases. A number of recently discovered disease loci appear to act as “genetic pivot points” between pathogen defence and autoimmune pathogenesis. It is anticipated that characterising these loci will provide novel insights regarding the interplay between immune and autoimmune responses, as well as reveal potential therapeutic targets for treating both infectious and autoimmune diseases. Murine models provide a complementary approach to human studies for investigating genetic and cellular mechanisms that underlie susceptibility to infectious and autoimmune diseases. The non-obese diabetic (NOD) mouse strain is one of the best-characterised models of type 1 diabetes (T1D), an autoimmune disease caused by the destruction of insulin-producing pancreatic β cells. Similar to humans, predisposition to T1D is attributed to multiple genetic loci in NOD mice. Intriguingly, non-diabetogenic mouse strains can also harbour diabetogenic alleles for some T1D susceptibility loci. Using congenic mice, our laboratory confirmed that non-diabetogenic C57BL/6 (B6) mice harbour a diabetogenic allele for a T1D susceptibility locus on chromosome (Chr) 13, termed Idd14. Coincidentally, the Idd14 locus overlapped with Listr2, a proposed susceptibility locus for Listeria monocytogenes, which is an extensively studied intracellular bacterial pathogen. Notably, B6 mice are resistant to L. monocytogenes infection, whereas NOD mice are reported as susceptible. We therefore hypothesised that allelic variation for this interval, which increases T1D risk, would confer resistance against L. monocytogenes in NOD mice. The goal of this study was to investigate the biological and genetic effects of allelic variation for Idd14/Listr2 in NOD mice for L. monocytogenes infection and T1D. Towards this goal, the first aim was to investigate the immunological basis of susceptibility to L. monocytogenes in NOD mice. In comparison to infected B6 mice, infected NOD mice exhibited reduced antigen-specific CD8+ T-cell responses, which was associated with apparent deficiencies in dendritic cells. Infected NOD mice also exhibited exacerbated neutrophilia, a potential compensatory mechanism in susceptible hosts. The second aim was to determine the role of neutrophils during L. monocytogenes infection. Specific depletion of neutrophils impaired bacterial clearance in susceptible NOD mice, but not in resistant B6 mice, demonstrating that neutrophils are essential for controlling L. monocytogenes infection in susceptible hosts. In addition, NOD macrophages exhibited impaired antimicrobial function. These results indicate that neutrophils may compensate for deficient T-cell and macrophage responses to ensure host survival during L. monocytogenes infection. The third aim was to confirm the Listr2 locus using congenic NOD mouse strains, which harbour different B6-derived Chr 13 interval, and determine if the refined intervals for Listr2 and Idd14 continue to co-localise. Concurrently, it was found that the Idd14 locus could be dissected into two sub-loci, Idd14.1 and Idd14.2. Listr2 was confirmed and co-localised with Idd14.1 to an overlapping interval (~ 18 Mb) on Chr 13. This co-localisation raises the intriguing possibility that allele variation for the same gene(s) within this interval affects both infection and autoimmune disease susceptibility.