This project aims to integrate knowledge of genetic susceptibility, immune cell function, and environmental modifiers in determining risk for type 1 diabetes (T1D) in the non-obese diabetic (NOD) mouse model. Similar to human T1D, disease risk in the NOD mouse is polygenic and has been mapped to multiple Idd loci. We have fine-mapped the Idd4.1 locus and identified Nlrp1b as its candidate gene. We report an alternatively spliced isoform in the diabetes-resistant Nrlp1b allele, resulting in a truncated NLRP1b protein that is unable to activate release of IL-1β. In another aspect of this project, we have characterized the critical contribution to T1D pathogenesis by γδ T cells. We report that CD27- γδT cells infiltrate islets of pre-diabetic NOD mice. Adoptive transfer of T1D to lymphocyte-deficient NOD.SCID recipients was potentiated when CD27- γδ T cells were transferred, compared to transfer of αβ T cells alone. Antibody-mediated blockade of IL-17 prevented T1D transfer in this setting. Moreover, introgression of genetic Tcrd deficiency onto the NOD background provided robust T1D protection. Finally, we report novel relationships between the gut microbiome, host sex hormones and metabolism, and T1D pathogenesis in the NOD mouse. Using germ-free, specific pathogen free, and microbiome-transplanted NOD mice, we show that colonization by commensal microbes elevated serum testosterone and protected NOD males from T1D. Transfer of gut microbiota from adult males to immature females altered the recipients’ microbiota, resulting in elevated testosterone and metabolomic changes, reduced islet inflammation and autoantibody production, and endowed robust T1D protection. Collectively, the data presented in this thesis describe a novel genetic lesion involved in T1D risk and its immunological consequences, demonstrate a potent role for IL-17-producing γδ T cells in NOD mouse model, and uncover a novel relationship between the gut microbiome, host hormonal and metabolic phenotypes, and autoimmunity risk.

This project aims to integrate knowledge of genetic susceptibility, immune cell function, and environmental modifiers in determining risk for type 1 diabetes (T1D) in the non-obese diabetic (NOD) mouse model. Similar to human T1D, disease risk in the NOD mouse is polygenic and has been mapped to multiple Idd loci. We have fine-mapped the Idd4.1 locus and identified Nlrp1b as its candidate gene. We report an alternatively spliced isoform in the diabetes-resistant Nrlp1b allele, resulting in a truncated NLRP1b protein that is unable to activate release of IL-1β. In another aspect of this project, we have characterized the critical contribution to T1D pathogenesis by γδ T cells. We report that CD27- γδT cells infiltrate islets of pre-diabetic NOD mice. Adoptive transfer of T1D to lymphocyte-deficient NOD.SCID recipients was potentiated when CD27- γδ T cells were transferred, compared to transfer of αβ T cells alone. Antibody-mediated blockade of IL-17 prevented T1D transfer in this setting. Moreover, introgression of genetic Tcrd deficiency onto the NOD background provided robust T1D protection. Finally, we report novel relationships between the gut microbiome, host sex hormones and metabolism, and T1D pathogenesis in the NOD mouse. Using germ-free, specific pathogen free, and microbiome-transplanted NOD mice, we show that colonization by commensal microbes elevated serum testosterone and protected NOD males from T1D. Transfer of gut microbiota from adult males to immature females altered the recipients’ microbiota, resulting in elevated testosterone and metabolomic changes, reduced islet inflammation and autoantibody production, and endowed robust T1D protection. Collectively, the data presented in this thesis describe a novel genetic lesion involved in T1D risk and its immunological consequences, demonstrate a potent role for IL-17-producing γδ T cells in NOD mouse model, and uncover a novel relationship between the gut microbiome, host hormonal and metabolic phenotypes, and autoimmunity risk.

No description available.

Type 1 Diabetes (T1D) is an autoimmune disease where the immune system destroys the insulin-producing beta cells within the pancreas. Due to the difficulty of obtaining relevant tissue samples from patients at risk of disease, many researchers have utilized the nonobese diabetic (NOD) mouse as a model of T1D due to their natural high susceptibility for this disease which shares many characteristics with human patients. This model has been critical for uncovering many mechanisms involved in the pathogenesis of T1D including the key roles played by autoreactive T cells in the destruction of beta cells. More recently, NOD mice have shown that self-reactive B cells act as important antigen presenting cells for activating and amplifying the T cell response against beta cells. In order to identify faulty self-tolerance mechanisms causing production and activation of B cells recognizing beta cell proteins, we have developed a transgenic mouse model whereby elevated numbers of B cells are made specific for a neo-self antigen whose expression is restricted to beta cells on the T1D-prone NOD genetic background and compared it to that of transgenic mice of the non-autoimmune prone C57BL/6 (B6) genetic background. These studies revealed that NOD and B6 B cells can both be effectively tolerized to the model beta cell-restricted antigen. However, provision of help from activated T cells readily overturned this tolerance on the NOD but not the B6 background. Prior evidence has associated Idd5 (chromosome 1) and Idd9/11 (chromosome 4) diabetes susceptibility loci in NOD mice with the development of self reactive B cells contributing to T1D. The gene encoding CTLA-4 has been identified as the major candidate susceptibility gene within Idd5, thus leading to our studies comparing B cell expression of this molecule in NOD and diabetes-resistant strains. Although almost always associated with down-modulating T cells responses, our studies and that of others confirm expression of CTLA-4 by activated B cells. We encountered B cell expression of CTLA-4 to vary from that of T cells, being expressed earlier and predominantly on the cell surface rather than within intracellular vesicles. Our studies also showed aberrant expression of different splice variants of CTLA-4 by NOD B cells compared to diabetes-resistant mice controlled by genes within and outside the Idd5 genetic locus. Hence, these studies raise the possibility that CTLA-4 may contribute to T1D through its actions on both T and B cells. Given the large nature of the Idd9/11 susceptibility locus in NOD mice and the absence of any strong candidate genes that may influence the diabetogenic capacity of B cells in this strain, we resorted to microarray technology to reveal putative genes within this genomic region with the potential to control the B cell phenotype. We focused our microarray studies on the first transitional (T1) B cell population in the spleen given that it is an important stage of tolerance to peripherally expressed self-antigens which have been found to possess various defects in NOD mice. Comparing gene expression profiles of NOD T1 B cells that expressed susceptibility or resistance alleles at the Idd9/11 locus identified 20 differentially expressed genes with the potential to contribute to development of diabetogenic B cells. Overall, data presented in this thesis provides a greater understanding of the molecular and cellular mechanisms underlying B cell contribution to T1D in NOD mice. These data are hoped to eventually lead to the development of selective strategies for removing or inhibiting only those B cells that contribute to development of T1D while ensuring that humoral immunity to foreign pathogens remains intact in human patients at risk of developing disease.

Research Doctorate - Doctor of Philosophy (PhD) / Background: Autoimmune diseases arise from the breakdown of central tolerance resulting in the escape of self reactive T-lymphocytes from the thymus to the periphery. As a group of conditions, autoimmune diseases occur in approximately 5% of the general population and represent the third most common cause of morbidity, placing considerable expenses on the health care system and society. Understanding the underlying pathogenesis and pathophysiology of these diseases is therefore important for the correct diagnosis and treatment of these patients. While some autoimmune diseases have been paid particular attention, little is known about the pathogenesis of the pituitary autoantibodies. Aims: To identify target autoantigens in the pituitary autoimmune disease lymphocytic hypophysitis and autoantigen(s) relating to pituitary manifestations in APS1 patients. Methods: A pituitary cDNA expression library was immunocreened with lymphocytic hypophysitis and APS1 patient sera to identify target autoantigens. These were then tested in an ITT assay for autoantigen specificity to relating to the disorders. Immunofluorescence of pituitary tissue was performed to determine the cell types targeted in the disorders. Results: Two APS1 autoantigens were identified, a major autoantigen ECE-2 and a minor autoantigen TSGA10, although neither apparently correlated to pituitary manifestations in APS1. T-box 19 was also identified as a significant minor autoantigen in 10.5% of lymphocytic hypophysitis patients. Immunoreactivity in a single lymphocytic hypophysitis patient against cells of the intermediate lobe of the guinea pig pituitary is also reported. Discussion: Immunoscreening a target organ cDNA expression library is a valuable method for identifying novel autoantigens, with immunopreciptation assay a quick and reliable method for analysing a large cohort of patients for autoantibodies. We have identified another two APS1 autoantigens and the first significant autoantigen in lymphocytic hypophysitis. While further characterisation of these autoantigens are required, these novel findings broaden our current understanding of pituitary autoimmunity.

Thesis (M. D.)--University of Hong Kong, 2007. / Also available in print.

Diss. Linköping : Linköpings universitet, 2006.

Thesis (Ph. D.)--University of Virginia, 1998. / Includes bibliographical references (130-147). Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 96-139) Also available in print.

Thesis (Ph. D.)--University of Chicago, Dept. of Pathology. / Includes bibliographical references. Also available on the Internet.