Functional studies of candidate genes contributing to type 1 diabetes in the NOD mouse

Type 1 Diabetes (T1D) is an autoimmune disorder caused by both genetic and environmental factors. The non-obese diabetic (NOD) mouse is one of the best and most commonly studied animal models for T1D. This mouse strain spontaneously develops diabetes through a process that closely resembles the human pathogenesis. More than 20 insulin dependent susceptibility (Idd) loci have been identified in the NOD mouse, contributing to disease susceptibility; however, the contribution of each of the various factors to disease pathogenesis is largely unknown. The aim of this thesis was to identify and functionally characterize candidate genes mediating susceptibility to murine T1D. Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a negative regulator of T-cell activation and has been shown to be associated with autoimmune diseases. Genetic analyses of the NOD mouse have identified the Ctla-4 gene as a major candidate for the Idd5.1 diabetes susceptibility locus and NOD mice have been found to display an impaired expression of CTLA-4 upon anti-CD3 stimulation in vitro. In Paper I, we showed that a novel locus (Ctex) in the distal part of the chromosome 1 together with the Idd3 (Il-2) locus on chromosome 3, constitute the major factors conferring the observed difference in CTLA-4 expression levels. Moreover, we also demonstrated that the defective expression of CTLA-4 in NOD T-cells can in part be overcome by the addition of exogenous interleukin-2 (IL-2). In Paper II, using congenic mice, we confirmed that the Ctex locus contributes to decreased expression of CTLA-4 observed in NOD mice and restricted the region of interest to a 28.8 Mb region containing the Cd3ζ gene. We also demonstrated a phenotypic correlation between strains carrying the NOD versus C57BL/6 alleles of Cd3ζ, respectively and showed that expression of CD3ζ is impaired in activated NOD CD4+ T cells. The NOD allele of the Cd3ζ region was found to confer impaired T cell activation and the defective CD3 signalling could be surpassed by PMA plus ionomycin stimulation supporting the notion of CD3ζ as a prime candidate gene for Ctex. NOD lymphocytes display relative resistance to various apoptosis-inducing signals, which have been proposed to contribute to the pathogenesis of diabetes. Resistance to dexamethasone-induced apoptosis in NOD immature thymocytes has been mapped to the Idd6 locus. In Paper III we restricted the Idd6 locus to an 8 cM region on the telomeric end of chromosome 6 using a set of congenic mice. In addition, we could confirm that the Idd6 region controls apoptosis resistance in immature thymocytes and restricted the control of apoptosis resistance to a 3 cM region within the Idd6 locus. In Paper IV, we further restricted the Idd6 locus to a 3 Mb region and excluded the region controlling the resistance to apoptosis as directly mediating susceptibility to diabetes. We also showed that defective expression of the Lrmp/Jaw1 gene, encoding an endoplasmatic reticulum resident protein, is controlled by the Idd6 locus making it the prime candidate for Idd6. Together, these results contribute to the identification and functional characterization of candidate genes that may confer susceptibility to T1D in the NOD mouse. These results offer important insights into the pathophysiological processes underlying this disease