T cell responses and NK cell function in experimental autoimmune diabetes

Abstract

The immune system is regulated by several complex mechanisms to maintain self tolerance and prevent autoimmune diseases. However, genetic as well as environmental factors can increase the risk of breaking self tolerance. Autoimmune diabetes is a polygenic disorder characterized by the selective destruction of pancreatic beta cells. Self-reactive T cells mediate the disease, but other cells including B cells, macrophages, dendritic cells (DC) , natural killer (NK) cells, NKT cells and regulatory T cells have been suggested to influence disease development.

The aim of this thesis has been to characterize the regulation of T cell responses and the function of NK cells in autoimmune diabetes. CD8+ T cells are major producers of IFN-gamma, a key cytokine in autoimmune diabetes. The regulation of IFN-gamma production by CD8+ T cells was studied in relation to stimulatory strength. IFN-gamma secretion in stimulated naïve CD8+ T cells varied inversely with proliferation. Low concentrations of antigenic peptide and low levels of costimulation resulted in weak T cell proliferation, but substantial IFN-gamma production. Our unpublished data showed that CD8+ T cells from NOD mice (that spontaneously develop diabetes) are predisposed to increased IFNgamma responses, suggesting a possible role for IFN-gamma produced by self-reactive CD8+ T cells in the initiation phase of autoimmune diabetes.

We also evaluated the role of the signaling adapter molecule DAP12 in a CD4+ T cell transgenic diabetes mouse model. Previously, it was shown that DAP 12 knock out mice were protected from experimental autoimmune encephalitis (EAE). Protection from EAE was associated with diminished priming of autoreactive CD4+ T cells in the mutant mice. We showed that DAP12 had an adverse effect in our model of autoimmune diabetes. In the absence of a functional DAP12 molecule, both diabetes and the activation of autoreactive CD4+ T cells were increased. This increased disease pathogenicity was accompanied by reduced suppression by antigen-specific CD4+CD25+ regulatory T cells. DAP 12 mutated mice had a different composition of DC, with an increased frequency of plasmacytoid DC (pDC), suggesting an interesting link between pDC and activation of regulatory T cells.

NK cells may be involved in autoimmmunity and have been suggested to serve as regulatory cells in T cell mediated autoimmune diseases. A comprehensive analysis of NK cell function in NOD mice was made. NK cells in NOD mice could be identified by the combination of two monoclonal antibodies, TMbeta-1 (recognizing the beta-chain of the IL-2 receptor) and DX5 (recognizing CD49b integrin). Using these antibodies we demonstrated that there were no abnormalities in the size of NK cell populations in NOD mice compared to non-diabetogenic strains. However, NOD NK cell functions were broadly impaired due to defects in multiple activation pathways. This NK cell deficiency could be partially restored by NK cell activation in vivo, and after IL-12 and IL-18 stimulation in vitro.

Furthermore, treating NOD mice with the TMbeta-1 antibody prevented disease. We confirmed, by flow cytometry, that all NK cells were depleted by this treatment. NK cell depletion was also confirmed by the complete lack of NK killing ability in antibody treated mice. A fraction of NKT cells was also depleted. In addition, a small population of CD8+ T cells, some of which co-expressed NKG2D, disappeared after treatment. TMbeta-1 treated mice could mount a normal primary CD8+ T cell response, but it is still possible that this small population of CD8+ T cells is important for diabetes development.

A particularly interesting finding in this study was that disease was blocked when the antibody was administered late in disease pathogenesis. Even when given to prediabetic mice at 15 weeks of age, most mice were protected from diabetes, suggesting that the TMbeta-1 antibody targets effector cell/s responsible for a late, perhaps direct beta cell cytotoxic, effect in disease. This finding opens up the possibility for future development of new late-acting drugs against this disease.