Autoantibodies to the thyrotropin receptor can either activate thyroid gland causing hyperthyroidism or block TSH mediated activation of thyroid and cause hypothyroidism. Until several years ago, it was not possible to develop an animal mode due to the unavailability of large quantities of purified TSHR. Subsequent to cloning of human TSHR, several laboratories, including our own, have used human recombinant proteins to induce the disease in mice. These studies have provided new insights on the requirements for an optimal immune response to TSHR, resulting in thyroid perturbation. Earlier, we expressed the ectodomain of mouse TSHR (mTSHR) and showed that it is antigenically distinct from human TSHR. Recently, we expressed mTSHR on M12 cells (H-2D) and used them to immunize BALB/c mice. These mice showed significant TBII activity with concomitant raise in T4 levels. In the present study, we propose to use a soluble ectodomain of mTSHR and various cell lines expressing mTSHR, Class-II and Co-stimulatory molecules to define optimal conditions required to induce autoimmunity to TSHR. Sera will be tested for antibody production and hormonal perturbations, and thyroids will be evaluated for pathology and radioiodine uptake. We will carryout studies to evaluate the importance of CD4+ vs. CD8+ and Th1 vs. Th2 T cells. To do this, we will use selective depletion and adoptive transfer experiments, determine the relevance of cytokines, and test the ability of the protein to induce disease in Class-I and II, IFNgamma, and IL4 knockout mice available on BALB/c background. To define TSHR epitopes to which pathogenic antibodies bind, we will carryout epitope mapping studies. For this, we will employ recombinant fragments of TSHR, ectodomains of TSHR-LH/CGR chimeric proteins and cells expressing these chimeras. These proteins or their fragments will be tested in a number of different serological and bioassays. Together these studies are expected to allow establishment of an appropriate animal model to study autoimmunity to TSHR. Such a model would facilitate a thorough understanding of the regulation of the immune response to TSHR with implications for the development of novel therapeutics.
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