In this project we propose to determine the role of candidate G protein-coupled receptors (GPCRs) in enforcing deletion of developing auto-reactive T cells in the thymus. In order for T cells to recognize and respond to the wide array of pathogens encountered throughout life, each cell expresses a unique antigen receptor, the T cell receptor (TCR), that is capable of recognizing and responding to individual pathogens. These vastly diverse receptors are generated by random and imprecise rearrangements of gene segments encoding the TCR. Unfortunately, this random recombination process also yields autoreactive TCRs that could induce autoimmune diseases. To prevent overt autoimmunity, developing T cells are educated in the thymus through interactions with thymic stromal cells: thymocytes expressing overtly autoreactive TCRs are induced to undergo apoptosis. This process of thymic self-tolerance induction is referred to as central tolerance. Central tolerance i imposed largely within the interior, medullary region of the thymus, where thymocytes encounter a wide array of self-antigens expressed on stromal cells, namely dendritic cells and/or medullary thymic epithelial cells. It is critical that developing thymocytes enter the medulla and interact with stromal cells therein to eliminate autoreactive cells. From studies of Autoimmune Polyglandular Syndrome-I patients along with mouse models of this disorder, we know that if central tolerance induction in the medulla is impaired, multi-organ autoimmunity ensues. Based on our previous studies, GPCR signaling is required for thymocyte medullary entry. In this proposal, the role of candidate GPCRs in promoting medullary entry and self-tolerance will be evaluated. Using 2-photon microscopy, live thymocytes deficient for candidate GPCRs will be imaged to determine whether their ability to enter the medulla or to interact with medullary stromal cells is impaired. In a complementary set of traditional immunological approaches, murine models deficient for candidate GPCRs will be tested for overt autoimmunity, as well as for an inability to induce central tolerance to model medullary self- antigens. Finally, unbiased gain-of-function screens will identify additional molecular candidates that promote thymocyte medullary entry and self-tolerance. These studies will illuminate molecular mechanisms that promote thymocyte migration into the medulla and interactions with medullary stromal cells to promote central tolerance, thus broadening our understanding of the etiology of autoimmune diseases.