Thymocytes require stromal-derived signals, incluiding activation of chemokine receptors EBI2 and GRP146, for proper differentiation and selection



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The thymus is the primary lymphoid organ where immature thymocytes differentiate and mature to become functional T cells that are self-MHC restricted and self-tolerant. In addition to developing T cells, the thymus is comprised of stromal cells, subdivided into cortical and medullary regions, which form a component of the microenvironment for thymocyte development. Throughout their development, thymocytes undergo bidirectional crosstalk with the thymic epithelial cells, which is essential for proper development of both the thymocyte and epithelial subsets. Thymic involution is a detrimental process of aging which results in degeneration of the thymic microenvironment, reduction in T cell output, and contraction of the naïve T cell pool, thus contributing to impaired immune responses in aging individuals. Molecular and cellular changes that drive thymic atrophy during aging are not well understood. To address this gap in knowledge, I analyzed transcriptional changes in purified thymic stromal subsets early in the process of age-associated thymic involution (Chapter 2). These studies revealed that there is gradual down-regulation of cell cycle genes and E2F3 transcriptional targets in thymic epithelial cell subsets as the thymus begins to involute. This suggests that diminished proliferative activity in a subset of thymic epithelial cells is a major contributing factor to age-associated involution. I also identified an increasingly proinflmamatory signature in thymic dendritic cells early in the involution process. These results have provided novel insights into the mechanisms behind thymic atrophy and identify cellular targets for thymic rebound strategies. Chemokine receptors, a subset of G protein coupled receptors (GPCRs), have been implicated in guiding thymocytes between different thymic regions, thus enabling interactions with local stromal subsets that support T cell differentiation. For example, chemokine receptors play an essential role in driving thymocyte migration into the medulla, where thymocytes scan numerous self-antigens to establish central tolerance. Importantly, failure of thymocytes to enter the thymic medulla results in impaired self-tolerance, leading to autoimmunity. We have identified EBI2 and GPR146 as candidate GPCRs that could contribute to thymocyte accumulation in the medulla, interactions with antigen presenting cells therein, and thus the induction of tolerance. In chapter 3, I report that EBI2 is essential for negative selection toward some self-antigens and restrains regulatory T cell generation in the thymus. Through two-photon imaging, I identified reduced motility and medullary accumulation as mechanisms by which EBI2 deficiency could impair negative selection. In chapter 4, I describe generation of a GPR146 deficient mouse strain, and initial studies indicating GPR146 is involved in thymocyte differentiation and selection. Through these studies we have revealed novel functions for GPCRs in promoting thymocyte migration and the induction of self-tolerance.