The contribution of GPCRs to thymocyte medullary entry and central tolerance

描述（由申请人提供）：在本项目中，我们建议确定候选G蛋白偶联受体（GPCR）在强制删除胸腺中发育中的自身反应性T细胞中的作用。为了使T细胞识别和响应在整个生命过程中遇到的各种病原体，每个细胞表达独特的抗原受体，T细胞受体（TCR），能够识别和响应单个病原体。这些差异极大的受体是由编码TCR的基因片段的随机和不精确重排产生的。不幸的是，这种随机重组过程也会产生可诱导自身免疫性疾病的自身反应性TCR。为了防止明显的自身免疫，发育中的T细胞通过与胸腺基质细胞的相互作用在胸腺中接受训练：诱导表达明显的自身反应性TCR的胸腺细胞进行细胞凋亡。胸腺自身耐受诱导的这一过程被称为中枢耐受。中枢耐受性主要施加在胸腺的内部、髓质区域内，其中胸腺细胞遇到在基质细胞（即树突状细胞和/或胸腺髓质上皮细胞）上表达的广泛的自身抗原。发育中的胸腺细胞进入髓质并与其中的基质细胞相互作用以消除自身反应细胞是至关重要的。从自身免疫性多腺综合征-I患者沿着这种疾病的小鼠模型的研究中，我们知道，如果髓质中的中枢耐受诱导受损，则多器官自身免疫增强。根据我们以前的研究，GPCR信号是胸腺细胞进入髓质所必需的。在本提案中，将评价候选GPCR在促进骨髓进入和自身耐受性方面的作用。使用双光子显微镜，将对缺乏候选GPCR的活胸腺细胞进行成像，以确定它们进入髓质或与髓质基质细胞相互作用的能力是否受损。在一组互补的传统免疫学方法中，将测试候选GPCR缺陷的鼠模型的明显自身免疫性，以及不能诱导对模型髓质自身抗原的中枢耐受性。最后，无偏见的功能获得筛选将确定额外的分子候选人，促进胸腺细胞髓质进入和自我耐受。这些研究将阐明促进胸腺细胞向髓质迁移的分子机制，以及与髓质基质细胞相互作用以促进中枢耐受，从而拓宽我们对自身免疫性疾病病因的理解。

DESCRIPTION (provided by applicant): 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.