Signals That Control Thymocyte Migration

描述（由申请人提供）：双光子显微镜与遗传编码的荧光蛋白（例如GFP）一起使用，使得直接可视化三维活组织中的细胞行为成为可能。我们一直在使用这种方法来研究胸腺细胞迁移和相互作用与胸腺基质细胞，以及这些事件如何涉及到积极和消极的选择T细胞抗原受体（TCR）的剧目。在之前的资助期间，我们意外地观察到许多经历负选择的胸腺细胞不会迅速停滞和死亡，而是采用高度受限的迁移模式，同时与限制区周围的树突状细胞进行重复的短暂接触。在目的1中，我们计划1）确定自身反应性髓质胸腺细胞限制区的解剖学特征，2）测试大比例的多克隆髓质胸腺细胞由于阴性选择而进行限制迁移的假设，3）探索AIRE在建立阴性选择的解剖学小生境中的作用，4）确定阴性选择配体的丰度如何影响阴性选择期间自身反应性胸腺细胞的行为。在目的2中，我们计划通过以下方式研究迁移、趋化因子和TCR库选择之间的关系：1）检查在不同模式的胸腺细胞选择期间胸腺细胞迁移和TCR信号传导之间的平衡，2）检查调节趋化因子梯度对胸腺细胞-基质细胞相互作用和胸腺细胞选择的影响，和3）研究在缺乏衔接蛋白GIT-2的胸腺细胞中改变的趋化因子应答和受损的正选择之间的关系。通过深入了解产生功能性和自我耐受的成熟T细胞的机制，这些研究可能会改善自身免疫，免疫缺陷的治疗，并提高我们操纵对肿瘤或病原体的免疫反应的能力。

DESCRIPTION (provided by applicant): Two-photon microscopy, together with the use of genetically encoded fluorescent proteins (e.g. GFP), has made it possible to directly visualize cellular behavior in 3- dimensional living tissues. We have been using this approach to investigate thymocyte migration and interactions with thymic stromal cells, and how these events relate to positive and negative selection of the T cell antigen receptor (TCR) repertoire. In the previous funding period we made the unexpected observation that many thymocytes undergoing negative selection do not rapidly arrest and die, but rather adopt a highly confined migration pattern while make repeated, transient contacts with dendritic cells surrounding confinement zones. In Aim 1 we plan to 1) determine the anatomical features of confinement zones for auto-reactive medullary thymocytes, 2) test the hypothesis that a large proportion of polyclonal medullary thymocytes are undergoing confined migration as the result of negative selection, 3) explore the role of AIRE in establishing anatomical niches for negative selection and, 4) determine how the abundance of negative selecting ligands affects the behavior of auto-reactive thymocytes during negative selection. In Aim 2, we plan to investigate the relationship between migration, chemokines and TCR repertoire selection by 1) examining the balance between thymocyte migration and TCR signaling during different modes of thymocyte selection, 2) examining the impact of modulating chemokine gradients on thymocyte-stromal cell interactions and thymocyte selection, and 3) investigating the relationship between altered chemokine response and impaired positive selection in thymocytes lacking the adaptor protein, GIT-2. By providing insight into the mechanisms that generate mature T cells that are functional and self-tolerant, these studies may lead to improved treatments for autoimmunity, immunodeficiency, and improve our ability to manipulate immune response to tumors or pathogens.