Signals That Control Thymocyte Migration

描述(申请人提供)：双光子显微镜与基因编码的荧光蛋白(例如GFP)的使用一起，使直接观察三维活组织中的细胞行为成为可能。我们一直使用这种方法来研究胸腺细胞的迁移和与胸腺基质细胞的相互作用，以及这些事件如何与T细胞抗原受体(TCR)谱系的阳性和阴性选择有关。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，同时与禁闭区周围的树突状细胞进行重复的、短暂的接触。在目标1中，我们计划1)确定自身反应性髓质胸腺细胞的限制区的解剖学特征，2)检验由于负选择的结果，很大比例的多克隆髓质胸腺细胞正在进行受限迁移的假设，3)探索AIRE在建立负选择解剖学生态位中的作用，4)确定负选择配体的丰度如何影响负选择期间自身反应性胸腺细胞的行为。在目标2中，我们计划通过1)在不同的胸腺细胞选择模式中检测胸腺细胞迁移和TCR信号之间的平衡，2)检测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，以及3)研究缺乏适配蛋白GIT-2的胸腺细胞中趋化因子反应的改变和正选择受损的关系，来研究迁移、趋化因子和TCR谱系选择之间的关系。通过提供对产生功能和自我耐受的成熟T细胞的机制的洞察，这些研究可能导致改进对自身免疫、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.