Mechanistic analysis of T cell MTOC reorientation with a photoactivatable antigen

描述（由申请人提供）：T细胞反应是由T细胞受体（TCR）通过识别抗原呈递细胞（APC）表面显示的外来抗原而激活，并导致某些细胞因子或细胞溶解因子向APC定向分泌。细胞溶解因子导致被感染的靶细胞死亡，细胞因子刺激参与免疫反应的其他细胞。定向分泌使T细胞能够特异性地与靶细胞交流，对T细胞的有效功能至关重要。这在Griscelli综合征和Chediak-Higashi综合征等疾病中得到了清楚的证明，在这些疾病中，T细胞定向分泌受损导致免疫缺陷。此外，错误的分泌可能导致未感染细胞的死亡或免疫反应的不适当激活。定向分泌需要T细胞微管组织中心（MTOC）重新定位到与靶细胞接触的免疫突触（IS）。我们对MTOC重定向的理解受到直接观察过程的困难的限制。MTOC重新定位发生在TCR激活后的几分钟内，然而大多数关于这一过程的研究都是基于在设定的时间点对T细胞和apc之间的固定偶联物进行染色。这种方法不能提供对这种快速过程进行机理研究所需的时间精度。这个项目的目标是更清楚地定义负责T细胞MTOC重新定向的机制，使用光激活抗原，直到暴露在短脉冲紫外线下才失去活性。该方法能够对T细胞活化进行精确的时空控制，并将应用于单细胞活成像实验中，同时观察各种荧光标记蛋白的募集行为和MTOC重定向。它还将允许观察RNAi敲除或候选蛋白质的化学抑制如何影响活T细胞中MTOC的重新定向和其他蛋白质的招募行为。最后，定向细胞因子分泌测定将用于确定MTOC重定向缺陷是如何由敲低或抑制相关蛋白引起的，从而影响T细胞功能。公共卫生相关性：T细胞在抗原特异性免疫反应中起核心作用。定向地向靶细胞分泌某些细胞因子或细胞溶解因子的能力对于有效的T细胞功能至关重要，因为它可以实现与靶细胞的特定通信。定向分泌需要微管组织中心（MTOC）重新定位到与靶细胞的接触部位；然而，人们对这一过程知之甚少。我将研究MTOC重新定向的机制，使用光激活抗原，提供对T细胞激活的精确控制，使分析具有高空间和时间分辨率。

DESCRIPTION (provided by applicant): A T cell response is initiated by T cell receptor (TCR) activation through recognition of foreign antigen displayed on the surface of an antigen presenting cell (APC) and results in the directional secretion of certain cytokines or cytolytic factors towards the APC. Cytolytic factors cause death of an infected target cell, and cytokines stimulate other cells involved in the immune response. Directional secretion enables a T cell to specifically communicate with a target cell and is critical for effective T cell function. This is clearly demonstrated in diseases such as Griscelli syndrome and Chediak-Higashi syndrome where impaired directional secretion by T cells results in immunodeficiency. In addition, misdirected secretion could result in the death of uninfected cells or inappropriate activation of immune responses. Directional secretion requires reorientation of the T cell microtubule organizing center (MTOC) to the immune synapse (IS), the contact site with the target cell. Our understanding of MTOC reorientation has been limited by the difficulty of directiy observing the process. MTOC reorientation occurs within minutes of TCR activation, however most studies of this process are based on staining of fixed conjugates between T cells and APCs at set time points. This approach does not provide the temporal precision necessary for mechanistic study of such a rapid process. The goal of this project is to more clearly define the mechanism responsible for T cell MTOC reorientation using a photoactivatable antigen that is inactive until exposure to a short pulse of UV light. This method enables precise spatial and temporal control over T cell activation, and will be applied in single cell live imaging experiments to simultaneously observe recruitment behaviors of various fiuorescently labeled proteins and MTOC reorientation. It will also allow observation of how RNAi knockdown or chemical inhibition of a candidate protein affects MTOC reorientation and recruitment behaviors of other proteins in live T cells. Finally, a directional cytokine secretion assay will be used to determine how MTOC reorientation defects caused by knockdown or inhibition of proteins involved affects T cell function. PUBLIC HEATLH RELAVENCE: T cells play a central role in the antigen specific immune response. The ability to directionally secrete certain cytokines or cytolytic factors towards a target cell is critical for effective T cell function as it enables specific communication with a target cell. Directional secretion requires reorientation of the microtubule organizing center (MTOC) to the contact site with the target cell; however this process is poorly understood. I will investigate the mechanism of MTOC reorientation using a photo activatable antigen that provides precise control over T cell activation enables analysis with high spatial and temporal resolution.