Directing Function at the Natural Killer Cell Secretory Immunological Synapse

描述(由申请者提供)：自然杀伤(NK)免疫细胞是人体抵御感染和恶性疾病的重要细胞。NK细胞以基线武装来调节细胞毒作用，它研究它们的环境，精确地瞄准它们对单个癌细胞或病毒感染细胞的杀伤作用。NK细胞通过一个被称为“定向分泌”的严格调控的分步过程，将高度特化的大细胞器(裂解颗粒)动员到NK/靶细胞界面，称为免疫突触(IS)。这项拟议的工作解决了以下三个关键挑战：1)组织裂解颗粒以集中和有效地传递；2)协调皮质细胞骨架以允许裂解颗粒流出；3)促进裂解颗粒向突触膜的转运。首先，溶解颗粒聚集到微管组织中心(MTOC)，然后极化到NK细胞IS。这项拟议的工作将确定推动裂解颗粒融合的信号和细胞输入，并具体确定融合是否使NK细胞更有效地杀戮，不太可能杀害“无辜的旁观者”，以及更有能力调解连环杀戮。其次，目前的范例表明，溶解颗粒通过肌动蛋白皮质中的一个大间隙发生出口。然而，高分辨率成像模式表明，溶解颗粒利用丝状(F)-肌动蛋白网络中较小的、特定生成的低密度管道。结合高定量成像、新的脱颗粒指示剂和创新的人类NK细胞操作技术，拟议的工作将进一步定义这些脱颗粒管道的性质和要求。最后，最近发现的基于NK细胞裂解颗粒的运动复合体是裂解颗粒最终进入突触膜所必需的。使用裂解颗粒和NK细胞皮质的活细胞成像和过继转移式实验将确定裂解颗粒相关肌球蛋白IIA使颗粒能够找到、穿过和/或形成F-肌动蛋白管道的机制。肌球蛋白-IIA尾巴和肌球蛋白-IIA磷酸化在这些功能中的具体作用将使用免疫学和生化分析相结合的方法进行测试。在没有严格控制定向分泌的情况下，NK细胞会失去效率，不分青红皂白地杀死邻近组织和健康细胞，对人类宿主构成危险。这项拟议的工作将重新定义如何控制获得细胞毒性的基本范式，定义新的NK细胞功能的细胞检查点，并为控制免疫的基本细胞生物学过程提供新的见解。更好地了解免疫调节机制，精确地控制细胞毒的逐步进展，将使在人类疾病背景下对NK细胞功能的治疗性操作成为可能。

DESCRIPTION (provided by applicant): Natural killer (NK) immune cells are essential for human defense against infection and malignancy. Armed at baseline to mediate cytotoxicity, NK cells survey their environment, precisely targeting their cytolytic effects to individual cancerous or virus-infected cells. Through a tightly regulated step-wise process termed "directed secretion", NK cells mobilize highly specialized large organelles (lytic granules) to the NK/target cell interface, known as the immune synapse (IS). The proposed work addresses the following three critical challenges to accessing and controlling directed secretion for NK cell cytotoxicity: 1) organizing lytic granules for focused and efficient delivery; 2) orchestrating the cortical cytoskeleton to permit lytic granule egress; and 3) facilitating transit of lytic granules to the synaptic membrane. First, lytic granules converge to the microtubule organization center (MTOC) prior to polarizing to the NK cell IS. The proposed work will identify the signal and cellular inputs that drive lytic granule convergence and specifically determine whether convergence makes NK cells more efficient killers, less likely to kill "innocent bystanders", and more capable of mediating serial kills. Second, current paradigms suggest that lytic granule egress occurs through a single large clearance in the actin cortex. High-resolution imaging modalities, however, suggest that lytic granules exploit smaller, specifically generated hypodense conduits in the filamentous (F)-actin network. Combining highly quantitative imaging with a novel degranulation indicator and innovative human NK cell manipulation techniques, the proposed work will further define the nature and requirement for these conduits for degranulation. Lastly, recently identified NK cell lytic granule-based motor complexes are required for the lytic granule's final approach to the synaptic membrane. Live-cell imaging and adoptive transfer-style experiments using lytic granules and NK cell cortices will define the mechanisms by which lytic granule-associated myosin-IIA enables granules to find, move through, and/or form F-actin conduits. The specific role of the myosin-IIA tail and myosin-IIA phosphorylation in these functions will be tested using a combination of immunological and biochemical assays. In the absence of strict control of directed secretion, NK cells would lose efficiency and indiscriminately kill neighboring tissue and healthy cells, posing a danger to the human host. The proposed work will redefine fundamental paradigms as to how access to cytotoxicity is controlled, define new cellular checkpoints in NK cell function, and provide novel insights into the basic cell biological processes that govern immunity. Better understanding the immunoregulatory mechanisms controlling the precise step-wise progression to cytotoxicity will enable therapeutic manipulation of NK cell function in the context of human disease.