Directing Function at the Natural Killer Cell Secretory Immunological Synapse

描述（由申请人提供）：自然杀伤（NK）免疫细胞对于人类抵御感染和恶性肿瘤至关重要。NK细胞以介导细胞毒性为基线，调查其环境，精确地将其细胞溶解作用靶向单个癌细胞或病毒感染的细胞。通过被称为“定向分泌”的严格调节的逐步过程，NK细胞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）。提出的工作解决了以下三个关键挑战，以访问和控制NK细胞细胞毒性的定向分泌：1）组织裂解颗粒集中和有效的交付; 2）协调皮质细胞骨架，以允许裂解颗粒出口;和3）促进裂解颗粒的运输到突触膜。首先，溶解颗粒在极化至NK细胞IS之前会聚至微管组织中心（MTOC）。拟议的工作将确定驱动溶解颗粒收敛的信号和细胞输入，并具体确定收敛是否使NK细胞更有效的杀手，不太可能杀死“无辜的旁观者”，以及更有能力介导连环杀人。其次，目前的范例表明，溶解颗粒出口发生通过一个单一的大清除肌动蛋白皮质。然而，高分辨率成像模式表明，溶解颗粒利用更小的，专门产生的低密度管道中的丝状（F）-肌动蛋白网络。将高度定量成像与新型脱粒指示剂和创新的人类NK细胞操作技术相结合，拟议的工作将进一步定义这些脱粒管道的性质和要求。最后，最近确定的NK细胞裂解颗粒为基础的电机复合体所需的裂解颗粒的最后办法的突触膜。使用裂解颗粒和NK细胞皮质的活细胞成像和过继转移型实验将定义裂解颗粒相关肌球蛋白IIA使颗粒能够找到、移动通过和/或形成F-肌动蛋白管道的机制。将使用免疫学和生物化学测定的组合来测试肌球蛋白-IIA尾和肌球蛋白-IIA磷酸化在这些功能中的特定作用。在没有严格控制定向分泌的情况下，NK细胞将失去效率并不加选择地杀死邻近组织和健康细胞，对人类宿主构成危险。拟议的工作将重新定义如何控制细胞毒性的基本范式，定义NK细胞功能中的新细胞检查点，并为管理免疫力的基本细胞生物学过程提供新的见解。更好地理解控制细胞毒性精确逐步进展的免疫调节机制将使人类疾病背景下的NK细胞功能的治疗操作成为可能。

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.