Directing Function at the Natural Killer Cell Secretory Immunological Synapse

自然杀伤细胞分泌免疫突触的指导功能

• 批准号: 8448575
• 负责人: Jordan Scott Orange
• 金额: $ 33.27万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8448575
• 关键词: Actins; Address; Adoptive Transfer; Area; Automobile Driving; Biochemical; Biological; Biological Assay; Biological Process; Caliber; Cancerous; Cell Degranulation; Cell membrane; Cell physiology; Cells; Clinical; Complex; Cytolysis; Cytoplasmic Granules; Cytoskeleton; Data; Dynein ATPase; Ensure; Environment; F-Actin; Human; Image; Imaging Techniques; Immune; Immunity; Individual; Infection; Lateral; Lead; Life; Lytic; Malignant Neoplasms; Mediating; Membrane; Microscopy; Microtubule-Organizing Center; Microtubules; Motor; Myosin ATPase; Myosin Type II; Natural Immunity; Natural Killer Cells; Nature; Nonmuscle Myosin Type IIA; Organelles; Penetration; Phosphorylation; Process; Resolution; Rest; Role; Signal Transduction; Surveys; Synapses; Synaptic Membranes; Tail; Techniques; Testing; Therapeutic; Tissues; Virus; Work; arm; base; cell cortex; cell killing; cell motility; cellular imaging; coronin protein; cytotoxicity; density; human disease; imaging modality; immunological synapse; innate immune function; innovation; insight; killings; novel; programs; research study

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 and mechanisms 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.

描述（由申请人提供）：自然杀伤（NK）免疫细胞对于人类防御感染和恶性肿瘤至关重要。 NK 细胞在基线时武装起来介导细胞毒性，它们会调查其环境，精确地将其细胞溶解作用瞄准个体癌变细胞 或病毒感染的细胞。通过称为“定向分泌”的严格调控的逐步过程，NK 细胞将高度特化的大型细胞器（裂解颗粒）动员至 NK/靶标 细胞界面，称为免疫突触（IS）。拟议的工作解决了获取和控制 NK 细胞毒性定向分泌的以下三个关键挑战： 1) 组织溶解颗粒以集中、高效地输送； 2) 协调皮质细胞骨架以允许裂解颗粒排出； 3) 促进溶解颗粒转运至突触膜。首先，裂解颗粒在极化至 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裂解颗粒收敛的信号和细胞输入以及机制，并具体确定收敛是否使 NK 细胞成为更有效的杀手，不太可能杀死“无辜的旁观者”，并且更有能力介导连环杀戮。其次，当前的范例表明，溶解颗粒的流出是通过肌动蛋白皮层中的单个大间隙发生的。然而，高分辨率成像模式表明，裂解颗粒利用丝状 (F)-肌动蛋白网络中较小的、专门生成的低密度导管。将高度定量成像与新型脱颗粒指示器和创新的人类 NK 细胞操作技术相结合，拟议的工作将进一步定义这些脱颗粒导管的性质和要求。最后，最近发现的基于 NK 细胞溶解颗粒的运动复合体是溶解颗粒最终到达突触膜所必需的。使用裂解颗粒和 NK 细胞皮层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、移动和/或形成 F-肌动蛋白导管的机制。肌球蛋白-IIA 尾部和肌球蛋白-IIA 磷酸化在这些功能中的具体作用将使用免疫学和生化测定的组合进行测试。在缺乏严格控制定向分泌的情况下，NK细胞会失去效率并无差别地杀死邻近组织和健康细胞，对人类宿主造成危险。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，并为控制免疫的基本细胞生物过程提供新的见解。更好地了解控制细胞毒性的精确逐步进展的免疫调节机制将使在人类疾病背景下对 NK 细胞功能进行治疗性操作成为可能。