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）：自然杀手（NK）免疫细胞对于针对感染和恶性肿瘤的人类防御至关重要。 NK细胞在基线以介导细胞毒性的基线以调查其环境 或病毒感染的细胞。通过被称为“定向分泌”的严格调节的踏板过程，NK细胞动员了高度专业的大型细胞器（裂解颗粒） 细胞界面，称为免疫突触（IS）。拟议的工作解决了以下三个关键挑战，以访问和控制NK细胞毒性的定向分泌： 1）组织裂解颗粒以进行聚焦和有效的递送； 2）编排皮质细胞骨架以允许裂解颗粒出口； 3）促进裂解颗粒向突触膜的转运。首先，裂解颗粒在偏振到NK细胞之前会收敛到微管组织中心（MTOC）。所提出的工作将确定驱动裂解颗粒收敛的信号和细胞输入和机制，并明确确定收敛是否使NK细胞更有效地杀死杀手，杀死“无辜旁观者”的可能性较小，并且更有能力介导连续杀伤。其次，当前的范式表明，裂解颗粒出口是通过肌动蛋白皮质中的单个大间隙发生的。但是，高分辨率成像方式表明，裂解颗粒利用丝状（F） - 肌动蛋白网络中特定生成的低调导管利用了裂解颗粒。提出的工作将高度定量的成像与新颖的脱粒指标和创新的人NK细胞操纵技术相结合，将进一步定义这些脱粒的吊导的性质和需求。最后，最近确定的NK细胞裂解颗粒的运动复合物是裂解颗粒对突触膜的最终方法所必需的。使用裂解颗粒和NK细胞皮质的实活细胞成像和过继的转移式实验将定义裂解颗粒相关的肌球蛋白-IIA使颗粒能够找到，通过F-肌动蛋白的抑制剂的机制。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通过免疫学和生化测定的组合进行测试。在没有严格控制定向分泌的情况下，NK细胞会失去效率并无与伦比杀死邻近的组织和健康细胞，从而对人类宿主构成危险。提出的工作将重新定义有关如何控制细胞毒性，定义NK细胞功能中新的细胞检查点的基本范式，并提供有关控制免疫力的基本细胞生物学过程的新见解。更好地了解控制精确的逐步发展到细胞毒性的免疫调节机制将使在人类疾病的背景下对NK细胞功能进行治疗操纵。