Immunofluorescent 3D-analysis of NK-target conjugates

描述（由申请人提供）：本提案的总体目标 目的是深入了解自然杀伤细胞（NK）的作用机制。 由MHC或非MHC分子调节，以及导致 颗粒胞吐作用评估在心脏病中的异常程度和性质 Wiskott-Aldrich综合征蛋白引起的信号转导途径 （WASP）缺陷将使我们能够定义缺陷的重要性 分子在这些途径。细胞骨架和信号传导的校正 在用野生型正常NK细胞进行逆转录病毒转导后的缺陷 WASP将确定该分子在NK信号传导中所起的作用。等 分析还可以揭示其他信号分子之间的相互作用， 它们的相互激活发生。颗粒的替代途径 细胞溶解性NK细胞中的胞吐作用可能存在， 单个信号分子可以提供这种信息。目前还不知道 当激活信号超过抑制信号时， 信号.我们将描述NKG 2D，2B 4和NKp 46激活 受体在神经母细胞瘤，特别是确定的差异， 发生在肿瘤细胞杀伤突触的分子事件与 保留肿瘤细胞的NK克隆。受体-配体相互作用的动力学和 在溶细胞NK细胞中SMAC的形成可能是快速的，并且只有 在真实的时间中进行的分子事件的时间分析， 在这些研究中提出的，将可能确定分子序列 activation. FRET分析将鉴定相互作用的蛋白质的共缔合。 分子从单纯的物理共定位。我们将解决这些问题 具体目标1： 比较MHC调控的超分子时空组织， 在与HLA I类缺陷靶相互作用期间，正常NK细胞的免疫突触中的激活簇（SMAC）与表达HLA I类缺陷靶的靶相互作用期间， 只有一个自身HLA I类分子。具体目标2： （WASP）在介导颗粒胞吐的NK细胞信号传导途径中的作用。具体 目的3：研究2B 4、NKp 46在不同时间和空间的分布特征 和NKG 2D在非MHC介导的NK细胞与SMAC的相互作用期间， 自体肿瘤靶向，神经母细胞瘤和识别招募的差异 信号分子与肿瘤杀伤免疫突触的关系 通过分析体外和体内缀合物，获得肿瘤保留NK细胞。 这样的分析将提高我们对细胞溶解的生物学的理解。 可能在免疫治疗中有潜在用途的效应淋巴细胞。

DESCRIPTION (provided by applicant): The overall objective for this proposal is to gain insight on the mechanisms by which Natural Killer (NK) cells are regulated either by MHC or non-MHC molecules and the mechanisms leading to granule exocytosis. Evaluating the extent and nature of abnormalities in the signal transduction pathways resulting from Wiskott-Aldrich Syndrome Protein (WASP) deficiency will enable us to define the essentiality of the defective molecule in these pathways. Correction of the cytoskeletal and signaling defects upon retroviral transduction of the NK cells with the wild type normal WASP will identify the role played by this molecule in NK signaling. Such analysis may also uncover the interplay between other signaling molecules and WASP for their mutual activation to occur. Alternative pathways for granule exocytosis in cytolytic NK cells are likely to exist and evaluating defects in a single signaling molecule could provide such information. It is not known what form the NK synapse takes when activating signals outweigh the inhibitory signals. We will describe the role of NKG2D, 2B4 and NKp46 activating receptors in Neuroblastoma, especially identifying the differences in molecular events occurring at the synapse of tumor-cell killing versus the tumor-cell sparing NK clone. The kinetics of receptor-ligand interactions and SMAC formation in the cytolytic NK cell are likely to be fast and only the temporal analysis of the molecular events performed in real time as are proposed in these studies, will likely identify the sequence of molecular activation. FRET analysis will identify co-association of the interacting molecules from the mere physical co-localization. We will address these issues through the following specific aims: Specific aim 1: To characterize and compare the MHC regulated temporal and spatial organization of supramolecular activation clusters (SMAC) in the immune synapse of normal NK cells during interaction with HLA class I deficient target versus the target expressing only one self- HLA class I molecule. Specific aim 2: To determine the role of (WASP) in NK cell signaling pathway(s) mediating granule exocytosis. Specific aim 3: To characterize the temporal and spatial redistribution of 2B4, NKp46 and NKG2D in the SMAC during non-MHC mediated NK cell interactions with the autologous tumor target, Neuroblastoma and identify differences in recruitment of signaling molecules to the immune synapse of the tumor-killing versus the tumor-sparing NK cell by analyzing both the in vitro and in vivo conjugates. Such analysis will improve our understanding of the biology of cytolytic effector lymphocytes that could be of potential use in immunotherapy.