Immunofluorescent 3D-analysis of NK-target conjugates

描述(由申请人提供)：本建议书的总体目标 是为了深入了解自然杀伤(NK)细胞 由MHC或非MHC分子调节，以及导致 颗粒胞吐。评估脑血管异常的程度和性质 Wiskott-Aldrich综合征蛋白的信号转导途径 (黄蜂)缺陷使我们能够定义有缺陷的人的重要性 分子在这些途径中。细胞骨架和信号的校正 野生型正常NK细胞逆转录病毒转导缺陷的研究 WASP将确定该分子在NK信号中所起的作用。是这样的 分析还可能揭示其他信号分子之间的相互作用 黄蜂使它们的相互激活发生。颗粒剂的另类路径 溶细胞NK细胞的胞吐作用可能存在，并评估缺陷 一个单一的信号分子就可以提供这样的信息。目前尚不清楚 当激活信号超过抑制信号时，NK突触采取什么形式 信号。我们将描述NKG2D、2B4和NKp46的激活作用 神经母细胞瘤中的受体，特别是识别 发生在肿瘤细胞杀伤突触上的分子事件与 肿瘤细胞节约型NK克隆。受体-配体相互作用的动力学和 溶细胞性NK细胞中Smac的形成很可能是快速的，只有 按原样实时执行分子事件的时间分析 在这些研究中提出的，将很可能识别出分子序列 激活。FRET分析将确定相互作用的共同关联 分子仅仅来自物理上的共局域化。我们将解决这些问题 通过以下具体目标：具体目标1：确定和 MHC调控的超分子时空组织的比较 正常NK细胞免疫突触中与HLAI类缺陷靶与表达靶相互作用时的激活簇(SMAC) 只有一个自身的人类白细胞抗原I类分子。具体目标2：确定 (黄蜂)在NK细胞信号通路(S)中介导颗粒胞吐。特定的 目的3：研究2B4、NKp46的时空再分布特征 和NKG2D在非MHC介导的NK细胞与Smac相互作用过程中的表达 自体肿瘤靶点、神经母细胞瘤及鉴别征集的差异 肿瘤杀伤的免疫突触的信号分子与 通过分析体外和体内的结合物来抑制肿瘤的NK细胞。 这样的分析将提高我们对细胞溶解生物学的理解 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.