The role of NK cells in human M. Tuberculosis infection

描述(申请人提供)：NK细胞有助于对病毒感染的免疫力，但关于它们在防御细胞内细菌方面的作用的信息有限。先天防御在对结核分枝杆菌的免疫反应中很重要，因为与结核病患者密切接触的人中有三分之二保持健康，而且结核菌素皮肤测试呈阴性。这表明，在T细胞识别分枝杆菌抗原和启动迟发性超敏反应之前，先天免疫控制了感染。了解对结核分枝杆菌的先天免疫将增强我们开发预防结核病的疫苗的能力，包括多药耐药结核病的生物恐怖威胁。最近，我们发现NK细胞利用NKp46受体来裂解结核分枝杆菌感染的单核细胞。这项提案将描述NK细胞通过以下目的促进针对结核分枝杆菌的天然免疫的机制。1)在结核分枝杆菌感染的单核巨噬细胞上鉴定新的NKp46配体。NKp46配体(S)将通过免疫沉淀和质谱学进行鉴定。该配体将被克隆和表达，从而生产重组蛋白和抗体。这些工具将用于证实NKp46与其配体(S)在体内的相互作用；2)表征NK细胞裂解感染的单核巨噬细胞的机制。我们将评估介导感染细胞裂解的分枝杆菌因子和效应机制，如Fas、穿孔素/颗粒酶和ATP介导的裂解；3)描述NKp46介导结核分枝杆菌感染的单核巨噬细胞裂解的信号通路。我们将使用特定的途径抑制剂，测量酶的磷酸化和激酶活性，进行免疫沉淀和Western blotting，并通过使用表达阳性或显性阴性结构的病毒表达载体改变单个信号分子的水平；4)表征介导结核分枝杆菌感染的单核吞噬细胞裂解的NK细胞亚群。我们将使用多参数细胞分选方法研究NK细胞亚群表达细胞表面标记和细胞因子的特定模式。这些研究将提供有关先天免疫反应的基本信息，并为开发利用NK细胞功能的疫苗奠定基础。这些发现将促进免疫调节策略的发展，以提高对结核分枝杆菌的免疫反应，包括耐多药结核病。

DESCRIPTION (provided by applicant): NK cells contribute to immunity against viral infection, but information on their role in defense against intracellular bacteria is limited. Innate defenses are important in the immune response to Mycobacterium tuberculosis because two-thirds of close contacts of tuberculosis patients remain healthy and have negative tuberculin skin tests. This suggests that innate immunity controls the infection before T cells recognize mycobacterial antigens and mount a delayed type hypersensitivity response. Understanding innate immunity to M. tuberculosis will enhance our capacity to develop vaccines that protect against tuberculosis, including the bioterrorist threat of multidrug-resistant tuberculosis. Recently, we found that NK cells use the NKp46 receptor to lyse M. tuberculosis infected monocytes. This proposal will characterize the mechanisms by which NK cells contribute to innate immunity against M. tuberculosis, through the following aims. 1) Identify novel ligands for NKp46 on M. tuberculosis-infected mononuclear phagocytes. NKp46 ligand(s) will be identified by immunoprecipitation, followed by mass spectrometry. The ligand will be cloned and expressed, allowing production of recombinant protein and antibodies. These tools will be used to confirm the interactions between NKp46 and its ligand(s) in vivo; 2) characterize the mechanisms by which NK cells lyse infected mononuclear phagocytes. We will evaluate the mycobacterial factors and effector mechanisms that mediate lysis of infected cells such as Fas, perforin/granzyme and ATP-mediated lysis; 3) Delineate the signaling pathways by which NKp46 mediates lysis of M. tuberculosis-Infected mononuclear phagocytes. We will use specific pathway inhibitors, measure enzyme phosphorylation and kinase activity, perform immunoprecipitation and Western blotting, and alter levels of individual signaling molecules through use of viral expression vectors expressing positive or dominant negative constructs; 4) Characterize the NK cell subpopulations that mediate lysis of M. tuberculosis-infected mononuclear phagocytes. We will study NK cell subpopulations expressing specific patterns of cell surface markers and cytokines, using multiparameter cell sorting. These studies will provide fundamental information on the innate immune responses and lay the groundwork for development of vaccines that take advantage of the functional capacity of NK cells. These findings will facilitate development of immunomodulatory strategies to boost the immune response to M. tuberculosis, including multidrug-resistant tuberculosis.