The role of NK cells in human M. Tuberculosis infection

描述（由申请人提供）：NK细胞有助于免疫病毒感染，但有关其在防御细胞内细菌中的作用的信息有限。先天防御在对结核分枝杆菌的免疫反应中很重要，因为三分之二的结核病患者密切接触者保持健康，结核菌素皮肤试验呈阴性。这表明先天免疫在T细胞识别分枝杆菌抗原和发起延迟型超敏反应之前控制了感染。了解对结核分枝杆菌的先天免疫将提高我们开发预防结核病疫苗的能力，包括预防耐多药结核病的生物恐怖主义威胁。最近，我们发现NK细胞利用NKp46受体裂解结核分枝杆菌感染的单核细胞。该建议将通过以下目的描述NK细胞促进对结核分枝杆菌的先天免疫的机制。1)鉴定结核分枝杆菌感染单核吞噬细胞上NKp46的新配体。NKp46配体将通过免疫沉淀鉴定，然后进行质谱分析。该配体将被克隆和表达，从而产生重组蛋白和抗体。这些工具将用于确认NKp46及其配体在体内的相互作用；2)表征NK细胞裂解感染单核吞噬细胞的机制。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、穿孔蛋白/颗粒酶和atp介导的裂解；3)阐明NKp46介导结核分枝杆菌感染单核吞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个体信号分子的水平；4)表征介导结核分枝杆菌感染单核吞噬细胞裂解的NK细胞亚群。我们将研究NK细胞亚群表达细胞表面标志物和细胞因子的特定模式，使用多参数细胞分选。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。这些发现将有助于开发免疫调节策略，以增强对结核分枝杆菌（包括耐多药结核病）的免疫反应。

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.