NK cells and IL-15 in the human innate immune response to tuberculosis

描述(申请人提供)：尽管长期广泛接触结核分枝杆菌，但一些人仍然是结核杆菌阴性，这表明在T细胞识别结核分枝杆菌抗原之前，先天免疫控制了感染。我们发现来自健康供者的NK细胞通过NK细胞上的NKp46和NKG2D分别与感染细胞上的波形蛋白和ULBP1结合来杀伤结核分枝杆菌感染的单核巨噬细胞。我们的初步数据表明，在大量接触结核病的结核菌素阴性者(严重暴露于PPD-S)中，NK细胞比来自其他健康捐赠者的NK细胞更能溶解感染的单核细胞。严重接触PPD-S的单核细胞也会产生高浓度的IL-15。基于这些数据，我们假设：(1)NKp46、NKG2D及其配体介导了严重暴露的PPD-S增强的NK细胞的裂解能力；(2)NKp46、NKG2D及其配体受IL-15介导的信号上调。为了解决这些假设，我们提出了以下目标。 目的1.确定NKP46、NKG2D及其配体是否参与了PPD-S诱导的NK细胞裂解结核分枝杆菌感染细胞的能力。1.1.NK细胞受体及其配体的表面表达和中和。用流式细胞仪检测NKp46、NKG2D、Vimentin和ULBP1的表达。我们将使用针对这些受体和配体的siRNA和中和抗体来确定它们对感染细胞裂解的贡献。1.2.NK细胞受体和配体上调的机制。我们将确定在严重暴露于PPD-S的患者中，上调的NK细胞受体和配体是否显示出基因表达增加或蛋白质移位到细胞表面的增加。1.3.严重接触PPD-S是否显示感染其他病原体的细胞有更大的NK细胞裂解？我们将评估严重暴露于PPD-S的NK细胞对李斯特菌、隐孢子虫和Epstein Barr病毒感染细胞的杀伤能力。我们还将检测在AIM 1.1中发现上调的NK细胞受体和配体的表达。 目的2.检测IL-15对严重接触PPD-S的NK细胞杀伤感染细胞能力的影响。2.1.IL-15对NKp46、NKG2D、Vimentin和ULBP1表达的影响我们将利用IL-15及其受体的抗体以及siRNA来确定IL-15是否促进了重度暴露的PPD-S的NK细胞受体和配体的表达，并确定其是通过可溶性细胞因子还是通过与IL-15R结合并通过信号转导至NK细胞来实现的。2.2.探讨IL-15促进NK细胞受体和配体表达的信号转导途径。用免疫印迹法检测NK细胞和感染单核细胞中STAT3、STAT5和STAT6的表达。接下来，我们将使用siRNA和慢病毒载体来改变STAT水平，并确定IL-15诱导的增加NK细胞受体和配体表达的STAT。这些研究将提供对结核病先天耐药机制的洞察，这将对开发最大限度地提高先天免疫反应的疫苗至关重要。 结核病是一种传染病，每年导致全球190万人死亡，开发有效的疫苗是紧迫的公共卫生优先事项。有些人天生对结核病具有抵抗力，即使广泛接触传染性结核病患者，也不会被感染。这一提议将为这种耐药性增加的免疫学机制提供新的见解，利用这些机制将对开发最大限度地提高对结核病的天生耐药性的疫苗至关重要。

DESCRIPTION (provided by applicant): Despite extensive exposure to M. tuberculosis over long periods, some persons remain tuberculin-negative, suggesting that innate immunity controls the infection before T cells recognize M. tuberculosis antigens. We have found that NK cells from healthy donors lyse M. tuberculosis-infected mononuclear phagocytes through binding of NKp46 and NKG2D on NK cells to vimentin and ULBP1, respectively, on infected cells. Our preliminary data indicate that, in tuberculin-negative persons with heavy exposure to tuberculosis (heavily exposed PPD-s), NK cells lyse more infected monocytes than NK cells from other healthy donors. Monocytes from heavily exposed PPD-s also produce high concentrations of IL-15. Based on these data, we hypothesize that: (1) NKp46, NKG2D and their ligands mediate the enhanced lytic capacity of NK cells from heavily exposed PPD-s; (2) NKp46, NKG2D and their ligands are upregulated by IL-15-mediated signaling. To address these hypotheses, we propose the following aims. Aim 1. Determine if NKp46, NKG2D and their ligands contribute to the increased capacity of NK cells from heavily exposed PPD-s to lyse M. tuberculosis-infected cells. 1.1. Surface expression and neutralization of NK cell receptors and ligands. We will measure expression of NKp46, NKG2D vimentin and ULBP1 by flow cytometry. We will use siRNA and neutralizing antibodies to these receptors and ligands to determine their contribution to lysis of infected cells. 1.2. Mechanisms for upregulation of NK cell receptors and ligands. We will determine if upregulated NK cell receptors and ligands in heavily exposed PPD-s show increased mRNA expression or increased translocation of protein to the cell surface. 1.3. Do heavily exposed PPD-s show greater NK cell lysis of cells infected with other pathogens? We will evaluate the capacity of NK cells from heavily exposed PPD-s to lyse cells infected with Listeria, Cryptosporidium and Epstein Barr virus. We will also measure expression of NK cell receptors and ligands found to be upregulated in aim 1.1. Aim 2. Determine the contribution of IL-15 to the capacity of NK cells from heavily exposed PPD-s to lyse infected cells. 2.1. Effects of IL-15 on expression of NKp46, NKG2D, vimentin and ULBP1. We will use antibodies to IL-15 and its receptor, as well as siRNA, to determine if IL-15 increases expression of NK cell receptors and ligands in heavily exposed PPD-s, and to determine if it does so through soluble cytokine or by binding to IL-15R and signaling in trans to NK cells. 2.2. To delineate the signaling pathways by which IL- 15 increases expression of NK cell receptors and ligands. We will evaluate expression of STAT3, STAT5 and STAT6 in NK cells and infected monocytes by Western blot. Next, we will use siRNA and lentiviral vectors to alter STAT levels, and identify the IL-15-induced STATs that increase expression of NK cell receptors and ligands. These studies will provide insight into innate mechanisms of resistance to tuberculosis, which will be critical for development of vaccines that maximize innate immune responses. Tuberculosis is an infectious disease that kills 1.9 million people worldwide annually, and development of an effective vaccine is an urgent public health priority. Some persons have innate resistance to tuberculosis and do not become infected despite extensive exposure to infectious tuberculosis patients. This proposal will provide new insight into the immunologic mechanisms for this increased resistance, and harnessing of these mechanisms will be critical to develop a vaccine that maximizes innate resistance to tuberculosis.