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

描述（由申请人提供）：尽管广泛暴露于M。在长期的结核病中，有些人保持结核菌素阴性，这表明先天免疫在T细胞识别M之前控制感染。结核抗原我们已经发现，来自健康供体的NK细胞裂解M。通过NK细胞上的NKp 46和NKG 2D分别与感染细胞上的波形蛋白和ULBP 1结合，抑制结核病感染的单核吞噬细胞。我们的初步数据表明，在结核菌素阴性的人与结核病的严重暴露（严重暴露的PPD-s），NK细胞裂解更多的感染单核细胞比其他健康供体的NK细胞。来自高度暴露的PPD的单核细胞也产生高浓度的IL-15。基于这些数据，我们假设：（1）NKp 46、NKG 2D及其配体介导来自重度暴露的PPD-s的NK细胞的溶解能力增强;（2）NKp 46、NKG 2D及其配体通过IL-15介导的信号传导上调。为了解决这些假设，我们提出了以下目标。 目标1。确定NKp 46、NKG 2D及其配体是否有助于增加来自重度暴露PPD-s的NK细胞裂解M的能力。结核病感染细胞1.1. NK细胞受体和配体的表面表达和中和。我们将通过流式细胞术测量NKp 46、NKG 2D波形蛋白和ULBP 1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细胞受体和配体的上调机制。我们将确定是否在大量暴露的PPD中上调的NK细胞受体和配体显示mRNA表达增加或蛋白质向细胞表面的易位增加。1.3.重度暴露的PPD是否显示出对感染其他病原体的细胞的更大的NK细胞溶解？我们将评估来自高度暴露的PPD的NK细胞裂解李斯特菌、隐孢子虫和爱泼斯坦巴尔病毒感染的细胞的能力。我们还将测量发现在目标1.1中上调的NK细胞受体和配体的表达。 目标二。确定IL-15对来自重度暴露的PPD-s的NK细胞裂解感染细胞的能力的贡献。2.1. IL-15对NKp 46、NKG 2D、波形蛋白和ULBP 1表达的影响。我们将使用针对IL-15及其受体的抗体以及siRNA来确定IL-15是否增加重度暴露的PPD中NK细胞受体和配体的表达，并确定其是否通过可溶性细胞因子或通过结合IL-15 R和反式信号传导至NK细胞来这样做。2.2.描述IL- 15增加NK细胞受体和配体表达的信号通路。我们将通过Western blot评估STAT 3、STAT 5和STAT 6在NK细胞和感染的单核细胞中的表达。接下来，我们将使用siRNA和慢病毒载体来改变STAT水平，并鉴定IL-15诱导的STAT，其增加NK细胞受体和配体的表达。这些研究将提供对结核病抗性的先天机制的深入了解，这对于开发最大化先天免疫应答的疫苗至关重要。 结核病是一种传染病，每年在全世界造成190万人死亡，开发有效的疫苗是一项紧迫的公共卫生优先事项。有些人对结核病具有先天抵抗力，即使广泛接触传染性结核病患者也不会感染。这一提议将为这种增加的抗性的免疫机制提供新的见解，并且利用这些机制对于开发最大化对结核病的先天抗性的疫苗至关重要。

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.