Functions of the KLRG1 molecule on NK cells and T cells

描述(申请人提供)：NK细胞在抵抗病原体如小鼠巨细胞病毒(MCMV)的先天防御中起着至关重要的作用。NK细胞表面既有激活受体又有抑制受体，它们与天然细胞因子协同作用，调节NK细胞的功能。最近，我们发现杀伤细胞凝集素样受体G1(KLRG1)表达在最成熟的NK细胞上，感染MCMV可诱导NK细胞和CD8+T细胞表面KLRG1分子表达上调。我们还证实了KLRG1的参与可以抑制NK细胞的效应器功能。此外，我们还构建了KLRG1四聚体，并通过流式细胞术鉴定了四聚体阳性的细胞系。基于这些初步数据，我们建议对KLRG1函数的几个方面进行研究。在具体目标1中，我们将以KLRG 1作为成熟NK细胞的标志，研究MCMV感染后NK细胞的间隔性扩增和收缩阶段。我们还将研究体内阻断KLRG1功能在小鼠感染MCMV的背景下的后果，以确定KLRG1功能是否阻止NK细胞和/或CD8v T细胞的失控激活。在特定的目标2中，我们将研究决定KLRG1分子抑制特性的生化机制。为此，将进行野生型和突变型KLRG1分子的转染/表达分析。在特定的目标3中，我们将使用报告细胞系方法，以证实我们的四聚体染色的特异性，并鉴定表达KLRG1配体的细胞。在特定的目标4中，将开发一种表达克隆策略来克隆KLRG1配体。这些新的研究将为深入了解KLRG1特别是NK受体在NK细胞和CD8+T细胞上的特异性和免疫调节作用提供见解，并为调节这一重要的免疫细胞反应提供策略。

DESCRIPTION (provided by applicant): NK cells play a crucial role in innate defense against pathogens such as murine cytomegalovirus (MCMV). The presence of both activating and inhibitory receptors on the surface of NK cells act collaboratively and in concert with innate cytokines to regulate NK cell functions. Recently, we showed that the killer cell lectin-like receptor G 1 (KLRG 1) is expressed on the most mature NK cells and that infection with MCMV induces an up-regulation of the KLRG1 molecule on the surface of both NK cells and CD8+ T cells. We also demonstrated that KLRG 1 engagement can inhibit NK cell effector functions. In addition, we generated the KLRG1 tetramer and have identified cell lines that are tetramer positive by flow cytometry. Based on these preliminary data, we therefore propose to study several aspects of KLRG 1 functions. In Specific Aim 1, we will use KLRG 1 as a marker of mature NK cells to investigate the NK cell compartmental expansion and contraction phases in response to MCMV infection. We will also examine the consequences of the in vivo blocking of KLRG 1 functions in the context of the MCMV infection of mice in order to determine whether KLRG1 function is to prevent the uncontrolled activation of both NK cells and/or CD8 v T cells. In Specific Aim 2, we will study the biochemical mechanism that dictates the inhibitory properties of the KLRG1 molecule. To do this, transfection/expression analyses of wild-type and mutant KLRG1 molecules will be performed. In Specific Aim 3, we will use a reporter cell line approach, to both confirm the specificity of our tetramer staining and identify cells that express the KLRG1 ligand. In Specific Aim 4, an expression cloning strategy will be developed to clone the KLRG1 ligand. These novel studies will provide insights into the specificity and immunoregulatory role of KLRG1 in particular and NK receptors in general on both NK cells and CD8+ T cells, as well as provide strategies for the regulation of this important category of immune cell responses.