Schönberg Kathrin, Hejazi Maryam, Uhrberg Markus
Institute for Transplantation Diagnostics and Cell Therapeutics, University Clinic, Heinrich-Heine University, Düsseldorf, Germany.
Methods Mol Biol. 2012;903:381-92. doi: 10.1007/978-1-61779-937-2_26.
Natural killer (NK) cells provide a first line of defense against viral infections and prepare the ground for subsequent action of virus-specific T cells in a concerted way. Human NK cells use a sophisticated system of inhibitory and stimulatory receptors of the killer cell immunoglobulin-like receptor (KIR) gene family, which are expressed in a clonally distributed manner. Several studies suggest that KIR play a critical role in NK cell-mediated protection against HCV and HIV infection. As each NK cell expresses an individual set of KIR receptors that enables them to sense differences in HLA class I expression, classical measurement of NK cell function by analysis of target cell killing does not enable one to define and isolate the clinically relevant NK cell effector subsets. Here, we have developed a flow cytometry-based protocol to measure cytolytic activity together with KIR expression at a clonal level. Combined analysis of KIR expression in conjunction with cell surface mobilization of CD107 enables precise enumeration of cytolytic NK cells with defined specificity for HLA class I. Moreover, via inclusion of intracellular perforin or alternatively granzyme B, NK cells with deficient loading of cytotoxic granula can be identified. The present protocol enables identification and isolation of cytotoxic NK cells on a clonal level and enables reliable measurement in healthy as well as in pathological settings such as virus infection and hematological disease.
自然杀伤（NK）细胞为抵抗病毒感染提供了第一道防线，并协同为病毒特异性T细胞的后续作用奠定基础。人类NK细胞利用杀伤细胞免疫球蛋白样受体（KIR）基因家族的一套复杂的抑制性和刺激性受体系统，这些受体以克隆分布的方式表达。多项研究表明，KIR在NK细胞介导的针对丙型肝炎病毒（HCV）和人类免疫缺陷病毒（HIV）感染的保护中起关键作用。由于每个NK细胞表达一组独特的KIR受体，使其能够感知HLA I类表达的差异，通过分析靶细胞杀伤来经典测量NK细胞功能并不能定义和分离临床相关的NK细胞效应亚群。在此，我们开发了一种基于流式细胞术的方案，用于在克隆水平上测量细胞溶解活性以及KIR表达。结合KIR表达与CD107细胞表面动员的分析，能够精确计数对HLA I类具有明确特异性的细胞溶解NK细胞。此外，通过检测细胞内穿孔素或颗粒酶B，可识别细胞毒性颗粒装载不足的NK细胞。本方案能够在克隆水平上识别和分离细胞毒性NK细胞，并能够在健康以及病毒感染和血液疾病等病理情况下进行可靠测量。
