Protocol for the clonal analysis of NK cell effector functions by multi-parameter flow cytometry

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.