A rapid method for assessment of natural killer cell function after multiple receptor crosslinking

NK cell function is regulated by the integration of signals from activating and inhibitory receptors. We developed an assay to study the effect of co-crosslinking NK cell receptors in pair-wise combinations without the need to purify NK cells. Monoclonal antibodies recognising inhibitory and activating receptors were coated to flat bottomed tissue culture plates and degranulation was measured within unfractionated, freshly isolated resting or cytokine activated peripheral blood mononuclear cells by flow cytometric analysis of CD107a expression. Measured degranulation responses were NK cell specific, since no expression of CD107a was induced in gated T cells. We detected enhancement of degranulation in response to combinations of antibodies against activating NK cell receptors, including CD16, NKG2D, NKp30 and NKp46 compared to each antibody when combined with an isotype matched control antibody. Co-crosslinking of NKG2A resulted in the inhibition of degranulation measured in response to anti-NKp30 or anti-NKp46 alone in both resting or cytokine pre-activated NK cells, but had no effect on CD16 or NKG2D mediated responses. Interferon gamma production was assayed by intracellular cytokine staining and in cell culture supernatants after receptor crosslinking. No IFN-γ could be detected from resting NK cells after receptor crosslinking whereas the pattern of IFN-γ production in cytokine pre-activated NK cells reflected that observed for degranulation. We conclude that this assay is suitable for the analysis of the impact of NK cell receptor co-crosslinking on multiple NK cell functions and has the potential for application to pathologic conditions where limited numbers of cells are available for study.