The γc chain cytokines share their ability to regulate the expression of the NKG2D-DAP10 receptor complex. Ex vivo isolated human NK cells were left untreated (medium) or were stimulated with IL-2, IL-7, IL-15, TGF-β1, or combinations as indicated for either 1 or 3 days. (A) Gray-filled histograms represent isotype control staining while black line histograms show staining with anti-NKG2D antibody from a representative experiment. Values in each histogram correspond to the median fluorescence intensity of NKG2D cell-surface staining. (B) Summary of the cell-surface expression of NKG2D on day 1 and day 3, following the indicated treatments. Bar graphs represent the average values of median fluorescence intensity from at least 5 donors; error bars represent SEM. Asterisks indicate the statistical significance (*P < .05; **P < .01; ***P < .005 paired Student t test). (C) NKG2D (top panel) and DAP10 (bottom panel) transcripts were analyzed by qRT-PCR with primer sets specific for each transcript of interest. mRNA expression levels, normalized to 18S rRNA, are presented in arbitrary units. Scatter plots show values for individual donors with mean ± SEM. Each symbol represents a donor. Statistical significance was tested by ANOVA for NKG2D and paired Student t test for DAP10 and indicated by asterisks (*P < .05; **P < .01; ***P < .005). (D) Total cell lysates of primary NK cells, left untreated or stimulated as indicated for 3 days, were immunoblotted with anti-DAP10 antibody. Actin was used as a loading control. The result shown is representative of 4 separate experiments with different donors.
