Down-regulation of CD16 and CD62L surface expression upon cytokine stimulation. (A) Representative fluorescence-activated cell sorter plot of CD16 expression before and after activation with PMA for 1 hour, with IL-12 and IL-18 overnight, or with the class I negative cell line K562 for 5 hours. (B) Representative fluorescence-activated cell sorter plot of CD16 expression before and after activation. CD16 expression was measured on CD56^dim NK cells after incubation in either media alone, IL-12 and IL-18, or with the cell line K562. NK cell activation was determined by expression of CD107a and production of IFN-γ. (C) PBMCs from healthy donors were incubated in either media alone, IL-12 and IL-18 ± IL-15, IL-15 or IL-2 (n = 5). CD16 surface expression was measured on CD56^dim NK cells. Bars represent the mean ± SEM. The percentage of NK cells expressing CD16 after cytokine treatment was compared with cells treated with media alone using the paired t test. Statistical significance is indicated as: *P ≤ .05; **P < .01; ***P < .001. (D) Purified NK cells were incubated in either media alone (white bar) or with IL-12 and IL-18 for 18 hours. Cells were washed, incubated in media alone, and CD16 expression was measured at 0, 24, 48, and 72 hours after IL-12 and IL-18 stimulation. Bars represent the mean ± SEM. CD16 expression after IL-12 and IL-18 stimulation was compared with media alone using the Student t test. Statistical significance is indicated as: *P ≤ .05; **P < .01. (E) CD62L expression was measured on CD56^bright (left panel) and CD56^dim (right panel) NK cells. (F) Intracellular IFN-γ production by CD56^bright (left panel) and CD56^dim (right panel) NK cells was measured by flow cytometry.