Receptor expression varies with serial engagement. (A–F) NK cells were sequentially stimulated through CD16 and NKG2D on surfaces coated with either rituximab (Rtx) or MICA and both with ICAM-1 as schematically represented in Fig. 1 A (1 h at each step). Then, CD16 and NKG2D levels were assessed by flow cytometry. Histograms represent surface expression of CD16 (A and B) and NKG2D (C and D) after each step as indicated. (A and B) The vertical line denotes the point at which NK cells were considered as CD16⁺. Numbers denote the percent of CD16⁺ cells from a representative donor. Graphs show the gMFI of CD16 expression of CD16⁺ populations normalized to unstimulated (US) control cells. (C and D) gMFI of NKG2D expression from the total NK cell population. (E and F) NK cell viability after each stimulation. n = 7; symbols represent different donors; mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 calculated by one-way ANOVA.