Effective restimulation of NK cells is receptor dependent. (A–I) NK cells were sequentially activated through CD16 and NKG2D on slides coated with either rituximab (Rtx) or MICA and both with ICAM-1. Slides were also coated with anti-perforin mAb to capture secreted perforin, which was visualized by a noncompeting Alexa Fluor 488–labeled anti-perforin mAb. (A) Schematic representation of experimental approach. NK cells were sequentially incubated for 1 h on differently coated surfaces as indicated. (B) Representative images of perforin secreted from one cell during sequential stimulations. Bars, 10 µm. (C and F) Quantitative analysis of perforin secreted by cells from a representative donor. Each point is the integrated fluorescence intensity (IFI) of perforin captured from one cell (median ± interquartile range [IQR]). (D and G) Median IFI values of perforin secretion from different donors (n = 6; mean ± SEM; symbols represent different donors). (E and H) Intracellular (IC) perforin levels were assessed by flow cytometry upon each round of stimulation. Graphs represent geometric mean fluorescence intensity (gMFI) values (n = 3; mean ± SD; symbols represent different donors). (I) Comparison of gMFI values of intracellular perforin upon two rounds of stimulation on either rituximab or MICA. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 calculated by Kruskal-Wallis test (C and F), Friedman test (D, E, G, and H), or Student’s t test (I). US, unstimulated. See also Figs. S1, S2, and S3.