Interrelationships between different NK-cell responses induced by target cell recognition. Resting NK cells were mixed with S2 cells expressing ligands for NK-cell receptors, as indicated, and incubated for 6 hours at 37°C. For some stimulations, S2 cells were preincubated with diluted anti–S2 cell serum (+ IgG). After stimulation, the cells were surface stained with fluorochrome-conjugated anti-CD56 and anti-CD107a mAbs, fixed, permeabilized, and stained intracellularly with fluorochrome-conjugated mAbs to cytokines and chemokines. (A) Lymphocytes were gated on forward scatter height (FSC-H) versus side scatter height plots (SSC-H). Single-cell events were gated on forward scatter height (FSC-H) versus forward scatter area plots (FSC-A). CD56^dim NK cells were gated on CD56 versus dead cell marker (DCM) plots. The second and third rows show MIP-1α, TNF-α, IFN-γ, and CD107a staining in relation to MIP-1β staining after stimulation with S2 cells, as indicated. The bottom row shows MIP-1β, MIP-1α, TNF-α, and IFN-γ staining in relation to CD107a staining, and IFN-γ staining in relation to TNF-α staining (right panel). Gates were set using fluorochrome-conjugated isotype control mAbs. The plots are derived from one representative donor. (B) CD56^dim NK cells were gated as described in panel A, and a Boolean gating strategy was used for analysis. Pie charts represent the frequency of cells positive for the given number of measured responses (MIP-1β, TNF-α, IFN-γ, and CD107a). Thus, cells can be categorized into the number of responses they display. Arcs depict the relative frequency of cells specifically positive for MIP-1β, TNF-α, IFN-γ, and/or CD107a staining, as indicated. Values represent the mean of 6 different donors.