CD56^dimDNAM-1^negNK cells present poor killing capabilities and downregulate the killing activity of CD56^dimDNAM-1^posNK cells. (A) NK cells were enriched from PBMCs by Ficoll density gradient centrifugation followed by magnetic bead negative cell selection. K562 target cells were added to wells containing total NK cells in a 10:1 effector:target ratio. After 4 hours, cells were stained and analyzed for degranulation by quantifying the percentages of CD107a-positive cells after gating on CD56^brightDNAM-1^pos (red), CD56^dimDNAM-1^pos (blue), or CD56^dimDNAM-1^neg (green) NK cell subsets. Results are shown as percentages of positive cells ± SEM from 8 individual healthy donors, pooled from 2 independent experiments. (B-E) Fluorescence-activated cell sorting NK cell subsets were stimulated overnight in IL-12 (10 ng/mL), IL-15 (100 ng/mL), and IL-18 (50 ng/mL) and added to wells containing K562 target cells in a 10:1 effector:target ratio. (B) After 4 hours of culture, cytotoxicity of NK cell subsets against K562 target cells was quantified by flow cytometry with dead target cells identified as carboxyfluorescein diacetate succinimidyl ester (CFSE)^neg Annexin V/propidium iodide (PI)^pos. Target cells alone were used as control (–). Data are presented as mean ± SEM of percentages of Annexin V⁺ and/or PI⁺ target cells from 8 individual donors run in duplicate. Each dot represents one technical replicate. Data were pooled from 5 independent experiments. (C) The conjugation time between NK cells and target cells leading to target cell death was analyzed by using time-lapse microscopy. Time to membrane ruffle corresponds to the time (in seconds) between initial cell contact between the NK cell and the target cell and the first sign of target cell membrane ruffle. Results are shown as mean ± SEM from 20 to 30 individual NK cell:target cell conjugations pooled from at least 6 time-lapse videos obtained from 3 independent experiments. (D-E) Cytotoxicity of NK cell subsets was assessed as in panel B, but cells were cultured with (+) or without (–) supernatant from CD56^dimDNAM-1^neg (D^neg), heat-inactivated CD56^dimDNAM-1^neg (HI D^neg), or CD56^brightDNAM-1^pos (CD56^bright) NK cells that had been stimulated overnight in IL-12 (10 ng/mL), IL-15 (100 ng/mL), and IL-18 (50 ng/mL). Each graph shows the mean ± SEM of percentages Annexin V⁺ and/or PI⁺ target cells from 4 individual donors pooled from 2 independent experiments. Individual dots represent the mean value of duplicate wells for 1 donor. Data obtained from 1 individual donor are depicted with the same symbol. Data were analyzed by 2-way (A) or 1-way ANOVA without pairing (B-C) or a 1-way ANOVA with pairing (D-E) followed by a Tukey multiple comparison post hoc test. *P < .05; **P<.01; ***P < .001; ****P < .0001.