CD56^dimDNAM-1^negNK cells proliferate poorly and produce limited amount of IFN-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF) in response to cytokine stimulation. (A) CD56^brightDNAM-1^pos (red), CD56^dimDNAM-1^pos (blue), and CD56^dimDNAM-1^neg (green) NK cell subsets were purified from healthy donor PBMCs and stained with carboxyfluorescein diacetate succinimidyl ester (CFSE). The proliferation of NK cell subsets was assessed by measuring CFSE dilution after 5 days of culture with IL-2 (500 IU) and IL-15 (10 ng/mL). One representative histogram from 4 individual donors is displayed. (B) CD56^brightDNAM-1^pos, CD56^dimDNAM-1^pos, and CD56^dimDNAM-1^neg NK cell subsets were purified from healthy donor PBMCs. Intracellular IFN-γ production was assessed after overnight stimulation with IL-12 (10 ng/mL), IL-15 (100 ng/mL), and IL-18 (50 ng/mL). Representative fluorescence-activated cell sorting plots and cumulative data are shown. Each data point represents the mean percentage of IFN-γ⁺ NK cells obtained from culture duplicates; data are shown as mean ± SEM from 4 donor samples. (C) Healthy donor NK cells were purified into 6 subsets based on their expression of CD16 (+ or –), CD56 (bright [b] or dim [d]) and DNAM-1 (+ or –) and were cultured overnight with (striped fill pattern) or without (no fill pattern) stimulation with IL-12 (10 ng/mL), IL-15 (100 ng/mL), and IL-18 (50 ng/mL). Concentrations of indicated cytokines in the culture supernatant were analyzed by cytometric bead array. Data were normalized to reflect the relative cytokine/chemokine production per 10 000 cells and are presented as mean ± SEM of duplicate wells from 20 individual donors over 10 independent experiments. Data were analyzed with a 1-way (B) or 2-way (C) ANOVA followed by a Tukey multiple comparison post hoc test. *P <. 05; **P < .01; ***P < .001; ****P <.0001. No significant difference between subsets was found in the nonstimulated samples in panel C.