CD56^dimDNAM-1^negNK cells are terminally differentiated. (A-B) CD56^brightDNAM-1^pos, CD56^dimDNAM-1^pos, and CD56^dimDNAM-1^neg NK cell subsets were purified from healthy donor PBMCs and cultured for 4 days in the presence of IL-2 (600 IU) (A) or IL-12 (10 ng/mL), IL-15 (100 ng/mL), and IL-18 (50 ng/mL) (B). At days 1, 2, and 4, NK cells were analyzed by using flow cytometry for surface expression of CD56 and DNAM-1. Results are displayed as the percentage of cells falling within CD56^brightDNAM-1^pos, CD56^dimDNAM-1^pos, or CD56^dimDNAM-1^neg gates. Data are shown as the mean ± SEM of duplicate wells from 5 individual donors pooled from 3 independent experiments. (C) Telomere length was determined by hybridization of a fluorescein isothiocyanate–labeled peptide nucleic acid probe to telomeric repeats in the DNA of NK cell subsets. For each NK cell subset, mean fluorescence intensity (MFI) values of the incorporated probe was normalized to K562 control cell fluorescence. Data are shown as mean ± SEM values obtained for 6 individual donors and are pooled from 3 independent experiments, with each symbol representing one individual donor. No significant difference was found by using a 1-way ANOVA on paired values. (D-E) Purified CD56^brightDNAM-1^pos, CD56^dimDNAM-1^pos, and CD56^dimDNAM-1^neg NK cells were cultured overnight in the absence (D) or presence (E) of IL-12 (10 ng/mL), IL-15 (100 ng/mL), and IL-18 (50 ng/mL). Apoptosis was determined by measuring Annexin V uptake by using flow cytometry. Data are shown as mean ± SEM from 5 individual donors. Individual dots represent the mean value of duplicate wells, with each symbol representing one individual donor. Data were analyzed by using a 1-way ANOVA on paired values followed by a Tukey multiple comparison post hoc test. *P < .05; **P < .01.