IFN-γ production by NK-cell subsets after cytokine stimulation. Sorted CD56^bright (black), CD56^dim CD62L⁺ (dark gray) and CD56^dim CD62L⁻ cells (light gray; 10⁶/mL) were stimulated with 50 ng/mL IL12+50 ng/mL IL-18, 50 ng/mL IL-12+50ng/mL IL-15 (A-E) or with PB-derived mDCs in the presence of 100 ng/mL lipopolysaccharide and 10 μg/mL R848 (C). Induction of IFN-γ transcripts by real-time quantitative reverse-transcribed polymerase chain reaction (A) and IFN-γ protein expression by enzyme-linked immunosorbent assay (B) was measured after 16 hours of stimulation. Intracellular staining of IFN-γ was analyzed by FACS (C-E) after 24 hours of stimulation in the presence of BrefeldinA for the final 8 hours. (A) Mean values ± SEM of IFN-γ mRNA fold induction in stimulated CD56^bright and CD56^dim CD62L⁺ compared with CD56^dim CD62L⁻ cells of 3 independent experiments is shown. (B) Mean levels of IFN-γ protein ± SEM of 3 donors. (C) One representative experiment of 16 (IL-12 + IL-18), 7 (IL-12 + IL-15) or 5 (DCs) is shown. Stimulated (open histograms) and unstimulated (gray-filled histograms) cells as well as percentage of IFN-γ producing cells are depicted for each condition. Mean percentage (D) and mean MFI (E) values ± SEM of IFN-γ expressing cells are shown. *P < .05, **P < .01; ***P < .0001 was calculated by the paired t test.