Preactivation with IL-12 + IL-18 results in human memory-like NK cells with enhanced IFN-γ productionafterrestimulation. (A) Schema of experimental approach with IL-12 + IL-18 preactivation. In later experiments, preactivated was varied as indicated. (B-C) PBMCs (containing 13% ± 5% CD56⁺CD3⁻ NK cells) were preactivated for 16 hours with control (IL-15, 1 ng/mL), or IL-12 (10 ng/mL) + IL-18 (50 ng/mL) + IL-15 (1 ng/mL). Cells were then washed 3 times to remove preactivating cytokines and cultured with low-dose IL-15 (1 ng/mL) to support survival for 7, 14, or 21 days. After each time point, cells were washed, restimulated for 6 hours with IL-12 (10 ng/mL) + IL-15 (100 ng/mL), and assayed for NK cell IFN-γ production by intracellular flow cytometry. (B) Representative bivariate flow plots from control and IL-12 + IL-18–preactivated NK cells showing IFN-γ production after IL-12 + IL-15 restimulation on days 7, 14, or 21 (gated on live CD45⁺CD56⁺CD3⁻ NK cells). (C) Summary data shown as means ± SEM percentage of IFN-γ⁺ NK cells. NK cells were assessed without restimulation immediately after preactivation (16 hours; n = 6; 3 independent experiments) to confirm that NK cells were preactivated or after 7 days of rest to demonstrate a return to a resting state (n = 8; 4 independent experiments). After 7 (n = 8; 4 independent experiments), 14 (n = 4; 2 independent experiments), or 21 (n = 4; 2 independent experiments) days, NK cells were restimulated with IL-12 + IL-15 for 6 hours and assayed for IFN-γ production. (D) Purified NK cells exhibit memory-like properties. Experiments were performed as in panels A-C, but with purified NK cells without restimulation (n = 6, 3 independent experiments for 16 hours; n = 12, 4 independent experiments for 7 days) and after IL-12 + IL15 restimulation for 7 days (n = 12; 4 independent experiments) or 14 days (n = 6; 3 independent experiments). Purified NK cells were ≥ 95% CD56⁺CD3⁻ with < 0.5% CD3⁺ T cells. *P < .05; **P < .01; ***P < .001.