Additionalpreactivation conditions lead to enhanced memory-like NK cell IFN-γ production. (A-B) Purified (≥ 95% CD56⁺CD3⁻) NK cells were stimulated for 16 hours with either low-dose IL-15 (1 ng/mL, control), IL-12 (10 ng/mL), high-dose IL-15 (100 ng/mL), IL-18 (50 ng/mL), or the indicated IL-12/IL-15/IL-18 combinations. After 16 hours of activation, the cells were washed as in Figure 1A and then rested for 7 days before restimulation with IL-12 + IL-15 for 6 hours to assess IFN-γ production. In all conditions in which high-dose IL-15 was not used, low-dose IL-15 was included to support survival during preactivation. These data show that preactivation with all combinations of IL-12, IL-15, and IL-18 lead to memory-like NK cell IFN-γ production on restimulation (n = 4; 2 independent experiments). (C) In a separate set of donors, purified (≥ 95% CD56⁺CD3⁻) NK cells were preactivated for 16 hours by cross-linking CD16 with plate-bound anti-CD16 mAb (or control mouse IgG1) or CD16 cross-linking in combination with IL-12 (10 ng/mL), IL-18 (50 ng/mL), or IL-12 (10 ng/mL) + IL-18 (50 ng/mL). The preactivated cells were washed as in Figure 1A, rested for 7 days, and then restimulated with IL-12 + IL-15 for 6 hours to assess IFN-γ production. Summary data are shown as means ± SEM percentage of IFN-γ⁺ CD56^dim NK cells. These data show that CD16 cross-linking in combination with cytokines (IL-12 and IL-12 + IL-18) results in enhanced IFN-γ production on restimulation (n = 6; 3 independent experiments). Statistical comparisons are with control conditions, except in panel C as indicated. *P < .05; **P < .01; ***P < .001.