Low IL-12 concentrations promote NK receptor ligand-specific expansion of long-term IL-2 culture primary NK cells. (A) Assays’ design: Purified human peripheral blood NK cells were primed with IL-2^high (300 U/ml) for 10 d before IL-12^low (2.5 ng/ml) stimulation. Cytokines were added only at the beginning of the culture, and IL-2 was washed out before the restimulation. Data are displayed as n = number of donors. Mean ± SD. Paired t test, parametric; *, P < 0.05; **, < P < 0.01; ***, P < 0.001. mIgG1-coated beads were used as a negative control (≥2 independent experiments). (B) Proliferation (CTV) assay. Long-term primed NK cells were co-cultured with IL-12^high, ^low, or ^ultra-low (25, 2.5, or 0.25 ng/ml, respectively) in the presence of increasing IL-18 concentrations (25, 2.5, or 0.25–0 ng/ml, respectively). IL-2^high (300 U/ml) or media without cytokines were used as positive or negative controls. Lower panel: relative cell viability. (C) Live NK cell numbers during IL-12 stimulation relative to IL-2^low (3 U/ml). (D) Proliferation (CTV) assay. Primed NK cells were stimulated using beads coated with antibodies against the indicated activating receptors with IL-2^high, IL-12^low, or media without cytokines. Upper left: activating NK receptor expression levels; upper right: CTV histograms; lower left: CTV gMFI; lower right: relative cell viability. (E) Live NK cell numbers of IL-2–primed NK cells after co-cultured for 6 d with Ba/F3 cells expressing NKp30 ligand (B7-H6), relative to no ligand in the presence of variable IL-2 or IL-12 concentrations.