IL-12–mediated NK cell proliferation during target cell co-culture. Purified human peripheral blood NK cells were primed with IL-2^high (300 U/ml) for 10 d (long-term) prior to the indicated assays. Cytokines were added at the beginning of the cell culture. IL-2 was washed out prior to target cell co-culture. n = number of donors, each data line represents one donor measurement. (i–iii) Primed NK cells were co-cultured with Ba/F3 cells expressing NKp30 ligand (B7-H6; i), NKG2D ligand (MICA; ii), or without ligands (WT; iii), supplemented with IL-12^low (2.5 ng/ml) or media without cytokine. (iv) Representative histograms for CTV levels during NK cell and Ba/F3 cells co-culture. (v) Percentage of live target cells during the assay. Unpaired t test; ***, P < 0.001. **, NK cells cultured without Ba/F3 cells (no target, repeated data for comparison) were supplemented with IL-2 (300 U/ml), IL-12^low, or media without cytokines. Effector target ratio = 1:2, 1:4, 1:8. Ba/F3 cell viability without effectors decreased over time due to culture conditions (200 µl media, U-shaped 96-well plate).