Activating NK receptor stimulation promotes differential IL-12 signaling leading to IL-2–primed NK cell expansion. (A) Assays’ design: Purified human peripheral blood NK cells were primed with IL-2^high (300 U/ml) for 3 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 subsequent stimulation. 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) of IL-2–primed NK cells induced by IL-12^low with or without anti-NKp30 beads. Left, CTV histograms from two representative donors; middle, CTV gMFI; right, relative cell viability. (C) pSTAT4^Y693 gMFI at 48 h (right: representative histograms, color-coded). (D) pSTAT^Y694 gMFI at 48 h (right: representative histograms, color-coded). (E) pS6^S235/236 gMFI at 48 h (right: representative histograms, color-coded). (F) Ki-67 gMFI at days 0, 2, 4, and 6. (G) Live NK cell numbers following the indicated stimulation. Fold-change in live cells was evaluated by cell counting and trypan blue stain exclusion. Integrated data from two independent experiments, n = 4/experiment (total of n = 8).