Activating NK receptor stimulation promotes mTOR activation by IL-12. (A) 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) and NKp30 stimulation. IL-2 was washed out before the subsequent stimulation, and cytokines were added only at the beginning of the culture. n = number of donors. Mean ± SD. Paired t test, parametric; *, P < 0.05; **, < P < 0.01; ***, P < 0.0001. mIgG1-coated beads were used as a negative control. (A.i) pSTAT4^Y693. (A.ii) pSTAT5^Y694. (A.iii) pS6^S235/236 (≥2 independent experiments). (B) NK92 cells were stimulated with IL-12 (2.5 ng/ml; B.i) or IL-2 (200 U/ml; B.ii) for the indicated amount of time and tested for pSTAT4^Y693 or pSTAT5^Y694. Cytokines were added first at 240 min and then as indicated. Mean ± SEM; unpaired t test, parametric; *, P < 0.05; **, < P < 0.01 (≥2 independent experiments). (C) 3-d IL-2^high primed NK cells were stimulated by anti-NKp30 beads or control mIgG1 beads and/or IL-12 (2.5 ng/ml), or IL-2 (300 U/ml) or media without cytokines in the presence of different inhibitors against mTOR, STAT3, or STAT5, or with TGFβ^{high or low} (12.5–1.25 ng/ml) for 6 d. (C.i) Proliferation (CTV gMFI) assay. (C.ii) Relative cell viability. (C.iii) CTV histograms during TGFβ co-stimulation. Statistical analysis was calculated relative to media. n = number of donors, mean ± SD. Paired t test, parametric; *, P < 0.05; **, < P < 0.01; ***, P < 0.0001 (≥2 independent experiments). STAT3 inhibitors were used as a control for STAT5 inhibitors.