IL-2, IL-15, IL-18, or IFNα, and NKp30 contribution to ex vivo human NK cell expansion, proliferation, and viability by IL-12. (A) Primary NK cell relative numbers. Samples were measured by flow cytometry on day 6 of the indicated stimulation. n = 4 (number of donors). (B) Purified human peripheral blood NK cells (n = 8, number of donors) were cultured for 6 d with IL-2^high (300 U/ml), IL-12^low (2.5 ng/ml), or media without cytokines. Expression levels of the indicated Bcl2 family protein were assessed by intracellular staining and flow cytometry. Mean ± SD. Paired t test, parametric; *, P < 0.05; **, < P < 0.01; ***, P < 0.001. (B.i) Live NK cell number per reading (left) and matched live NK cells percentage (right). (B.ii) Expression of anti-apoptotic proteins (left to right): Mcl1, Bcl-X_L, Bcl2, pBad^S112, and representative histograms. (C) Proliferation (CTV) assay: ex vivo NK cells were stimulated with IL-12^low (2.5 ng/ml) and/or IL-2^low (3 U/ml) with or without IL-18 (C.i) or IFNα (C.ii). Upper panels: cell proliferation measured by CTV; lower panels: cell viability measured by fixable near-infrared viability dye. (C.iii) Representative CTV histograms. Stimulation was done in the presence of mIgG1- or anti-NKp30–coated beads. Mean ± SD. Paired t test, parametric; *, P < 0.05; **, < P < 0.01. n = 4 (≥2 independent experiments). (D) Cell proliferation (CTV) assays: ex vivo NK cells were stimulated with anti-NKp30 beads with or without anti-DNAM1 beads in the presence of the indicated IL-15 concentrations and IL-12 (2.5 ng/ml) as indicated. n = number of donors, mean ± SD. n = 4, mean ± SD.