Fig. 1.
Cytotoxic activity of IL-12/15/18 activated NK cells. a–c 2 × 105 purified NK cells were stimulated overnight in a 96 well plate with IL-12 (10 ng/ml), IL-15 (20 ng/ml) and IL-18 (100 ng/ml) and IL-2 (100 iu/ml) added on alternate days and then assayed on day 8. For anti-CD137 experiments, plates were pre-coated with anti-CD137 at a concentration of 10 μg/ml. a, b Cytotoxicity of IL-12/15/18 and IL-2-activated NK cells from healthy controls before and after cytokine stimulation. NK cells were tested against control 721.221 (221) cells and 7 different human liver cancer cell lines, SNU387, SNU398, SNU423, SNU475, Huh7, HepG2, PLC. One representative cytotoxicity assay is shown in a and the means and SEM from six donors are shown in b. All experiments were performed at an effector:target ratio of 2:1. c Expression of receptors on NK cells before and after stimulation with the cytokine cocktail (n = 5). For b and c significance was tested using the non-parametric Kruskal–Wallis one-way ANOVA test (Graph Pad Prism™). Tests were performed individually for each cell line tested. p values where shown compare unstimulated cells with cells stimulated either with cytokines alone, or with cytokines plus anti-CD137. For all panels *p < 0.05. d Expression of NKG2D ligands on the HCC cell lines by flow cytometry. NKG2D ligands are shown by red lines and isotype control by black lines. Positive control cells lines were K562 (MICA, MICB), Jurkat (ULBP1), HEK293T (ULBP2, ULBP3). Comparison of cytotoxicity (e) and IFNγ secretion (f) of cultured IL-12/15/18 primed NK cells and unprimed NK cells following incubation with the indicated cell lines for four hours (n = 6 donors). Primed NK cells were stimulated as for a–c and unprimed NK cells were cultured in 100 iu/ml IL-2 on day 0, and subsequently alternate days. Cells were tested at day 8 of culture and experiments were performed at an effector:target ratio of 2:1. Means and SEMs are shown. A paired t test was performed for each cell line comparing paired, primed and unprimed, NK cells from each donor (Graph Pad Prism™). For e and f *p < 0.05, **p < 0.001