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. 2015 Feb 5;45(4):1159–1169. doi: 10.1002/eji.201445132

Figure 1.

Figure 1

K3 CpG and cGAMP (TLR9 and STING agonists, respectively) synergistically induce innate IFN-γ production by human NK cells. (A) hPBMCs from two healthy donors were incubated with K3 CpG (10 μg/mL), cGAMP (10 μM), or K3 CpG (10 μg/mL) + cGAMP (10 μM) for 24 h and the supernatant IFN-γ concentrations were measured by ELISA. Data are representative of at least two independent experiments, and are shown as the mean + SD of duplicates from one experiment, representative of at least two performed. *p < 0.05; **p < 0.01 (one-way ANOVA with Bonferroni's multiple comparison test). (B) hPBMCs from three healthy donors were stimulated with K3 CpG, cGAMP, or K3 CpG + cGAMP for 16 h, with the last 4 h in the presence of Brefeldin A. After stimulation, cells were analyzed by flow cytometry for the detection of IFN-γ-producing cells. The percentage of IFN-γ-producing CD3+CD8+ T cells, CD3+CD8− T cells (including CD4+ T cells), and CD3−CD56+CD16+ NK cells are indicated in the quadrants. Data from one donor, which is representative of three donors, is shown. (C) hPBMCs from two healthy donors were treated with 5 μg/mL of isotype control, type-I IFN neutralizing, IL-12/23p40 neutralizing, or type-I IFN + IL-12/23p40 neutralizing antibodies 30 min prior to 24 h of stimulation with K3 CpG, cGAMP, or K3 CpG + cGAMP. IFN-γ production was measured by ELISA. Data are representative of at least two independent experiments, and are shown as the mean + SD of duplicates from one experiment, representative of at least two performed. *p < 0.05; **p < 0.01 (one-way ANOVA with Bonferroni's multiple comparison test).