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. 2016 Mar 22;7:101. doi: 10.3389/fimmu.2016.00101

Figure 2.

Figure 2

IL-15 and IL-18 can synergize to drive IFN-γ in absence of IL-12 or IL-2. PBMCs were stimulated for 6 or 18 h in vitro and production of intracellular IFN-γ by NK cells was measured in response to Med (medium alone), IL-2, IL-12, IL-15, IL-18, or IL-21. Representative flow cytometry plots show gating of CD3−CD56+ NK cells and percentage of CD56+ cells that are positive for intracellular IFN-γ on unstimulated and IL-12-stimulated NK cells (5 ng/ml) (A). IFN-γ production by NK cells was measured after stimulation with Med, IL-2, IL-12, IL-15, IL-18, or IL-21 (concentrations in nanograms per milliliter as labeled) for 18 h (B) (n = 4–9, data from one to three experiments). Concentrations in boxes indicate those used in following graphs. IFN-γ production by NK cells was also measured after stimulation with a titration of IL-2 (0, 5, and 50 ng/ml) in combination with IL-12 (12.5 pg/ml), IL-15 (0.75 ng/ml), and/or IL-18 (10 ng/ml) for 6 h (C) or 18 h (D) (n = 7–8, data from two experiments). IFN-γ production by NK cells was also measured following stimulation with 5 ng/ml IL-21 in combination with 5 ng/ml IL-2, 0.75 ng/ml IL-15, or 10 ng/ml IL-18 after 18 h (E) (n = 8, data from one experiment). IFN-γ expression after stimulation for 18 h with a combination of IL-18 (10 ng/ml) and γc cytokines was re-plotted to facilitate comparison between IL-2 (5 ng/ml), IL-15 [0.75 ng/ml; both from (D)], and IL-21 [5 ng/ml; from (E)] for 18 h (F) (n = 7–8, data from one to two experiments). Box plots show the 5th to 95th percentile range. Data were analyzed using paired Wilcoxon signed-rank tests [(B): no lines; lowest concentration compared to Med; (E) capped lines] or ANOVA tests for linear trend for trend analysis across increasing cytokine concentrations including Med [(B–D), uncapped lines]. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. n ≥ 8, other than for IL-21 titration (B) where n = 4.