KIR3DL2⁺ NK cells release cytokines in response to ODN C stimulation. (A) IL2-activated NK cell populations were cultured for 1 hour in medium supplemented with 1 ng/mL IL12 either in the absence or in the presence of ODN C (5 μg/mL) and then assessed by double fluorescence analysis for IFN-γ or TNF-α production and KIR3DL2 expression. In the upper-right quadrants is indicated the percentage of KIR3DL2⁺ cells that produce cytokines. Results are representative of 5 independent experiments. (B) IFN-γ production by KIR3DL2⁺ or KIR3DL2⁻ NK cell clones was assessed by specific enzyme-linked immunosorbent assay after treatment for 2 hours with 1 ng/mL IL12 used alone () or together with 0.5 μg/mL (▭), 5 μg/mL (), or 100 μg/mL () ODN C. Results are representative of 3 independent experiments. (C) ODN C uptake by IL2-activated NK cell populations was analyzed in the absence or presence of chloroquine pretreatment. The upper histograms represent the FITC–ODN C fluorescence intensity, whereas the lower histograms represent the expression of KIR3DL2 under the same experimental conditions. The percentage of positive cells and the MFI of the positive peaks are indicated in each histogram. Results are representative of 4 distinct experiments. (D) ODN C–mediated IFN-γ production by IL2-activated NK cell populations was analyzed in the absence or presence of pretreatment with chloroquine 0.04 mM (CLQ). IFN-γ content was assessed by specific enzyme-linked immunosorbent assay. Data are represented as medians and interquartile ranges (IQRs) of 4 independent experiments.