Analysis of KIR2DL3⁺and KIR2DL3^–NK cell IFN-γ production upon interaction with HLA-Cw*0702. Purifed NK cells from donor 49 were incubated with (bottom row) or without IL-2 (1000 U/mL) (top row) in the presence of 221 or 221-Cw*0702 cells for 12 hours with brefeldin A. To detect the frequency of KIR2DL3⁺ IFN-γ–positive cells, NK cells were stained with anti-KIR2DL3–PE (GL-183) and anti-IFN-γ–FITC mAbs and analyzed by flow cytometry (A). Cells analyzed were 30 000 for NK cells incubated with target cells and 20 000 for NK cells incubated alone. The gate was drawn to exclude dead NK cells and B-cell targets. The values reported in the upper quadrants are the frequencies of KIR2DL3⁺ NK cells producing IFN-γ; shown in the lower quadrants are the proportions of KIR2DL3^– NK cells producing IFN-γ. The percentage of NK cells inhibited by HLA-Cw*0702 was calculated from the frequencies of KIR2DL3⁺ or KIR2DL3^– NK cells producing IFN-γ observed following culture with 221 cells compared with those observed following culture with 221-Cw*0702 (B). Top graph, no cytokine; bottom graph, incubation with IL-2. For donor 49 the frequency of KIR2DL3⁺ cells (34%) was determined by flow cytometry using anti-KIR2DL3 (GL-183). The frequency did not change upon a 12-hour incubation with IL-2 or when 221 or 221-Cw*0702 was present (C). Top row, no cytokine; bottom row, incubation with IL-2. Data are representative of results obtained with 3 donors (44, 46, and 49; Figure 3).