Functional characterization of IL-2–cultured primary NK cells.

<p>(<b>A</b>) Intracellular Ca²⁺-mobilization induced by activating NK cell receptors. The following monoclonal antibodies (mAbs) were used for NK cell stimulation: negative controls, IgG isotype (cIgG, red) and anti-CD56 (dark blue); receptor stimulation, anti-2B4 (orange), anti-DNAM-1 (light blue), anti-CD16 (green) and anti-2B4 and anti-DNAM-1 (purple). Polyclonal IL-2–cultured NK cells were pre-incubated with the indicated mAbs on ice and loaded with Fluo-4 and Fura Red dyes. Ca²⁺-mobilization was assessed by flow cytometry. After 30 sec, secondary F(ab′)2 goat anti-mouse IgG crosslinking antibody was added (black arrow). FL-1/FL-3 ratios are plotted as a function of time. (<b>B</b>) Comparison of degranulation by freshly isolated (open bars) and IL-2–cultured (black bars) NK cells. NK cells were incubated with the following target cells: murine P815 cells (negative control), P815 cells coated with mAbs to the indicated receptors (Fc receptor-positive for redirected ADCC; IgG isotype and anti-CD56 mAb coated P815 cells were used as negative controls) or with NK cell-susceptible human K562 cells (positive control). Subsequently, NK cells were stained with fluorochrome-conjugated anti-CD56 and anti-CD107a mAbs and analyzed by flow cytometry. The percentage of CD107a-positive NK cells is shown as mean ± standard deviation (SD) of at least eight independent experiments. Differences between two groups were examined using the Student's t-test (***, p<0.001). Correlation between degranulation by freshly isolated (open bars) and IL-2–cultured (black bars) NK cells were determined according to Pearson (r = 0.95).