As described in Ref. (19), whole blood was diluted with PBS and the mononuclear cells were recovered using Lymphoprep® lymphocyte separation medium (Axis-Shield, Oslo, Norway). For antibody staining, ~0.5 × 106 fresh mononuclear cells were washed and incubated with human IgG for 15 min at room temperature to block Fc-receptor binding and then stained on ice for 30 min with combinations of fluorescently labeled mAb, including those specific for CD3, CD16, and CD56 to allow for identification of CD56bright and CD56dim subsets (19). After washing, the cells were analyzed on a LSR-II flow cytometer (BD, San Jose, CA, USA), and data were processed using FlowJo software. Fresh mononuclear cells (0.5 × 106) were rested overnight and then stimulated with 1 × 106 K562 cells for 3 h at 37°C. Alternatively, mononuclear cells were cultured overnight with 0.5 µg/L IL-12 and then transferred to polystyrene plates coated with anti-NKp46 mAb for 3 h. Cells were stained with mAb to CD3, CD16, CD56, and CD107a. Cells also were fixed in 2% paraformaldehyde solution, then permeabilized (1× Permeabilization buffer, eBioscience) and stained with anti-IFN-γ and anti-MIP-1β mAb.