IVIg-DCs trigger NK-cell activation, IFN-γ production, and degranulation. (A) IVIg-DCs, HSA-DCs, or CTRL-DCs were cultured for 48 hours with allogeneic NK cells (ratio DC/NK was 1:6) after which CD69 (top plot) and CD25 expression (bottom plot) on CD56⁺ cells were determined. Depicted are NK cells activated by IVIg-DCs (gray histogram) and by CTRL-DCs (black histogram), and staining with an irrelevant isotype control mAb of NK cells activated by IVIg-DCs (dotted histogram). Similar NK activation was observed when DC/NK ratio was changed to 1:3 or 1:1 (data not shown). (B) IVIg-DCs, HSA-DCs, or CTRL-DCs were washed to remove additives, and cultured for 48 hours with allogeneic NK cells (■). CD69 expression (N = 6; Wilcoxon test for paired data, *P < .05) and CD25 expression (N = 5; Wilcoxon test for paired data, *P < .05) on NK cells were significantly enhanced after coculture with IVIg-DCs. Treatment of NK cells with 10 mg/mL IVIgs or HSA in absence of DCs (□) had only minor activating effects on NK cells. (C) IFN-γ concentration was determined in cell-free supernatants of DC-NK cocultures. In the supernatants of NK cells stimulated with CTRL-DCs or HSA-DCs, no IFN-γ could be detected, while in the supernatants of NK cells stimulated with IVIg-DCs, high IFN-γ levels were detected (N = 7; Wilcoxon test for paired data, *P < .01). (D) Expression of the lytic granule membrane protein CD107a on the NK-cell surface after 6 hours of coculture of matured allogeneic CTRL-DCs, IVIg-DCs, or HSA-DCs (N = 5; Wilcoxon test for paired data, *P < .05). Error bars in panels B-D are SD. (E) Representative dot plots showing CD107a expression on NK cells cultured for 6 hours without DCs (negative control), NK cells stimulated with PMA and ionomycin (2.5 μg/mL and 0.5 μg/mL, respectively) (positive control), NK cells cocultured with CTRL-DCs, and NK cells cocultured with IVIg-DCs. Numbers on plots are the percentage of cells in that quadrant.