IL-10 is involved in diffDC-induced activation of NK cells. (A) DCs or TLR agonist-DCs (1 × 105) were incubated with purified splenic resting NK cells. After 24 hours of coculture, the supernatants were collected and analyzed by ELISA for IFN-γ production. (B) Intracellular staining for IFN-γ expression in splenic NK cells cocultured with various DCs in the presence of 10 μg/mL Brefeldin A for 18 hours. (C-F) Before being incubated with NK cells, 1.0 × 105 diffDCs or LPS-diffDCs were pretreated with anti–IL-10, anti–IL-12, or both anti–IL-10 and anti–IL-12 neutralizing mAbs (5 μg/mL) for 1 hour, then were cocultured with splenic resting NK cells. Similar to diffDC-NK coculture, 1.0 × 105 IL-10–/– diffDCs (with or without 500 ng/mL LPS stimulation) were cocultured with splenic resting NK cells. In some experiments, the exogenous 300 pg/mL (left) or 700 pg/mL (right) IL-10 was added into IL-10–/– diffDC-NK-cell or IL-10–/– LPS-diffDC-NK-cell coculture, respectively. After 24 hours of coculture, the supernatants were collected to measure IFN-γ production by ELISA. The data shown here were obtained in mean of triplicates. The diffDC/NK ratio in all these experiments was 1:5. *P < .05.
