IL-10 is partially required for the stimulation of NK cytotoxicity by diffDCs. (A-B) Splenic NK cells (5 × 105) were cocultured with diffDCs, LPS-diffDCs, CpG-diffDCs, LTA-diffDCs, HSP70-diffDCs, maDCs, or imDCs for 18 hours. In some experiments, before being incubated with NK cells, 1.0 × 105 diffDCs or LPS-diffDCs were pretreated with anti–IL-10 antibody (5 μg/mL) for 1 hour, then 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 NK cells. Then the cytotoxicity of NK cells against the target cells (YAC-1) was tested in a standard 4-hour 51Cr release assay at various effector-to-target ratios. The data shown here were obtained in mean of triplicates. The DC/NK ratio in these experiments was 1:5. (C) FACS analysis of NK killing of DCs. After 24 hours of coculture, cells were harvested and then stained with specific immunofluorescence-conjugated anti-CD11c mAb and 7-AAD. In the maDC plus IL-10 group, the exogenous 700 pg/mL IL-10 was added into maDC-NK-cell coculture. The dot plots were derived from gated events with forward- and side-light scatter characteristics of DCs.
