IL-12–pulsed NK cells enhance the IFN-α release by pDCs, in the presence of CpG-A, in a contact-dependent manner. (A) Purified resting NK cells were cultured overnight in the presence or in the absence of rhIL-12 or, as control, with rhIL-2. Cells were then washed and cocultured with autologous pDCs in the presence of CpG-A at 2 different concentrations (5 μg/mL or 0.5 μg/mL). After 24 hours of culture, supernatants from pDCs alone and from NK/pDC cocultures were collected and analyzed for IFN-α content by specific ELISA. ▪, IFN-α content in supernatants collected from cell cultures stimulated by CpG-A at the final concentration of 5 μg/mL; □, IFN-α content in supernatants collected from cell cultures stimulated by CpG-A at the final concentration of 0.5 μg/mL. The NK/pDC ratio was 10:1. A representative experiment of 10 performed is shown. (B) pDCs were cultured, in the presence of CpG-A (5 μg/mL), with either resting or short-term IL-12–activated NK cells together or separated by a semipermeable membrane. ▪, supernatants collected from NK cells and pDCs cultured together in a 24-well plate; ▦, supernatants harvested from transwell. NK/pDCs ratio was 10:1. A representative experiment of 4 performed is shown.