IL-12 delivery by mDCs is critical for NK cell activation. (A) mDCs promote the secretion of IFN-γ in NK cells. Purified human NK cells were cultured in the presence of iDCs or mDCs. IFN-γ was measured in the supernatants of the DC/NK cell cocultures by ELISA at 24 hours. mDCs and iDCs alone did not produce detectable amounts of IFN-γ (not shown). The depicted data represent means of triplicates ± SEM in one representative experiment of 5. (B-D) IL-12 is involved in the mDC-mediated NK cell activation. mDCs were pretreated with neutralizing anti-IL-12 or anti-IL-15 mAb prior to coculture (B) or electroporated with siRNA specific for IL-12 p40 or NF-κB p50 subunit (or control siRNA) prior to LPS stimulation and incubated with resting NK cells (at a 1:10 DC/NK cell ratio) in 96-well plates for 24 hours (C). It is noteworthy that accumulation of IL-12 in the supernatant of the 10⁴ mDCs cultured alone or together with 10⁵ NK cells could not be detected by ELISA (not shown). The RNA interference was functional because IL-12 was barely detectable in mDCs pretreated with siRNAp40 or siRNA NF-κBp50 using confocal microscopy and was not produced in the DC/NK cell coculture supernatants at 1:1 DC/NK cell ratios (while produced in the absence of siRNA; not shown). Mouse BM-DCs propagated for 7 days from wild-type or IL-12p35^-/- mice were cocultured with wild-type NK cells at 1:2 and 1:10 DC/NK cell ratios for 18 hours. The levels of IFN-γ measured in the mDC, NK, or mDC/NK cell coculture supernatants in the human (B-C) or murine system (D) are shown as means ± SEM of triplicate wells of a representative experiment (of 3).