IFN-γ induction is IL15 dependent. (A) Total B cells from Ii^−/− (immature) mice were incubated in the presence or absence of 120 μM of Syk inhibitor (piceatannol; pic) or with 6 μL of DMSO for 3 hours, and RT-PCR was carried out using primers for IL-15 and HPRT. (B) Total B cells from Ii^−/− (immature) were incubated in the presence or absence of piceatannol (120 μM) or 6 μL of DMSO with or without IL-12 (20 ng/mL), IL-18 (200 ng/mL), and anti-Ly49D for 3 hours. RT-PCR was carried out using primers for IL-15 and HPRT. (C) Total B cells from Ii^−/− (immature) mice were incubated in the presence or absence of 1 μg/mL of the IL-15 inhibitor SolIL-15Rα overnight, and RT-PCR was carried out using primers for IFN-γ and HPRT. (D,E) Total B cells from Ii^−/− (immature) were incubated in the presence or absence of 1 μg/mL of SolIL-15Rα overnight with or without anti-Ly49D (D), or IL-12 (20 ng/mL; E) and IL-18 (200 ng/mL; E). RT-PCR was carried out using primers for IFN-γ and HPRT. (F) Total lymphocytes from Ii^−/− mice (immature) were incubated overnight in the presence or absence of IL-12 (20 ng/mL) and IL-18 (200 ng/mL) in the presence or absence of SolIL-15Rα. The cells were then stained with anti-B220, and IFN-γ protein levels were analyzed by intracellular staining. Histograms show IFN-γ expression in B220⁺ cells. (G) Graph showing the fold increase in IFN-γ⁺ cells following the various treatments minus the background of the isotype control. (H) Total B cells from Ii^−/− mice (immature) were incubated in the presence or absence of SolIL-15Rα (1 μg/mL) overnight. Their actin polymerization was analyzed as described in “Cytoskeleton rearrangement.” The results presented are representative of 3 separate experiments. Error bars represent SD.