IL-15–deficient B cells produce low levels of IFN-γ. (A) Total B cells from Ii^−/− (immature) control (mature) or IL-15^−/− (mature) mice were purified. Total RNA was isolated, and RT-PCR was carried out using primers for IFN-γ and HPRT as described in “RNA isolation and reverse transcription.” (B) Total B cells from control (mature) or IL-15^−/− mice were purified. Total RNA was isolated, and quantitative real-time PCR was carried out using primers for IFN-γ and HPRT as described in “Quantitative real-time RT-PCR.” (C) Total B cells from IL-15^−/− mice were suspended in 5 mL RPMI plus 10% FCS and incubated in the presence or absence of IL-15 (30 ng/mL) for various periods of time, as indicated. RT-PCR using primers for IFN-γ and HPRT was performed. (D) Immature B cells (CD21⁻, B220⁺) from control and IL-15^−/− mice were purified, and their actin polymerization was analyzed as described in “Cytoskeleton rearrangement.” (E,F) Transwell migration assay. Total cells from control and IL15^−/− mice were placed in the upper well of a 24-well transwell plate in the presence or absence of CXCL12 (1 μg/mL). After 4 hours, the total and migrating cells were immunostained: immature (B220⁺AA4.1⁺; E); mature (B220⁺AA4.1⁻; F), and the percentage of each population was evaluated by FACS analysis. Percentage of migration was calculated as the number of migrating cells in the lower chamber in the presence of CXCL12 minus the number of migrating cells in the lower chamber without CXCL12, as a fraction of the input cells in the upper chamber. The results presented are representative of 3 separate experiments. Error bars represent SD.