Immature B cells produce and secrete higher levels of IL-15 compared with mature cells. (A-D) RT-PCR analysis. (A) Total B cells (B220⁺) from Ii^−/− (immature) or control mice (mature). (B) Purified immature (IgD⁻ B220⁺) and mature (IgD⁺) cells from control mice. (C) Purified immature (CD21⁻ B220⁺) and mature (IgD⁺) B cells derived from control mice. (D) T1, T2, and mature B cells were purified as described in “Separation of B cells.” Total RNA was isolated, and reverse transcription using primers for IL-15 and HPRT was carried out using Superscript II RT. The results presented are representative of 3 different experiments. (E) Intracellular IL-15 expression. Total lymphocytes from control (mature), Ii^−/− (immature), and IL-15^−/− mice were stained for B220 marker and analyzed for IL-15 protein levels by intracellular staining as described in “Intracellular staining.” Histograms show IL-15 expression in B220⁺ cells. (F) IL-15 secretion. Total B cells (B220⁺) from Ii^−/− (immature) or control mice (mature) were incubated overnight at 37°C, and IL-15 levels in the conditioned medium derived from both populations were analyzed by ELISA. The results presented are representative of 3 separate experiments. Data shown represent means (± SE).