SWAP-70 regulates IRF4 function during IL-21–induced plasma cell differentiation. (A) Purified B cells from indicated 8–10-wk-old female mice were stimulated with continuous αIgM (cIgM) and αCD40 in the presence or absence of 50 ng/ml IL-21. After 96 h of stimulation, proliferation of B cells was measured by thymidine incorporation. The data are representative of three independent experiments. (B) Purified B cells were stimulated with cIgM and αCD40 in presence or absence of IL-21 (5 or 50 ng/ml, as indicated). After 4 d of culture, in vitro plasma cell differentiation was measured by quantifying the percentage of CD138⁺ cells by FACS. The data are representative of three independent experiments. (C) Purified B cells were stimulated as in A. 72 h after culture, cells were harvested, and mRNA levels of Prdm1 were measured by real-time PCR. The data are representative of three independent experiments. (D) Purified B cells were stimulated as in A. At the times indicated, cells were harvested and mRNA levels of Prdm1 were measured by real-time PCR. The data are representative of two independent experiments. (E) Purified B cells from WT or S70^−/− female mice were stimulated as indicated. 48 h after stimulation, nuclear extracts were prepared and immunoprecipitated with an anti-IRF4 antibody (αIRF4 IP) or with a control antibody (Ctrl IP). The immunoprecipitates were resolved by 8% SDS-PAGE, and then analyzed by Western blot using an anti–SWAP-70 antiserum (top). The blot was reprobed with an anti-IRF4 antibody (bottom). The data are representative of two independent experiments. (F) Purified B cells were stimulated for 48 h, as indicated in A. ChIP assays were performed with either an anti-IRF4 antibody or a control antibody. Real-time PCR for the IL-21 response element in the Prdm1 gene was performed as indicated. The data are representative of two independent experiments. (G) Purified B cells were stimulated as indicated in A. 48 h after stimulation, Irf4 mRNA expression was evaluated by real-time PCR. The data are representative of two independent experiments. (H) Purified B cells from WT and S70^−/− female mice were stimulated as indicated. The phosphorylation of STAT3 at the different time points was evaluated by Western blot with an antibody recognizing phospho-STAT3 (Y705; top). The blot was reprobed with an antibody recognizing total STAT3 (bottom). The data are representative of two independent experiments. (I) Purified B cells were stimulated as in A. 48 h after culture, ChIP assays were performed with either an anti-STAT3 antibody or a control antibody, and real-time PCR for the Prdm1 IL-21 response element was performed as indicated. The data are representative of two independent experiments. (J) Purified B cells were stimulated as in A. 72 h after culture, cells were harvested, and mRNA levels of Prdm1 were measured by real-time PCR. The data are representative of three independent experiments. (K) Purified B cells from female WT and S70^−/− mice were retrovirally transduced with a control vector or with vectors expressing either a full-length form of SWAP-70 (FL SWAP-70) or SWAP-70 (1–332) in presence of stimulation with cIgM+αCD40. 5 d after stimulation, cells were stained for CD138 and then analyzed by FACS for CD138 expression. The data are representative of two independent experiments. Throughout the figure white bars represent unstimulated cells, gray bars represent cells stimulated with cIgM+αCD40, and black bars represent cells stimulated with cIgM+αCD40+IL-21. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ns, not statistically significant.