A r t i c l e Figure 5. DEF6 regulates the expression of AID in antigen-engaged B cells by con- trolling IRF4 function. (A) Purified B cells from indicated 8–10-wk-old female mice were stimulated with single-round pulse IgM (pIgM) followed by stimulation with an CD40 antibody in the presence or absence of 50 ng/ml IL-21. 48 h after stimulation, Aicda mRNA expression was measured by real-time RT-PCR. The data are representative of three independent experiments. (B) Purified B cells from female WT and Def6tr/tr mice were stim- ulated as in A. 48 h later, extracts were ob- tained and analyzed by Western blot using an anti-AID antibody (top). The blot was reprobed with an antibody against -actin as a loading control (bottom). The data are representative of three independent experiments. (C) Purified B cells from female WT mice were stimulated as in A. Nuclear extracts were then prepared and immunoprecipitated with anti-IRF4 (IRF4 Ab IP) or with a control antibody (Contrl IP). The immunoprecipitates were then analyzed by immunoblotting using an anti- serum against DEF6 (top). The blot was later stripped and reprobed with an IRF4 antibody (bottom). The data are representative of four independent experiments. (D) ChIP assays were performed on purified B cells from fe- male WT and Def6tr/tr mice stimulated as in A with either an anti-IRF4 antibody or a control antibody, and real-time PCR for the Aicda promoter was performed as indicated. The data are representative of two independent experiments. (E) Purified B cells from female WT and Def6tr/tr mice were stimulated as in A. 48 h later, extracts were obtained and ana- lyzed by Western blot using an antibody rec- ognizing phospho-STAT3 (Y705; top). The blot was reprobed with an antibody recognizing total STAT3 (bottom). The data are representa- tive of two independent experiments. (F) ChIP assays were performed on purified B cells from female WT and Def6tr/tr mice stimulated as in A with either an anti-STAT3 antibody or a control antibody, and real-time PCR for the Aicda promoter was performed as indicated. The data are representative of two independent experiments. (G) Purified B cells from 8–10-wk-old WT, Def6tr/tr, Irf4/, and Def6tr/trIrf4/ female mice were stimu- lated as described in A. 48 h after stimulation, Aicda mRNA expression was measured by real-time PCR. The data are representative of three independent experiments. (H) Purified B cells from female WT and Def6tr/tr mice were retrovirally transduced with a control vector or with vectors expressing either a full-length form of DEF6 (DEF6 FL) or DEF6 (1–385) in presence of stimulation with pIgM+CD40. 5 d after stimulation, cells were harvested and sorted for YFP+ cells. Sorted YFP+ cells were then subjected to real-time RT-PCR for Aicda mRNA expression. The data are representative of two independent experiments. Throughout the figure white bars represent unstimulated cells, gray bars represent cells stimulated with pIgM+CD40 and black bars repre- sent cells stimulated with pIgM+CD40+IL-21. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ns, not statistically significant. observed (Fig. 6 A). Interestingly, although no differences in IL-21R expression could be observed in B cells from the 4 genotypes (unpublished data), B cells lacking SWAP-70 were found to generate a greater number of plasma cells than WT B cells especially when the cultures were supplemented with IL-21 (Fig. 6 B). This effect could be observed even at early time points in the culture (unpublished data). In line with the transfer experiments, B cells from DKO female mice JEM Vol. 209, No. 3 behaved similarly to Swap-70/ B cells, whereas Def6tr/tr B cells generated an equivalent number of plasma cells as WT B cells. Consistent with the known role of Blimp-1 as the master regulator of plasma cell differentiation, Swap-70/ and DKO B cells expressed higher levels of Prdm1 mRNA than WT or Def6tr/tr B cells (Fig. 6, C and D). Thus, these data indicate that SWAP-70 inhibits IL-21–driven plasma cell differentiation. 589