again particularly evident in the presence of IL-21 (Fig. 5 B). These data suggest that DEF6, but not SWAP-70, regulates AID induction during the early phases of the T–B interaction. We had previously found that nuclear DEF6 can physi- cally interact with IRF4 and inhibit IRF4 function in CD4+ T cells (Chen et al., 2008). Given that IRF4 regulates the expression of AID in B cells (Klein et al., 2006; Sciammas et al., 2006), we explored the possibility that DEF6 could also in- teract with IRF4 in B cells and regulate its function in this compartment. Coimmunoprecipitation experiments, indeed, confirmed that nuclear DEF6 physically interacts with IRF4 in B cells (Fig. 5 C). Because the Aicda promoter contains potential IRF-binding sites (Lee et al., 2006), chromatin im- munoprecipitation (ChIP) assays with an anti-IRF4 antibody were conducted next. When compared with WT B cells, stimulated Def6tr/tr B cells exhibited increased binding of IRF4 to the Aicda promoter, although WT and Def6tr/tr B cells ex- pressed similar levels of IRF4 (Fig. 5 D; and not depicted). Intriguingly, the enhanced binding of IRF4 to the Aicda pro- moter was only minimally affected by the presence of IL-21 (Fig. 5 D). Given the known ability of IL-21 to activate STAT3 and the broad cooperativity between IRF4 and STAT3 (Kwon et al., 2009), we reasoned that the aberrant binding of IRF4 observed in the absence of DEF6 may result in in- creased STAT3 recruitment to the AICDA promoter upon exposure to IL-21. Although WT and DEF6-deficient B cells activated STAT3 at similar levels in response to IL-21 stimu- lation (Fig. 5 E), ChIP assays with a STAT3 antibody dem- onstrated that Def6tr/tr B cells exhibited enhanced binding of STAT3 to the Aicda promoter upon stimulation with IL-21 (Fig. 5 F). These data therefore suggest that the hyperrespon- siveness of DEF6-deficient B cells to IL-21 is caused not only by deregulated binding of IRF4 but also by the subsequent ability of the bound IRF4 to recruit STAT3. To further confirm the role of IRF4 in the abnormal activation of DEF6-deficient B cells, we used B cells from mice doubly deficient in DEF6 and IRF4, which we had previously generated (Chen et al., 2008). As shown in Fig. 5 G, the concomitant lack of IRF4 abolished the aberrant induc- tion of Aicda mRNA observed in Def6tr/tr B cells. Consistent with a mechanism whereby DEF6 can sequester IRF4 and prevent it from binding to the Aicda promoter, retroviral transduction experiments furthermore revealed that reintro- duction of full-length DEF6 (DEF6 FL), but not of a DEF6 mutant (DEF6 1–385) that is unable to interact with IRF4 (Chen et al., 2008), could rescue the abnormal induction of AID detected in Def6tr/tr B cells (Fig. 5 H), although both constructs were expressed at similar levels (unpublished data). Collectively, these data support the notion that nuclear DEF6 can sequester IRF4 in activated B cells and inhibit its function. SWAP-70 regulates IL-21–induced plasma cell differentiation by controlling the activity of IRF4 In line with previous findings (Quemeneur et al., 2008), the transfer experiments had indicated that SWAP-70 plays a key role in regulating plasma cell formation and that its absence in B cells was a major contributor to the plasma cell accumula- tion observed in the DKO female mice. To explore the mole- cular mechanisms by which SWAP-70 regulates T cell–driven plasma cell differentiation, we employed a standard in vitro culture system where resting B cells are stimulated in the pres- ence of continuous IgM signaling (cIgM) and CD40 stimula- tion. In light of the importance of IL-21 in the abnormalities that we had observed, these stimulations were conducted in the presence or absence of this cytokine. Under these stimu- latory conditions, no significant differences in the prolifera- tion of WT, Swap-70/, Def6tr/tr, or DKO B cells were Table 1. Percentages of B220+PNA+ cells after transfer into Rag1/ mice Table 2. Percentages of B220loCD138+ cells after transfer into Rag1/ mice B cells WT WT S70-/- S70-/- Def6tr/tr Def6tr/tr DKO DKO B220+ PNA+ % 1.2 ± 0.6 1.4 ± 0.4 0.5 ± 0.2 1.2 ± 0.2 2.0 ± 0.6 3.9 ± 1.8 3.8 ± 1.5 5.8 ± 1.0 T cells WT Def6tr/tr WT Def6tr/tr WT Def6tr/tr WT Def6tr/tr B cells WT WT S70-/- S70-/- Def6tr/tr Def6tr/tr DKO DKO %B220loCD138+ % 0.3 ± 0.2 0.3 ± 0.3 0.8 ± 0.3 1.5 ± 0.7 0.2 ± 0.4 0.3 ± 0.8 0.7 ± 0.2 1.5 ± 0.7 T cells WT Def6tr/tr WT Def6tr/tr WT Def6tr/tr WT Def6tr/tr 588 Percentage of B220+PNA+ cells after transfer of indicated combinations of T and B cells into Rag1/ mice followed by immunization with SRBCs. B220+PNA+ cells were evaluated 7 d after immunization by flow cytometry. P values are as follows: WT T+WT B vs. Def6tr/tr T+WT B, NS; WT T + S70/ B vs. Def6tr/tr T + S70/ B, P < 0.005; WT T + Def6tr/tr B vs. Def6tr/tr T + Def6tr/tr B, P < 0.05; WT T + DKO B vs. Def6tr/tr T + DKO B, P < 0.01; WT T+WT B vs. WT T + S70/ B cells, P < 0.005; WT T + WT B vs. WT T + Def6tr/tr B cells, P < 0.05; WT T + WT B vs. WT T + DKO B cells, P < 0.005; Def6tr/tr T + WT B vs. Def6tr/tr T + S70/ B cells, NS; Def6tr/tr T + WT B vs. Def6tr/tr T + Def6tr/tr B cells, P < 0.01; Def6tr/tr T + WT B vs. Def6tr/tr T + DKO B cells, P < 0.005. Percentage of B220loCD138+ cells after transfer of indicated combinations of T and B cells into Rag1/ mice followed by immunization with SRBCs. B220loCD138+ cells were evaluated 7 d after immunization by flow cytometry. P values are as follows, WT T+WT B vs. Def6tr/tr T+WT B, NS; WT T + S70/ B vs. Def6tr/tr T + S70/ B, P < 0.05; WT T + Def6tr/tr B vs. Def6tr/tr T + Def6tr/tr B, NS; WT T+DKO B vs. Def6tr/tr T + DKO B, P < 0.01; WT T+WT B vs. WT T + S70/ B cells, P < 0.01; WT T + WT B vs. WT T + Def6tr/tr B cells, NS; WT T + WT B vs. WT T+ DKO B cells, P < 0.005; Def6tr/tr T + WT B vs. Def6tr/tr T + S70/ B cells, P < 0.05; Def6tr/tr T + WT B vs. Def6tr/tr T + Def6tr/tr B cells, NS; Def6tr/tr T + WT B vs. Def6tr/tr T + DKO B cells, P < 0.005. IRF4 in autoimmunity | Biswas et al.