Because IRF4 can control plasma cell generation by reg- ulating the expression of Blimp-1 (Kwon et al., 2009), we explored the possibility that SWAP-70, given its high degree of structural homology to DEF6 (Gupta et al., 2003b; Tanaka et al., 2003), could also interact with IRF4 and thereby reg- ulate its capacity to promote plasma cell differentiation in response to T cell help. We first performed coimmunopre- cipitation experiments to assess whether SWAP-70 and IRF4 could interact in B cells. These experiments demonstrated that nuclear SWAP-70 can indeed physically interact with IRF4 in B cells stimulated with cIgM and CD40 (Fig. 6 E). Interestingly, the interaction between SWAP-70 and IRF4 was weaker when these experiments were conducted in the presence of IL-21 or in LPS-stimulated B cells, suggesting that stimuli known to strongly drive plasma cell differentia- tion may be able to modulate this interaction. ChIP assays were subsequently conducted to assess the ef- fects of the absence of SWAP-70 on the recruitment of IRF4 to the IL-21 response element, a crucial functional element in the Prdm1 gene that is cooperatively targeted by IRF4 and STAT3 (Kwon et al., 2009). Stimulated Swap70/ B cells exhibited increased binding of IRF4 to this crucial regulatory element when compared with WT B cells, despite expressing similar IRF4 levels (Fig. 6, F and G). Presence of IL-21 fur- ther augmented this effect. In contrast, the absence of DEF6 alone did not alter the binding of IRF4 to the Prdm1 IL-21 response element. B cells from the DKO mice exhibited a pat- tern similar to the one displayed by B cells lacking SWAP-70 alone. Given the known ability of STAT3 to bind to this ele- ment together with IRF4 (Kwon et al., 2009), we also exam- ined the recruitment of STAT3 to this region. No STAT3 phosphorylation was observed in the absence of IL-21 stimu- lation in either WT or Swap-70/ B cells (unpublished data). Although, in response to IL-21, STAT3 was phosphorylated to a similar extent in WT and Swap-70/ B cells (Fig. 6 H), Swap-70/ B cells exhibited enhanced binding of STAT3 to the Prdm1 gene in the presence of IL-21 (Fig. 6 I). The ab- normalities displayed by the DKO B cells mirrored those of B cells lacking SWAP-70 alone, whereas DEF6-deficient B cells behaved similarly to WT B cells. Thus, SWAP-70 restricts the recruitment of IRF4 and STAT3 to the Prdm1 IL-21 response element. A r t i c l e To gain additional support for the role of IRF4 in medi- ating the enhanced IL-21–driven plasma cell differentiation observed in Swap-70/ B cells, we explored the effects of concomitantly deleting IRF4 in Swap-70/ B cells (Fig. 6 J). Absence of IRF4 in Swap-70/ B cells prevented the aber- rant induction of Blimp-1 observed in these cells upon expo- sure to T cell–dependent stimuli. Because a deletional analysis revealed that the C-terminal portion of SWAP-70 is critical for its interaction with IRF4 (unpublished data), we next used a mutant of SWAP-70 that is unable to interact with IRF4 (SWAP-70 1–332) in retroviral transduction experi- ments (Fig. 6 K). These experiments demonstrated that the increased generation of plasma cells observed in Swap-70/ B cells could be corrected by full-length SWAP-70, but not by SWAP-70 (1–332). Altogether, these data support the no- tion that SWAP-70 regulates the ability of IRF4 to regulate T cell–induced plasma cell differentiation. DISCUSSION The molecular mechanisms that ensure the precise orchestra- tion of T and B cell interactions and the factors that perturb such regulatory mechanisms are not fully understood. Here we demonstrate that mice deficient in both DEF6 and SWAP-70, the only two members of a unique family of molecules, develop aberrant T-dependent humoral responses and a lupus-like disor- der that, similarly to human SLE, preferentially affects the female gender. One of the key mechanisms by which DEF6 controls T-dependent humoral responses is via its ability to tightly regu- late the production of IL-21 by CD4+ T cells, an effect that we have previously shown to be dependent on the ability of DEF6 to control IRF4 function (Biswas et al., 2010; Chen et al., 2008). Consistent with studies in other autoimmune models (Bubier et al., 2009; Jang et al., 2009), deletion of IL-21 corrects the B cell abnormalities observed in our mice supporting the idea that, in this system, deregulated production of IL-21 primarily exerts its effects by driving aberrant B cell responses. Although we have not observed any in vitro abnormalities in the DKO B cells upon exposure to IL-17 (unpublished data) it is possible that the aberrant production of IL-17 by Def6tr/tr T cells might also augment the autoimmune responses observed in the DKO mice as previously shown in other autoimmune models (Hsu et al., 2008; Wu et al., 2010). sponse 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 indepen- dent 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 fe- male 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+CD40. 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+CD40, and black bars represent cells stimulated with cIgM+CD40+IL-21. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ns, not statistically significant. JEM Vol. 209, No. 3 591