Biswas et al., 2010), we first explored the effects of the simul- taneous lack of DEF6 and SWAP-70 on the production of these cytokines. When cultured under neutral conditions, DKO T cells from young female mice aberrantly produced IL-17 and IL-21 to an extent similar to that exhibited by Def6tr/tr T cells (Fig. 4 A). In contrast, Swap-70/ CD4+ T cells behaved similarly to WT CD4+ T cells (Fig. 4 A). Moreover, the lack of SWAP-70 either by itself or in combi- nation with the absence of DEF6 did not affect the produc- tion of IFN- or IL-4 (Fig. 4 A; and not depicted). Consistent with these findings, intracellular staining for IL-17 and IL-21 demonstrated that, unlike WT and Swap-70/ mice, 24-wk-old Def6tr/tr and DKO female mice contained comparable popu- lations of T cells that aberrantly produced both IL-17 and IL-21 (Fig. 4 B).These data suggest that the lack of SWAP-70 does not affect the aberrant cytokine production observed in DEF6-deficient CD4+ T helper cells. In view of the enhanced ability of Def6tr/tr CD4+ T cells to produce IL-21 and the key role that IL-21 plays in driving B cell responses, we also explored the effects of IL-21 defi- ciency on the humoral abnormalities observed in the absence of DEF6. For these experiments, we crossed IL-21/ mice with Def6tr/tr mice on a mixed background where DEF6 defi- ciency alone is sufficient to mediate exaggerated T and B cells responses (Fanzo et al., 2006). Control mice, Def6tr/tr, IL-21/, and Def6tr/trIL-21/ mice were then immunized with SRBCs and splenic B cell responses were quantified 7 d after 586 Figure 3. Abnormal responses to immu- nization with a TD antigen in young DKO female mice. (A and B) 8–10-wk-old female mice (n = 6–7/group) were immunized with NP-KLH emulsified in CFA. Unimmunized con- trol mice received CFA only (not depicted). At day 7 after immunization, spleens were ana- lyzed for the numbers of B220+NP+ cells (A) and of B220+NP+PNA+ cells (B) by flow cy- tometry. (C and D) ELISPOT analysis of total (C) and high-affinity (D) IgG1 secretion by splenocytes from NP-KLH–immunized mice at day 14 after immunization. (E and F) Serum responses to NP-KLH immunization in the indicated mice. 14 d after immunization, serum samples were collected and total NP- specific anti-IgG1 (E) and IgG2a (F) antibody titers were determined by ELISA with NP14- BSA as capture antigen. Each dot represents individual mice, and horizontal bar represents mean for each group. Data are representative of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.005. immunization (Fig. 4, C and D). As expected, in response to immuniza- tion, Def6tr/tr mice exhibited higher numbers of GC B cells and plasma cells as compared with control mice. These effects were clearly inhibited by the absence of IL-21. IL-21 defi- ciency did not exert any effects on the expansion of effector (CD62LloCD44hi) CD4+ T cell and ICOS+ CD4+ T cells, which is detected in Def6tr/tr mice (Fig. 4, E and F). These re- sults thus support the notion that aberrant IL-21 production by DEF6-deficient CD4+ T cells plays a key role in the exag- gerated B cell responses observed in mice that lack DEF6. Differential requirements for DEF6 and SWAP-70 in B cells during T cell–dependent humoral responses Given that the lack of DEF6 in CD4+ T helper cells appeared to be primarily responsible for the cytokine abnormalities de- tected within the T cell compartment, we next used WT or Def6tr/tr CD4+ T cells in a series of transfer experiments together with B cells purified from each of the four geno- types. Different combinations of WT and Def6tr/tr CD4+ T cells and resting B cells from WT, Def6tr/tr, Swap-70/, or DKO mice were transferred into Rag1/ mice, and, after appropriate reconstitution, the recipient mice were immu- nized with SRBCs. 7 d after immunization, the frequencies of PNA+ GC B cells and plasma cells were assessed by FACS. Transfer of either WT T cells or DEF6-deficient T cells with WT B cells led to comparable frequencies of GC B cells and plasma cells (Tables 1 and 2). Consistent with previous re- sults (Quemeneur et al., 2008), transfer of Swap-70/ B cells with WT T cells resulted in lower numbers of GC B cell, but increased numbers of plasma cells, as compared with WT B cells. Interestingly, transferring Swap-70/ B cells with IRF4 in autoimmunity | Biswas et al.