A r t i c l e Figure 4. Aberrant production of IL-21 by DEF6-deficient T cells plays a key role in the humoral abnormalities observed in mice lacking DEF6. (A) Purified CD4+ T cells from 8–10-wk-old WT, S70/, Def6tr/tr, and DKO female mice were subjected to a primary stimulation, and then restimulated with CD3 and CD28 for 48 h. Supernatants were then collected and assayed for IL-17 (left), IL-21 (middle), and IFN- (right) production by ELISA. The data are representative of three independent experiments. The error bars rep- resent mean ± SD. (B) Splenocytes from 24-wk-old WT, S70/, Def6tr/tr, and DKO fe- male mice were stimulated with PMA (50 µg/ml) and Ionomycin (1 µM) for 5 h. After stimula- tion, cells were subjected to intracellular staining for IL-17 and IL-21, and then ana- lyzed for cytokine production by flow cytom- etry. The data are representative of three independent experiments. (C–F) 8–10-wk-old female WT, Def6tr/tr, IL-21/, and Def6tr/tr IL-21/ mice were immunized with SRBC (n = 4–7/group). Unimmunized control mice (not depicted) received PBS only. At day 7 after immunization, splenocytes were ana- lyzed for numbers of B220+PNA+ cells (C), B220loCD138+ cells (D), CD4+CD62LloCD44hi cells (E), and CD4+ICOS+ cells (F). Each dot represents individual mice and horizontal bars indicate mean for each group. The data are representative of two independent experi- ments. *, P < 0.05; **, P < 0.01; ***, P < 0.005. Def6tr/tr T cells rather than WT T cells further increased their ability to differentiate into plasma cells. In contrast to the findings with Swap-70/ B cells, Def6tr/tr B cells exhibited an enhanced ability to differentiate into GC B cells when co- transferred with Def6tr/tr T cells, whereas the differentiation of Def6tr/tr B cells into plasma cells was similar to that observed in WT B cells. Strikingly, DKO B cells exhibited an increased capacity to form both GC B cells and plasma cells, which was particularly evident when these cells were co-transferred with Def6tr/tr T cells rather than WT T cells. Collectively, these studies suggest that the absence of DEF6 in B cells plays a key role in driving the aberrant GC formation observed in DKO mice, whereas the major contribution of SWAP-70 defi- ciency to the B cell abnormalities that occur in the DKO mice occurs during the terminal differentiation of B cells into plasma cells, and that both of these abnormalities are mark- edly augmented by the lack of DEF6 in T cells. DEF6 regulates IL-21 responsiveness of antigen-engaged B cells by controlling IRF4 function The transfer experiments suggested that the absence of DEF6 in B cells leads to an enhanced capacity of these cells to respond to help provided by T cells during their initial encounter at the T–B cell border. One of the critical features of such productive JEM Vol. 209, No. 3 interactions is the ability of B cells to up-regulate the expres- sion of AID (Ramiro et al., 2007; Delker et al., 2009). To in- vestigate whether the lack of DEF6 in B cells results in aberrant AID induction upon exposure to T cell signals, we used a re- cently developed in vitro culture system in which a single- round IgM signal is provided to B cells (Damdinsuren et al., 2010). Such a system is believed to more closely resemble the short-term physiological exposure to antigen encountered in vivo by B cells that have migrated to the T–B border. Ac- cordingly, purified resting B cells (CD43) from 8–10-wk-old WT, Swap-70/, Def6tr/tr, or DKO female mice were stimu- lated with a single round of anti-IgM treatment, followed by anti-CD40 stimulation (Fig. 5 A). Given that increased pro- duction of IL-21 by Def6tr/tr T cells plays a crucial role in this system, these stimulations were conducted in the presence or absence of IL-21. Under these culture conditions, B cells from Def6tr/tr mice expressed higher levels of Aicda mRNA as com- pared with WT B cells, an effect that was augmented by the presence of IL-21. Consistent with the transfer experiments, DKO B cells expressed a pattern of abnormalities similar to those observed in Def6tr/tr B cells, whereas Swap-70/ B cells behaved similarly to WT B cells. Western blotting experiments with an anti-AID antibody confirmed the aberrant induc- tion of AID observed in the absence of DEF6, which was 587