Although Tfh cells are normally viewed as the key source of IL-21 in the course of T cell–dependent B cell responses (Crotty, 2011), in autoimmune models such as the BXSB- Yaa and the MRL/lpr mice, one can observe the expansion of extrafollicular T helper cells, which express high levels of ICOS and IL-21 but do not express high-levels of CXCR5 (Odegard et al., 2008; Bubier et al., 2009). Because the DKO mice exhibited increased numbers of Tfh cells and an ex- panded population of CXCR5/loPD-1hiBcl-6int T cells that might represent extrafollicular T helper cells, this latter subset may also contribute to the aberrant production of IL-21 in the DKO mice. Interestingly, in some settings, PD-1 on Tfh cells can regulate the survival of GC B cells and the produc- tion of long-lived plasma cells (Good-Jacobson et al., 2010; Hams et al., 2011), raising the possibility that abnormal up- regulation of PD-1 by these extrafollicular T helper cells may also participate in promoting the aberrant humoral responses detected in the female DKO mice. Experiments are presently being conducted to gain additional insights into the precise localization of these distinct subsets in the lymphoid organs of DKO female mice. Although B cells express both DEF6 and SWAP-70, our studies now provide evidence that DEF6 and SWAP-70 do not appear to function in a redundant manner in this com- partment. Our in vivo findings support the idea that DEF6 regulates the early phases of activation that are triggered upon the encounter of an antigen-specific B cell with its cognate T helper cell. One of the key outcomes of these productive engagements, which are critically mediated by the CD40L– CD40 interaction and the production of cytokines, is the up-regulation of AID expression (Ramiro et al., 2007; Delker et al., 2009). Consistent with the known role of IRF4 in the control of AID expression (Klein et al., 2006; Sciammas et al., 2006), our studies demonstrate that DEF6 inhibits the ex- pression of AID by restricting the ability of IRF4 to bind to the Aicda promoter, suggesting that the DEF6–IRF4 inter- action is a new component of the complex regulatory net- works, which have recently been implicated in the control of AID expression (Park et al., 2009; Tran et al., 2010; Ise et al., 2011). Interestingly, the expression of IRF4 can be regulated by estrogen (Carreras et al., 2010), suggesting that deregula- tion of IRF4 activity could exert more profound pathogenic consequences in the female gender. Whether this effect plays a role in the known estrogen-mediated regulation of AID expression (Pauklin et al., 2009; Mai et al., 2010), and whether it contributes to the striking gender bias observed in the de- velopment of lupus-like disease in the DKO mice, however, remains to be established. Studies are now in progress to fur- ther delineate the role of estrogen in the pathophysiology that develops in DKO mice. Although DEF6 in B cells appears to primarily control the early phases of B cell differentiation during T cell–dependent responses, the presence of SWAP-70, as shown by these and previous studies (Quemeneur et al., 2008), is required for the proper control of terminal B cell differentiation in response to T cell–dependent antigens. In line with these results, we 592 have found that expression of DEF6 decreases in GC B cells and that plasma cells express lower levels of both DEF6 and SWAP70 (unpublished data). Our findings demonstrate that SWAP-70, similarly to DEF6, can physically interact with IRF4 and that this interaction is critical for the ability of SWAP-70 to regulate T cell–driven plasma cell differentia- tion because it restricts the ability of IRF4 to access the Prdm1 regulatory regions, thus limiting the expression of Blimp-1. Thus, in addition to the known requirement for high lev- els of IRF4 expression (Sciammas et al., 2006), plasma cells may also need to decrease the expression of SWAP- 70 to ensure that IRF4 is fully functional. We have re- cently observed that, in T cells, DEF6 can also regulate the phosphorylation of IRF4 by ROCK2 (Biswas et al., 2010), but addition of ROCK inhibitors did not affect the dif- ferentiation abnormalities observed in DEF6-deficient or SWAP-70–deficient B cells in our in vitro systems (un- published data). One of the striking features of B cells lacking either DEF6 or SWAP-70 is that these cells become hyperresponsive to IL-21. One of the key mechanisms responsible for this effect is that, in the absence of DEF6 or SWAP-70, deregulated binding of IRF4 to its target genes also results in enhanced recruitment of STAT3 upon exposure to IL-21. Thus, by in- hibiting IRF4, DEF6 and SWAP-70 can prevent its known capacity to recruit and/or cooperate with STAT3 (Kwon et al., 2009), and thus regulate the IL-21 responsiveness of B cells. Importantly, the differential ability of DEF6 and SWAP-70 to control IRF4 function at distinct stages of B cell differen- tiation can help ensure that IRF4, and subsequently STAT3, only access restricted sets of target genes that are appropriate for the specific differentiation step. This, in turn, can enable IL-21 to act in a stage-specific manner, and thus play a multi- faceted role in humoral responses. One of the major benefits of using a complex system of regulators and counter-regulators is that it can help ensure that only appropriately selected B cells proceed along specific differentiation pathways and that this process is tightly con- trolled in time and space. The remarkable alterations in splenic lymphoid architecture and the spontaneous development of a lupus-like syndrome in mice lacking both DEF6 and SWAP-70 support the idea that these two molecules are key compo- nents of the regulatory networks that ensure the precise orches- tration of these processes. Intriguingly, although the lack of DEF6 alone can result in the spontaneous development of auto- immune syndromes in other strains (Chen et al., 2008; Fanzo et al., 2006) it fails to do so in C57BL/6 mice, where the concomitant lack of SWAP-70 is required. Given the pro- found B cell abnormalities observed in the DKO mice, the known ability of B cells to promote T helper responses (Crotty, 2011), and the known differences in thresholds for maintain- ing B cell tolerance among strains (Putterman and Diamond, 1998), a likely scenario is that, although the lack of DEF6 alone predisposes toward autoimmunity, this predisposition can only convert to full-blown autoimmunity in the context of a “per- missive” B cell environment. Interestingly, Swap-70/ mice IRF4 in autoimmunity | Biswas et al.