on a 129/Sv background developed autoantibodies with age (Borggrefe et al., 2001) suggesting that the absence of SWAP-70 alone can also predispose to autoimmunity in spe- cific genetic settings. The concomitant absence of DEF6 and SWAP-70 altered some of the B cell phenotypes detected in mice lacking SWAP-70 alone. Indeed, in contrast to Swap-70/ mice, which exhibit decreased GC formation upon TD immunizations (Quemeneur et al., 2008), DKO mice dis- played enhanced GC formation. In view of our findings that SWAP-70 inhibits the ability of IRF4 to up-regulate Blimp-1, B cells lacking SWAP-70 alone would be pre- dicted to prematurely up-regulate Blimp-1 upon exposure to T cell help and might thus preferentially commit to an extrafollicular plasma cell fate. Such a commitment would be modified by the simultaneous absence of DEF6 because the lack of DEF6 imparts B cells with an increased capability of becoming a GC B cell. Thus, consistent with the phenotype of the DKO, the simultaneous absence of DEF6 and SWAP-70 would lead to both increased GC formation and plasma cell differentiation upon exposure to TD antigens. Although our studies have mostly focused on the inhibi- tory roles of DEF6 and SWAP-70 on lymphocyte function, it is important to note that DEF6 and SWAP-70 do not ex- clusively function as negative regulators of T and B cell activation. For instance although DEF6 restricts the ability of T cells to produce IL-17 and IL-21 under neutral condi- tions (Chen et al., 2008; Biswas et al., 2010), it promotes Th17 differentiation upon exposure to a proinflammatory environment (Canonigo-Balancio et al., 2009). A similar complex role is emerging for SWAP-70. Although, as dem- onstrated here and in a previous study (Quemeneur et al., 2008), it functions as an inhibitor of plasma cell differentia- tion under T cell–dependent conditions, it can promote the initiation of plasma cell differentiation under T cell– independent conditions (Chopin et al., 2011). Interestingly, this effect appears to be caused by the ability of SWAP-70 to fine-tune IRF4 expression, highlighting the intricate cross-talk between IRF4 and this family of molecules. In ad- dition to regulating gene expression, DEF6 and SWAP-70 can also regulate cytoskeletal reorganization as a result of their ability to control the activation of Rac and Cdc42 (Shinohara et al., 2002; Gupta et al., 2003a; Bécart et al., 2008). The employment of molecules like DEF6 and SWAP-70, which can simultaneously control gene expres- sion programs and cytoskeletal dynamics, might thus provide lymphocytes with a unique molecular system to precisely couple the acquisition of specific differentiation programs with their localization and even with their antigen respon- siveness. These molecules thus likely do not act simply as “inhibitors” of lymphocyte activation but rather might function as “sensors,” helping ensure that lymphocyte acti- vation is regulated in a dynamic and plastic manner, and thus enabling immune responses to be appropriately tailored to the offending pathogen. JEM Vol. 209, No. 3 A r t i c l e MATERIALS AND METHODS Mice. DEF6-deficient (Def6tr/tr) mice were generated by Lexicon Pharma- ceuticals (OmniBank) using a gene trapping strategy as previously described (Fanzo et al., 2006). These mice were backcrossed onto a C57BL/6 background for >10 generations. Swap-70/ mice were generated by R. Jessberger (In- stitute of Physiological Chemistry, Dresden University of Technology, Dresden, Germany; Borggrefe et al., 2001) and were also backcrossed onto a C57BL/6 background for >10 generations. Irf4/ mice on a C57BL/6 background were obtained from T. Mak at University of Toronto (Toronto, Canada; Mittrücker et al., 1997). To generate Def6tr/trSwap-70/ (DKO) mice, Def6tr/tr mice were crossed with Swap70/ mice that had been back- crossed onto the C57BL/6 background. To generate Def6tr/trIrf4/ and Swap-70/Irf4/, Irf2/ mice were crossed with either Def6tr/tr or Swap- 70/ mice, respectively. IL21/ mice on mixed strain background were obtained from the Mutant Mouse Regional Resource Centers (Lexicon strain ID 011723-UCD), and then crossed with Def6tr/tr129/SvJ mice to generate Def6tr/trIL21/ mice. All mice used in the experiment were kept under specific pathogen–free conditions. The experimental protocols were approved by the Institutional Animal Care and Use Committee of Columbia University and The Hospital for Special Surgery. Flow cytometry. Single-cell suspensions from thymus, spleen, and lymph nodes were isolated, stained with Biotin- or Fluorochrome-conjugated CD3e (eBioscience), CD4 (eBioscience), CD8 (eBioscience), CD25 (eBio- science), CD44 (eBioscience), CD62L (eBioscience), CD69 (eBioscience), ICOS (eBioscience), PD-1 (eBioscience), CXCR5 (eBioscience), Foxp3 (eBioscience), B220 (eBioscience), CD21 (BD), CD23 (BD), IgM (eBiosci- ence), IgD (eBioscience), CD138 (BD), IgG1 (eBioscience), and PNA (Jackson ImmunoResearch Laboratories) and analyzed by FACS. To enumerate the number of NP-specific B cells in spleen, spleen cells were stained with NP-PE (Biosearch Technologies) 7 and 14 d after NP-KLH immunization, as previously described (Quemeneur et al., 2008). Data were analyzed using FlowJo (Tree Star) software, as previously described (Chen et al., 2008). For intracellular staining, splenocytes were stimulated with 50 µg/ml PMA and 1 µM Ionomycin (EMD) in the presence of 1 µM Monensin (eBioscience) for 5 h. After stimulation, cells were subjected to intracellular staining for IL-17 and IL-21 cytokines using anti–mouse IL-17 PE (BD) and anti– human IL-21R Fc (R&D Systems) antibodies. FITC-conjugated anti– human antibody (Jackson ImmunoResearch Laboratories) was used as a secondary antibody. Immunizations. In SRBC immunizations, 8–10-wk-old female mice were immunized once intraperitoneally with 1 × 105 sheep red blood cells in PBS (Cocalico Biologicals), as per standard protocol (Klein et al., 2006). Control mice received PBS only. 7 d after immunization, control and immunized mice were sacrificed and spleens were harvested for flow cytometric analysis. In NP-KLH immunizations, 8–10-wk-old female mice were immunized once intraperitoneally with NP-KLH (100 µg per mouse; Biosearch Tech- nologies) in complete Freund’s adjuvant, as per standard protocol (Klein et al., 2006). 7 and 14 d after immunization, control and immunized mice were sacri- ficed and spleens and bone marrow were analyzed for anti–NP-KLH re- sponse. ELISPOT assays were performed as previously described (Quemeneur et al., 2008). Histopathology, immunohistochemical, and immunofluorescence staining. Tissue specimens were fixed in 10% neutral buffered formalin and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin (H&E) and analyzed by light microscopy.The severity of glomerulo- nephritis was assessed as previously described (Atkinson et al., 2008). Immuno- fluorescence analysis on frozen kidney sections was performed by staining with FITC-labeled goat anti–mouse IgG or anti-C3 (Jackson ImmunoResearch Laboratories) and specimens were analyzed with a LSM 510 laser scanning confocal microscope (Carl Zeiss, Inc.). The severity of immune complex deposition was determined in a blinded manner according to previously described parameters (Atkinson et al., 2008). 593