For immunofluorescence staining of the spleens, the spleen tissue was embedded in Tissue-tek (OCT compound; Sakura) and snap-frozen. 8-µm cryostat sections were air dried for an hour before staining. The sections were fixed for 10 min with ice-cold acetone, and then incubated for 1 h at room temperature with the primary antibody (biotinylated peanut agglutinin [Vector Laboratories], rabbit anti–mouse IgG (H+L)-biotin [Southern Bio- tech], rat anti–mouse IgM-biotin [BD], and PE anti–mouse CD45R/B220 [BD]). Primary antibodies were diluted in PBS/0.5% BSA. Sections were then incubated for 1 h with Alexa Fluor 488–conjugated Streptavidin (Invit- rogen) before mounting in Gel/Mount (Biomeda). Samples were analyzed using a microscope (Eclipse E600; Nikon) and images were captured by Q capture software (version 2.68.4). The channels were overlaid using Photo- shop software (Adobe). Serum autoantibodies. Serum levels of anti-dsDNA were determined by ELISA (Alpha Diagnostics) as previously described (Chen et al., 2008; Fanzo et al., 2006). Autoantibody array was performed in UTSW Genomics and Microarray Core Facility as previously described (Li et al., 2005). For intergroup comparisons, the Student’s t test was used. Heat map diagrams with row-wise and column-wise clustering were generated using Cluster and TreeView software. In these diagrams, fluorescence intensities that were higher than the row mean were colored red, those that fell below the row mean were colored green, and cells with signals close to the mean were left black. In vitro B cell assays. Resting B cells (CD43 B cells) were purified from mice by negative selection using CD43 microbeads (Miltenyi Bio- tec). B cell assays were performed as previously described (Kuchen et al., 2007). In brief, for short stimulation, B cells were incubated with 10 µg/ml goat anti–mouse IgM F(ab)2 (Jackson ImmunoResearch Laboratories) at 4°C for 30 min. Unbound anti-IgM was removed from the medium by washing and centrifuging the cells at 4°C. The cells were resuspended in chilled complete medium and shifted to 37°C by placement in an incuba- tor or in water bath. 4 h later, 5 µg/ml monoclonal anti-CD40 (BD) in the presence or absence of 50 ng/ml mouse IL-21 (R&D Systems) was added, and the cells were further incubated for various time periods depending on the assay. For plasma cell studies, B cells (106/well) were stimulated with IgM (5 µg/ml; Jackson ImmunoResearch Laboratories) and CD40 (2 µg/ml) for 96 h in the presence or absence of recombinant mouse IL-21 (50 ng/ml). For proliferation assays, cultures were pulsed with [3H]thymidine (1 µCi/well; PerkinElmer) for the last 12 h of culture. In vitro differentia- tion of plasma cells was determined by FACS after staining with anti-CD138 antibody (BD). Real-time RT-PCR. Total RNA was isolated from cells or organs using RNeasy Mini kit (QIAGEN). cDNAs were prepared and analyzed for the expression of the gene of interest by real-time PCR using a SYBR Green PCR master mix kit (Applied Biosystems). The primers for Prdm1, Aicda were purchased from QuantiTect Primer Assay (QIAGEN). The expression of each gene was normalized to the expression of -actin. Cell extracts, Western blotting, and coimmunoprecipitation. Nuclear and cytoplasmic extracts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagent kit, as previously described (So et al., 2006). The purity of the nuclear and cytoplasmic fractions was verified by probing with antibodies against lamin B (Santa Cruz Biotechnology, Inc.) and -tubulin (Sigma-Aldrich). For coimmunoprecipitation experiments, cell lysates were immunoprecipitated with an anti-IRF4 antibody, as previ- ously described (Chen et al., 2008). The immunoprecipitates were resolved by 8% SDS-PAGE, transferred to a nitrocellulose membrane, and then im- munoblotted with either an anti–SWAP-70 antibody (Santa Cruz Biotech- nology, Inc.) or anti-DEF6 antiserum (Chen et al., 2008). For the AID Western blots, the blots were probed with an anti-AID antibody, which was a gift from J. Chaudhuri (Memorial Sloan-Kettering Cancer Center, New York, NY; Chaudhuri et al., 2003). 594 ChIP assays. ChIP assays were performed as previously described (Chen et al., 2008). In brief, CD43 B cells were purified and either left unstimulated or stimulated for 48 h. After harvesting, the cells were cross-linked with formaldehyde, and chromatin extracts were prepared by standard methods. The DNA–protein complexes were immunoprecipitated with an IRF4- specific antibody or a control antibody. After cross-linking was reversed and proteins were digested, the DNA was purified from the immunoprecipitates as well as from input extracts, and then analyzed by quantitative PCR using primers specific for the murine AICDA promoter (Lee et al., 2006), and the IL-21 response element in the Prdm1 gene (Kwon et al., 2009; forward, 5-AACCGTTGAAAGACGGTGACTGA-3; reverse, 5-CACATCATC- TGTGCTGGAGGCA-3) as previously described (Chen et al., 2008). Adoptive transfer experiments. For transfer experiments, total CD4+ T cells purified from WT and Def6tr/tr mice were mixed with CD43 B cells from all the four genotypes (WT, S70/, Def6tr/tr, and DKO) at 1:2 ratio and transferred in Rag1/ recipient mice. 21 d after transfer, recipient mice were checked for successful reconstitution followed by immunization with 2% SRBC. 7 d after immunization, spleens of recipient mice were analyzed for B cell response to SRBC immunization. We would like to thank Dr. Jayantha Chaudhuri for the generous gift of the AID antibody. The research was supported by the Alliance for Lupus Research, the Mary Kirkland Center for Lupus Research, and grants from the National Institutes of Health (HL62215 and AI076474 to A. Pernis). The authors have no conflicting financial interests. Submitted: 13 June 2011 Accepted: 23 January 2012 REFERENCES Atkinson, C., F. Qiao, H. Song, G.S. Gilkeson, and S. Tomlinson. 2008. Low-dose targeted complement inhibition protects against renal disease and other manifestations of autoimmune disease in MRL/lpr mice. J. Immunol. 180:1231–1238. Bécart, S., A.J. Balancio, C. Charvet, S. Feau, C.E. Sedwick, and A. Altman. 2008. Tyrosine-phosphorylation-dependent translocation of the SLAT protein to the immunological synapse is required for NFAT tran- scription factor activation. Immunity. 29:704–719. http://dx.doi.org/ 10.1016/j.immuni.2008.08.015 Biswas, P.S., S. Gupta, E. Chang, L. Song, R.A. Stirzaker, J.K. Liao, G. Bhagat, and A.B. Pernis. 2010. Phosphorylation of IRF4 by ROCK2 regulates IL-17 and IL-21 production and the development of auto- immunity in mice. J. Clin. Invest. 120:3280–3295. http://dx.doi.org/ 10.1172/JCI42856 Borggrefe, T., M. Wabl, A.T. Akhmedov, and R. Jessberger. 1998. A B-cell-specific DNA recombination complex. J. Biol. Chem. 273:17025– 17035. http://dx.doi.org/10.1074/jbc.273.27.17025 Borggrefe, T., L. Masat, M. Wabl, B. Riwar, G. Cattoretti, and R. Jessberger. 1999. Cellular, intracellular, and developmental expres- sion patterns of murine SWAP-70. Eur. J. Immunol. 29:1812–1822. http://dx.doi.org/10.1002/(SICI)1521-4141(199906)29:06<1812:: AID-IMMU1812>3.0.CO;2-J Borggrefe, T., S. Keshavarzi, B. Gross, M. Wabl, and R. Jessberger. 2001. Impaired IgE response in SWAP-70-deficient mice. Eur. J. Immunol. 31: 2467–2475. http://dx.doi.org/10.1002/1521-4141(200108)31:8<2467:: AID-IMMU2467>3.0.CO;2-P Brüstle, A., S. Heink, M. Huber, C. Rosenplänter, C. Stadelmann, P. Yu, E. Arpaia, T.W. Mak, T. Kamradt, and M. Lohoff. 2007. The develop- ment of inflammatory T(H)-17 cells requires interferon-regulatory factor 4. Nat. Immunol. 8:958–966. http://dx.doi.org/10.1038/ni1500 Bubier, J.A., T.J. Sproule, O. Foreman, R. Spolski, D.J. Shaffer, H.C. Morse III, W.J. Leonard, and D.C. Roopenian. 2009. A critical role for IL-21 receptor signaling in the pathogenesis of systemic lupus ery- thematosus in BXSB-Yaa mice. Proc. Natl. Acad. Sci. USA. 106:1518– 1523. http://dx.doi.org/10.1073/pnas.0807309106 IRF4 in autoimmunity | Biswas et al.