Defects in germinal center B cells and T_FH cells in Batf^−/− mice. (a) Germinal center B cells in spleen or Peyer’s patches from unimmunized or SRBC-immunized mice were stained with anti-B220, anti-GL7, and anti-Fas. Shown are two-color histograms gated on B220⁺ cells. Numbers indicate the percentage Fas⁺GL7⁺ cells in the indicated gate. Data are representative of three experiments. (b) Splenocytes were stained on day 7 for CD4 and CXCR5 from mice unimmunized mice or mice immunized with SRBC. Data are representative of three experiments. (c) Flow cytometry of CD4 and CXCR5 expression in spleens of SJL mice that received transfers of either CD4⁺CD62L⁺ CD45.2⁺ cells from Batf^+/+ and Batf^−/− mice and immunized with SRBC 7 days before staining. The data are gated on CD45.2⁺ cells. Data are representative of two experiments.

Batf directly binds to conserved elements in the c-Maf and Bcl-6 loci in vivo. (a) Purified Batf^+/+ or Batf^−/− CD4⁺ T cells were cultured with anti-CD3 and anti-CD28 with IL-6 for 24 h and treated with PMA/ionomycin for last 4 hours. Nuclear extracts were analyzed by EMSA using a consensus AP-1 probe². Annealed double-stranded oligonucleotides of 29 to 32 bp in length from the indicated regions of c-Maf locus were used as competitors as described². Data are representative of two experiments. (b) CD4⁺ T cells were stimulated as in a and ChIP analysis of the indicated regions was performed using anti-BATF antibody². *P<0.05 and **P<0.005 (unpaired student t-test). Data are from two independent experiments (mean and s.e.m.). (c) EMSA was performed as in a using annealed double-stranded oligonucleotides from the indicated regions of Bcl-6 locus as competitors. Data are representative of two experiments. (d) CD4⁺ T cells were stimulated as in a and ChIP analysis of the indicated regions of the Bcl-6 locus was performed. *P<0.05 and **P<0.005 (unpaired student t-test). Data are from two independent experiments (mean and s.e.m.). (e) CD4⁺ T cells were cultured with anti-CD3 and anti-CD28 in the presence of IL-6, anti-IL-4, anti-IFN-γ, and anti-TGF-β and infected with hCD4-pA GFP-RV or hCD4-pA GFP-Bcl-6-promoter-RV. hCD4 and GFP expression was examined on day 3 after stimulation. Shown are the percentages of GFP⁺ cells within the hCD4⁺ cell gate. *P<0.05(unpaired student t-test). Data are from two independent experiments (mean and s.e.m.). (f) Phoenix E cells were transfected with the Bcl-6 reporter plasmid hCD4-pA GFP-Bcl-6-promoter-RV, or Bcl-6 reporter plasmid containing the indicated 5′ conserved regions of Bcl-6 locus (ECR1, ECR2, ECR3, ECR4). hCD4 and GFP expression was examined 2 days after transfection. Shown is a single color histogram of GFP expression for hCD4⁺ cells. Data are representative of three experiments.

Co-expression of Bcl-6 and c-Maf partially restores T_FH activity in Batf^−/− T cells. CD4⁺ T cells purified from Batf^+/+ mice (a, b) or Batf^−/− mice (c, d) were activated with anti-CD3 and anti-CD28 and infected with the indicated retroviruses. Infected T cells were mixed with BALB/c B cells and transferred into Rag2^−/− mice subsequently immunized with SRBC. CXCR5 expression by GFP⁺CD4⁺ T cells and Fas and GL7 expression by B was analyzed by FACS at 7 days after immunization. (a) FACS analysis for CD4 and CXCR5 (upper panels) expression by Batf^+/+ T-cells infected with the empty retrovirus GFP-RV (Empty), BATF-expressing (BATF) or Bcl-6-expressing (Bcl-6) retrovirus. Data shown is gated on GFP⁺ cells. FACS analysis for Fas and GL7 (lower panels) gated on co-transferred B220⁺ cells. Data are representative of two experiments. Transferred T cells were between 60% to 80% were GFP⁺. (b) Frequency of CXCR5⁺ Batf^+/+ CD4⁺ T cells (left) or Fas⁺GL7⁺ B220⁺ cells (right) 7 days after immunization. **P<0.005, versus empty-RV control (unpaired student t-test). NS, not significant. Data are from two independent experiments (mean and s.e.m.). (c) FACS analysis of CD4 and CXCR5 (upper panels) expression by Batf^−/− T cells infected with the indicated retroviruses; GFP-RV (Empty); BATF-GFP; Bcl-6-GFP; c-Maf-GFP; CD40L-GFP; Bcl-6-hCD4 and c-Maf-GFP; or Bcl-6-hCD4 and CD40L-GFP RV. Data shown is gated on GFP⁺ cells (for single infections) or GFP⁺hCD4⁺ cells (for double infections). FACS analysis of Fas and GL7 expression (lower panels) is gated on B220⁺ cells. Data are representative of two experiments. In double retroviral infections, roughly 50% of transferred T cells were hCD4⁺GFP⁺. (d) Frequency of CXCR5⁺ Batf^+/+ CD4⁺ T cells (left) or Fas⁺GL7⁺ B220⁺ cells (right) 7 days after immunization. *P<0.05 and **P<0.005, versus empty-RV control (unpaired student t-test). NS, not significant. Data are from two independent experiments (mean and s.e.m.).

Batf directly regulates AID mRNA expression. (a) Relative expression levels of genes involved in class switch recombination determined by microarray analysis of Batf^+/+ or Batf^−/− B cells activated for 2 days with LPS. Expression is shown as a ratio of expression in Batf^−/− B cells compared to Batf^+/+ B cells. Data are from one microarray experiment. (b) Quantitative RT-PCR analysis of AID mRNA expression in Batf^+/+ or Batf^−/− B-cells stimulated for 2 or 4 days with LPS. Results are normalized to hprt expression and are presented as arbitrary unit (AU). *P<0.05 (unpaired student t-test). Data are from three independent experiments. (c) EMSA analysis was performed using B-cell nuclear extract from Batf^+/+ B cells (WT) or Batf^−/− B cells (KO) stimulated with LPS plus IL-4 for 24 hours and either a consensus AP-1 probe or +17 kb (2) AID probe (Supplementary Table 2) in the presence (+) or absence (−) of anti-BATF antibody. (d) B cells were stimulated as in c and ChIP performed with DNA precipitated using anti-BATF antibody and amplified using primers specific to −1.5 kb or +17 kb regions of AID locus. *P<0.05 (unpaired student t-test). NS, not significant. Data are from two independent experiments (mean and s.e.m.). (e) ChIP was performed as in d but DNA was precipitated using anti-acetylated histone H3. ***P<0.005 (unpaired student t-test). NS, not significant. Data are from two independent experiments (mean and s.e.m.). (f) FACS analyses (left) was performed on wild-type, Batf^−/− or AID^−/− B cells on the C57/BL6 background, activated with LPS and IL-4 and infected with the indicated retrovirus, and analyzed after 3 days. Data are representative data of two experiments. Frequency (right) of IgG1 positive B220⁺ B cells as determined in (f) from three biological replicates (mean and s.e.m.). *P<0.05, **P<0.01, and ***P<0.005, versus empty-RV control value (unpaired student t-test). NS, not significant.

Batf is required for c-Maf and Bcl-6 expression by in vitro activated T cells. (a) Relative expression levels of the indicated genes involved in follicular helper T cell development or function were determined for Batf^−/− T cells by microarray analysis of Batf^−/− and Batf^+/+ CD4⁺ T cells stimulated for 3 days with anti-CD3 and anti-CD28 in the presence of IL-6, anti-IL-4, anti-IFN-γ, and anti-TGF-β. Relative values are presented from one microarray experiment with the expression in Batf^+/+ CD4⁺ T cells defined as 1. (b) Quantitative RT-PCR analysis of c-Maf, Bcl-6, Blimp-1, CXCR5, IL-21, and IRF4 mRNA expression in Batf^+/+ or Batf^−/− T cells stimulated as in a. Results are normalized to HPRT mRNA expression and are presented as arbitrary unit (AU). *P<0.05 (unpaired student t-test). Data are combined from three independent experiments (mean and s.e.m.).

Batf is required for optimal CD40 ligand expression. (a) Purified naïve Batf^+/+ or Batf^−/− CD4⁺ T cells were cultured with anti-CD3 and anti-CD28 for 12 h (CD40L analysis) or 24 h (ICOS and OX40 analysis) and analyzed by FACS. Shown are single color histograms for the indicated protein gated on CD4⁺ cells. Shaded histogram represents isotype control staining. Data are representative of three experiments. (b) CD40L expression by cells as described in (a) was analyzed by FACS at 3, 6, 12, and 24 hour after stimulation. Shown is the percentage of CD40L⁺ cells (left) and Mean fluorescence intensity (MFI) (right). *P<0.05, **P<0.01, and ***P<0.005 (unpaired student t-test). NS, not significant. Data are from three independent experiments (mean and s.e.m.). (c) CD40L expression by cells as described in (a) was analyzed by real-time PCR at 1, 3, 6, 12, and 24 hour after stimulation. *P<0.05 and ***P<0.005 (unpaired student t-test). NS, not significant. Data are from two independent experiments (mean and s.e.m.). (d) EMSA was performed as described in Figure 3a using the indicated double-stranded oligonucleotides from the CD40L locus as competitors. Data are representative of two experiments.

Impaired class switching in Batf^−/− B cells. (a) FACS analysis for IgA, IgM and B220 expression by lymphocytes from Peyer’s patches (PP) or Lamina propria (LPL) of Batf^+/+ or Batf^−/− mice was performed. Data are representative of two experiments. (b) Quantitative RT-PCR analysis of post-switched Iμ-C_H transcripts (PST) in Batf^+/+ or Batf^−/− B-cells stimulated for 4 days as in Supplementary figure 10a, b. Expression is normalized to hprt expression and is presented relative to the expression in Batf^+/+ B cells, set at 1. **P<0.0001 (unpaired student t-test). Data are from three independent experiments. (c) Digestion-circularization PCR assay of genomic DNA isolated from Batf^+/+ or Batf^−/− B-cells unstimulated (d0) or stimulated with LPS plus IL-4 for 4 days (d4) for measurement of Sμ-Sγ1 recombination. Data are representative of two experiments.

Batf is required for germline transcription. (a) Quantitative RT-PCR analysis was carried out for the indicated germline I_H-C_H transcripts (GLT) in Batf^+/+ (black) or Batf^−/− (white) B cells stimulated for 2 days as in Fig. 6d. Expression is normalized to hprt expression and is presented as relative to the expression in Batf^+/+ B cells defined as 1. ***P<0.005 and ****P<0.0001 (unpaired student t-test). NS, not significant. Data are from three independent experiments. (b) Batf^+/+ or Batf^−/− B cells were activated with LPS, infected with empty-GFP RV (white) or BATF-GFP retrovirus (black), infected GFP⁺ cells purified by sorting and cultured for 2 days with LPS plus IFN-γ (γ2a GLT), or LPS plus TGF-β (γ2b GLT and αGLT) as indicated, and then quantitative RT-PCR analysis for the indicated germline I_H-C_H transcripts (GLT) was carried out. Expression is normalized to hprt expression. *P<0.05 and **P<0.01 (unpaired student t-test). NS, not significant. Data are from two independent experiments. (c) Batf^+/+ or Batf^−/− B cells were stimulated with αCD40 plus IL-4 and TGF-β and infected with either the empty reporter virus (hCD4-GFP) or the Iα promoter-reporter virus (hCD4-GFP-αp). FACS analysis for hCD4 and GFP expression on day 4 after stimulation is shown. Numbers represent the percentages of cells in indicated gate. (d, e) ChIP analysis of BATF binding to the indicated I promoters (d) or indicated regions of the 3′ IgH enhancer (e) in Batf^+/+ (black) or Batf^−/− (white) B cells cultured for 2 days with LPS. *P<0.05 and **P<0.01 (unpaired student t-test). NS, not significant. Data are from two independent experiments.