The AP-1 transcription factor Batf controls T(H)17 differentiation

Abstract

Activator protein 1 (AP-1, also known as JUN) transcription factors are dimers of JUN, FOS, MAF and activating transcription factor (ATF) family proteins characterized by basic region and leucine zipper domains. Many AP-1 proteins contain defined transcriptional activation domains, but BATF and the closely related BATF3 (refs 2, 3) contain only a basic region and leucine zipper, and are considered to be inhibitors of AP-1 activity. Here we show that Batf is required for the differentiation of IL17-producing T helper (T(H)17) cells. T(H)17 cells comprise a CD4(+) T-cell subset that coordinates inflammatory responses in host defence but is pathogenic in autoimmunity. Batf(-/-) mice have normal T(H)1 and T(H)2 differentiation, but show a defect in T(H)17 differentiation, and are resistant to experimental autoimmune encephalomyelitis. Batf(-/-) T cells fail to induce known factors required for T(H)17 differentiation, such as RORgamma t (encoded by Rorc) and the cytokine IL21 (refs 14-17). Neither the addition of IL21 nor the overexpression of RORgamma t fully restores IL17 production in Batf(-/-) T cells. The Il17 promoter is BATF-responsive, and after T(H)17 differentiation, BATF binds conserved intergenic elements in the Il17a-Il17f locus and to the Il17, Il21 and Il22 (ref. 18) promoters. These results demonstrate that the AP-1 protein BATF has a critical role in T(H)17 differentiation.

Figure 1. Loss of IL-17 production in Batf^−/− T cells

a, DO11.10⁺CD4⁺ T cells from CD2-N-FLAG-Batf transgenic mice or littermates were cultured with OVA/APCs under T_H2 conditions for 7 days, and stained with antibodies to CD4 and FLAG. b,Batf^+/+ and Batf^−/− CD4⁺CD62L⁺CD25 T cells cultured under T_H17 conditions were restimulated with PMA/ionomycin on days 7 (left panel) or 3 (middle and right panels) and stained for IL-17, IFN-γ, IL-2 and IL-10. c, IL-17 and IFN-γ expression in DO11.10⁺CD4⁺ T cells from Batf^+/+Batf ^+/− and Batf^−/− mice activated with OVA/APCs under T_H17 conditions. Data are representative of at least 2 independent experiments.

Figure 2. Batf^−/− mice are resistant to EAE

a,Batf^+/+ (n=12) and Batf^−/− (n=13) mice were immunized with MOG_33–35 peptide. (Mean clinical EAE scores +/− s.e.m, representative of two independent experiments). b, 13 days after EAE induction, CNS-infiltrating lymphocytes were stimulated with PMA/ionomycin, gated on CD4⁺ cells and stained for intracellular IL-17 and IFNγ (Clinical scores are in parentheses, data are representative of 2–3 mice per group). c,Batf^+/+ and Batf^−/− mice were injected with control PBS buffer (n=5) or 1×10⁷Batf^+/+ CD4⁺ T cells (n=6) four days prior to EAE induction. Mean clinical scores are shown.

Figure 3. Batf controls multiple T_H17-associated genes

a, IL-21 expression in Batf^+/+ or Batf^−/− T cells cultured under T_H17 conditions determined by qRT-PCR and ELISA. (mean + s.d. 3 mice). b, IL-17 and IFN-γ expression of CD4⁺CD62L⁺CD25⁻ T cells cultured in a in the presence or absence of IL-21. c, Microarray analysis of anti-CD3/CD28-activated T cells at 72h, presented as heat maps of genes 5-fold-induced in Batf^+/+ T cells under T_H17 conditions. d, IL-17 and IL-22 expression in Batf^+/+ or Batf^−/− CD4⁺ T cells activated under T_H17 conditions for 3 days. e, Anti−CD3/CD28-activated Batf^+/+ or Batf^−/− CD4⁺ T cells were left uninfected or infected with RORγt-GFP-RV or control-GFP-RV, and stained for IL-17.

Figure 4. Batf directly regulates IL-17 expression

a,Batf^+/+ and Batf^−/− CD4⁺ T cells cultured under T_H17 conditions were infected with hCD4-pA-GFP-RV-IL-17p reporter virus. GFP expression after PMA/ionomycin restimulation is shown. b, Batf^+/+ and Batf^−/− CD4⁺ T cells cultured under T_H17 conditions for 5 days were subjected to ChIP analysis of the indicated regions using anti-Batf antibody (mean + s.d.). c, d, f, EMSA supershift analysis of T_H17 whole cell extracts using a consensus AP-1 (c, f) or the IL-17_{(−155 to − 187)} probe (d). (Batf^+/+ (WT), Batf^−/− (KO), CD2-N-FLAG-Batf transgenic (TG), IL-17_{(−155 to − 187)} and RORE probes were used as competitors).