Fig 2: ATP-Linked OXPHOS Controls BATF Induction by Regulating TCR Signaling(A and B) Naive CD4 T cells were differentiated under Th1 or Th2 polarizing conditions in the presence of vehicle or oligomycin for 24 h, and expression of (A) BATF, IRF4, and (B) GATA3 was determined by flow cytometry (4 to 5 independent experiments).(C and D) Naive CD4 T cells from Nur77/GFP spleens were activated under Th17 conditions in the presence of vehicle or oligomycin for indicated period of time.(C) Graph shows the expression of Nur77/GFP. (D) Representative plots are gated on live CD4 T cells (representative of 3 independent experiments).(E) Naive CD4 T cells were activated under Th17 conditions with vehicle or oligomycin. Representative histogram overlay shows the phosphorylation of ZAP70 at 24 h (representative of 5 independent experiments).(F) Naive CD4 T cells from Nur77/GFP reporter spleens were stimulated with Th17 cytokines for 6 h. BATF expression in GFP+ and GFP− cells were determined by flow cytometry (3 independent experiments).Graphs show the average ± SD; (A and C) two-way ANOVA; (B, E and F) unpaired t test *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: Mitochondrial OXPHOS Regulates Th17 Differentiation by Promoting BATF Expression(A and B) Naive CD4 T cells were activated under Th17 conditions in the presence of vehicle or oligomycin for 24 h. Cells were then washed and cultured with Th17-differentiating cytokines for an additional 48 h. (A) Experimental design. (B) Frequency of IL-17A+ and Foxp3+ live CD4 T cells was determined by flow cytometry (4 independent experiments).(C and D) Naive CD4 T cells were activated under Th17 conditions in the presence of vehicle or oligomycin for 24 h; (C) relative mRNA expression of Batf and Irf4 and (D) BATF and RORγt protein expression are shown (3 to 4 independent experiments).(E) Vehicle- or oligomycin-treated activated CD4 T cells were retrovirally transduced with hNGFR-BATF overexpressing vector (OE) or hNGFR-empty vector (EV) at 24 h. The expression of IL-17A+ and RORγt+ cells (gated on CD4+ hNGFR+ T cells) were analyzed on days 3 to 4 (4 independent experiments).Graphs show the average ± SD; (B) unpaired t test; (C) ratio-paired t test; (E) one-way ANOVA *p < 0.05, **p < 0.01, ns, not significant.

Fig 4: IRF4 is indispensable for chromatin remodeling, but not for BATF binding, during effector differentiation of CTLsWT recipient mice were adoptively transferred with 1 × 106 of CD4Cre and IRF4cKO P14 cells, followed by infection with LCMV Arm. Effector P14 cells were collected at day 3 post-infection and subjected to ATAC-seq and BATF CUT&RUN (n = 2).(A) Volcano plot represents differences in chromatin accessibility between CD4Cre and IRF4cKO effector P14 cells.(B) Scatterplots showing relationship between differences in chromatin accessibilities between WT and Batf−/− effector P14 cells (horizontal axis) in Figure 2 and between CD4Cre and IRF4cKO effector P14 cells (vertical axis). OCRs that were differentially accessible between either WT and Batf−/− P14 cells or CD4Cre and IRF4cKO P14 cells were analyzed.(C) Representative ATAC-seq signal tracks for WT, Batf−/−, CD4Cre, and IRF4cKO effector P14 cells.(D) PCA of ATAC-seq data of WT and Batf−/− effector P14 cells in Figure 2 and CD4Cre and IRF4cKO effector P14 cells.(E) Volcano plot showing differences in chromatin accessibilities between Batf−/− and IRF4cKO effector P14 cells.(F) Volcano plot represents differences in BATF binding between CD4Cre and IRF4cKO effector P14 cells.(G) BATF CUT&RUN signal coverages at OCRs differentially accessible or shared between CD4Cre and IRF4cKO effector P14 cells.(H) Scatterplots showing relationship between differences in BATF bindings (horizontal axis) and those in chromatin accessibilities (vertical axis) between CD4Cre and IRF4cKO effector P14 cells.

Fig 5: IRF4 is indispensable for chromatin remodeling, but not for BATF binding, during effector differentiation of CTLs(A–D) Flow cytometric analysis of splenocytes of WT recipient mice infected with LCMV Arm and adoptively transferred with WT or Batf−/− P14 cells (1 × 105 each cell) transduced with indicated RV after 1 day of stimulation in vitro (Figure S4), assessed at day 8 post-infection of recipient mice. Data are representative of two independent experiments, with 3–5 mice in each experiment.(A) Plots are gated on total CTLs and numbers indicate the percentage of RV-transduced violet-excited fluorochromes (VEX)+ cells among total CTLs (black) or total P14 cells (green).(B) Percentage of VEX+ P14 cells among total CTLs and total P14 cells (mean ± SD).(C) Expression of KLRG1 and CD127 on RV-transduced P14 cells; plots are gated on VEX+ P14 cells.(D) Percentage of KLRG1lo CD127hi MPEC populations among total RV+ P14 cells (mean ± SD).(E–G) Batf−/− effector P14 cells transduced with indicated RV were subjected to ATAC-seq at day 4 post-infection (n = 2).(E) Volcano plots showing differences in chromatin accessibility between Batf RV- and empty RV-transduced cells, Batf RV- and H55Q RV-transduced cells, and empty RV- and H55Q RV-transduced cells.(A) PCA of ATAC-seq data of Batf−/− effector P14 cells transduced with indicated RV and that of WT and Batf−/− effector P14 cells in Figure 2.(G) ATAC-seq signal coverages of Batf−/− effector P14 cells transduced with indicated RV at OCRs differentially accessible or shared between WT and Batf−/− P14 cells in Figure 2 (top) or between CD4Cre and IRF4cKO effector P14 cells in Figure 4 (bottom).(H–J) Batf−/− P14 cells transduced with indicated RV were cultured in vitro and subjected to CUT&RUN (H and I) or EMSA (J) at day 3 post-RV transduction. (H and I) Representative BATF (H) or IRF4 (I) CUT&RUN signal coverages. Data from biological replicates (n = 2) were merged and analyzed.(J) Nuclear extracts of P14 cells were analyzed using the AICE1 probe31 in the presence or absence of antibodies against BATF and IRF4 as indicated. Data are representative of two independent experiments.(B and D) *p < 0.05, **p < 0.01, and ***p < 0.001 (Kruskal-Wallis test).