Figure 5. c-Maf acts synergistically with Blimp-1 to induce IL-10 in Th1 cells. (A and B) Naive CD4+ T cells were treated either with a nontargeting control siRNA (siRNA Ctl) or with a c-Maf targeting siRNA (siRNA c-Maf) before Th1 differentiation. (A) Relative Maf expression was measured by qPCR for 48 h and c-Maf protein levels were determined by immunoblot 72 h after siRNA treatment. Indication of protein size is in kD. **, P = 0.0021 (B) Relative Il10 expression determined by qPCR and IL-10 ELISA measured 72 h after the start of Th1 culture and siRNA treatment. From left to right: *, P = 0.017; *, P = 0.032. (C) ChIP analysis of c-Maf binding to CNS upstream of Il10 in Th1 cells 72 h after activation. *, P = 0.027. (D) ChIP analysis of c-Maf binding to CNS upstream of Il10 in WT or Prdm1-deficient (CKO) Th1 cells. From left to right: *, P = 0.023; *, P = 0.042. (E) WT or CKO Th1 cells were transduced with either control (GFP RV) or with a c-Maf (Maf RV) retroviral construct. Frequency of IL-10 and IFN-γ producers (gated on CD4+GFP+ cells) was determined by flow cytometry 96 h after priming of Th1 cells after PMA/ionomycin restimulation. A representative dot plot is shown. From left to right: **, P = 0.0025; **, P = 0.007; *, P = 0.021. (F) Luciferase reporter assay in HEK 293T cells transfected with indicated CNS reporter constructs together with empty vector (control), Blimp-1, and/or c-Maf expression plasmids. Firefly luciferase activity was measured 18 h after transfection and is presented relative to constitutive renilla luciferase activity. From left to right: *, P = 0.013; *, P = 0.024. (G) Relative Maf expression as determined by qPCR after exposure of naive Th cells to increasing amounts of rmIL-12 (0, 0.1, 1, 10, and 100 ng/ml) for 48 h and in WT or STAT4−/− Th0 and Th1 cells 72 h after priming of naive T cells. *, P = 0.012. (H) Relative Prdm1 expression and Blimp-1 and c-Maf Protein level in Th0 and Th1 cells after retroviral transduction with GFP RV or c-Maf RV measured by qPCR and immunoblot, respectively. Cells were FACS-sorted for GFP expression 48 h after transduction. Indication of protein size is in kD. *, P = 0.042. (I) ChIP analysis of c-Maf binding to CNS (MARE) sites in the Prdm1 locus in Th1 cells 72 h after activation. *, P = 0.033. Data are representative of four (A and B) or three (C–I) independent experiments (mean ± SEM).
Figure 5.

c-Maf acts synergistically with Blimp-1 to induce IL-10 in Th1 cells. (A and B) Naive CD4+ T cells were treated either with a nontargeting control siRNA (siRNA Ctl) or with a c-Maf targeting siRNA (siRNA c-Maf) before Th1 differentiation. (A) Relative Maf expression was measured by qPCR for 48 h and c-Maf protein levels were determined by immunoblot 72 h after siRNA treatment. Indication of protein size is in kD. **, P = 0.0021 (B) Relative Il10 expression determined by qPCR and IL-10 ELISA measured 72 h after the start of Th1 culture and siRNA treatment. From left to right: *, P = 0.017; *, P = 0.032. (C) ChIP analysis of c-Maf binding to CNS upstream of Il10 in Th1 cells 72 h after activation. *, P = 0.027. (D) ChIP analysis of c-Maf binding to CNS upstream of Il10 in WT or Prdm1-deficient (CKO) Th1 cells. From left to right: *, P = 0.023; *, P = 0.042. (E) WT or CKO Th1 cells were transduced with either control (GFP RV) or with a c-Maf (Maf RV) retroviral construct. Frequency of IL-10 and IFN-γ producers (gated on CD4+GFP+ cells) was determined by flow cytometry 96 h after priming of Th1 cells after PMA/ionomycin restimulation. A representative dot plot is shown. From left to right: **, P = 0.0025; **, P = 0.007; *, P = 0.021. (F) Luciferase reporter assay in HEK 293T cells transfected with indicated CNS reporter constructs together with empty vector (control), Blimp-1, and/or c-Maf expression plasmids. Firefly luciferase activity was measured 18 h after transfection and is presented relative to constitutive renilla luciferase activity. From left to right: *, P = 0.013; *, P = 0.024. (G) Relative Maf expression as determined by qPCR after exposure of naive Th cells to increasing amounts of rmIL-12 (0, 0.1, 1, 10, and 100 ng/ml) for 48 h and in WT or STAT4−/− Th0 and Th1 cells 72 h after priming of naive T cells. *, P = 0.012. (H) Relative Prdm1 expression and Blimp-1 and c-Maf Protein level in Th0 and Th1 cells after retroviral transduction with GFP RV or c-Maf RV measured by qPCR and immunoblot, respectively. Cells were FACS-sorted for GFP expression 48 h after transduction. Indication of protein size is in kD. *, P = 0.042. (I) ChIP analysis of c-Maf binding to CNS (MARE) sites in the Prdm1 locus in Th1 cells 72 h after activation. *, P = 0.033. Data are representative of four (A and B) or three (C–I) independent experiments (mean ± SEM).

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