TGF-β antagonizes Blimp-1–dependent IL-10 induction in T cells. (A) Naive CD4⁺ T cells from wild-type (WT) or conditional Prdm1-deficient (CKO) mice were cultured in vitro under Th1, Th2, or Th17 skewing conditions. Production of IL-10 was measured by ELISA 72 h after priming of Th cells. *, P = 0.024. (B) Relative Prdm1 expression measured by qPCR and Blimp-1 protein levels determined by immunoblot in WT and STAT4-deficient (STAT4^−/−) T cells upon stimulation with rmIL-27 for 48 h. Indication of protein size is in kD. (C) ELISA of IL-10 produced by WT or CKO T cells stimulated with rmIL-27 for 72 h. **, P = 0.0089. (D) Relative Prdm1 and Maf expression as well as Blimp-1 and c-Maf protein level in Th1 cells cultured in the presence of increasing amounts of rhTGF-β (0, 0.04, 0.2, 1, and 5 ng/ml) for 72 h as determined by qPCR and immunoblot, respectively. Indication of protein size is in kD. (E) Frequency of IL-10⁺ and IFN-γ⁺ T cells cultured under Th1 skewing conditions in the presence of increasing amounts of rhTGF-β was measured by flow cytometry after PMA/ionomycin restimulation 72 h after Th1 priming (gated on CD4⁺). A representative dot plot of Th1 cells cultured in the absence (w/o) or the presence (w/; 5 ng/ml) of rhTGF-β is shown. (F) Relative Prdm1 and Maf expression in T cells after stimulation with IL-27 (w/o rhTGF-β) or IL-27/rhTGF-β (w/TGF-β; 2 ng/ml) 48 h after activation as determined by qPCR. From left to right: **, P = 0.0011; **, P = 0.009. (G) ELISA of IL-10 produced by IL-27–stimulated T cells in the absence (w/o) or presence (w/) of rhTGF-β (2 ng/ml) measured 72 h after stimulation. **, P = 0.007. (H) IL-10 production by WT or CKO T cells after exposure to increasing amounts of rmIL-27 (1, 5, and 25 ng/ml) in the absence (w/o) or presence (w/; 2ng/ml) of rhTGF-β for 72 h was measured by ELISA. Data are representative of three independent experiments (mean ± SEM).