Blimp-1 is strictly IL-12/STAT-4–dependent in Th1 cells. (A) Relative Il10 expression after exposure of naive Th cells to increasing amounts of rmIL-12 (0, 0.1, 1, 10, and 100 ng/ml) for 48 h as determined by qPCR. (B) Naive CD4⁺ T cells from wild-type (WT) or STAT4-deficient (STAT4^−/−) mice were cultured in vitro under Th0 or Th1 skewing conditions, respectively. IL-10 production was measured by ELISA 72 h after Th cell priming. *, P = 0.024. (C) ChIP analysis of STAT4 binding to CNS upstream of Il10 in Th1 cells 72 h after activation. **, P = 0.0074; *, P = 0.027. (D) ChIP analysis of histone modifications at CNS upstream of Il10 in WT and STAT4^−/− Th1 cells 72 h after activation. From left to right: *, P = 0.019; *, P = 0.021. (E) Relative Prdm1 expression measured by qPCR after exposure of naive Th cells to increasing amounts of rmIL-12 for 48 h. (F) Relative Prdm1 expression and Blimp-1 protein level in WT and STAT4^−/− Th0 or Th1 cells 72 h after activation as determined by qPCR and immunoblot, respectively. Indication of protein size is in kD. *, P = 0.046. (G) Schematic representation of CNSs upstream of Prdm1. Positions of CNS are given in kb relative to TSS. (H) ChIP analysis of STAT4 binding to CNS upstream of Prdm1 in Th1 cells 72 h after activation. STAT4^−/− Th1 cells and a region known to harbor no STAT binding sites (neg) served as negative controls. Data are presented as fold enrichment to isotype control. From left to right: *, P = 0.01; *, P = 0.019; *, P = 0.0062. Data are representative of at least 2 independent experiments (error bars, mean ± SEM).