Blimp-1 is crucial for IL-10 expression by Th1 cells in vitro. Naive CD4⁺ T cells from wild-type (WT) or conditional Prdm1-deficient (CKO) mice were cultured in vitro under Th1 skewing conditions. (A and B) Relative Il10 expression in WT or CKO Th1 cells and frequency of IL-10– (A) and IFN-γ–producing Th1 cells (gated on CD4⁺; B) 96 h after T cell priming by qPCR or intracellular staining after PMA/ionomycin restimulation, respectively. A representative dot plot is shown. From left to right: **, P = 0.0042; **, P = 0.0031. (C and D) Th1 cells were transduced with either a GFP control (GFP RV) or with a Blimp-1 (Prdm1 RV) retroviral construct. Relative Il10 expression in GFP-sorted cells and frequency of IL-10 and IFN-γ producers (gated on CD4⁺GFP⁺) 96 h after activation after PMA/ionomycin restimulation as determined by qPCR or flow cytometry, respectively. A representative dot plot is shown. From left to right: **, P = 0.0037; **, P = 0.0046. (E) Schematic representation of CNSs in the Il10 locus upstream of the TSS. Positions of CNS are given in kb relative to TSS. (F) ChIP analysis of Blimp-1 binding to CNS upstream of Il10 in Th1 cells. Th1 cells were fixed 72 h after the start of culture. *, P = 0.038. (G) ChIP analysis of histone modifications at CNS upstream of Il10 in WT and CKO Th1 cells 72 h after activation. Data are representative of three (A and B) or two (C and D) independent experiments, or data summarize three (F; duplicates/experiment) or two (G; duplicates/experiment) independent experiments, respectively, (mean ± SEM).