Identification of a conserved distant T-bet enhancer region. (A) ChIP assay–based genomic scanning. Twenty primer pairs were used for these experiments. (B) Sequences precipitated by anti-Stat4 mAbs. Purified CD8 and CD4 T cells were stimulated with anti-CD3 plus anti-CD28 mAbs, with or without IL-12 for 24 hours, and ChIP assays were performed with anti-Stat4. Sequences of T-bet regulatory regions precipitated by anti-Stat4 were quantitated by real-time PCR. Data are expressed as the ratio of TCR/IL-12–mediated to TCR alone–mediated enrichments. (C) Sequences precipitated by anti-Stat1 mAbs. Purified IFNγ-deficient CD4 and CD8 T cells were stimulated with anti-CD3 plus anti-CD28 mAbs, with or without IFNγ (10 ng/mL) for 24 hours, and ChIP assays were performed with anti-Stat1. Sequences of T-bet regulatory regions precipitated by anti-Stat1 were quantitated by real-time PCR. Data are expressed as the ratio of TCR/IFNγ-mediated to TCR alone–mediated enrichments. (B,C) Data shown are the means plus or minus SD from 3 independent experiments. (D) Alignment of mouse and human T-bet enhancer. Nucleotides differing between mouse and human are boxed. Potential STAT-binding motifs TTCGAGGAA and TTAGCGGAA are underlined. (E) IL-12 also induces IFNγ-independent T-bet in human CD8 T cells. Human CD8 T cells were stimulated with anti-CD3 plus anti-CD28 Abs, with or without anti-IFNγ Abs, in combination with human IL-12 or human IFNγ. T-bet expression was normalized and expressed as percentage relative to GAPDH. Data shown are one representative of 3 independent experiments; standard errors of PCR triplicates are shown.