Figure 2. Twist1 induction requires IL-12 signaling via STAT4, but not IFN-γ or T-bet. (A) CD62Lhi DO11.10 Th cells were stimulated for 5 d under Th1-polarizing conditions (5 ng/ml IL-12, anti-IL4), reduced IL-12 (1/25:200 pg/ml; 1/625:8 pg/ml, anti-IL4), in the absence of IL-12 (anti–IL-12, anti-IL4), in the presence of IFN-γ (10 ng/ml IFN-γ, anti-IL-12, anti-IL-4), or under Th2-polarizing conditions. Twist1 mRNA in Th cells activated for 3 h with PMA/ionomycin and IL-2 was quantified by RT-PCR. The amount of twist1 transcripts induced under Th1-polarizing conditions was set to 1. Data are presented as the mean ± the SD of at least three experiments. (B) CD62Lhi Th cells of T-bet−/− mice and syngenic BALB/c mice were stimulated with anti-CD3/CD28 and BALB/c APCs under Th1 (IL-12 and IFN-γ), or under Th2-polarizing conditions for 6 d. Data represent the mean ± the SD of three experiments. The amount of twist1 transcripts induced in activated wt Th1 cells was set to 1. (C) CD4+ cells of STAT4−/− and syngenic BALB/c mice were stimulated with anti-CD3/CD28, and BALB/c APCs under Th1 (IL-12 and IFN-γ) or under Th2-polarizing conditions for 5 d. Data represent the mean ± SD (four mice each). (D) The binding of STAT4 to the proximal promoter of twist1 was analyzed by ChIP. 6-d-old Th1 cells were restimulated with PMA/ionomycin in the presence of 10 ng/ml IL-12 for 3 h or left unstimulated. The immunoprecipitated DNA was quantified by RT-PCR using primers specific for the proximal twist1 promoter. The precipitated DNA was normalized to the amount of input DNA. The amount of twist1 transcripts precipitated in the presence of IL-12 was set to 1. Data represent the mean ± SD of three experiments. (E) Naive DO11.10 Th cells were stimulated for 5 d with APCs and OVA327-339 under Th1-polarizing conditions. Cells were restimulated under the same conditions (Th1), or in the presence of anti–IL-12. Twist1 transcripts were quantified on d 11. The amount of twist1 mRNA on d 5 was set to 1. Data represent mean ± SD of three experiments.
Figure 2.

Twist1 induction requires IL-12 signaling via STAT4, but not IFN-γ or T-bet. (A) CD62Lhi DO11.10 Th cells were stimulated for 5 d under Th1-polarizing conditions (5 ng/ml IL-12, anti-IL4), reduced IL-12 (1/25:200 pg/ml; 1/625:8 pg/ml, anti-IL4), in the absence of IL-12 (anti–IL-12, anti-IL4), in the presence of IFN-γ (10 ng/ml IFN-γ, anti-IL-12, anti-IL-4), or under Th2-polarizing conditions. Twist1 mRNA in Th cells activated for 3 h with PMA/ionomycin and IL-2 was quantified by RT-PCR. The amount of twist1 transcripts induced under Th1-polarizing conditions was set to 1. Data are presented as the mean ± the SD of at least three experiments. (B) CD62Lhi Th cells of T-bet−/− mice and syngenic BALB/c mice were stimulated with anti-CD3/CD28 and BALB/c APCs under Th1 (IL-12 and IFN-γ), or under Th2-polarizing conditions for 6 d. Data represent the mean ± the SD of three experiments. The amount of twist1 transcripts induced in activated wt Th1 cells was set to 1. (C) CD4+ cells of STAT4−/− and syngenic BALB/c mice were stimulated with anti-CD3/CD28, and BALB/c APCs under Th1 (IL-12 and IFN-γ) or under Th2-polarizing conditions for 5 d. Data represent the mean ± SD (four mice each). (D) The binding of STAT4 to the proximal promoter of twist1 was analyzed by ChIP. 6-d-old Th1 cells were restimulated with PMA/ionomycin in the presence of 10 ng/ml IL-12 for 3 h or left unstimulated. The immunoprecipitated DNA was quantified by RT-PCR using primers specific for the proximal twist1 promoter. The precipitated DNA was normalized to the amount of input DNA. The amount of twist1 transcripts precipitated in the presence of IL-12 was set to 1. Data represent the mean ± SD of three experiments. (E) Naive DO11.10 Th cells were stimulated for 5 d with APCs and OVA327-339 under Th1-polarizing conditions. Cells were restimulated under the same conditions (Th1), or in the presence of anti–IL-12. Twist1 transcripts were quantified on d 11. The amount of twist1 mRNA on d 5 was set to 1. Data represent mean ± SD of three experiments.

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