Increased TGF-β sensitivity is reflected in decreased IL-2 levels during Th17 differentiation but has no effect on iTreg differentiation. Naive CD4⁺ T cells isolated from TRAF6-ΔT and control mice were stimulated with plate-coated anti-CD3 and soluble anti-CD28 in Th17 (TGF-β1 [0, 0.1, 1.0 ng/mL] plus IL-6) or iTreg (TGF-β1 [0, 0.1, 1.0 ng/mL] plus IL-2) conditions. (A) Levels of IL-2 mRNA were analyzed by real-time PCR after 48-hour incubation. (B) Levels of RORγt mRNA were analyzed by real-time PCR after 5-day culture. (C) After 5 days of culture, cells were restimulated, and the amount of IL-17–producing cells was determined by flow cytometry. (D) After 5 days of culture, Foxp3 protein levels were determined by intracellular staining. Numbers and gates in the flow cytometry plots indicate percentages of Foxp3-expressing cells. (C-D) Right panels depict data from triplicate cultures. (E) Relative Foxp3 mRNA levels were determined by real-time PCR analysis after 5 days of culture. All experiments were repeated at least 2 times. *P < .05, **P < .005.