TRAF6-deficient CD4⁺ T cells are more sensitive to SMAD-mediated TGF-β signaling. CD4⁺ naive T cells were stimulated in 96-well flat-bottom plates with plate-coated anti-CD3 and soluble anti-CD28 with the indicated concentrations of TGF-β1. (A) Cell proliferation was measured by tritium incorporation during the last 8 hours of a 72-hour culture. Error bars represent SD from triplicate wells in 1 of 3 experiments. (B) IL-2 RNA levels were determined by real-time PCR analysis after 48 hours of stimulation. The experiment was repeated with similar results. (C) IL-2 protein levels in the supernatants were determined by ELISA after 72 hours of culture. Error bars represent SD from triplicate wells in 1 of 2 experiments. (D) CD4⁺ T cells were stimulated for 24 hours with plate-coated anti-CD3 and soluble anti-CD28. After 16 hours of resting, cells were stimulated with TGF-β1 (1 ng/mL) for the indicated times. Phosphorylated and total Smad2/3 proteins were detected in Western blot assays of cell lysates (left). Graphs represent relative amounts of phospho-Smad2 and phospho-Smad3 after normalization to total Smad2 and Smad3, respectively. Receptor levels were determined in cells before TGF-β1 stimulation (right). Figure shows a representative result from 3 independent experiments. (E) CFSE-stained naive CD4⁺ T cells were stimulated with plate-coated anti-CD3 and soluble anti-CD28 in the presence of TGF-β1 (1 ng/mL) with or without IL-2. CFSE dilution due to cell proliferation was determined by flow cytometric analysis after 3 days of stimulation. Numbers and gates in the flow cytometry plots indicate percentage of cells with lower CFSE staining levels than undivided cells. The experiment was repeated with similar results. *P < .01, **P < .05.