Phosphorylation of Smad2/3 was assessed in cells (0.2 × 106) previously fixed for 20 min at 37°C with fixation/permeabilization solution (BD Biosciences), permeabilized for 30 min at 4°C with Phosflow Perm Buffer III (BD Biosciences), and washed twice with phosphate-buffered saline (PBS) by staining for 30 min with Alexa Fluor 647–conjugated anti-pSmad2/3 (clone O72-670, BD Biosciences), further washing, and analysis. For assessment of IFN-γ production by intracellular cytokine staining, cells (0.2 × 106) were restimulated for 16 hours with soluble anti-CD3 and anti-CD28 (1 and 5 μg/ml, respectively) under culture conditions as indicated in the figure legends and then treated for 4 hours with phorbol 12-myristate 13-acetate (10 ng/ml; Sigma-Aldrich) and ionomycin (500 ng/ml; Sigma-Aldrich). During the final 2 hours of activation, the cells were treated with monensin (BioLegend) to block cytokine secretion. The cells were then washed and fixed for 20 min at 37°C with fixation/permeabilization solution, washed with permeabilization buffer before being stained for 45 min with PE-conjugated anti–IFN-γ (clone B27, ImmunoTools), and undergoing further washing and analysis. FoxP3 abundance was assessed in cells (0.2 × 106) previously fixed for 20 min at 37°C in FoxP3 fixation/permeabilization solution (eBioscience) and washed with FoxP3 permeabilization buffer (eBioscience) by staining for 45 min with anti-human FoxP3-PE (clone PCH101, eBioscience), which was followed by further washing and analysis.

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