Total CD4+ T cells were isolated from mouse spleens using the EasySep Mouse CD4+ T Cell Isolation Kit (STEMCELL Technologies). Purified CD4+ T cells were then cultured in tissue culture plates coated with anti-CD3 (5 μg/ml; clone 145-2C11). Unless otherwise indicated, cells were cultured at 1.35 × 106 cells per well in 24-well plates, and anti-CD28 (2 μg/ml; clone 37.51) was added to all cultures. TH1 cells were differentiated using anti–IL-4 (10 μg/ml; clone 11B11), hIL-2 (10 ng/ml), and IL-12 (10 ng/ml). TH2 cells were differentiated using anti–IFN-γ (10 μg/ml; clone R4-6A2), hIL-2 (20 ng/ml), and IL-4 (50 ng/ml), whereas for TH9 cells, anti–IFN-γ (10 μg/ml) was used together with hTGFβ1 (10 ng/ml), hIL-2 (20 ng/ml), and IL-4 (250 ng/ml). TH17 cells were differentiated using anti–IFN-γ (10 μg/ml), anti–IL-4 (10 μg/ml), anti-CD25 (5 μg/ml; clone PC61), and anti-CD122 (4 μg/ml; clone TM-β1) antibodies, hTGFβ1 (2 ng/ml), IL-6 (20 ng/ml), and IL-1β (10 ng/ml). For Tregs, antibody (10 μg/ml) against IFN-γ was used together with hTGFβ1 (5 ng/ml) and IL-2 (5 ng/ml). In cytokine competition experiments (Fig. 8B), cytokines used were IL-2 (20 ng/ml), IL-4 (50 ng/ml), TGFβ1 (2 ng/ml), IL-6 (20 ng/ml), and IL-1β (10 ng/ml). SMs AT-406 (Selleckchem) at 10 μM, LCL161 (Selleckchem) at 1 μM, or DMSO control was added to the cultures directly after addition of polarizing cytokines and antibodies.

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