Retroviruses were produced in Plat-E cells. Plat-E cells were transfected with pMX-IRES-GFP plasmids containing the indicated genes, and the medium was replaced twice with 3 ml of fresh medium every 10 hours after transfection. The retrovirus-containing supernatant was collected 72 hours after the medium was replaced for the second time and used to infect T cells.

Sorted naive CD4+ T cells were differentiated into TH17 cells in the presence of TGF-β1 and IL-6 (48-well plate, 0.5 × 106 cells per well); 20 to 24 hours later, the cells were transfected with 1 ml of the indicated retrovirus in the presence of polybrene (10 μg/ml) and 10 mM Hepes and infected for 2 hours at 1500g at 32°C. After transfection, the cells were resuspended in TH17 differentiation medium and cultured for 3 days. The indicated cytokines (e.g., IL-17A and IL-17F) and other TFs (e.g., Foxp3 and RORγt) were measured by gated CD4+GFP+ cells after retrovirus infection for 72 hours.

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