Inducing Naive CD4+ T Cells into Treg Cells

Naive CD4⁺ T cells were isolated from the spleen of female mice at the age of 6–8 weeks with MojoSort Mouse CD4⁺ Naïve T Cell Isolation Kit (Biolegend) according to the manufacturer’s instructions. For Th0 induction, 2 to 3 × 10⁵ naive CD4⁺ T cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (Biological Industries), penicillin-streptomycin (500 U; PAN biotech), and β-mercaptoethanol (50 μM; Sigma-Aldrich) and stimulated with Dynabeads Mouse T-Activator CD3/CD28 (Gibco) at a cell:bead ratio of 1:1 in the presence of rmIL-2 (200 U/ml; Peprotech) (Wang et al., 2019 (link)). To obtain iTreg cells, rhTGFβ1 (2 or 0.5 ng/ml; Peprotech) was simultaneously added along with the reagents used for inducing Th0 cells. ACLY inhibitor: SB204990 (Tocris Bioscience), ACC inhibitor: PF05175157 (Tocris Bioscience), FASN inhibitor: cerulenin (Biovision), protein synthesis inhibitor: CHX (Tocris Bioscience), proteasome inhibitor: MG132 (Selleck), lysosome inhibitor: leupeptin (Selleck).