Induction of T reg cell differentiation in vitro was conducted according to published protocols (Feng et al., 2015 (link); Yue et al., 2016 (link)). Briefly, cell culture plates or dishes were precoated with 1 µg/ml anti-CD3 (clone 145-2C11; Bio X Cell) and anti-CD28 (clone 37.51; Bio X Cell) antibodies in PBS at 37°C for 2 h and washed with PBS once before cell culture. CD4 Tn cells were grown in complete RPMI1640 supplemented with 100 U/ml recombinant human IL-2 (PeproTech) and 1 ng/ml recombinant human TGF-β (R&D Systems) with or without 0.25 mM ASC-2-phosphate (Sigma) for 4 d.
To assess the stability of Foxp3 expression in vitro, iT reg cells sorted based on GFP-Foxp3 expression after 4 d of induction were further cultured on plates coated with both anti-CD3 (1 µg/ml) and anti-CD28 (1 µg/ml) antibodies and either recombinant human IL-2 or 25 µg/ml IL-2 neutralization antibodies (JES6-54H and S4B6-1; Bio X Cell) for 4 d more. To culture nT reg cells, FACS-sorted nT reg cells were cultured in complete RPMI1640 supplemented with 10% FBS in the presence of Dynabeads Mouse T-Activator CD3/CD28 Beads (Thermo Fisher Scientific) with 500 U/ml recombinant human IL-2.
To assess the stability of Foxp3 expression in vitro, iT reg cells sorted based on GFP-Foxp3 expression after 4 d of induction were further cultured on plates coated with both anti-CD3 (1 µg/ml) and anti-CD28 (1 µg/ml) antibodies and either recombinant human IL-2 or 25 µg/ml IL-2 neutralization antibodies (JES6-54H and S4B6-1; Bio X Cell) for 4 d more. To culture nT reg cells, FACS-sorted nT reg cells were cultured in complete RPMI1640 supplemented with 10% FBS in the presence of Dynabeads Mouse T-Activator CD3/CD28 Beads (Thermo Fisher Scientific) with 500 U/ml recombinant human IL-2.