Monoclonal anti cd3

Ectopic ESCs were cultured in 24-well plates (Corning, Steuben County, NY, USA) at a density of 1 × 10⁵ cells/well. The co-culture systems were established by incubating 2 × 10⁵ monocytes with ESCs or alone, adding of estrogen (10⁻⁸ M; Sigma), 1-MT (0.05 mM; Sigma), or estrogen (10⁻⁸ M; Sigma) plus 1-MT (0.05 mM; Sigma). Meanwhile, naive CD4⁺ T cells were cultured in 24-well plates that coated with monoclonal anti-CD3 (5 μg/ml; Biolegend) and monoclonal anti-CD28 (1 μg/ml; Biolegend), in the presence of recombinant human IL-2 (50 ng/ml; Biolegend). The monocytes-derived macrophages and 2 × 10⁵ naive CD4⁺ T cells were collected to co-culture with ESCs in 1 ml medium/well for 5 days (Supplementary Figure 1).

Peripheral blood mononuclear cells (PBMCs) were isolated by Lymphoprep (Stemcell Technologies Inc.) density gradient centrifugation. CD4⁺CD25⁺ T cells were obtained by positively selection using CD4⁺CD25⁺ T cells (Regulatory T cells) micro-magnetic beads according to the manufacturer instructions (Miltenyi Biotec). CD4⁺CD25⁻ cells were obtained by negatively selection according to the manufacturer instructions (Miltenyi Biotec). CD4⁺CD25⁺ T cells were cultured in 96-well plates that coated with monoclonal anti-human CD3 (5 μg/ml; Biolegend, clone: OKT3) and monoclonal anti-human CD28 (1 μg/ml; Biolegend, clone: CD28.2), in the presence of recombinant human IL-2 (50 ng/ml; Biolegend), while CD4⁺CD25⁻ cells were cultured in 24-well plates that coated with monoclonal anti-CD3 (5 μg/ml; Biolegend) and monoclonal anti-CD28 (1 μg/ml; Biolegend). T_reg cells were collected to culture with non-treated ESCs, 1-MT-pretreated ESCs, vector-pretreated ESCs, and MRC2-silenced ESCs or not for 48 h after T_reg cells proliferation for two weeks. After that, T_reg cells from these groups respectively cultured with paired CFSE-labeled CD4⁺CD25⁻ T cells (T_eff) for 48 h and then detected by flow cytometry.