eGFP+Foxp3− T cells lack suppressive activity.

FACS-sorted CD4+eGFP−, CD4+eGFP+CD25lo and CD4+eGFP+CD25hi populations were added at varying ratios to responder T cells (Tresp) with simultaneous anti-CD3 stimulation. Ex vivo isolated CD4+CD25+eGFP+ cells (nTregs) from DEREG mice were used as control. (A) Dot plots demonstrate the purity of various FACS-sorted iTreg populations. Sorting was performed on the basis of eGFP and CD25 expression. (B) Comparison of Foxp3 expression on sorted iTreg sub-populations. Dotted line represents Foxp3 expression on live CD4+eGFP− T cells, solid gray line represent Foxp3 expression on live CD4+eGFP+CD25lo and solid black line represent Foxp3 expression on CD4+eGFP+CD25hi T cells. (C) Representative histograms for dilution of proliferation dye on gated live CD4+ Tresp cells (left panel). Quantification of proliferated Tresp cells under various conditions (right panel). Stimulated and non-stimulated Tresp cells served as positive (Pos) and negative (Neg) controls, respectively. Error bars designate SD of triplicates from one representative of three individual experiments.

本实验将经荧光激活细胞分选（FACS）分选的CD4⁺eGFP⁻、CD4⁺eGFP⁺CD25lo及CD4⁺eGFP⁺CD25hi细胞群体以不同比例添加至应答T细胞（Tresp）中，同时施加抗CD3刺激。采用来自DEREG小鼠的体外分离CD4⁺CD25⁺eGFP⁺细胞（天然调节性T细胞，nTregs）作为对照。(A) 散点图展示了各类FACS分选的诱导性调节性T细胞（iTreg）群体的纯度，分选基于eGFP与CD25的表达水平进行。(B) 分选得到的iTreg亚群的叉头框蛋白P3（Foxp3）表达水平比较：虚线代表活CD4⁺eGFP⁻T细胞的Foxp3表达，实心灰线代表活CD4⁺eGFP⁺CD25lo T细胞的Foxp3表达，实心黑线代表CD4⁺eGFP⁺CD25hi T细胞的Foxp3表达。(C) 门控活CD4⁺应答T细胞的增殖染料稀释情况代表性直方图（左图）；不同条件下增殖的应答T细胞的定量分析（右图）。经刺激与未刺激的应答T细胞分别作为阳性（Pos）与阴性（Neg）对照。误差棒代表三次重复实验的标准差（SD），数据取自3次独立实验中的1次代表性实验。