Phenotype of Treg and Tcon, and suppressive activity of Treg. (A) Representative contour plot showing expression of CD4 in the lymphocyte gate, and expression of CD25 and CD127 in the CD4⁺ lymphocyte gate. PBMCs were stained with CD4, CD25, and CD127 antibody. CD4⁺ lymphocytes were isolated into CD25^neg-lowCD127^med-high Tcon (dotted line), and CD25^med-highCD127^low Treg (solid line). (B) Expression of Foxp3 in Treg and Tcon. Surface-stained PBMCs were stained with intracellular anti-Foxp3 antibody or isotype IgG (filled histograms). Foxp3 expression was measured in Tcon (dotted line) and Treg (solid line). (C-D) Functional capacity of isolated Treg. Tcon and Treg were purified by flow cytometric cell sorting. A total of 2 × 10⁴ Tcon alone or cocultured with 2 × 10⁴ autologous Treg were incubated with 10⁵ irradiated allogeneic PBMCs in the presence of anti-CD3 antibody. Tcon cultured alone in the absence of allogeneic PBMCs and anti-CD3 antibody was used as a negative control. Tcon cultured with allogeneic PBMC and anti-CD3 antibody was used as the positive control. Proliferation of Tcon was determined by ³H-thymidine incorporation (C) and CFSE dilution assay (D). Representative FACS profile of Tcon only (dotted line), Tcon cocultured with Treg (solid line), and Tcon only without stimulation (filled histograms) is shown. Both assays were performed in triplicate.