HLA-DR and CD45RA expression delineates 3 regulatory T-cell subpopulations. (A) CD4⁺ T cells from cord blood, thymus, and adult peripheral blood of healthy individuals were analyzed by flow cytometry. Plots shown were gated on CD4⁺FOXP3⁺ T cells. After staining for extracellular antigens, cells were fixed, permeabilized, and intracellular staining of CTLA4, FOXP3, Helios, Ki-67 was performed using eBioscience FOXP3 Staining Buffer Set, following manufacturer instructions. (B) Plots show (left panel) the percentage of each Treg subpopulation in adult peripheral blood samples (n = 13) and cord blood samples (n = 12) and (right panel) the percentage of total FOXP3⁺ cells within CD4⁺ T cells. P values were calculated using a 2-tailed nonparametric Mann-Whitney test. (C) Expression of FOXP3, CD25, and CTLA4 were assessed in each Treg subpopulation by flow cytometry (left panel). Bar histograms represent the mean fluorescence intensity of FOXP3 and CTLA4 for adult peripheral blood (n = 13) and cord blood (n = 12). (D) Expression of Ki-67 and Helios in each Treg subpopulation was analyzed in cord blood, thymus, and adult peripheral blood of healthy individuals by flow cytometry. (E) Sorted CD4⁺CD25⁻CD45RA⁺ Tconvs (Tresp) were used as responder cells, stained with 1mM CFSE (CellTrace CFSE Cell Proliferation Kit; Life Technologies) and 10⁴ CFSE-labeled responder CD4⁺CD25⁻CD45RA⁺ T cells were cultured alone or cocultured with 10⁴ unlabeled Treg cells at different ratio Treg:Tresp (1:1; 1:4; 1:8) in triplicates. (Tresp:Treg, white bar, 1:1; gray bar, 4:1; black bar, 8:1) in the presence of CD3/CD28 beads. Proliferation of CFSE-labeled cells was assessed by flow cytometry after 84 to 90 hours of culture. Histograms represent the percentage of suppression of Tresp proliferation. (F) CCR6 and CCR7 expression on Treg subpopulations, naive CD4 T cells (Naive), and RA⁻DR⁻ CD4 Tconvs (Tconv) were assessed by flow cytometry. Histograms are representative of 4 different healthy individuals. Tresp, T responder.