Characterization of CD25^highCD27^posCD45RA^negCD4^pos Tregs. (A) Flow cytometry of peripheral CD25^high and CD25^neg cells within the PBMC CD4^posCD27^posCD45RA^neg gate (n = 6). Percentages of cells positive for the indicated marker (top) are given. (B) Flow cytometry of sorted CD25^highCD27^posCD45RA^negCD4^pos (CD25^highCD27^pos) Tregs and CD25^negCD27^posCD45RA^negCD4^pos (CD25^negCD27^pos) Teffs. CD4 cells were isolated by magnetic bead–based negative selection. The enriched cells were labeled with anti-CD25, anti-CD27, and anti-CD45RA mAb and next, CD25^highCD27^posCD45RA^neg and CD25^negCD27^posCD45RA^neg cell populations were isolated by high-purity flow cytometric cell sorting. Density plots show surface staining of CD127 (x-axis) and intracellular staining of Foxp3 (y-axis). (C) Real-time quantitative reverse-transcriptase (RT)–PCR of the mRNA expression of Tbet, GATA3, RORγt, FOXP3, and TGFβ in freshly sorted CD25^highCD27^pos Tregs and CD25^negCD27^pos Teffs. The RT-PCR data shown were normalized to human HPRT1 levels, and expression in nonstimulated conditions of CD25^negCD27^pos cells was set as 1.0, and fold reduction or increase in gene expression was calculated. Cells obtained from 3 to 5 different donors were used. Mean and SD from different experiments are shown. * indicates statistically significant differences. (D) ³H-thymidine incorporation–based suppression assay of sorted CD25^highCD27^pos Tregs and CD25^negCD27^pos Teffs. Sorted cells were titrated (x-axis) into cultures with CD4^pos responder T cells (25 × 10³) and subsequently stimulated with anti-CD3 plus anti-CD28 mAb–coated beads. Proliferation (y-axis) was measured at day 3 after the start of the culture. A representative experiment is shown. Mean and SD of triplicate measurements are shown. Data are from n = 6 different healthy volunteers (A), or show representative experiments of 2 (C,D) or 3 (D) separate experiments performed with cells obtained from different cell donors.