Phenotypic characterization of regulatory T cells. Phenotypic analysis was performed on healthy donors (n = 32) and patients with leukemia (n = 21) followed longitudinally from baseline (before SCT), days +30, +45, +60, +90, and +120 after SCT. Representative data on one individual are presented here. (A) PBMCs were stained initially with anti-CD3, anti-CD4, and anti-CD25 antibodies followed by intracellular staining with anti–CTLA-4 and anti-FOXP3. (B) The histogram was gated on CD4⁺CD25⁺ T cells and represents the proportion of CD4⁺CD25⁺ T cells that are FOXP3⁺. (C) The dot plot was gated on CD4⁺ T cells: the upper right quadrant represents CD25⁺FOXP3⁺ T-cell population, and the lower right quadrant, the CD25^–FOXP3⁺ population. (D-F) CTLA-4 and FOXP3: histograms are gated on CD4⁺FOXP3⁺ and CD4⁺FOXP3^– populations. Whereas CD4⁺FOXP3⁺ T cells also co-express CTLA-4 (E), CD4⁺FOXP3^– T cells were essentially CTLA-4 negative (F). (G) Correlation between FOXP3 mRNA copies and CD4⁺FOXP3⁺ (%). Dots correspond to individual samples tested from healthy donors and patients with leukemia. A significant positive correlation was seen between the frequency of CD4⁺FOXP3⁺ T cells and FOXP3 mRNA copies, as shown by the trendline.