Isolation of untouched Treg cells from CD4⁺ T cells by CD49d/CD127 depletion. CD4⁺ cells of human PBMCs were depleted of CD127/CD49d-expressing cells by MACS. (A) FACS analysis before and after depletion. FACS plots are shown for the CD4⁺ cell population before the depletion (left panels) and for the cells remaining after CD49d/CD127 depletion (right panels). Data are shown for the staining CD49d versus CD127 (top panels) and CD25 versus Foxp3 (bottom panels). Numbers refers to the percentage of cells in each quadrant. (B) Suppressive capacity. CD49d⁻CD127⁻ cells isolated by MACS depletion were used as suppressor cells (Treg) in a FACS-based in vitro assay to inhibit the proliferation of CFDA-labeled CD4⁺ cells (ie, CD49d⁺CD127⁺ cells removed from the Treg population by the depletion). The panels indicate the CFDA staining without any stimulation (top panel), after stimulation (middle panel) or after stimulation in the presence of CD49d⁻CD127⁻ Treg cells at a ratio of 1:1 (bottom panel). Numbers represent the percentage of dividing CD4⁺ cells. Data are representative of 8 independent experiments with blood from healthy donors (average inhibition, 78% ± 11%).