Suppressive capacity segregates with the CD49d⁻CD127⁻ CD4⁺ T-cell subset. (A) CD49d/CD127 FACS sorting. CD4⁺ cells isolated from PBMC with a commercial MACS-depletion kit were stained with α-CD49d and α-CD127 and sorted by FACS into the CD49d⁻CD127⁻ and the CD49d⁺CD127⁻ subset (top panels). The Foxp3 versus CD25 staining is shown in the bottom panels. Numbers represent percentages of cells per quadrant. (B) In vitro suppression assay. CD49d⁻CD127⁻ and CD49d⁺CD127⁻ cells were incubated with CD4⁺ cells. Proliferation of CD4⁺ cells was induced by stimulation; suppressor cells were added at a ratio of 1:1. Inhibition of the proliferative response of CD4⁺ cells was determined in a FACS-based suppression assay as shown in Figure 4B. Suppression is expressed as “% inhibition” based on the fraction of dividing cells in reference to noninhibited stimulated CD4⁺ cells. Summarized data of 4 independent experiments are shown (% inhibition CD49d⁻CD127⁻ cells, 76.9% ± 12.8%; CD49d⁺CD127⁻ cells, 27.8% ± 7%).