Absence of CD49d and CD127 defines Foxp3⁺ Treg cells free of contaminating effector cells. (A) Foxp3 expression of CD127⁺, CD49d⁻CD127⁻, and CD49d⁺CD127⁻ subsets of CD4⁺ T cells. Human PBMC were stained for CD4, CD25, CD127, CD49d, and Foxp3 and gated for CD4⁺ T cells. (Top panel) Costaining of CD49d and CD127. Gates and percentages of the 3 major populations are indicated. (Bottom panels) Costaining of CD25 and Foxp3 is shown for CD127⁺ (left), CD49d⁻CD127⁻ (middle), and CD49d⁺CD127⁻ cells (right). Numbers represent the percentages of CD25⁺Foxp3⁺ Treg cells in the indicated gate. Mean fluorescence intensity (MFI) of Foxp3 refers to the cells of the respective gate. One of 6 independent experiments is shown. (B) Release of IFN-γ and IL-17. Purified CD4⁺ T cells were stimulated in vitro with PMA/ionomycin to measure the cytokine production. The FACS analysis was carried out as in panel A, except that the cells were stained intracellularly with α–IFN-γ and α–IL-17.