CD49d removes Th1- and Th17-like cells from CD25^high Treg preparations. (A) Double staining of human PBMC for CD4 and CD25. Percentage of CD4⁺ cells of total PBMCs is indicated. (B) Inverse correlation of CD49d with Foxp3 expression. Costaining of Foxp3 with CD25 (left panel), CD127 (middle panel), and CD49d (right panel) is shown for CD4⁺ cells gated according to Figure 1A. Numbers indicate percentage of cells in each quadrant. (C) Cytokine-secreting CD4⁺ cells express CD49d. CD4⁺ T cells were stimulated in vitro with PMA/ionomycin and analyzed by FACS. Staining is shown for CD49d versus IL-17 or IFN-γ. Numbers represent percentages of cells per quadrant. (D) Segregation of IFN-γ and IL-17 secretion with CD49d expression within the Foxp3⁺ subset. CD4⁺ cells were activated as described. After activation the cells were stained with α-Foxp3, α-CD49d, α–IFN-γ, and α–IL-17. Cells were gated on Foxp3. Density plots show the cytokine staining versus CD49d. Numbers refer to the percentage of cells in the quadrant. Average percentages of cytokine producers within the subsets (n = 6) were IL-17⁺Foxp3⁺CD49d⁺ (13% ± 6.8%), IL-17⁺Foxp3⁺CD49d⁻ (2.6% ± 0.6%); IFN-γ⁺Foxp3⁺CD49d⁺ (13.5% ± 3.9%), IFN-γ⁺Foxp3⁺CD49d⁻ (2% ± 1.2%). (E) Separation of cytokine-secreting effector cells from CD4⁺CD25^high Treg cells. PBMC were stained with α-CD4, α-CD25, and α-CD49d and sorted by FACS into the 3 CD4⁺ subsets CD25^low, CD49d⁺CD25^high, and CD49d⁻CD25^high. The sorted cell subsets were activated with PMA/ionomycin and stained intracellular for IFN-γ and IL-17. Numbers represent the percentage of cells in the indicated quadrant.