Detection of CCR6⁺ regulatory memory-like T cells in human PBMCs. (A) FACS analysis. PBMCs were stained with antibodies specific for CD4, CD25, CCR6, and CD45RO. Contour plots are shown for the CD4⁺ populations gated on high expression of CD25 (CD4⁺CD25^high, right panel), which essentially consists only of Treg cells,⁵⁰  and the population of CD4⁺ cells, that express either no or only low levels of CD25 (CD4⁺CD25^neg/low, left panel), which consists mainly of naive and effector cells. The plots show the staining of CCR6 versus CD45RO; the percentage of cells is indicated for each quadrant. One of 5 independent experiments with PBMCs from healthy donors is shown. (B) Suppression assay. CCR6^-CD25^high (•) and CCR6⁺CD25^high T cells (○) were isolated by FACS from human PBMCs. Their ability to suppress proliferation of CD4⁺ T cells stimulated by αCD3 was tested as described in Figure 2E. Proliferation in the absence of regulatory CD25^high T cells is represented as a bar. Line plot indicates proliferation in presence of increasing amounts of CD25⁺ T cells. One of 2 independent experiments is shown.