In vitro–expanded CD4⁺CD25^high T cells are enriched in CD62L⁺CCR7⁺ cells that coexpress FOXP3 and CTLA-4. (A) Sorting gates (left dotplot) and FOXP3 expression of CD4⁺CD25^high (tinted histogram) and CD4⁺CD25^– T cells (solid line) within human PBMCs. (B) CD62L, CCR7, FOXP3, and CTLA-4 coexpression of CD4⁺CD25^high and CD4⁺CD25^– T cells after in vitro expansion. FACS-sorted cell populations were expanded in vitro for 2 weeks, rested for 4 days in medium with IL-2, and stained for CCR7 and CD62L, followed by intracellular staining for FOXP3 (top right histograms) or CTLA-4 (bottom right histograms), respectively. Numbered histograms on the right correspond to the marked quadrants in the dotplot of CD4⁺CD25^high cells shown on the left. Shaded histograms represent expression by all CD4⁺CD25^high cells, whereas solid lines represent the expression profile of the subpopulation within the respective quadrant; short dashed lines show the expression level of expanded CD4⁺CD25^– T cells, and lines with long dashes represent isotype controls. Numbers in dot plots indicate percent of cells within gates or quadrants.