Blood Tregs and non-Tregs both proliferate when cultured with fibroblasts and IL-15. (A) T cells were isolated from the blood, and the highest 5% and lowest 10% of CD25 expressers were isolated by cell sorting and labeled with CFSE. (B) CD25^hi T cells contained 58% FOXP3⁺ Tregs, and a subset of these cells proliferated when cultured for 3 days with IL-15 and fibroblasts. (C) CD25^lo T cells contained less than 1% FOXP3⁺ Tregs; many of these cells proliferated when cultured with IL-15 and dermal fibroblasts. (D) Most CD25^hi proliferative cells (P1) were FOXP3⁺ Tregs, whereas nonproliferative T cells (P0) were purely non-Tregs. (E) Nonregulatory FOXP3⁻ T cells proliferated but did not up-regulate FOXP3 to levels seen in regulatory T cells. For scatterplots, numbers indicate the percentage of cells in each quadrant. (F) Both CLA⁺ and CLA⁻ blood Tregs expand when cultured with fibroblasts, IL-2, and IL-15. T cells were isolated as above, and CD25^hi T cells were further separated into CLA⁺ and CLA⁻ subsets. The number of FOXP3⁺ T cells before and after 1 week of culture with fibroblasts, IL-15, and IL-2 are shown.