CD25^hiCD69^lo T cells isolated from skin contain a population of natural regulatory T cells. (A) Skin-resident T cells isolated from skin cultured in IL-2 and IL-15 contained increased numbers of FOXP3⁺ T cells that also expressed high levels of CD25 and low levels of CD69. (B) Skin-resident T cells isolated from explant cultures were sorted into CD25^hiCD69^lo and CD25^lo populations. (C) CD25^hiCD69^lo T cells (CD25^hi) were anergic to stimulation with soluble anti-CD3 and anti-CD28 antibodies (αCD3, CD28) and suppressed the proliferation of CD25^lo T cells (CD25^lo) isolated from the same sample of skin. (D) Suppression was not affected by neutralizing antibodies to IL-10 (αIL-10) and/or TGF-β (αTGF-β) but was dependent upon cell-cell contact. Suppression was prevented by separation of the CD25^hi and CD25^lo T-cell populations by a 0.4-μm pore membrane (transwell). (E) A subpopulation of sorted CD25^hi T cells retain high expression of CD25 and FOXP3 after 1 week of culture on fibroblast monolayers in the presence of IL-2 and IL-15. Sorted CD25^lo cells lack FOXP3⁺ T cells and FOXP3⁺ T cells do not develop after 1 week of culture under the same conditions. (F) FOXP3 was up-regulated in both Treg and non-Tregs with cell activation, but this did not obscure identification of Tregs. T cells isolated from skin were examined for FOXP3 expression before and after stimulation with IL-2 and IL-15. Dotplots demonstrate that a clear population of Tregs was discernible under both conditions. The mean fluorescent intensities for each peak are shown on the histograms. FOXP3 expression increased in both groups with stimulation, but the Treg population remained separated from non-Tregs by at least a log increase in FOXP3 staining intensity. For scatterplots, numbers indicate the percentage of cells in each quadrant. For bar graphs, error bars indicate standard deviation.