IL-15 and dermal fibroblasts induce the proliferation of skin-resident FOXP3⁺ Tregs. (A) CD25^hiCD69^lo (CD25^hi) skin-resident T cells did not proliferate when treated with IL-2 and IL-15 alone (IL2/15) but proliferated at low levels when stimulated with soluble anti-CD3 and anti-CD28 antibodies (CD3/28). CD25^lo skin-resident T cells isolated from the same skin sample also did not proliferate when treated with IL-2 and IL-15 alone, but proliferated robustly after treatment with CD3 and CD28 antibodies. Error bars indicate standard deviation. (B) Sorted CD25^hi skin-resident T cells with regulatory activity proliferated when cultured on monolayers of dermal fibroblasts for 1 week in the presence of IL-2 and IL-15. (C) Sorted CFSE-labeled CD25^hi skin-resident T cells with regulatory activity cultured on fibroblasts without IL-2 and IL-15 did not proliferate (fib alone) but cells cultured on fibroblast monolayers with IL-2 and IL-15 (fib + IL-2, IL-15) did proliferate. (D) Unsorted CFSE-labeled skin-resident T cells cultured on fibroblast monolayers with IL-2 and IL-15 showed preferential expansion of the CD25^hiCD69^lo subset. (E) IL-15 and culture on dermal fibroblasts is necessary and sufficient to induce preferential expansion of FOXP3⁺ skin Tregs. Skin-resident T cells isolated from 2-week explant cultures were labeled with CFSE and cultured for 1 week on fibroblast monolayers with the indicated cytokines. CFSE-low cells have undergone proliferation. All results shown have been replicated using T cells from a minimum of 3 different skin donors. Numbers indicate the percentage of cells in each quadrant.