(A) Scheme of the experimental protocol is as follows: the dorsal skin of Kaede/Foxp3^hCD2/hCD52 was sensitized, and 5 days thereafter the abdominal skin was challenged. 2 days after challenge, the painted areas were photoconverted, and 24 hours after photoconversion, cells from the skin DLNs were analyzed by flow cytometry. (B and C) The frequency of Kaede-red and Kaede-green cells among CD4⁺ cells, and the frequencies of hCD2/Foxp3+ cells in total, Kaede-green, and Kaede-red cells among CD4⁺ cells were analyzed. Numbers within plots or histograms indicate percentage of cells in the respective areas. (D) The numbers of CD44^mid naive (M), CD44^hi memory (H), and naive plus memory (H/M) phenotypes of hCD2^–CD4⁺ non-Tregs (–), hCD2⁺CD4⁺ Tregs (+), and total (hCD2^– and hCD2⁺; +/–) CD4⁺ T cells among total CD4⁺ cells and Kaede-red cells in the DLNs were counted. (E) Number of Tregs and non-Tregs in the skin. The mice were painted with DNFB or vehicle on the abdomen, followed by DNFB or vehicle application on the ears. The number of CD4⁺ Tregs and CD4⁺ non-Tregs and the percentage ratio of Tregs among CD4⁺ T cells in the ears were measured. (F) Transwell assay. The number of hCD2⁺CD4⁺ cells and CD11c⁺ cells of skin-cell suspensions from Foxp3^hCD2/hCD52 mice that migrated to the lower chamber was analyzed. Data are presented as means ± SD (D–F) and are representative of 3 independent experiments. Student’s t test was performed between the indicated groups. *P < 0.05 (D–F).