Cell migration from the skin to DLN during a cutaneous immune response.
(A) Scheme of the experimental protocol is as follows: the dorsal skin of Kaede/Foxp3hCD2/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 CD44mid naive (M), CD44hi 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 Foxp3hCD2/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).