Immunosuppressive effect of Kaede-red Tregs in the skin.
(A) Suppression of CHS response by Kaede-red Tregs. Kaede-red or Kaede-green Tregs (4 × 103 cells/ear) of Kaede/Foxp3hCD2/hCD52 mice sensitized, challenged, and photoconverted as in Figure 3A were injected into ear skin of mice sensitized with DNFB 5 days prior. Immediately after injection, the mice were challenged, and the ear thickness change was measured at 48, 72, and 96 hours after challenge. (B–D) The mice were sensitized, challenged, and photoconverted as in Figure 3A. Twenty-four hours after photoconversion, 20 μl of 0.3% DNFB (challenge; +) or vehicle (challenge; –) (B) or 20 μl of 0.3% DNFB or 20 μl of 1% TNCB (C) was painted onto the ear. Twenty-four hours later, the ear skin and blood (D) were collected and dissociated for flow cytometry. The number of Kaede-red Tregs in the skin and the frequency of Kaede-red Tregs in CD4+ T cell subset of the blood were evaluated (n = 3, each group). Data are presented as means ± SD and representative of 3 independent experiments (A–C). Student’s t test was performed between the indicated groups. *P < 0.05. Numbers within plots indicate percentage of cells in the respective areas (D).