Kinetics and suppression activity of CD25hi Kaede-red migratory Tregs.
(A–C) Characterization of CD25hi subset. Kaede/Foxp3hCD2/hCD52 mice were treated as in Figure 3A, and the expression levels of hCD2/Foxp3 and CD25 on CD4+hCD2/Foxp3+ Tregs in total, Kaede-red, and Kaede-green DLN cells and in non-DLN cells (A), the frequency of Kaede-red populations in each population (B), and the expression levels of surface markers on Kaede-red or Kaede-green Tregs in the DLNs (C) were analyzed. (D) Kinetics of T cell migration. Kaede/Foxp3hCD2/hCD52 mice were sensitized and challenged as in Figure 3A and photoconverted immediately (day 0), 1 (day 1), 2 (day 2), or 3 (day 3) days after challenge. The number of each subset migrating for 24 hours after photoconversion and the frequency of Kaede-red cells among each subset were measured. (E) Foxp3hCD2/hCD52 mice were sensitized with DNFB (S+) and challenged with DNFB (C+) or vehicle (C–). Skin suspensions were evaluated for the expression of hCD2/Foxp3 and CD25. (F) Skin DLNs cells of sensitized B6 mice were stimulated in the absence or presence of Kaede-red total hCD2+ Tregs (25hi/int), CD25hi Tregs (25hi), or CD25int Tregs (25int). (G) mRNAs for Il10 (IL-10), Tgfb1 (TGF-β), and Ctla4 (CTLA-4) of Kaede-green CD25int or CD25hi Tregs, Kaede-red CD25int or CD25hi Tregs, or Kaede-green CD25int Tregs in DLNs (D) or non-DLNs (N) were evaluated. The expression level in Kaede-green CD25int Tregs was normalized to 1. Data are presented as means ± SD (n = 3) (D, F, and G). *P < 0.05 between indicated groups. (F and G). Numbers within plots or histograms indicate percentage of cells (A, B, and E).