Enhanced ear swelling response by Treg depletion and immunosuppressive activity of Treg subsets on T cell proliferation in vitro.
(A) The number of Tregs in the LNs after administration of Campath-1G Ab. (B) CHS: the Kaede/Foxp3hCD2/hCD52 mice were sensitized, and injected with vehicle or Campath-1G Ab before challenge (n = 8 for each group). (C–F) Immunosuppressive activity of Tregs. Kaede-red and Kaede-green Tregs were sorted from the Kaede/Foxp3hCD2/hCD52 mice, sensitized, challenged, and photoconverted. (C) Skin DLN cells of mice sensitized with DNFB were stimulated with DNBS in the presence or absence of Kaede-red Tregs or Kaede-green Tregs in vitro (n = 3). (D) Suppressive effect of Tregs in vitro. Kaede-red and Kaede-green Tregs were prepared as above and added to T cells stimulated with plate-bound anti-CD3 Ab. (E) Antigen specificity of Treg functions. LN cells from DNFB-sensitized or TNCB-sensitized mice were stimulated with DNBS or TNBS in vitro. Kaede-red and Kaede-green Tregs were added, and percentage inhibition of cell proliferation was evaluated as follows: (cell proliferation with DNBS or TNBS) – (cell proliferation with DNBS or TNBS in the presence of Tregs)/(cell proliferation with DNBS or TNBS) – (cell proliferation with vehicle) × 100. (F) Quantitative RT-PCR analysis on mRNA for Il10 (IL-10), Tgfb1 (TGF-β), and Ctla4 (CTLA-4) of Kaede-red Tregs and Kaede-green Tregs. The expression of each gene was normalized by the expression of Gapdh, and those in Kaede-green non-Tregs were normalized to 1 (n = 3). Data are representative of 3 independent experiments and presented as means ± SD (A–F). *P < 0.05 between the indicated groups (Student’s t test, A, B, E, and F; 1-way ANOVA followed by Dunnett multiple comparison test, C and D).