IMCs lack suppressive activity. (A) Suppressive activity of Gr-1⁺ cells isolated from skin of S100A9Tg C57BL/6 mice treated for 6 wk with TPA. IMCs were added to splenocytes from OT-1 mice at the indicated ratios. Cells were incubated for 3 d in the presence of specific (SIINFEKL) or control (gp100) peptide. Proliferation was measured in triplicates by [³H]thymidine incorporation. Each experiment included three mice. (right) Antigen-specific suppressive assay with Gr-1⁺ MDSCs isolated from spleens of EL-4 tumor-bearing mice. Mean and SD are shown. **, P < 0.01 from no MDSC control. (B) Suppression activity of Gr-1⁺CD11b⁺ IMCs isolated from BM of mice treated with TPA. Allogeneic mixed lymphocyte reactions were performed using CD3-depleted irradiated FVB/N splenocytes as stimulators and BALB/c T cells as responders, mixed at 1:1 ratio. IMCs were added to the mix at the indicated ratios. Proliferation was measured in triplicates by [³H]thymidine incorporation (n = 3). Mean and SD are shown. (C) The phenotype of DCs isolated from skin of TPA-treated WT and S100A9Tg mice. Each group included four mice. Mean and SD are shown. ***, P < 0.001. (D) LCs in epidermis of mice. (top) Representative image of LCs. Bars, 50 µm. (bottom) Bar graph shows cumulative result of the number of LCs per 1 mm² of epidermis. Each group included four mice (mean and SD are shown). **, P < 0.01. (E) Migration of skin DCs to draining LNs. Dorsal shaved skins of WT and S100A9Tg mice previously treated with acetone or TPA were painted with DDAO, and 24 h later DDAO⁺CD11c⁺ cells were evaluated in draining LNs by flow cytometry. Each experiment was performed three times. Mean and SD are shown. *, P < 0.05. (F) T cells from OT1 mice were labeled with DDAO fluorescent dye and injected i.v. into TPA-treated WT and S100A9Tg C57BL/6 mice. OVA was applied to the skin 24 h later, and LNs and CD8⁺CD45.1⁺ T cell spleens were evaluated by flow cytometry 3 d after the application. A typical example of the CD8⁺CD45.1⁺ T cell proliferation is shown on the left, and cumulative results (mean ± SD) of three mice in each group are shown on the right.