IMCs recruit CD4⁺ T cells to the skin. (A) The number of CD4⁺ T cells in the skin of WT and S100A9Tg FVB/N mice. The number of cells was evaluated by IHC and counted per square millimeter of tissue. Each experiment included five mice. (B) The number of γδ T cells in skin of TPA-treated WT and S100A9Tg C57BL/6 mice. The number of cells was evaluated by IHC and counted per square millimeter of tissue (n = 3). (C) The number of CD4⁺ T cells in the skin of WT and S100A9KO C57BL/6 mice evaluated by IHC and counted per square millimeter of tissue. Each experiment included five mice. (D) The proportion of CD4⁺ cells among CD45⁺ hematopoietic cells in WT and S100A9Tg mice treated with TPA and evaluated by flow cytometry. Six mice per group. (E) Intracellular staining of different cytokines in cells isolated from the skin of WT and S100A9Tg mice treated with TPA. CD4⁺ cells were gated. Each group included three to six mice. (F) Polarization of naive CD62L⁺CD4⁺ T cells by IMCs in vitro. T cells were cultured with BM IMCs from WT and S100A9Tg mice at a 1:1 ratio for 4 d in the presence of CD3/CD28 beads. Cells were then stimulated for 4 h with TPA/ionomycin in the presence of GolgiStop. Intracellular cytokines were evaluated within the population of CD4⁺ T cells by flow cytometry (n = 5). (A–F) Mean and SD are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (G) Localization of Gr-1⁺ and CD4⁺ T cells or γδ T cells in skin of TPA-treated S100A9Tg mice. Immunofluorescent microscopy with the indicated antibodies is performed. Merge staining included DAPI (blue). Typical example of three experiments is shown. Epidermis and dermis are marked with E and D. Please note that to see accumulated CD4⁺ T cells in the skin, the field had to be moved further down and epidermis was outside the area. Bars, 50 µm.