PU.1 and ID2 are essential for the steady-state differentiation of LCs. (A) Left, reporter mice of the indicated genotypes were assessed for the expression of GFP in epidermis (green) and migratory LN (red). Gray histograms show autofluorescence in LCs isolated from wild-type (wt) epidermis. Right, IRF4 expression was analyzed intracellular by flow cytometry from the same populations from a wild-type mouse. Gray histogram shows the background staining from a goat IgG isotype control. Results are representative of three experiments. (B) Epidermal sheets from mice of the indicated genotype were analyzed for CD11c and MHCII expression. Numbers represent the frequency of CD11c⁺MHCII⁺ LCs in the epidermis of a representative mouse of each genotype. Graph shows the mean proportion of epidermal CD11c⁺MHCII⁺ LCs ± SD from at least six mice per genotype. **, P < 0.01; ***, P < 0.001 (unpaired Student’s t test) compared with wild-type sample. (C) Epidermal sheets from mice of the indicated genotype stained for CD3ε (red, indicative of T cells) and Langerin (green, LCs) and analyzed by immunofluorescence microscopy. Bars, 80 µM. Data are representative of three experiments. Numbers below represent the mean density of LCs/mm² ± SD from 20 fields per genotype. (D) Flow cytometric of CD11c and MHCII expression in peripheral LN of the indicated genotype. Number represents the frequency of CD11c⁺MHCII^high migratory DCs. Results are representative of six mice. (E) Flow cytometry of Langerin and CD103 expression in CD11c⁺MHCII^high gated cells (as in D) from the peripheral LN cells of the indicated genotype. Numbers represent the frequency of cells within the indicated quadrant. Data are representative of three independent experiments. (F and G) Bar graphs show the mean proportion ± SD of CD103⁺langerin⁺ DCs (F) and CD103⁻langerin⁺ LCs (G), gated as in E, from at least six mice per genotype. **, P < 0.01; ***, P < 0.001 (unpaired Student’s t test) compared with wild-type sample. ns, not significant, P > 0.05.