ATG5 deficiency in Langerhans cells disrupts autophagosomes and depletes their epidermal network. (A) Representative immunofluorescent staining of MAP1LC3B (LC3) on LCs obtained by in vitro emigration from epidermal sheets of Atg5^WT (Video 1) and Atg5^ΔCd207 (Video 2) mice. LC3: green; CD207: red; DAPI: blue. Scale bars: 10 µm. (B and C) Representative histogram plot of LC3B staining and (C) quantification of autophagy flux in immature or mature LCs of 3-wk-old Atg5^WT and Atg5^ΔCd207 mice, treated or not with chloroquine. Autophagy flux was calculated as a ratio between MFI for LC3B in treated and untreated cells. (D) Representative dot plots for the identification of MHCII⁺ TCRγδ⁻ Langerhans cells (LCs; all CD207⁺) and MHCII⁻ TCRγδ⁺ dendritic epidermal T Cells (DETCs) among CD45⁺ cells in freshly digested back skin epidermal suspension of 6-mo-old Atg5^WT and Atg5^ΔCd207 mice. (E) Comparison over time of the percentage of LCs among live CD45⁺ epidermal cells for control (Atg5^WT and Atg5^WT/Δ) and Atg5^ΔCd207 mice. (F) Percentage of LCs among live CD45⁺ cells in freshly digested back skin epidermis of 10–20 wk-old Atg7^WT and Atg7^ΔCd207 mice. (G) Percentage of CD45⁺ MHCII+ CD207+ LCs among live CD45⁺ cells obtained from fresh epidermal cell suspensions of 2-mo-old Rubicn^−/− and Rubicn^+/+ mice. (H) Immunofluorescence staining of CD207+ LCs in ear epidermal sheets of 2-mo-old Rubicn^−/− and Rubicn^+/+ mice. Images are representative of two independent experiments. Insets 1 and 2: close-up views. (I) Representative immunofluorescent staining of CD207 on epidermal sheets of ear skin from 3 wk (upper panels) and 6-mo-old (lower panels) Atg5^WT and Atg5^ΔCd207 mice. Scale bars: 100 µm. (J) Percentages of epidermal LCs stained for BrdU incorporation and Ki67 expression for 4-wk (top) and 6-mo-old (bottom) Atg5^WT and Atg5^ΔCd207 mice. All data are pooled from at least three independent experiments, with each point representing one individual mouse (except [E]: n ≥ 4 mice per time-point). Statistical analysis: Mann–Whitney U test (except [C]: Kruskal–Wallis one-way ANOVA followed by Dunn’s multiple comparison test; and (E): two-way ANOVA followed by Tukey’s multiple comparison test). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, P > 0.05.