Two developmental pathways give rise to langerin⁺ skin-derived DCs found in CLNs. (A) Staining of epidermal sheets from Lang-DTR-EGFP → B6 (CD45.1) chimeras, wild-type (WT), and Lang-DTR-EGFP mice with an anti-langerin antibody (red). As expected, the langerin⁺ cells found in the epidermis of Lang-DTR-EGFP mice differ from those of WT mice and of Lang-DTR-EGFP → B6 (CD45.1) chimeras in that they express EGFP. Genotypes are indicated for each column and markers on the left. Results are representative of at least 10 mice of each genotype. (B) Tissue-derived DCs found in CLNs were defined on the basis of their CD11c^{inter to high} MHCII^high phenotype (Fig. S2), and analyzed for the expression of langerin versus CD45.2, langerin versus EGFP, and EGFP versus CD45.2. Contour plots from Lang-DTR-EGFP CLNs are also included for comparison. The percentages indicated for each window correspond to the percentage of cells among CD11c^{inter to high}, MHCII^high DCs. Results are representative of at least five experiments. (C) Staining of a CLN section from a Lang-DTR-EGFP → B6 (CD45.1) chimera with an anti-langerin antibody (red) shows that host-derived (EGFP⁻ langerin⁺) LCs colocalize with donor-derived (EGFP⁺ langerin⁺) LCs. A stained section from Lang-DTR-EGFP mice is shown for comparison. Bars: (A) 50 μm; (C) 200 μm.

Donor-derived langerin⁺ DCs are found in skin explants. (A) Confocal microscopic analysis of epidermal sheets from B6 (CD45.1) → Lang-DTR-EGFP chimera that were left untreated (untreated) or had received 3 injections of DT (DT 3 X) a week apart were analyzed 1 d after the last DT injection. Staining with anti-langerin antibody (red) revealed the presence of langerin⁺ EGFP⁺ LCs in untreated sheets and their complete absence 1 d after the last DT injection. Bars: (untreated panel) 50 μm; (DT-treated panel) 300 μm. Results are representative of at least two experiments. (B) Ear explants from B6 (CD45.1) → Lang-DTR-EGFP chimera that had received 3 injections of DT (DT 3 X) were prepared 1 d after the last injection and cultured in the presence of CCL21. CD11c⁺ MHCII⁺ cells migrating out of the ear explants were analyzed by using langerin-CD45.2, langerin-EGFP, and EGFP-CD45.2 plots. Ear explants from untreated B6 (CD45.1) → Lang-DTR-EGFP chimera and Lang-DTR-EGFP mice were also included for comparison. The percentages indicated for each window corresponds to the percentage of cells among CD11c⁺, MHCII⁺ cells. Results are representative of at least two experiments.

Donor-derived langerin⁺ DCs are located in the skin dermis. (A) Staining of sections of ear from B6 (CD45.1) → Lang-DTR-EGFP chimera that had received 3 injections of DT and were prepared 1 d after the last injection were stained with anti-langerin (red) and anti–keratin 5 (blue) antibodies. Staining with the anti–keratin 5 antibody allowed visualization of the epidermis. Donor-derived, langerin⁺ DCs (EGFP⁻, langerin⁺) are exclusively found in the dermis. Bar, 30 μm. Ep, epidermis; D, dermis. Results are representative of at least two experiments. (B) CLNs of DT-treated B6 (CD45.1) → Lang-DTR-EGFP chimera were collected 1 d after the last DT injection. Tissue-derived (CD11c^{inter to high}, MHCII^high) DCs were analyzed as described in the legend of Fig. 1 for the expression of langerin-CD45.2, langerin-EGFP, and EGFP-CD45.2. The percentage indicated for each window corresponds to the percentage of cells among CD11c^{inter to high}, MHCII^high DCs. Results are representative of at least 4 experiments.

Kinetics of reappearance of donor-derived langerin⁺ DCs after DT ablation. Ear explants and CLNs from Lang-DTR-EGFP → B6 (CD45.1) chimeras that had received 3 injections of DT 1 wk apart were prepared at the specified time points after the last DT treatment. For ears, analysis was performed on cells migrating out of CCL21-treated ear explants. Gated DCs (ears, CD11c⁺ MHCII⁺; CLNs, CD11c^{inter to high} MHCII^high) were analyzed using langerin-EGFP plots. A window corresponding to the langerin⁺ EGFP⁺ subset is shown. The percentage indicated for this window corresponds to the percentage of donor-derived, langerin⁺, EGFP⁺ cells within the DC gates specified above. Also shown is a confocal microscopic analysis of a section of ear from a DT-treated Lang-DTR-EGFP → B6 (CD45.1) chimera 14 d after the last DT treatment. Staining with anti-langerin (red) and anti-keratin 5 (blue), revealed that donor-derived (EGFP⁺), langerin⁺ DCs are located exclusively in the dermis, whereas host-derived (EGFP⁻), langerin⁺ DCs are found in the epidermis. Some donor-derived, langerin⁺ DDCs (marked with a star) are found adjacent to hair follicles. Results are representative of at least two experiments. Ep, epidermis; D, dermis. Bar, 30 μm.

BrdU incorporation of host- and donor-derived langerin⁺ DCs found in the skin.Lang-DTR-EGFP→ B6 (CD45.1) chimeras were exposed to BrdU for 1 wk, and ear explants were prepared. DCs migrating out of the ear explants were gated on CD11c⁺, MHCII⁺ cells and analyzed. Three subsets can be distinguished using langerin versus CD45.2 dot plots: host-derived LCs (subset I: langerin⁺, CD45.2⁻, EGFP⁻), donor-derived, langerin⁺ DDCs (subset II: langerin⁺, CD45.2⁺, EGFP⁺), and donor-derived, langerin⁻ DDCs (subset III: langerin⁻, CD45.2⁺, EGFP⁻). Histograms show BrdU incorporation for each subset. Gray-filled histograms correspond to control staining profile of mice that received no BrdU. Percentages of BrdU⁺ cells are indicated. Results are representative of at least two experiments.

Wild-type langerin⁺ DDCs express higher levels of CD45.2 than wild-type epidermal LCs. Wild-type C57BL/6 (CD45.2⁺) mice were left untreated or exposed to BrdU for 1 wk, and ear explants were prepared and cultured in the presence of CCL21. The MHCII⁺, CD11c⁺ DCs migrating out of the ear explants were analyzed using langerin-CD45.2 plots. Two discrete subsets can be readily discriminated among langerin⁺ DCs according to the levels of CD45.2 they express. Subset I corresponds to LCs and expresses intermediate levels of CD45.2, whereas subset II corresponds to langerin⁺ DDCs and expresses high levels of CD45.2. Subset III corresponds to langerin⁻ DDCs. Histograms show BrdU incorporation for each subset. Gray-filled histograms correspond to control staining profile of mice that received no BrdU. Percentages of BrdU⁺ cells are indicated. Results are representative of at least two experiments.

Donor-derived LCs colonize emptied epidermal niches. B6 (CD45.1) → Lang-DTR-EGFP chimeras that had received 3 injections of DT 1 wk apart were analyzed at various time points after the last injection. (A) Epidermal sheets 1 (a), 4 (b), and 7 wk (c) after the last DT injection were stained with anti-langerin antibody (red) and ToPro3 blue. Rare patches of donor-derived LCs (langerin⁺ EGFP⁻) were found 1 wk after the last injection (arrow). An enlarged view (206.8 × 206.8 μm) of one of these patches is shown in the top right corner of a. d corresponds to a section of an ear from a DT-treated B6 (CD45.1) → Lang-DTR-EGFP chimera 7 wk after the last DT injection costained with anti-langerin (red) and –keratin 5 (blue) antibodies. Donor-derived LCs are found in both the dermis and epidermis. Bars: (a–c) 200 μm; (d) 50 μm. Results are representative of at least two experiments with two to three mice per experiment. Ep, epidermis; D, dermis. (B) Analysis of tissue-derived (CD11c^{inter to high}, MHCII^high) DCs present in the CLNs from untreated and DT-treated B6 (CD45.1) → Lang-DTR-EGFP chimera. In the case of the DT-treated mice, CLNs were analyzed 7 wk after the last DT-injection. Dot plots show langerin-EGFP distribution. Consistent with the results shown in A, 7 wk after the last DT injection, the CLNs of DT-treated B6 (CD45.1) → Lang-DTR-EGFP chimera are deprived of host-derived LCs and contain an abundant population of donor-derived LCs. Results are representative of at least two experiments.