PMC

Kinetics of langerin⁺ repopulation in the dermis. Kinetics of reappearance of langerin⁺ DCs was followed by flow cytometry in the dermis. (A) Dots plots show langerin expression (EGFP) among live (DAPI⁻) CD45⁺I-A^b+ cells in the dermis at different times after DT treatment. (B) Graph in log scale presents the percentage of I-A^b+ langerin⁺ DCs among total dermal DAPI⁻CD45⁺ cells (•) versus epidermal LCs (▪) at different times after DT treatment. (right) Histogram shows the percentage of I-A^b+ langerin⁺ DCs among DAPI⁻CD45⁺ cells before and after DT treatment (DAY14). Mean values (± the SD) are shown. Representative data from three independent experiments are shown. Each experiment included at least three separately analyzed mice per time point; error bars represent the SD between the results obtained from each of the three mice.

Phagocytic processing and migration capacities of blood-derived langerin⁺ DCs. Lethally irradiated F1 (C57BL/6 I-A^b−/−/BALB/c I-E^d+) mice were reconstituted with BM cells isolated from donor Langerin-EGFP CD45.1⁺ C57BL/6 I-A^b+ mice. (A, top) Overlaid histograms show YAe staining on gated host LCs (CD45.2⁺CD11c⁺Langerin-EGFP⁻), donor langerin⁺ dermal DCs (CD45.1CD11c⁺Langerin-EGFP⁺), and donor langerin⁻ dermal DCs (CD45.2⁺CD11c⁺Langerin-EGFP⁻). LCs isolated from F1 (C57BL/6 I-A^b+/+/BALB/c I-E^d+) mice were used as controls. (A, bottom) YAe staining is shown for host LC-derived DCs (CD45.2⁺CD11c⁺Langerin-EGFP⁻), donor langerin⁺ DCs (CD45.1⁺CD11c⁺CD8a⁻Langerin-EGFP⁺), and donor langerin⁻ DCs (CD45.2⁺CD11c⁺CD8⁻langerin⁻) in the skin DLNs. Corresponding YAe isotype control (blue) and WT F1-positive control (green) are shown. (B) Mean values (± the SD) of the percentage of YAe⁺ cells among donor langerin⁺ or langerin⁻ DCs in the dermis and skin DLNs. Representative data from two independent experiments are shown. Each experiment included at least three separately analyzed mice; error bars represent the SD between the results obtained from each of the three mice. (C) Host LCs in the epidermis, donor langerin⁺, or langerin⁻ DCs in the dermis, LC-derived DCs, or donor langerin⁺ or langerin⁻ DCs in the skin DLNs were purified by cell sorter and stained on cytospin for YAe and langerin as described in Materials and methods. Insets show YAe isotype control for each tested DC population. Dashed white lines indicate where cells were spliced from different fields to present more cells within a panel. Bar, 10 μm.

Origin of langerin⁺ DC in mice. This diagram illustrates a hypothetical view on cutaneous DC differentiation pathways in mice in the steady state. Epidermal LCs are maintained by radioresistant hematopoietic progenitors that have taken residence in the skin before birth, whereas the majority of dermal DCs derive from circulating DC precursors. Our results now suggest that circulating blood-derived CCR2⁺ DCs are constitutively recruited to the dermis. E- and/or P-selectin direct this recruitment in transplanted animals, and studies are conducted in the laboratory to examine if this is also true in nontransplanted mice. In response to cutaneous factors, CCR2⁺ DCs differentiate into dermal langerin⁺ DCs, but might also be able to differentiate into dermal langerin⁻CX3CR1^+/− DCs. Dermal langerin⁺ DCs transit, but do not accumulate in the dermis, where they capture skin antigens, before emigrating to the skin DLNs to present skin-derived peptides in the context of MHC molecules. Both LCs and dermal DCs require CCR7 to migrate to the T cells area of skin DLNs. In the skin DLNs, dermal langerin⁺ DCs are undistinguishable from LC-derived DCs, and they are characterized as CD8⁻langerin⁺CD11c^lo, but differ from blood-derived CD8⁺ langerin^loCD11^hi DCs. Whether CCR2⁺ blood precursors that give rise to dermal langerin⁺ DCs and contribute to CD8⁻langerin⁺ CD11c^lo in the skin DLNs represent a committed DC precursor or a circulating monocyte is currently under study in the laboratory.

Homeostasis of langerin⁺ DCs in parabiotic mice. Each parabiotic pair consisted of one CD45.1⁺ C57BL/6 mouse with either a CD45.2⁺ C57BL/6 mouse (A–D) or CD45.2⁺ Langerin-DTR/EGFP C57BL/6 mouse for DT depletion experiments (E–H). (A–D) Skin and skin DLN chimerism was examined by flow cytometry in untreated CD45.2⁺ parabiont of a CD45.1/CD45.2 parabiotic pair at 2 mo after the initiation of parabiosis. (A) Dot plots show the chimerism of gated LCs (CD45⁺I-A^b+ langerin⁺) in the epidermis (top) and the chimerism of gated langerin⁻ and langerin⁺ DCs (CD45⁺I-A^b+) in the dermis (bottom). (B) Bar graphs show the percentages of donor (CD45.1⁺)-derived cells among gated LCs (top) and langerin⁻ or langerin⁺ dermal DCs (bottom). (C) Dot plots show the percentage of host and donor cells among gated CD11c⁺ I-A^b+ DCs, langerin⁻CD8⁻ DCs and langerin⁺CD8⁻ DCs. (D) Mean values of langerin⁻ versus langerin⁺ DCs chimerism in CD45.2⁺ parabionts. Similar data are found in the skin and skin DLNs of untreated CD45.2 Langerin-DTR/EGFP parabionts (unpublished data). Representative data from 5 separate experiments are shown. Each experiment included two to four mice. Skin was separately analyzed for each mouse, whereas skin DLNs were pooled (pool of two mice); error bars represent the SD between the results obtained from each mouse (skin) or from pooled DLNs (skin DLN). (E–H) Similar data to that found in A–D was obtained from DT-treated CD45.2⁺ Langerin-DTR/EGFP parabiont of a CD45.1/CD45.2 Langerin-DTR/EGFP parabiotic pair. Data presented are from 18 d after DT treatment, a time when LCs are still depleted (top), whereas langerin⁺ dermal DCs are recovered. Representative data from two separate experiments are shown. Each experiment included two separately analyzed mice, except for the skin DLNs that were pooled. For the skin, error bars represent the SD between the results obtained from each of the two mice.

Mechanisms involved in the recruitment to the dermis and the migration to the skin DLNs of dermal langerin⁺ DCs. (A–D) Host congenic CD45.1⁺C57BL/6 were lethally irradiated and transplanted with donor CD45.2⁺ C57BL/6 (Control), CCR7-deficient (CCR7 KO), or CD62L-deficient (CD62L KO) BM. After complete donor engraftment, the chimerism of total langerin⁺ cells in the dermis and in the skin DLNs was measured by flow cytometry. (A) Dot plots show CD45.1 (host) versus CD45.2 (donor) expression in langerin⁺ dermal DCs from control, CCR7KO, and CD62LKO chimeric mice. (B) Mean values (± the SD) of langerin⁺ dermal DCs chimerism. (C and D) Same as in A and B, but for CD8⁻langerin⁺ DCs in the skin DLNs of control, CCR7 KO, or CD62L KO groups. (E) Lethally irradiated host wild-type CD45.2⁺C57BL/6 (Control) or CD45.2⁺ P/E-selectin deficient (P/E-selectin KO) were lethally irradiated and transplanted with congenic CD45.1⁺C57BL/6 BM. After complete donor engraftment, the chimerism of total CD45⁺I-A^b+ langerin⁺ DCs in the dermis was measured by flow cytometry. Bar graph shows the chimerism of langerin⁺ dermal DCs in control group (black bars) versus P/E-selectin KO group (white bars) 2 mo after transplantation. Mean values (± the SD) are shown. (F) CD45.1⁺ C57BL/6 were lethally irradiated and reconstituted with a mixture of CD45.1⁺ WT BM and CD45.2⁺ BM isolated from WT, CCR2 KO, or CCR6 KO BM, as described in the Materials and methods. 2 mo after transplant, chimerism of total langerin⁺ DCs in the dermis was measured. Bar graph shows the mean value of the percentage of CD45.2⁺ cells among langerin⁺ and langerin⁻ dermal DCs in control group (black bars), CCR2 KO group (white bars), and CCR6 KO group (gray bars). Mean values (± the SD) are shown. Representative data from two independent experiments are shown. Each experiment included at least three separately analyzed mice; error bars represent the SD between the results obtained from each of the three mice.

Kinetics of langerin⁺ repopulation in the epidermis and in skin DLNs. 6–8-wk-old Langerin-DTR/EGFP mice received a single intraperitoneal injection of DT (1 μg) and the kinetics of reappearance of langerin⁺ was followed by flow cytometry in the epidermis and the skin DLNs at different time points after depletion (Fig. S1). (A, top) Dot plots show I-A^b and langerin expression (EGFP) among living (DAPI⁻) CD45⁺ cells in the epidermis at different times after DT treatment. The remaining CD45⁺I-A^b−langerin-EGFP⁻ cells represent dendritic epidermal T cells (DETC). (A, bottom) The percentage of cells expressing CD8 and langerin among DAPI⁻CD11c⁺I-A^b+ DCs is shown for skin DLNs. (B) Graphs in log scale present the kinetics of reappearance of LCs in the epidermis and CD8⁻langerin⁺ (▪) versus CD8⁺langerin^lo (•) in the skin DLNs. Mean values (± the SD) are shown. (right) Histogram shows the percentage of CD8⁻langerin⁺ (mean values ± the SD) among CD11c⁺I-A^b+ cells before and after DT treatment (DAY14). (C) Overlaid histograms show the langerin levels of expression measured either by EGFP fluorescence (top) or langerin intracellular staining (bottom) in skin DLN CD11c⁺I-A^b+ CD8⁻ langerin⁺ cells before and after DT treatment (DAY14). C57BL/6 control and isotype control are shown in black for Langerin-EGFP or langerin intracellular staining, respectively. Representative data from three independent experiments are shown. Each experiment included at least three separately analyzed mice per time point; error bars represent the SD between the results obtained from each of the three mice. (D) Double immunostaining (B220 in blue and langerin in red) of skin DLNs from Langerin-DTR/EGFP (Langerin-EGFP in green) before and after DT treatment (DAY14). Fig. S1 is available at http://www.jem.org/cgi/content/full/jem.20071733/DC1. Bars: (top) 200 mm; (bottom) 50 mm.

Homeostasis of langerin⁺ DCs after congenic BM transplantation. Lethally irradiated host CD45.2⁺C57BL/6 were transplanted with congenic donor CD45.1⁺C57BL/6 BM (CD45.1 into CD45.2 chimeric mice). 2 mo after reconstitution, host versus donor chimerism was measured in the langerin⁺ DC population in the skin (A and B) and in skin DLNs (D and E). (A) Dot plots show I-A^b and langerin expression among dermal CD45⁺ cells, and the host versus donor chimerism of langerin⁺ dermal DCs (CD45⁺I-A^b+). (B) Mean values of langerin⁺ DCs chimerism in the dermis. LC (CD45⁺I-A^b+ langerin⁺) chimerism in the epidermis is shown as control. (C) Expression of different markers was compared by flow cytometry between host remaining (CD45.2⁺I-A^b+ langerin⁺) LCs and donor-derived langerin⁻ or langerin⁺ dermal DCs. (D) Dot plots show CD8 versus langerin expression among CD11c⁺I-A^b+ cells in the skin DLNs and host versus donor chimerism of CD8⁻ langerin⁺ DCs. (E) Mean values of langerin⁺ DC chimerism. (F) Phenotype of donor-derived CD8⁻ DC langerin⁺, CD8⁺ DC langerin⁻, and host remaining LCs. (G) Back skin cross-sections isolated from CD45.2 Langerin-EGFP into CD45.1 chimeric mice (left) or the reverse CD45.1 into CD45.2 Langerin-EGFP chimeric mice (right) were stained with anti-langerin antibody. Nuclei were counterstained with DAPI. Bar, 50 μm. (H) Dermal cell suspensions were prepared from CD45.2 Langerin-EGFP into CD45.1 chimeric mice. Donor langerin⁺ dermal DCs were sorted on the criteria of CD45.2, I-A^b, and EGFP expression and harvested onto cytospin slides for langerin staining. Inset shows langerin staining control. Bar, 10 μm. Representative data from four independent experiments are shown. Each experiment included at least three separately analyzed mice; error bars represent the SD between the results obtained from each of the three mice.