Generation of the langerin^−/− mouse. (a) Schematic diagram of the mouse langerin gene locus (Langerin^wt), langerin^−/− targeting construct, the neomycin-containing allele (Langerin^neo), and the langerin^−/− allele (Langerin^null). Filled black boxes, exons; filled dark grey box, 3′ untranslated region; filled black triangles, LoxP sequences; filled grey boxes, neomycin (NEO) and herpes simplex virus thymidine kinase (TK) expression cassettes; arrows, location of PCR oligonucleotide binding sequences; hatched black box, 5′ external EcoRV-KpnI single-copy probe. Restriction enzymes: B, BglII; E, EcoRV; K, KpnI; X, XhoI. (b) Southern blot analysis of EcoRV-digested genomic DNA from one of the injected ES clones (+/Neo) and a wild-type ES cell clone (WT). (c) PCR of genomic tail DNA from wild-type (WT), heterozygote (+/−) and langerin^−/− homozygote (−/−) mice. M, 100-bp DNA ladder (Invitrogen).

Langerhans cells are present in the epidermis of langerin^−/− mice. (A) Epidermal sheets from C57BL/6 control (a to c) and langerin^−/− (d to f) mice fixed in acetone were stained with rat isotype antibody (a and d), rat anti-langerin MAb clone 929F3 (b and e), or hamster anti-CD3+ rat anti-I-A^b (c and f), followed by goat anti-hamster antibody-Alexa Fluor 488 (green) and goat anti-rat antibody-Alexa Fluor 594 (red). Original magnification, ×400. (B) LC were sorted from the epidermis after ear sheet trypsin treatment. Cells were stained for CD11b (PerCP-Cy5), I-A^b (PE), and cell surface (MAb 205C1 FITC) or intracellular (MAb 929F3 FITC) langerin and analyzed by flow cytometry. Cells were gated on CD11b (gate R2 in left panels) and then analyzed for I-A^b and the presence of langerin, analyzed either at the cell surface (middle panels) or intracellularly (right panels).

Epidermal Langerhans cells in langerin^−/− mice do not have BG. Native ear skin of langerin^−/− mice (A, C, and D) and normal control mice (B and E) was processed for transmission electron microscopy. Rod-shaped BG were readily detected in LC from normal mice (panel B, lower left region enlarged in panel E; BG are marked with arrowheads). In the epidermis of langerin^−/− mice, none of the inspected LC possessed BG. Two high-power examples from two different cells are given in panels C and D. Magnifications and bars: A, ×7,000 and 1 μm; B, ×9,800 and 1 μm; C, ×51,000 and 200 nm; D, ×25,000 and 200 nm; E, ×40,000 and 200 nm.

In vivo migration of epidermal Langerhans cells is unaffected in langerin^−/− mice painted with FITC. C57BL/6 controls and langerin^−/− mice were painted with FITC on the dorsa of both ears. Two days later, mice were sacrificed and cells from draining LN were harvested, enriched for CD11c-expressing cells with magnetic beads, and immunolabeled for flow cytometry analysis. (A) Dot plots show FITC versus CD11c from the LN draining the FITC-painted ears of both mouse lines; (B) langerin/CD207 versus FITC dot plots on CD11c⁺-gated LN cells stained for intracellular langerin. Percentages for each quadrant are noted.

Protein antigen presentation and cross-presentation is not altered in langerin^−/− mice. (A) In vitro OVA presentation. Langerhans cells were allowed to crawl out from the epidermis of C57BL/6 control and langerin^−/− mice. After 72 h, cells were counted and placed in 96-well plates with different concentrations of OVA protein and OVA-specific CD8⁺ T lymphocytes from OT-I (MHC class I restricted) or CD4⁺ T lymphocytes from OT-II (MHC class II restricted) mice for 48 h. Triplicate supernatants were collected and added to the IL-2-dependent CTLL2 cell line, and proliferation was assessed with [³H]thymidine. (B) In vivo OVA presentation. C57BL/6 control and langerin^−/− mice were reconstituted with CFSE-labeled spleen cells from OT-I or OT-II mice. After 48 h, the recipient mice were injected with OVA protein or PBS in the footpads. Four days later, draining LN cells were harvested and analyzed for CFSE staining by flow cytometry to assess the specific proliferation of the adoptively transferred T lymphocytes. (C) OVA cross-presentation. C57BL/6 and langerin^−/− mice were injected intravenously with OVA protein and sacrificed 18 h later for spleen recovery. DC were enriched from spleens, and CD8⁺ or CD4⁺ subpopulations were sorted. Graded numbers of each DC subpopulation were then cultured in duplicate for 3 days with 10,000 OVA-specific T lymphocytes from OT-I or OT-II mice. Each well was pulsed with [³H]thymidine for 8 h, and the incorporated radioactivity was counted.

langerin^−/− and C57BL/6 mice are equally susceptible to pathogenic microorganisms. (A) K. pneumoniae. K. pneumoniae (10³ CFU) or PBS was delivered directly into the tracheas of anesthetized C57BL/6 control and langerin^−/− mice. The survival curves are presented. (B) M. tuberculosis. C57BL/6 control and langerin^−/− mice were exposed to M. tuberculosis-loaded aerosols. Five mice per group weresacrificed on days 1, 8, 21, and 98. Spleens and lungs were homogenized, and M. tuberculosis CFU were counted after culture on solid medium. Mean CFU ± standard deviations are represented. (C) L. major. L. major metacyclic promastigotes (10³) were delivered intradermally into the right ear of C57BL/6 control and langerin^−/− mice (ventral side). The thickness of both ears was recorded weekly and plotted ± standard deviation. Pictures show that epidermally isolated LC from both langerin^−/− and C57BL/6 control mice were able to capture parasites. Cells (2 × 10⁶) were cultured at 34°C in 5% CO₂ overnight with 2 × 10⁷ L. major promastigotes. After adhesion to poly-l-lysine-coated coverslips and fixation, cells were stained with an anti-I-A^b biotinylated MAb and L. major was stained with a hamster polyclonal immune serum. Primary antibodies were detected with streptavidin-Alexa Fluor 488 (green) and anti-hamster antibody-Texas Red (red). Original magnification, ×1,000.

Chemical skin carcinogenesis is not modified in langerin^−/− mice. C57BL/6 control and langerin^−/− mice were subjected to a two-stage chemical carcinogenesis protocol with DMBA as the initiator and TPA as the promoter. Skin tumors were counted biweekly, and the graph represents the percentage of mice bearing tumors.