Production of monoclonal antibodies that recognize the extracellular domain of mouse langerin/CD207

Abstract

Langerin CD207 is a type II transmembrane protein. It is responsible for the formation of Birbeck granules, which are intracellular organelles within Langerhans cells, the dendritic cells of stratified squamous epithelia like the epidermis. Because current anti-CD207 antibodies have limitations, we prepared new monoclonals by immunizing rats with the extracellular region of mouse Langerin followed by a boost with enriched Langerhans cells (LCs). We secured a large panel of mAbs, most of which reacted with the carboxy terminal carbohydrate recognition domain. These mAbs could be used to immunoblot and immunoprecipitate mouse Langerin and to stain the cell surface and intracellular pools of CD207 by FACS analysis. Labeling of Birbeck granules was also achieved by immunoelectron microscopy. Anti-CD207 identified LCs in the epidermis and skin draining lymph nodes of BALB/c and C57BL/6 mice, but BALB/c mice had an additional Langerin(+) population in spleen, thymus and mesenteric lymph node. This additional subset had higher levels of CD8 and CD205 than epidermal LCs, and also had a less mature phenotype, i.e., lower MHC II, CD40 and CD86. Subcutaneous injection of IgG but not IgM forms of these new anti-CD207 mAbs led to rapid and selective labeling of the Langerin(+) cells in skin draining lymph nodes as well as spleen. The new IgG anti-CD207 mAbs should be useful for further research on LCs and dendritic cells including an evaluation of the consequences of antigen delivery within anti-CD207 mAbs in vivo.

Figure 1. Soluble forms of mouse Langerin and rat IgM mAbs

(A) Purification of hIgG1Fc-mLangerinECD and hIgG1Fc-mSIGN-R1ECD proteins. The recombinant proteins were stably expressed in CHO cells and purified from the culture medium by Protein A Sepharose. The purified recombinant proteins were analyzed on 10 % SDS-PAGE by Coomassie blue staining (upper panel) or transferred onto PVDF membrane and immunoblotted using anti-human IgG (lower panel). (B) Purification of SF-mLangerinECD and SF-mSIGN-R1ECD proteins. The recombinant proteins were stably expressed in CHO cells and purified from the culture medium by anti-FLAG M1 affinity gels. The purified recombinant proteins were analyzed on 10 % SDS-PAGE by Coomassie blue staining (upper panel) or transferred onto PVDF membrane and immunoblotted using anti-FLAG (lower panel). (C) Screening of rat anti-mLangerin immune serum by Western blot. A CHO cell-line stably expressing full-length mSIGN-R3 (negative control) and 4 different CHO cell-lines stably expressing full-length mLangerin were lysed and used to screen diluted sera from rats immunized with soluble mLangerin. The blots were incubated with the sera in 1:2000 dilutions. (D) MAb ML11 stains Langerin⁺ cells in situ. Ear epidermis (left panels) was separated from dermis, fixed with acetone for 20 min at RT, and double stained with anti-MHC II-PE and ML11 mAb followed by biotinylated anti-rat IgM and Alexa 488-labeled streptavidin. Skin draining lymph nodes (right panels) were embedded in OCT, sectioned at 10 μm, and stained with anti-B220-PE and ML11 mAb followed by biotinylated anti-rat IgM and Alexa 488-labeled streptavidin.

Figure 2. Characterization of rat IgG mAb to mouse Langerin

(A) FACS analysis of stable CHO/mLangerin cells with new rat IgG anti-CD207 mAbs (filled histograms) or with rat IgG isotype control III-10 mAb (open histogram), detected with PE-labeled anti-rat IgG Ab. (B) Binding of anti-mLangerin mAbs to different domains of Langerin. Whole cell lysates of 293T cells transfected with full-length mouse Langerin (Full), or with its extracellular domain (ECD) or its carbohydrate recognition domain (CRD), were applied to 10 % SDS-PAGE and blotted with the 10 new rat IgG anti-CD207 mAbs (L1 to L37) followed by HRP-conjugated anti-rat IgG. (C) Whole cell lysates of skin draining LNs (from C57BL/6 mice) were immunoprecipitated with mAbs of L31, L20 or III-10 isotype control. Samples were resolved in 10 % SDS-PAGE and immunoblotted with L31.

Figure 3. Staining of epidermal Langerhans cells with L31 mAb

(A) Ear epidermis was obtained, fixed with acetone for 20 min, and double labeled with anti-MHC II-biotin, followed by streptavidin-Alexa 555 and L31 followed by anti-rat IgG Alexa 488. DAPI staining was performed to detect the nucleus of the cells. (B) Freshly isolated LCs were surface-stained for MHC II-PE, CD11b-FITC and biotin conjugated L31 (filled histogram) or the corresponded isotype control (open histogram) followed by streptavidin-APC. Plots are gated on MHC II⁺ CD11b⁺ cells. (C) Ear skin explants were cultured in RPMI with 5 % FBS for 16 hrs before fixation, and stained for MHC II and Langerin as described in (A). (D) Sections of peripheral LNs from BALB/c mice were labeled for Langerin with L31 followed anti-rat IgG Alexa 488 (green) and anti-B220 Alexa 647 (red).

Figure 4. Birbeck granules can be visualized by immuno EM with L31

(A) CHO cells stably transfected with mLangerin (CHO/mLangerin) were processed for electron microscopy and analyzed for the presence of Birbeck granules (middle and right panels, filled arrow). No BGs were detected in control CHO/Neo cells (left panel). (B) CHO cells transfected with mLangerin (middle and right panels) were processed for electron microscopy and incubated with L31 (right panel) or the matching isotype control (middle panel) followed by 12 nm colloidal gold particles conjugated anti-rat mAbs (open arrow). CHO cells transfected with irrelevant cDNA as negative control (left panel, CHO/Neo) did not display membrane superimposition and were not stained with L31 mAb.

Figure 5. Phenotype of Langerin⁺ cells in lymphoid cell suspensions of BALB/c mice

(A) Skin draining LNs (pLN), spleen, mesenteric LNs (mLN) and thymus were treated with collagenase D. Low density fractions from a 30 % BSA gradient were used to enrich DCs. The expression of Langerin was evaluated after fixation and permeabilization. After gating out autofluorescent cells on the FL3 channel, large cells (defined by FSC versus SSC) were analyzed with L31 mAb and CD11c mAb. (B) Langerin⁺ cells (boxes in upper panels in A) were analyzed for the expression of CD8α, MHC II and DEC205. (C) Phenotype of the two types of Langerin⁺ cells found in peripheral LNs in the steady state and the Langerin⁺ cells in spleen, thymus and mesenteric LNs. Levels of surface MHC-II, CD40, CD86, CD80, B7-DC, B7-H1 and CCR7 expressed in CD11c^hi are compared to those found on the surface of CD11c^inter Langerin⁺ cells in peripheral LN. Langerin⁺ cells are identified after fixation and permeabilization. Dotted open histogram corresponds to the isotype control.

Figure 6. Expression of Langerin in different mouse strains

(A) The indicated organs from BALB/c mice were lysed and 30 μg were applied to 10 % SDS-PAGE, transferred onto PVDF membrane and immunoblotted using L31 mAb and anti-tubulin. (B) Elevated expression levels of Langerin in different tissues of BALB/c (“B”) mouse are detected in comparison to C57BL/6 (“C”) mouse by Western blot analyses with L31 mAb and anti-tubulin as described in (A). (C) F1 (BALB/c × C57BL/6) mice maintain the CD11c^hi Langerin^low population that is detected in BALB/c but not C57BL/6 mice. Peripheral LNs and spleen were treated with Collagenase D and the low density fractions from a 30 % BSA gradient were used to enrich DCs. After gating out autofluorescent cells by using the FL3 channel, large cells (defined by FSC versus SSC) were analyzed by flow cytometry for the expression of total Langerin after fixation and permeabilization (with L31), and surface CD11c and MHC II.

Figure 7. Surface expression of mLangerin as detected with L31 mAb

DCs from BALB/c mice were enriched by negative selection. In brief, spleen and skin draining LN cell suspensions were incubated with biotin-labeled anti-CD3, B220, CD19, DX5, Gr1 and Thy1.2 mAbs. The non-DCs were removed by the application of a magnetic field after the incubation with streptavidin-beads. Cells were stained with Alexa 647 labeled L31 mAb, or the corresponded isotype control, and CD11c FITC. For the total staining, the cells were fixed and permeabilized with cytofix/cytoperm buffer (BD Biosciences) following the manufacture’s instructions.

Figure 8. Langerin targeting of mAb L31 after s.c. injection

(A) BALB/c mice were injected s.c. with 10 μg of Alexa 647-conjugated rat IgG mAbs L31 or III-10, the isotype-matched control. At the indicated time points, DCs were enriched from the draining lymph nodes or spleen by floating in 30 % BSA and analyzed by FACS. The frequencies of DCs capturing the injected, labeled III-10 (top panels) or L31 are indicated. (B) Langerin⁺ cells in skin draining lymph nodes are targeted following s.c. injection of 25 μg of L31 or III-10 isotype control. Peripheral LNs were harvested 3 hrs post-inoculation and the injected mAbs visualized by staining with anti-rat IgG Alexa 555 (green) and anti-B220-Alexa 647 (red).