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.

Comparable T helper 1 (Th1) and CD8 T-cell immunity by targeting HIV gag p24 to CD8 dendritic cells within antibodies to Langerin, DEC205, and Clec9A

“…Only 25% of CD8 + DCs in B6 mice were targeted with α-Langerin mAb (Fig. 1B), however, which is consistent with data showing that Langerin is only expressed at low levels in CD8 + DCs in B6 mice (33,34). Labeling of CD8 + DCs was abrogated in Langerin B6 KO mice.…”

“…All fluorochrome-labeled antibodies are listed in Table S1. mAbs to Langerin (L31) (33), DCIR2 (33D1) (35), DEC205 (NLDC145) (36,37), Clec9A (10B4) (28), control Ig (GL117) (38), and CD40 (IC10) were produced from hybridoma supernatants; purified on protein G (Pierce); and, when necessary, labeled with Alexa 647 (Invitrogen) per the manufacturer's instructions. Anti-CD11c beads (N418), anti-CD8 beads (Ly-2), and anti-CD19 beads were from Miltenyi Biotec.…”

“…When we performed similar experiments in C57BL/6 mice, α-DEC205-gag-p24 led to higher CD4 + T-cell responses than α-Langerin-gag-p24 ( Fig. S4 A and B), possibly because Langerin was less expressed on CD8 + DCs in C57BL/6 mice (33,34). Fewer responding CD4 + T cells were seen with one dose of α-DCIR2-gagp24 or nontargeted HIV gag-p24 protein given along with α-CD40 and poly IC.…”

Cutting Edge: Langerin/CD207 Receptor on Dendritic Cells Mediates Efficient Antigen Presentation on MHC I and II Products In Vivo

“…Langerin/CD207 is a type II transmembrane protein (5). Originally, Langerin was thought to be LC-specific, but it is expressed on other DCs, especially CD8 ϩ DEC ϩ DCs in spleen (7) and skin draining lymph nodes (pLN), although at lower levels (6,8,9). The expression of Langerin by CD8 ϩ DEC ϩ DCs is primarily seen in BALB/c mice but is much reduced in C57BL/6 mice (8).…”

“…In this study, we use a newly developed mAb to the extracellular domain of mouse Langerin to begin to study the consequences of Ag uptake via Langerin in vivo (8). We have cloned the H and L chains of this mAb and engineered the H chain to encode OVA.…”