Generation of anti-human DEC205/CD205 monoclonal antibodies that recognize epitopes conserved in different mammals

Abstract

DEC205/CD205 is a C-type multilectin receptor, expressed highly in dendritic cells (DCs). Previous efforts to generate anti-human DEC205 (anti-hDEC205) monoclonal antibodies (mAbs) from mice immunized with subdomain proteins of hDEC205 resulted in a few mAbs. Recently, we expressed and utilized a full-length extracellular domain protein of hDEC205 to successfully generate 5 strong anti-hDEC205 mAbs from mice. In this study, DEC205 knockout (KO) mice were immunized with this full-length extracellular domain protein of hDEC205. One of the 3 immunized DEC205 KO mice was chosen for the highest anti-hDEC205 titer by flow cytometric analysis of serum samples on CHO cells stably expressing hDEC205 (CHO/hDEC205 cells) and used for hybridoma fusion. From a single fusion, more than 400 anti-hDEC205 hybridomas were identified by flow cytometric screen with CHO/hDEC205 cells, and a total of 115 hybridomas secreting strong anti-hDEC205 mAb were saved and named HD1 through HD115. To characterize in detail, 10 HD mAbs were chosen for superior anti-hDEC205 reactivity and further subjected to cloning and purification. Interestingly, out of those 10 chosen anti-hDEC205 HD mAbs, 5 mAbs were also strongly reactive to mouse DEC205 while 8 mAbs were found to stain DEC205(+) DCs on monkey spleen sections. In addition, we also identified that HD83, one of the 10 chosen HD mAbs, stains DEC205(+) DCs in rat spleen and lymph node. Therefore, by immunizing DEC205 KO mice with a full-length extracellular domain protein of hDEC205, we generated a large number of strong anti-hDEC205 mAbs many of which are cross-species reactive and able to visualize DEC205(+) DCs in lymphoid tissues of other mammals.

Figure 1. Reactivity of HD mAbs to human and mouse DEC205 on transfectant cells

(A) Two of the 10 chosen HD mAbs, HD30 and HD83, were compared with anti-hDEC205 MG38.2 and anti-mDEC205 NLDC145 mAbs for their reactivities to human and mouse DEC205. CHO cells stably expressing hDEC205 or mDEC205 were stained with HD30, HD83, cloned MG38.2 (MG38.2 with the constant regions of mouse IgG1 heavy chains), or cloned NLDC145 (NLDC145 with the constant regions of mouse IgG1 heavy chains). All antibodies were used at 1 µg/ml followed by detection with PE-conjugated anti-mIgG1. Mouse IgG1 antibody was used as a negative isotype control. (B) Reactivities of the 10 chosen HD mAbs to human and mouse DEC205 were compared by FACS analysis with CHO/hDEC205 and CHO/mDEC205 cells as in (A) but binding of each mAb to the cells was detected with PE-conjugated anti-mIgG.

Figure 2. Western blot analysis of anti-DEC205 mAbs

(A) Control CHO, CHO/mDEC205, and CHO/hDEC205 cells were lysed and each lysate equivalent to 5 × 10⁵ cells was subjected to the western blot analyses by HD30, HD83, NLDC145, and anti-actin antibodies. (B) A series of deletion constructs in the extracellular domain of hDEC205 were generated in fusion with hIgG1 Fc domain, and each deletion construct was expressed in 293T cells. Lysates of cells expressing different subdomains of hDEC205 were subjected to western blot analyses with HD30, HD83, or anti-hIgG.

Figure 3. Immunohistochemical stains of HD mAbs on the splenic tissues from human and monkey

(A) Cells expressing DEC205 at high levels were detected in the white pulp of the human spleen by HD30. Double staining of human spleen sections was carried out with anti-DEC205 mAb HD30 (in green) and anti-CD11c antibody (DCs in red). (B) HD83 stained DEC205⁺ cells in the T-cell areas of the white pulps in monkey spleen. Double staining of monkey spleen sections was carried out with anti-DEC205 mAb HD83 (in red) and antibodies to CD206 (red pulp macrophages in green; upper panel) or CD8 (T cells in green; lower panel).

Figure 4. HD83 stained DEC205⁺ DCs in the T-cell areas of rat lymphoid tissues

(A) Rat spleen was stained triple-immunofluorescently for HD83 (red), class II MHC (green), and tissue framework (IV collagen, white). Inset shows dendritic shape of HD83⁺ class II MHC⁺ cells in high magnification. Arrow indicates central artery. B: B-cell area; T: T-cell area; Scale bar = 80 µm (10 µm in high magnification). (B) Rat cervical lymph node was stained as in (A).