Altered expression and endocytic function of CD205 in human dendritic cells, and detection of a CD205-DCL-1 fusion protein upon dendritic cell maturation

Abstract

CD205 (DEC-205) is a member of the macrophage mannose receptor family of C-type lectins. These molecules are known to mediate a wide variety of biological functions including the capture and internalization of ligands for subsequent processing and presentation by dendritic cells. Although its ligands await identification, the endocytic properties of CD205 make it an ideal target for those wishing to design vaccines and targeted immunotherapies. We present a detailed analysis of CD205 expression, distribution and endocytosis in human monocyte-derived dendritic cells undergoing lipopolysaccharide-induced maturation. Unlike other members of the macrophage mannose receptor family, CD205 was up-regulated upon dendritic cell maturation. This increase was a result of de novo synthesis as well as a redistribution of molecules from endocytic compartments to the cell surface. Furthermore, the endocytic capacity of CD205 was abrogated and small amounts of the recently identified CD205-DCL-1 fusion protein were detected in mature DC. Our results suggest that CD205 has two distinct functions -- one as an endocytic receptor on immature dendritic cells and a second as a non-endocytic molecule on mature dendritic cells -- and further highlight its potential as an immuno-modulatory target for vaccine and immunotherapy development.

Figure 1

CD205 is ubiquitously expressed amongst human peripheral blood mononuclear cells (PBMC) but is highest on antigen-presenting cells. (a) Fresh human PBMC were analysed by flow cytometry. Dot-plots show PBMC staining for CD205 (x-axis) and a lineage specific marker (y-axis) compared with isotype-matched antibody staining. Numbers are mean fluorescence intensity (MFI) values for lineage⁺ CD205⁺ cells. Data are representative of three experiments. (b) Mean MFI of CD205 staining on CD3⁺, CD19⁺ and CD14⁺ cells from three healthy donors + standard deviations.

Figure 2

Comparison of Endo180, macrophage mannose receptor (MMR) (CD206), and CD205 expression on dendritic cells (DC). Monocytes (Mono), and immature and mature DC (48 hr with 100 ng/ml of lipopolysaccharide), were stained with an isotype-matched control (black histograms) or antibodies specific for DC markers and C-type lectins (white histograms). CD205 is up-regulated upon maturation, whereas the MMR is down-regulated. Endo180 is unaffected by DC maturation. Data are representative of five experiments.

Figure 3

CD205 is up-regulated on mature dendritic cells (DC) over 48 hr as a result of de novo synthesis. (a) Flow cytometry histogram showing how CD205⁺ cells are divided into CD205^low or CD205^high (shaded) populations. (b) The percentage of CD205^high cells in a maturing DC population over a period of 48 hr. The results represent the mean of three experiments ± standard deviations. (c) Real-time polymerase chain reaction analysis of cDNA samples from monocytes, immature DC and mature DC, normalized against values for the housekeeping gene, hypoxyanthine-guanine phosphoribosyl transferase (HGPRT), and expressed as fold induction over monocyte values. The results represent the mean of triplicate samples + standard deviations. Data are representative of five experiments.

Figure 4

Mature dendritic cells (DC) translocate CD205 to the cell surface and down-regulate CD205-mediated endocytosis. Saponin-treated cells were stained with an isotype-matched control (a, d and g; MOPC21) and the MR6 antibody (b, e and h). Antibodies were detected with an rabbit anti-mouse immunoglobulin-tetra-methyl rhodamine iso-thiocyanate (TRITC) secondary antibody. A flow cytometry-based assay was used to analyse the rate of internalization of intercellular adhesion molecule-1 (ICAM-1) (6.5B5), Transferrin receptor (TfnR) (CD71), and anti-CD205 (MR6) in each cell type (c, f, and g). Internalization was plotted as a percentage of the total surface bound antibody at time zero. Data are representative of five experiments.

Figure 5

Detection of mRNA transcripts for putative CD205–DCL-1 fusion(s) in dendritic cells (DC). (a) Primers specific for CD205, DCL-1, the CD205–DCL-1 fusions, interleukin (IL)-10, IL-12p40 and β-actin were used to detect mRNA transcripts in cDNA from monocyte-derived DC. CD205 and DCL-1 transcripts were detected in all samples. Primers for the CD205–DCL-1 fusions produced two bands. Results are representative of five experiments. wt, wild type. (b) Schematic representation of predicted protein structures encoded by detected mRNA transcripts. CD, cytoplasmic domain; CR, cysteine-rich domain; CTLD, C-type lectin domain; FnII, fibronectin type II domain; TM, transmembrane domain.

Figure 6

Immunoprecipitated CD205–DCL-1 fusion protein can only be detected in mature dendritic cell (DC) samples. CD205 molecules were immunoprecipitated by MR6 monoclonal antibody (mAb) from the lysates of 107 monocytes, and immature or mature DC, under non-reducing conditions. Proteins were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and detected on a western blot using biotinylated MG38 or MOPC21 (control). A single band at ≈ 205 000 molecular weight (MW) was detected in the monocyte and immature DC sample corresponding to wild-type CD205. A similar band was detected in the mature DC sample. A weaker band, at ≈ 210 000–215 000 MW, corresponding to a CD205–DCL-1 fusion protein, was also detected in this sample. Data are representative of three experiments.