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Keywords:

  • dendritic cell;
  • c-type lectin;
  • expression;
  • endocytosis

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

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.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

CD205 (also known as DEC-205 and gp200-MR6) is perhaps the least understood member of the macrophage mannose receptor (MMR) family of C-type lectins.1,2 Both human and mouse molecules share the same multidomain structure: an N-terminal cysteine-rich domain, a fibronectin type II domain and a series of 10 tandemnly repeated C-type lectin-like domains (CTLDs).3–5 CD205 is predominantly expressed by the thymic cortical epithelium and by dendritic cells (DC) in mice and humans (and other species6–10), but can also be detected at low levels on T and B lymphocytes and several other epithelial cell types.11–15 Whereas the MMR (CD206) mediates calcium-dependent interactions with carbohydrate ligands, such as mannose, fucose and glucose, CD205 possesses none of the amino acid motifs associated with carbohydrate binding, and ligands await identification.1,16 Despite this, much is known about the biology of CD205. Like the MMR, it is believed to function as an antigen-uptake receptor, binding and internalizing ligands and delivering them to endosomal compartments where major histocompatibility complex (MHC) class II loading occurs.3,17,18 Indeed, a number of studies have shown that two amino acid internalization motifs within the cytoplasmic tail of CD205 direct its trafficking via clathrin-coated pits (tyrosine-based motif) through late endosomes and lysosomes (tri-acidic motif) and back to the cell surface.3,18 Crucially, the CD205 endocytic pathway is non-stimulatory and has been elegantly utilized in vitro in humans,19 and in vivo, in mice,20–22 to deliver specific antigens to DC without inducing maturation. This is particularly useful since antigen delivery in the absence of maturational stimuli results in presentation of antigens by immature, or ‘semimature’ DC and the induction of specific T-cell tolerance.20 Tolerance induction can be overcome if antibodies to CD40 are given at the same time as the antigen to induce DC maturation.21 CD205 can also deliver antigens into both the MHC class II and MHC class I presentation pathways, indicating that CD205-mediated endocytosis might be coupled to a mechanism for cross-presentation.22 Thus, by targeting antigens to CD205 on immature DC, researchers could potentially switch off both specific CD4+ and CD8+ T-cell responses. Alternatively, targeting antigens to CD205 on maturing DC, perhaps by co-administering a maturation signal, such as a Toll-like receptor (TLR) agonist or antibodies to CD40, might induce a potent adaptive immune response. These properties make CD205 an extremely appealing target for those wishing to enhance specific T-cell responses,19,23,24 or extinguish them (as in autoimmunity or transplantation).

As CD205 carries obvious appeal as a therapeutic target, we undertook a detailed study of its expression, intracellular distribution and endocytic function, during human monocyte-derived DC development and maturation. In the course of our investigation, we analysed these cells for the possible expression of an intergenic splice variant of CD205 and a novel C-type lectin, DCL-1. The new protein, previously detected in Hodgkin lymphoma cell lines, carries the extracellular domains of CD205 plus an additional CTLD and a transmembrane and cytoplasmic domain derived from DCL-1.25 Significantly, the cytoplasmic domain of DCL-1 contains different endocytic and endosomal targeting motifs from those found in the wild-type CD205 molecule and could potentially mediate altered intracellular signalling or direct the receptor along new endocytic pathways.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Cell purification and generation of monocyte-derived dendritic cells

Fresh peripheral blood was collected from healthy volunteers after obtaining informed consent. Peripheral blood mononuclear cells (PBMC) were separated by centrifugation on a Ficoll–hypaque (Nycomed, Roskilde, Denmark) density gradient (according to the manufacturer's instructions). CD14+ monocytes were isolated by positive selection using magnetic antibody cell sorting (MACS) beads (Miltenyi Biotech, Bergisch Gladbach, Germany) and cultured in RPMI-1640 (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS) (BioSera, Ringmer, UK), 25 ng/ml of interleukin (IL)-4 and 50 ng/ml of granulocyte–macrophage colony-stimulating factor (GM-CSF) (First Link Ltd, Birmingham, UK). Cytokines were added on days 0 and 4 to induce DC differentiation. DC were activated to a mature phenotype on day 6 by the addition of 100 ng/ml of lipopolysaccharide (LPS) (from Salmonella minnesota; Sigma-Aldrich, Poole, UK), or 25 ng/ml of tumour necrosis factor-α (TNF-α) and 25 ng/ml of IL-1β (First Link), for a further 48 hr.

Fluorescence-activated cell sorter analysis

Fresh PBMC or DC were washed three times in cold fluorescence-activated cell sorter (FACS) buffer [phosphate-buffered saline (PBS) containing 3% FCS and 0·02% NaN3) and then stained with the appropriate antibodies – CD14 (Diaclone, Besancon, France), MHC class II (Pharmingen, Becton Dickinson; BD Biosciences, New Jersey, NJ, USA), CD80 (BD Biosciences), CD86 (BD Biosciences), CD40 (Diaclone), Endo180 (a kind gift from Professor C. M. Isacke, Institute of Cancer Research, London, UK), MR6–fluorescein isothiocyanate (FITC),11 CD206 (mannose receptor)–FITC (BD Biosciences), CD3–phycoerythrin (PE) (DakoCytomation Ltd, Glostrup, Denmark), CD14-PE (Diaclone), CD19-PE (Sigma-Aldrich) – at a final concentration of 10 mg/ml for 30 min on ice and in the dark. Isotype-control antibodies were mouse IgG1 (MOPC21; Sigma-Aldrich) and mouse IgG2a (Sigma-Aldrich). Cells were washed three times in FACS buffer and analysed immediately by flow cytometry (FACScalibur; Becton Dickinson).

Intracellular staining and microscopy

Cells were first fixed in 4% paraformaldehyde for 10 min on ice before being treated with 0·05% saponin in PBS. Cells were then stained intracellularly with 1 µg/ml of MR6 monoclonal antibody (mAb) for 30 min and then washed three times in a 0·025% solution of saponin in PBS. MR6 binding was detected using rabbit anti-mouse IgG–tetramethyl rhodamine iso-thiocyanate (TRITC) at a 1 : 40 dilution (BD Biosciences). Cells were washed a further three times before cytospinning onto clean slides using a Cytospin 3 centrifuge (Thermo-Shandon Inc., Pittsburgh, PA, USA) and mounting with fluorescent mounting medium and a coverslip. Slides were stored at 4°, in the dark, until ready for analysis. Staining was visualized using a Zeiss Axiovert S100 TV microscope (Carl Zeiss International, Welwyn Garden City, UK) and images were analysed by PC using polymorph™ software. Images were acquired using the ×100 oil-immersion objective.

Endocytosis assay

Cells were harvested and diluted to 2 × 105 cells per 100 ml in X-VIVO 15 serum-free medium and aliquoted in a 96-well, round-bottomed, plate. These were allowed to equilibrate for 30 min at 37°. After this time, 10 mg/ml (in 20 ml of serum-free medium) of MR6 (anti-CD205), M-A712 (anti-CD71; Transferrin receptor; BD Biosciences), and 6.5B5 [anti-intercellular adhesion molecule-1 (anti-ICAM-1); ATCC, American Type Culture Collection, Manassas, VA, USA] was added. At the end of each time point, the cells were washed with FACS buffer and fixed with cold 4% paraformaldehyde in PBS for 10 min. Untreated, fixed cells, representing the zero time point, were then stained with MR6, CD71, or 6.5B5 (ICAM-1) for 30 min at 4°. Antibodies at the cell surface were detected with rabbit anti-mouse IgG–FITC (BD) diluted 1 : 40 with FACS buffer. Cells were then incubated for a further 30 min before being washed three times with cold PBS. Finally, cells were analysed by flow cytometry for bound antibody. Samples at various time points were compared with the signal acquired from the zero time point sample and converted to a percentage.

Real-time polymerase chain reaction

Levels of CD205 expression between freshly isolated monocytes, day 5 immature DC, and mature DC (48 hr with 100 ng/ml of LPS) were compared by real-time polymerase chain reaction (PCR) using the iTaqTM SYBR® Green Supermix with ROX (Biorad, Hercules, CA, USA). A 0·25 µl sample of cDNA, corresponding to 1 ng of mRNA, was amplified for the C-terminal portion of CD205, or the housekeeping gene, hypoxanthine-guanine phosphoribosyl transferase (HGPRT), using 500 nm of the following primers: CD205 carboxyl terminus of protein (C-term) 5′-GGATCCATCTGGCCGCGCAGCTAATGAC, CD205 C-term 3′-AAGCTTACCGTTTTCAGGCTTTAAGCAG, HGPRT 5′-GCGTCGTGATTAGCGATG and HGPRT 3′-GCTTATATCCAACACTCCGTGG (MWG-Biotech, Ebersberg, Germany). Real-time PCR was performed in triplicate using the Prism 7700 sequence detector (Applied Biosystems, Foster City, CA, USA) following the manufacturer's instructions. A melt (dissociation curve) was performed at the end of each run to confirm the absence of primer-dimers.

Reverse transcription–PCR

A total of 5 × 106 immature DC or LPS-matured DC were washed with PBS and resuspended in RNALater (Qiagen, Hilden, Germany). mRNA was extracted using an RNeasy mini kit (Qiagen), according to the manufacturer's instructions, and the resulting mRNA was treated with DNAse (Qiagen) for 20 min prior to elution from the mini kit columns. The recovered RNA was diluted to a concentration of 200 mg/ml RNA and stored at −20°. A 1 mg sample of isolated mRNA was used to synthesize cDNA using the First-strand cDNA synthesis kit (GE Healthcare UK, Chalfont St. Giles, UK) and the bifunctional pd(N)6 primer. cDNA was then used as a template for a PCR reaction using one of the following sets of primers: IL-10, 5′-TCAGCACTGCTCTGTTGCCTG and 3′-AATCGTTCACAGAGAAGCTCA; IL-12p40, 5′-CGTAGAATTGGATTGGTATCCGG and 3′-GCTCTTGCCCTGGACCTGAACGC; CD205 tail, 5′-AGAATGGGTCACGGTGGATC and 3′-AGTCATGGAAAGAAGGAAGCA; DCL-1 tail, 5′-GAAGCCATCAAAGTAGAAAGC and 3′-CTTCTCCAACTACCAAACAC; CD205–DCL-1 tail, 5′-AGAATGGGTCACGGTGGATC and 3′-CTTCTCCAACTACCAAACAC; and β-actin 5′-ATCTGGCACCACACCTTCTACAATGAGC TGCG and 3′-CGTCATACTCCTGCTTGCTGATCCACATCTGC. (MWG-Biotech).

PCR amplification was carried out using a Primus 96 Plus thermocycler (MWG-Biotech) set to run 35 cycles of the following: 94° for 30 seconds, 55° for 30 seconds and 72° for 1 min. Samples were run on a 1% agarose gel containing ethidium bromide and visualized under ultraviolet (UV) light.

Cell lysis and immunoprecipitation

A total of 5 × 106 immature DC, and the same number of mature DC, were lysed for 30 min on ice in 150 mm NaCl, 10 mm Tris–HCl (pH 8·0), 1% Nonidet P40, supplemented with protease inhibitors. Lysates were then centrifuged (10 min, 10 000 g, 4°) to sediment cell debris and equilized for protein content by the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL, USA). Lysates were incubated with 5 µg/ml of MR6 and 100 µl of protein G microbeads (Miltenyi Biotech) overnight at 4° on a roller. The microbeads were passed through a prepared µMACS column on a µMACS magnetic separator. After washing with lysis buffer, proteins were eluted in 50 µl of preheated (95°) 1 × sodium dodecyl sulphate (SDS) gel loading buffer (50 mm Tris–HCl, 1% SDS, 0·005% bromophenol blue, 10% glycerol). Immunoprecipitated proteins were analysed on a 4–15% gradient SDS polyacrylamide gel, which was run for 3 hr to separate the bands of interest. Electrophoresed proteins were then blotted onto a 0·45 µm pore poly(vinylidene difluoride) membrane (Immobilon-P; Millipore, Billerica, MA, USA). CD205 molecules were detected using 10 µg/ml of biotinylated MG38 (anti-human CD205; ATCC) followed by extravidin peroxidase conjugate (diluted 1 : 1000; Sigma-Aldrich), and then visualized chemiluminescently by the enhanced chemiluminescence (ECL) substrate (Amersham Biosciences) and exposure to X-ray film (Hyperfilm ECL; Amersham). Ethical permission for this work was approved by the research ethics committee of the Hammersmith, Queen Charlotte's, and Chelsea and Acton Hospitals, London, UK.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

CD205 is expressed by all human PBMCs but is highest on antigen-presenting cells

In both human and mouse, CD205 is expressed by thymic cortical epithelium and DC but can also be detected on lymphocytes.3,5,11–13,15,26 A similar distribution pattern was seen in human PBMC when stained with the monoclonal antibody, MR6.8,15,26 Three populations of CD205+ cells were present in peripheral blood (Fig. 1a). The CD205low group proved to consist of CD3+ T cells, the CD205intermediate population was CD19+ B cells, and the CD205high cells were characterized as the CD14+ monocyte population. In addition, all cell subsets carried mRNA for CD205 (results not shown). These data suggest that CD205 is widely expressed amongst haematopoietic subsets, but higher levels tend to be detected on cells with antigen-presenting capability – in keeping with its putative role in antigen capture (Fig. 1b). CD205 (protein and mRNA) was also detected on B cells, natural killer (NK) cells, neutrophils, eosinophils and all intestinal epithelial cell lines tested, but was not detectable in human sapheous vein endothelial cells (data not shown). This distribution corresponds with, and extends, published data.11,27,28

image

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.

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Expression of Endo180, MMR (CD206) and CD205 on developing DC

As the MMR family of C-type lectins all possess endocytic activity, we wanted to investigate how these structurally related receptors are regulated during DC development and maturation (Fig. 2). Freshly isolated monocytes were defined as Endo180low, MMRneg and CD205low. Following culture with IL-4 and GM-CSF for 5 days to generate immature DC, cells were Endo180low, MMRhigh and CD205high. When these cells were stimulated for 48 hr with 100 ng/ml of LPS to induce maturation, they became Endo180low, MMRneg and CD205veryhigh. Although all of the MMR family members have similar endocytic properties, they each displayed distinct expression patterns on DC. Endo180 expression was low on monocytes and was essentially unaffected by DC development and maturation; the MMR was up-regulated on immature DC but was down-regulated upon maturation;29 and CD205, although present on all cells, showed low expression on monocytes, increased expression on immature DC, and was greatly up-regulated on mature DC. This occurred when the DC were stimulated with either LPS, or with the inflammatory cytokines, TNF-α and IL-1β (results not shown).

image

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.

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Mature DC up-regulate CD205 over 48 hr as a result of de novo synthesis

During flow cytometric analysis of maturing DC, we observed a gradual transition of cells moving from a CD205high phenotype (immature DC) to a CD205veryhigh phenotype (mature DC). We undertook a time-course analysis and verified that this transition takes ≈ 48 hr to affect the whole population (Fig. 3a,b). We next sought to identify whether the up-regulation of CD205 was the result of de novo synthesis or translocation of molecules from an intracellular pool. We conducted real-time PCR analysis of CD205, normalized against a housekeeping gene, HGPRT (Fig. 3c). Immature DC were shown to carry more mRNA for CD205 than freshly isolated monocytes (1·3 ± 0·3-fold induction), whereas mature DC showed greatly increased levels of CD205 mRNA (8·3 ± 1·3-fold induction). This indicated that at least some of the CD205 up-regulation seen in mature DC was indeed the result of de novo synthesis.

image

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 CD205low or CD205high (shaded) populations. (b) The percentage of CD205high 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.

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Mature DC translocate CD205 to the cell surface and down-regulate CD205-mediated endocytosis

We next used fluorescence microscopy to analyse the cellular localization of CD205 within cells. Monocytes and immature DC possessed extensive intracellular compartments containing CD205 (Fig. 4b,e). Indeed, the majority of the CD205 appeared to be intracellular with comparatively small amounts at the cell surface. However, an analysis of mature DC revealed CD205 staining predominantly at the cell surface with very little staining in intracellular spaces (Fig. 4h). It thus appeared that translocation from intracellular pools also contributed to the increased surface expression of CD205. Cells were next analysed for their CD205 endocytic activity (Fig. 4c,g,i). Monocytes and immature DC rapidly (< 20 min) internalized CD205 upon binding of MR6 antibody, whereas antibody to ICAM-1 (6.5B5) remained at the cell surface throughout the same time course. Similar results were observed when using the other CD205 antibody, MG38 (not shown). These data corresponded with observations made by Mahnke et al., who showed rapid internalization of CD16, coupled to the cytoplasmic tail of CD205, upon antibody ligation.18 However, unlike this group, we did not observe significant recycling of internalized anti-CD205 back to the cell surface – possibly because the MR6 antibody was degraded or removed in endosomal compartments during receptor recycling. In contrast, mature DC were unable to endocytose CD205 during the 60 min of the experiment. This response appeared to be specific to CD205 because the activity of the Transferrin receptor (CD71) – a well-known endocytic molecule – was unaffected by DC maturation. Of all the subsets tested (including CD3+ T cells, CD19+ B cells and granulocytes) mature DC were the only ones to express the non-endocytic form of CD205. This phenotype arose when the DC were stimulated with either LPS or with inflammatory cytokines (TNF-α and IL-1β; not shown). We were unable to replicate the results using LPS-treated T cells, B cells, or granulocytes.

image

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.

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Detection of CD205–DCL-1 fusion mRNA transcripts in monocytes, immature DC and mature DC

How might such a loss of endocytic activity be controlled? We hypothesized that the lack of CD205 endocytic activity on mature DC could be the result of increased expression of a novel CD205–DCL-1 fusion protein recently described in Hodgkin's lymphoma cell lines.25 This hybrid protein carries the outer domains of CD205 plus the C-type lectin, and transmembrane and cytoplasmic domains of DCL-1, and thus has a different cytoplasmic tail from that of wild-type CD205. The role of DCL-1, and the functional significance of CD205–DCL-1 fusion proteins, is not known. Primer pairs were designed to detect mRNA transcripts for CD205 (between the CTLD10 and the cytoplasmic domain) and DCL-1 (CTLD and cytoplasmic domain). By combining the CD205 forward primer and the DCL-1 reverse primer, the presence of CD205–DCL-1 fusion transcripts was detected. As controls, we used primers specific for IL-10 and IL-12p40 to confirm the expected phenotype of our samples. The IL-10 transcript was detected in all three samples, whereas IL-12p40 was only detected in mature DC samples. Wild-type CD205 and DCL-1 transcripts were detected in all samples. Perhaps surprisingly, two transcripts for the putative CD205–DCL-1 fusion were also detected in all samples (Fig. 5a). Upon sequence analysis, these transcripts were found to correspond to two transcripts previously detected by Kato et al. (Fig. 5b). The larger transcript is produced by splicing of exon 34 of CD205 and exon 2 of DCL-1 such that CD205 CTLD10 is fused to the CTLD of DCL-1. This molecule is predicted to contain the extracellular domains of CD205 plus the CTLD, transmembrane and cytoplasmic domains of DCL-1. The smaller transcript is similar (splicing exon 33 of CD205 and exon 2 of DCL-1), but with 186 bp deleted from the C-terminus of CD205 CTLD10 (CTLD10 is truncated). Because this analysis was not quantitative, we were unable to confirm relative increases/decreases in mRNA.

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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.

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CD205–DCL-1 fusion protein can be detected only in mature DC

Transcripts for the DCL-1–CD205 fusion appeared in samples from DC, suggesting that both types of CD205 molecule might be expressed as protein. One possibility was that, of these mRNAs, those coding for the DCL-1–CD205 fusion were selectively translated in mature DC, leading to a dominance of this protein at the cell surface and possibly explaining the lack of endocytosis in mature DC. To test this, CD205 molecules from monocytes, immature DC, or mature DC lysates were immunoprecipitated using the anti-CD205 mAb, MR6.12 Proteins were separated by SDS–polyacrylamide gel electrophoresis and detected by western blot using a biotinylated MG38 antibody.30 In monocytes and immature DC, a band corresponding to wild-type CD205 [≈ 205 000 molecular weight (MW)] was detected (Fig. 6). However, an additional band (≈ 215 000 MW), corresponding to the predicted molecular weight for a CD205–DCL-1 fusion protein, was also detected in the mature DC sample. These results indicate that mature DC expressed both wild-type CD205 and a small amount of the novel form of CD205 simultaneously. However, wild-type CD205 remained the dominant protein and it would thus seem likely that abrogation of CD205 endocytic activity on mature DC is not the result of CD205–DCL-1 expression.

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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.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Using the anti-human CD205 mAb, MR6, we have observed CD205 expression and endocytic activity in all subsets of human T cells, all subsets of B cells, monocytes, all subsets of granulocytes, monocyte-derived dendritic cells, and epithelial cells derived from human colon, breast, and kidney.4,12,15,26–28,31 (M. Butler and M. Ritter unpublished observations). Our observations were recently confirmed by Kato et al., who also found that CD205 expression was highest in those cells associated with antigen capture and presentation.11 Although CD205 is broadly expressed in humans, it is increasingly being targeted by those wishing to deliver antigens or drugs to DC. We thus chose to investigate CD205 expression and endocytosis during the course of monocyte-derived DC development and maturation and evaluate how this might affect the viability of CD205-targeted vaccines or immunomodulatory therapies.

CD205 on DC displayed a completely different pattern of expression to that displayed by two other members of the MMR family – Endo18032–35 and the MMR itself.36 Both molecules are extremely endocytic and a number of groups have targeted antigens to DC and macrophages via the MMR, with some success.37–39 However, the MMR appears to be less efficient at targeting antigens to MHC class II loading compartments.18,22,29 We found that MMR expression on DC was down-regulated on maturation, as previously reported,36 Endo180 was unaffected, but CD205 was massively up-regulated on DC maturation, also as previously reported.29 This expression pattern is unlike that of a classical pattern recognition receptor (like the MMR or Toll-like receptors), which all tend to be down-regulated upon DC maturation.36,40 Real-time PCR and intracellular microscopy further revealed that up-regulation of CD205 by mature DC was probably the result of both de novo synthesis and the translocation of CD205 molecules from intracellular spaces to the cell surface. Lastly, we found that this change was associated with a loss of CD205 endocytic activity on mature DC.

Taken together, these data strongly suggest an additional function for CD205 that is unrelated to its endocytic activity and antigen internalization. This second function could be associated with cellular interactions within secondary lymphoid organs because this is the destination of the majority of maturing DC,41,42 and CD205 takes some time (≈ 48 hr) to be fully up-regulated – perhaps reflecting the time it takes for a DC to leave the tissue and migrate to the local lymph nodes. In addition, CD205 expression closely parallels that of MHC class II molecules, which are mainly found in the intracellular compartments of immature DCs but are redistributed to the cell surface upon maturation.43,44 Alternatively, CD205 could mediate interactions with extracellular matrix proteins, endothelium, or perhaps enhance DC–T-cell interactions in lymph nodes. These roles are all efficiently carried out by other C-type lectins, such as DC-SIGN.45–50 However, antibody to CD205 does not affect T-cell proliferation in mixed lymphocyte reactions (M. Butler, unpublished observations), implying that this molecule is not essential for DC-induced T-cell activation.

CD205 has recently been implicated in the capture of apoptotic thymocytes by thymic epithelial cells.51 Cortical thymic epithelial cells express large amounts of CD205 and capture of apoptotic cells could provide the rich source of peptides required for positive and negative selection in the thymus.12,15,31 If CD205 also plays this role in DC, then mature DC might use this receptor to trap intact antigens at the cell surface – perhaps in the form of apoptotic bodies or exosomes – to form an antigenic ‘depot’. This type of activity has been observed in follicular dendritic cells (FDC), which can capture exosomes, native antigens and immune complexes via complement receptors (CD21 and CD35) and Fc receptors.52–54 Crucially, these receptors do not internalize antigens, but retain them at the cell surface, allowing maturing B cells to sample unprocessed antigen and hone their receptor affinities. Mature DC could thus utilize CD205 to deliver unprocessed antigens to the lymph node where they would present intact antigen or exosomes directly to lymph node B cells in the same way that FDC do. In addition, unprocessed antigens carried by CD205 on mature DC would be made available to DC permanently resident in the lymph node. These cells maintain their antigen-processing ability and can process and present antigens to naïve T cells and B cells. Trapping of antigen at the surface of mature DC, via CD205, might also help to explain why CD205-targeted antigens provide a long-lasting stimulus for the generation of specific immunity, as shown by Bonifaz et al. when they administered CD205-targeted antigens to mice along with anti-CD40 antibodies to induce DC maturation.22

How might the switch of CD205 function on maturing DC be controlled? We hypothesized that the loss of CD205 endocytic activity on mature DC resulted from the expression of the CD205–DCL-1 fusion protein in place of wild-type CD205 molecules. The CD205–DCL-1 fusion switches the cytoplasmic tail of CD205 to that of DCL-1. Sequence analysis suggests that this cytoplasmic domain has different amino acid motifs from that of CD205, and expressed CD205–DCL-1 fusion proteins might have altered endocytic properties.25 We were able to detect two transcripts of CD205–DCL-1 fusion mRNA in all of our DC types. Products corresponded with published observations of two CD205–DCL-1 mRNA transcripts in Hodgkin's lymphoma cell lines.25 However, we could only detect one putative CD205–DCL-1 protein in small quantities in mature DC. Again, this reflected data from Kato et al. – although it is possible that the other transcript was also expressed but was present at levels too low to be detected. CD205–DCL-1 expression was accompanied by a large increase in wild-type CD205 protein expression, implying that mature DC express both fusion and wild-type CD205.

It is possible that CD205–DCL-1 fusion proteins are expressed by other cell types, but are present at concentrations below the detection limits of our assay. If this proves to be the case, then CD205–DCL-1 fusion proteins might be found at low levels in all cells that express CD205 and DCL-1. Intergenic splicing events such as this are extremely rare and because the genes for CD205 (LY75) and DCL-1 (KIAA0022) lie adjacent to each other on chromosome 2, it is possible that the CD205–DCL-1 fusion protein is the result of ‘leaky’ transcriptional termination by RNA polymerases.55 However, Kato et al. found that CD205–DCL-1 fusion protein transcripts were detected in Hodgkin lymphoma cell lines, even in the absence of DCL-1 expression, suggesting that these molecules could be biologically relevant rather than caused by errors in transcription.25 As the fusion protein substitutes the cytoplasmic domain of CD205 for that of DCL-1, ligands binding to CD205–DCL-1 receptors, rather than wild-type CD205, might generate different intracellular signals, leading to modified cellular responses or altered intracellular trafficking. Little is known about the intracellular signalling activity of CD205 and DCL-1, and the functional relevance of CD205–DCL-1 proteins will require a much greater understanding of DCL-1 itself. However, in our study it seems unlikely that CD205–DCL-1 expression could account for the complete abrogation of CD205 endocytic activity on mature DC.

Surprisingly, the mechanisms behind reduced endocytic activity following DC maturation is a relatively unexplored field. Upon stimulation there is a transient increase in macropinocytic activity and TLR expression followed by a rapid down-regulation of these processes and reduced expression of several groups of receptors associated with antigen capture.36,40,56 These include the TLRs,40 Fc receptors and C-type lectins, such as the MMR.29 Many of these are not just down-regulated, but are often lost altogether – even at the mRNA level.40 Despite this, mature DCs do maintain a low level of endocytic activity, and receptors such as CD71 (transferrin receptor),57 CD83,58 CD89 (IgA receptor)59 and CCR760 have been shown to mediate endocytic activity in these cells. Consequently, it seems unlikely that the loss of CD205 endocytic capacity in mature DC is simply the result of an overall decreased rate of endocytosis, as has previously been proposed.61 Indeed, CD205 appears to be the first example of a C-type lectin exhibiting both endocytic and non-endocytic behaviour during DC maturation. Furthermore, our experiments indicate that this is not the result of altered intracellular signalling via the CD205–DCL-1 fusion protein. Loss of endocytic activity is perhaps more likely to be related to the large-scale cytoskeletal changes associated with DC maturation. Indeed, endocytic activity in maturing DC has been shown to be partly dependent on the activity of Rho family GTPases,62 and this area may offer a route to unravelling the mechanisms behind CD205 endocytosis.

The properties of CD205 on monocyte-derived DC, described here, further enhance its appeal as a vaccine target. The endocytic nature of CD205 in immature DC (and many other non-professional antigen-presenting cells) ensures that antigens targeted to CD205, in the absence of an inflammatory signal, are presentated by ‘regulatory’ DC,21 or by cells lacking adequate costimulatory molecules, and hence generate regulatory T-cell responses. Alternatively, targeting antigens to CD205 in the presence of a DC maturation signal leads to persistent and potent adaptive immunity – perhaps partly resulting from retention of antigens (e.g. apoptotic fragments, exosomes, immune complexes) at the surface of mature DC in lymph nodes. Thus, while the biological role of CD205 and the functional significance of its broad expression urgently require a greater understanding, its potential as an immunomodulatory target is clear.

Acknowledgement

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

This work was funded by the Medical Research Council.

References

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  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
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