Correspondence to: Ralph M. Steinman, Laboratory of Cellular Physiology and Immunology, The Rockefeller University, 1230 York Ave., New York, NY, 10021-6399. Tel:(212) 327-8106 Fax:(212) 327-8875
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Abstract |
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Many receptors for endocytosis recycle into and out of cells through early endosomes. We now find in dendritic cells that the DEC-205 multilectin receptor targets late endosomes or lysosomes rich in major histocompatibility complex class II (MHC II) products, whereas the homologous macrophage mannose receptor (MMR), as expected, is found in more peripheral endosomes. To analyze this finding, the cytosolic tails of DEC-205 and MMR were fused to the external domain of the CD16 Fc receptor and studied in stable L cell transfectants. The two cytosolic domains each mediated rapid uptake of human immunoglobulin (Ig)G followed by recycling of intact CD16 to the cell surface. However, the DEC-205 tail recycled the CD16 through MHC IIpositive late endosomal/lysosomal vacuoles and also mediated a 100-fold increase in antigen presentation. The mechanism of late endosomal targeting, which occurred in the absence of human IgG, involved two functional regions: a membrane-proximal region with a coated pit sequence for uptake, and a distal region with an EDE triad for the unusual deeper targeting. Therefore, the DEC-205 cytosolic domain mediates a new pathway of receptor-mediated endocytosis that entails efficient recycling through late endosomes and a greatly enhanced efficiency of antigen presentation to CD4+ T cells.
Key Words: dendritic cell, antigen presentation, DEC-205, MHC class II, endocytosis
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Introduction |
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Several cell surface receptors deliver ligands to the endocytic system for purposes of extensive intracellular digestion within lysosomes. Many of these receptors function exclusively in endocytosis, such as the prototype low-density lipoprotein receptor (LDLR)1 (
Adsorptive endocytosis receptors, like the macrophage mannose receptor (MMR), FcR, and B cell antigen receptor (BCR), are used in the immune system to facilitate antigen capture and presentation of peptides to T cells (
To dissect the role of the cytosolic domain in these findings, we studied a totally heterologous system: L cells stably transfected with a fusion receptor formed between the external domains of human CD16 and different cytosolic tails. We find that the DEC-205 cytosolic domain has a distinct distal region with an acidic EDE triad. The distal region and its acidic sequence are required for recycling beyond early endosomes through deeper major histocompatibility complex class IIpositive (MHC II+), late endosomes and lysosomes. Such distal targeting is unique for adsorptive endocytosis receptors that have been analyzed to date, and proves to be necessary for presentation of antigenic peptides at low doses of ligand. This new pathway for receptor-mediated uptake is therefore a hybrid of the two known endocytic pathways discussed above, targeting deeper digestive compartments but recycling efficiently to produce biologically active peptides.
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Materials and Methods |
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Cells
The MHC II (I-Ek)expressing cell line DCEK.ICAM.Hi7 was maintained in RPMI with 7.5% Ig-depleted FCS (Atlanta Biologicals), 10 IU/ml penicillin/streptomycin, 200 µM/ml glutamine (GIBCO BRL), and 50 µM 2-mercaptoethanol (Sigma-Aldrich) (R7 medium). To maintain MHC II and intracellular adhesion molecule (ICAM)-1 expression, every other week the cells were kept in MXH medium (R7 containing 6 µg/ml mycophenolic acid, 250 µg/ml xanthine, 15 µg/ml hypoxanthine [all from Sigma-Aldrich], and 800 µg/ml G418 [GIBCO BRL]). CD16 chimeric receptor transfectants were kept in MXH medium additionally supplemented with 5 µg/ml puromycin (Calbiochem). DCs were generated from mouse bone marrow cultured in the presence of GM-CSF as described previously (
Cloning and Transfection
The cDNAs coding for the intracellular domains of DEC-205 and MMR were cloned separately from the extracellular expressed domains into a TA vector (Invitrogen), and oligonucleotide directed mutagenesis using QuikChange® (Stratagene) was performed. STOP codons and a tyrosine to alanine exchange were introduced into different parts of the DEC tail (see Fig 1 A). All mutations were verified by sequencing (DNA Technology Center, The Rockefeller University). Using standard methods, the mutated DEC tails were cloned into the BamH1-EcoR1 site of the pApuro vector (gift of Dr. Kurosaki, American Cynamid Company, Lederle Laboratories, Department of Cardiovascular Molecular Biology, Pearl River, NY) behind the sequence coding for the extracellular domain of the human FcIII receptor (
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Flow Cytometric Analysis of Surface Markers
Cells were harvested with trypsin (Boehringer), then incubated with cold PBS/7% normal goat serum (vol/vol) followed by incubation for 45 min on ice with the following hybridoma supernatants: antiMHC II (14-4-4S; American Type Culture Collection), antiICAM-1 (YN-1; American Type Culture Collection), and anti-CD16 (3G8). B7 expression was assayed with antiB7-1 antibody (1G10; BD PharMingen). After washing with cold PBS/1% FCS (vol/vol), FITC-labeled mouse antirat Ig or goat antimouse Ig (Jackson ImmunoResearch Laboratories) secondary reagents at a final dilution of 1 µg/ml in PBS/1% FCS (vol/vol) were added. After 45 min on ice, cells were washed and resuspended in PBS containing 1 µM propidium iodide (Sigma-Aldrich) to gate out dead cells during analysis for fluorescence using a FACScanTM (Becton Dickinson).
Surface Binding and Internalization of Human IgG
Human IgG (HuIgG; Jackson ImmunoResearch Laboratories) was diluted to 1 mg/ml in PBS. Aggregates were formed by incubation at 65°C for 30 min. Thereafter, aliquots were stored at 4°C until use, but no longer than 1 wk. In some antigen-presenting assays, HuIgG was aggregated by incubation with goat anti-HuIgG antibodies for 4 h at room temperature. To assay surface binding of HuIgG, cells were harvested and washed with cold PBS/1% FCS (vol/vol) and incubated for 45 min on ice with monomeric or aggregated HuIgG (aggHuIgG) at 0.1100 µg/ml. After washes with cold PBS/1% FCS (vol/vol), FITC-labeled goat antihuman IgG antibodies (Jackson ImmunoResearch Laboratories) at a final concentration of 1 µg/ml in PBS/1% FCS (vol/vol) were added. After 45 min on ice, cells were washed, resuspended in PBS, and analyzed on a FACScanTM. To measure internalized HuIgG, the DCEK.ICAM.Hi7 cells were seeded into 96-well plates (20,000 cells/well), cultured overnight, and transferred on ice followed by addition of HuIgG at a final concentration of 50 µg/ml. After 45 min, unbound HuIgG was removed by washing with medium, and aliquots of cells were harvested and fixed with 4% paraformaldehyde (PFA). The remaining cells were further incubated in R7 at 37°C after which the cells were harvested, fixed, and stained with FITC-labeled goat antihuman IgG antibodies as described above. To detect internalized HuIgG, cells were permeabilized before adding FITC-labeled goat antihuman antibodies, using a 10-min incubation with PBS/0.1% (vol/wt) saponin (Sigma-Aldrich). In time course experiments, the amount of internalized HuIgG was calculated by subtracting the mean fluorescence in fixed cells (surface-bound HuIgG) from that recorded with fixed and permeabilized cells (internalized and surface-bound HuIgG).
Receptor Recycling
Cells were cultured for 2 h in 10 µg/ml cycloheximide (CHX; Calbiochem), sufficient to block protein biosynthesis as assessed by [35S]methionine labeling (not shown). Surface CD16 then was saturated by incubating the cells in presence of CXH with aggHuIgG (100 µg/ml) at 37°C for 10 min, followed by chilling the cells for 1 h on ice. Thereafter, cells were cultivated in R7 supplemented with CXH, and at different times, recycled CD16 receptors were detected by incubation with 125I-HuIgG at 10 µg/ml on ice. Bound HuIgG was measured by counting (Wallac).
Confocal Immunofluorescence Microscopy of L Cells and DCs
L cells were seeded into LabTek tissue culture chambers (Nunc) and incubated overnight. Cells were washed twice with warm RPMI medium, fixed with 4% paraformaldehyde/PBS (wt/vol) for 20 min at room temperature, and permeabilized by incubation with permeabilization buffer (RPMI containing 10% normal goat serum [GIBCO BRL], 0.05% saponin [Sigma-Aldrich], 10 mM glycine [Sigma-Aldrich]) for 15 min at room temperature. Alternatively, DCs were grown from bone marrow precursors using GM-CSF as described (
Antigen Presentation to T Cells
To obtain HuIgG- or RbIgG-specific T cells, 68-wk-old B10.BR mice (The Jackson Laboratory) were primed to HuIgG or RbIgG by subcutaneous injection of 50 µg HuIgG or RbIgG emulsified in complete Freund's adjuvant (Difco). In some experiments, mice were primed to RbIgG using antiDEC-205 Rb antiserum or anti-MMR Rb antiserum. 8 d later, draining lymph nodes were removed and single cell suspensions were prepared by teasing with forceps and forcing the nodes through a nylon mesh. T cells were purified by passage over nylon wool columns and incubating the eluted cells with antibodies directed against MHC II (M5/114; American Type Culture Collection) and CD45 (B220; American Type Culture Collection) on ice for 30 min. In some experiments, anti-CD8 antibodies (TIB 211; American Type Culture Collection) were added to obtain purified CD4+ T cells. Cells were washed and incubated with goat antirat Dynabeads (Dynal) at a ratio of 4 beads to 1 target cell for an additional 30 min at 4°C to remove non-T cells. For antigen presentation assays with DCEK.ICAM.Hi7 cells, transfected and untransfected DCEK.ICAM.Hi7 cells were irradiated with 5,000 rads and seeded into 96-well plates (15,000 cells/well). HuIgG was added to the cells in graded doses. After overnight culture, unbound HuIgG was removed by washing the plates with warm R7 medium. Thereafter, 250,000 T cells/well in 200 µl R7 were added in triplicate, and the plates were incubated for 34 d. For antigen presentation assays with DCs, developing bone marrow DCs were harvested after 6 d of culture in GM-CSF, placed on ice, and incubated with graded doses of rabbit antiDEC-205, anti-MMR, and preimmune serum for 1 h. Unbound antibodies were washed away and aliquots of the DCs were seeded in triplicates into 96-well plates. Then 200,000 T cells/well were added and cultured for 34 d. To assay T cell proliferation, [3H]thymidine (1 µCi/well; Amersham Pharmacia Biotech) was added for the last 12 h before harvesting. Incorporation of radioactivity was determined by scintillation counting. Data are shown are means of triplicates where the standard deviation was <10% of the mean cpm.
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Results |
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Unique Localization and Function of DEC-205 in DCs
DEC-205 belongs to the group VI family of lectins (
By confocal laser scan microscopy, both the MMR and DEC-205 were abundant in intracellular granules (Fig 1 A, red). As expected for a recycling endocytic receptor, very little of the MMR was found in late endosomes or lysosomes that were labeled for LAMP-1 or for MHC II (Fig 1 A, green and merge images). In marked contrast, the bulk of the intracellular DEC-205 was localized in perinuclear MIICs, as demonstrated by colocalization with LAMP-1 and with MHC II (Fig 1 A).
To detect a functional consequence for the distinct targeting of the MMR and DEC-205, we used the rabbit polyclonal antibodies as surrogate antigens for T cells primed to rabbit Ig, as
Expression of Chimeric DEC-205/CD16 Receptors in Transfected DCEK.ICAM.Hi7 Cells
The targeting of endocytic receptors is determined by amino acid sequences within their intracellular domains (for review see
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To determine which components of the cytoplasmic tail were required for efficient targeting and presentation via late endosomes or lysosomes (Fig 1), we constructed chimeric receptors containing the external IgG binding domain of the HuFcIII receptor or CD16 and different DEC-205 cytosolic tails (Fig 2 B). In addition to the wild-type (WT) DEC-205 tail, we made truncations to remove the terminal three amino acids (long tail, LT), residues 1931 with the putative EDE distal targeting sequence (intermediate tail, IT), and residues 631 with the coated pit sequence (short tail, ST). We also made mutants of the DEC-205 tail, converting tyrosine to alanine in the coated pit sequence (altered tail or AT), and EDE to alanines in the putative distal targeting sequence. We used the wild-type MMR tail for comparison.
The CD16 chimeras were transfected into a murine fibroblast-like line, DCEK.ICAM.Hi7, which expresses MHC II as well as the T cell costimulatory molecules B7-1 (CD80) and ICAM-1 (CD54) (
Binding, Uptake, and Recycling of HuIgG by CD16 Chimeric Receptors
To test the function of the chimeric receptors, we first measured binding of HuIgG as illustrated for WT-DEC:CD16 (Fig 3 A). The transfectants all bound ligand. Saturation occurred at 10 µg/ml, whereas the untransfected DCEK.ICAM.Hi7 cell line (Fig 3 A, none) did not bind HuIgG. Binding required that the human IgG be heat aggregated, as expected for functional FcRIII (
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To monitor endocytosis, aggHuIgG was bound to each transfectant on ice, and aliquots were transferred to 37°C for 2 h. The FACS® was used to follow uptake at the single cell level. Binding of FITC anti-HuIgG to fixed cells detected residual surface HuIgG, and to fixed permeabilized cells, detected surface and intracellular HuIgG. With WT-DEC:CD16expressing cells, HuIgG was no longer detectable at the surface after the 2-h chase at 37°C (Fig 3 D), but strong staining was obtained in permeabilized cells, indicating that the DEC tail mediated uptake of ligands bound to a heterologous receptor.
Similar results, i.e., endocytosis of bound HuIgG, were obtained with cells expressing the LT-, IT-, and AT-DEC:CD16 chimera (not shown). In contrast, cells transfected with the short sixamino acid tail (ST-DEC:CD16), lacking the putative coated pit localization sequence, showed no significant loss of surface IgG (Fig 3 D), indicating that deletion of amino acid residues 631 prevents endocytosis of the DEC:CD16 chimeric receptor. In a more detailed time course study (Fig 3 E), we included cells expressing AAA-DEC:CD16 and MMR:CD16. Again, the ST-DEC:CD16 chimera did not internalize, whereas half the surface Ig entered the cells via the other CD16 chimeras within 15 min. The AT-DEC:CD16 chimera, which contained a mutation of the tyrosine residue in the coated pit localization site, was capable of endocytosis although at a somewhat slower rate (Fig 3 E). Therefore, all the chimeric CD16 receptors bound aggHuIgG, and all except the ST-DEC:CD16 mediated rapid adsorptive uptake of ligand.
To examine recycling of the DEC:CD16 chimeric receptors, we blocked protein synthesis using CHX at 10 µg/ml for 2 h. We then added a saturating dose of aggHuIgG, placed the cells on ice, washed, and transferred the L cells to 37°C. This procedure was sufficient to saturate all of the CD16 chimeric receptors (data not shown). Thereafter, at subsequent time points, we added 125I-labeled aggHuIgG to detect a reappearance of functional CD16 receptors. The intact cytosolic tails (WT, MMR) recycled within 1 h after internalization, but the truncated intermediate DEC tail (IT) and the AAA-DEC:CD16 chimera recycled less rapidly (Fig 3 F). The 1-h recycling time could underestimate the speed of recycling via the DEC-205 tail, because the pH of the vacuolar system in L cells may be insufficiently low to quickly elute all of the bound HuIgG ligand. As expected, the ST tail did not recycle, i.e., regenerate functional CD16, because endocytosis was not occurring. To rule out replenishment of receptors from endocytic pools, rather than recycling, we repeated the experiments in cells exposed for 1 h at 37°C to aggHuIgG to occupy intracellular stores. Again, recycling of new HuIgG binding receptors took place (data not shown). We conclude that the DEC-205 and MMR tails are each capable of mediating ligand uptake, discharge, and recycling to the cell surface, and that these activities can be carried out by all of the mutant cytosolic tails we had prepared, except for the short tail lacking the coated pit localization and uptake sequence.
Intracellular Compartments Targeted by CD16 Chimeric Receptors
At this stage of the studies, the DEC-205 and MMR tails seemed similar. Distinctive features of the DEC-205 tail became apparent upon examining the intracellular targeting of CD16 chimeric receptors and HuIgG ligand, and in tests of antigen presentation to HuIgG-primed T cells. First, we did confocal immunofluorescence microscopy to simultaneously identify MHC II and LAMP-1 or the early endosomal marker TfR. In all transfectants (WT shown here), MHC II largely colocalized with LAMP-1, a marker for late endosomes/lysosomes in the perinuclear region (Fig 4, top), and not with TfR in the periphery (Fig 4, bottom).
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In either the absence or presence of IgG ligand, WT-DEC:CD16 chimeric receptors efficiently localized to late endosomal/lysosomal compartments, as shown by colocalization with LAMP-1 and MHC II in the perinuclear region (left columns of Fig 5A and Fig B, respectively). In contrast, the MMR:CD16 did not colocalize with LAMP-1 and was found primarily in the peripheral cytoplasm (Fig 5 A). Receptor targeting to late endosomes was mediated by sequences found in the distal portion of the cytoplasmic tail of DEC-205, since DEC-IT:CD16 chimeras, which lacked amino acids 1831 in the cytoplasmic tail of DEC-205, failed to accumulate in MHC II+ lysosomes and were found in vesicles throughout the cytoplasm (Fig 5A and Fig B). The ST-DEC:CD16 chimera failed to mediate endocytosis completely, and was distributed along the cell membrane (Fig 5 A).
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To follow the targeting of bound ligand, we incubated the L cells with aggHuIgG for 30 min on ice, followed by a 30-min chase at 37°C. Surface-bound HuIgG was found on all transfectants after incubation on ice (Fig 6 A, top), confirming the FACS® data (Fig 3 A). After incubation at 37°C, HuIgG was mainly found in lysosomes in WT-DEC:CD16 transfectants, colocalizing with LAMP-1 in the perinuclear region (Fig 6 A, bottom left). In contrast, HuIgG endocytosed by either IT-DEC:CD16 or MMR:CD16 transfected cells, colocalized with TfR in early endosomes (data not shown), indicating that ligands bound to these receptors failed to reach lysosomes efficiently (Fig 6). HuIgG bound to the ST-DEC:CD16 chimera remained surface bound during the 30-min chase period. From these results, we conclude that the DEC-205 cytoplasmic domain targets CD16 chimeras and their ligands to MHC II+LAMP-1+ vacuoles, whereas the cytoplasmic domain of the MMR targets primarily to early endosomes. Sequences for targeting to late endosomes lie between positions 18 and 31 in the DEC-205 tail. The different targeting routes of MMR:CD16 and AAA-DEC:CD16 versus WT-DEC:CD16 should be reflected in a different time course for their recycling of these receptors, but in fact unoccupied receptors reappeared with a similar pace. However, the speed of recycling via early endosomal compartments is likely to have been underestimated because the pH of the early endosomes may be insufficiently low to quickly elute all of the bound HuIgG ligand.
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Presentation of Ligands Internalized by Chimeric CD16 Receptors to CD4+ T Cells
To determine whether antigen bound to CD16 chimeric receptors was processed and presented, we pulsed the transfected cells with aggHuIgG for 6 h, and assayed for presentation to primed T lymphocytes. Cells expressing WT-DEC:CD16 induced strong T cell proliferation, with saturation concentrations of 1 µg/ml aggHuIgG (Fig 7 A). Immune complexes formed with anti-HuIgG and soluble HuIgG were also presented efficiently, whereas nonaggregated HuIgG, which did not bind to CD16 receptors (Fig 3 A), only induced occasional T cell proliferation at high doses (100 µg/ml; Fig 7B and Fig C).
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When the mutant cytosolic tails were studied, LT-DEC:CD16 chimeras were indistinguishable from WT-DEC:CD16 in inducing T cell proliferation at low antigen concentrations. AT-DEC:CD16 chimeras (tyrosine 15 substituted by alanine) showed only a slightly diminished response. In contrast, IT-DEC:CD16, which mediated endocytosis and recycling (Fig 3E and Fig F) but failed to target to MHC II+LAMP-1+ compartments (Fig 5), was inefficient for antigen presentation. Cells expressing MMR:CD16 chimeras resembled those expressing IT-DEC:CD16 in that they were only able to stimulate T cell proliferation at high concentrations of HuIgG (10100 µg/ml; Fig 7). ST-DEC:CD16 (lacking the 25 most distal amino acids in the DEC-205 cytoplasmic domain) did not stimulate T cell proliferation above the background obtained with untransfected DCEK.ICAM.Hi7 cells. These data indicate that sequences required to target antigens for efficient presentation differ from those required for endocytosis and recycling. IT-DEC:CD16 and MMR:CD16 chimeras mediate endocytosis and recycling, but additional information found between amino acids 18 and 28 in the DEC-205 cytoplasmic domain is required for targeting to antigen processing compartments.
Importance of the Distal EDE Sequence in the Distinct Targeting of the DEC-205 Tail
Acidic amino acids are implicated in intracellular targeting, e.g., the movement of HIV-1 nef protein to lysosomes (
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Discussion |
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A Specialized Receptor for Antigen Uptake and Presentation on DCs
High doses of protein antigens, e.g., 1001,000 µg/ml, are typically added to antigen-presenting cells to generate the MHC IIpeptide complexes that are ligands for CD4+ T cells. The efficiency of antigen binding and uptake is greatly enhanced by receptor-mediated endocytosis, as occurs with the MMR (
Additional work will be needed to pursue the physiological implication of these findings, i.e., that DEC-205 is used by DCs to improve antigen presentation. The following types of future experiments would be of value. First, to assess the role of the distal cytoplasmic tail in the context of a full length receptor, it would be important to mutate full length DEC-205; currently, it is not yet feasible to obtain high level expression of this large receptor. Second, the studies of CD16-DEC fusion receptors could be extended into DCs using different vectors, and such experiments are underway. Third, ligands for DEC-205 and MMR need to be identified, so that the presentation of natural ligands rather than surrogates can be tested. Interestingly, DEC-205 and not the MMR is readily detected on DCs within the T cell areas of mouse and human lymphoid tissues (
Two Functional Regions of the DEC-205 Cytosolic Domain
The membrane-proximal region of the DEC-205 tail contains the sequence FSSVRY, which resembles the coated pit localization sequences described in many other receptors (
The more intriguing region of the DEC-205 cytosolic tail was the distal region. Residues 2831 seemed superfluous, but residues 1827 were critical for several functions. In the absence of this "distal targeting sequence," the DEC-205 tail did not target either receptor or ligand to late endosomes and lysosomes (Fig 5) and did not mediate efficient antigen presentation (Fig 7). However, the distal targeting sequence was not required for uptake (Fig 3 E) and membrane recycling (Fig 3 F).
A cluster of acidic amino acids (EDE) in the distal DEC-205 tail proved critical for its distinct intracellular movements (Fig 5 and Fig 7). Acidic clusters also mediate trans-Golgi network retrieval of the mannose-6-phosphate receptor and furin, the latter interacting with a cytosolic sorting molecule PACS-1 (2-macroglobulin, and complement component C3 into lysosomes (
A Novel Pathway for an Adsorptive Endocytosis Receptor
As mentioned above (see Introduction), some receptors mediate ligand uptake and discharge in early endosomes, followed by recycling of intact receptors to the surface and further rounds of uptake. Other receptors signal cell activation and growth, and then uptake is followed by ligand and receptor digestion in lysosomes. The new pathway, illustrated by DEC-205, is a hybrid between these two. The cytosolic domain of this receptor can mediate uptake into deeper vacuoles, followed by recycling of ostensibly intact receptor. Concomitantly, there is a marked improvement in the efficiency with which peptides are salvaged and displayed as MHCpeptide complexes. It will be important now to follow the distribution and function of DEC-205 in epithelia and brain endothelium, where this receptor is also abundant. It is possible that in epithelia, as in antigen-presenting cells, DEC-205 will target to deeper proteolytic vacuoles and lead to the production of biologically active peptides.
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Footnotes |
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1 Abbreviations used in this paper: aggHuIgG, aggregated human IgG; BCR, B cell antigen receptor; CHX, cycloheximide; DC, dendritic cell; ICAM, intracellular adhesion molecule; LAMP-1, lysosome-associated membrane protein 1; LDLR, low-density lipoprotein receptor; MMR, macrophage mannose receptor; MHC II, major histocompatibility complex class II; MIIC, MHC II compartment; PLA2R, phospholipase A2 receptor; TfR, transferrin receptor.
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Acknowledgements |
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We thank the many colleagues who gave us valuable advice during preparation of the manuscript.
These experiments were supported by a fellowship from the Deutsche Forschungsgemeinschaft to K. Mahnke (MA 1924/1-1), grants to R.M. Steinman from the Juvenile Diabetes Foundation and the National Institutes of Health (AI13013 and AI39672), to M. Nussenzweig from the Human Science Frontiers Program, and to S. Lee (National Institutes of Health Medical Scientist Training Program grant GM07739).
Submitted: 11 July 2000
Revised: 15 September 2000
Accepted: 15 September 2000
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References |
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