Unique Localization and Function of DEC-205 in DCs
DEC-205 belongs to the group VI family of lectins (Drickamer and Taylor 1993
) that contain 8–10 extracellular, contiguous C-type lectin domains and include the MMR. Both DEC-205 and MMR can be expressed on DCs (Jiang et al. 1995
; Engering et al. 1997
). Rabbit polyclonal antibodies to mouse DEC-205 (Swiggard et al. 1995
) and the mouse MMR (our unpublished data) were used to localize these receptors in developing mouse DCs, generated from bone marrow progenitors with GM-CSF (Inaba et al. 1992
). By day 6, the cultures were predominantly immature DCs with endocytic activity and abundant, intracellular, late endosomes or lysosomes (Pierre et al. 1997
). These compartments were rich in MHC II products and are called MIICs.
By confocal laser scan microscopy, both the MMR and DEC-205 were abundant in intracellular granules ( 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 ( 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 ( 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 Chesnut and Grey 1981
first did to show presentation via the BCR. When antibodies were added to immature DCs in the cold, the cells bound comparable amounts of anti-MMR and DEC-205 Ig ( B, top). When the same cells were added to cultures of primed T cells, the anti–DEC-205 was presented with much higher efficiency ( B, bottom). To rule out that the quantitative difference is due to different allotypic determinants within the MMR and DEC-205 antibodies, additional experiments were performed using T cells from mice primed to RbIgG with either anti–DEC-205 or anti-MMR antiserum ( C). Here again the anti–DEC-205 was presented with much higher efficiency regardless of whether the T cells were derived from animals primed with either DEC-205 or MMR antibodies. Thus, DEC-205 is normally found in MIICs, whereas MMR is predominantly found in early endosomes; when rabbit antibodies are bound to these two receptors, DEC-205 is much more efficient at presenting peptides to rabbit Ig–primed, CD4+
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 Bonifacino and Dell'Angelica 1999
). A reexamination of the cytosolic domains (“tails”) of three homologous lectins—the MMR, DEC-205, and the phospholipase A2 receptor (PLA2R)—showed the tails of the MMR and PLA2R to be very similar to each other but different from DEC-205 (gray shading in A). All three tails contained a membrane-proximal, putative coated pit localization sequence ( A, underlined) for uptake (Collawn et al. 1990
), but the distal region of DEC-205 was distinct and included a sequence of three acidic amino acids ( A, EDE). Interestingly, acidic sequences in other receptors are implicated in intracellular sorting (Matter et al. 1993
; Voorhees et al. 1995
; Wan et al. 1998
; Piguet et al. 1999
; Simmen et al. 1999
Figure 2 Cytosolic domains used to study the targeting of endocytosis receptors. (A) Sequences of the cytoplasmic domain of three multilectin receptors for adsorptive endocytosis: DEC-205, PLA2R, and MMR. Underlining indicates a consensus sequence for endocytosis (more ...)
To determine which components of the cytoplasmic tail were required for efficient targeting and presentation via late endosomes or lysosomes (), we constructed chimeric receptors containing the external IgG binding domain of the HuFcγIII receptor or CD16 and different DEC-205 cytosolic tails ( 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 19–31 with the putative EDE distal targeting sequence (intermediate tail, IT), and residues 6–31 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) (Dubey et al. 1995
). Comparable surface expression of each CD16 chimeric receptor was obtained ( C) in the stable transfectants, without altering expression of MHC II ( C) or B7-1 (not shown).
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 ( A). The transfectants all bound ligand. Saturation occurred at 10 μg/ml, whereas the untransfected DCEK.ICAM.Hi7 cell line ( A, none) did not bind HuIgG. Binding required that the human IgG be heat aggregated, as expected for functional FcγRIII (Unkeless et al. 1981
). On PAGE, <10% of the HuIgG aggregated (molecular mass > 200,000 D) upon heating to 65°C for 30 min ( B), so that saturable binding of the expressed chimeric receptors was occurring at <1 μg/ml of aggHuIgG. When the sensitivity of bound ligand to pH was measured, aggHuIgG began to elute at pH 5, and only 40% remained at pH 4 ( C).
Figure 3 Ligand binding, uptake, and recycling via CD16 DEC-205 chimeric receptors. (A) Binding of aggHuIgG to transfected cells. WT-DEC/CD16 transfected cells (WT) and untransfected cells (None) were incubated on ice for 1 h with graded doses of aggHuIgG. After (more ...)
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:CD16–expressing cells, HuIgG was no longer detectable at the surface after the 2-h chase at 37°C ( 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 six–amino acid tail (ST-DEC:CD16), lacking the putative coated pit localization sequence, showed no significant loss of surface IgG ( D), indicating that deletion of amino acid residues 6–31 prevents endocytosis of the DEC:CD16 chimeric receptor. In a more detailed time course study ( 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 ( 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 ( 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 (, top), and not with TfR in the periphery (, bottom).
Figure 4 Presence of MHC II+ lysosomal compartments in CD16 transfectants (WT-DEC:CD16 shown here). Stably transfected DCEK.ICAM.Hi7 cells on tissue culture chamber slides were fixed and double labeled for MHC II and lysosomal (LAMP-1) proteins (top) or TfR (bottom). (more ...)
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 and , respectively). In contrast, the MMR:CD16 did not colocalize with LAMP-1 and was found primarily in the peripheral cytoplasm ( 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 18–31 in the cytoplasmic tail of DEC-205, failed to accumulate in MHC II+ lysosomes and were found in vesicles throughout the cytoplasm ( and ). The ST-DEC:CD16 chimera failed to mediate endocytosis completely, and was distributed along the cell membrane ( A).
Figure 5 Typical distribution of CD16 chimeric receptors in DCEK.ICAM.Hi7 cells in the absence (shown here) or presence of ligand, 10 μg/ml aggHuIgG. (A) CD16 and LAMP-1 double labeling. Cells on tissue culture chamber slides were fixed and stained for (more ...)
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 ( A, top), confirming the FACS® data ( 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 ( 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 (). 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.
Figure 6 Intracellular localization of endocytosed HuIgG in CD16 transfectants. (A) Double labeling for endocytosed HuIgG and LAMP-1. Cells, grown on tissue chamber slides, were incubated with 10 μg/ml HuIgG for 1 h on ice. Unbound HuIgG was washed away (more ...)
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 ( A). Immune complexes formed with anti-HuIgG and soluble HuIgG were also presented efficiently, whereas nonaggregated HuIgG, which did not bind to CD16 receptors ( A), only induced occasional T cell proliferation at high doses (100 μg/ml; and ).
Figure 7 Antigen presentation by DEC-CD16 transfected cells. (A) Presentation of aggHuIgG. Transfected and untransfected (none) DCEK.ICAM.Hi7 cell lines were incubated for 6 h with aggHuIgG, washed, and used to present antigen to 250,000 purified lymph node T (more ...)
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 ( and ) but failed to target to MHC II+LAMP-1+ compartments (), 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 (10–100 μg/ml; ). 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 (Piguet et al. 1999
), the movement of furin to the trans-Golgi network (Voorhees et al. 1995
; Simmen et al. 1999
), and the retrieval of the LDLR from apical to basolateral membranes of epithelial cells (Matter et al. 1993
). To assess if acidic amino acids in the distal part of the DEC-205 tail were required for late endosomal targeting, we mutated the EDE residues to alanines (AAA-DEC:CD16). The AAA-DEC:CD16 chimera was fully competent for adsorptive uptake of aggHuIgG and recycling back to the surface ( and ), but failed to target aggHuIgG or CD16 to lysosomes ( B). Consistent with the absence of late endosomal targeting, presentation of antigen to IgG-primed T cells was greatly reduced ( A) and occurred only at antigen levels comparable to those needed for IT-DEC:CD16– and MMR:CD16-mediated presentation. Thus, the EDE in the DEC-205 tail is required for its unique lysosomal targeting and antigen presenting functions.