HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Saudrais, C. Articles by Salamero, J. Search for Related Content PubMed PubMed Citation Articles by Saudrais, C. Articles by Salamero, J.

Intracellular Pathway for the Generation of Functional MHC Class II Peptide Complexes in Immature Human Dendritic Cells¹

Cédric Saudrais^*, Danièle Spehner, Henri de la Salle, Alain Bohbot^§, Jean-Pierre Cazenave, Bruno Goud^*, Daniel Hanau and Jean Salamero^2,*

^* Unité Mixte de Recherche, Centre National de la Recherche Scientifique 144, Laboratoire "Mécanismes Moléculaires du Transport Intracellulaire," Institut Curie, Paris, France; and Contrat Jeune Formation (CJF) INSERM 94-03, Laboratoire d’Histocompatibilité, INSERM U.311, Etablissement de Transfusion Sanguine, and ^§ Service d’Onco-Hématologie, Hopital de Hautepierre, Strasbourg, France

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Binding of antigenic peptides to MHC class II (MHC-II) molecules occurs in the endocytic pathway. From previous studies in B lymphocytes, it is believed that most but not all of the newly synthesized MHC-II molecules are directly targeted from the trans-Golgi network to endosomal compartments. By using pulse-chase metabolic labeling followed by cell surface biotinylation, we show here that in contrast to an EBV-transformed B cell line and human monocytes, the majority of newly synthesized MHC-II molecules (at least 55 ± 13%) are first routed to the plasma membrane of dendritic cells derived from human monocytes. They reach the cell surface in association with the invariant chain (Ii), a polypeptide known to target MHC-II to the endosomal/lysosomal system. Following rapid internalization and degradation of Ii, these ßIi complexes are converted into ß-peptide complexes as shown by their SDS stability. These SDS-stable dimers appear as soon as 15 to 30 min after internalization of the ßIi complexes. More than 80% of ß dimers originating from internalized ßIi complexes are progressively delivered to the cell surface within the next 2 h. Depolymerization of microtubules, which delays the transport to late endosomal compartments, did not affect the kinetics of conversion of surface ßIi into SDS-stable and -unstable ß dimers. Altogether, these data suggest that newly liberated class II ß heterodimers may bind peptides in different compartments along the endocytic pathway in dendritic cells derived from human monocytes.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Immune response against exogenous Ag is initiated by presentation of antigenic peptides in the context of MHC class II (MHC-II)³ molecules. The specificity and efficiency of Ag presentation depend on the intracellular transport of MHC molecules (1). The and ß subunits of MHC-II molecules are assembled in the endoplasmic reticulum, where they associate with the invariant chains (Ii) (1). Ii MHC-II (ßIi) complexes then travel through the Golgi complex and reach the endocytic pathway, where degradation of Ii allows MHC-II to bind immunogenic peptides generated by proteolytic cleavage of internalized proteins. At the steady state, intracellular MHC-II molecules are predominantly located in endosomal compartments (2). In human APC, these intracellular compartments share characteristics with lysosomes (3). They appeared as multilamellar or multivesicular structures called MHC-II compartments (MIIC). Similar compartments have also been identified in human dendritic epidermal Langerhans cells (4, 5) and mouse dendritic cells (DCs) (6). MIIC are thought to represent a specialization of the prelysosomal or lysosomal compartments for efficient peptide loading onto newly synthesized MHC-II molecules (3, 7). They seem to be distinct from conventional early or late endosomal compartments and contain the majority of HLA-DM, a protein that catalyzes loading of peptide on MHC-II molecules (8, 9). This and current biochemical data, essentially obtained in B cells, have led to the generally accepted view of a direct transport of ßIi complexes from the trans-Golgi network (TGN) to the MIIC, before cell surface expression of peptide-loaded ß dimers (7, 10). However, other studies report the presence of MHC-II ß dimers in other compartments (11) or even throughout the whole endocytic route in B cells (12). The precise pathway taken by MHC-II molecules en route to peptide-loading compartments thus remains unclear.

All cell types that express MHC-II and Ii chains have the capacity to present Ags. Among them, DCs display unique characteristics for Ag presentation: 1) they synthesize high levels of MHC-II molecules (13, 14), 2) they express specialized receptors thought to potentiate the capture of diverse Ags and their specific delivery to the processing compartments (15, 16), 3) they efficiently present Ags in situ (17), and 4) they can prime virgin T lymphocytes in vitro and even in vivo (18). Moreover, low numbers of immature DCs and small amounts of Ag are sufficient to induce T cell stimulation in vitro (19). Upon maturation, these cells become even better APCs, when primed with Ags before their in vitro differentiation into mature DCs, while they lose their capacity to capture and process new Ags (19). In vivo, DCs are widely distributed in the body. They constitute a trace population of cells circulating between nonlymphoid and lymphoid tissues. In the nonlymphoid tissues, where they reside in an "immature" state, DCs are ideally placed to perform a "sentinel" function for the immune system (20). There, they capture Ags, process them into an immunogenic form, and present MHC-II-peptide complexes to sensitized T cells. Under some conditions, which remain to be defined, DCs can migrate from the nonlymphoid tissues to the T cell-dependent areas of the lymphoid organs and undergo maturation (21). Consequently, in the lymphoid tissues, "mature" DCs can initiate the sensitization of naive/resting T cells.

A number of techniques have been developed allowing the isolation of relatively low quantities (1 to 2.10⁶ cells) of tissue human DCs or even highly purified lymphoid DCs (22, 23). Long-term mouse cell lines are also available (24, 25). However, due to the relative difficulty of isolating large quantities of immature human DCs, the precise dynamic of the intracellular transport of MHC-II molecules, which requires following the behavior of a small proportion of newly synthesized molecules from their site of synthesis to their final destination, has been less studied in these cells than in other MHC-II expressing cells. There have been conflicting reports on the nature of DC precursors in peripheral blood (for review see 26 . One emerging explanation is that more than one cell type can develop into cells with the morphologic and functional characteristics of DCs (27, 28). Depending on the original cells and on the in vitro method used for derivation, DCs with different phenotypes can also be obtained (29).

Recently, it has been shown that low density mononuclear cells cultured with a combination of granulocyte-macrophage (GM)-CSF and IL-4 develop into cells that are highly efficient in the processing and presentation of diverse Ags to T cells. These cells also show phenotypical characteristics of immature DCs that can be further differentiated into mature DCs (19, 30). As already suggested (31), we found that these very same immature DCs can be differentiated into activated macrophages upon addition of macrophage-CSF (our unpublished observations), implying that these immature DCs are pluripotent cells.

We wanted to biochemically investigate the intracellular process leading to peptide formation in these cells. We thus compared these monocyte-derived immature DCs with other APCs for the intracellular trafficking of MHC-II molecules.

We report that in strong contrast to an EBV-transformed B (BEBV) cell line and freshly isolated human monocytes, a large proportion (more than 55%) of functional MHC-II first traffic through the plasma membrane of human immature DCs in association with Ii. These ßIi complexes, transiently expressed at the plasma membrane of DCs, are rapidly internalized and are converted into ß dimers in a microtubule-independent manner. Then, the ß dimers can bind peptide, as assessed by their SDS stability (32), and are progressively delivered to the cell surface within the 2 h following synthesis of the polypeptide chains.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells, Abs, cell culture, and chemicals

DCs were derived from human blood monocytes using human recombinant (hr)GM-CSF and IL-4 (both generously provided by Schering-Plough, Union, NJ), as previously described (19). Monocytes were isolated by continuous flow centrifugation leukapheresis and counterflow centrifugation elutriation as previously described (33). They were maintained in RPMI 1640 medium supplemented with 2 mM glutamine, 1% sodium pyruvate, 1% nonessential amino acids, 10% heat-inactivated FCS (all from Life Technologies, Paisley, U.K.), 50 ng/ml hrGM-CSF, and 200 U/ml hrIL-4 (referred to as complete medium). Differentiated DCs were always used at day 7. Differentiation of monocytes was followed by flow cytometry analysis of different surface markers. For FACS analysis, cells were washed once in PBS and resuspended in PBS, 3% FCS, and 0.1% NaN₃ in 96-well plates. Cells were incubated with different primary Abs as specified, followed by species-specific FITC-labeled secondary Abs. After washing, cells were analyzed with a FACScan (Becton Dickinson, Mountain View, CA).

At day 7, DCs express high levels of the MHC-II molecules HLA-DR and CD74 (Ii); MHC class I molecules (HLA-A, -B, -C); and the Ags CD1a, CD1b, CD1c, CD40, CD80 (B7-1), CD11b, CD11c, CD18 (ß₂ integrin), CD54 (ICAM-1), and CD44. They express moderate amounts of CD32 (FcRII) and express only small amounts of the CD86 (B7-2) Ag. DCs do not express CD3, CD14, CD25, CD16 (FcRIII), CD64 (FcRI), or E-cadherin. These results are in agreement with previously published observations (19) (data not shown). EBV-transformed B lymphocytes used in this study have been described elsewhere (34). The different Abs used were: L243 (35) (IgG2a, anti-DRß dimers; Becton Dickinson); W6/32 (IgG2a, anti-HLA class I; American Type Culture Collection, Rockville, MD); BU 45 (IgG1, anti-CD74 (Ii); Serotec, Kidlington, U.K.); MAB89 (IgG1, anti-CD40; Immunotech, Marseille, France); IT2.2 and BB1 (IgG2b, anti-CD86 (B7.2) and IgM, anti-CD80 (B7-1); PharMingen, San Diego, CA); BL6, 4A7.6, and L161 (IgG1, IgG2a, IgG1, anti-CD1a, anti-CD1b, and anti-CD1c, respectively; all from Immunotech); Leu-15 (IgG2a, anti-CD11b; Becton Dickinson); Leu M5 (IgG2b, anti-CD11c; Becton Dickinson); Leu CD-54 (IgG2b, anti-CD54; Becton Dickinson); Leu M3 (IgG2b, anti-CD14; Becton Dickinson); Leu-4 (IgG1, anti-CD3; Becton Dickinson); 10.1, FLI8.26, and 3G8 (IgG1, IgG2b, IgG1, anti-CD64, anti-CD32, and anti-CD16, respectively; PharMingen); and G44-26 (IgG2b, anti-CD44; PharMingen). Mouse IgG1, IgG2a, and IgG2b (all from Sigma Chemical Co., St. Louis, MO) were used for isotype controls and an FITC-conjugated affinity-isolated F(ab')₂ fraction of a sheep anti-mouse Ig Ab (Silenus, Hawthorn, Victoria, Australia) for the labeling procedures. The mouse hybridoma cell line DA6.147 (IgG2a, anti-HLA-DR-chain) has been described (36). The rabbit antiserum, RHuIi, was prepared by immunizing rabbits with the 191–212 peptide (PKESLELEDPSSGLGVTKQDLG) from human Ii coupled to keyhole limpet hemocyanin (37). For electron microscopy studies, the following Abs were used: a polyclonal rabbit anti-DR-chain Ab (kindly provided by Dr. J. Neefjes, The Netherlands Cancer Institute, Amsterdam, The Netherlands), 20-nm gold-labeled goat anti-rabbit Ab (British BioCell Laboratories, Cardiff, U.K.), and gold-labeled F(ab')₂ fragments of the mAb anti-CD1b (Immunotech). F(ab')₂ fragments were prepared as described (38), and the labeling of the F(ab')₂ fragments with gold particles was performed as previously reported (38). Most chemicals used in this study were obtained from Sigma Chemical Co.

Radiolabeling, biotinylation, immunoprecipitation, and electrophoresis

Cells were pulse labeled with ³⁵S Promix (Amersham France, Les Ulis, France) for either 5 or 10 min at 37°C and chased for various periods of time, essentially as described previously (39). At indicated times, 20.10⁶ cells were chilled in cold PBS and cell surface biotinylated with a solution containing 25 mg of NHS-SS-biotin (Pierce Chemical Co., Rockford, IL) in 1 ml of cold PBS for 5 min at 4°C. The reaction was quenched with 50 mM of ice-cold glycine in PBS. Cells were solubilized in 1% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl, 5 mM EDTA, 0.2% BSA, and protease inhibitors. Postnuclear lysates were precleared for 2 h with protein A-Sepharose at 4°C. Precleared lysates were immunoprecipitated with the Abs RHuIi, L243, W6.32, and DA6.147 previously bound on protein A-Sepharose CL-4B (Pharmacia, Uppsala, Sweden). Immunoprecipitates were washed as published elsewhere (39) and eluted in 10 ml of 10% SDS either at 95°C for 5 min or at room temperature for 30 min when indicated. Eluted material was then resuspended in 100 ml of lysis buffer without BSA. The efficiency of biotinylation being about 10 to 15% (data not shown), biotinylated proteins were recovered with streptavidin agarose on 90% of the eluted material, and the remaining 10% were left untreated. Except when specified, samples were boiled in Laemmli’s sample buffer (40) for electrophoresis on a 10 to 15% SDS-PAGE and run under reducing conditions (100 mM DTT) as described previously (41). Gels were processed for fluorography.

Quantification of sequential immunoprecipitations

All quantifications were based on the signal for a radiolabeled HLA-DR ß-chain. To distinguish between the two forms of MHC-II molecules (ßIi precursor forms and ß dimers), a first immunoprecipitation was performed with L243 mAb, which recognized ß dimers dissociated from intact Ii chains (42, 43). Supernatants were then submitted to a second immunoprecipitation with the anti-DR DA6.147 mAb (36) to recover residual DR molecules corresponding to ßIi complexes. A hypothetical background of ß dimers dissociated from Ii but recovered in the DA6.147 immunoprecipitation would be most detectable at a late time point in our pulse chase experiments (4 h), when the signal for these dimers is at a maximum. In B cells, the relative proportion between the DA6.147 and L243 immunoprecipitated ß-chain at this time point was only 3%. In DCs, some Ii chains associated with ß dimers were still found at this late time point. They reach a proportion of 17% of the maximum signal for Ii, obtained at a chase time of 30 min. We thus subtracted 17% of the signal for the ß-chain at the 30-min time point from the remaining signal detected with DA6.147 in the second immunoprecipitation after 4 h of chase. We calculated that only 12% of the ß-chain signal detected in the DA6.147 immunoprecipitation could represent residual ß dimers.

Autoradiographies exposed in the linear range of detection were scanned with a video camera (Bio-print system; Vilber Lourmat, Marne la Vallée, France). Optical densitometry was performed using the Bio-1D software (Vilber Lourmat). The background signal was calculated for each lane and subtracted from the ODs of the area corresponding to protein bands.

Internalization and recycling experiments

For internalization experiments, cells were pulse labeled, chased, and biotinylated as described above. They were then incubated for various times at 37°C to allow biotinylated material to endocytose. At the end of the chase period, ice-cold PBS was added and the cells were washed twice at 4°C. Lysis, immunoprecipitation, and streptavidin precipitation were performed as above. When specified, 20 µM of nocodazole was added to the cells after biotinylation at 4°C and before further incubation at different temperatures to allow for internalization of the biotinylated material.

For recycling experiments, cells were pulse labeled, chased, biotinylated, and reincubated at 37°C for further transport. Reduction of remaining or recycled surface NHS-SS-biotin was performed with 20 mM 2-mercaptoethanesulfonic acid (MESNA; Sigma Chemical Co.), 50 mM Tris, pH 8.6, 100 mM NaCl, and BSA 0.2% for 10 min at 4°C. The cells were solubilized, cell lysates were immunoprecipitated, and biotinylated immunoprecipitates were separated as described above.

Immunogold-labeling procedure of DCs

DCs (incubated in complete medium with or without 20 µM nocodazole) were cooled to 4°C for 10 min and incubated at 4°C for 60 min, in the presence of 10 nm of gold-labeled F(ab')₂ fragments of the mAb anti-CD1b (final dilution, 1%). Thereafter, DCs were warmed to 20°C for 30 min, and either fixed at 20°C for electron microscopy or washed at 4°C, incubated at 37°C for 5 min in previously warmed complete medium (with or without 20 µM nocodazole), and fixed at 37°C for electron microscopy.

Preparation of DCs for transmission electron microscopy

DCs maintained at either 20°C or 37°C in 1 ml of the above-mentioned medium were fixed by adding an equal volume of fixative solution, previously warmed to 20°C or 37°C, respectively, and composed of 3% glutaraldehyde (Electron Microscopy Sciences, Euromedex, Strasbourg, France) in 0.1 M sodium cacodylate buffer containing 2% sucrose (305 mOsm, pH 7.3) (both from Merck, Darmstadt, Germany). After 5 min, the mixture was centrifuged, the supernatant discarded, and the pellet resuspended and further fixed for 45 min with the same fixative solution maintained at 20°C or 37°C, respectively. DCs were then washed in the 0.1 M sodium cacodylate buffer and postfixed for 1 h, at 4°C, with 1% osmium tetroxid (Merck) in 0.1 M sodium cacodylate buffer. After additional washing in the 0.1 M sodium cacodylate buffer, DCs were dehydrated in successively increasing (50, 70, 80, 95, and 100%) ethanol concentrations. Finally, the cells were incubated overnight in Epon (Electron Microscopy Sciences)-absolute alcohol (1:1, v/v) and embedded in Epon. Ultrathin sections, stained with lead citrate (Leica, Bron, France) and uranyl acetate (Merck), were examined under a Philips CM 120 BioTwin electron microscope (120 kV).

To characterize the MIIC, unlabeled DCs were fixed by adding for 10 min at room temperature and for 50 min at 4°C a fixative solution containing 2.5% paraformaldehyde, 0.1% glutaraldehyde (both Electron Microscopy Sciences) diluted in 0.1 M sodium cacodylate buffer (305 mOsm, pH 7.3). Thereafter, samples were washed once in 0.1 M sodium cacodylate buffer at 4°C, concentrated in agar, and further washed overnight at 4°C in 0.1 M sodium cacodylate buffer. After 30 min of staining at 4°C with 2% uranyl acetate diluted in Michaelis buffer, dehydration and Lowicryl K4 m embedding procedures were conducted as reported previously (44). DCs were then processed for immunoelectron microscopy essentially as described (44). Briefly, ultrathin sections of Lowicryl K4 m were collected on Formvar carbon-coated nickel grids (Agar, Stansted, U.K.). Grids were pretreated for 1 h at room temperature with PBS containing 1% goat serum and incubated overnight with the rabbit anti-DR-chain Ab (dilution 1/400). Thereafter, the sections were washed with PBS/1% goat serum and further incubated for 1.5 h at room temperature with the gold-labeled goat anti-rabbit Ab (dilution 1/20). Finally, the grids were washed two times with PBS/0.5% goat serum, two times with PBS, postfixed for 30 min at room temperature with 2.5% glutaraldehyde in PBS, washed with distilled water, and stained with 1.8% uranyl acetate/0.2% methylcellulose. Sections were then examined under a Philips CM 120 BioTwin electron microscope (120 kV). Controls were performed by substituting nonimmune rabbit IgG for the primary Ab.

Immunofluorescence and confocal microscopy

DCs were first allowed to adhere on glass coverslips precoated with a 0.1% polyL-lysine solution in water for 30 min at 37°C. Adherent cells were incubated with 10 µg/ml of IgGs purified from the RaHuIi antiserum for 30 min at 4°C. They were then incubated for 30 min at 37°C for the indicated times in the presence or absence of 20 µM nocodazole. Cells were fixed in 3% paraformaldehyde for 10 min. After permeabilization with 0.05% saponin in PBS supplemented with 0.2% BSA, they were incubated with the L243 mAb and finally stained with both FITC and Texas Red-donkey antisera directed against mouse and rabbit IgGs (Jackson ImmunoResearch, West Grove, PA). The coverslips were then mounted in Mowiol (Merck, Darmstadt, Germany).

Confocal laser scanning microscopy and immunofluorescence analysis were performed using a TCS4D confocal microscope based on a DM microscope interfaced with an Argon/Krypton laser. Simultaneous double fluorescence acquisitions were performed using the 488- and 568-nm laser lines to excite FITC and Texas Red dyes using a 100x oil immersion Plan Apo objective (numerical aperture = 1.4). The fluorescence was selected with the appropriate double fluorescence dichroic mirror and band pass filters and measured with blue-green sensitive and red side sensitive-one photomultipliers.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
A high proportion of ßIi complexes reaches the plasma membrane of DCs

We first compared the cell surface appearance of newly synthesized MHC-II molecules in BEBV cells, in freshly isolated blood monocytes and in DCs obtained by culturing the latter cells in the presence of hrGM-CSF and hrIL-4. These three types of cells express both MHC-II molecules (not shown) and Ii chain at their cell surface. As shown in Figure 1A, larger amounts of Ii were always detected at the cell surface of immature DCs. We found that most if not all cell surface Ii was associated with ß dimers of MHC II molecules (not shown). MHC-II molecules exist as two major forms where ß dimers can be complexed with diverse isoforms of Ii in a nonameric precursor structure (45) and as free ß dimers from which Ii has been released by proteolytic cleavage. To distinguish between these two forms of MHC-II molecules, we took advantage of the restricted specificity of the L243 mAb for the ß dimers of HLA-DR devoid from intact Ii chains (42, 43). Cells were surface biotinylated at chosen times of a pulse-chase experiment (Fig. 1B). Lysates were first immunoprecipitated with the L243 mAb. Supernatants were then submitted to immunoprecipitation with the DA6.147 mAb to recover residual DR molecules corresponding to ßIi complexes. The partition between ß and ßIi forms of MHC-II molecules using sequential immunoprecipitations was asserted by our quantification analysis, as described in Materials and Methods. Biotinylated cell surface molecules were compared with the total immunoprecipitated material.

View larger version (47K): [in this window] [in a new window]   FIGURE 1. Maturation and cell surface appearance of MHC-II molecules in BEBV cells, monocytes, and DCs. A, Surface expression of Ii on BEBV cells, monocytes, and DCs was analyzed using the BU 45 mAb. Thin lines correspond to isotypic control. Note the enhancement of the cell surface expression of Ii during the differentiation of monocytes into DCs. B, Cells were pulse labeled for 10 min. After the indicated times of chase, the cell surface was biotinylated at 4°C. The L243 mAb was used for the first immunoprecipitation (left panel). Surface proteins were separated on streptavidin-agarose before analysis by SDS-PAGE (Biot.) and compared with an aliquot representing the total immunoprecipitated material (Total; see Materials andMethods). Proteins in the supernatants of the first immunoprecipitation were then immunoprecipitated with the DA6.147 mAb (right panel). Immunoprecipitates were treated as for L243 immunoprecipitation (Total vs Biot.). Precipitated polypeptide chains are indicated with arrowheads. Autoradiographs for BEBV and DC were exposed for different times to show comparable intensities of protein bands for p33 and ß-chain in the total fractions. C, The third gel (DC) of Bwas exposed for a longer time (four times). Note that a high proportion of the mature ß-chain is found at the cell surface within 30 min to 1 h after synthesis in the DA6.147 immunoprecipitates.

In contrast, the analysis of remaining biotinylated DR molecules immunoprecipitated with the DA6.147 mAb showed that much higher amounts of ß dimers complexed with Ii chains were detected at the surface of DCs, compared with monocytes and BEBV cells (Fig. 1B, Biot. DA6.147). In human cells, Ii is present in four different isoforms: two alternatively spliced forms, p33 and p41 and, due to two different initiation sites, a p35 and a p43 form (46). All isoforms can assemble with MHC-II molecules (46). As previously shown in human B cells (47), trace amounts of ßIi complexes devoid of the p35 Ii isoform were detected at the plasma membrane of BEBV cells and monocytes (Fig. 1, Biot. DA6.147). The transient expression of ßIi complexes at the plasma membrane of DCs reached its maximum within 30 min to 1 h after their synthesis. A polypeptide chain of 47 kDa (p47) (Fig. 1) was also strongly detected at the plasma membrane of DCs. It represents the mature sialylated p41 form (48), as assessed by neuraminidase desialylation after immunoprecipitation with a rabbit antiserum directed against human Ii (not shown). This isoform was barely detectable in BEBV cells and monocytes. During further chase times, all Ii chains, including cell surface p47 and the 35-kDa proteins (corresponding to comigrating sialylated p33 (mp33 for mature p33) and MHC-II DR-chains), progressively declined (Fig. 1). Immature ß-chain signals also declined over the time course of the experiment. In fact, as is better seen after a longer exposure (Fig. 1C), this molecule is first converted into a more mature form, which migrates between immature ß-chain and p33 Ii. Then, it is transiently expressed at the plasma membrane in an ßIi complex. Finally, ß dimers are formed as detected here in the first immunoprecipitation with the L243 mAb (Fig. 1, left panel).

A quantitative analysis, performed on DCs from six blood donors, showed that more than half (55 ± 13%) of the newly synthesized ßIi complexes gained access within 30 min to the DCs surface, where they are transiently expressed (Fig. 2A). The disappearance of ßIi complexes from the plasma membrane of DCs correlates with the formation of free ß dimers and the subsequent arrival of these dimers at the plasma membrane (Fig. 2A). Using the same quantitative analysis, we found that only 7.8% of the newly synthesized ßIi complexes also gained access to the plasma membrane of BEBV cells (Fig. 2B).

View larger version (15K): [in this window] [in a new window]   FIGURE 2. Transient expression of ßIi complexes at the cell surface precedes the appearance of mature ß dimers in DCs. A, The signals corresponding to the ß-chain present at the cell surface in ßIi complexes (biotinylated ß-chain immunoprecipitated with DA6.147, circles), the ß-chain in total ß dimers (immunoprecipitated with L243, squares), and the ß-chain in plasma membrane ß dimers (biotinylated ß-chain immunoprecipitated with L243, triangles) were quantified by optical densitometry of autoradiographs obtained in four separate experiments for DCs. Independent experiments showed that after 4 h, all ß-chains (100%) detected with L243 were already present at the cell surface of DCs (data not show). Consequently, biotinylation efficiency was estimated for each experiment by comparing total and biotinylated - and ß-chains at this time point. Results are expressed as percent of the maximum of ß-chain expression at the cell surface. SDs were also calculated. B, Same as in A for BEBV cells expressed as a mean of three separate experiments. As not all ß dimers were present at the cell surface after 4 h in these cells, a mean biotinylation efficiency obtained from other experiments was used to establish this curves. The signal for ß after 4 h, detected with L243, is represented by 100%.

View larger version (56K): [in this window] [in a new window]   FIGURE 3. Comparative kinetics of cell surface arrival of ßIi complexes and MHC class I molecules in DCs. A, DCs were pulse labeled (5 min) and biotinylated at 4°C at indicated times. Cell lysates were first immunoprecipitated with L243 mAb (not shown) before a second immunoprecipitation with DA6.147 mAb. B, Cell lysates were finally submitted to a third immunoprecipitation with W6/32 mAb. Proteins were separated before analysis on SDS-PAGE as above (Total vs Biot.). Arrowheads indicate the respective positions of the different polypeptide chains. H.C., MHC class I heavy chain.

ßIi complexes transiently expressed at the plasma membrane of DCs are converted into SDS-stable ß dimers

We analyzed in further detail the conversion of ßIi complexes transiently expressed at the plasma membrane into ß-peptide complexes. This was possible by taking advantage of the capacity of L243 mAb to recognize undissociated ß-peptide complexes in an SDS stability assay (32). DCs were labeled for 10 min, chased for 30 min, and surface biotinylated at 4°C. As already shown, this chase time corresponds to the arrival of significant amounts of metabolically labeled ßIi complexes at the plasma membrane (Figs. 1, 2A, and 3). However, at this time no radiolabeled ß dimers were detected on the cell surface (Figs. 1 and 2A). After given times of further incubation at 37°C, cells were lysed and the lysates immunoprecipitated with L243 mAb. The immunoprecipitates were then dissociated in the presence of SDS for 30 min at room temperature and adsorbed onto streptavidin-agarose beads. To test the SDS stability of the ß dimers, the final material was eluted from the beads in the presence of SDS and the reducing agent DTT at room temperature. After the first immunoprecipitation with the L243 mAb, the lysates were reimmunoprecipitated with the DA6.147 mAb. Surface-biotinylated ßIi complexes rapidly disappeared following their internalization at 37°C. At the same time, newly synthesized p41, present in the total fraction of ßIi complexes, still matured into the p47 sialylated form (compare Fig. 4A total vs biotinylated, DA6.147). This showed that an excess of free-labeled Ii can still assemble with nonradioactive - and ß-chains in the endoplasmic reticulum during subsequent chase times, while biotinylation of cell surface proteins synchronizes further transport of radiolabeled biotinylated ßIi complexes. Consequently, the biotinylated ß dimers detected in this experiment (Fig. 4B) were generated from ßIi complexes previously expressed at the plasma membrane (Fig. 4A Biot., 0'). Newly formed biotinylated-ß dimers, including both SDS-stable and -unstable forms, appeared within 15 (data not shown) to 30 min (Fig. 4B, Biot.) after internalization of biotinylated ßIi complexes. The proportion of SDS-stable dimers in DCs widely differed from one blood donor to the next (not shown), probably reflecting the various levels of SDS stability for the different DR molecules associated with peptides. However, the rate of conversion of ßIi complexes into ß dimers did not vary between donors. These molecules, which appeared as both SDS-sensitive and -resistant dimers, increased in concentration over the next 30 min. Interestingly, the kinetics of formation for biotinylated and total ß dimers were undistinguishable.

View larger version (59K): [in this window] [in a new window]   FIGURE 4. Conversion of cell surface-biotinylated ßIi complexes into ß dimers. DCs were pulse labeled (10 min), chased for 30 min, and surface biotinylated. Cell surface molecules were then allowed to internalize by further incubation at 37°C for the indicated times in min. The first immunoprecipitation was performed with the L243 mAb (B). Proteins in the supernatants of the first immunoprecipitation were then immunoprecipitated with DA6.147 mAb (A). Proteins were separated before analysis on SDS-PAGE as described in Materials and Methods (Total vs Biot.). The SDS stability of L243-immunoprecipitated ß dimers was tested (see Materials andMethods). Samples were separated at room temperature and were either boiled (B) or not (NB) before gel loading. The result of boiling is represented for the last chase time (90 min) in the Total fraction. The two autoradiographs shown in this Figure (DA6.147 and L243) correspond to different exposure times.

Intracellular ß dimers generated from internalized ßIi complexes return to the plasma membrane of DCs

We next addressed whether ß dimers generated from internalized ßIi complexes underwent further transport to the DCs plasma membrane. Cells were pulse labeled, chased for 30 min, and biotinylated with cleavable NHS-SS-biotin at 4°C as described above. The cells were then warmed to 37°C (Fig. 5), allowing biotinylated proteins to internalize. At given times, cells were cooled to 4°C and one-half of each sample was treated with MESNA, a membrane-impermeant reducing agent, to strip the biotin present at the cell surface. Cells were lysed and cell lysates were immunoprecipitated first with the L243 mAb, and second with the DA6.147 mAb. The immunoprecipitates were dissociated in the presence of SDS for 5 min at 95°C and adsorbed onto streptavidin-agarose beads. As shown in Figure 4, biotinylated ßIi complexes were present at the plasma membrane as early as 30 min after their synthesis (Fig. 5A, -MESNA). At this time (0'), no significant amount of newly formed ß dimers were detected at the surface of DCs (Fig. 5B) and about 80% of the biotin linked to ßIi complexes was reduced by MESNA treatment (Fig. 5A, +MESNA). Within 30 min after internalization, 75% of biotinylated ßIi complexes were converted into biotinylated ß dimers (Fig. 5B, lower panel and Fig. 6), 20% of which had already reached the plasma membrane. The ß dimers appearing within the next hour in the total fraction can be generated, either from ßIi complexes reaching the plasma membrane later than 30 min after their synthesis (Figs. 5 and 6), or from ßIi complexes directly routed from the TGN to the endosomal compartments. Most of the ß dimers (74%) originating from cell surface-biotinylated ßIi complexes were progressively delivered to the plasma membrane of DCs within 2 h. However, a small amount of biotinylated ß dimers still remained resistant to the MESNA treatment even after 4 h of internalization (Figs. 5B and Fig. 6). This might represent either ß dimers retained in the endocytic compartments, or ß dimers cycling continuously between plasma membrane and endosomes. Alternatively, it might also reflect the incomplete efficiency of the biotin reduction.

View larger version (53K): [in this window] [in a new window]   FIGURE 5. ß dimers generated from internalized ßIi complexes return to the cell surface. DCs were pulse labeled (10 min), chased for 30 min, and surface biotinylated with cleavable NHS-SS-biotin. Before lysis, half of the samples were treated with MESNA, a membrane-impermeant reducing agent. Immunoprecipitations were conducted as in Figure 4. A, Only the biotinylated material found in DA6.147 immunoprecipitates is shown. The time 0' was used to calculate the reduction efficiency of the MESNA treatment, which was estimated at about 80%. B, Both total (Total) and biotinylated (Biot.) material immunoprecipitated with L243 were run on SDS-PAGE. Note that very little material is present at time 0' in both Total and Biot. samples. MESNA treatment does not lead to a loss of L243-immunoprecipitated material, as seen in the upperpart of the figure (compare -MESNA and +MESNA in Total).

View larger version (16K): [in this window] [in a new window]   FIGURE 6. Kinetics of internalization of ßIi complexes and the return to the cell surface of the corresponding ß dimers. Quantifications were performed on the signal for ß-chains as described in Materials and Methods. Results were obtained from four separate experiments conducted as described in Figure 5. The biotinylated ß-chain in ßIi complexes detected with DA6.147 (diamonds) at time 0' is represented by 100%. The disappearance of these complexes is followed by the generation of ß dimers inside the cell (circles). These biotinylated dimers are detected with L243 and are resistant to the reducing treatment. Biotinylated ß-chains returning to the cell surface in ß dimers (squares) are sensitive to the MESNA reducing treatment. Their quantity was estimated by subtracting MESNA-resistant ß-chains in ß dimers from total biotinylated ß-chains in ß dimers.

Microtubules are not required for the conversion of cell surface ßIi complexes into ß dimers

It is already known that presentation of different peptides may involve distinct pools of MHC II molecules and may depend on the way Ags are delivered into APC (49, 50). The rather fast generation of ß dimers from internalized ßIi complexes in DCs is compatible with the loading of ß dimers in different endosomal compartments with different proteolytic and physiologic properties (12). This led us to better characterize where, in the endocytic pathway of DCs, cell surface ßIi can be converted into ß dimers. For this purpose, we took advantage of the fact that migration of transmembrane proteins along the endocytic pathway can be inhibited, or modulated, by incubating cells under two different experimental conditions. First, cells can be incubated at relatively low temperatures. At this temperature, prelysosomal late endosomes no longer fuse with newly formed endosomes, at least as observed in Chinese hamster ovary cells (51). Second, delivery of ligands (52, 53) or transmembrane proteins (54, 55) from early endosomes to degradative compartments of the endocytic pathway, probably lysosomes, can be slowed down by inducing depolymerization of microtubules in the presence of nocodazole.

The effect of low temperature incubation or nocodazole on the migration of transmembrane proteins in DCs was first checked by following under the electron microscope the fate of CD1b molecules. Indeed, we have recently demonstrated that CD1b molecules expressed at the DC surface are internalized by receptor-mediated endocytosis and reach successively early endosomes, multivesicular/late endosomes, and finally the MIIC (our manuscript in preparation) (56). Thus, DCs were incubated, for 60 min at 4°C then for 30 min at 20°C, with gold-labeled anti-CD1b F(ab')₂. Progression of CD1b was blocked in the early endosomes as illustrated by the fact that gold-labeled anti-CD1b F(ab')₂ accumulated in these compartments (Fig. 7). When cells were warmed up to 37°C for only 5 min, clear appearance of gold-labeled anti-CD1b molecules in MIIC was detected (Fig. 8, A–C, G, and H; and Table I). We then attempted to inhibit the transport of CD1b molecules toward MIIC using nocodazole. We first confirmed that complete depolymerization of the DC microtubules was achieved using 20 µM nocodazole. This treatment did not interfere with the accumulation of gold-labeled anti-CD1b F(ab')₂ in early endosomes, when DCs were incubated for 60 min at 4°C and for 30 min at 20°C (data not shown). However, when DCs were incubated in the presence of both gold-labeled anti-CD1b F(ab')₂ and 20 µM nocodazole for 60 min at 4°C and 30 min at 20°C, washed at 4°C, and finally incubated for 5 min at 37°C, the transport of CD1b molecules from early endosomes to later endosomal compartments and to the MIIC was considerably reduced (Table I and Fig. 8, D–F).

View larger version (94K): [in this window] [in a new window]   FIGURE 7. Accumulation of an internalizing transmembrane protein in early endosomes at 20°C. DCs were incubated, in the presence of gold-labeled F(ab')2 fragments of an anti-CD1b mAb, for 60 min at 4°C, then 30 min at 20°C, in the absence of nocodazole. Note the gold particles accumulating in endosomes, some of which contain small intraluminal vesicles. The same findings were observed when DCs were incubated at 20°C in the presence of nocodazole.

View larger version (59K): [in this window] [in a new window]   FIGURE 8. Localization along the endocytic pathway of an internalizing transmembrane protein in the presence or absence of 20 µM nocodazole. A–C, DCs were incubated, in the presence of gold-labeled F(ab')2 fragments of an anti-CD1b mAb, for 60 min at 4°C, 30 min at 20°C, and finally 5 min at 37°C in the absence of nocodazole. A, Visualization of gold-labeling of an early endosome (center) as well as a multivesicular/late endosome (left) and a MIIC (right). B and C show a gold-labeled multivesicular/late endosome and a typical gold-labeled multilaminar MIIC, respectively. D–F, DCs incubated as in A–C but in the presence of nocodazole show gold-labeling of early endosomes (D) and of some multivesicular/late endosomes (E), while gold-labeled MIIC are only rarely observed (F). Note in F the abundance of homogenous-appearing unlabeled MIIC. These MIIC, whose content in MHC-II molecules has not been determined in this experiment, are clearly visible in G and H.G–H, Lowicryl K4 m-embedded DCs were incubated successively with 1) a rabbit anti-DR-chain Ab and 2) a gold-labeled goat anti-rabbit Ab. Homogenous-appearing MIIC are numerous in the monocyte-derived DCs (G), which contain, in some areas, typical multilaminar MIIC (H).

View larger version (60K): [in this window] [in a new window]   FIGURE 9. Effect of microtubule depolymerization on transport of internalized ßIi complexes. DCs were incubated with RHuIi at 4°C for 30 min (a and b), then washed and incubated at 37°C for 30 min without (c andd) or with (e andf) nocodazole (see Materials and Methods). Cells were processed for immunofluorescence staining as described in Materials andMethods. They were then processed for confocal laser scanning microscopy. a, c, and e represent Texas Red labeling (showing internalized RHuIi); b, d, andf represent FITC labeling (showing MHC-II molecule staining). Representative images of three independent experiments corresponding to superpositions of four medial optical slices are shown (bars: 10 µm).

View larger version (48K): [in this window] [in a new window]   FIGURE 10. Depolymerization of microtubules do not affect the kinetics of conversion of surface ßIi into SDS-stable and unstable ß dimers. DCs were pulse labeled (10 min), chased for 30 min, and surface biotinylated. Cell surface molecules were then allowed to internalize by further incubation with or without nocodazole (+ or -NOCO) at 20°C for 30 min. MHC-II molecules behavior was then observed as described in Figure 4, DCs being incubated at 37°C for the indicated time with half of the cells treated with nocodazole. A, The samples were boiled before run on the SDS-PAGE. Total and biotinylated fractions are shown (Total and Biot.). B, The samples were not boiled to test the SDS stability of ß dimers. Only the biotinylated material is shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The kinetics of delivery of ßIi complexes on the plasma membrane of DCs were very similar to those of MHC class I molecules. This suggests a direct transport from the Golgi complex to the plasma membrane. We cannot exclude an arrival at the plasma membrane after targeting from the TGN to early endosomes, as recently shown for newly synthesized transferrin receptor (58). The ßIi complexes reaching early endosomes could either enter a recycling pathway to the cell surface or be transported to late endocytic compartments. However, Ii has been shown to be very sensitive to proteases (59, 60). The fact that cell surface Ii is intact, as well as the rapid kinetics of transport from the TGN to the plasma membrane of mature sialylated Ii forms, would imply that newly synthesized ßIi complexes reside for a very short time in endosomes before cell surface appearance in DCs.

Interestingly, we demonstrate that within 30 min after the internalization, most of the cell surface ßIi complexes (74%) are already converted into ß dimers, part of them being stable in SDS. However, we observed that, although the conversion of most cell surface ßIi complexes into ß dimers was relatively rapid, the deposit of these dimers at the plasma membrane of DCs was progressive, reaching its completion only 2 h after ßIi internalization. These results are consistent with a rapid sorting and active targeting of cell surface ßIi complexes to processing compartments such as MIIC. In these compartments, the rapid dissociation of Ii from internalized ßIi complexes will give rise to ß dimers. Then, the progressive return of newly formed ß dimers to the plasma membrane would suggest that egress from MIIC is a rate-limiting step in the delivery of MHC-II molecules at the cell surface. Alternatively, endocytosed ßIi complexes could also be converted into ß dimers in different endocytic compartments (11), including early endosomes (12). The fact that nocodazole depolymerization of microtubules blocks the access of ßIi complexes to ß dimer-rich compartments without affecting the conversion of ßIi into ß-peptides complexes favors this latter hypothesis. Although recent studies performed on another type of MHC-II-expressing cell do not argue in favor of the transport of ß dimers from different endocytic compartments to the plasma membrane (61), it was shown that peptide loading by itself can take place in endocytic compartments upstream from the MIIC (12), independently of polymerized microtubules (62). A process in which a large proportion of newly formed ß dimers originating from cell surface-internalized ßIi complexes can bind peptides in endocytic compartments with different proteolytic and physiologic properties would extend the range of peptides presented to CD4⁺ T cells by these APC. Consequently, this would reinforce the suspected "sentinel" role of DCs throughout the body by helping these cells to identify and activate trace amounts of Ag-reactive T cell clones. This latter hypothesis does not exclude a predominant accumulation of most endocytic MHC-II molecules in the MIIC of DCs at the steady state, nor does it preclude the possibility that ß dimers continuously recycling between endocytic compartments and the plasma membrane of these cells can bind newly encountered peptides (30).

It was already known that newly synthesized ßIi complexes can reach the plasma membrane before rapid internalization and targeting to the endosomal compartments. Support for this hypothesis was obtained in studies showing that endosomal targeting signals present in the cytoplasmic domains of all Ii isoforms also function as internalization signals (63, 64). In B lymphocytes, this results in the rapid endocytosis of class II ßIi from the cell surface (57). The fraction of the ßIi complexes reaching the cell surface does not contain p35, revealing that isoforms of the Ii chain may modulate the different transport pathways taken by newly synthesized ßIi complexes en route to processing compartments (47). However, the relative importance of these different pathways in B cells remains to be accurately quantified.

Remarkably, we found a differential level of expression for the three main isoforms of Ii in the three different MHC-II-expressing cell types. We show that, like murine Langerhans cells (65, 66), human immature DCs derived in vitro from blood monocytes naturally express higher levels of the alternatively spliced p41 isoform of Ii. An attractive hypothesis would be that the exon 6b, which codes for additional luminal amino acid residues exclusively present in p41, accounts for the enhancement of the transport of MHC-II molecules between Golgi and the plasma membrane in DCs. In this respect, p41 has been found to act as a better potentiator of Ag presentation than p31 (67). It has been suggested that p41 could do so by modifying the transport and/or processing of MHC-II molecules (68). However, such a hypothesis is not consistent with the generally accepted models of intracellular targeting of membrane proteins in which sorting or retention signals are found in cytoplasmic tails. Moreover, if the presence of p41 in the nonameric complexes leaving the TGN (45) was responsible for delivery at the cell surface, nonamers reaching the plasma membrane should contain at least one p41. From our estimation this should lead to a twofold enrichment of p41 in the surface fraction of ßIi complexes, which did not occur (not shown). Finally, we cannot formally exclude that the direct trafficking of newly synthesized ßIi complexes to the cell surface of immature DCs is due to their high level of synthesis saturating the specific machinery that targets them to MIIC. However, overexpression of these complexes in HeLa cells (37), corresponding to about 10 times the rate of synthesis in DCs, does not lead to an equivalent amount of ßIi transiently expressed at the plasma membrane. A better knowledge about the cytosolic machinery that interacts with the Ii cytoplasmic tails and that mediates the intracellular targeting of MHC-II molecules will clarify the respective roles of the different Ii isoforms and/or the differences in the intracellular trafficking of MHC II molecules in different cell types.

The assay described here, which allowed us to follow and quantify the conversion of cell surface ßIi into functional ß-peptide complexes and their arrival to the plasma membrane, designates the human monocyte-derived DCs as the appropriate material to study the mechanisms underlying the transport of newly synthesized MHC-II molecules via the plasma membrane and its role in Ag presentation.

	Acknowledgments

 
We thank Huguette Bausinger, Dominique Fricker, Jean-Claude Garaud, Philippe Ohlmann, and Fabienne Proamer for expert technical assistance and Gerard Vetter, Dominique Morineau, and Daniel Meur for photographic works. We are grateful to Gordon Langsley for reviewing the English and Catherine Dargemont for critical reading of the manuscript. We also thank the Schering-Plough company for kindly providing hrIL-4 and hrGM-CSF.

	Footnotes

 
¹ This work was supported by Grant CJF 94-03 from INSERM, the Etablissement de Transfusion Sanguine de Strasbourg, the Agence Française du Sang (Paris) (F0RTS 96), the Fondation pour la Recherche Médicale, Grant No. 1405 from the Association de la Recherche contre le Cancer, Grant No. ERB CHR XCT 940592 from the European Community, and a grant from La Ligue Nationale contre le Cancer.

² Address correspondence and reprint requests to Dr. Jean Salamero, UMR CNRS 144, Institut Curie, 26 rue d’Ulm, 75248 Paris Cedex 05, France. E-mail address:

³ Abbreviations used in this paper: MHC-II, MHC class II; BEBV, EBV-transformed B cells; DCs, dendritic cells; Ii, invariant chain; MIIC, MHC-II compartments; TGN, trans-Golgi network; ßIi, Ii MHC-II complexes; GM, granulocyte-macrophage; hr, human recombinant; MESNA, mercaptoethanesulfonic acid.

Received for publication July 10, 1997. Accepted for publication November 17, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Cresswell, P.. 1994. Assembly, transport, and function of MHC class II molecules. Annu. Rev. Immunol. 12:259.[Medline]
2. Riberdy, J. M., R. R. Avva, H. J. Geuze, P. Cresswell. 1994. Transport and intracellular distribution of MHC class II molecules and associated invariant chain in normal and antigen-processing mutant cell lines. J. Cell Biol. 125:1225.[Abstract/Free Full Text]
3. Peters, P. J., J. J. Neefjes, V. Oorschot, H. L. Ploegh, H. J. Geuze. 1991. Segregation of MHC class II molecules from MHC class I molecules in the Golgi complex for transport to lysosomal compartments. Nature 349:669.[Medline]
4. Kleijmeer, M. J., V. M. Oorschot, H. J. Geuze. 1994. Human resident Langerhans cells display a lysosomal compartment enriched in MHC class II. J. Invest. Dermatol. 103:516.[Medline]
5. Mommaas, A. M., A. A. Mulder, C. J. Out, G. Girolomoni, H. K. Koerten, B. J. Vermeer, F. Koning. 1995. Distribution of HLA class II molecules in epidermal Langerhans cells in situ. Eur. J. Immunol. 25:520.[Medline]
6. Kleijmeer, M. J., M. A. Ossevoort, C. J. van Veen, J. J. van Hellemond, J. J. Neefjes, W. M. Kast, C. J. Melief, H. J. Geuze. 1995. MHC class II compartments and the kinetics of antigen presentation in activated mouse spleen dendritic cells. J. Immunol. 154:5715.[Abstract]
7. Neefjes, J. J., V. Stollorz, P. J. Peters, H. J. Geuze, H. L. Ploegh. 1990. The biosynthetic pathway of MHC class II but not class I molecules intersects the endocytic route. Cell 61:171.[Medline]
8. Denzin, L. K., P. Cresswell. 1995. HLA-DM induces CLIP dissociation from MHC class II alpha beta dimers and facilitates peptide loading. Cell 82:155.[Medline]
9. Sherman, M. A., D. A. Weber, P. E. Jensen. 1995. DM enhances peptide binding to class II MHC by release of invariant chain-derived peptide. Immunity 3:197.[Medline]
10. Glickman, J. N., P. A. Morton, J. W. Slot, S. Kornfeld, H. J. Geuze. 1996. The biogenesis of the MHC class II compartment in human I-cell disease B lymphoblasts. J. Cell Biol. 132:769.[Abstract/Free Full Text]
11. Amigorena, S., J. R. Drake, P. Webster, I. Mellman. 1994. Transient accumulation of new class II MHC molecules in a novel endocytic compartment in B lymphocytes. Nature 369:113.[Medline]
12. Castellino, F., R. N. Germain. 1995. Extensive trafficking of MHC class II-invariant chain complexes in the endocytic pathway and appearance of peptide-loaded class II in multiple compartments. Immunity 2:73.[Medline]
13. Schuler, G., R. M. Steinman. 1985. Murine epidermal Langerhans cells mature into potent immunostimulatory dendritic cells in vitro. J. Exp. Med. 161:526.[Abstract/Free Full Text]
14. Witmer-Pack, M. D., J. Valinsky, W. Olivier, R. M. Steinman. 1988. Quantitation of surface antigens on cultured murine epidermal Langerhans cells: rapid and selective increase in the level of surface MHC products. J. Invest. Dermatol. 90:387.[Medline]
15. Jiang, W., W. J. Swiggard, C. Heufler, M. Peng, A. Mirza, R. M. Steinman, M. C. Nussenzweig. 1995. The receptor DEC-205 expressed by dendritic cells and thymic epithelial cells is involved in antigen processing. Nature 375:151.[Medline]
16. Sallusto, F., M. Cella, C. Danieli, A. Lanzavecchia. 1995. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J. Exp. Med. 182:389.[Abstract/Free Full Text]
17. Guery, J. C., F. Ria, L. Adorini. 1996. Dendritic cells but not B cells present antigenic complexes to class II-restricted T cells after administration of protein in adjuvant. J. Exp. Med. 183:751.[Abstract/Free Full Text]
18. Inaba, K., J. P. Metlay, M. T. Crowley, R. M. Steinman. 1990. Dendritic cells pulsed with protein antigens in vitro can prime antigen-specific, MHC-restricted T cells in situ. J. Exp. Med. 172:631.[Abstract/Free Full Text]
19. Sallusto, F., A. Lanzavecchia. 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J. Exp. Med. 179:1109.[Abstract/Free Full Text]
20. Steinman, R. M.. 1991. The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9:271.[Medline]
21. Austyn, J. M.. 1996. New insights into the mobilization and phagocytic activity of dendritic cells. J. Exp. Med. 183:1287.[Free Full Text]
22. Thirdborough, S. M., N. J. London, R. F. James, P. R. Bell. 1993. A method for the isolation of human lymphoid dendritic cells from the spleen.