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International Immunology, Vol. 11, No. 1, 99-111, January 1999
© 1999 Japanese Society for Immunology

Cellular distribution of a mixed MHC class II heterodimer between DR{alpha} and a chimeric DOß chain

Angela Samaan, Jacques Thibodeau2, Wahib Mahana, Flora Castellino1, Pierre A. Cazenave2 and Thomas J. Kindt

1 Laboratories of Immunogenetics and Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA
2 Laboratory of Analytical Immunochemistry, Institut Pasteur, 25 Rue du Dr Roux, 75724 Paris Cedex 15, France

Correspondence to: J. Thibodeau, Laboratory of Molecular Immunology, Department of Microbiology and Immunology, University of Montreal, CP 6128, Succ. Centre-Ville, Montreal, Quebec H3C 3J7, Canada


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Human MHC class II antigens include HLA-DR, -DQ, and -DP molecules that present antigens to CD4+ T cells, as well as the non-classical molecules HLA-DM and -DO. HLA-DM promotes peptide binding to class II molecules in endocytic compartments and HLA-DO, which is physically associated with HLA-DM in B lymphocytes, regulates HLA-DM function. Antibodies specific for the DOß chain were obtained by immunization of mice with a heterodimer consisting of a chimeric DOß chain (DR/DOß), containing 18 N-terminal residues of DRß, paired with the DR{alpha} chain and isolated from transfected murine fibroblasts. The specificity of this serum for the DOß chain and the lysosomal expression of the HLA-DO protein was confirmed using mutant human B cell lines lacking DR or DO molecules. The lysosomal localization of HLA-DO in human B cells contrasts with the cell surface expression of the mixed pair in transfected murine fibroblasts and raises questions concerning the role of the putative targeting motifs in HLA-DO. Transfection of the chimeric DR/DOß chain along with DR{alpha} into human epithelial HeLa cells resulted in high levels of expression of the mixed isotypic pair at the surface of transfectants as well as in lysosomes. The same pattern was observed in HeLa cells transfected with the DOß chimera and a DR{alpha} chain lacking the cytoplasmic tail. Taken together, these results suggest that functional sorting motifs exist in the DOß chain but that the tight compartmentalization of HLA-DO observed inside B lymphocytes is controlled by the HLA-DO{alpha} chain and HLA-DM.

Keywords: class II, DR{alpha}, DOß, MHC


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
MHC class II molecules are heterodimeric cell surface glycoproteins that bind peptides derived from exogenous antigens and present them to CD4+ T cells (1). In the endoplasmic reticulum (ER), newly synthesized class II {alpha} and ß chains form a complex with the invariant chain (Ii), which is then transported to the trans-Golgi network and subsequently sorted to endosomal/lysosomal compartments (2). Efficient peptide loading of class II molecules requires additional class II-like molecules, such as HLA-DM, that catalyze the dissociation of class II-associated invariant chain peptide (CLIP) from class II {alpha}ß–CLIP complexes (3–5). DM is a resident of the endosomal system and in particular of the MHC class II compartment (MIIC) (6–8), a lysosomal structure where class II molecules acquire antigenic peptides (9,10).

The classical class II proteins are encoded by genes in the MHC located on human chromosome 6; this region also encompasses genes encoding additional proteins that are involved in antigen-processing function such as TAP1, TAP2, LMP2, LMP7 and HLA-DM (2). There is an additional pair of polypeptide chains designated HLA-DO with a structure similar to classical class II molecules and encoded in this class II region of the MHC. In contrast to classical class II genes and reminiscent of HLA-DM, HLA-DO genes exhibit limited polymorphism (11–13). Very recently, it was shown that HLA-DO is retained in the ER and exits only in the presence of HLA-DM (14). Moreover, this stable interaction allows HLA-DO to modulate the activity of HLA-DM in a pH-dependent manner (15–18). In melanoma cells, it was reported that HLA-DO is expressed only in early compartments of the antigen-processing pathway while lysosomal localization was observed in other cell types (14,19). Although the mouse H-2O molecule was detected at the surface of B lymphocytes as well as in intracellular compartments (12), surface expression of HLA-DO has not been reported (14,19).

The present report describes the generation of a polyclonal mouse antibody against DOß. Using this antiserum, we have analyzed the intracellular localization of HLA-DO in human B cells, and the expression in transfected cells of a mixed pair formed between the classical DR{alpha} chain and a chimeric DOß chain. Our results show that the cytoplasmic sequence of DOß contains targeting signals but is not sufficient for exclusive sorting of the mixed pair to the lysosomes of fibroblasts and epithelial cells.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Cells and antibodies
The mutant cell lines 721.82 (.82) and 721.61 (.61), derived from the Epstein–Barr virus-transformed HLA-hemizygous B cell line 721.45, have been described (20). Mutant .82 has a large homozygous deletion in MHC class II which includes the DR region but not the genes for antigen processing or the DOB gene. Mutant .61 carries a homozygous deletion in the MHC region which includes antigen-processing genes and DOB (21). These cells were cultured in modified ISCOVE'S (Biofluid, Rockville, MD), with 25 mM HEPES, 10% FCS, 2 mM glutamine and 10 mg/ml gentamycin.

L243 is an anti-HLA-DR mAb (ATCC, Rockville, MD). For use in affinity purification, L243 was purified from ascites and coupled at 5 mg/ml to CNBr-activated Sepharose 4B (Pharmacia, Piscataway, NY). Anti-LAMP-1 (CD107a) is a mAb (IgG1,{kappa}) which reacts with the heavily glycosylated 110 kDa lysosomal-associated membrane protein (PharMingen, San Diego, CA). XD5.117 is an anti-DRß (IgG1) mAb (22). DA6.147 is an anti-DR{alpha} (IgG1) mAb (23).

DR/DOß chimeric construction and transfectants
cDNAs encoding HLA-DOß and -DRß have been described (11). A DR/DO chimera containing the signal peptide and the first 18 amino acids of DR1ß, fused to a fragment encoding DOß beginning at amino acid 19, was made by the overlap extension technique (24). First, a fragment was generated by PCR from DRß cDNA in vector RSV.3 (25) using a pair of oligonucleotides, one matching the RSV.LTR at the 5' end of the cDNA and the other sequence centered on codon 18 (oligonucleotide DOß 18B: 5'-TGT CCC GTT GAA GAA ATG ACA TTC AAA-3') as described (26). A complementary oligonucleotide (DOß 18C: 5'-TCT GAA GAG GCT GTC GAC TTT ACT GTC-3') was used to generate a second fragment from DOB cDNA in RSV.3 ending in the 3' non-coding region. The two overlapping fragments were used to generate the chimeric DR/DO cDNA by PCR. The PCR product of the overlap extension was cut with SacI and cloned into the SacI site of the RSV.3 vector where the SacI–HindIII fragment of DRB had been previously replaced by the 3' SacI–HindIII fragment of DOB. The final construct was verified by sequencing. The RSV.3 DR/DOB construct was transfected by the calcium phosphate technique (27) into DAP 2.3 Dd cells along with the DRA cDNA cloned in the RSV.5 vector containing a hygromycin-resistance gene (28). Stable transfectants were selected in 25 µg/ml hygromycin. Stable transfectant cell lines: DAP 2.3 (DR{alpha}) and DAP 2.3 (DR{alpha} + DRß) have been described (25,29). The truncated DR{alpha} cDNA encodes a stop codon at position 217 and was cloned into RSV.5 neo (30). HeLa cells were transfected with DR{alpha} wild-type or DR{alpha}{Delta}CYT and the chimeric DR/DOß chain, selected in G418 and sorted using L 243. HeLa cells and the DR{alpha}{Delta}CYT cDNA were a gift from Dr R. P. Sékaly.

Protein purification
DOß was purified from 8x109 DAP2.3 transfectants expressing DR{alpha} + DR/DOß. Cells were lysed for 1 h on ice at 2x107 cells/ml in lysis buffer consisting of 1% NP-40 in Tris–saline (10 mM Tris–HCl pH 7.5/150 mM NaCl) containing 5 mM iodoacetamide, 0.5 mM PMSF and 0.1 mM TLCK. Nuclei were removed by centrifugation for 1 h at 10,000 r.p.m. at 4°C. The lysate was passed over serially connected columns containing the following beads: Sepharose 4B (Pharmacia, Piscataway, NY), mouse IgG–agarose (Sigma, St Louis, MO) and, finally, L243-conjugated Sepharose 4B. The columns were disconnected and the L243 column was washed overnight with 0.5% NP-40 in Tris–saline, followed by another wash with several columns volumes of 0.5% NP-40 in Tris–saline supplemented with 300 mM NaCl and a final wash with 1% N-octylglucoside (OG) in Tris–saline. Bound proteins were eluted with 1% OG, 0.02% NaN3, 5% glycerol and 50 mM diethylamine, pH 11.2. Fractions were immediately neutralized by adding 80 µl 2M Tris buffer, pH 7.4. Fractions containing the chimeric DR/DOß were identified by Coomassie blue staining of SDS–PAGE gels, pooled and concentrated (67 µg/ml) with a Centricon 10 (Amicon, Beverly, MA).

Generation of anti-DOß antibodies and flow cytometric analysis
BALB/c mice were immunized s.c. and in multiple sites with affinity-purified DR{alpha}-DR/DO chimeric protein (10 µg/100 µl) mixed 1:1 with complete Freund's adjuvant for the first injection. One month later, and for all subsequent immunizations, mice received 10 µg of the chimeric protein mixed 1:1 with incomplete Freund's adjuvant. One week after each injection, blood was collected and sera screened for reactivity with DAP cells transfected with the chimeric DR/DO chain by flow cytometry analysis on a FACScan (Becton Dickinson, San Jose, CA). Bleedings were taken at regular intervals and sera with the best response to DOß were pooled and used for further experiments.

For flow cytometry analysis, 106 cells were incubated with pooled anti-DOß sera at different dilutions for 30 min at 4°C. Viable cells are shown after gating on forward and side scatter. For intracellular staining, 106 cells were washed in PBS and fixed in 4% paraformaldehyde for 20 min at room temperature. Cells were washed twice in PBS and incubated in NH4Cl (50 mM in PBS) for 15 min. Cells were washed and permeabilized for 15 min at room temperature in 0.05% saponin/1% BSA in PBS. mAb DA6.147 coupled to FITC was added to the cell suspension for 20 min at room temperature. After washing, the samples were analyzed by flow cytometry on a FACScan (Becton Dickinson).

Metabolic labeling and immunoprecipitations
Transfected DAP 2.3 fibroblasts (2x107) were radiolabeled with 0.5 mCi [35S]methionine translabel (ICN, Costa Mesa, CA) in methionine-free EMEM supplemented with 3% dialysed FCS for 8 h at 37°C. B cells were labeled in methionine-free RPMI medium for 4 h at 37°C. Metabolically labeled B cells were lysed and the lysates were loaded over a 1 ml column of lentil lectin–Sepharose 4B (Pharmacia, Piscataway, NY). After extensive washing with loading buffer, glycosylated material was eluted with the same buffer containing 0.1 M {alpha}-methyl mannoside. This material, as well as lysates of the DAP 2.3 transfected cells was precleared overnight with normal mouse serum and Protein G–Sepharose (Pharmacia) at 4°C. The lysates were then immunoprecipitated with the anti-DOß mouse polyclonal antibody or with L243 and Protein G–Sepharose. Sepharose pellets were washed and immunoprecipitated material was analyzed either by conventional 10% SDS–PAGE or two-dimensional gels. Two-dimensional gel analysis with non-equilibrium pH gradient gel electrophoresis (NEPHGE) in the first dimension and SDS–PAGE in the second dimension was performed as previously described (31,32).

Electrophoresis and immunoblotting
Samples were electrophoresed on 10% SDS–PAGE and electroblotted onto Immobilon PVDF membranes (Millipore, Bedford, MA) using a Novex transfer cell (96 mM glycine, 12 mM Tris and 10% methanol). Membranes were processed for fluorescent detection using the Vistra ECF Western blotting kit (Amersham, Piscataway, NJ). Non-specific sites were blocked (membrane blocking reagent; Amersham) in TBS/Tween buffer (25 mM Tris–HCl, pH 7.8/190 mM NaCl/0.15% Tween 20). Membranes were incubated with either anti-DOß serum or anti-DRß mAb, followed by fluorescein-linked anti-mouse Ig. Fluorescence (linear range) was detected with a Fluorimager 595 (Molecular Dynamics, Sunnyvale, CA).

Subcellular fractionation
Normal or mutant B cell lines (4x109) were disrupted by nitrogen cavitation in 10 ml of homogenizing buffer (0.25 M sucrose/1 mM EDTA, pH 6.8). After low-speed centrifugation, the postnuclear supernatant was layered onto a 27% Percoll/sucrose gradient as previously described (33,34), and spun for 1 h at 34,500 g at 4°C in a Vti50 vertical rotor (Beckman Instruments, Palo Alto, CA). Fractions were collected from the bottom of the gradient using a Beckman fraction recovery system and those containing ß-hexosaminidase activity (peak 1 high density) were pooled.

Cell surface and lysosomal preparation radiolabeling
Approximately 2x107 .45, .82 or .61 cells were surface labeled with 1.0 mCi of Na125I (1 Ci = 37 Gbq) in cold HBSS by lactoperoxidase-catalyzed iodination (35). The iodinated cells were >98% viable as determined by Trypan blue exclusion. Lysosomal fractions from each cell type were pooled and the lysosomal membranes were separated after centrifugation at 18,000 g at 4°C by using a TL-100 Beckman ultramicrofuge. The lysosomal membranes from each sample were labeled with 125I following the same procedure as for the surface iodination (35). Iodinated lysosomal fractions were lysed in 1% NP-40 and immunoprecipitated with antibodies against lamp-1 (36) or anti-DOß antibody.

Internalization assay
Cells were incubated for 30 min at 4°C with saturating concentrations of the L243 Fab fragment coupled to biotin using the sulfo-NHS-LC-biotin as described by the manufacturer (Pierce, Rockford IL). After washing at 4°C, cells were transferred to 37°C. Aliquots were removed at regular time intervals and transferred to a 10-fold excess cold PBS. Cells were spun and stained using phycoerythrin (PE)-coupled streptavidin before flow cytometry analysis.

Fluorescence microscopy
Cells were plated on cover slips and cultivated for 3 days before intracellular staining. The cover slips were rinsed in PBS and the cells were fixed in 4% formaldehyde for 20 min at 20°C. After two washes in PBS, the cover slips were immersed in 50 mM NH4Cl for 15 min and washed in PBS. Cells were permeabilized for 15 min in 0.05% saponin in PBS containing 1% BSA. Intracellular staining was performed by adding biotinylated L243- and Lamp-1-specific antibodies. After 20 min at 20°C, cells were washed twice in saponin buffer and incubated for 20 min with Texas red-coupled streptavidin (1/800) (Harlan) and anti-mouse IgG1 (1/100) coupled to fluorescein (Southern Biotechnology, Birmingham, AL) (37). The cells were washed twice and fixed in 1% formaldehyde before the cover slips were mounted for analysis by fluorescence microscopy.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Generation and specificity of anti-DO antibody
In the absence of a specific antibody for HLA-DO, we designed a mixed class II molecule that would allow the expression and purification of the DOß chain. First, a chimeric class II ß chain (DR/DOß) was constructed by replacing the sequence encoding the extracellular amino acids 1–18 of HLA-DOß with the corresponding 1–18 sequence of DRß. This was done to maximize the pairing with the DR{alpha} chain in the membrane distal domains (29,38). Then, the DR/DOß construct was expressed in murine fibroblasts following co-transfection with the HLA-DR{alpha} cDNA. The cell surface expression of this mixed class II molecule was detected using the anti-DR mAb L243 which is directed against an epitope in the DR{alpha} chain that is dependent upon {alpha}ß pairing (39). The mixed heterodimer (DR{alpha} + DR/DOß) was purified from the transfected fibroblasts by affinity chromatography on an L243 affinity column and analyzed by SDS–PAGE (Fig. 1AGo).



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  		Fig. 1. Specificity of polyclonal anti-DOß antiserum. (A) Affinity purified DR{alpha} + DR/DOß was analyzed on a 10% SDS–PAGE gel under non-reducing conditions and stained with Coomassie blue. (B) DAP 2.3 transfectants were metabolically labeled for 8 h with [35S]methionine and cysteine. Cell lysates were subjected to immunoprecipitation analysis using normal mouse serum (NMS), monoclonal L243 or the new antiserum. Lanes indicated by DO represent immunoprecipitates formed from DAP 2.3 fibroblasts expressing DR{alpha} and DR/DOß chains, while lanes marked DR show immunoprecipitates formed from fibroblasts expressing the DR{alpha} and DRß chains. The mAb L243 shows the position of DR{alpha}/ß migration in the appropriate transfectants and is a reference for comparison with immunoprecipitates formed with the new serum. The two lanes at the right show immunoprecipitates formed from DAP 2.3 expressing DR{alpha} only (–) using either the monoclonal L243 or the new antiserum. (C) Lysates from the transfectants described in B were immunoprecipitated with the anti-DR{alpha} mAb (DA6.147) and immunoblotted either with new serum or with anti-DRß (XD5.117) respectively. The new serum reacts with a 30 kDa protein precipitated from DR{alpha} + DR/DOß transfectants and does not react with proteins expressed in DR{alpha} + DRß transfectants (left panel). The anti-DRß mAb recognized only the DRß chain immunoprecipitated from the DR{alpha} + DRß transfectants (right panel).

 
An antiserum was raised by injecting mice with the column eluate and tested by immunoprecipitation analysis using transfected fibroblasts expressing either DR{alpha} + DRß or DR{alpha} + DR/DOß. Cell lysates were precipitated with either normal mouse serum, mAb L243, or with the new polyclonal antiserum after metabolic labeling with [35S]methionine and cysteine. Samples from each immunoprecipitation were separated under non-reducing conditions by SDS–PAGE. The new antiserum precipitated proteins of 35 (DR{alpha}) and 30 (DR/DOß) kDa from the DR{alpha} + DR/DOß transfectant, and proteins of 35 (DR{alpha}) and 28 (DRß) kDa from the DR{alpha} + DRß transfectant. Immunoprecipitation of DR{alpha} + DRß dimers with the new serum was expected since mice were immunized with both the chimeric DOß and the DR{alpha} chains. The mol. wt of the ß chain from the DR/DO immunoprecipitates formed with the new antiserum corresponds to the predicted mol. wt of the chimeric molecule. To further ascertain the specificity of this antiserum, we analyzed immunoprecipitates formed using extracts prepared from transfectants expressing DR{alpha} alone. The failure to precipitate a 35 kDa {alpha} chain (Fig. 1BGo) indicates that this antiserum does not recognize epitopes on unpaired DR{alpha} chains. As predicted, L243 does not precipitate DR{alpha} in the absence of a ß chain. Normal mouse serum did not precipitate any of these proteins.

The specificity of this antiserum was confirmed by western blot analysis using the transfectants described above. After immunoprecipitation with anti-DR{alpha} mAb (DA6.147), the samples were blotted with the new antiserum or with anti-DRß mAb (XD5.117). As seen in Fig. 1(C)Go, the new antiserum specifically recognizes the 30 kDa DR/DOß chimeric chain immunoprecipitated from (DR{alpha} + DR/DOß) transfectants. Similarly, the anti-DRß mAb (XD5.117) only recognized the DRß chain immunoprecipitated from the (DR{alpha} + DRß) transfected cells. This result demonstrates that the new antiserum specifically recognizes DOß epitopes and that it is not cross-reactive with DRß chains. Henceforth, we will refer to this serum as anti-DOß.

Expression of endogenous HLA-DOß in normal and mutant human B cell lines
The expression of DOß was evaluated in mutant cell lines .82 and .61, which were derived from the B cell line .45 by immunoselection (20) and which lack different segments of the MHC region. Mutant .82 lacks the entire DR region and mutant .61 lacks the genomic region encoding antigen-processing genes as well as DOB. These three cell lines were metabolically labeled, lysed and fractionated on a lentil lectin column. After elution, the bound fraction was analyzed by comparing immunoprecipitates formed with anti-DOß or with mAb L243 by two-dimensional gel electrophoresis (Fig. 2Go). The DOß chain from the .82 mutant migrated as a group of three basic spots indicated by arrowheads in Fig. 2(C)Go. This assignment is based upon mol. wt, pI and the fact that DO proteins are not present in the .61 cell line (Fig. 2EGo). Although the strong signal obtained from the DRß chains partially obscures the pattern of DOß spots, .45 cells have essentially the same DOß pattern (Fig. 2AGo) as .82 cells (Fig. 2CGo). In .45 cells the most prominent DOß spot is indicated by an arrowhead. The presence of DR{alpha} and DRß spots in the samples immunoprecipitated with anti-DOß is due to the precipitation of paired {alpha} and ß chains caused by the reactivity against the DR{alpha} chain which was present during immunization (see above). The protein tentatively identified as DO{alpha} chain (based on its migration and its presence in .45 and .82, and absence in .61) migrated as a group of more acidic spots (indicated by two arrows) (Fig. 2CGo). Consistent with this assignment, these spots were not detected in .61 mutant cells (Fig. 2EGo). When the same samples were immunoprecipitated with mAb L243, neither DO{alpha} nor ß spots were observed in the .45, .61 and .82 cells. These results show that in human B cells, anti-DOß precipitates a complex of molecules consistent with the mol. wt and isoelectric points of DO{alpha} and DOß.



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  		Fig. 2. Expression of endogenous HLA-DOß in normal and mutant B cell lines. Human 721 .45 B cells and mutant derivatives .82 and .61 were metabolically labeled ([35S]methionine + cysteine) for 4 h and cell lysates passed over a lentil lectin column. The eluate was immunoprecipitated with anti-DOß (A, C and E) or with mAb L243 (B, D and F). Two-dimensional gel analysis was performed by NEPHGE from right to left (acidic to basic) and the second dimension by SDS–PAGE under reducing conditions. Arrowheads indicate the position of DOß spots visible in .45 and .82 cells, but not in .61 cells. Open arrowheads indicate the anticipated position in cells that do not express DOß. In the absence of DR molecules (mutant .82), three DOß and two DO{alpha} arrows spots are visible. These spots were also visible on the original autoradiograph of .45 cells. The open arrowheads indicate the absence of DOß in the anti- DR{alpha} (L243) precipitated samples for .45, .82 and .61 cells.

 
Localization of HLA-DO in human B cell lines
Flow cytometry analysis of the .45 and .82 B cell lines with the anti-DOß antiserum failed to detect surface expression of HLA-DO (data not shown). To improve the sensitivity of detection, surface proteins of parent and mutant cell lines were radiolabeled with 125I using lactoperoxidase. DO molecules could not be detected in the lysates of the iodinated .45 and .82 cell lines (data not shown).

In order to determine the localization of HLA-DO, subcellular fractionation was carried out. Equal numbers of .45, .82 and .61 B cells were disrupted by nitrogen cavitation and the distinct intracellular compartments were separated on the basis of their differential density with a 27% Percoll/sucrose gradient, essentially as described previously (33,34). The distribution of the ß-hexosaminidase enzyme defines the presence of two distinct peaks (Fig. 3Go): a high-density peak [1] containing lysosomes and mitochondria, and a low-density peak [2] containing ER, Golgi, early endosomes, late endosomes and plasma membranes (34). The identity of peak 1 as the lysosomal fraction was confirmed by the presence of Lamp-1 following immunoprecipitation (data not shown). The individual fractions comprising peak 1 were pooled, labeled with 125I and lysed with NP-40. Proteins immunoprecipitated from peak 1 with anti-DOß were analyzed by two-dimensional gel electrophoresis (Fig. 4Go). Specific spots, corresponding to DOß, observed in the lysosomal preparation from .45 and .82 cells, were absent in .61 cells. The arrowheads in Fig. 4Go indicate the migration of DOß spots detected by biosynthetic labeling, as in Fig. 2Go. The putative DO{alpha} spots were not visible in the iodinated lysosomal fraction, probably because the DO{alpha} chain is not labeled efficiently by this procedure as is the case for DP{alpha} (40). Again, the presence of DR{alpha} and DRß spots in the .45 and .61 immunoprecipitates is due to the reactivity against the DR{alpha} chain present during immunization. These spots are absent in .82 immunoprecipitates due to DR deletion in these cells.



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  		Fig. 3. Percoll gradient fractionation of cellular compartments from normal and mutant human B cell lines. Distribution of the endocytic marker ß-hexosaminidase in .45 .82 and .61 cells after fractionation on a 27% Percoll gradient. Fraction 1 corresponds to the bottom of the gradient.

 


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  		Fig. 4. HLA-DO resides in a lysosomal compartment. The lysosomal fractions from each cell type were pooled: fractions 2–8 of .45 cells, fractions 3–6 of .82 cells and fractions 2–7 of .61 cells. Separately, the pooled fractions were 125I radiolabeled and extracted with detergent. Immunoprecipitates were formed with anti-DOß serum and analyzed by two-dimensional gel electrophoresis. The position of DOß spots is indicated by arrowheads in normal (.45) and mutants (.82) cells.

 
Trafficking of the DR{alpha} + DR/DOß heterodimer
We have analyzed the expression of the mixed DR{alpha} + DR/DOß heterodimer in the murine fibroblast cell line DAP 2.3. Combining an {alpha} chain that is normally expressed at the cell surface with a ß chain retained in the ER may yield insights regarding the sorting signals displayed by the different molecules. Surface expression was examined by flow cytometry using DAP 2.3 transfectants expressing DR{alpha} + DR/DOß, DR{alpha} + DRß or DR{alpha} alone. Interestingly, Fig. 5Go shows that the antiserum against DOß recognized the mixed pair at the cell surface. At optimum dilution (1:100), the antiserum reacted more strongly with fibroblasts expressing the DR{alpha} + DR/DOß (mean fluorescence intensity 28.52) than with those expressing wild-type DR molecules (mean fluorescence intensity 13.64) (Fig. 5Go, right panel). At optimum dilution (1:100), the DR{alpha}-specific L243 mAb also recognized the mixed pair at the cell surface, but reacted more strongly with fibroblasts expressing the DR{alpha} + DRß molecules (mean fluorescence intensity 37) than with those expressing DR/DOß (mean fluorescence intensity 20) (Fig. 5Go, left panel). No staining was detected using fibroblasts transfected with DR{alpha} alone (Fig. 5Go, lower panel).



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  		Fig. 5. Surface expression of the DR{alpha} + DR/DOß chimeric protein. Cell suspensions of DAP 2.3 transfected with DRA-DR\DOB (top panel), with DRA-DRB (middle panel) or with DRA (bottom panel) were incubated with anti-DOß serum at dilutions of 1:10 (solid line), 1:100 (broken line) and 1:1000 (dotted line), or with monoclonal L243, at dilutions of 1:10 (solid line) and 1:100 (broken line). The shaded profiles represent reactivity with FITC-labeled goat anti-mouse Ig alone.

 
Surface expression of the DR{alpha} + DR/DOß molecule might have been predicted in murine cells since it was previously reported that surface iodination results in labeling of H2-O (12). Therefore, we tested for surface expression of the DR{alpha} + DR/DOß molecule in human epithelial HeLa cells. It was previously shown in these cells that, in the absence of HLA-DM, transfected HLA-DO is mostly found in the ER, with minute amounts detected in the lysosomes (14). Interestingly, transfection of the DR{alpha} chain together with the DR/DOß chain resulted again in surface expression (Fig. 6Go). Positive staining with the L243 antibody and with anti-DOß confirmed that the mixed pair is at the surface, and resulted in the same ratio of the mean fluorescence values as compared to DAP 2.3 cells (Fig. 5Go); at optimum dilution (1:100), L243 reacted more strongly with HeLa cells expressing wild-type DR molecules (mean fluorescence intensity 55.3) than with those expressing DR/DOß (mean fluorescence intensity 33.7), unlike the case of anti-DOß, at optimum dilution (1:100) reacted more strongly with HeLa cells expressing DR/DOß (mean fluorescence intensity 28.7) than with those expressing DR1 (mean fluorescence intensity 9.6). As expected, the chimeric ß chain does not react with the anti-DRß (XD5.117) antibody (Fig. 6Go, lower panel). These results show that the mixed pair is expressed at the surface of human HeLa cells just as on murine fibroblasts.



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  		Fig 6. The DR/DO heterodimer is expressed at the surface of human epithelial cells. HeLa cells were stably transfected with DRA-DRB (left panels) or DRA-DR/DOB (right panels) cDNAs. Cells that were positive for staining with L243 were sorted and their cell-surface expression profile was compared to HeLa cells expressing DR1 (HeLa DR1) using the DRA-specific L243 antibody (upper), anti-DOß antibody (middle) or the DRß-specific XD5.117 antibody (lower).

 
We then compared endocytosis of the wild-type DR1 or DR{alpha} + DR/DOß heterodimers (Fig. 7Go). Surface MHC molecules were tagged using a biotinylated L243 Fab fragment and allowed to internalize at 37°C before staining with PE-coupled streptavidin. Results show that the mixed pair behaves very much like the wild-type DR1 molecules in these cells: surface molecules are slowly internalized and a plateau is attained after 60 min, corresponding to the steady-state equilibrium where molecules recycle back to the cell surface (41).



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  		Fig. 7. DOß-containing molecules behave the same as wild-type DR1 at the surface of HeLa cells. HeLa cells expressing class II molecules were incubated with biotinylated Fab fragment of L243, washed and transferred to 37°C. Aliquots were removed at regular intervals and quenched on ice. Cells were incubated with PE-coupled streptavidin and analyzed by flow cytometry. The mean fluorescence value of control cells kept all the time at 4°C was considered as 100%.

 
To analyze sorting of the mixed pair in human epithelial cells, we performed fluorescence microscopy on HeLa cells labeled intracellularly with L243. Figure 8Go(A) shows that expression of the control wild-type DR1 in the absence of the Ii chain results in a diffuse intracellular labeling, corresponding to the intense reticular ER and perinuclear trans-Golgi localization described previously (42–44). On the other hand, the cells transfected with the mixed pair showed a very precise vesicular staining with the L243 antibody (Fig. 8CGo). In order to identify these compartments, we analyzed the same cells labeled using a Lamp-1-specific antibody. Figure 8(D)Go shows a pattern similar to the one obtained with L243. This co-localization of the two markers in all the cells examined confirms the sorting of the mixed pair in the lysosomes. Interestingly, these compartments have a very different morphology in cells transfected with DOß where a significant degree of `swelling' is observed, reminiscent of the situation in HeLa cells transfected with the Ii (45).



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  		Fig. 8. The mixed DR/DO molecules accumulate in lysosomal structures in HeLa cells. Cells were analyzed by fluorescence microscopy. HeLa cells expressing the mixed pair were stained with a mAb (IgG1) specific for the Lamp-1 molecule and with the biotinylated L243 (IgG2a) antibody. The specific signal was obtained by using Texas red-coupled streptavidin (A, C and E) and an IgG1-specific secondary goat anti-mouse antibody coupled to FITC (B, D and F). Arrows point to a few swollen vesicles where the co-localization occurs between class II molecules and the Lamp-1 marker.

 
The DR{alpha} cytoplasmic tail is not required for surface expression
We next addressed the possible role of the DR{alpha} cytoplasmic tail in the cell-surface expression of the mixed pair. The DR/DOß construct was co-transfected into HeLa cells with a cDNA encoding a DR{alpha} chain truncated at position 217, which removes the entire cytoplasmic domain. Positive cells (DR{alpha}{Delta}CYT + DR/DOß) were purified by flow cytometry and cell-surface expression of a heterogeneous population is illustrated in Fig. 9Go. The presence of the DOß chimera at the cell surface is demonstrated by positive staining with anti-DOß antibody. These cells were saponin-permeabilized and analyzed using an anti-DR{alpha} antibody (DA6.147) that is specific for the cytoplasmic tail of the DR{alpha} chain (46). These analyses confirm the expression in these cells of an {alpha} chain devoid of an intracellular sequence (Fig. 9Go, lower panel). By contrast, the anti-DR{alpha} (DA6.147) antibody is positive for HeLa (DR{alpha} + DR/DOß) transfectants permeabilized in the same manner (Fig. 9Go, middle panel). These results show cell-surface expression of the (DR{alpha} + DR/DOß) pair even in the absence of the DR{alpha} chain cytoplasmic sequence.



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  		Fig. 9. Surface expression of DR{alpha} + DR/DOß is independent of the DR{alpha} cytoplasmic tail. HeLa cells were stably transfected with DR{alpha}{Delta}CYT and DR/DOß. DR{alpha}{Delta}CYT encodes an {alpha} chain truncated immediately after the transmembrane domain and thus lacks the cytoplasmic tail. Cells that were positive for staining with L243 were sorted and their cell-surface expression profile was compared to HeLa DR{alpha} + DR/DOß transfectants using the anti-DOß antibody or the DA6.147 antibody which recognizes an epitope in the cytoplasmic tail of the DR{alpha} chain. Cells were saponin-permeabilized for staining with DA6.147.

 
Fluorescence microscopy on these cells revealed a pattern of expression similar to the mixed pair with the intact DR{alpha} cytoplasmic tail and confirmed the swelling of the lysosomes in the presence of the DOß chain. Taken together, these results show that the mixed pair, in contrast with wild-type HLA-DO, is not retained in the ER of human cells in the absence of HLA-DM but rather accumulates at the cell surface and in the lysosomes.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
An antibody specific for HLA-DO was obtained and used to study the cellular expression of HLA-DOß. Polyclonal antiserum was raised by immunizing mice with purified DR{alpha} + DR/DOß molecules. A chimeric DOß chain, in which the first ß strand in the membrane-distal domain was replaced by that of DRß, was used to maximize the pairing with DR{alpha}. Indeed, as revealed by the crystal structure of HLA-DR1 (47), the antiparallel juxtaposition of the first ß strand of the {alpha} and ß chains constitutes an important contact region in the interface of the class II heterodimer. The antiserum reacted strongly with DOß on Western blots, was able to recognize the dimeric structure expressed on the cell surface and did not cross-react with DRß. For unclear reasons, hybridomas secreting DOß-specific antibodies could not be isolated in several attempts involving fusion of spleen cells from mice producing the anti-DOß antibody. Using the anti-DOß serum we were unable to detect HLA-DO at the surface of human B cell lines by either flow cytometry analysis or by immunoprecipitation of radioiodinated surface proteins. The lysosomal localization of HLA-DO in B lymphocytes is in accordance with previous results (14,16).

Although the expression of mixed isotypic pairs involving chains of classical and non-classical class II molecules has not been reported, it is interesting that we detected the DR{alpha} + DR/DOß heterodimer at the surface of transfected murine fibroblasts. However, the plasma membrane expression in HeLa cells was not predicted, since a DO{alpha} and ß heterodimer was reported to reside in the ER of these cells (14). At least three possibilities might explain the lack of ER retention for the mixed pair. First, the DR{alpha} chain may mask a retention motif in DOß allowing egress through the default pathway. Alternatively, DR{alpha} may simply lack such motifs normally encoded in the DO{alpha} chain. Finally, the DR{alpha} and DR/DOß chains, unlike HLA-DO, might fold efficiently together in the absence of HLA-DM. To investigate a potential role for the DR{alpha} chain in our observations, we truncated the cytoplasmic tail of DR{alpha} and tested for surface expression of the DR{alpha}{Delta}CYT + DR/DOß heterodimer (Fig. 9Go). Cell surface expression of this truncated chimera excluded a role of the DR{alpha} cytoplasmic tail in masking an ER retention motif in DOß. Since there is no known ER-retention motif in the short cytoplasmic tail of HLA-DO{alpha} (48), we favor the last possibility, at least in B cells, where DM acts as a chaperone and assists {alpha}ß pairing to prevent degradation of HLA-DO. Indeed, recent data also obtained using HeLa transfectant cells suggest that association of DO with DM is required for efficient exit of DO from the ER (14). Consistent with this interpretation, we observed spots in two-dimensional gels (Fig. 4Go) corresponding to the reported migration of DMß (14). The putative DMß spots were detected after iodination of lysosomal compartments and immunoprecipitation with anti-DOß antibody, suggesting a stable DO–DM association. These results and other recently presented data are consistent with the interpretation that this interaction modulates DM function (15,16). The situation could be different in thymic epithelial cells where Karlsson et al. observed the accumulation of the H-2Oß chain even in the absence of H-2O{alpha} or H-2M (18).

In addition to bringing answers as to the potential mechanisms of HLA-DO retention in the ER, our experiments address the existence of sorting motifs encoded in this molecule. In human HeLa cells, the mixed pair and the wild-type DR1 seemed to behave the same after reaching the cell surface (Fig. 7Go). Nevertheless, lysosomal localization was evident only for the DOß-containing molecules in these HLA-DM-negative cells. Future experiments should clarify if the mixed pair reaches the lysosomes directly from the Golgi and if they gain access to the cell surface from these compartments before being internalized and recycled.

From our results, we make the following observations regarding sorting motifs of HLA-DO. The cytoplasmic domain of DOß is exceptionally long for a class II molecule and contains two putative signals for lysosomal sorting; a GYVRT sequence as well as a di-leucine motif (LL). The former motif is reminiscent of the classical GYXXZ motif, where X is any amino acid, Z is a hydrophobic amino acid and G is responsible for the sorting to lysosomes directly from the trans-Golgi (49). This tyrosine-based signal is most probably utilized in HeLa cells where a strong lysosomal staining was observed. However, a completely efficient targeting signal may be formed only in the presence of both the DO{alpha} and DOß cytoplasmic tails, as described for the internalization signal in HLA-DR, (41) explaining the cell-surface expression of the mixed DR{alpha} + DR/DOß pair.

The second motif, LL, in the DOß chain may be involved in the internalization, sorting and lysosomal retention as well. Although a functional leucine-based motif was also identified in the ß chain of classical class II molecules such as HLA-DR, it was proposed to function from the cell surface and it is clear that it cannot substitute for the Ii chain (50). Taken together with these results, our observations point to a critical role for the tyrosine-based motif in the peculiar behavior of the DOß-containing molecules. A thorough mutagenesis analysis and intracellular localization studies will be needed to confirm the role and the relative importance of the di-leucine- and putative tyrosine-based motifs in the lysosomal sorting in HeLa cells. The system described here allowing the pairing between different isotypes of classical and non-classical class II molecules will be very useful to answer these questions.

The presence of functional sorting motifs in HLA-DO raises some interesting questions about their physiological role and the need for DM in transporting DO to lysosomes. It remains possible that the sorting signals resulting from the DM-DO association are cumulative and different than those displayed by HLA-DM alone. Indeed, DOß contains a di-leucine motif which could affect trafficking of the complex, allowing the sorting to earlier compartments than HLA-DM. Alternatively, these motifs may be a safety retrieval mechanism preventing the `leaking' of HLA-DO to the surface of B cells upon dissociation from HLA-DM. Indeed, it was recently shown in HeLa cells using a dominant-negative mutant of clatherin that HLA-DM (and thus most probably DO) traffics from endosomal compartments to the plasma membrane and back to intracellular compartments (51). The tight control operated by HLA-DM over HLA-DO's egress from the ER and trafficking in B cells may be necessary if the HLA-DO molecule can exert detrimental functions for these cells in addition to the inhibition of HLA-DM activity. An additional function for HLA-DO was inferred from its peculiar pattern of tissue expression. Since HLA-DO is found in thymic epithelial cells independently of other class II molecules, it may need its own sorting signals to gain access to lysosomes from the Golgi or the plasma membrane (19,52). The function of HLA-DO and H-2O in the thymus, however, remains enigmatic (18,19,53).

Another interesting characteristic of DOß-expressing HeLa cells is the presence of enlarged Lamp-1-containing lysosomal compartments. (Fig. 8Go). A similar phenomenon was observed with early endosomal compartments (devoid of Lamp-1) in COS cells transfected with the Ii chain (54) and was imputed to the N-terminal cytoplasmic tail of the molecule (54,55). More recently, it was postulated that these large intracellular compartments were the result of oligomerization of the cytoplasmic tails of Ii, thus inhibiting fission or budding at the membrane level (56). This idea comes from the resolution of the three-dimensional structure of the 27 amino acids peptide corresponding to the cytoplasmic tail of Ii and which forms a triple-stranded bundle (56). Apart from the similarities in the length of the Ii and DOß cytoplasmic tails, there is no obvious homology in their primary sequences and it will be interesting to determine the three-dimensional structure of the DOß cytoplasmic tail.


   Acknowledgments
 
We are especially grateful to Eric Long for discussion and Jon Shuman for critical reading of the manuscript. We thank Roy Teller and Jean-Philippe Corre for technical assistance, and Nancy Cogan for secretarial assistance. We thank Howard Anderson for help with two-dimensional gels, Rafick P. Sékaly for transfected cells, Robert Demars for mutant B cells and Ronald Germain for use of lab facilities to prepare cellular fractions. We also thank Leila Gasmi and Anne Bieth in Professor Gérard Buttin's laboratory (Pasteur) for helping with fluorescence microscopy, Anne Louise for cell sorting, and Andreas Alcover for reagents. This work was financed in part by the FRM-Sidaction (France).


   Abbreviations
 
CLIPclass II-associated invariant chain peptide
ERendoplasmic reticulum
Iiinvariant chain
NEPHGEnon-equilibrium pH gradient gel electrophoresis
OGN-octylglucoside
PEphycoerythrin

   Notes
 
The first two authors contributed equally to this work

Transmitting editor: K. Eichmann

Received 4 June 1998, accepted 6 October 1998.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Germain, R. N. 1994. MHC-dependant antigen processing and peptide presentation: providing ligands for T lymphocyte activation. Cell 76:287.[Web of Science][Medline]
  2. Cresswell, P 1994. Assembly, transport, and function of MHC class II molecules. Annu. Rev. Immunol. 12:259.[Web of Science][Medline]
  3. Sloan, V. S., Cameron, P., Porter, G., Gammon, M., Amaya, M., Mellins, E. and Zaller, D. M. 1995. Mediation by HLA-DM of dissociation of peptides from HLA-DR. Nature 375:802.[Medline]
  4. Denzin, L. K. and Cresswell, P. 1995. HLA-DM induces CLIP dissociation from MHC class II {alpha}ß dimers and facilitates peptide loading. Cell 82:155.[Web of Science][Medline]
  5. Sherman, M. A., Weber, D. A. and Jensen, P. E. 1995. DM enhances peptide binding to class II MHC by release of invariant chain-derived peptide. Immunity 3:197.[Web of Science][Medline]
  6. Pierre, P., Denzin, L. K., Hammond, C., Drake, J. R., Amigorena, S. and Cresswell, P. 1996. HLA-DM is localized to conventional and unconventional MHC class II-containing endocytic compartments. Immunity 4:229.[Web of Science][Medline]
  7. Peters, P. J., Neefjes, J. J., Oorschot, V., Ploegh, H. L. and Geuze, H. J. 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]
  8. Sanderson, F., Kleijmeer, M. J., Kelly, A., Verwoerd, D., Tulp, A., Neefjes, J. J., Geuze, H. J. and Trowsdale, J. 1994. Accumulation of HLA-DM, a regulator of antigen presentation, in MHC class II compartments. Science 266:1566.[Abstract/Free Full Text]
  9. Tulp, A., Verwoerd, D., Dobberstein, B., Ploegh, H. L. and Pieters, J. 1994. Isolation and characterization of the intracellular MHC class II compartment. Nature 369:120.[Medline]
  10. West, M. A., Lucocq, J. and Watts, C. 1994. Antigen processing and class II MHC peptide-loading compartments in human B-lymphoblastoid cells. Nature 369:147.[Medline]
  11. Tonnelle, C., DeMars, R. and Long, E. O. 1985. DOß: a new ß chain gene in HLA-D with a distinct regulation of expression. EMBO J. 4:2839.[Web of Science][Medline]
  12. Karlsson, L., Surh, C. D., Sprent, J. and Peterson, P. A. 1991. A novel class II MHC molecule with unusual tissue distribution. Nature 351:485.[Medline]
  13. Chouchane, L., Brown, T. J. and Kindt, T. J. 1993. Structure and expression of a nonpolymorphic rabbit class II gene with homology to HLA-DOB. Immunogenetics 38:64[Web of Science][Medline]
  14. Liljedahl, M., Kuwana, T., Fung-Leung, W. P., Jackson, M., Peterson, P. A. and Karlsson, L. 1996. HLA-DO is a lysosomal resident which requires association with HLA-DM for efficient intracellular transport. EMBO J. 15:4817.[Web of Science][Medline]
  15. Denzin, L. K., Sant'Angelo, D. B., Hammond, C., Surman, M. J. and Cresswell, P. 1997. Negative regulation by HLA-DO of MHC Class II-restricted antigen processing. Science 278:106.[Abstract/Free Full Text]
  16. Van Ham, S. M., Tjin, E. P. M., Lillemeir, B. F., Gruneberg, U., Van Meijgaarden, K. E., Pastoors, L., Verwoerd, D., Tulp, A., Canas, B., Rahman, D., Ottenhoff, T. H. M., Pappin, D. J. C., Trowsdale, J. and Neefjes, J. 1997. HLA-DO is a negative regulator of HLA-DM-mediated MHC class II peptide loading. Curr. Biol. 7:950.[Web of Science][Medline]
  17. Kropshofer, H., Vogt, A. B., Thery, C., Armandola, E. A., Li, B. C., Moldenhauer, G., Amigorena, S. and Hammerling, G. J. 1998. A role for HLA-DO as a co-chaperone of HLA-DM in peptide loading of MHC class II molecules. EMBO J. 17:2971.[Web of Science][Medline]
  18. Liljedahl, M., Winqvist, O., Surh, C. D., Wong, P., Ngo, K., Teyton, L., Peterson, P. A., Brunmark, A., Rudensky, A. Y., Fung-Leung, W. P. and Karlsson, L. 1998. Altered antigen presentation in mice lacking H2-O. Immunity 8:233.[Web of Science][Medline]
  19. Douek, D. C. and Altmann, D. M. 1997. HLA-DO is an intracellular class II molecule with distinctive thymic expression. Int. Immunol. 9:355.[Abstract/Free Full Text]
  20. DeMars, R., Chang, C. C., Shaw, S., Reitnauer, P. J. and Sondel, P. M. 1984. Homozygous deletions that simultaneously eliminate expression of class I and class II antigens of EBV-transformed B-lymphoblastoid cells. I. Reduced proliferative responses of autologous and allogeneic T cells to mutant cells that have decreased expression of class II antigens. Hum. Immunol. 11:77.[Web of Science][Medline]
  21. Ceman, S., Petersen, J. W., Pinet, V. and DeMars, R. 1994. Gene required for normal MHC class II expression and function is localized to approximately 45 kb of DNA in the class II region of the MHC. J. Immunol. 152:2865.[Abstract]
  22. Radka, S. F., Machamer, C. E. and Cresswell, P. 1984. Analysis of monoclonal antibodies reactive with human class II beta chains by two-dimensional electrophoresis and western blotting. Hum. Immunol. 10:177.[Web of Science][Medline]
  23. Guy, K., Van Heyningen, V., Cohen, B. B., Deane, D. L. and Steel, C. M. 1982. Differential expression and serologically distinct subpopulations of human Ia antigens detected with monoclonal antibodies to Ia alpha and beta chains. Eur. J. Immunol. 12:942.[Web of Science][Medline]
  24. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. and Pease, L. R. 1989. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51.[Web of Science][Medline]
  25. Jacobson, S., Sekaly, R.-P., Jacobson, C. L., McFarland, H. E. and Long, E. O. 1989. HLA class II-restricted presentation of cytoplasmic measles virus antigens to cytotoxic T cells. J. Virol. 63:1756.[Abstract/Free Full Text]
  26. Herman, A., Labrecque, N., Thibodeau, J., Marrack, P., Kappler, J. W. and Sekaly, R.-P. 1991. Identification of the staphylococcal enterotoxin A superantigen binding site in the ß1 domain of the human histocompatibility antigen HLA-DR. Proc. Natl Acad. Sci. USA 88:9954.[Abstract/Free Full Text]
  27. Graham, F. L. and van der Eb, A. J. 1973. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456.[Web of Science][Medline]
  28. Long, E. O., Rosen-Bronson, S., Karp, D. R., Malnati, M., Sekaly, R.-P. and Jaraquemada, D. 1991. Efficient cDNA expression vectors for stable and transient expression of HLA-DR in transfected fibroblast and lymphoid cells. Hum. Immunol. 31:229.[Web of Science][Medline]
  29. Karp, D. R., Teletski, C. L., Jaraquemada, D., Maloy, W. L., Coligan, J. E. and Long, E. O. 1990. Structural requirements for pairing of {alpha} and ß chains in HLA-DR and HLA-DP molecules. J. Exp. Med. 171:615.[Abstract/Free Full Text]
  30. Robadey, C., Ammerlaan, W., Muller, C., Cloutier, I., Sékaly, R. P., Haefliger, J. A. and Demotz, S. 1997. The processing routes determined by negatively charged residues in DR1-restricted T cell determinants. J. Immunol. 159:3238.[Abstract]
  31. O`Farrell, P. Z., Goodman, H. M. and O'Farrell, P. H. 1977. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12:1133.[Web of Science][Medline]
  32. Machamer, C. E. and Cresswell, P. 1982. Biosynthesis and glycosylation of the invariant chain associated with HLA-DR antigens. J. Immunol. 129:2564.[Abstract]
  33. Robbins, A. R. and Roff, C. F. 1987. Isolation of mutant Chinese hamster ovary cells defective in endocytosis. Methods Enzymol. 138:458[Web of Science][Medline]
  34. Castellino, F. and Germain, R. N. 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.[Web of Science][Medline]
  35. Morrison, M. 1980. Lactoperoxidase-catalyzed iodinations as a tool for investigation of proteins. Methods Enzymol. 70:214.[Medline]
  36. Fukuda, M. 1991. Lysosomal membrane glycoproteins. Structure, biosynthesis, and intracellular trafficking. J. Biol. Chem. 266:21327–21330.[Free Full Text]
  37. Niedergang, F., San Jose, E., Rubin, B., Alarcon, B., Dautry-Varsat, A. and Alcover, A. 1997. Differential cytosolic tail dependence and intracellular fate of T-cell receptors internalized upon activation with superantigen or phorbol ester. Res. Immunol. 148:231.[Web of Science][Medline]
  38. Lechler, R. I., Sant, A. J., Braunstein, N. S., Sekaly, R., Long, E. O. and Germain, R. N. 1990. Cell surface expression of hybrid murine/human MHC class II {alpha}ß dimers. J. Immunol. 144:329.[Abstract]
  39. Altman, D. A., Heyes, J. M., Ikeda, H., Sadler, A. M., Wilkinson, D., Madrigal, J. A., Bodmer, J. G. and Trowsdale, J. 1990. Fine mapping of HLA class II monoclonal antibody specificities using transfected L cells. Immunogenetics 32:51[Web of Science][Medline]
  40. Watson, A. J., DeMars, R., Trowbridge, I. S. and Bach, F. H. 1983. Detection of a novel human class II HLA antigen. Nature 304:358.[Medline]
  41. Pinet, V., Vergelli, M., Martin, R., Bakke, O. and Long, E. O. 1995. Antigen presentation mediated by recycling of surface HLA-DR molecules. Nature 375:603.[Medline]
  42. Lotteau, V., Teyton, L., Peleraux, A., Nilsson, T., Karlsson, L., Schmid, S. L., Quaranta, V. and Peterson, P. A. 1990. Intracellular transport of class II MHC molecules directed by invariant chain. Nature 348:600.[Medline]
  43. Lamb, C. A., Yewdell, J. W., Bennink, J. R. and Cresswell, P. 1991. Invariant chain targets HLA class II molecules to acidic endosomes containing internalized influenza virus. Proc. Natl Acad. Sci. USA 88:5998.[Abstract/Free Full Text]
  44. Simonsen, A., Momburg, F., Drexler, J., Hammerling, G. J. and Bakke, O. 1993. Intracellular distribution of the MHC class II molecules and the associated invariant chain (Ii) in different cell lines. Int. Immunol. 5:903.[Abstract/Free Full Text]
  45. Stang, E. and Bakke, O. 1997. MHC class II associated invariant chain induced enlarged endosomal structures. A morphological study. Exp. Cell Res. 235:79.[Web of Science][Medline]
  46. Gruneberg, U., Rich, T., Roucard, C., Marieke van Ham, S., Charron, D. and Trowsdale, J. 1997. Two widely used anti-DR alpha monoclonal antibodies bind to an intracellular C-terminal epitope. Human Immunol. 53:34.[Web of Science][Medline]
  47. Brown, J. H., Jardetzky, T. S., Gorga, J. C., Stern, L. J., Urban, R. G., Strominger, J. L. and Wiley, D. C. 1993. Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1. Nature 364:33.[Medline]
  48. Pelham, H. R. 1995. Sorting and retrieval between the endoplasmic reticulum and Golgi apparatus. Curr. Opin. Cell Biol. 7:530.[Web of Science][Medline]
  49. Sandoval, I. V. and Bakke, O. 1994. Targeting of membrane proteins to endosomes and lysosomes. Trends Cell Biol. 4:292.[Medline]
  50. Zhong, G., Romagnoli, P. and Germain, R. N. 1997. Related leucine-based cytoplasmic targeting signals in invariant chain and major histocompatibility complex class II molecules control endocytic presentation of distinct determinants in a single protein. J. Exp. Med. 185:429.[Abstract/Free Full Text]
  51. Liu, S. H., Marks, M. S. and Brodsky, F. M. 1998. A dominant-negative clathrin mutant differentially affects trafficking of molecules with distinct sorting motifs in the class II major histocompatibility complex (MHC) pathway. J. Cell Biol. 140:1023.[Abstract/Free Full Text]
  52. Surh, C. D., Gao, E.-K., Kosaka, H., Lo, D., Ahn, C., Murphy, D. B., Karlsson, L., Peterson, P., Sprent, J. and Gao, E. K. 1992. Two subsets of epithelial cells in the thymic medulla. J. Exp. Med. 176:495.[Abstract/Free Full Text]
  53. Karlsson, L., Surh, C. D., Sprent, J. and Peterson, P. A. 1992. An unusual class II molecule. Immunol. Today 13:469.[Web of Science][Medline]
  54. Romagnoli, P., Layet, C., Yewdell, J., Bakke, O. and Germain, R. N. 1993. Relationship between invariant chain expression and major histocompatibility complex class II transport into early and late endocytic compartments. J. Exp. Med. 177:583.[Abstract/Free Full Text]
  55. Pieters, J., Bakke, O. and Dobberstein, B. 1993. The role of the cytoplasmic tail of MHC class II-associated invariant chain in targeting to and retention in endocytic compartments. J. Cell. Sci. 106:831.[Abstract]
  56. Motta, A., Amodeo, P., Fucile, P., Castiglione Morelli, M., Bremnes, B. and Bakke, O. 1997. A new triple-stranded {alpha}-helical bundel in solution: the assembling of the cytosolic tail of MHC associated invariant chain. Structure 5:1453.[Medline]

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