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The Journal of Immunology, 2001, 167: 884-892.
Copyright © 2001 by The American Association of Immunologists

Regulation of MHC Class II Antigen Presentation by Sorting of Recycling HLA-DM/DO and Class II within the Multivesicular Body1

Marcel van Lith2,*, Marieke van Ham2,3,*, Alexander Griekspoor*, Esther Tjin*, Desiree Verwoerd*, Jero Calafat{dagger}, Hans Janssen{dagger}, Eric Reits*, Liesbeth Pastoors{ddagger} and Jacques Neefjes*

Divisions of * Tumor Biology, {dagger} Cell Biology, and {ddagger} Cellular Biochemistry, Netherlands Cancer Institute, Amsterdam, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
MHC class II molecules bind antigenic peptides in the late endosomal/lysosomal MHC class II compartments (MIIC) before cell surface presentation. The class II modulatory molecules HLA-DM and HLA-DO mainly localize to the MIICs. Here we show that DM/DO complexes continuously recycle between the plasma membrane and the lysosomal MIICs. Like DM{beta} and the class II-associated invariant chain, the DO{beta} cytoplasmic tail contains potential lysosomal targeting signals. The DO{beta} signals, however, are not essential for internalization of the DM/DO complex from the plasma membrane or targeting to the MIICs. Instead, the DO{beta} tail determines the distribution of both DM/DO and class II within the multivesicular MIIC by preferentially localizing them to the limiting membrane and, in lesser amounts, to the internal membranes. This distribution augments the efficiency of class II antigenic peptide loading by affecting the efficacy of lateral interaction between DM/DO and class II molecules. Sorting of DM/DO and class II molecules to specific localizations within the MIIC represents a novel way of regulating MHC class II Ag presentation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Antigenic fragments from exogenous pathogens are taken up by APCs and are presented to the immune system by MHC class II molecules. After association with the invariant chain (Ii),4 that contains a lysosomal targeting motif (1), class II molecules are targeted to the major peptide loading compartment, termed MIIC, where it contacts Ags generated in the endocytic pathway. Upon degradation of the Ii a small fragment (class II-associated invariant chain peptide (CLIP)) remains associated to the class II peptide-binding groove. In professional APCs, CLIP release is catalyzed by the class II-like molecule HLA-DM (2, 3, 4). As DM also disrupts other unstable class II-peptide complexes, it acts as a peptide editor, eventually promoting presentation of tightly binding antigenic peptides (5, 6).

In B cells the composition of the class II antigenic peptide repertoire is controlled by yet another class II-like molecule, termed HLA-DO (7, 8, 9). DO tightly associates with DM, which is required for endoplasmic reticulum (ER) exit and interferes with its catalytic function (10, 11, 12). DO-expressing cells accumulate class II/CLIP complexes at the cell surface (10, 11, 12), yet formation of stable class II/peptide complexes is only reduced by half. The efficacy of DM inhibition by DO is pH dependent, being more effective at endosomal pH than at the acidic pH of the MIICs. The specific inhibition of DM by DO in endosomes results in a modulation of the class II peptide repertoire by preventing the presentation of certain peptides while inducing the presentation of others (12, 13).

Whereas class II molecules only transiently linger in the MIICs to pick up their Ags on their way to the cell surface (14, 15), both DM (16) and DO (9) are considered to be MIIC residents. The mechanism behind this bifurcation in the intracellular trafficking pathways of class II molecules and DM/DO is still poorly understood. Internalization and intracellular targeting of transmembrane proteins is mediated by discrete motifs in their cytoplasmic domains. In general, these motifs are either tyrosine based (YxxØ or NpxY, where x is any amino acid, and Ø is an amino acid with a bulky hydrophobic group) or di-leucine-based (17, 18). Class II molecules use the Ii di-leucine-based motif to reach the MIICs (1). Lysosomal targeting of DM is dependent on a DM{beta} tyrosine-based motif (YTPL) (19, 20). Interestingly, the cytoplasmic domain of human DO{beta} contains two potential targeting motifs (21, 22), the function of which is as yet unclear.

The cellular trafficking pathway of DM/DO complexes is investigated using the green fluorescent protein (GFP) tagged to DO. We show that DM/DO complexes are constitutively delivered from MIICs to the cell surface together with class II molecules, and are immediately internalized to recycle back to the MIICs. Although we demonstrate that two potential lysosomal targeting motifs in the DO{beta} cytoplasmic domain are functional in a chimera with CD8, they are not of importance for cellular trafficking of the DM/DO complex. However, the cytoplasmic domain of DO{beta} redistributes class II molecules and DM/DO complexes within the multivesicular MIIC, which strikingly affects the efficiency of peptide exchange. Thus, we define a novel sorting mechanism that regulates the efficacy of the class II-mediated immune response by specialized protein sorting within the MIICs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines, transfectants, and Abs

The human melanoma cell line Mel JuSo (HLA-A1, HLA-B8, HLA-Cw7, HLA-DR3, and HLA-DQ2 by DNA typing) and its stable transfectants are grown in IMEM with 10% FCS in the presence or the absence of 2000 µg/ml G418, 600 µg/ml hygromycin (Life Technologies, Gaithersburg, MD), and/or 500 µg/ml Zeocin (Invitrogen, Breda, The Netherlands). Continuous expression of the GFP-tagged proteins is ensured by regular selection of the GFP-positive cells by FACS.

The rabbit polyclonal antiserum against class II was previously described (15), and the sera against DO{alpha} (A10 3.0) and DO{beta} (A64 5.1) were raised against fusion proteins of GST with DO{alpha} or DO{beta}, respectively. The HLA-DR-specific mAb L243 (American Type Culture Collection, Manassas, VA) (23) and 1B5 (24), the class II/CLIP-specific Ab CERCLIP.1 (25), the DR3-antigenic peptide specific mAb 16.23 (26, 27), the DM{alpha}-specific mAb 5C1 (28), and the anti-transferrin receptor mAb (B3/25; Roche, Indianapolis, IN) were previously described. The anti-actin mAb Ab-1 was purchased from Oncogene Research Products (San Diego, CA), the anti-human CD8 OKT8 mAb was obtained from the American Type Culture Collection (CRL-8014), and the Texas Red-conjugated secondary Abs were purchased from Molecular Probes (Eugene, OR).

Quantification of Western blots was performed with the Alpha Innotech (San Leandro, CA) digital imaging system and Phoretix 1D Advanced software (Alpha Innotech).

DNA constructs

The DO{alpha}, DO{beta}-GFP, and nuclear localization signal (NLS)-GFP constructs have been previously described (10, 29). The cDNAs encoding human dynamin (type 1), both wild-type (wt) and dominant negative (K44A, negative for nucleotide binding of the GTPase), provided by A. M. van der Bliek (30), were recloned into pcDNA3. DNAs were introduced into the Mel JuSo cell line using calcium phosphate or via microinjection.

The CD8-DM{beta}, CD8-DO{alpha}, and CD8-DO{beta} chimeras were generated by ligation of SalI/BamHI-digested PCR products encoding the cytoplasmic tails of DM{beta}, DO{alpha}, or DO{beta} to SalI/BamHI-digested pCMUIV-CD8 vector (31). The CD8-DO{beta} chimeras containing the tyrosine to alanine and/or the di-leucine to alanine substitution were generated by cloning hybridized complementary primers encoding the mutated cytoplasmic DO{beta} tail into the SalI/BamHI site of pCMUIV-CD8.

The pcDNA3 IRES-NLS-GFP vector was generated by ligation of part of the LZRSneo vector (containing a multiple stop sequence, a multiple cloning site, and an internal ribosomal entry site (IRES)) into the BamHI and EcoRI sites of the pcDNA3 NLS-GFP vector. The cDNA of DO{beta} was cloned into the XhoI site of pcDNA3 IRES-NLS-GFP. The DO{beta}-CD8 chimeric construct was generated by a fusion PCR between the extracellular to transmembrane region of DO{beta} and the cytoplasmic domain of CD8, followed by cloning into the EcoRI and XhoI sites of pcDNA3 IRES-NLS-GFP.

Subcellular fractionation by density gradient electrophoresis

Subcellular fractionation on wtDO and DOCD8 transfectants incubated with 2 mg/ml HRP (type VIA; Sigma, St. Louis, MO) for 30 min at 37°C was essentially performed as previously described (32). The fractions collected were assayed for total protein (bicinchoninic acid; Pierce, Rockford, IL), HRP activity, and {beta}-hexosaminidase. Plasma membrane fractions were separated from ER fractions using wheat-germ agglutinin (32). Proteins from the different fractions were precipitated with TCA and analyzed by SDS-PAGE and immunoblotting with A64 5.1 ({alpha}DO{beta}).

FACS analysis

For FACS analysis, 1 x 106 cells were stained with saturating amounts of unlabeled primary Ab and PE-conjugated F(ab')2 rabbit anti-mouse IgG (H+L; Zymed, San Francisco, CA) and analyzed on a FACScan flow cytometer (BD Biosciences, Mountain View, CA).

Microinjection and confocal microscopy

Fifty to 100 cells were typically microinjected intranuclearly with a mix of the cDNA encoding NLS-GFP (0.2 ng/ml) and the cDNAs indicated (0.1–0.2 µg/µl) on a heated xy stage of a Zeiss Anxiovert 135 M microscope (New York, NY) equipped with an Eppendorf manipulator 5171/transjector 5246 system as previously described (29). After injection, cells were allowed to recover overnight at 37°C for expression of the microinjected cDNAs.

Living cells were analyzed in a heated tissue culture chamber at 37°C. Brefeldin A (BFA; 5 µg/ml; Sigma) was added 1.5–4.5 h before confocal laser scanning microscopy (CLSM) analysis. CLSM analyses were performed using a Bio-Rad MRC600 confocal microscope equipped with an argon/krypton laser (Bio-Rad, Hercules, CA). Single-channel green fluorescence was detected at {lambda} >515 nm after excitation at {lambda} = 488 nm. For dual analyses, green fluorescence was detected at {lambda} = 520–560 nm, and red fluorescence was detected at {lambda} >585 nm after excitation at {lambda} = 568 nm.

For immunofluorescence labeling, cells were fixed with 3.7% formaldehyde in PBS, permeabilized (0.1% Triton X-100 in PBS), and incubated for 1 h with Abs diluted in PBS containing 0.5% BSA, followed by washing and 1-h incubations with Texas Red-conjugated secondary Abs and additional repeated washing.

Cell surface Ag presentation assay

Cells were incubated in medium with 200 µM cycloheximide at 37°C for 4 h. After trypsinization, cells were washed twice in phosphate-citrate buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.14 mM citric acid, adjusted to pH 5 or 7, supplemented with 0.05% NaN3, 50 µM 2-deoxyglucose, and 0.1% BSA). Subsequently, cells were incubated in the appropriate phosphate-citrate buffer with protease inhibitors in the absence or the presence of 20 µM biotinylated ApoB2877–2894 at 37°C for 2.5 h. After washing with cold PBS/0.1% BSA, bound biotinylated peptides were detected by streptavidin-conjugated PE (Molecular Probes) followed by FACS analysis.

Immunoelectron microscopy

Cells were fixed in 0.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) and processed for ultrathin cryosectioning as previously described (14). For immunolabeling, sections were incubated with purified mAb anti-human DM{alpha} (5C1), followed by subsequent incubations with rabbit anti-mouse IgG and 10 nm protein A-conjugated colloidal gold. Next, the sections were treated with 1% glutaraldehyde, followed by further incubation with rabbit anti-human class II serum and 15 nm protein A-conjugated colloidal gold. After embedding in a mixture of methyl-cellulose and uranyl acetate, sections were analyzed with a Philips CM10 electron microscope (Eindhoven, The Netherlands). For quantification of class II and DM distribution patterns in the multivesicular bodies (MVB), gold label in apparent contact with the perimeter membrane was considered perimeter membrane labeling.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DM/DO complexes recycle between plasma membrane and MIIC

To study the intracellular transport of the DM/DO complex we used a stable transfectant of the melanoma cell line Mel JuSo expressing a heterodimeric complex of DO{alpha} and a chimera of GFP fused to the DO{beta} cytoplasmic tail (10). The DO{alpha}{beta}GFP complex quantitatively associates with DM without affecting endogenous class II and DM levels (10). Confocal analysis shows that DM/DO{alpha}{beta}GFP complexes accumulate in perinuclear, lysosome-like MIICs (10), which is indistinguishable from that of DR{alpha}{beta}GFP (Fig. 1Go, -BFA).



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  		FIGURE 1. Bifurcation of cellular trafficking pathways of DR{alpha}{beta}GFP and DO{alpha}{beta}GFP/DM. Confocal analysis of living Mel JuSo cells expressing DR{alpha}{beta}GFP or DO{alpha}{beta}GFP was performed at 37°C with control cells (-BFA) or cells cultured with BFA for 4.5 h (+BFA).

 
The primary localization of DM/DO in the MIICs enables these molecules to take part in the peptide loading process of nascent class II molecules colocalizing in these compartments. After Ag binding, the class II/peptide complexes need to be transported to the cell surface for Ag presentation. We previously visualized direct transport of DR{alpha}{beta}GFP-containing vesicles to the cell surface (14). Although DM/DO resides in similar vesicles, it is not observed at the cell surface (Fig. 1Go). DM/DO may be selectively retained in MIICs, which implies a differential intracellular sorting of class II and DM/DO complexes. Alternatively, DM/DO is cotransported to the cell surface and immediately reinternalized. The route of entry into the MIICs of DM/DO{alpha}{beta}GFP is compared with that of DR{alpha}{beta}GFP in living cells by addition of BFA to block the transport of newly synthesized molecules out of the ER. One and a half hours of BFA incubation do not visibly affect the quantity and distribution of the DO{alpha}{beta}GFP/DM-containing vesicles (data not shown). This indicates that the vesicles do not form part of the constitutive secretory pathway from the ER to the cell surface, as the secretory vesicles get depleted of newly synthesized material within this time frame. Similar observations were made for DR{alpha}{beta}GFP-containing vesicles (14). Prolonged BFA incubation for an additional 3 h enhances fluorescent staining of the reticulate ER network due to accumulation of newly synthesized GFP-tagged proteins and depletes the cells from intracellular vesicular DR{alpha}{beta}GFP (Fig. 1Go, +BFA). Interestingly, DM/DO{alpha}{beta}GFP complexes remain present in intracellular vesicles, albeit in slightly reduced numbers (Fig. 1Go, +BFA). Similar findings were obtained using cycloheximide (data not shown). Thus, whereas DR{alpha}{beta}GFP is transported through the endosomal/lysosomal compartments and incorporated into the plasma membrane within 4.5 h, DM/DO{alpha}{beta}GFP complexes continue to prevail in intracellular compartments.

To discriminate between selective retention of DM/DO in MIICs and deposition at the plasma membrane followed by rapid internalization, cells are microinjected with cDNAs encoding wild-type dynamin or a dominant negative dynamin mutant that inhibits clathrin-mediated endocytosis (30). Coinjection with a cDNA encoding GFP tagged with a strong NLS (33) allows identification of microinjected cells by nuclear GFP fluorescence and simultaneously assesses the viability of the cells. The wt dynamin does not affect the distribution of either DM/DO{alpha}{beta}GFP or MHC class II complexes (Fig. 2GoA, wtDyn). Overexpression of dominant negative dynamin induces membrane ruffling, typical for cells inhibited in endocytosis. The distribution of fluorescence in DR{alpha}{beta}GFP-expressing cells is not significantly affected by mutant dynamin. Fluorescent DM/DO{alpha}{beta}GFP complexes, however, accumulate at the plasma membrane (indicated by arrows) of the microinjected cells, but not in control cells (Fig. 2GoA, asterisks, DN Dyn-BFA). Cell surface expression of HLA-DO was confirmed by staining intact nonpermeabilized dominant-negative dynamin-expressing cells with {alpha}DO{beta}, followed by CLSM analysis (data not shown). These results demonstrate that in normal cells DM/DO is exported to the cell surface together with class II, followed by immediate reinternalization by clathrin-coated vesicles, whereas the majority of class II complexes remain at the cell surface. The very transient appearance of low amounts of DM/DO{alpha}{beta}GFP complexes at the plasma membrane can be confirmed by cell surface iodination and recovery of iodinated DM/DO{alpha}{beta}GFP via immunoprecipitation (data not shown), subcellular fractionation (see below, Fig. 4GoD) and FACS analysis (see later). Iodinated DM/DO{alpha}{beta}GFP is still recovered after 4-h cycloheximide treatment, confirming that the DM/DO pool that transiently appears at the plasma membrane is not derived from the constitutive secretory pathway, as this pathway gets depleted of proteins within 1.5 h (data not shown). Effective inhibition of clathrin-mediated endocytosis by mutant dynamin was demonstrated by immunofluorescence detection of the transferrin receptor, as it accumulates at the plasma membrane of microinjected cells instead of in early endosomes (Fig. 2GoB). Uptake of the fluid phase marker sulforhodamine 101 was not affected by mutant dynamin, indicating that it specifically blocks clathrin-mediated endocytosis (data not shown).



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  		FIGURE 2. DM/DO{alpha}{beta}GFP complexes recycle between plasma membrane and intracellular compartments. A, Confocal analysis of living Mel JuSo transfectants expressing DM/DO{alpha}{beta}GFP or DR{alpha}{beta}GFP after nuclear microinjection with NLS-GFP in combination with either wt dynamin (wtDyn) or dominant negative dynamin (DN Dyn). Microinjected cells were identified by fluorescent NLS-GFP expression in the nucleus. Arrows indicate cell surface expression of DO{alpha}{beta}GFP in dominant negative dynamin-injected cells. Three noninjected cells are indicated by asterisks. Cells were subjected to 4.5-h BFA treatment when indicated (+BFA). -BFA, Control. Images are shown in false colors to better visualize differences in relative fluorescence intensity. B, Localization of the transferrin receptor in fixed DO{alpha}{beta}GFP-expressing Mel JuSo cells (DO{alpha}{beta}GFP signal, left) by specific immunostaining (right) after microinjection with dominant negative dynamin, NLS-GFP, and BFA treatment. Dominant negative dynamin retains the recycling transferrin receptor at the plasma membrane.

 


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  		FIGURE 4. Expression levels of DR, DM, and DO in wtDO and DOCD8 transfectants. A, Schematic representation of the DO{beta} constructs stably expressed in wtDO and DOCD8 Mel JuSo. B, Cell lysates from Mel JuSo, wtDO, and DOCD8 are analyzed by SDS-PAGE and immunoblotting with A10 3.0 ({alpha}DO{alpha}), A64 5.1 ({alpha}DO{beta}), 5C1 ({alpha}DM{alpha}), and 1B5 ({alpha}DR{alpha}). Expression levels are quantified, and loading is corrected by actin expression levels. The data represented are ratios relative to wtDO (DO{alpha} and DO{beta}) or Mel JuSo (DM{alpha} and DR{alpha}). C, CLSM analysis of wtDO and DOCD8 transfectants after fixation with methanol and specific immunostaining with {alpha}DO{beta} (affinity-purified A64 5.2; green) and 1B5 (red). D, wtDO and DOCD8 transfectants incubated with HRP for 30 min are subjected to subcellular fractionation by density gradient electrophoresis. Top, The different fractions are analyzed for the presence of the endosomal fluid phase marker, HRP, the lysosomal marker {beta}-hexosaminidase ({beta}-hex), and total protein levels (plasma membrane and ER). Protein-abs, Protein absorbance. Bottom, Fractions are separated by SDS-PAGE and immunoblotted with {alpha}DO{beta}. Underlined fractions represent the lysosomes, ER, and plasma membrane peaks indicated in the top panels.

 
The ongoing presence of vesicles containing fluorescent DM/DO and class II in cells blocked in endocytosis indicates that the majority of newly synthesized DM/DO and class II molecules is targeted directly to the endocytic pathway without prior cell surface deposition (Fig. 2GoA, DN Dyn-BFA). Indeed, additional BFA treatment depletes cells blocked in endocytosis of all vesicular DM/DO{alpha}{beta}GFP, whereas control cells retain a pool of fluorescent vesicles as observed previously (Fig. 2GoA, DN Dyn + BFA). In contrast, BFA treatment depletes both microinjected cells and control cells from intracellular DR{alpha}{beta}GFP at similar rates, indicating that replenishment of the endocytic pathway via recycling DR{alpha}{beta}GFP is not a major class II trafficking route in these cells (Fig. 2GoA, DN Dyn + BFA).

Together, these data suggest that after deposition into MIICs all DM/DO{alpha}{beta}GFP complexes continuously recycle between the cell surface and intracellular Ag loading compartments. DM/DO thus enters the MIICs via two major routes, a direct input from the biosynthetic pathway and reuse of exocytosed material via continuous recycling through the endocytic pathway.

The cytoplasmic tail of DO{beta} contains lysosomal targeting signals

Targeting of DM to the MIICs is dependent on a tyrosine-based motif in the cytoplasmic tail of DM{beta} (19, 20). Although this signal may well suffice to target the DM/DO complex to the MIICs, the putative targeting motifs in the DO{beta}-chain (Fig. 3GoA) could contribute to cellular trafficking of DM/DO. The functionality of these motifs is investigated using a chimera in which the DO{beta} cytoplasmic tail is fused to the extracellular and transmembrane domains of CD8{alpha} (Fig. 3GoA). In addition, CD8-DM{beta} and CD8-DO{alpha} chimeras are generated. The localization of the chimeras is examined after expression of the constructs in the Mel JuSo DO{alpha}{beta}GFP transfectant. The wt CD8 localizes to the plasma membrane (Fig. 3GoB), consistent with the lack of a functional targeting motif that could deviate the protein from the default secretory pathway. Likewise, the CD8-DO{alpha} chimera lacks a targeting signal, as it primarily localizes to the plasma membrane (Fig. 3GoB). In contrast, the CD8-DM{beta} chimera is targeted to perinuclear vesicles and colocalizes with DO{alpha}{beta}GFP (Fig. 3GoB). In agreement with previous studies (19, 20), these vesicles are CD63 positive, mannose-6-phosphate negative, and negative for the early endosome marker EEA1 (data not shown), emphasizing the lysosomal nature of these compartments. The CD8-DO{beta} chimera is also targeted to perinuclear vesicles that are positive for DO{alpha}{beta}GFP. Thus, the DO{beta} cytoplasmic tail contains a functional targeting motif.



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  		FIGURE 3. The cytoplasmic domain of DO{beta} contains lysosomal targeting information. A, Schematic representation of the CD8 chimeras used for CLSM analysis and amino acid sequences of DO{alpha} and DO{beta} cytoplasmic tails. Potential targeting motifs are in bold. B and C, CLSM analysis of transiently expressed (chimeric) CD8 proteins in Mel JuSo cells stably expressing DO{alpha}{beta}GFP after fixation of the cells in methanol and specific immunostaining (DO{alpha}{beta}GFP in green, {alpha}CD8 in red).

 
The DO{beta} cytoplasmic tail contains a membrane-proximal tyrosine residue and two membrane-distal leucine residues (Fig. 3GoA), both potential lysosomal targeting motifs. To investigate whether either of these residues is involved in targeting the CD8-DO{beta} chimera, constructs are made in which the tyrosine and/or the two leucines are mutated into alanine residues (Fig. 3GoA). Elimination of the tyrosine, the two leucine residues, or both results in accumulation of the chimeras at the plasma membrane (Fig. 3GoC). Thus, both the tyrosine residue and the two leucines are involved in lysosomal targeting of the CD8-DO{beta} chimera.

The DO{beta} cytoplasmic tail is not essential for lysosomal targeting of the DM/DO complex

As DM and DO tightly associate, the benefit of a targeting signal in addition to the DM{beta} signal is unclear. Possibly the additional sorting information in DO{beta} distinguishes the subcellular targeting of the DM/DO complex from that of DM alone. To investigate the contribution of the DO{beta} motif to trafficking of the DM/DO complex, the targeting information is removed by replacing the DO{beta} tail in the DM/DO complex by that of CD8 (Fig. 4GoA). The wt DO{beta} and chimeric DO{beta}CD8 chains are expressed from one bicistronic transcript together with the NLS-GFP protein, allowing a correlation of DO{beta} expression levels to those of GFP. Stable clonal Mel JuSo transfectants are generated expressing DO{alpha} in conjunction with either wtDO{beta} or the DO{beta}CD8 chimera. Similar expression levels of the DO complexes are obtained by FACS sorting of the transfectants for equal GFP levels. Western blot analysis and subsequent quantification confirms the similar DR, DM, and DO expression levels (Fig. 4GoB).

To examine whether the tail of DO{beta} has an effect on subcellular localization of the DM/DO complex, wtDO and DOCD8 transfectants are subjected to subcellular fractionation by density gradient electrophoresis. To correct for experimental variation, the localization of lysosomes ({beta}-hexosaminidase), endosomes (HRP), and ER/plasma membrane (total protein) in the gradient was determined in each independent experiment. Subsequent Western blot analysis of the different fractions revealed that in both wtDO and DOCD8 transfectants DO (and DM, data not shown) primarily localizes to {beta}-hexosaminidase-positive fractions (lysosomes), and only a small portion is detectable in plasma membrane fractions (Fig. 4GoD). Since DO requires DM for ER exit (9), part of the DO pool remains in the ER due to relative overexpression. This assures quantitative association of DM with DO, as validated by immunoprecipitation (10). Lysosomal localization of wtDO and DOCD8 is confirmed by CLSM analysis (Fig. 4GoC). Thus, the relative intracellular distribution of DM/DO is not affected by DO{beta} tail modification. This indicates that the DM/DO complex does not require the additional DO{beta} sorting signal for proper targeting to the MIICs.

The cytoplasmic tail of DO{beta} influences peptide exchange

To investigate whether the DO{beta} tail has an effect on Ag presentation, the composition of the class II/peptide complexes was analyzed in the Mel JuSo, wtDO, and DOCD8 transfectants by FACS. The total amount of class II molecules at the cell surface was relatively unvaried (L243, Fig. 5GoA). In line with the DM modulatory function of DO (10, 11, 12), a large portion of these class II molecules was occupied by CLIP in the wtDO transfectant (CERCLIP, Fig. 5GoA). Surprisingly, however, the DOCD8 transfectant showed a substantial further increase in class II/CLIP levels (CERCLIP; Fig. 5GoA). The relative amount of class II/CLIP complexes was even more than doubled in the DOCD8 transfectant compared with the wtDO transfectant (ratio CERCLIP/L243, Fig. 5GoA). The reduced release of CLIP from class II in the DOCD8 transfectant was paralleled by a 50% reduction in the relative amount of class II molecules presenting stably binding peptides, as demonstrated by the mAb 16.23 that recognizes these complexes (ratio 16.23/L243, Fig. 5GoA).



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  		FIGURE 5. The DO{beta} cytoplasmic tail affects the efficacy of class II/antigenic peptide loading. A, FACS analysis of 5000 Mel JuSo cells either expressing or not expressing wtDO or DOCD8 using the class II mAb L243, the DR/CLIP mAb CERCLIP.1, and the DR3/peptide mAb 16.23 ({blacksquare}, Mel JuSo; , Mel JuSo/wtDO; black line, Mel JuSo/DOCD8; gray line, secondary Ab only). The relative ratio of median values obtained by CERCLIP.1 and 16.23 vs total class II levels (L243) is plotted in the right panel. Shown are representative results from three independent experiments, with mean fluorescence intensities depicted in arbitrary units on a logarithmic scale. B, Total cell lysates of Mel JuSo, Mel JuSo/wtDO, and Mel JuSo/DOCD8 were boiled (b) in reducing sample buffer or left at room temperature (nb). Samples were subsequently analyzed by SDS-PAGE and Western blotting. SDS-stable class II complexes and the DR{alpha}-chain were detected with mAb 1B5. The arrow indicates the presence of DR{alpha}{beta} dimers.

 
HLA-DR3 molecules that have acquired a stably binding peptide are resistant to dissociation in SDS-containing buffer under nonboiling conditions, whereas HLA-DR3 molecules with CLIP readily dissociate. Indeed, lysates of the wtDO transfectant contain less SDS-stable class II dimer/peptide complexes compared with untransfected Mel JuSo (Fig. 5GoB). Again, the DOCD8 transfectant shows an even greater reduction in the amount of stable class II/peptide complexes vs unstable class II molecules (Fig. 5GoB), demonstrating that omission of the DO{beta} cytoplasmic tail is deleterious for the efficiency of CLIP-antigenic peptide exchange on class II molecules. The observed differences in Ag presentation between the wtDO and DOCD8 transfectant are consistent for all other wtDO and DOCD8 clones (n > 5) tested. Furthermore, the catalytic activity of DM/DO complexes is not affected by the absence of the DO{beta} tail as determined by an in vitro peptide exchange assay (data not shown). Taken together, these results demonstrate that the tail of DO{beta} affects the efficiency of DM/DO-mediated catalysis of class II antigenic peptide loading, although it does not influence the subcellular localization or the catalytic activity of the DM/DO complex.

The DO{beta} tail alters the distribution of DR and DM/DO within the multivesicular body

How does the cytoplasmic tail of DO{beta} affect the process of Ag presentation, as it is not involved in the localization of the DM/DO complex to the MIICs? It could be that DO affects localization of the DM/DO complex at a level that cannot be detected by subcellular fractionation studies. Therefore, the intracellular localization of DM/DO and class II in Mel JuSo and both transfectants is analyzed in more detail by immunoelectron microscopy. Intracellularly both DM(/DO) and class II primarily localize to MVB, a characteristic morphology for the MIICs in this cell line (Fig. 6GoA). Strikingly, the distribution between the limiting perimeter membrane and the internal vesicles of both DM(/DO) and class II within the multivesicular MIICs is altered in the wtDO transfectant compared with the DOCD8 transfectant or Mel JuSo itself (Fig. 6GoA). Quantification of the distribution demonstrates that in Mel JuSo and the DOCD8 transfectant DR is equally distributed over perimeter membrane and internal membranes, whereas DR is enriched on the perimeter membrane in the wtDO transfectant (Fig. 6GoB). The redistribution to the perimeter membrane is also seen for DM(/DO), going from 30% in Mel JuSo and the DOCD8 transfectant to 50% in the wtDO transfectant (Fig. 6GoB). The redistribution seems to be specific for DR and DM/DO complexes, since the distribution of the lysosomal marker CD63 residing in the same MIICs is not altered (data not shown). Apparently, the DO{beta} tail determines the intracompartment localization of the DM/DO complex as well as that of the interacting class II molecules (34, 35).



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  		FIGURE 6. Cytoplasmic tail of DO{beta} redistributes DR and DM to the perimeter membrane of the MVB and does not affect the efficacy of DM/DO action on class II peptide loading in a planar membrane. A, Immunoelectron microscope image of DM (10 nm gold) and class II (15 nm gold) distribution in a representative MVB in Mel JuSo, Mel JuSo/wtDO, and Mel JuSo/DOCD8 cells. Bar = 100 nm. B, Quantification of DM and class II distribution on intravesicular membranes vs the perimeter membrane of MVB. Gold particles representing class II and DM molecules were counted in 70–180 different MVBs within one experiment, and the experiment was repeated three times. C, Efficacy of class II/peptide loading at the plasma membrane of Mel JuSo, Mel JuSo/wtDO, and Mel JuSo/DOCD8 cells after treatment of the cells with 200 µM cycloheximide for 4 h in the absence or the presence of 20 µM biotinylated ApoB at the indicated pH for 2.5 h. Bound biotinylated ApoB was detected by streptavidin-conjugated PE, followed by FACS analysis.

 
How can the preferential localization of class II and DM(/DO) to the perimeter membrane of the multivesicular MIICs result in a situation that favors efficient class II peptide exchange? The interaction of class II molecules and DM (/DO) in vivo probably requires the presence of both proteins in the same cellular membrane (28). Since lateral transport between internal vesicles or from internal vesicles to the limiting membrane of MVBs is restrained (J. Calafat et al., manuscript in preparation), this implies that DM/DO on the internal vesicles can only interact with class II molecules within the same internal vesicle. On the other hand, the concurrent presence of class II and DM/DO on the much larger single-perimeter membrane allows a more efficient and less restrained lateral interaction between both molecules. To investigate the efficiency of catalysis of class II peptide loading of DM/DO complexes possessing or not possessing the DO{beta} tail in the same planar membrane, wtDO and DOCD8 transfectants were used in a cell surface peptide exchange assay. Cell surface class II and DM/DO reside in the same planar membrane and should therefore have the same capability for lateral interaction in both wtDO and DOCD8 transfectants. To enrich cell surface DM/DO complexes, cells were treated for 4 h with cycloheximide, as we noted previously that this impedes the efficiency of DM/DO internalization (data not shown), but not of exocytosis. FACS analysis demonstrated equal levels of DM/wtDO and DM/DOCD8 at the plasma membrane (data not shown). Cells were then incubated with or without the biotinylated antigenic peptide ApoB2877–2894 at the indicated pHs. Bound biotinylated peptides were detected with streptavidin-conjugated PE in FACS analysis. At pH 7, Mel JuSo, wtDO, and DOCD8 bind ApoB with similar efficiency (Fig. 6GoC), in line with the low catalytic activity of both DM and DM/DO at neutral pH (13). When incubated at pH 5, Mel JuSo shows a strong increase in the loading of ApoB, while the wtDO or DOCD8 transfectant show a reduced increase (Fig. 6GoC), in concordance with the inhibitory activity of DO on DM and the in vitro peptide exchange assays published (13). When wtDO and DOCD8 are compared, both are equally efficient in loading ApoB (Fig. 6GoC), again confirming that the catalytic activity of DM/DO is not affected by the absence of the DO{beta} tail. Thus, the difference in class II loading between wtDO and DOCD8 (Fig. 5Go) is relieved when the ability for lateral interaction between class II and DM/DO is the same. This strongly suggests that the DO{beta}-mediated redistribution of class II and DM toward the perimeter membrane of MIICs provides an optimal setting for lateral interaction between the DM/DO complex and class II molecules. As a consequence, this results in a more efficient class II peptide exchange.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The class II Ag presentation pathway presents an intriguing dilemma for the cellular sorting machinery, as class II molecules are expressed at the cell surface, while the function of the modulatory proteins DM and DO primarily lies within the MIIC. Here, we show that DM/DO complexes are constitutively transported to the plasma membrane along with class II molecules, rather than being actively retained in the MIIC. In contrast to class II, DM/DO complexes are rapidly internalized and routed back to the MIIC. The transient cell surface exposure of DM/DO explains why small amounts of DM are present at the cell surface of B cells and immature dendritic cells (36). Internalization of DM/DO complexes recaptures cell surface deposited complexes, thereby ensuring continuous replenishment of the DM/DO pool in the MIIC. However, complexes are also entering the MIIC from the biosynthetic route, and only blockage of both pathways ensues to empty the MIIC.

An important consequence of the continuous recycling of DM/DO is that these proteins traverse all compartments of the endocytic pathway before they accumulate in the MIIC. Thus, the modulatory activity of DM(/DO) on class II peptide loading is not confined to the MIIC, but extends to both the transport pathway from MIIC to plasma membrane as well as the entire endocytic pathway. This observation is reminiscent of the presence of DM in compartments other than the classical MIIC (37). The formation of class II/peptide complexes occurs in different compartments of the endosomal/lysosomal pathway; some epitopes are preferentially loaded onto class II molecules in early compartments, others in later compartments (38, 39, 40, 41). Although DM can potentially catalyze the loading of peptides in all these compartments, to date early endosomal class II peptide loading has been reported to be DM independent (40, 42, 43, 44). The present study demonstrates that this cannot be explained by the absence of DM from these compartments. The suboptimal pH in early endosomes for DM catalysis, and the high transit rate of proteins through early endosomes may account for the apparent DM-independent peptide loading in these compartments. B cells that express DO may have an additional mechanism to prevent DM-catalyzed peptide loading in early endosomes by the pH-dependent inhibition of DM by DO (12, 13). Indeed, peptide loading of Ags that are taken up by the B cell receptor occurs in acidified compartments and is dependent on newly synthesized class II molecules (45, 46, 47). B cells may therefore use DO to confine DM function to these compartments, as previously suggested (12).

Both class II molecules and DM/DO are directly sorted from the trans-Golgi network into the endosomal pathway. The class II molecules are targeted into the endosomal/lysosomal pathway via a di-leucine-based lysosomal targeting signal in the associated Ii (1). DM uses a different targeting signal (tyrosine-based) present in the DM{beta} cytoplasmic tail for lysosomal targeting (19, 20). We here demonstrate that DO{beta} contains two targeting motifs that have the potential to mediate lysosomal targeting, only when acting in consortium. This in contrast to the tyrosine- and di-leucine-based motifs described to date that are individually sufficient for lysosomal targeting (48). Recently, Brunet et al. (49) reported an exclusive role of the DO{beta} di-leucine motif for intracellular targeting of their class II reporter protein in HeLa cells. Their data also show that elimination of this di-leucine motif resulted in some cell surface expression of the class II reporter molecule, with a considerable fraction still colocalizing with LAMP-1 (e.g., Fig. 5Go, G–L) (49). It is possible that further elimination of the tyrosine residue would have resulted in exclusive cell surface expression. Our data clearly show that elimination of either the tyrosine or di-leucine motif is sufficient for cell surface expression of the CD8 reporter molecules with a concomitant exclusion from DO{alpha}{beta}GFP vesicles (Fig. 3GoC). Thus, the DM/DO complex may represent a unique example of a protein complex with multiple targeting signals, a tyrosine-based motif for DM{beta} and both a tyrosine and a di-leucine motif in the DO{beta}-chain.

The targeting information in DO{beta} is not required for correct lysosomal targeting of the DM/DO complex, since replacing the DO{beta} cytoplasmic domain for that of CD8 does not abolish lysosomal localization of the DM/DO complex. The lysosomal targeting signal of DM is probably sufficient for proper intracellular sorting of the DM/DO complex. These observations were recently confirmed by Brunet et al. (49). Although not required for targeting, the cytoplasmic tail of DO{beta} influences the efficiency of peptide exchange. These effects on Ag presentation correlate with the unique ability of DO to determine the localization of class II and DM within the multivesicular MIIC. Since DO is stably associated to DM (35), DO can easily codistribute DM molecules within the MVB. Apparently, class II molecules interact with DM/DO complexes at least transiently, because they are also redistributed.

How can the observed redistribution of class II molecules and DM, induced by the cytoplasmic domain of DO{beta} be beneficial for peptide exchange? The presence of a protein on either the perimeter membrane or the internal vesicles can determine further intracellular trafficking of such a protein. The epidermal growth factor receptor is internalized upon ligand binding and sorted toward the internal vesicles of MVBs, resulting in its degradation. Kinase-dead mutants of the epidermal growth factor receptor are also internalized, but are sorted to the perimeter membrane of MVBs, followed by recycling to the plasma membrane (50). Thus, the redistribution of class II molecules and DM/DO toward the perimeter membrane would be beneficial for directing class II molecules to the cell surface. The cell surface levels of class II and the total amount of class II, however, do not significantly differ between wtDO and the DOCD8 transfectant, probably because class II is resistant to lysosomal conditions.

Another explanation for the enhanced efficiency of peptide exchange in wtDO cells may be that redistribution of class II and DM/DO toward the perimeter membrane enhances the probability of interaction through free lateral mobility. Previous studies demonstrated that class II and DM need to physically interact to catalyze peptide exchange (28, 51). Given the total number of class II molecules (~100,000 as suggested for dendritic cells (52)) expressed by an APC and the 20:1 molar ratio of total class II and DM (53), a single MVB (~100/cell) contains approximately 50 DM molecules. This implies that only a subset of the internal vesicles will contain DM. Since the internal vesicles of a MVB may be relatively secluded subcompartments with restrained transport between internal vesicles (J. Calafat et al., manuscript in preparation), lateral interaction between class II and DM/DO on internal vesicles may be severely restricted. On the other hand, the concurrent presence of class II and DM/DO on the same perimeter membrane allows free lateral mobility and thereby enhances the probability of interaction. As demonstrated here, DM and DR indeed laterally interact, resulting in peptide exchange, consistent with the observation of small amounts of catalytically active DM at the cell surface (36). The redistribution of class II and DM(/DO) toward the perimeter membrane of the MIICs is reminiscent of a similar redistribution of class II molecules in Chediak-Higashi syndrome B cells (54). This redistribution correlates to an enhanced interaction between DM and class II molecules and a more efficient removal of CLIP (55).

In conclusion, we have now demonstrated that DM/DO complexes constitutively recycle between the plasma membrane and the MIIC. Although the DM/DO complex contains multiple targeting signals, retrieval of DM/DO complexes from the plasma membrane is primarily controlled by the lysosomal targeting signal in DM{beta}. The targeting information of DO{beta} is instead used for the distribution of class II and DM/DO within the MVB. This distribution determines the efficiency of peptide exchange and thereby forms a novel way to regulate class II peptide loading. Thus, we have defined here for the first time that a cytoplasmic domain (of DO{beta}) determines the localization of class II and DM within the MVB and regulates class II loading at the ultrastructural level.


   Acknowledgments
 
We thank P. Cresswell for the CERCLIP.1 Ab and the T2.DR3 cells, J. P. Johnson for the 16.23 Ab, and L. Karlsson for the anti-DM antiserum.


   Footnotes
 
1 This work was supported by a Pioneer Grant from Netherlands Organization for Scientific Research and Grant CT960069 from the European Community. Back

2 M.v.L. and M.v.H. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Marieke van Ham, Unit of Experimental Oncopathology, Department of Pathology, Free University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. E-mail address: sm.vanham@vumc.nl or jneefjes{at}nki.nl Back

4 Abbreviations used in this paper: Ii, invariant chain; ApoB, apolipoprotein B; BFA, brefeldin A; CLIP, class II-associated invariant chain peptides; CLSM, confocal laser scanning microscopy; ER, endoplasmic reticulum; GFP, green fluorescent protein; IRES, internal ribosomal entry site; MIIC, MHC class II compartment; MVB, multivesicular body; NLS, nuclear localization signal; wt, wild type. Back

Received for publication November 9, 2000. Accepted for publication May 11, 2001.


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 Materials and Methods
 Results
 Discussion
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