MHC class II compartments in human dendritic cells undergo profound structural changes upon activation

Immature dendritic cells efficiently capture exogenous antigens in peripheral tissues. In an inflammatory environment, dendritic cells are activated and become highly competent antigen-presenting cells. Upon activation, they lose their ability for efficient endocytosis and gain capability to migrate to secondary lymphoid organs. In addition, peptide loading of MHC class II molecules is enhanced and MHC class II/peptide complexes are redistributed from an intracellular location to the plasma membrane. Using immuno-electron microscopy, we show that activation of human monocyte-derived dendritic cells induced striking modifications of the lysosomal multilaminar MHC class II compartments (MIICs), whereby electron-dense tubules and vesicles emerged from these compartments. Importantly, we observed that MHC class II expression in these tubules/vesicles transiently increased, while multilaminar MIICs showed a strongly reduced labeling of MHC class II molecules. This suggests that formation of the tubules/vesicles from multilaminar MIICs could be linked to transport of MHC class II from these compartments to the cell surface. Further characterization of endocytic organelles with lysosomal marker proteins, such as the novel dendritic cell-specific lysosomal protein DC-LAMP, HLA-DM and CD68, revealed differential sorting of these markers to the tubules and vesicles .     

Reorganization of multivesicular bodies regulates MHC class II antigen presentation by dendritic cells

Immature dendritic cells (DCs) sample their environment for antigens and after stimulation present peptide associated with major histocompatibility complex class II (MHC II) to naive T cells. We have studied the intracellular trafficking of MHC II in cultured DCs. In immature cells, the majority of MHC II was stored intracellularly at the internal vesicles of multivesicular bodies (MVBs). In contrast, DM, an accessory molecule required for peptide loading, was located predominantly at the limiting membrane of MVBs. After stimulation, the internal vesicles carrying MHC II were transferred to the limiting membrane of the MVB, bringing MHC II and DM to the same membrane domain. Concomitantly, the MVBs transformed into long tubular organelles that extended into the periphery of the cells. Vesicles that were formed at the tips of these tubules nonselectively incorporated MHC II and DM and presumably mediated transport to the plasma membrane. We propose that in maturing DCs, the reorganization of MVBs is fundamental for the timing of MHC II antigen loading and transport to the plasma membrane.     

MHC class II molecules traffic into lipid rafts during intracellular transport

There have been many studies demonstrating that a portion of MHC class II molecules reside in detergent-insoluble membrane domains (commonly referred to as lipid rafts). We have proposed that the function of raft association is to concentrate specific MHC class II-peptide complexes in plasma membrane microdomains that can facilitate efficient T cell activation. We now show that MHC class II becomes lipid raft associated before binding antigenic peptides. Using pulse-chase radiolabeling techniques, we find that newly synthesized MHC class II and MHC class II-invariant chain complexes initially reside in a detergent-soluble membrane fraction and acquire detergent insolubility as they traffic to lysosomal Ag processing compartments. Monensin, an inhibitor of protein transport through the Golgi apparatus, blocks association of newly synthesized MHC class II with lipid rafts. Treatment of cells with leupeptin, which inhibits invariant chain degradation, leads to the accumulation of MHC class II in lipid rafts within the lysosome-like Ag-processing compartments. Raft fractionation of lysosomal membranes confirmed the presence of MHC class II in detergent-insoluble microdomains in Ag-processing compartments. These findings indicate that newly synthesized MHC class II complexes are directed to detergent-insoluble lipid raft microdomains before peptide loading, a process that may facilitate the loading of similar peptides on MHC class II complexes in these microdomains.     

The biogenesis of the MHC class II compartment in human I-cell disease B lymphoblasts

The localization and intracellular transport of major histocompatibility complex (MHC) class II molecules nd lysosomal hydrolases were studied in I-Cell Disease (ICD) B lymphoblasts, which possess a mannose 6-phosphate (Man-6-P)-independent targeting pathway for lysosomal enzymes. In the trans-Golgi network (TGN), MHC class II- invariant chain complexes colocalized with the lysosomal hydrolase cathepsin D in buds and vesicles that lacked markers of clathrin-coated vesicle-mediated transport. These vesicles fused with the endocytic pathway leading to the formation of "early" MHC class II-rich compartments (MIICs). Similar structures were observed in the TGN of normal beta lymphoblasts although they were less abundant. Metabolic labeling and subcellular fractionation experiments indicated that newly synthesized cathepsin D and MHC class II-invariant chain complexes enter a non-clathrin-coated vesicular structure after their passage through the TGN and segregation from the secretory pathway. These vesicles were also devoid of the cation-dependent mannose 6-phosphate (Man-6-P) receptor, a marker of early and late endosomes. These findings suggest that in ICD B lymphoblasts the majority of MHC class II molecules are transported directly from the TGN to "early" MIICs and that acid hydrolases cam be incorporated into MIICs simultaneously by a Man-6-P-independant process.     

Regulation of MHC class II antigen presentation by sorting of recycling HLA-DM/DO and class II within the multivesicular body.

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 DMbeta and the class II-associated invariant chain, the DObeta cytoplasmic tail contains potential lysosomal targeting signals. The DObeta signals, however, are not essential for internalization of the DM/DO complex from the plasma membrane or targeting to the MIICs. Instead, the DObeta 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.     

CD1 molecules efficiently present antigen in immature dendritic cells and traffic independently of MHC class II during dendritic cell maturation

Upon exposure to Ag and inflammatory stimuli, dendritic cells (DCs) undergo a series of dynamic cellular events, referred to as DC maturation, that involve facilitated peptide Ag loading onto MHC class II molecules and their subsequent transport to the cell surface. Besides MHC molecules, human DCs prominently express molecules of the CD1 family (CD1a, -b, -c, and -d) and mediate CD1-dependent presentation of lipid and glycolipid Ags to T cells, but the impact of DC maturation upon CD1 trafficking and Ag presentation is unknown. Using monocyte-derived immature DCs and those stimulated with TNF-alpha for maturation, we observed that none of the CD1 isoforms underwent changes in intracellular trafficking that mimicked MHC class II molecules during DC maturation. In contrast to the striking increase in surface expression of MHC class II on mature DCs, the surface expression of CD1 molecules was either increased only slightly (for CD1b and CD1c) or decreased (for CD1a). In addition, unlike MHC class II, DC maturation-associated transport from lysosomes to the plasma membrane was not readily detected for CD1b despite the fact that both molecules were prominently expressed in the same MIIC lysosomal compartments before maturation. Consistent with this, DCs efficiently presented CD1b-restricted lipid Ags to specific T cells similarly in immature and mature DCs. Thus, DC maturation-independent pathways for lipid Ag presentation by CD1 may play a crucial role in host defense, even before DCs are able to induce maximum activation of peptide Ag-specific T cells.     

Double labelling of major histocompatability complex molecules and lysosomal protein lamp-1 on human dendritic cells

Summary In this study, double labelling for major histocompatability complex (MHC) class I and class II molecules and for MHC molecules and the lysosomal membrane protein lamp-1 on ultrathin cryosections of dendritic cells isolated from human peripheral blood was performed. The plasma membrane proved to be positive for both MHC class I and MHC class II molecules and was labelled for only a very few lamp-1 molecules. MHC class I and MHC class II molecules did not co-localize intracellularly except in some peripherally located vesicles. However, many MHC class II-labelled vesicles were present in a juxtanuclear position but only some of them were co-labelled for lamp-1. These results indicate the presence of a separate, non-lysosomal compartment for class II molecules in dendritic cells.     

MHC class II-associated invariant chain contains a sorting signal for endosomal compartments

The invariant chain (Ii) is a transmembrane protein that associates with the MHC class II molecules in the endoplasmic reticulum. Expression of Ii in MHC class II-negative CV1 cells showed that it acquired complex-type oligosaccharide side chains and was retained in endosomal compartments. To search for a sorting signal, we made progressive deletions from the cytoplasmic N-terminus of Ii. Deleting 11 amino acid residues resulted in a protein that was still sorted and retained in endosomal vesicles, whereas deletion of 15 or more amino acid residues resulted in a protein that became resident in the plasma membrane. Amino acids 12-15 are thus essential for intracellular transport to endosomal compartments. As Ii is intracellularly associated with the MHC class II molecules, it is proposed that Ii determines the intracellular transport route of these molecules.     

Transport and intracellular distribution of MHC class II molecules and associated invariant chain in normal and antigen-processing mutant cell lines

We have compared the intracellular transport and subcellular distribution of MHC class II-invariant chain complexes in a wild-type HLA-DR3 homozygous cell line and a mutant cell line, T2.DR3. The latter has a defect in antigen processing and accumulates HLA-DR3 molecules associated with an invariant chain-derived peptide (CLIP) rather than the normal complement of peptides derived from endocytosed proteins. We find that in the wild-type cells, CLIP is transiently associated with HLA-DR3 molecules, suggesting that the peptide is a normal class II- associated intermediate generated during proteolysis of the invariant chain. In the mutant cell line proteolysis of the invariant chain is less efficient, and HLA-DR3/CLIP complexes are generated much more slowly. Examination of the mutant cell line by immunoelectronmicroscopy shows that class II-invariant chain complexes accumulate intracellularly in large acidic vesicles which contain lysosomal markers, including beta-hexosaminidase, cathepsin D, and the lysosomal membrane protein CD63. The markers in these vesicles are identical to those seen in the class II-containing vesicles (MIICs) seen in the wild- type cells but the morphology is drastically different. The vesicles in the mutant cells are endocytic, as measured by the internalization of BSA-gold conjugates. The implication of these findings for antigen processing in general and the nature of the mutation in particular are discussed.     

Functional early endosomes are required for maturation of major histocompatibility complex class II molecules in human B lymphoblastoid cells.

Major histocompatibility complex (MHC) class II molecules are targeted together with their invariant chain (Ii) chaperone from the secretory pathway to the endocytic pathway. Within the endosome/lysosome system, Ii must be degraded to enable peptide capture by MHC class II molecules. It remains controversial exactly which route or routes MHC class II/Ii complexes take to reach the sites of Ii processing and peptide loading. We have asked whether early endosomes are required for successful maturation of MHC class II molecules by using an in situ peroxidase/diaminobenzidine compartment ablation technique. Cells whose early endosomes were selectively ablated using transferrin-horseradish peroxidase conjugates fail to mature their newly synthesized MHC class II molecules. We show that whereas transport of secretory Ig through the secretory pathway is virtually normal in the ablated cells, newly synthesized MHC class II/Ii complexes never reach compartments capable of processing Ii. These results strongly suggest that the transport of the bulk of newly synthesized MHC class II molecules through early endosomes is obligatory and that direct input into later endosomes/lysosomes does not take place.     

Antigen loading of MHC class I molecules in the endocytic tract

Major histocompatibility complex (MHC) class I molecules bind antigenic peptides that are translocated from the cytosol into the endoplasmic reticulum by the transporter associated with antigen processing. MHC class I loading independent of this transporter also exists and involves peptides derived from exogenously acquired antigens. Thus far, a detailed characterization of the intracellular compartments involved in this pathway is lacking. In the present study, we have used the model system in which peptides derived from measles virus protein F are presented to cytotoxic T cells by B-lymphoblastoid cells that lack the peptide transporter. Inhibition of T cell activation by the lysosomotropic drug ammoniumchloride indicated that endocytic compartments were involved in the class I presentation of this antigen. Using immunoelectron microscopy, we demonstrate that class I molecules and virus protein F co-localized in multivesicular endosomes and lysosomes. Surprisingly, these compartments expressed high levels of class II molecules, and further characterization identified them as MHC class II compartments. In addition, we show that class I molecules co-localized with class II molecules on purified exosomes, the internal vesicles of multivesicular endosomes that are secreted upon fusion of these endosomes with the plasma membrane. Finally, dendritic cells, crucial for the induction of primary immune responses, also displayed class I in endosomes and on exosomes.     

CD1 and major histocompatibility complex II molecules follow a different course during dendritic cell maturation

The maturation of dendritic cells is accompanied by the redistribution of major histocompatibility complex (MHC) class II molecules from the lysosomal MHC class II compartment to the plasma membrane to mediate presentation of peptide antigens. Besides MHC molecules, dendritic cells also express CD1 molecules that mediate presentation of lipid antigens. Herein, we show that in human monocyte-derived dendritic cells, unlike MHC class II, the steady-state distribution of lysosomal CD1b and CD1c isoforms was unperturbed in response to lipopolysaccharide-induced maturation. However, the lysosomes in these cells underwent a dramatic reorganization into electron dense tubules with altered lysosomal protein composition. These structures matured into novel and morphologically unique compartments, here termed mature dendritic cell lysosomes (MDL). Furthermore, we show that upon activation mature dendritic cells do not lose their ability of efficient clathrin-mediated endocytosis as demonstrated for CD1b and transferrin receptor molecules. Thus, the constitutive endocytosis of CD1b molecules and the differential sorting of MHC class II from lysosomes separate peptide- and lipid antigen-presenting molecules during dendritic cell maturation.     

Endosomal sorting of MHC class II determines antigen presentation by dendritic cells

Dendritic cells (DCs) initiate primary immune responses by presenting pathogen-derived antigens in association with major histocompatibility Class II molecules (MHC II) to T cells. In DCs, MHC II is constitutively synthesized and loaded at endosomes with peptides from hydrolyzed endogenous proteins or exogenously acquired antigens. Whether peptide loaded MHC II (MHC II-p) is subsequently recruited to and stably expressed at the plasma membrane or degraded in lysosomes is determined by the status of the DC. In immature DCs, MHC II-p is ubiquitinated after peptide loading, driving its sorting to the luminal vesicles of multivesicular bodies. These luminal vesicles, and the MHC II-p they carry, are delivered to lysosomes for degradation. MHC II-p is inefficiently ubiquitinated in DCs that are activated by pathogens or inflammatory stimuli, thus allowing its transfer to and stable expression at the plasma membrane.     

Regulation of intracellular trafficking of human CD1d by association with MHC class II molecules

CD1 family members are antigen-presenting molecules capable of presenting bacterial or synthetic glycolipids to T cells. Here we show that a subset of human CD1d molecules are associated with major histocompatibility complex (MHC) class II molecules, both on the cell surface and in the late endosomal/lysosomal compartments where class II molecules transiently accumulate during transport. The interaction is initiated in the endoplasmic reticulum with class II-invariant chain complexes and appears to be maintained throughout the class II trafficking pathway. A truncated form of CD1d which lacks its cytoplasmic YXXZ internalization motif is transported to late endosomal/lysosomal compartments in the presence of class II molecules. Furthermore, the same CD1d deletion mutant is targeted to lysosomal compartments in HeLa cells expressing class II molecules and invariant chain by transfection. The deletion mutant was also found in lysosomal compartments in HeLa cells expressing only the p33 form of the invariant chain. These data suggest that the intracellular trafficking pathway of CD1d may be altered by class II molecules and invariant chain induced during inflammation.     

Assembly and transport of MHC class II molecules.

At the surface of antigen-presenting cells MHC class I and class II molecules present peptides to respectively CD8+ and CD4+ T cells. MHC class I molecules acquire peptides right after synthesis in the endoplasmic reticulum. MHC class II molecules do not acquire peptides in the endoplasmic reticulum but instead associate with a third chain, the invariant chain which impedes peptide binding. Subsequently the invariant chain takes MHC class II molecules to the endosomal/lysosomal compartment thanks to a targeting signal retained in its cytoplasmic tail. It then dissociates from the MHC class II dimer to allow it to bind peptides.     

Identification of a lysosomal peptide transport system induced during dendritic cell development

The delivery of protein fragments to major histocompatibility complex (MHC)-loading compartments of professional antigen-presenting cells is essential in the adaptive immune response against pathogens. Apart from the crucial role of the transporter associated with antigen processing (TAP) for peptide loading of MHC class I molecules in the endoplasmic reticulum, TAP-independent translocation pathways have been proposed but not identified so far. Based on its overlapping substrate specificity with TAP, we herein investigated the ABC transporter ABCB9, also named TAP-like (TAPL). Remarkably, TAPL expression is strongly induced during differentiation of monocytes to dendritic cells and to macrophages. TAPL does not, however, restore MHC class I surface expression in TAP-deficient cells, demonstrating that TAPL alone or in combination with single TAP subunits does not form a functional transport complex required for peptide loading of MHC I in the endoplasmic reticulum. In fact, by using quantitative immunofluorescence and subcellular fractionation, TAPL was detected in the lysosomal compartment co-localizing with the lysosome-associated membrane protein LAMP-2. By in vitro assays, we demonstrate a TAPL-specific translocation of peptides into isolated lysosomes, which strictly requires ATP hydrolysis. These results suggest a mechanism by which antigenic peptides have access to the lysosomal compartment in professional antigen-presenting cells.     

Functional characterization of a lysosomal sorting motif in the cytoplasmic tail of HLA-DObeta

HLA-DO is an intracellular non-classical class II major histocompatibility complex molecule expressed in the endocytic pathway of B lymphocytes, which regulates the loading of antigenic peptides onto classical class II molecules such as HLA-DR. The activity of HLA-DO is mediated through its interaction with the peptide editor HLA-DM. Here, our results demonstrate that although HLA-DO is absolutely dependent on its association with DM to egress the endoplasmic reticulum, the cytoplasmic portion of its beta chain encodes a functional lysosomal sorting signal. By confocal microscopy and flow cytometry analysis, we show that reporter transmembrane molecules fused to the cytoplasmic tail of HLA-DObeta accumulated in Lamp-1(+) vesicles of transfected HeLa cells. Mutagenesis of a leucine-leucine motif abrogated lysosomal accumulation and resulted in cell surface redistribution of reporter molecules. Finally, we show that mutation of the di-leucine sequence in DObeta did not alter its lysosomal sorting when associated with DM molecules. Taken together, these results demonstrate that lysosomal expression of the DO-DM complex is mediated primarily by the tyrosine-based motif of HLA-DM and suggest that the DObeta-encoded motif is involved in the fine-tuning of the intracellular sorting.     

Polarized transport of MHC class II molecules in Madin-Darby canine kidney cells is directed by a leucine-based signal in the cytoplasmic tail of the beta-chain.

MHC class II molecules are found on the basolateral plasma membrane domain of polarized epithelial cells, where they can present Ag to intraepithelial lymphocytes in the vascular space. We have analyzed the sorting information required for efficient intracellular localization and polarized distribution of MHC class II molecules in stably transfected Madin-Darby canine kidney cells. These cells were able to present influenza virus particles to HLA-DR1-restricted T cell clones. Wild-type MHC class II molecules were located on the basolateral plasma membrane domain, in basolateral early endosomes, and in late multivesicular endosomes, the latter also containing the MHC class II-associated invariant chain and an HLA-DM fusion protein. A phenylalanine-leucine residue within the cytoplasmic tail of the beta-chain was required for basolateral distribution, efficient internalization, and localization of the MHC class II molecules to basolateral early endosomes. However, distribution to apically located, late multivesicular endosomes did not depend on signals in the class II cytoplasmic tails as both wild-type class II molecules and mutant molecules lacking the phenylalanine-leucine motif were found in these compartments. Our results demonstrate that sorting information in the tails of class II dimers is an absolute requirement for their basolateral surface distribution and intracellular localization.     

Interaction of Mycobacterium avium-containing phagosomes with the antigen presentation pathway

Pathogenic mycobacteria infect macrophages where they replicate in phagosomes that minimize contact with late endosomal/lysosomal compartments. Loading of Ags to MHC class II molecules occurs in specialized compartments with late endosomal characteristics. This points to a sequestration of mycobacteria-containing phagosomes from the sites where Ags meet MHC class II molecules. Indeed, in resting macrophages MHC class II levels decreased strongly in phagosomes containing M. avium during a 4-day infection. Phagosomal MHC class II of early (4 h) infections was partly surface-derived and associated with peptide. Activation of host macrophages led to the appearance of H2-M, a chaperon of Ag loading, and to a strong increase in MHC class II molecules in phagosomes of acute (1 day) infections. Comparison with the kinetics of MHC class II acquisition by IgG-coated bead-containing phagosomes suggests that the arrest in phagosome maturation by mycobacteria limits the intersection of mycobacteria-containing phagosomes with the intracellular trafficking pathways of Ag-presenting molecules.     

MHC class II invariant chain homologues in rainbow trout (Oncorhynchus mykiss)

The MHC class II invariant chain (Ii or CD74) in higher vertebrates is necessary for normal MHC class II loading in endosomal compartments. Detection of an Ii chain in fish would greatly support the idea that MHC class II function in fish and higher vertebrates is similar. Before this study only Ii homologues had been reported in fish that are unlikely to perform true Ii function. In the present study two Ii-like genes, Onmy-Iclp-1 and Onmy-Iclp-2, were detected in rainbow trout. Conservation of elements, particularly in Onmy-Iclp-1, suggests that the encoded proteins may be involved in MHC class II transport and peptide loading as is the Ii protein. The expression pattern of both rainbow trout genes was similar to that of the MHC class II beta chain, with strong expression in the lymphoid tissues, gills and intestine. Analysis of separated peripheral blood leucocyte fractions indicated that expression of Onmy-Iclp-1, Onmy-Iclp-2 and the MHC class II beta chain were all highest in B lymphocytes. This agrees with the expectation that the functions of the products of the new genes are closely associated with MHC class II. It is interesting why in rainbow trout there are two proteins that may function similar to Ii in higher vertebrates.