A role for HLA-DO as a co-chaperone of HLA-DM in peptide loading of MHC class II molecules

In B cells, the non-classical human leukocyte antigens HLA-DO (DO) and HLA-DM (DM) are residents of lysosome-like organelles where they form tight complexes. DM catalyzes the removal of invariant chain-derived CLIP peptides from classical major histocompatibility complex (MHC) class II molecules, chaperones them until peptides are available for loading, and functions as a peptide editor. Here we show that DO preferentially promotes loading of MHC class II molecules that are dependent on the chaperone activity of DM, and influences editing in a positive way for some peptides and negatively for others. In acidic compartments, DO is engaged in DR-DM-DO complexes whose physiological relevance is indicated by the observation that at lysosomal pH DM-DO stabilizes empty class II molecules more efficiently than DM alone. Moreover, expression of DO in a melanoma cell line favors loading of high-stability peptides. Thus, DO appears to act as a co-chaperone of DM, thereby controlling the quality of antigenic peptides to be presented on the cell surface.     

How HLA-DM affects the peptide repertoire bound to HLA-DR molecules

Considerable progress has been made in the field of major histocompatibility complex (MHC) class II-restricted antigen presentation. The analysis of mutant cell lines defective in antigen presentation revealed a central role for the nonclassical MHC class II molecule HLA-DM. Cell biological and biochemical characterization of HLA-DM provided deeper insight into the molecular mechanisms underlying the loading process: HLA-DM accumulates in acidic compartments, where it stabilizes classical class II molecules until a high-stability ligand occupies the class II peptide binding groove. Thus, HLA-DM prevents empty alpha beta dimers from functional inactivation at low endosomal/lysosomal pH in a chaperone-like fashion. In the presence of peptide ligands, HLA-DM acts as a catalyst for peptide loading by releasing CLIP, the residual invariant chain-derived fragment by which the invariant chain is associated with the class II molecules during transport from the endoplasmic reticulum to the loading compartments. Finally, there is accumulating evidence that HLA-DM functions as a peptide editor that removes low-stability ligands, thereby skewing the class II peptide repertoire toward high-stability alpha beta: peptide complexes presentable to T cells.     

Variation in HLA-DM expression influences conversion of MHC class II alpha beta:class II-associated invariant chain peptide complexes to mature peptide-bound class II alpha beta dimers in a normal B cell line

The maturation of invariant chain (Ii):MHC class II complexes into peptide-loaded alpha beta dimers occurs by proteolytic removal of Ii chain and binding of antigenic peptides derived from exogenous and endogenous Ags. A fragment of the Ii chain (class II-associated invariant chain peptide (CLIP) remains associated with class II alpha beta and is an intermediate in this process. Conversion of alpha beta:CLIP complexes into alpha beta:peptide complexes is facilitated by HLA-DM. Two unique mAbs, specific for I-Ab bound to human CLIP and I-Ab bound to DR alpha peptide, were used to assess the formation of these peptide:class II complexes in a human B lymphoblastoid cell line (B-LCL) (Swei) transfected with I-A(b). In multiple independent Swei:I-Ab transfectants, the amount of human CLIP (hCLIP):I-Ab expressed was inversely proportional to the amount of DR alpha 52-68:I-Ab; quantitative differences in HLA-DM expression accounted for this phenotype. In the low DM transfectant, a substantial proportion of I-Ab, but not DR molecules, was altered structurally and unable to present native protein Ags. Addition of DM transgenes to the DM-low cells resulted in an increase in DR alpha 52-68:I-Ab coupled with a decrease in hCLIP:I-Ab complexes and restoration of exogenous protein Ag presentation. The DR5 molecules in Swei cells, which have a lower affinity for hCLIP than I-Ab, were not affected by low DM expression, suggesting that the amount of DM required for conversion of CLIP:class II to peptide:class II may depend on the affinity of the class II molecules for CLIP or DM.     

Aberrant intermolecular disulfide bonding in a mutant HLA-DM molecule: implications for assembly, maturation, and function

HLA-DM (abbreviated DM) is an MHC-encoded glycoprotein that catalyzes the selective release of peptides, including class II-associated invariant chain peptides, from MHC class II molecules. To perform its function, DM must assemble in the endoplasmic reticulum (ER), travel to endosomes, and interact productively with class II molecules. We have described previously an EBV-transformed B cell line, 7.12.6, which displays a partial Ag presentation defect and expresses a mutated DM beta-chain with Cys79 replaced by Tyr. In this study, we show that HLA-DR molecules in 7.12.6 have a defect in peptide loading and accumulate class II-associated invariant chain peptides (CLIP). Peptide loading is restored by transfection of wild-type DMB. The mutant DM molecules exit the ER slowly and are degraded rapidly, resulting in greatly reduced levels of mutant DM in post-Golgi compartments. Whereas wild-type DM forms noncovalent alphabeta dimers, such dimers form inefficiently in 7.12.6; many mutant DM beta-chains instead form a disulfide-bonded dimer with DM alpha. Homodimers of DM beta are also detected in 7.12.6 and in the alpha-chain defective mutant, 2.2.93. We conclude that during folding of wild-type DM, the native conformation is stabilized by a conserved disulfide bond involving Cys79beta and by noncovalent contacts with DM alpha. Without these interactions, DM beta can form malfolded structures containing interchain disulfide bonds; malfolding is correlated with ER retention and accelerated degradation.     

DR/CLIP (class II-associated invariant chain peptides) and DR/peptide complexes colocalize in prelysosomes in human B lymphoblastoid cells

In APCs, MHC class II molecules (MHC class II) bind antigenic peptides after HLA-DM mediated removal of CLIP. To characterize intracellular sites of peptide loading in human B lymphoblastoid cell lines, we conducted immunoelectron microscopy studies with Abs recognizing MHC class II associated with CLIP or bound peptide, respectively, together with Abs to HLA-DM and endocytic markers. The distribution of these molecules indicates that peptide binding occurs in compartments with characteristics of normal late endosomes, and in compartments that show characteristics of late endosomes, but are not detectably accessed by endocytosed BSA-gold. The latter compartments may represent or give rise to recycling vesicles that deliver peptide-loaded class II molecules to the cell surface. In addition, we have compared cells in which HLA-DM and HLA-DR interaction is defective with cells in which this interaction is intact, and find that DM/DR interaction is not required for the proper localization of either molecule to peptide-loading compartments.     

MHC class II transport from lysosomal compartments to the cell surface is determined by stable peptide binding, but not by the cytosolic domains of the alpha- and beta-chains

Inside APCs, MHC class II molecules associate with antigenic peptides before reaching the cell surface. This association takes place in compartments of the endocytic pathway, more related to endosomes or lysosomes depending on the cell type. Here, we compared MHC class II transport from endosomal vs lysosomal compartments to the plasma membrane. We show that transport of MHC class II molecules to the cell surface does not depend on the cytosolic domains of the alpha- and beta-chains. In contrast, the stability of the alphabeta-peptide complexes determined the efficiency of transport to the cell surface from lysosomal, but not from endosomal, compartments. In murine B lymphoma cells, SDS-unstable and -stable complexes were transported to the cell surface at almost similar rates, whereas after lysosomal relocalization or in a cell line in which MHC class II molecules normally accumulate in lysosomal compartments, stable complexes were preferentially addressed to the cell surface. Our results suggest that when peptide loading occurs in lysosomal compartments, selective retention and lysosomal degradation of unstable dimers result in the expression of highly stable MHC class II-peptide complexes at the APC surface.     

Invariant chain cleavage and peptide loading in major histocompatibility complex class II vesicles

B lymphocytes contain a novel population of endocytic vesicles involved in the transport of newly synthesized major histocompatibility complex (MHC) class II alpha beta chains and alpha beta peptide complexes to the cell surface. We now present evidence that these class II-enriched vesicles (CIIV) are also likely to be a site for the loading of immunogenic peptides onto MHC molecules. We used the serine protease inhibitor leupeptin to accumulate naturally occurring intermediates in the degradation of alpha beta-invariant chain complexes and to slow the intracellular transport of class II molecules. As expected, leupeptin caused an accumulation of Ii chain and class II molecules (I-A(d)) in endosomes and lysosomes. More importantly, however, it enhanced the selective accumulation of a 10-kD invariant chain fragment associated with sodium dodecyl sulfate (SDS)-labile (empty) alpha beta dimers in CIIV. This was followed by the dissociation of the 10-kD fragment, formation of SDS-stable (peptide-loaded) alpha beta dimers, and their subsequent appearance at the cell surface. Thus, CIIV are likely to serve as a specialized site, distinct from endosomes and lysosomes, that hosts the final steps in the dissociation of invariant chain from class II molecules and the loading of antigen-derived peptides onto newly synthesized alpha beta dimers.     

A recombinant single-chain human class II MHC molecule (HLA-DR1) as a covalently linked heterotrimer of alpha chain, beta chain, and antigenic peptide, with immunogenicity in vitro and reduced affinity for bacterial superantigens

Major histocompatibility complex (MHC) class II molecules bind to numerous peptides and display these on the cell surface for T cell recognition. In a given immune response, receptors on T cells recognize antigenic peptides that are a minor population of MHC class II-bound peptides. To control which peptides are presented to T cells, it may be desirable to use recombinant MHC molecules with covalently bound antigenic peptides. To study T cell responses to such homogeneous peptide-MHC complexes, we engineered an HLA-DR1 cDNA coding for influenza hemagglutinin, influenza matrix, or HIV p24 gag peptides covalently attached via a peptide spacer to the N terminus of the DR1 beta chain. Co-transfection with DR alpha cDNA into mouse L cells resulted in surface expression of HLA-DR1 molecules that reacted with monoclonal antibodies (mAb) specific for correctly folded HLA-DR epitopes. This suggested that the spacer and peptide did not alter expression or folding of the molecule. We then engineered an additional peptide spacer between the C terminus of a truncated beta chain (without transmembrane or cytoplasmic domains) and the N terminus of full-length DR alpha chain. Transfection of this cDNA into mouse L cells resulted in surface expression of the entire covalently linked heterotrimer of peptide, beta chain, and alpha chain with the expected molecular mass of approximately 66 kDa. These single-chain HLA-DR1 molecules reacted with mAb specific for correctly folded HLA-DR epitopes, and identified one mAb with [MHC + peptide] specificity. Affinity-purified soluble secreted single-chain molecules with truncated alpha chain moved in electrophoresis as compact class II MHC dimers. Cell surface two-chain or single-chain HLA-DR1 molecules with a covalent HA peptide stimulated HLA-DR1-restricted HA-specific T cells. They were immunogenic in vitro for peripheral blood mononuclear cells. The two-chain and single-chain HLA-DR1 molecules with covalent HA peptide had reduced binding for the bacterial superantigens staphylococcal enterotoxin A and B and almost no binding for toxic shock syndrome toxin-1. The unique properties of these engineered HLA-DR1 molecules may facilitate our understanding of the complex nature of antigen recognition and aid in the development of novel vaccines with reduced superantigen binding.     

In vitro and in vivo evidence for high frequency of I-Ab-reactive CD4+ T cells in HLA-DQ or HLA-DRA transgenic mice lacking endogenous MHC class I and/or class II expression

Although T cells are educated to recognize foreign antigenic peptides in the context of self MHC molecules during their development in the thymus, peripheral T cells also recognize allo- and xeno-MHC molecules. The lower frequency of xeno-MHC-reactive T cells than that of allo-MHC-reactive T cells is often explained by the difference in the degree of homology between xeno- or allo-MHC and self MHC molecules, as well as by the species barrier of the molecules involved in immune recognition. To distinguish these two possibilities, we estimated the frequency of I-Ab-reactive CD4+ T cells selected by HLA-DQ or DR alpha E beta b molecules, using HLA-DQ6 and HLA-DRA transgenic C57BL/6 (B6) mice lacking endogenous MHC class I and/or class II molecules (DQ6A0/0 and DR alpha 30A0/0 beta 20/0). CD4+ lymph node T cells from DQ6A0/0 and DR alpha 30A0/0 beta 20/0 showed the strong proliferative response to I-Ab molecules. In addition, DQ6A0/0 and DR alpha 30A0/0 beta 20/0 rejected the skin graft from mice expressing I-Ab molecules irrespective of MHC class I expression, indicating that the CD4+ T cells recognizing I-Ab molecules are directly involved in this rejection. The estimated frequency of I-Ab-reactive CD4(+)CD8- thymocytes in DR alpha 30A0/0 beta 20/0 and DQ6A0/0 was comparable with that observed in the MHC class II-disparate strains. Our findings thus indicate that CD4+ T cells selected to mature on xeno-MHC class II molecules such as HLA-DQ6 or DR alpha E beta b, when these molecules are expressed in mice, recognize I-Ab molecules as allo-MHC class II, despite the less structural homology.     

Craniopharyngioma in children. Surgical treatment by a transbasal anterior approach

The proper folding and assembly of major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum (ER) is an intricate process involving a number of components. Nascent heavy chains of MHC class I molecules, translocated into the ER membrane, are rapidly glycosylated and bind the transmembrane chaperone calnexin. In humans, after dissociation from calnexin, fully oxidized MHC class I heavy chains associate with beta 2-microglobulin (beta 2m) and the soluble chaperone calreticulin. This complex interacts with another transmembrane protein, tapasin, which is believed to assist in MHC class I folding as well as in mediating the interaction between assembling MHC class I molecules and the transporter associated with antigen processing (TAP). The TAP heterodimer (TAP1-TAP2) introduces the final component of the MHC class I molecule by translocating peptides, predominately generated by the proteasome, from the cytosol into the ER where they can bind dimers of beta 2M and the MHC class I heavy chain. Recently, the thiol oxidoreductase ERp57--also known as GRP58, ERp61, ER60, Q2, HIP-70, and CPT and first misidentified as phospholipase C-alpha--has been shown to bind in conjunction with calnexin or calreticulin to a number of newly synthesized ER glycoproteins when their N-linked glycans are trimmed by glucosidases I and II. It was speculated that ERp57 is a generic component of the glycan-dependent ER quality control system. Here, we show that ERp57 is a component of the MHC class I peptide-loading complex. ERp57 might influence the folding of MHC class I molecules at a critical step in peptide loading.     

Indices of carbohydrate and lipid metabolism in vasopressin-replete and -deficient New Zealand genetically hypertensive rats

Intracellular antigens are continually presented to cytotoxic T lymphocytes by major histocompatibility complex (MHC) class I molecules, which consist of a polymorphic 43 kDa heavy chain and a 12 kDa soluble subunit beta 2-microglobulin (beta 2m), and which bind an 8-10 amino-acid antigenic peptide. The assembly of this trimolecular complex takes place in the lumen of the endoplasmic reticulum (ER) and almost certainly requires cofactors. Most MHC class I molecules in the ER that have not yet acquired peptide are simultaneously bound to the transporter associated with antigen processing (TAP), to the 48 kDa glycoprotein tapasin and to the lectin-like chaperone calreticulin, in a multicomponent 'loading complex'. Previous studies have shown that a mutant MHC class I molecule T134K (in which Thr134 was changed to Lys) fails to bind to TAP. Here, we show that this point mutation also disrupted, directly or indirectly, the interaction between MHC class I molecules and calreticulin. T134K molecules did not present viral antigens to T cells even though they bound peptide and beta 2m normally in vitro. They exited the ER rapidly as 'empty' MHC class I complexes, unlike empty wild-type molecules which are retained in the ER and degraded. We show here that, paradoxically, the rapid exit of empty T134K molecules from the ER was dependent on a TAP-derived supply of peptides. This implies that MHC class I assembly is a two-stage process: initial binding of suboptimal peptides is followed by peptide optimisation that depends on temporary ER retention.     

The tetraspan protein CD82 is a resident of MHC class II compartments where it associates with HLA-DR, -DM, and -DO molecules

In specialized APCs, MHC class II molecules are synthesized in the endoplasmic reticulum and transported through the Golgi apparatus to organelles of the endocytic pathway collectively called MHC class II compartments (MIICs). There, the class II-associated invariant chain is degraded, and peptides derived from internalized Ag bind to empty class II in a reaction that is facilitated by the class II-like molecule HLA-DM. An mAb raised to highly purified, immunoisolated MIICs from human B lymphoblastoid cells recognized CD82, a member of the tetraspan family of integral membrane proteins. Subcellular fractionation, immunofluorescence microscopy, and immunoelectron microscopy showed that CD82 is highly enriched in MIICs, particularly in their internal membranes. Coprecipitation analysis showed that CD82 associates in MIICs with class II, DM, and HLA-DO (an inhibitor of peptide loading that binds DM). Similar experiments showed CD63, another tetraspan protein found in MIICs, also associates with these molecules in the compartment and that CD82 and CD63 associate with each other. Preclearing experiments demonstrated that both CD82 and CD63 form complexes with DM-associated class II and DM-associated DO. The ability of CD82 and CD63 to form complexes with class II, DM, and DO in MIICs suggests that the tetraspan proteins may play an important role in the late stages of MHC class II maturation.     

Enhanced interaction of HLA-DM with HLA-DR in enlarged vacuoles of hereditary and infectious lysosomal diseases

Following biosynthesis, class II MHC molecules are transported through a lysosome-like compartment, where they acquire antigenic peptides for presentation to T cells at the cell surface. This compartment is characterized by the presence of HLA-DM, which catalyzes the peptide loading process. Here we report that the morphology and function of the class II loading compartment is affected in diseases with a phenotypic change in lysosome morphology. Swollen lysosomes are observed in cells from patients with the hereditary immunodeficiency Chediak-Higashi syndrome and in cells infected with Coxiella burnetii, the rickettsial organism that causes Q fever. In both disease states, we observed that HLA-DR and HLA-DM accumulate in enlarged intracellular compartments, which label with the lysosomal marker LAMP-1. The distribution of class I MHC molecules was not affected, localizing disease effects to the endocytic pathway. Thus, cellular mechanisms controlling lysosome biogenesis also affect formation of the class II loading compartment. Analysis of cell surface class II molecules revealed that their steady-state levels were not reduced on diseased cells. However, in both disease states, enhanced interaction between HLA-DR and HLA-DM was detected. In the Chediak-Higashi syndrome cells, this correlated with more efficient removal of the CLIP peptide. These findings suggest a mechanism for perturbation of Ag presentation by class II molecules and consequent immune deficiencies in both diseases.     

Bacterial enteritides of poultry

We report an experimental system for abundant expression of specific peptide-class II complexes in vivo and in vitro. We have constructed a cassette which allows for the replacement of the CLIP region of invariant chain (Ii) with an antigenic peptide. In fibroblasts expressing an altered Ii protein, in which CLIP has been replaced with peptide 52-68 from the class II I-E alpha chain (pEalpha), pEalpha-I-Ab complexes are formed with high efficiency. This peptide loading occurs in the endoplasmic reticulum (ER) when the Ii:pEalpha fusion protein associates with the I-Ab alpha and beta chains. The trimeric complexes of Ii:pEalpha and I-Ab molecules are stable in SDS and can be detected by the pEalpha-I-Ab-specific mAb, YAe, indicating that pEalpha is bound in the class II groove in the context of full-length Ii. These data strongly suggest that the CLIP region of intact Ii prevents peptide loading in the ER by binding in the peptide binding groove of newly synthesized class II alphabeta dimers.     

Quantitative three-dimensional assessment of face-lift with an optical facial surface scanner

We have established a strain of transgenic mice in which the HLA-DRA gene was integrated into the X-chromosome and the xenogeneic mixed isotype molecule, DR alpha E beta b, was expressed in a cell type-specific manner, although the transgenic DRA gene contained only 268 base pairs of the 5'-flanking region. The DR alpha E beta b molecules expressed in the transgenic mice functioned as major histocompatibility complex (MHC) class II to select T-cell repertoire, and to stimulate mixed lymphocyte reaction. In female transgenic mice homozygous for HLA-DRA (DR alpha-B6-F-homo) and male transgenic mice (DR alpha-B6-M), DR alpha E beta b molecules were expressed in almost all of the MHC class II Ab-positive cells. In contrast, the expression of DR alpha E beta b molecules in female transgenic mice hemizygous for HLA-DRA (DR alpha-B6-F-hemi) was found only in part of the Ab positive cells, and the proportion of cells expressing the DR alpha E beta b molecules varied due to random inactivation of one of the X-chromosomes. Clonal deletions of the T cells and mature thymocytes bearing Tcrb-V5 and Tcrb-V11, which are eliminated from the peripheral repertoire in mice expressing self-superantigen and MHC class II E molecules, were incomplete in DR alpha-B6-F-hemi as compared with those in DR alpha-B6-F-homo, and were correlated with the proportion of DR alpha E beta b-positive spleen cells. These observations suggested that the number of bone marrow-derived cells expressing DR alpha E beta b molecules was critical for clonal deletions of Tcrb-V5+ and Tcrb-V11+ T cells in the thymus.     

Degradation of mouse invariant chain: roles of cathepsins S and D and the influence of major histocompatibility complex polymorphism

Antigen-presenting cells (APC) degrade endocytosed antigens into peptides that are bound and presented to T cells by major histocompatibility complex (MHC) class II molecules. Class II molecules are delivered to endocytic compartments by the class II accessory molecule invariant chain (Ii), which itself must be eliminated to allow peptide binding. The cellular location of Ii degradation, as well as the enzymology of this event, are important in determining the sets of antigenic peptides that will bind to class II molecules. Here, we show that the cysteine protease cathepsin S acts in a concerted fashion with other cysteine and noncysteine proteases to degrade mouse Ii in a stepwise fashion. Inactivation of cysteine proteases results in incomplete degradation of Ii, but the extent to which peptide loading is blocked by such treatment varies widely among MHC class II allelic products. These observations suggest that, first, class II molecules associated with larger Ii remnants can be converted efficiently to class II-peptide complexes and, second, that most class II-associated peptides can still be generated in cells treated with inhibitors of cysteine proteases. Surprisingly, maturation of MHC class II in mice deficient in cathepsin D is unaffected, showing that this major aspartyl protease is not involved in degradation of Ii or in generation of the bulk of antigenic peptides.     

The assembly and stability of MHC class II-(alpha beta)2 superdimers

X-ray crystallography of several MHC class II molecules revealed a structure described as a dimer of heterodimers, or a superdimer. This discovery led to the hypothesis that MHC class II molecules may interact with the TCR and CD4 as an (alpha beta)2 superdimer, potentially providing more stable and stimulatory interactions than can be provided by the simple alpha beta heterodimer alone. In this study, using chemical cross-linking, we provide evidence for the existence of the superdimers surface of B cells. We further characterize the superdimers and demonstrate that in lysates of B cells, I-Ek dimers and superdimers are derived from the same population of I-Ek molecules. Purified, I-Ek molecules in solution also exist as a mixture of 60-kDa dimers and 120-kDa superdimers, indicating that I-Ek has an intrinsic ability to form 120-kDa complexes in the absence of other cellular components. Peptide mapping showed that the alpha beta and (alpha beta)2 complexes are closely related and that the superdimers do not contain additional polypeptides not present in the dimers. The (alpha beta)2 complex displays thermal and pH stability similar to that of the alpha beta complex, both being denatured by SDS at temperatures above 50 degrees C and at a pH below 5. These data support the model that MHC class II has an intrinsic ability to assume the (alpha beta)2 superdimeric conformation, which may be important for interactions with the TCR and CD4 coreceptor.     

Intracellular pathway for the generation of functional MHC class II peptide complexes in immature human dendritic cells

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