Functional HLA-DM on the surface of B cells and immature dendritic cells

HLA-DM (DM) plays a critical role in antigen presentation through major histocompatibility complex (MHC) class II molecules. DM functions as a molecular chaperone by keeping class II molecules competent for antigenic peptide loading and serves as an editor by favoring presentation of high-stability peptides. Until now, DM has been thought to exert these activities only in late endosomal/lysosomal compartments of antigen-presenting cells. Here we show that a subset of DM resides at the cell surface of B cells and immature dendritic cells. Surface DM engages in complexes with putatively empty class II molecules and controls presentation of those antigens that rely on loading on the cell surface or in early endosomal recycling compartments. For example, epitopes derived from myelin basic protein that are implicated in the autoimmune disease multiple sclerosis are down-modulated by DM, but are presented in the absence of DM. Thus, this novel concept of functional DM on the surface may be relevant to both protective immune responses and autoimmunity.     

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.     

Editing of an immunodominant epitope of glutamate decarboxylase by HLA-DM

HLA-DM stabilizes peptide-receptive class II alphabeta dimers and facilitates the capture of high affinity peptides, thus influencing the peptide repertoire presented by class II molecules. Variations in DM levels may therefore have a profound effect on the antigenic focus of T cell-mediated immune responses. Specifically, DM expression may influence susceptibility and resistance to autoimmune diseases. In this study the role of DM in HLA-DR4-restricted presentation of an insulin-dependent diabetes mellitus autoantigen, glutamate decarboxylase (GAD), was tested. Presentation of immunodominant GAD epitope 273-285 was regulated by endogenous DM levels in human B lymphoblasts. T cell responses to exogenous GAD as well as an endogenous cytoplasmic form of this Ag were significantly diminished with increasing cellular expression of DM. Epitope editing by DM was observed only using Ag and not small synthetic peptides, suggesting that this process occurred within endosomes. Results with cytoplasmic GAD also indicated that peptides from this compartment intersect class II proteins in endocytic vesicles where DM editing was facilitated. Changes in DM levels within APC may therefore influence the presentation of autoantigens and the development of autoimmune disorders such as type I diabetes.     

Endocytic recycling is required for the presentation of an exogenous peptide via MHC class II molecules

Exogenous antigenic peptides captured and presented in the context of major histocompatibility (MHC) class II molecules on APC, have been employed as potent vaccine reagents capable of activating cellular immune responses. Binding and presentation of select peptide via surface class II molecules has been reported. Here, a role for endocytosis and early endosomes in the presentation of exogenous peptides via MHC class II molecules is described. T cell recognition of a 14 amino acid human serum albumin-derived peptide in the context of HLA-DR4 was observed only with metabolically active APC. The delayed kinetics and temperature dependence of functional peptide presentation via APC, were consistent with a requirement for peptide internalization to early endosomal compartments prior to T cell recognition. Ablating endocytosis by exposing cells to inhibitors of ATP production completely blocked the display of functional peptide:class II complexes on the surface of the APC. Presentation of the peptide was also found to be sensitive to primaquine, a drug that perturbs the recycling of transport vesicles containing endocytic receptors and mature class II complexes. Functional presentation of the endocytosed peptide was dependent upon these mature class II complexes, as inhibitor studies ruled out a requirement for newly synthesized class II molecules. N-terminal processing of the endocytosed peptide was observed upon trafficking through endosomal compartments and linked to the formation of functional peptide:class II complexes. These findings establish a novel mechanism for regulating class II-restricted peptide presentation via the endocytic pathway.     

Role of APC in the selection of immunodominant T cell epitopes

Following antigenic challenge, MHC-restricted T cell responses are directed against a few dominant antigenic epitopes. Here, evidence is provided demonstrating the importance of APC in modulating the hierarchy of MHC class II-restricted T cell responses. Biochemical analysis of class II:peptide complexes in B cells revealed the presentation of a hierarchy of peptides derived from the Ig self Ag. Functional studies of kappa peptide:class II complexes from these cells indicated that nearly 20-fold more of an immunodominant epitope derived from kappa L chains was bound to class II DR4 compared with a subdominant epitope from this same Ag. In vivo, T cell responses were preferentially directed against the dominant kappa epitope as shown using Ig-primed DR4 transgenic mice. The bias in kappa epitope presentation was not linked to differences in class II:kappa peptide-binding affinity or epitope editing by HLA-DM. Rather, changes in native Ag structure were found to disrupt presentation of the immunodominant but not the subdominant kappa epitope; Ag refolding restored kappa epitope presentation. Thus, Ag tertiary conformation along with processing reactions within APC contribute to the selective presentation of a hierarchy of epitopes by MHC class II molecules.     

Cutting edge: H-2DM is responsible for the large differences in presentation among peptides selected by I-Ak during antigen processing

We quantitated the amounts of peptides from hen egg-white lysozyme presented by I-A(k) molecules in APC lines. The large chemical gradient of presentation of the four hen egg-white lysozyme epitopes observed in cell lines expressing HLA-DM or H-2DM (referred to in this study as DM) was significantly diminished in the T2.A(k) line lacking DM. Differences in levels of presentation between wild-type and DM-deficient APC were observed for all four epitopes, but differences were most evident for the highest affinity epitope. As a result of these quantitative differences in display, presentation of all four epitopes to T cells was impaired in the line lacking DM. The binding affinity of the pool of naturally processed peptides from DM-expressing lines was higher than that from the DM-deficient line. Thus, using a direct biochemical approach in APC, we demonstrate that DM influences the selection of peptides bound to MHC class II by favoring high affinity peptides.     

Formation of two peptide/MHC II isomers is catalyzed differentially by HLA-DM

Major histocompatability class II proteins are transmembrane alphabeta-heterodimers that present peptides to T-cells. MHC II may bind exogenous peptides directly at the cell surface. Alternatively, peptides derived from processing of endosomal protein may bind to MHC II in endosomal compartments. There, HLA-DM catalyzes the formation of peptide/MHC complexes, which are then transported to the cell surface. Here we report evidence that the peptide Ii CLIP 81-104 binds to DR*0404 in two alternate registries, whose dissociation rates, while kinetically indistinguishable at pH 5.3 and 37 degrees C, are kinetically resolved in the presence of HLA-DM. In one registry isomer, CLIP Met 91 is placed in the N-terminal P1 pocket of DR*0404, and peptide dissociation is readily catalyzed by HLA-DM. In a second proposed registry, likely with CLIP Leu 97 in the P1 pocket, the complex is substantially less sensitive to HLA-DM catalysis. Without HLA-DM, or at pH 7, the fraction of each isomer formed in solution is relatively insensitive to the duration of incubation with peptide. However, with HLA-DM, the fraction of the DM-insensitive isomer is dramatically influenced by peptide incubation time. The mechanism of isomer formation appears to be determined by the HLA-DM-modified relative association to the two registries, followed by HLA-DM-catalyzed dissociation of each isomer and rebinding, leading to a final isomer composition determined by these kinetic constants. Intramolecular isomer interconversion does not appear to be involved. The behavior of these complexes may provide a model for peptide editing by DM in endosomes.     

Ubiquitination of human leukocyte antigen (HLA)-DM by different membrane-associated RING-CH (MARCH) protein family E3 ligases targets different endocytic pathways

HLA-DM plays an essential role in the peptide loading of classical class II molecules and is present both at the cell surface and in late endosomal peptide-loading compartments. Trafficking of DM within antigen-presenting cells is complex and is, in part, controlled by a tyrosine-based targeting signal present in the cytoplasmic tail of DMβ. Here, we show that DM also undergoes post-translational modification through ubiquitination of a single lysine residue present in the cytoplasmic tail of the α chain, DMα. Ubiquitination of DM by MARCH1 and MARCH9 induced loss of DM molecules from the cell surface by a mechanism that cumulatively involved both direct attachment of ubiquitin chains to DMα and a functional tyrosine-based signal on DMβ. In contrast, MARCH8-induced loss of surface DM was entirely dependent upon the tyrosine signal on DMβ. In the absence of this tyrosine residue, levels of DM remained unchanged irrespective of whether DMα was ubiquitinated by MARCH8. The influence of MARCH8 was indirect and may have resulted from modification of components of the endocytic machinery by ubiquitination.     

Editing of the HLA-DR-peptide repertoire by HLA-DM.

Antigenic peptide loading of classical major histocompatibility complex (MHC) class II molecules requires the exchange of the endogenous invariant chain fragment CLIP (class II associated Ii peptide) for peptides derived from antigenic proteins. This process is facilitated by the non-classical MHC class II molecule HLA-DM (DM) which catalyzes the removal of CLIP. Up to now it has been unclear whether DM releases self-peptides other than CLIP and thereby modifies the peptide repertoire presented to T cells. Here we report that DM can release a variety of peptides from HLA-DR molecules. DR molecules isolated from lymphoblastoid cell lines were found to carry a sizeable fraction of self-peptides that are sensitive to the action of DM. The structural basis for this DM sensitivity was elucidated by high-performance size exclusion chromatography and a novel mass spectrometry binding assay. The results demonstrate that the overall kinetic stability of a peptide bound to DR determines its sensitivity to removal by DM. We show that DM removes preferentially those peptides that contain at least one suboptimal side chain at one of their anchor positions or those that are shorter than 11 residues. These findings provide a rationale for the previously described ligand motifs and the minimal length requirements of naturally processed DR-associated self-peptides. Thus, in endosomal compartments, where peptide loading takes place, DM can function as a versatile peptide editor that selects for high-stability MHC class II-peptide complexes by kinetic proofreading before these complexes are presented to T cells.     

Human cytomegalovirus disrupts constitutive MHC class II expression

CD8(+) and CD4(+) T lymphocytes are important in controlling human CMV (HCMV) infection, but the virus has evolved protean mechanisms to inhibit MHC-based Ag presentation and escape T lymphocyte immunosurveillance. Herein, the interaction of HCMV with the MHC class II Ag presentation pathway was investigated in cells stably transfected with class II transactivator. Flow cytometry experiments demonstrate that HCMV infection decreases cell-surface MHC class II expression. HCMV down-regulates MHC class II surface expression without a significant effect on class II RNA or steady-state protein levels. SDS-stability and confocal microscopy experiments demonstrate normal levels of steady-state peptide-loaded class II molecules in infected cells and that class II molecules reach late endosomal and HLA-DM positive peptide-loading compartments. However, MHC class II positive vesicles are retained in an abnormal perinuclear distribution. Finally, experiments with a mutant HCMV strain demonstrate that this novel mechanism of decreased MHC class II expression is not mediated by one of the known HCMV immunomodulatory genes. These defects in MHC class II expression combined with previously identified CMV strategies for decreasing MHC class I expression enables infected cells to evade T lymphocyte immunosurveillance.     

An insertion mutant in DQA1*0501 restores susceptibility to HLA-DM: implications for disease associations

HLA-DM (DM) catalyzes CLIP release, stabilizes MHC class II molecules, and edits the peptide repertoire presented by class II. Impaired DM function may have profound effects on Ag presentation events in the thymus and periphery that are critical for maintenance of self-tolerance. The associations of the HLA-DQ2 (DQ2) allele with celiac disease and type 1 diabetes mellitus have been appreciated for a long time. The explanation for these associations, however, remains unknown. We previously found that DQ2 is a poor substrate for DM. In this study, to further characterize DQ2-DM interaction, we introduced point mutations into DQ2 on the proposed DQ2-DM interface to restore the sensitivity of DQ2 to DM. The effects of mutations were investigated by measuring the peptide dissociation and exchange rate in vitro, CLIP and DQ2 expression on the cell surface, and the presentation of α-II-gliadin epitope (residues 62-70) to murine, DQ2-restricted T cell hybridomas. We found that the three α-chain mutations (α+53G, α+53R, or αY22F) decreased the intrinsic stability of peptide-class II complex. More interestingly, the α+53G mutant restored DQ2 sensitivity to DM, likely due to improved interaction with DM. Our data also suggest that α-II-gliadin 62-70 is a DM-suppressed epitope. The DQ2 resistance to DM changes the fate of this peptide from a cryptic to an immunodominant epitope. Our findings elucidate the structural basis for reduced DQ2-DM interaction and have implications for mechanisms underlying disease associations of DQ2.     

Presentation by recycling MHC class II molecules of an influenza hemagglutinin-derived epitope that is revealed in the early endosome by acidification

We investigated the roles of nascent and recycling MHC class II molecules (MHC II) in the presentation of two well-defined I-E(d)-restricted epitopes that are within distinct regions of the influenza virus hemagglutinin (HA) protein. The site 3 epitope (S3; residues 302-313) lies in the stalk region that unfolds in response to mild acidification, while the site 1 epitope (S1; residues 107-119) is situated in the stable globular domain. In a murine B lymphoma cell line and an I-E(d)-transfected fibroblast cell line, presentation from inactivated virus of S3 is inhibited by primaquine, a compound that prevents recycling of cell surface proteins, including MHC II, while S1 presentation is unaffected. In contrast, brefeldin A, an agent that inhibits exit of proteins from the endoplasmic reticulum, selectively inhibited S1 presentation without affecting S3 presentation, suggesting that S1 presentation requires nascent MHC II. The use of agents that perturb endosomal function revealed a requirement for acidification of internalized viral particles for presentation of both epitopes. Notably, all compounds tested had similar effects on presentation of the two epitopes derived from endogenously synthesized HA. Thus, recycling I-E(d) molecules appear to be crucial for capturing and presenting an epitope that is revealed in mild acidic conditions following the uptake of virions or the synthesis of Ag, while nascent I-E(d) molecules are required for presentation of a second epitope located in a structurally constrained region of the same polypeptide. Viral glycoproteins, such as HA, may have been a major impetus for the evolutionary establishment of this recycling pathway.     

The DMalpha and DMbeta chain cooperate in the oxidation and folding of HLA-DM

HLA-DM (DM) is a heterodimeric MHC molecule that catalyzes the peptide loading of classical MHC class II molecules in the endosomal/lysosomal compartments of APCs. Although the function of DM is well-established, little is known about how DMalpha and beta-chains fold, oxidize, and form a complex in the endoplasmic reticulum (ER). In this study, we show that glycosylation promotes, but is not essential for, DMalphabeta ER exit. However, glycosylation of DMalpha N15 is required for oxidation of the alpha-chain. The DMalpha and beta-chains direct each others fate: single DMalpha chains cannot fully oxidize without DMbeta, while DMbeta forms disulfide-linked homodimers without DMalpha. Correct oxidation and subsequent ER egress depend on the unique DMbeta C25 and C35 residues. This suggests that the C25-C35 disulfide bond in the peptide-binding domain overcomes the need for stabilizing peptides required by other MHC molecules.     

New insights in antigen processing and epitope selection: development of novel immunotherapeutic strategies for cancer, autoimmunity and infectious diseases

Current evidence suggests that MHC class II-restricted CD4+ T-cells play a crucial role in orchestrating host immune responses against cancer as well as autoimmune and infectious diseases. Antigens must be processed within endosomal and lysosomal compartments of antigen presenting cells (APC) before binding to MHC class II molecules for display to T-cells. Only a limited number of processed peptides termed immunodominant are selected for display by MHC class II molecules and prove capable of inducing strong T-cell responses. Thus processing reactions within APC are of central importance for the development of effective vaccines as they modulate the number of peptide: class II complexes by enhancing or disrupting epitope formation and display. Studies suggest that there are substantial gaps in our knowledge of how antigen processing and presentation by APC regulates epitope selection and immunodominance in disease situations. Here we describe new insights in antigen processing and epitope selection with relevance to immunotherapeutic strategies for cancer, autoimmunity and infectious diseases.     

What to do with HLA-DO?

Antigenic peptide binding to MHC class II molecules in the endocytic pathway occurs via a multifactorial process that requires the support of a specialized lysosomal chaperone called HLA-DM. DM shows both in primary amino acid sequence and quaternary structure a high homology to both MHC class I and class II molecules. Like the peptide presenting class II molecules, DM is expressed in all professional antigen presenting cells. DM catalyzes the dissociation of peptides that do not bind stably to the class II peptide-binding groove, thereby leading to the preferential presentation of stably binding antigenic peptides. The recently discovered HLA-DO molecule is mainly expressed in B cells and associates with DM, thereby markedly affecting DM function. Like DM, the genes encoding the HLA-DO heterodimer lie within the MHC class II region and exhibit strong homology to classical class II molecules. This review evaluates the unique effects of DO on DM-mediated antigen presentation by MHC class II molecules and discusses the possible physiological relevance for the B cell-specific expression of DO and its function.     

Disruption of Hydrogen Bonds between Major Histocompatibility Complex Class II and the Peptide N-Terminus Is Not Sufficient to Form a Human Leukocyte Antigen-DM Receptive State of Major Histocompatibility Complex Class II

Peptide presentation by MHC class II is of critical importance to the function of CD4+ T cells. HLA-DM resides in the endosomal pathway and edits the peptide repertoire of newly synthesized MHC class II molecules before they are exported to the cell surface. HLA-DM ensures MHC class II molecules bind high affinity peptides by targeting unstable MHC class II:peptide complexes for peptide exchange. Research over the past decade has implicated the peptide N-terminus in modulating the ability of HLA-DM to target a given MHC class II:peptide combination. In particular, attention has been focused on both the hydrogen bonds between MHC class II and peptide, and the occupancy of the P1 anchor pocket. We sought to solve the crystal structure of a HLA-DR1 molecule containing a truncated hemagglutinin peptide missing three N-terminal residues compared to the full-length sequence (residues 306–318) to determine the nature of the MHC class II:peptide species that binds HLA-DM. Here we present structural evidence that HLA-DR1 that is loaded with a peptide truncated to the P1 anchor residue such that it cannot make select hydrogen bonds with the peptide N-terminus, adopts the same conformation as molecules loaded with full-length peptide. HLA-DR1:peptide combinations that were unable to engage up to four key hydrogen bonds were also unable to bind HLA-DM, while those truncated to the P2 residue bound well. These results indicate that the conformational changes in MHC class II molecules that are recognized by HLA-DM occur after disengagement of the P1 anchor residue.     

Early endosomal maturation of MHC class II molecules independently of cysteine proteases and H-2DM

Major histocompatibility complex (MHC) class II molecules bind and present to CD4(+) T cells peptides derived from endocytosed antigens. Class II molecules associate in the endoplasmic reticulum with invariant chain (Ii), which (i) mediates the delivery of the class II-Ii complexes into the endocytic compartments where the antigenic peptides are generated; and (ii) blocks the peptide-binding site of the class II molecules until they reach their destination. Once there, Ii must be removed to allow peptide binding. The bulk of Ii-class II complexes reach late endocytic compartments where Ii is eliminated in a reaction in which the cysteine protease cathepsin S and the accessory molecule H-2DM play an essential role. Here, we here show that Ii is also eliminated in early endosomal compartments without the intervention of cysteine proteases or H-2DM. The Ii-free class II molecules generated by this alternative mechanism first bind high molecular weight polypeptides and then mature into peptide-loaded complexes.     

HLA-DO is a negative modulator of HLA-DM-mediated MHC class II peptide loading

Background: Class II molecules of the major histocompatibility complex become loaded with antigenic peptides after dissociation of invariant chainderived peptides (CLIP) from the peptide-binding groove. The human leukocyte antigen (HLA)-DM is a prerequisite for this process, which takes place in specialised intracellular compartments. HLA-DM catalyses the peptide-exchange process, simultaneously functioning as a peptide ‘editor’, favouring the presentation of stably binding peptides. Recently, HLA-DO, an unconventional class II molecule, has been found associated with HLA-DM in B cells, yet its function has remained elusive.Results: The function of the HLA-DO complex was investigated by expression of both chains of the HLA-DO heterodimer (either alone or fused to green fluorescent protein) in human Mel JuSo cells. Expression of HLA-DO resulted in greatly enhanced surface expression of CLIP via HLA-DR3, the conversion of class II complexes to the SDS-unstable phenotype and reduced antigen presentation to T-cell clones. Analysis of peptides eluted from HLA-DR3 demonstrated that CLIP was the major peptide bound to class II in the HLA-DO transfectants. Peptide exchange assays in vitro revealed that HLA-DO functions directly at the level of class II peptide loading by inhibiting the catalytic action of HLA-DM.Conclusions: HLA-DO is a negative modulator of HLA-DM. By stably associating with HLA-DM, the catalytic action of HLA-DM on class II peptide loading is inhibited. HLA-DO thus affects the peptide repertoire that is eventually presented to the immune system by MHC class II molecules.     

Structural factors contributing to DM susceptibility of MHC class II/peptide complexes

Peptide loading of MHC class II (MHCII) molecules is assisted by HLA-DM, which releases invariant chain peptides from newly synthesized MHCII and edits the peptide repertoire. Determinants of susceptibility of peptide/MHCII complexes to DM remain controversial, however. Here we have measured peptide dissociation in the presence and the absence of DM for 36 different complexes of varying intrinsic stability. We found large variations in DM susceptibility for different complexes using either soluble or full-length HLA-DM. The DM effect was significantly less for unstable complexes than for stable ones, although this correlation was modest. Peptide sequence- and allele-dependent interactions along the entire length of the Ag binding groove influenced DM susceptibility. We also observed differences in DM susceptibility during peptide association. Thus, the peptide repertoire displayed to CD4(+) T cells is the result of a mechanistically complicated editing process and cannot be simply predicted from the intrinsic stability of the complexes in the absence of DM.     

Invariant chain modulates HLA class II protein recycling and peptide presentation in nonprofessional antigen presenting cells

The expression of MHC class II molecules and the invariant chain (Ii) chaperone, is coordinately regulated in professional antigen presenting cells (APC). Ii facilitates class II subunit folding as well as transit and retention in mature endosomal compartments rich in antigenic peptides in these APC. Yet, in nonprofessional APC such as tumors, fibroblasts and endocrine tissues, the expression of class II subunits and Ii may be uncoupled. Studies of nonprofessional APC indicate class II molecules access antigenic peptides by distinct, but poorly defined pathways in the absence of Ii. Here, investigations demonstrate that nonprofessional APC such as human fibroblasts lacking Ii internalize antigenic peptides prior to the binding of these ligands to recycling class II molecules. By contrast, fibroblast lines expressing Ii favor exogenous peptides binding directly to cell surface class II molecules without a need for ligand internalization. Endocytosis of class II molecules was enhanced in cells lacking Ii compared with Ii-expressing APC. These results suggest enhanced reliance on the endocytic recycling pathway for functional class II presentation in nonprofessional APC.