Detection of aberrant association of DM with MHC class II subunits in the absence of invariant chain

Human HLA-DM or mouse H2-DM plays a vital role for presentation of antigenic sequences by MHC class II peptide receptors. These non-classical MHC class II molecules catalyze the release of the invariant chain (Ii) fragment CLIP from the class II cleft and facilitate acquisition of antigenic peptides by MHC class II peptide receptors. H2-DM- or Ii-deficient mice display an impaired ability of their antigen-presenting cell to present peptides to CD4+ T cells and a molecular link between the immunodeficiencies of these mouse strains may exist. We show that in transfected cells the presence of HLA-DM molecules in endocytic vesicles was strongly reduced when HLA-DM was accompanied by HLA-DR. Exclusion of HLA-DM from endocytic vesicles is explained by mixed association of HLA-DM with HLA-DR subunits and retention of the aggregates in the endoplasmic reticulum. Expression of Ii, however, impairs formation of mixed HLA-DR and HLA-DM complexes and directs matched pairing of HLA-DR and HLA-DM heterodimers. In Ii gene-deficient mice, aberrant association of H2A with H2-DM polypeptides was detected. Low expression of Ii in transgenic mice inhibits interaction of H2A with H2-DM subunits and facilitates formation of H2-DM alphabeta heterodimers. A role of Ii for assembly of H2-DM heterodimers partially explains the immunodeficient phenotype of Ii-/- mice.     

Conformational alterations during biosynthesis of HLA-DR3 molecules controlled by invariant chain and HLA-DM

HLA-DM is known to catalyze the exchange of class II-associated invariant chain (Ii) peptide (CLIP) for cognate peptide during biosynthesis. In DM-negative cells HLA-DR3 molecules have been shown to predominantly present CLIP and to lack the DR3-specific mAb epitope 16.23, which has led to the assumption that CLIP prevents binding of mAb 16.23. In the present study we show that CLIP does not prohibit 16.23 epitope expression, but that the formation of this epitope is directly influenced by interactions of the DR molecule with Ii and DM. Detergent solubilized DR3 from wild-type as well as DM(-) cells bound CLIP in a 16.23(+) mode. On cells, however, neither CLIP nor antigenic peptide bound to DR3 in a 16.23(+) conformation, unless HLA-DM was expressed. Thus, HLA-DM appears to alter the conformation of DR3 in a peptide-independent fashion. Since in DM-deficient cells that also lack Ii, DR3 molecules assembled in a 16.23(+) conformation, we conclude that during biosynthesis Ii and DM exert opposing conformational constraints, characterized by suppressing or releasing 16.23 epitope expression. These results imply that DR3/peptide complexes, including DR3/ CLIP, can exist in two conformations depending on previous interaction with DM, but independent of the nature of the peptide bound. We show that these naturally occurring class II conformers can be selectively recognized by T cells.     

HLA-DM can partially replace the invariant chain for HLA-DR transport and surface expression in transfected endocrine epithelial cells

The function of HLA class II molecules as peptide presenters to CD4+ T cells depends on the expression of associated molecules such as the invariant chain (Ii) and DM responsible for the correct transport of and high-stability peptide binding to the class II dimers. In organs affected by autoimmune diseases, endocrine epithelial cells express class II molecules, which presumably are involved in the presentation of self-peptides to autoreactive T cells. We have transfected the rat insulinoma cell line RINm5F with different combinations of HLA-DR, Ii and HLA-DM cDNAs and have studied how Ii and DM affect the transport and stability of class II molecules expressed by the different transfectants. Immunofluorescence and biochemical analysis showed that cells transfected with DR and DM in the absence of Ii expressed mostly stable molecules in their surface, and showed a lower accumulation of DR molecules in the endoplasmic reticulum (ER) than cells expressing only DR. This suggests that, in the absence of invariant chain, DM molecules can not only exchange peptides other than class II-associated invariant chain peptide (CLIP) but may also be involved in the transport of class II molecules out of the ER towards the endosomal route. In addition, these data confirm that expression of DR alone or DR+Ii do not allow the formation of sodium dodecyl sulphate (SDS)-stable complexes, that cells expressing DR+Ii have most DR molecules occupied by CLIP and that Ii and DM molecules allow regular routing and peptide loading of class II molecules.     

MHC class II loading of high or low affinity peptides directed by Ii/peptide fusion constructs: implications for T cell activation

CD4(+) T cells recognize peptides presented on the cell surface of antigen presenting cells in the MHC class II context. The biosynthesis and transport of MHC class II molecules depend on the type II transmembrane invariant chain (Ii) and are tightly regulated processes. Ii is known to bind to the MHC class II peptide-binding groove via its class II-associated Ii peptide (CLIP) region early in the biosynthetic pathway to prevent premature peptide binding. In this study we have genetically exchanged CLIP with peptides of either high or low affinity for the class II peptide binding groove and utilized the properties of Ii to manipulate MHC class II loading. An inducible promoter controlled expression of the Ii/peptide fusion constructs, and presentation at different expression levels was studied. Both peptides were excised from Ii and presented on MHC class II molecules as shown by liquid chromatography-tandem mass spectrometry, but the high affinity peptide was presented more efficiently than the low affinity peptide. Both peptides were efficient in eliciting T cell responses at high Ii/peptide concentration independent of the duration of T cell stimulation. The peptides were also able to elicit an IL-2 response at low expression levels; however, the kinetic differed as the T cells required longer duration of T cell contact to reach a significant T cell response. This probably reflects the number of class II/peptide complexes at the cell surface and is discussed.     

The role of H2-O and HLA-DO in major histocompatibility complex class Il-restricted antigen processing and presentation

Summary: The function of major histocompatibility complex (MHC) class II molecules is to sample exogenous antigens for presentation to CD4⁺ T helper cells. After synthesis in the endoplasmic reticulum, class II molecules are directed into the endosomal system by association with the invariant chain (Ii), which is sequentially cleaved, generating class II dimers loaded with Ii-derived peptides (CLIP). These class Il-peptide complexes are physiological substrates for H2-M/HLA-DM, a resident of the endosomal/lysosomal system which facilitates the removal of CLIP from newly synthesised class II αβ dimers. Exchange of CLIP for antigenic class Il-binding peptides is also promoted by the action of H2-M/HLA-DM, resulting in stable peptide-class II complexes that are transported to the cell surface for presentation to CD4⁺ T cells. Recent evidence suggests that this H2-M/HLA-DM-mediated 'peptide editing' is influenced by another MHC class Il-encoded molecule, H2-O/HLA-DO. This non-polymorphic αβ heterodimer is associated with H2-M/HLA-DM during intra-cellular transport and within the endosomal system of B cells, H2-0/HLA-DO alters the peptide exchange function of H2-M/HLA-DM in a pH-dependent manner, so that H2-M/HLA-DM activity is limited to more acidic conditions, corresponding to lysosomal compartments. Indeed, H2-O/HLA-DO may serve to limit the presentation of antigens after fluid phase uptake by B cells, while augmenting presentation of antigens internalised via membrane Ig receptors. Such a mechanism may maintain the fidelity of the B-cell-CD4⁺ T-cell interaction, counteracting self reactivity arising from less stringent lymphocyte activation. Here, data evaluating the role of H2-O/HLA-DO shall be reviewed and its putative function discussed.     

Expression and function of HLA-DR3 and DQ8 in transgenic mice lacking functional H2-M

H2-M or HLA-DM are non-classical class II molecules encoded by the MHC and play an important role during antigen presentation. They catalyze exchange of CLIP (Class II-associated invariant chain peptide) or other low-affinity peptides bound to class II molecules for peptides capable of more efficient binding. The phenotype of mice lacking H2-M is determined by the allotype of the MHC class II molecules expressed. In general, H2-M deficiency does not affect the surface expression of mature class II molecules. The class II molecules in such cases predominantly contain CLIP in their peptide-binding groove. In some mice strains, H2-M deficiency results in defective CD4+ T-cell development accompanied by defective responses to conventional antigens and superantigens. Even though the HLA class II molecules show similar dependency for HLA-DM for presenting antigens in vitro, their interaction in vivo is not known. By using transgenic approach we show here that DQ8 and DR3 are expressed at normal levels in H2-M-deficient mice and the CD4+ T-cell development is unaltered. However, the ability of DQ8 molecules to present peptide antigens is compromised in a H2-M-deficient state. Presentation of exogenous bacterial superantigens by both DQ8 and DR3 is unaffected in H2-M-deficient mice. Unexpectedly, Staphylococcal Enterotoxin B-induced systemic IFN-gamma production was significantly higher in H2-M-deficient DQ8/DR3 transgenic mice and these mice were susceptible to SEB-induced toxic shock at doses that are non-lethal to H2-M-sufficient counterparts.     

A study of complexes of class II invariant chain peptide: Major histocompatibility complex class II molecules using a new complex-specific monoclonal antibody

Complexes of major histocompatibility complex (MHC) class II molecules containing invariant chain (Ii)-derived peptides, known as class II-associated invariant chain peptides (CLIP), are expressed at high levels in presentation-deficient mutant cells. Expression of these complexes in mutant and wild-type antigen-presenting cells suggests that they represent an essential intermediate in the MHC class II antigen-presenting pathway. We have generated a monoclonal antibody, 30-2, which is specific for these complexes. Using this antibody, we have found quantitative differences in CLIP: MHC class II surface expression in mutant and wild-type cells. Our experiments also show that CLIP: MHC class II complexes are preferentially expressed on the cell surface similar to total mature MHC class II molecules. These complexes are found to accumulate in the endosomal compartment in the process of endosomal Ii degradation. Analysis of the fine specificity of the antibody indicates that these complexes have Ii peptide bound to the peptide-binding groove.     

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 β 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 β:peptide complexes presentable to T cells.     

Achieving stability through editing and chaperoning: regulation of MHC class II peptide binding and expression

Summary: In antigen‐presenting cells (APCs), loading of major histocompatibility complex class II (MHC II) molecules with peptides is regulated by invariant chain (Ii), which blocks MHC II antigen‐binding sites in pre‐endosomal compartments. Several molecules then act upon MHC II molecules in endosomes to facilitate peptide loading: Ii‐degrading proteases, the peptide exchange factor, human leukocyte antigen‐DM (HLA‐DM), and its modulator, HLA‐DO (DO). Here, we review our findings arguing that DM stabilizes a globally altered conformation of the antigen‐binding groove by binding to a lateral surface of the MHC II molecule. Our data imply changes in the interactions between specificity pockets and peptide side chains, complementing data from others that suggest DM affects hydrogen bonds. Selective weakening of peptide/MHC interactions allows DM to alter the peptide repertoire. We also review our studies in cells that highlight the ability of several factors to modulate surface expression of MHC II molecules via post‐Golgi mechanisms; these factors include MHC class II‐associated Ii peptides (CLIP), DM, and microbial products that modulate MHC II traffic from endosomes to the plasma membrane. In this context, we discuss possible mechanisms by which the association of some MHC II alleles with autoimmune diseases may be linked to their low CLIP affinity.     

The impact of the non-classical MHC proteins HLA-DM and HLA-DO on loading of MHC class II molecules

Summary: Peptide binding to classical major histocompatibility complex (MHC) class II molecules is known to be determined by the properties of the class ii peptide binding groove but recently it turned out to be co-controlled by the activity of the non-classical MHC molecules HLA-DM and HLA-DO: HLA-DM functions as a mediator of peptide exchange. In addition, HLA-DM is a chaperone for MHC class II molecules in endosomal and lysosomal loading compartments because it stabilizes the empty MHC class Ii peptide binding groove and keeps it receptive for peptide loading until appropriate peptide ligands are captured. Since HLA-DM favors the generation of high-stability peptide-MHC class Ii complexes by releasing low-stability peptide ligands, DM activity affects the peptide repertoire presented on the ceil surface of antigen-presenting cells. HLA-DO is expressed mainly in B cells and binds tightly to HLA-DM thereby modulating its activity Together, HLA-DM and HLA-DO are critical factors in shaping the MHC class Il-associated self or foreign peptide repertoire of antigen presenting cells and, hence, govern initiation or prevention of an immune response.     

Structural principles of MHC class II antigen presentation

Normal immune surveillance depends on the ability of MHC class II molecules to bind peptide antigens and carry them to the cell surface for display to T cells. To do this efficiently, class II molecules must be able to bind peptides from a broad array of antigen sequences and retain them at the cell surface long enough for T-cell recognition to occur. Class II molecules accomplish this task through a combination of clever structural biochemistry and the help of at least two different molecular chaperones: the class II-associated invariant chain (Ii); and a non-peptide binding class II molecule termed H2-DM in mouse and HLA-DM in man (DM). Here, we compare the existing 3-dimensional structures of class II-peptide complexes in order to review the general principles of peptide binding and presentation. We extend this analysis to include the structures of proteins known to interact with MHC class II, focusing primarily on the Ii chain and DM.     

Identification and characterization of major histocompatibility complex class II compartments in cortical thymic epithelial cells

Previous studies have concentrated on elucidating the subcellular localization of major histocompatibility (MHC) class II molecules mainly in B cells, macrophages, and dendritic cells. Despite very rich cell-surface expression of MHC class II molecules by cortical thymic epithelial cells (cTECs), little is known regarding the expression of these molecules by cTECs at the subcellular level. In the present study we focused on the identification and characterization of MHC class II compartments (MIICs) in cTECs in situ by immunogold electron microscopy (IEM). We found that MHC class II molecules were located exclusively in the cytoplasmic vacuoles, and we identified these MHC class II molecule-containing cytoplasmic vacuoles as MIICs in cTECs. These MIICs were immunopositive for early endosomal, late endosomal, and lysosomal markers. Moreover, in these MIICs, MHC class II molecules were colocalized with cathepsin L, H2-DM, class II-associated invariant chain (Ii), and class II-associated invariant chain peptide (CLIP). Similarly, Ii molecules were colocalized with endosomal and lysosomal markers, cathepsin L, and H2-DM in the vacuoles. Taken together, these results suggest that MIICs in cTECs represent conventional endocytic compartments. The colocalization of MHC class II molecule or Ii with cathepsin L and H2-DM in the MIICs suggests that MIICs in cTECs may be sites of Ii degradation and peptide loading.     

HLA-DM and the MHC class II antigen presentation pathway

The MHC class II antigen processing pathway provides a mechanism to selectively present peptides generated in the endosomal compartments of antigen presenting cells to CD4⁺ T cells. Transport of newly synthesized class II molecules to the endosomal pathway requires the function of an accessory protein, invariant chain, which contains a region that interacts directly with the class II peptide binding site. Release of invariant chain and peptide loading by class II molecules are facilitated by a second accessory protein, HLA-DM. This MHC-encoded membrane protein catalyzes peptide exchange reactions, influencing the repertoire of peptides that are available for recognition by T cells.     

HLA-DM and the MHC class II antigen presentation pathway

The MHC class II antigen processing pathway provides a mechanism to selectively present peptides generated in the endosomal compartments of antigen presenting cells to CD4+ T cells. Transport of newly synthesized class II molecules to the endosomal pathway requires the function of an accessory protein, invariant chain, which contains a region that interacts directly with the class II peptide binding site. Release of invariant chain and peptide loading by class II molecules are facilitated by a second accessory protein, HLA-DM. This MHC-encoded membrane protein catalyzes peptide exchange reactions, influencing the repertoire of peptides that are available for recognition by T cells.     

Intracellular and cell surface heterotypic associations of human leukocyte antigen-DR and human invariant chain

The intracellular and cell-surface heterotypic associations of HLA-DR in the presence and absence of the invariant chain were investigated. Simultaneous confocal microscopy imaging of the Golgi apparatus and HLA-DR molecules revealed that cells transfected only with HLA-DR and not the invariant chain or HLA-DM, accumulate class II molecules mostly in the Golgi apparatus, proximal to the cell nucleus. In contrast, in cells transfected with both HLA-DR and the invariant chain, or HLA-DR, the invariant chain and HLA-DM, the class II molecules are more evenly distributed in intracellular compartments. Confocal microscopy and flow cytometry revealed that in the absence of the invariant chain, a greater number of HLA-DR molecules are transported to the cell surface. Biochemical experiments and nonequilibrium pH gradient electrophoresis revealed that HLA-DR associates with surface invariant chain in the presence of HLA-DM. In cells that lack HLA-DM, no cell-surface association of HLA-DR and Ii was observed. Taken together, these results reveal two separate and distinct functions for surface and intracellular invariant chain subsets. The intracellular invariant chain "arrests" the class II molecules in the endocytic pathway. In contrast, cell-surface invariant chain associates with class II molecules at the cell surface, possibly playing a role in recycling empty class II molecules or as an accessory molecule.     

Determination of the HLA-DM interaction site on HLA-DR molecules

HLA-DM removes CLIP and other loosely bound peptides from MHC class II molecules. The crystal structures of class II molecules and of HLA-DM have not permitted identification of their interaction sites. Here, we describe mutations in class II that impair interactions with DM. Libraries of randomly mutagenized DR3 alpha and beta chains were screened for their ability to cause cell surface accumulation of CLIP/DR3 complexes in EBV-B cells. Seven mutations were associated with impaired peptide loading in vivo, as detected by SDS stability assays. In vitro, these mutant DR3 molecules were resistant to DM-catalyzed CLIP release and showed reduced binding to DM. All mutations localize to a single lateral face of HLA-DR, which we propose interacts with DM during peptide exchange.     

Surface-expressed invariant chain (CD74) is required for internalization of human leucocyte antigen-DR molecules to early endosomal compartments

Transport of major histocompatibility complex (MHC) class II molecules to the endocytic route is directed by the associated invariant chain (Ii). In the endocytic pathway, Ii is proteolytically cleaved and, upon removal of residual Ii fragments, class II alpha beta dimers are charged with antigenic peptide and recognized by CD4+ T cells. Although distinct peptide-loading compartments such as MIIC (MHC class II loading compartment) and CIIV (MHC class II vesicles) have been characterized in different cells, there is growing evidence of a multitude of subcellular compartments in which antigenic peptide loading takes place. We employed a physiological cellular system in which surface Ii (CD74) and surface human leucocyte antigen (HLA)-DR were induced either alone or in combination. This was achieved by transient exposure of HT-29 cells to recombinant interferon-gamma (rIFN-gamma). Using distinct cellular variants, we showed that: (i) the majority of Ii molecules physically associate on the cell membrane with class II dimers to form DR alpha beta:Ii complexes; (ii) the presence of surface Ii is a prerequisite for the rapid uptake of HLA-DR-specific monoclonal antibodies into early endosomes because only the surface DR+/Ii+ phenotype, and not the DR+/Ii- variant, efficiently internalizes; and (iii) the HLA-DR:Ii complexes are targeted to early endosomes, as indicated by co-localization with the GTPase, Rab5, and endocytosed bovine serum albumin. Internalization of HLA-DR:Ii complexes, accommodation of peptides by DR alphabeta heterodimers in early endosomes and recycling to the cell surface may be a mechanism used to increase the peptide repertoire that antigen-presenting cells display to MHC class II-restricted T cells.     

MHC class II-associated invariant chain peptide replacement by T cell epitopes: engineered invariant chain as a vehicle for directed and enhanced MHC class II antigen processing and presentation

Proteolysis of the invariant chain (Ii) leads to the generation of abundant MHC class II-associated invariant chain peptides (CLIP), which bind in the MHC class II binding groove via supermotifs in a manner similar to that of antigenic peptides. We have engineered an Ii vector with the capacity to express any antigenic peptide of interest instead of CLIP, for T cell stimulation. When peripheral blood mononuclear cells (PBMC) were pulsed with Ii hybrids encoding T cell epitopes of tetanus toxin or acetylcholine receptor, stimulation of T cells was dramatically enhanced compared to stimulation after priming with either the native or recombinant proteins. Site-specific insertion of antigenic sequences into the CLIP region promoted enhanced antigenicity of Ii hybrids which were shown to be processed intracellularly in a chloroquine-sensitive compartment. Naturally processed T helper epitopes were visualized directly on the surface of PBMC and identified as analogs of CLIP associated with MHC class II molecules. This novel Ii vector provides a flexible and efficient system for the delivery of defined peptide epitopes to T cells which might be useful in the development of specific vaccines and in the study of intracellular processing.     

Differential up-regulation of HLA-DM, invariant chain, and CD83 on myeloid and plasmacytoid dendritic cells from peripheral blood

Two main dendritic cell (DC) subsets have been described in peripheral blood, the myeloid subset or DC1 that is characterized by the presence of CD11c and the plasmacytoid subset or DC2 negative for this marker. The two subsets may perform different functions and have been defined as immunogenic (the myeloid subset) or tolerogenic (the plasmacytoid subset). The expression of human leukocyte antigen (HLA)-DM molecules, which act as peptide editors in the antigen presentation process, was studied in freshly isolated plasmacytoid and myeloid DCs from peripheral blood. The expression of the invariant chain (Ii), the major histocompatibility complex class II (MHC-II) : class II-associated Ii peptide (CLIP) complex, and CD83 was also investigated. The results showed that intracellular expression of HLA-DM and the Ii was significantly higher in the plasmacytoid than in the myeloid DC subset. In contrast, a higher fraction of cell expressing MHC-II : CLIP complex was found in the myeloid than in the plasmacytoid DC subpopulation. CD83 was not detected in any of these two subsets. Following culture of these cells with interleukin-3 (IL-3), tumor necrosis factor-alpha (TNFalpha) and/or heat shock protein-70 (HSP-70), the expression of intracellular HLA-DM was up-regulated in the myeloid DCs to levels similar to those found in the plasmacytoid DCs, whilst the Ii was down-regulated in the plasmacytoid subset to similar levels to those expressed in the myeloid DCs. In addition, CD83 was up-regulated in the myeloid (CD11c+) but not in the plasmacytoid (CD11c-) DCs. The expression pattern of these antigen-processing molecules could be related to the immaturity and function attributed to these DC subsets.     

Quality control of MHC class II associated peptides by HLA-DM/H2-M

For many years the crucial components involved in MHC class II mediated antigen presentation have been thought to be known: polymorphic MHC class II molecules, the monomorphic invariant chain (li) and a set of conventional proteases that cleave antigenic proteins thereby generating ligands able to associate with MHC class II molecules. However, in 1994 it was found that without an additional molecule, HLA-DM (DM), efficient presentation of protein antigens cannot be achieved. Biochemical studies showed that DM acts as a molecular chaperone protecting empty MHC class II molecules from functional inactivation. In addition, it serves as a peptide editor: DM catalyzes not only the release of the invariant chain remnant CLIP, but of all sorts of low-stability peptides, and simultaneously favors binding of high-stability peptides. Through this quality control of peptide loading, DM enables APCs to optimize MHC restriction and to display their antigenic peptide cargo on the surface for prolonged periods of time to be scrutinized by T cells.