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.     

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.     

Cellular distribution of a mixed MHC class II heterodimer between DRalpha and a chimeric DObeta chain

Human MHC class II antigens include HLA-DR, -DQ, and -DP molecules that present antigens to CD4+ T cells, as well as the non-classical molecules HLA-DM and -DO. HLA-DM promotes peptide binding to class II molecules in endocytic compartments and HLA-DO, which is physically associated with HLA-DM in B lymphocytes, regulates HLA-DM function. Antibodies specific for the DObeta chain were obtained by immunization of mice with a heterodimer consisting of a chimeric DObeta chain (DR/DObeta), containing 18 N-terminal residues of DRbeta, paired with the DRalpha chain and isolated from transfected murine fibroblasts. The specificity of this serum for the DObeta chain and the lysosomal expression of the HLA-DO protein was confirmed using mutant human B cell lines lacking DR or DO molecules. The lysosomal localization of HLA-DO in human B cells contrasts with the cell surface expression of the mixed pair in transfected murine fibroblasts and raises questions concerning the role of the putative targeting motifs in HLA-DO. Transfection of the chimeric DR/DObeta chain along with DRalpha into human epithelial HeLa cells resulted in high levels of expression of the mixed isotypic pair at the surface of transfectants as well as in lysosomes. The same pattern was observed in HeLa cells transfected with the DObeta chimera and a DRa chain lacking the cytoplasmic tail. Taken together, these results suggest that functional sorting motifs exist in the DObeta chain but that the tight compartmentalization of HLA-DO observed inside B lymphocytes is controlled by the HLA-DOalpha chain and HLA-DM.     

Human duodenal epithelial cells constitutively express molecular components of antigen presentation but not costimulatory molecules

Constitutive expression of major histocompatibility complex (MHC) class II molecules by duodenal epithelial cells (EC) suggests that they can present antigen to CD4(+) T cells. However, other molecular components including invariant chain (Ii), HLA-DM, and costimulatory molecules CD80, CD86 and CD40, are required for efficient T-cell activation. We have investigated whether normal human duodenal EC possess these molecules and whether they can mediate MHC class II antigen presentation. EC were isolated from duodenal biopsies from patients in whom pathology was excluded. Freshly-isolated duodenal EC did not stimulate autologous T-cell proliferation against purified protein derivative of tuberculin. Flow cytometry and immunoblot analysis revealed that duodenal EC constitutively express HLA-DR, Ii, and HLA-DM. Surface MHC class II associated invariant chain peptide (CLIP) was not detectable, suggesting that HLA-DM functions normally in CLIP removal. Duodenal EC expressed SDS-stable HLA-DR alphabeta heterodimers, indicating that peptide binding had occurred. Surface expression of CD80, CD86 or CD40 was not detected although mRNA for these costimulatory molecules was present in all samples. These results suggest that nondiseased human duodenal EC can process and present antigen by the MHC class II pathway, but that they may induce anergy, rather than activation, of local T cells.     

HLA-DR-restricted presentation of purified myelin basic protein is independent of intracellular processing

Antigen presentation to CD4⁺ T cells involves intracellular antigen processing and loading of peptides onto newly synthesized major histocompatibility complex (MHC)-class II molecules. Some antigens, such as the lipid-bound, native form of myelin basic protein (LB-MBP) can also be presented by recycling of cell surface MHC class II molecules. The data reported here demonstrate that a preparation of highly purified, delipidated MBP (HP-MBP) follows yet another presentation pathway. Similar to LB-MBP, presentation of HP-MBP to HLA-DR1-restricted T cells was independent of HLA-DM, of newly synthesized proteins, and of invariant chain expression. However, in contrast to LB-MBP, presentation of HP-MBP was also independent of internalization of surface HLA-DR molecules. The different requirements for the presentation of the two molecular forms of MBP were further confirmed by use of the protease inhibitor E64: presentation of LB-MBP but not of HP-MBP was inhibited after treatment of target cells with E64. Furthermore, intact HP-MPB bound to isolated HLA-DR molecules in vitro with an association rate that was considerably faster than that of short peptides. These results show that presentation of HP-MBP is independent of intracellular processing and suggest that it may be presented to T cells by direct binding to surface HLA-DR molecules.     

Evidence for a human leucocyte antigen-DM-induced structural change in human leucocyte antigen-DOβ

Human leucocyte antigen (HLA)-DO is a non-classical major histocompatibility complex class II molecule which modulates the function of HLA-DM and the loading of antigenic peptides on molecules such as HLA-DR. The bulk of HLA-DO associates with HLA-DM and this interaction is critical for HLA-DO egress from the endoplasmic reticulum. HLA-DM assists the early steps of HLA-DO maturation presumably through the stabilization of the interactions between the N-terminal regions of the α and β chains. To evaluate a possible role for HLA-DM in influencing the conformation of HLA-DO, we made use of a monoclonal antibody, Mags.DO5, that was raised against HLA-DO/DM complexes. Using transfected cells expressing mismatched heterodimers between HLA-DR and -DO chains, we found that the epitope for Mags.DO5 is located on the DOβ chain and that Mags.DO5 reactivity was increased upon cotransfection with HLA-DM. Our results suggest that HLA-DM influences the folding of HLA-DO in the endoplasmic reticulum. A mutant HLA-DO showing reduced capacity for endoplasmic reticulum egress was better recognized by Mags.DO5 in the presence of HLA-DM. On the other hand, an HLA-DO mutant capable of endoplasmic reticulum egress on its own was efficiently recognized by Mags.DO5, irrespective of the presence of HLA-DM. Taken together, our results suggest that HLA-DM acts as a private chaperone, directly assisting the folding of HLA-DO to promote egress from the endoplasmic reticulum.     

Spatial separation of HLA-DM/HLA-DR interactions within MIIC and phagosome-induced immune escape

Major Histocompatibility Complex (MHC) class II molecules, including Human Leukocyte Antigen (HLA)-DR, present peptide fragments from proteins degraded in the endocytic pathway. HLA-DR is targeted to late-endocytic structures named MHC class II-containing Compartments (MIIC), where it interacts with HLA-DM. This chaperone stabilizes HLA-DR during peptide exchange and is critical for successful peptide loading. To follow this process in living cells, we have generated cells containing HLA-DR3/Cyan Fluorescent Protein (CFP), HLA-DM/Yellow Fluorescent Protein (YFP), and invariant chain. HLA-DR/DM interactions were observed by Fluorescence Resonance Energy Transfer (FRET). These interactions were pH insensitive, yet occurred only in internal structures and not at the limiting membrane of MIIC. In a cellular model of infection, phagosomes formed a limiting membrane surrounding internalized Salmonella. HLA-DR and HLA-DM did not interact in Salmonella-induced vacuoles, and HLA-DR was not loaded with antigens. The absence of HLA-DR/DM interactions at the limiting membrane prevents local loading of MHC class II molecules in phagosomes. This may allow these bacteria to successfully evade the immune system.     

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.     

HIV-1 gag polyprotein rescues HLA-DR intracellular transport in a human CD4+ cell line

Major histocompatibility complex class II HLA-DR molecules are plasma-membrane integral heterodimers, constitutively expressed in antigen-presenting cells. Their expression is known to be upregulated in peripheral T lymphocytes upon cell activation and to be constitutive in T cell lines. In H78-C10.0, a subclone of the human CD4+ T cell line HUT-78, the transport of MHC class II HLA-DR molecules is blocked, resulting in their localization within internal vesicular compartments rather than at the cell surface. In this article, we show that HIV-1(HX10) infection of H78-C10.0 cells induces HLA-DR surface expression. Moreover, the produced infectious viruses harbor the heterodimer molecules in their envelopes. To define which of the viral proteins was involved in this phenomenon, we infected H78-C10.0 cells with recombinant vaccinia vectors containing either the gag-pro coding sequence or the entire env gene. Only gag expression was able to induce HLA-DR cell-surface localization in H78-C10.0 cells. RT-PCR analysis of the infected cells revealed no significant alteration in the amount of HLA-DRalpha-specific RNA compared to untreated cells. This implies that Gag acts on downstream events. When the env viral gene, coding for the precursor glycoprotein gp160, was expressed in H78-C10.0, the Env protein targeted to the cell surface was poorly processed to its final mature forms gp120 and gp41. However, coexpression of the env and gag genes led to restoration of this phenotype. Although the mechanism is unknown, the data compiled in this study strongly suggest that the viral Gag protein can interact with the cellular trafficking apparatus. Moreover, in a specific cell type as H78-C10.0 this interaction can even reverse intracellular transport defects.     

Class II transactivator-induced expression of HLA-DO(beta) in HeLa cells

HLA-DO is an intracellular nonclassical MHC class II molecule expressed in the endocytic pathway of B lymphocytes. It shapes the repertoire of peptides bound to classical class II molecules such as HLA-DR by regulating the activity of HLA-DM. Using a peptide corresponding to the cytoplasmic tail of HLA-DO(beta), we have developed a mouse monoclonal antibody, HKC5. Immunofluorescence microscopy revealed that HKC5 recognizes HLA-DO molecules present in the endoplasmic reticulum as well as those in vesicular compartments of the endocytic pathway. In addition, the antibody detects the isolated beta chain on Western blots. Using mutants of the DO(beta) cytoplasmic tail fused to a reporter molecule and expressed in epithelial cells, we showed by flow cytometry that the antibody epitope includes one or both of the leucine residues forming the lysosomal sorting signal. Finally, we have used HKC5 to evaluate the presence of the HLA-DO(beta) chain in HeLa cells expressing the class II transactivator protein CIITA. Our flow cytometry and confocal microscopy analyses showed a marked expression of DO(beta) suggesting that HLA-DO could accumulate under the influence of CIITA in non-B cells.     

HLA-Class II Bound Self-Peptides From a Non-Professional APC are Derived from Intracellular Proteins.

Myeloid progenitor cells temporarily express HLA Class II molecules during development. As a first step of inquiry into the function of Class II on these cells, the profile of major self peptides bound to KG-1 myeloid leukemia cells was characterized. Searches of protein data bases revealed that all self peptides corresponded to intracellular, rather than exogenous or transmembrane, precursor proteins. Because the absence of a conventional self-peptide repertoire could be related to altered trafficking of Class II molecules, the biosynthesis of HLA-DR and the invariant chain proteins was determined. The MHC Class II associated invariant chain protein is synthesized normally in KG-1 cells, but processed fragments of invariant chain, CLIPs, occupy the antigen binding groove of KG-1 Class II molecules at a much lower frequency compared to mature APCs. Low CLIP occupancy of HLA-DR is a characteristic shared by KG-1 cells, normal CD 34 + progenitor cells and HLA-DR + breast carcinoma cells. The unusual profile of MHC Class II bound peptides and the low level of CLIP bound to HLA-DR suggest that the antigen processing pathway of KG-1 is different from that characterized in professional APCs and that exogenous antigen processing may be a developmentally acquired characteristic in the myeloid lineage.     

Peptide loading onto recycling HLA-DR molecules occurs in early endosomes

Presentation of exogenous antigens to MHC class II-restricted T cells can follow two different processing pathways. The classical pathway requires newly synthesized MHC class II molecules, invariant chain and HLA-DM expression, whereas the alternative pathway is independent of protein synthesis, invariant chain and HLA-DM. In both cases, MHC class II molecules associate with peptides derived from exogenous antigens that have been processed in endocytic compartments. Different endosomal/prelysosomal compartments where peptide/MHC class II complexes and HLA-DM molecules accumulate have been described. We show here that the alternative pathway uses an earlier compartment than the classical pathway. Experiments with chemically cross-liniked antigen suggest that recycling MHC class II molecules present rapidly degraded antigens, leading to a rapid immune response to exogenously added influenza virus proteins.     

HLA-DM negatively regulates HLA-DR4-restricted collagen pathogenic peptide presentation and T cell recognition

Rheumatoid arthritis, an autoimmune disease, is significantly associated with the HLA class II allele HLA-DR4. While the etiology of rheumatoid arthritis remains unknown, type II collagen (CII) is a candidate autoantigen. An immunodominant pathogenic epitope from this autoantigen, CII(261-273), which binds to HLA-DR4 and activates CD4+ T cells, has been identified. The non-classical class II antigen, HLA-DM, is also a key component of class II antigen presentation pathways influencing peptide presentation by HLA-DR molecules expressed on professional antigen-presenting cells (APC). Here, we investigated whether the HLA-DR4-restricted presentation of the pathogenic CII(261-273) epitope was regulated by HLA-DM expression in APC. We show that APC lacking HLA-DM efficiently display the CII(261-273) peptide/epitope to activate CD4+ T cells, and that presentation of this peptide is modulated dependent on the level of HLA-DM expression in APC. Mechanistic studies demonstrated that the CII(261-273) peptide is internalized by APC and edited by HLA-DM molecules in the recycling pathway, inhibiting peptide presentation and T cell recognition. These findings suggest that HLA-DM expression in APC controls class II-mediated CII(261-273) peptide/epitope presentation and regulates CD4+ T cell responses to this self epitope, thus potentially influencing CII-dependent autoimmunity.     

The MHC class II beta chain cytoplasmic tail overcomes the invariant chain p35-encoded endoplasmic reticulum retention signal

The human-specific p35 isoform of the invariant chain (Ii) includes an R-X-R endoplasmic reticulum (ER) retention motif that is inactivated upon HLA-DR binding. Although the masking is assumed to involve the cytoplasmic tails of class II molecules, the mechanism underlying this function remains to be investigated. Moreover, in light of the polymorphic nature of the class II cytosolic tails, little is known about the capacity of various isotypes or alleles to overcome the retention signal of Iip35. To gain further insights into these issues, we first addressed the proposed role of the HLA-DR cytoplasmic tails. As shown by flow cytometry, the presence of Iip35 in transfected HeLa cells prevented surface expression of HLA-DR molecules lacking their cytoplasmic tails (DRalphaTM/betaTM). These truncated class II molecules and Iip35 accumulated in the ER, and co-localized with calnexin, as determined by confocal microscopy. Sensitivity of DRalphaTM/betaTM to endoglycosidase H treatment confirmed that these molecules do not reach the trans-Golgi network when associated with Iip35. Further characterization revealed that the beta chain cytosolic tail is critical for efficient ER egress of class II/Iip35 complexes. Interestingly, our results clearly demonstrate for the first time that DP and DQ isotypes can also overcome the retention motif of Iip35 through a mechanism involving their very distinctive polymorphic beta chain cytoplasmic tails. Altogether, these results further dissect the masking of di-basic retention signals, and emphasize the interplay between class II molecules and Ii for the transport of the complex to the endocytic pathway.     

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.     

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.