MHC Class I Antigen Processing Pathways

The multistep process that culminates in major histocompatibility complex (MHC) class I presentation of foreign or self-peptides begins in the last phases of protein catabolism. Although the individual roles of many key molecules—such as proteasomes, the transporter associated with antigen processing, and various endoplasmic reticulum chaperones—have recently been elucidated, there still remain many questions regarding processing of proteins into MHC class I bound peptides. This review summarizes the recent developments in antigen processing for MHC class I molecules, with a focus on how proteins are believed to be sampled and selected for degradation.     

MHC Class II-Dependent Peptide Antigen Versus Superantigen Presentation to T Cells

T lymphocytes expressing the CD4 coreceptor can be activated by two classes of major histocompatibility complex (MHC) class II-bound ligands. The elaboration of a conventional T-cell mediated immune response involves recognition of an antigenic peptide bound to the MHC class II molecules by a T-cell receptor (TCR) specific to that particular antigen. Conversely, superantigens (SAgs) also bind to MHC class II molecules and activate T cells, leading to a completely different functional outcome; indeed, SAg-responsive T cells die through apoptosis following stimulation. Superantigens are proteins that are secreted by various bacteria. They interact with the TCR using molecular determinants that are distinct from the residues involved in the recognition of nominal antigenic peptides. Despite the similarities between the recognition of the two classes of ligands by the TCR, considerable structural difference is observed. Here, we discuss the current knowledge on the presentation of SAgs to T cells and compare the different aspects of the SAg response with the recognition of antigenic peptide/MHC complexes.     

Antigen Presentation by MHC Class II Molecules: Invariant Chain Function, Protein Trafficking, and the Molecular Basis of Diverse Determinant Capture

Major histocompatibility complex class II molecules are heterodimeric integral membrane proteins whose primary function is the presentation of antigenic peptides derived from proteins entering the endocytic pathway to CD4⁺ T lymphocytes. To accomplish this physiologic function, class II molecules must assemble in the secretory pathway without undergoing irreversible ligand association at that site, traffic efficiently to the endocytic pathway, and productively interact with protein ligands in these organelles before their ultimate expression on the plasma membrane. Here we review our work describing how invariant chain promotes the assembly and transport process, the complex itinerary of class II–invariant chain complexes through the endocytic pathway, the role of large protein fragments as substrates for class II binding, and the existence of a second pathway for antigen capture by mature class II molecules that complements that involving newly synthesized dimers. We integrate these observations into a coherent model for the operation of a class II-dependent antigen processing and presentation system able to capture diverse antigenic determinants present in proteins of varying structure.     

Delivery of Antigens to the MHC Class I Pathway Using Bacterial Toxins

Cytotoxic T lymphocytes (CTL) recognize antigens derived from endogenously expressed proteins presented on the cell surface in the context of major histocompatibility complex (MHC) class I molecules. Because CTL are effective in antiviral and antitumor responses, the delivery of antigens to the class I pathway has been the focus of numerous efforts. Generating CTL by immunization with exogenous proteins is often ineffective because these antigens typically enter the MHC class II pathway. This review focuses on the usefulness of bacterial toxins for delivering antigens to the MHC class I pathway. Several toxins naturally translocate into the cytosol, where they mediate their cytopathic effects, and the mechanisms by which this occurs has been elucidated. Molecular characterization of these toxins identified the functional domains and enabled the generation of modified proteins that were no longer toxic but retained the ability to translocate into the cytosol. Thus, these modified toxins could be examined for their ability to carry peptides or whole proteins into the cytosolic processing pathway. Of the toxins studied—diphtheria, pertussis, Pseudomonas, and anthrax—the anthrax toxin appears the most promising in its ability to deliver large protein antigens and its efficiency of translocation.     

Peptide–MHC Complexes Assembled Following Multiple Pathways:: An Opportunity for the Design of Vaccines and Therapeutic Molecules

Antigen degradation and peptide loading to major histocompatibility complex class I and class II molecules are described with special emphasis on “noncanonical” pathways. Examples of specific peptide loading for measles proteins are provided. In addition, characterization of defined epitopes presented to T cells can lead to the design of products of special interest in medicine and, in particular, in development of vaccines.     

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.     

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.     

Parameters Affecting the Immunogenicity of Recombinant T Cell Epitopes Inserted into Hybrid Proteins

In the past few years a considerable number of studies have focused on the mechanisms of antigen presentation by classical major histocompatibility complex (MHC) class I and class II encoded molecules. Among different approaches, the engineering of recombinant chimeric genes and proteins has provided new tools to analyze the parameters influencing the intracellular processing of antigenic determinants. This review will summarize and discuss the different models of recombinant genes and molecules that have been used to analyze the influence of the molecular environment of a T cell determinant on its efficient processing and MHC presentation. This approach may also represent an interesting tool for developing new vaccine strategies for inducing T cell responses against pathogens.     

Binding domain regulation of MHC class II molecule assembly, trafficking, fate, and function

Major histocompatibility complex class II molecules are heterodimeric type I integral membrane glycoproteins whose primary function is the capture of fragments of antigen in the endocytic pathway, and the presentation of these peptides to CD4+ αβ TCR-bearing T cells. The biochemical features of the class II peptide binding domain optimize it for this function by allowing interaction with denatured proteins prior to extensive degradation in endosomes and lysosomes. These same properties pose problems for αβ heterodimer assembly, avoidance of non-productive interactions with self-proteins, intracellular transport and dimer stability. This review discusses how coevolution of α and β chain binding domain polymorphism and the extrinsic control of binding site function by invariant chain occupancy deal with these problems and permit the efficient functioning of the class II presentation system.     

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.     

Major histocompatibility class II molecules prevent destructive processing of exogenous peptides at the cell surface of macrophages for presentation to CD4 T cells

We studied factors affecting major histocompatibility complex class II (MHC-II)-restricted presentation of exogenous peptides at the surface of macrophages. We have previously shown that peptide presentation is modulated by surface-associated proteolytic enzymes, and in this report the role of the binding of MHC-II molecules in preventing proteolysis of exogenous synthetic peptides was addressed. Two peptides containing CD4 T-cell epitopes were incubated with fixed macrophages expressing binding and non-binding MHC-II, and supernatants were analysed by high-performance liquid chromatography and mass spectrometry to monitor peptide degradation. The proportion of full-length peptides that were degraded and the number of peptide fragments increased when non-binding macrophages were used, leading to reduction in peptide presentation. When MHC-II molecules expressed on the surface of fixed macrophages were blocked with monoclonal antibody and incubated with peptides and the supernatants were transferred to fixed macrophages, a significant reduction in peptide presentation was observed. Peptide presentation was up-regulated at pH 5.5 compared to neutral pH, and the latter was found to be the pH optimum of the proteolytic activity of the surface enzymes involved in the degradation of exogenous peptides and proteins. The data suggest that MHC-II alleles that bind peptides protect them from degradation at the antigen-presenting cell surface for presentation to CD4 T cells and we argue that this mechanism could be particularly pronounced at sites of inflammation.     

Linkage disequilibrium between HLA class II (DR, DQ, DP) and antigen processing (LMP, TAP, DM) genes of the major histocompatibility complex

TAP, LMP and DM genes map within the major histocompatibility complex (MHC) class II region between the DQB1 and DPB1 loci, and are involved in the processing of peptides bound to HLA class I or class II molecules. In order to determine the various linkage disequilibria existing between these genes and HLA class II genes, we have analyzed TAP1, TAP2, LMP2, DMA, DMB, DRB1, DQA1, DQB1 and DPB1 polymorphisms in 162 unrelated healthy Caucasian individuals. Many positive or negative associations were observed between alleles at these loci, such as between DR/DQ and TAP2, DM or LMP, between DP and DMB, and between TAP2 and DM, TAP2 and LMP. Conversely, no linkage disequilibrium was detected between some closely related genes (DR/DQ and TAP1, TAP1 and TAP2, LMP2 and DM), in agreement with the existence of recombination hot spots in this region. Other weak linkage disequilibria are likely to exist in this region. These data allow to define some conserved MHC class II haplotypes including HLA class II and TAP, LMP and DM alleles. Furthermore, the knowledge of such linkage disequilibria is of outstanding importance in order to avoid misinterpretation of the data when studying MHC class II associations with autoimmune diseases.     

Pretransplant Exposure to Donor HLA-DR Antigen in Random Transfusion Units and the Development of Donor Antigen-Specific Hyporeactivity

Our previous studies have shown that the in vitro assay of donor antigen-specific hyporeactivity is a useful marker for identifying solid organ transplant recipients (kidney, lung and heart) at low risk for immunologic complications (i.e., late acute rejection episodes and chronic rejection). Donor antigen-specific hyporeactivity is defined as a significantly decreased post- vs. pretransplant proliferative response to donor antigens while response to third-party controls remains unchanged. We analyzed whether exposure to the same HLA-DR antigen pretransplant via random blood transfusion and posttransplant via the transplanted organ influenced the development of hyporeactivity. Thirty previously nontransfused recipients, each receiving two 150 ml pretransplant random blood transfusions, were assessed for hyporeactivity at 1 year posttransplant. Of the 12 recipients with pretransplant exposure to kidney HLA-DR via transfusions, 6 (50%) developed hyporesponsiveness; in contrast, of the 18 recipients who were not preexposed, only 3 (15%) exhibited this form of immunomodulation. Of interest, 2 of the 3 hyporesponsive recipients who were not preexposed, received units containing HLA-DR antigens previously shown to share crossreactive epitopes with the kidney HLA-DR. In conclusion, these results suggest a increased incidence in the development of hyporeactivity in patients receiving pretransplant transfusions which share an HLA-DR antigen with the transplanted kidney.     

Class II Antigen Processing Compartments and the Function of HLA-DM

The DMα and DMβ genes encode a nonpolymorphic, class II-like molecule which functions by an, as yet, undefined mechanism in the assembly of Major Histocompatibility Complex class II-peptide complexes. Indeed, mutant cells which express class II molecules but fail to express DM are unable to process and present native protein antigens. A striking phenotype of the mutation is class II molecules that contain almost exclusively a nested set of invariant chain peptides, termed CLIP, for class II associated Ii peptides, instead of the normal array of endogenously and exogenously derived peptides. Thus, DM appears to be required for the correct assembly of processed antigen-class II complexes. Recently, the subcellular compartments that contain DM and in which functional processed antigen-class II complexes are first formed have been described. Here, the evidence for the function of DM in the antigen-processing compartments is reviewed.     

PRBAM: a new tool to analyze the MHC class I and HLA-DR anchor motifs

Major histocompatibility complex (MHC) genes are highly polymorphic, which makes each MHC molecule different regarding their peptide repertoire, so they can bind and present to T lymphocytes. The increasing importance of immunopeptidomics and its use in personalized medicine in different fields such as oncology or autoimmunity demand the correct analysis of the peptide repertoires bound to human leukocyte antigen type 1 (HLA-I) and HLA-II molecules. Purification of the peptide pool by affinity chromatography and individual peptide sequencing using mass spectrometry techniques is the standard protocol to define the binding motifs of the different MHC-I and MHC-II molecules. The identification of MHC-I binding motifs is relatively simple, but it is more complicated for MHC-II. There are some programs that identify the anchor motifs of MHC-II molecules. However, these programs do not identify the anchor motif correctly for some HLA-II molecules and some anchor motifs have been deduced using subjective interpretation of the data. Here, we present a new software, called PRBAM (Peptide Repertoire-Based Anchor Motif) that uses a new algorithm based on the peptide–MHC interactions and, using peptide lists obtained by mass spectrometry sequencing, identifies the binding motif of MHC-I and HLA-DR molecules. PRBAM has an easy-to-use interface, and the results are presented in graphics, tables and peptide lists. Finally, the fact that PRBAM uses a new algorithm makes it complementary to other existing programs.     

Effective antigen presentation to helper T cells by human eosinophils

Although eosinophils are inflammatory cells, there is increasing attention on their immunomodulatory roles. For example, murine eosinophils can present antigen to CD4⁺ T helper (Th) cells, but it remains unclear whether human eosinophils also have this ability. This study determined whether human eosinophils present a range of antigens, including allergens, to activate Th cells, and characterized their expression of MHC class II and co‐stimulatory molecules required for effective presentation. Human peripheral blood eosinophils purified from non‐allergic donors were pulsed with the antigens house dust mite extract (HDM), Timothy Grass extract (TG) or Mycobacterium tuberculosis purified protein derivative (PPD), before co‐culture with autologous CD4⁺ Th cells. Proliferative and cytokine responses were measured, with eosinophil expression of HLA‐DR/DP/DQ and the co‐stimulatory molecules CD40, CD80 and CD86 determined by flow cytometry. Eosinophils pulsed with HDM, TG or PPD drove Th cell proliferation, with the response strength dependent on antigen concentration. The cytokine responses varied with donor and antigen, and were not biased towards any particular Th subset, often including combinations of pro‐ and anti‐inflammatory cytokines. Eosinophils up‐regulated surface expression of HLA‐DR/DP/DQ, CD80, CD86 and CD40 in culture, increases that were sustained over 5 days when incubated with antigens, including HDM, or the major allergens it contains, Der p I or Der p II. Human eosinophils can, therefore, act as effective antigen‐presenting cells to stimulate varied Th cell responses against a panel of antigens including HDM, TG or PPD, an ability that may help to determine the development of allergic disease.     

DM influences the abundance of major histocompatibility complex class II alleles with low affinity for class II-associated invariant chain peptides via multiple mechanisms

DM catalyses class II-associated invariant chain peptide (CLIP) release, edits the repertoire of peptides bound to major histocompatibility complex (MHC) class II molecules, affects class II structure, and thereby modulates binding of conformation-sensitive anti-class II antibodies. Here, we investigate the ability of DM to enhance the cell surface binding of monomorphic antibodies. We show that this enhancement reflects increases in cell surface class II expression and total cellular abundance, but notably these effects are selective for particular alleles. Evidence from analysis of cellular class II levels after cycloheximide treatment and from pulse-chase experiments indicates that DM increases the half-life of affected alleles. Unexpectedly, the pulse-chase experiments also revealed an early effect of DM on assembly of these alleles. The allelically variant feature that correlates with susceptibility to these DM effects is low affinity for CLIP; DM-dependent changes in abundance are reduced by invariant chain (CLIP) mutants that enhance CLIP binding to class II. We found evidence that DM mediates rescue of peptide-receptive DR0404 molecules from inactive forms in vitro and evidence suggesting that a similar process occurs in cells. Thus, multiple mechanisms, operating along the biosynthetic pathway of class II molecules, contribute to DM-mediated increases in the abundance of low-CLIP-affinity alleles.     

The Diversity of Antigen-Specific TCR Repertoires Reflects the Relative Complexity of Epitopes Recognized

Antigen-selected T cell receptor (TCR) repertoires vary in complexity from very limited to extremely diverse. We have previously characterized two different CD8 T cell responses, which are restricted by the same mouse major histocompatibility complex (MHC) class I molecule, H-2 K^d. The TCR repertoire in the response against a determinant from Plasmodium berghei circumsporozoite protein (PbCS; region 252–260) is very diverse, whereas TCRs expressed by clones specific for a determinant in region 170–179 of HLA-CW3 (human) MHC class I molecule show relatively limited structural diversity. We had already demonstrated that cytolytic T lymphocyte (CTL) clones specific for the PbCS peptide display diverse patterns of antigen recognition when tested with a series of single Ala-substituted PbCS peptides or mutant H-2 K^d molecules. We now show that CW3-specific CTL clones display much less diverse patterns of recognition. Our earlier functional studies with synthetic peptide variants suggested that the optimal peptides recognized were 9 (or 8) residues long for PbCS and 10 residues long for CW3. We now present more direct evidence that the natural CW3 ligand is indeed a 10-mer. Our functional data together with molecular modeling suggest that the limited TCR repertoire selected during the CW3 response is not due to a paucity of available epitopes displayed at the surface of the CW3 peptide/K^d complex. We discuss other factors, such as the expression of similar self MHC peptide sequences, that might be involved in trimming this TCR repertoire.     

Cross-priming utilizes antigen to the direct presentation pathway

CD8+ T cells play a crucial role in protective immunity to viruses and tumours. Antiviral CD8+ T cells are initially activated by professional antigen presenting cells (pAPCs) that are directly infected by viruses (direct-priming) or following uptake of exogenous antigen transferred from virus-infected or tumour cells (cross-priming). In order to efficiently target each of these antigen-processing pathways during vaccine design, it is necessary to delineate the properties of the natural substrates for either of these antigen-processing pathways. In this study, we utilized a novel T-cell receptor (TCR) transgenic mouse to examine the requirement for both antigen synthesis and synthesis of other cellular factors during direct or cross-priming. We found that direct presentation required ongoing synthesis of antigen, but that cross-priming favoured long-lived antigens and did not require ongoing antigen production. Even after prolonged blockade of protein synthesis in the donor cell, cross-priming was unaffected. In contrast, direct-presentation was almost undetectable in the absence of antigen neosynthesis and required ongoing protein synthesis. This suggests that the direct- and cross-priming pathways may utilize differing pools of antigen, an observation that has far-reaching implications for the rational design of vaccines aimed at the generation of protective CD8+ T cells.     

Evasion and subversion of antigen presentation by Mycobacterium tuberculosis

Mycobacterium tuberculosis is one of the most successful of human pathogens and has acquired the ability to establish latent or progressive infection and persist even in the presence of a fully functioning immune system. The ability of M. tuberculosis to avoid immune-mediated clearance is likely to reflect a highly evolved and coordinated program of immune evasion strategies, including some that interfere with antigen presentation to prevent or alter the quality of T-cell responses. Here, we review an extensive array of published studies supporting the view that antigen presentation pathways are targeted at many points by pathogenic mycobacteria. These studies show the multiple potential mechanisms by which M. tuberculosis may actively inhibit, subvert or otherwise modulate antigen presentation by major histocompatibility complex class I, class II and CD1 molecules. Unraveling the mechanisms by which M. tuberculosis evades or modulates antigen presentation is of critical importance for the development of more effective new vaccines based on live attenuated mycobacterial strains.