Functional regulation of immunoproteasomes and transporter associated with antigen processing

The central event in the cellular immune response to invading pathogens is the presentation of non-self antigenic peptides by major histocompatibility complex (MHC) class I molecules to cytotoxic T lymphocytes (CTLs). As peptide binding and transport proteins, MHC class I molecules have evolved distinct biochemical and cellular strategies for acquiring antigenic peptides, providing CTLs an extracellular representation of the intracellular antigen content. Whereas efficient generation of MHC class I binding peptides depends on the intracellular, immunoproteasome-mediated proteolysis machinery, translocation of peptides into the lumen of the endoplasmic reticulum requires the endoplasmic reticulum-resident, adenosine 5'-triphosphate (ATP) binding cassette transporter associated with antigen processing (TAP). Here we show, for the first time, that immunoproteasomes, TAP complexes, and MHC class I molecules are physically associated, providing an effective means of transporting MHC class I binding peptides from their sites of generation into the lumen of the endoplasmic reticulum for loading onto MHC class I molecules. In this review, we assess the current understanding of the functional regulation of immunoproteasomes and transporter associated with antigen processing.     

Forcing Tumor Cells to Present Their Own Tumor Antigens to the Immune System： a Necessary Design for an Efficient Tumor Immunotherapy

The general principle for tumor cells to escape from immune surveillance is to prevent tumor antigens from being recognized by the immune system. Many methods have been developed to increase the immunogenecity of the tumor cells. The most efficient methods are able to force tumor cells to present their own tumor antigens to the immune system. Stimulating Th cells by converting tumor cells into MHC class II /Ii- antigen presenting cells is one of the most efficient technologies. Using antisense methods, we suppress the expression of the Ii protein that normally co-expresses with MHC class II molecules and blocks the antigenic peptide binding site of MHC class II molecules during synthesis in the endoplasmic reticulum. In such tumor cells, the“unprotected“ MHC class II molecules pick up endogenous tumor antigenic peptides, which have been transported into the ER for binding to MHC class I molecules. Simultaneous presentation of tumor antigens by both MHC class I and II molecules generates a robust and long-lasting anti-tumor immune response. MHC class II /Ii- tumor cells are potent tumor cell vaccines and also cure a significant number of animals with renal and prostate tumors. We have developed analogous human gene vectors that are suitable for most patients and cancers.     

Enhancing cytotoxic T cell responses with altered-peptide ligands.

Interest in class I MHC-mediated immunotherapy is growing rapidly. In order to fight a virus or cancer effectively, a successful immunotherapeutic must activate a large number of specific CD8+ T cells and also generate immunological memory. Attempts to generate immune responses towards tumor- or virus-derived peptides have frequently been frustrated by the nature of the peptide antigen itself. Either the peptide does not bind well to its cognate MHC, or the T cells directed towards it have been functionally inactivated in vivo. Altered-peptide ligands (APL) are an effective way to circumvent these problems. However, generating enhanced binding of altered peptides to class I MHC while still maintaining recognition of the wild-type peptide is not straightforward. Many groups design enhanced binding peptides by substituting the observed anchor residues with those that are most preferred by the class I MHC molecule. For many antigenic peptides, this approach does not work. Furthermore, if a higher affinity peptide is designed, the substitutions may result in reduced recognition by CD8+ T cells. Therefore, the design of APL requires careful testing of each candidate therapeutic in terms of affinity for class I MHC and immunological reactivity. Lastly, immunotherapy using class I MHC must also take into account the large genetic heterogeneity in the population. A therapeutic that is only effective for 5-10% of the population is not as attractive as one that works for over 90% of the population. The use of MHC supertypes (groups of class I MHC allotypes that share similar peptide-binding characteristics) shows great promise in overcoming this problem.     

Cut and trim: generating MHC class I peptide ligands

Progress in understanding how peptide ligands are generated for MHC class I molecules took several interesting leaps and twists in the past year. Two independent lines of evidence suggest that most peptides are generated by proteasomal digestion of nascent proteins. The amino-terminally extended cytosolic precursors of an antigenic peptide were identified, bound to a mysterious carrier protein. Knowledge about the role of immunoproteasomes in antigen processing was fortified, cellular locales specialized for proteasomal degradation (and possibly antigenic-peptide production) were discovered and novel cytosolic proteases potentially involved in generating and trimming antigenic peptides were identified. The field is poised for quantitative analysis of the various pathways that contribute to the pool of peptides presented to the immune system by MHC class I molecules.     

Direct analysis of peptide binding to cell-associated MHC class I molecules.

Exogenously added synthetic peptides can mimic endogenously produced antigenic peptides recognized on target cells by MHC class I-restricted cytolytic T lymphocytes. While it is assumed that exogenous peptides associate with class I molecules on the target cell surface, direct binding of peptides to cell-associated class I molecules has been difficult to demonstrate. Using a newly developed binding assay based on photoaffinity labeling, we have investigated the interaction of two antigenic peptides, known to be recognized in the context of H-2Kd or H-2Db, respectively, with 20 distinct class I alleles on living cells. None of the class I alleles tested, with the exception of H-2Kd or H-2Db, bound either of the peptides, thus demonstrating the exquisite specificity of peptide binding to class I molecules. Moreover, peptide binding to cell-associated H-2Kd was drastically reduced when metabolic energy, de novo protein synthesis or protein egress from the endoplasmic reticulum was inhibited. It is thus likely that exogenously added peptides do not associate with the bulk of class I molecules expressed at the cell surface, but rather bind to short-lived molecules devoid of endogenous peptides.     

Generating MHC class I ligands from viral gene products

Summary: MHC class I molecules function to present peptides comprised of eight to I 1 residues to CD8+ T lymphocytes. Here we review the efforts of our laboratory lo understand bow cells generate such peptides from viral gene products. We particularly focus on the nature of substrates acted on by cytosolic proteases, the contribution of proteasomes and nun-proteasomal proteases lo peptide generation, the involvement of ubiquitination in peptide generation, the intracellular localization of proteasome generation of antigenic peptides, and the trimming of peptides in the endoplasmic reticulum.     

Proteasome isoforms exhibit only quantitative differences in cleavage and epitope generation

Immunoproteasomes are considered to be optimised to process Ags and to alter the peptide repertoire by generating a qualitatively different set of MHC class I epitopes. Whether the immunoproteasome at the biochemical level, influence the quality rather than the quantity of the immuno‐genic peptide pool is still unclear. Here, we quantified the cleavage‐site usage by human standard‐ and immunoproteasomes, and proteasomes from immuno‐subunit‐deficient mice, as well as the peptides generated from model polypeptides. We show in this study that the different proteasome isoforms can exert significant quantitative differences in the cleavage‐site usage and MHC class I restricted epitope production. However, independent of the proteasome isoform and substrates studied, no evidence was obtained for the abolishment of the specific cleavage‐site usage, or for differences in the quality of the peptides generated. Thus, we conclude that the observed differences in MHC class I restricted Ag presentation between standard‐ and immunoproteasomes are due to quantitative differences in the proteasome‐generated antigenic peptides.     

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.     

Conformational heterogeneity of MHC class II induced upon binding to different peptides is a key regulator in antigen presentation and epitope selection

T cells bearing αβ receptors recognize antigenic peptides bound to class I and class II glycoproteins encoded in the major histocompatibility complex (MHC). Cytotoxic and helper T cells respond respectively to peptide antigens derived from endogenous sources presented by MHC class I, and exogenous sources presented by MHC II, on antigen presenting cells. Differences in the MHC class I and class II structures and their maturation pathways have evolved to optimize antigen presentation to their respective T cells. A main focus of our laboratory is on efforts to understand molecular events in processing of antigen for presentation by MHC class II. The different stages of MHC class II—interactions with molecular chaperons involved in folding and traffic from the ER through the antigen-loading compartments, peptide exchange, and transport to the cell surface have been investigated. Through intense research on biophysical and biochemical properties of MHC class II molecules, we have learned that the conformational heterogeneity of MHC class II induced upon binding to different peptides is a key regulator in antigen presentation and epitope selection, and a determinant of the ability of MHC class II to participate in peptide association or dissociation and interaction with the peptide editor HLA-DM.     

MHC class I antigen presentation--recently trimmed and well presented

Presentation of antigenic peptide to T cells by major histocompatibility complex (MHC) class I molecules is the key to the cellular immune response. Non-self intracellular proteins are processed into short peptides and transported into endoplasmic reticulum (ER) where they are assembled with class I molecules assisted by several chaperone proteins to form trimeric complex. MHC class I complex loaded with optimised peptides travels to the cell surface of antigen presentation cells to be recognised by T cells. The cells presenting non-self peptides are cleared by CD8 positive T cells. In order to ensure that T cells detect an infection or mutation within the target cells the process of peptide loading and class I expression must be carefully regulated. Many of the cellular components involved in antigen processing and class I presentation are known and their various functions are now becoming clearer.     

MHC class I molecules can direct proteolytic cleavage of antigenic precursors in the endoplasmic reticulum

The large set of peptides presented by MHC (major histocompatibility complex) class I molecules are generated by proteolysis of diverse precursors in the cytoplasm and possibly in the endoplasmic reticulum (ER). To define the potential peptide trimming events in the ER, we analyzed proteolytic products generated in isolated microsomes. The residues flanking the N terminus of the final antigenic peptide were rapidly removed within the microsomes but only in the presence of appropriate MHC molecules. Remarkably, the precursor peptide was bound to the MHC molecules in a distinct conformation and required an aminopeptidase activity to generate the optimal peptide. The MHC molecules are therefore not only the final repositories of antigenic peptides, but they can also direct their excision from longer precursors.     

Enhanced major histocompatibility complex class I binding and immune responses through anchor modification of the non-canonical tumour-associated mucin 1-8 peptide

Designing peptide-based vaccines for therapeutic applications in cancer immunotherapy requires detailed knowledge of the interactions between the antigenic peptide and major histocompatibility complex (MHC) in addition to that between the peptide-MHC complex and the T-cell receptor. Past efforts to immunize with high-affinity tumour-associated antigenic peptides have not been very immunogenic, which may be attributed to the lack of T cells to these peptides, having been deleted during thymic development. For this reason, low-to-medium affinity non-canonical peptides represent more suitable candidates. However, in addition to the difficulty in identifying such antigens, peptide binding to MHC, and hence its ability to induce a strong immune response, is limited. Therefore, to enhance binding to MHC and improve immune responses, anchor modifications of non-canonical tumour-associated peptides would be advantageous. In this study, the non-canonical tumour-associated peptide from MUC1, MUC1-8 (SAPDTRPA), was modified at the MHC anchor residues to SAPDFRPL (MUC1-8-5F8L) and showed enhanced binding to H-2Kb and improved immune responses. Furthermore, the crystal structure of MUC1-8-5F8L in complex with H-2Kb was determined and it revealed that binding of the peptide to MHC is similar to that of the canonical peptide OVA8 (SIINFEKL).     

The carboxypeptidase ACE shapes the MHC class I peptide repertoire

The surface presentation of peptides by major histocompatibility complex (MHC) class I molecules is critical to CD8(+) T cell-mediated adaptive immune responses. Aminopeptidases have been linked to the editing of peptides for MHC class I loading, but carboxy-terminal editing is thought to be due to proteasome cleavage. By analysis of wild-type mice and mice genetically deficient in or overexpressing the dipeptidase angiotensin-converting enzyme (ACE), we have now identified ACE as having a physiological role in the processing of peptides for MHC class I. ACE edited the carboxyl terminus of proteasome-produced MHC class I peptides. The lack of ACE exposed new antigens but also abrogated some self antigens. ACE had substantial effects on the surface expression of MHC class I in a haplotype-dependent manner. We propose a revised model of peptide processing for MHC class I by introducing carboxypeptidase activity into the process.     

ERAAP synergizes with MHC class I molecules to make the final cut in the antigenic peptide precursors in the endoplasmic reticulum

The major histocompatibility complex class I molecules display peptides (pMHC I) on the cell surface for immune surveillance by CD8(+) T cells. These peptides are generated by proteolysis of intracellular polypeptides by the proteasome in the cytoplasm and then in the endoplasmic reticulum (ER) by the ER aminopeptidase associated with antigen processing (ERAAP). To define the unknown mechanism of ERAAP function in vivo, we analyzed naturally processed peptides in cells with or without appropriate MHC I and ERAAP. In the absence of MHC I, ERAAP degraded the antigenic precursors in the ER. However, MHC I molecules could bind proteolytic intermediates and were essential for generation of the final peptide by ERAAP. Thus, ERAAP synergizes with MHC I to generate the final pMHC I repertoire.     

Structure of MHC class I and class II cDNAs and possible immunodeficiency linked to class II expression in the Mexican axolotl

Summary: Despite the fact that the axolotl (Ambystoma spp. a urodele amphibian) displays a large T-cell repertoire and a reasonable B-cell repertoire, its humoral immune response is slow (60 days), non-anamnestic, with a unique IgM class. The cytotoxic immune response is slow as well (21 days) with poor mixed lymphocyte reaction stimulation. Therefore, this amphibian can be considered as immunodeficient. The reason for this subdued immune response could be an altered antigenic presentation by major histocompatibility complex (MHC) molecules. This article summarizes our work on axolotl MHC genes. Class I genes have been characterized and the cDNA sequences show a good conservation of non-polymorphic peptide binding positions of the a chain as well as a high diversity of the variable amino acids positions, suggesting that axolotl class I molecules can present numerous antigenic epitopes. Moreover, class I genes are ubiquitously transcribed at the lime of hatching. These class 1 genes also present an important polylocism and belong to the same linkage group as the class II B gene, they can be reasonably considered as classical class 1a genes. However, only one class II B gene has been characterized so far by Southern blot analysis. As in higher vertebrates, this gene is transcribed in lymphoid organs when they start to be functional. The sequence analysis shows that the peptide binding region of this class II β chain is relatively well conserved, but most of all does not present any variability in the β domain in inbred as well as in wild axolotls presuming a limited antigenic presentation of few antigenic epitopes. The immunodeficiency of the axolotl could then be explained by an altered class 11 presentation of antigenic peptides, putting into question the existence of cellular co-operation in this lowervertebrate. It will be interesting to analyze the situation in other urodele species and to determine whether our observations in axolotl represent a normal feature in urodele amphibians. But already two different models in amphibians, Xenopus and axolotl, must be considered in our search for understanding immune system and MHC evolution.     

MHC Class Ⅰ Antigen Presentation-Recently Trimmed and Well Presented

Presentation of antigenic peptide to T cells by major histocompatibility complex (MHC) class I molecules is thekey to the cellular immune response.Non-self intracellular proteins are processed into short peptides andtransported into endoplasmic reticulum (ER) where they are assembled with class I molecules assisted by severalchaperone proteins to form trimeric complex.MHC class I complex loaded with optimised peptides travels to thecell surface of antigen presentation cells to be recognised by T cells.The cells presenting non-self peptides arecleared by CD8 positive T cells.In order to ensure that T cells detect an infection or mutation within the targetcells the process of peptide loading and class I expression must be carefully regulated.Many of the cellularcomponents involved in antigen processing and class I presentation are known and their various functions arenow becoming clearer.Cellular & Molecular Immunology.2004;1(1):22-30.     

Antigen‐loaded ER microsomes from APC induce potent immune responses against viral infection

Although matured DC are capable of inducing effective primary and secondary immune responses in vivo, it is difficult to control the maturation and antigen loading in vitro. In this study, we show that ER‐enriched microsomal membranes (microsomes) isolated from DC contain more peptide‐receptive MHC I and II molecules than, and a similar level of costimulatory molecules to, their parental DC. After loading with defined antigenic peptides, the microsomes deliver antigenic peptide–MHC complexes (pMHC) to both CD4 and CD8 T cells effectively in vivo. The peptide‐loaded microsomes accumulate in peripheral lymphoid organs and induce stronger immune responses than peptide‐pulsed DC. The microsomal vaccines protect against acute viral infection. Our data demonstrate that peptide–MHC complexes armed microsomes from DC can be an important alternative to DC‐based vaccines for protection from viral infection.     

MHC class I-associated peptides identified from normal platelets and from individuals with idiopathic thrombocytopenic purpura

Major histocompatibility complex (MHC) class I molecules bind and display peptide antigens on the cell surface. CD8(+) T lymphocytes recognize peptides in association with class I proteins to initiate a cytotoxic immune response. To understand the specificity of such immune responses and to facilitate the development of therapies for disease, it is important to identify MHC-presented peptides. In this study, platelets, easily obtainable and often associated with immune-mediated disease, were selected to identify MHC class I-associated peptides. MHC-associated peptides presented on platelets of normal individuals and individuals with idiopathic thrombocytopenic purpura (ITP) were characterized. ITP is characterized by the premature immune destruction of platelets. It is associated with the production of antiplatelet autoantibodies, most often targeting platelet membrane GPIIb/IIIa or GPIb/IX. In addition to characterizing five fully and several partially sequenced peptides from platelets, the peptide GPRGA(L/I)S(L/I)(L/I) was identified from four of the five ITP patients. The anchor motif of this peptide correlates with the presence of the HLA-B7 allele. A BLAST search identified this peptide as GPIb (4-12). In conclusion, platelets from normal and ITP individuals can present peptides from general cellular proteins and platelet specific proteins, such as GPIb, to the immune system via MHC class I.     

Carrier-mediated uptake and presentation of a major histocompatibility complex class I-restricted peptide

Antigenic peptides derived from endogenous or viral proteins can associate with class I or class II major histocompatibility complex (MHC) molecules, while exogenous antigens are endocytosed, processed intracellularly and presented on MHC class II molecules. Here we describe a method that allows the presentation of an MHC class I-restricted antigenic peptide on MHC class I molecules, although it was taken up from the outside. The HLA-A2-restricted influenza virus matrix protein-derived peptide (flu, 57–68) was used either in soluble form or coupled via an S-S bridge to transferrin (Tf-flu). Target cells were incubated with flu or Tf-flu and the effective antigen presentation was detected in a cytotoxicity assay using flu peptide-specific, HLA-A2-restricted CD8⁺ cytotoxic T lymphocytes. Sensitization of target cells with Tf-flu required 5 to 10 times higher molar concentrations of peptide compared to sensitization with soluble free peptide. The Tf-flu construct was taken up by the cells via the Tf receptor (CD71) as the binding of Tf-flu was blocked by an excess of Tf. In contrast to the flu peptide, cytotoxicity elicited by Tf-flu was blocked by brefeldin A but not by chloroquine nor inhibitors of intracellular reducing steps, like 1-buthionine-(s, r)-sulfoximine or n-ethylmaleimide. Presentation of the flu peptide derived from Tf-flu construct is not hindered in the mutant T2 cell line, which lacks genes coding for transporter proteins for antigenic peptides (TAP1/TAP2) and proteasomes subunits, suggesting that the processing pathway described in this report may involve TAP-independent steps.     

Need for cellular and humoral immune responses in bovines to ensure protection from foot-and-mouth disease virus (FMDV)—A point of view

The published studies on immunization of experimental animals, cattle, and sheep with synthetic peptides containing the antigenic domains in FMDV structural protein VP1 were analyzed. The results obtained with various FMDV synthetic peptides designed to stimulate the humoral immune response in bovines were compared to the current knowledge on MHC class I and class II, and the properties of the peptide binding grooves in each of them. X-ray crystallography of MHC class I proteins provided the three-dimensional structure of the peptide binding groove and led to the isolation and identification of self and viral peptides that naturally associate with the peptide binding grooves of both types of MHC and HLA molecules. The available knowledge of the amino acid motifs in MHC and HLA class I-bound viral peptides priming the CD8⁺ cytotoxic T cell responses must be coupled with the understanding of the three-dimensional structure of BoLA class I. This would aid in the development of an experimental approach to induce bovine anti-FMDV CD8⁺ cytotoxic cells to complement the humoral immune response to FMDV, which is currently achieved by a killed virus vaccine and, at the experimental level, by a peptide vaccine. Stimulation of both cellular and humoral immune responses against FMDV in cattle may reduce the risk of disease and virus shedding.