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.     

Development of non-live vectors and procedures (liposomes, pseudo-viral particles, toxin, beads, adjuvantsellipsis) as tools for cancer vaccines

Recombinant virus encoding tumor antigens are the most used vectors in human clinical trials of cancer vaccines because of their ability to target exogenous antigen in the endogenous MHC class I pathway and to elicit CTL. However, their use requires different constraining procedures to avoid their spreading. The immunosuppression of cancer patients may also increase their intrinsic toxicity. Therefore, the development of non-live vectors may avoid these drawbacks. Different groups now clearly demonstrated that particulate antigens when they are phagocytosed could be targeted in the MHC class I pathway. They also induce CTL in mice which when immunized with these particulate antigens were protected against a challenge with tumors expressing this antigen. Other strategies using toxins or antigens fused or incorporated into various oil or lipid based chemical adjuvants have also succeeded in the induction of CTL response and in some cases have been shown to be efficient as cancer vaccine.     

Major histocompatibility complex class I-restricted antigen processing and presentation

Major histocompatibility complex (MHC) class I molecules present antigenic peptides to CD8-expressing cytotoxic T lymphocytes (CTLs). This antigen recognition system is critically important for immune surveillance against viruses and tumors. Most class I-binding peptides are generated in the cytosol, as side products from the degradation of misfolded proteins by proteasomes. A subset of the resulting peptides are translocated across the endoplasmic reticulum (ER) membrane by a dedicated peptide transporter, and these peptides are then loaded onto peptide-receptive class I molecules in the ER. The stable assembly of class I molecules with peptides is controlled by a variety of accessory proteins, including chaperones with general housekeeping functions and factors with dedicated roles in class I assembly. Peptide-filled class I molecules are then delivered to the cell surface for recognition by CTLs. This highly regulated process permits the host to rapidly counter invading pathogens with strong and sustained CTL responses and, at the same time, avoid misguided attacks. Here, how the class I antigen processing machinery accomplishes this daunting task is reviewed.     

Lipopeptides: a novel antigen repertoire presented by major histocompatibility complex class I molecules

Post‐translationally modified peptides, such as those containing either phosphorylated or O‐glycosylated serine/threonine residues, may be presented to cytotoxic T lymphocytes (CTLs) by MHC class I molecules. Most of these modified peptides are captured in the MHC class I groove in a similar manner to that for unmodified peptides. N‐Myristoylated 5‐mer lipopeptides have recently been identified as a novel chemical class of MHC class I‐presented antigens. The rhesus classical MHC class I allele, Mamu‐B*098, was found to be capable of binding N‐myristoylated lipopeptides and presenting them to CTLs. A high‐resolution X‐ray crystallographic analysis of the Mamu‐B*098:lipopeptide complex revealed that the myristic group as well as conserved C‐terminal serine residue of the lipopeptide ligand functioned as anchors, whereas the short stretch of three amino acid residues located in the middle of the lipopeptides was only exposed externally with the potential to interact directly with specific T‐cell receptors. Therefore, the modes of lipopeptide–ligand interactions with MHC class I and with T‐cell receptors are novel and fundamentally distinct from that for MHC class I‐presented peptides. Another lipopeptide‐presenting MHC class I allele has now been identified, leading us to the prediction that MHC class I molecules may be separated on a functional basis into two groups: one presenting long peptides and the other presenting short lipopeptides. Since the N‐myristoylation of viral proteins is often linked to pathogenesis, CTLs capable of sensing N‐myristoylation may serve to control pathogenic viruses, raising the possibility for the development of a new type of lipopeptide vaccine.     

Fusogenic liposomes efficiently deliver exogenous antigen through the cytoplasm into the MHC class I processing pathway

Exogenous soluble proteins enter the endosomal pathway by endocytosis and are presented in association with MHC class II rather than class I. In contrast, the delivery of exogenous protein antigens (Ag) into the cytosol generates MHC class I-restricted cytotoxic T lymphocytes (CTL) responses. Although several immunization approaches, such as the utilization of liposomes, have induced the in vivo priming of MHC class I-restricted CTL responses to protein Ag, it remains unclear whether this priming results from the direct delivery of protein Ag to the cytosol. Here we report that fusogenic liposomes (FL), which are prepared by fusing simple liposomes with Sendai virus particles, can deliver the encapsulated soluble protein directly into the cytosol of cells cultured concurrently and introduce it into the conventional MHC class I Ag presentation pathway. Moreover, a single immunization with ovalbumin (OVA) encapsulated in FL but not in simple liposomes results in the potent priming of OVA-specific CTL. Thus, FL function as an efficient tool for the delivery of CTL vaccines.     

MHC class I antigen processing of Listeria monocytogenes proteins: implications for dominant and subdominant CTL responses

Summary: Listeria monocytogenes (L. monocytogenes) secretes proteins associated with its virulence into the cytosol of infected cells. These secreted proteins are degraded by host cell proteasomes and processed into peptides that are bound by MHC class I molecules in the endoplasmic reticulum. We have found that the MHC class I antigen-processing pathway is very efficient at generating the epitopes that are presented to cytolytic T lymphocytes (CTL). Depending on which antigen is investigated, from 3 to 30 % of degraded antigens are processed into nonamer peptides that are bound by MHC class I molecules. Surprisingly, neither the efficiency of epitope generation nor the absolute number of epitopes per infected cell determines the magnitude of the in vivo CTL response. One of the least prevalent epitopes, derived from an antigen that is virtually undetectable in infected cells, primes the immunodominant CTL response in L. monocytogenes -infected mice. Our studies suggest that immunodominant and subdominant T-cell responses cannot be predicted by the prevalence of antigens or epitopes alone, and that additional factors, yet to be determined, are involved.     

Priming of cytotoxic T lymphocytes by five heat-aggregated antigens in vivo: Conditions,efficiency, and relation to antibody responses

Mice were immunized i.p. with soluble or heat-denatured protein antigens [ovalbumin, β-galactosidase, or recombinant E7 protein of human papilloma virus type 16 (HBV)]. Heat-denatured (100°C) preparations of these proteins were able to induce cytotoxic T lymphocytes (CTL) that recognize cells expressing the respective genes, whereas native protein was either inefficient or required up to 30-fold higher doses. If the heat-treated proteins were separated into aggregated and soluble fractions by ultracentrifugation, only the aggregated fractions were able to induce specific CTL; this is probably because of the easier access to one of the major histocompatibility complex class I loading pathways for exogenous antigen. Addition of the adjuvant aluminium hydroxide (alum) to aggregated proteins abolished their ability to induce CTL; thus, a condition leading to a strong antibody response appeared to inhibit CTL induction. Interestingly, immunization with heat-denatured ovalbumin plus alum increased the IgM/IgG1 ratio compared to immunization with native ovalbumin and alum. Immunization of B6 mice transgenic for an HLA-A2/H-2K^b hybrid gene with heat-denatured, recombinant HPV 16-E7 protein induced D^b-restricted CTL specific for the peptide 49–57 of E7, indicating that this epitope is immunodominant over any A2-restricted E7 epitope in these mice. A whole influenza virus preparation heated to 100°C or even autoclaved was still able to induce virus-specific CTL and BALB/c spleen cells heated to 100°C could still cross-prime minor H-specific CTL in B6 mice, although with lower efficiency than fresh spleen cells. Thus, aggregated proteins can be considered as components for future vaccines.     

Genes encoded in the major histocompatibility complex affecting the generation of peptides for TAP transport

The B cell line 721.174 has lost the ability to present intracellular antigens to major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL). This phenotype results from a homozygous deletion in the MHC that includes the peptide transporter genes TAP1 and TAP2, and the proteasome subunits LMP2 and LMP7. Recent work has shown that such cells transfected with TAP genes load their class I molecules with endogenous peptides, and present several viral epitopes to class I-restricted CTL. These data implied that the LMP2 and LMP7 genes were not required for the presentation of most epitopes through class I molecules. By contrast, while confirming the previous reports, we have identified several epitopes that appear to require genes in the MHC in addition to the TAP for their presentation. Further analysis localizes the defect to proteolysis in the cytosol. In one case, presentation could be partially restored by re-expression of full-length LMP7. Control experiments with LMP7, from which the putative pro-region had been removed, failed to restore presentation, and this lack of effect correlated with failure of the shortened LMP7 to incorporate into the proteasome. These results suggest a role for LMP7 in the generation of a viral epitope, but leave open the possibility that additional genes within the .174 deletion are required for full restoration of antigen presentation.     

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.     

Pathogen evasion strategies for the major histocompatibility complex class I assembly pathway

Major histocompatibility complex (MHC) class I molecules bind and present short antigenic peptides from endogenously or exogenously derived sources to CD8(+) cytotoxic T lymphocytes (CTL), with recognition of a foreign peptide normally targeting the cell for lysis. It is generally thought that the high level of MHC polymorphism, which is concentrated mostly within the peptide-binding groove, is driven by the 'evolutionary arms race' against pathogens. Many pathogens have developed novel and intriguing mechanisms for evading the continuous sampling of the intracellular and intercellular environments by MHC molecules, none more so than viruses. The characterization of immunoevasion mechanisms has improved our understanding of MHC biology. This review will highlight our current understanding of the MHC class I biosynthetic pathway and how it has been exploited by pathogens, especially viruses, to potentially evade CTL recognition.     

TAP-dependent major histocompatibility complex class I presentation of soluble proteins using listeriolysin

Immunization of mice with mixtures of listeriolysin, a pore-forming hemolysin secreted by the pathogenic bacterium Listeria monocytogenes, together with soluble ovalbumin, nucleoprotein of influenza virus, or β-galactosidase of Escherichia coli, resulted in strong cytotoxic CD8 T cell responses to each of the respective passenger proteins in vivo. Also, the concomitant addition of either protein with listeriolysin to target cells elicited efficient sensitization of these cells which could be attributed to the pore-forming activity of listeriolysin. This response was dependent upon a functional TAP transporter and was inhibitable by brefeldin A, indicating the transfer of the soluble proteins into the cytosol and the classical major histocompatibility (MHC) class I presentation pathway. The treatment of target cells with listeriolysin under our experimental conditions did not affect cell viability and the pores generated by listeriolysin treatment were repaired within 60 min. Introduction of soluble proteins into the MHC class I presentation pathway by listeriolysin provides a powerful system to study the cytotoxic response towards intracellular pathogens and would allow for rapid screening of potential antigens in vaccine formulations.     

Presentation of viral antigens restricted by H-2Kb,Db or Kd in proteasome subunit LMP2-and LMP7-deficient cells

In the class II region of the major histocompatibility complex (MHC), four genes implicated in MHC class I-mediated antigen processing have been described. Two genes (TAP 1 and TAP 2) code for multimembrane-spanning ATP-binding transporter proteins and two genes (LMP 2 and LMP 7) code for subunits of the proteasome. While TAP 1 and TAP 2 have been shown to transport antigenic peptides from the cytosol into the endoplasmic reticulum, where the peptides associate with MHC class I molecules, the role of LMP 2/7 in antigen presentation is less clear. Using antigen processing mutant T2 cells that lack TAP 1/2 and LMP 2/7 genes, it was recently shown that expression of TAP 1/2 alone was sufficient for processing and presentation of the influenza matrix protein M1 as well as the minor histocompatibility antigen HA-2 by HLA-A2. To understand if presentation of a broader range of viral antigens occurs in the absence of LMP 2/7, we transfected T2 cells with TAP 1, TAP 2 and either of the H-2K^b, D^b or K^d genes and tested their ability to present vesicular stomatitis vires and influenza virus antigens to virus-specific cytotoxic T lymphocytes. We found that T2 cells, expressing TAP 1/2 gene products, presented all tested viral antigens restricted through either the H-2K^b, D^b or K^d class I molecules. We conclude that the proteasome subunits LMP 2/7 as well as other gene products in the MHC class II region, except from TAP 1/2, are not generally necessary for presentation of a broader panel of viral antigens to cytotoxic T cells. However, the present results do not exclude that LMP 2/7 in a more subtle way may, or in rare cases completely, affect processing of antigen for presentation by MHC class I molecules.     

Alternative processing for MHC class I presentation by immature and CpG-activated dendritic cells

Exogenous proteins can be processed by antigen-presenting cells for the generation of MHC class I-restricted T cell responses. Where this occurs is not clear, although both transfer of internalized antigen into the cytosol and alternative processing in endolysosomes and phagosomes have been reported. Here we have studied the capacity of bone marrow-derived mouse myeloid dendritic cells (DC) to process the OVA protein for peptide presentation by H2-K(b). We have found that immature DC (iDC), both wild-type and transporter associated with antigen processing (TAP)-deficient cells, can transiently process OVA in a pathway which is resistant to inhibitors of the classical MHC class I pathway including the Golgi inhibitor Brefeldin A (BFA) and the proteasome inhibitor lactacystin. This alternative pathway is not found in subcultured DC with an intermediate maturity (imDC) or in resting, IL-3 expanded macrophages but can be re-expressed in imDC if these are activated by an immunostimulatory CpG oligonucleotide. Both iDC and CpG-activated DC were found to process OVA by regurgitation. In addition, we found that iDC secrete proteolytic enzymes into the supernatant, which can process OVA in the extracellular phase. These results suggest that multiple pathways exist for the processing of exogenous protein antigens into MHC class I-binding peptides.     

Constitutive macropinocytosis allows TAP-dependent major histocompatibility compex class I presentation of exogenous soluble antigen by bone marrow-derived dendritic cells

Dendritic cells expanded from mouse bone marrow (BMDC) with granulocyte/macrophage-colony-stimulating factor have potent T cell-stimulatory properties both in vitro and in vivo. This has been well documented for major histocompatibility complex (MHC) class II-restricted responses, and more recently using peptide-loaded and protein-pulsed DC for CD8 responses following adoptive transfer in mice. An unresolved question concerns the capacity of BMDC to present exogenous antigen on MHC class I molecules, an unconventional mode of MHC class I loading for which there is now considerable evidence, particularly in macrophages. Here, we show that BMDC exhibit high levels of macropinocytosis driven by constitutive membrane ruffling activity. Up to one-third of actively ruffling and macropinocytosing BMDC transferred pinocytosed horseradish peroxidase into the cytosol following a 15-min pulse, suggesting that they might be capable of presenting exogenous soluble antigen on MHC class I molecules. We show that BMDC presented exogenous ovalbumin to a T cell hybridoma more effectively, more rapidly, and at lower exogenous antigen concentrations than BM macrophages on a cell-for-cell basis. Presentation was TAP dependent, brefeldin A sensitive, and blocked by inhibitors of proteasomal processing, demonstrating use of the classical MHC class I pathway. Although effective presentation of exogenous antigen by BMDC occurred in the absence of agents which stimulate macropinocytosis, treatment with phorbol myristate acetate (PMA) enhanced both pinocytosis and MHC class I presentation by BMDC. Finally, PMA-stimulated BMDC exposed to exogenous ovalbumin in vitro were able to prime an antigen-specific cytotoxic T lymphocyte response following adoptive transfer in vivo.     

Activation of human CD4+ T cells by targeting MHC class II epitopes to endosomal compartments using human CD1 tail sequences

Distinct CD4(+) T-cell epitopes within the same protein can be optimally processed and loaded into major histocompatibility complex (MHC) class II molecules in disparate endosomal compartments. The CD1 protein isoforms traffic to these same endosomal compartments as directed by unique cytoplasmic tail sequences, therefore we reasoned that antigen/CD1 chimeras containing the different CD1 cytoplasmic tail sequences could optimally target antigens to the MHC class II antigen presentation pathway. Evaluation of trafficking patterns revealed that all four human CD1-derived targeting sequences delivered antigen to the MHC class II antigen presentation pathway, to early/recycling, early/sorting and late endosomes/lysosomes. There was a preferential requirement for different CD1 targeting sequences for the optimal presentation of an MHC class II epitope in the following hierarchy: CD1b > CD1d = CD1c > > > CD1a or untargeted antigen. Therefore, the substitution of the CD1 ectodomain with heterologous proteins results in their traffic to distinct intracellular locations that intersect with MHC class II and this differential distribution leads to specific functional outcomes with respect to MHC class II antigen presentation. These findings may have implications in designing DNA vaccines, providing a greater variety of tools to generate T-cell responses against microbial pathogens or tumours.     

Cross-presentation: dendritic cells and macrophages bite off more than they can chew!

As immunologists, our knowledge of the molecular mechanisms which underlie the presentation of antigens derived from extracellular or 'exogenous' sources to CD8 cytotoxic lymphocytes (CTL) has been limited. This process, termed 'cross-presentation', has been linked to the elicitation of protective CTL responses against tumours and may be extremely important in generating immune responses against clinically relevant pathogens that do not infect tissues of haemopoietic origin. It is now known that cross-presentation of exogenous antigens on major histocompatibility complex (MHC) class I occurs through several distinct cellular pathways. In this review we outline and discuss some recent advances in our understanding of these pathways.     

Soluble major histocompatibility complex molecules in immune regulation: highlighting class II antigens

The involvement of major histocompatibility complex (MHC) antigens in the development and regulation of immune response has been well defined over the years, starting from maturation, antigenic peptide loading, migration to the cell membrane for recognition by the T‐cell receptor and recycling for immune response cessation. During this intracellular trafficking, MHC antigens find a way to be excreted by the cells, because they can be found as soluble MHC class I (sMHC‐I) and class II (sMHC‐II) molecules in all body fluids. Although secretion mechanisms have not been sufficiently studied, sMHC molecules have been shown to display important immunoregulatory properties. Their levels in the serum have been shown to be altered in a variety of diseases, including viral infections, inflammation, autoimmunities and cancer, etc. while they seem to be involved in a number of physiological reactions, including maintenance of tolerance, reproduction, as well as mate choice vis‐à‐vis species evolution. The present review aims to present the thus far existing literature on sMHC molecules and point out the importance of these molecules in the maintenance of immune homeostasis.     

Antigen processing mutant T2 cells present viral antigen restricted through H-2K

Cytotoxic T lymphocytes (CTL) recognize foreign antigens as short peptides presented by class I molecules of the major histocompatibility complex (MHC). T2 cells are profoundly defective in the presentation of endogenously synthesized antigens to CTL due to a deletion of MHC class II-encoded genes for transporters associated with antigen presentation (TAP1/TAP2). Surprisingly, we here demonstrate that T2 cells, after infection with Sendai virus, are readily killed by H-2K^b restricted CD8⁺ T cells. In contrast to classical class I-mediated antigen presentation, the presentation of Sendai virus antigen inT2K^b cells is brefeldin A (BFA) insensitive. The present findings may suggest the presence of an alternative pathway for MHC class I-mediated antigen presentation in T2 cells.     

Class II major histocompatibility complex and organ specific autoimmunity in man

T helper lymphocytes only recognize the antigen to which they are directed if it is 'presented' to them by a cell expressing the same class II major histocompatibility complex (MHC) antigen as themselves. Since the target cells of most organ specific autoimmune diseases do not normally express class II MHC proteins on their surface, any cell specific surface antigens they possess may never have been presented, and therefore immunological tolerance to them may not exist. The target cells in several organ specific autoimmune diseases have been shown to express class II MHC aberrantly and this expression may have allowed presentation of such cell specific surface antigens to potentially autoreactive T helper lymphocytes. It has therefore been proposed that aberrant expression of class II MHC antigens may be an initiating factor in certain autoimmune diseases.