MHC class I immune evasion in MCMV infection

Murine cytomegalovirus (MCMV) is a well-studied model of natural β-herpesvirus infection. However, many questions remain regarding its control by and evasion of the immune response it generates. CD8 and CD4 T cells have both unique and redundant roles in control of the virus that differ based on the immunocompetence of the infected mice. MCMV encodes major histocompatibility complex (MHC) class I immune evasion genes that can have an impact in vitro, but their role in infection of immunocompetent mice has been difficult to identify. This review addresses the evidence for their in vivo function and suggests why they may be evolutionarily conserved.     

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.     

The murine gamma-herpesvirus-68 MK3 protein causes TAP degradation independent of MHC class I heavy chain degradation

The murine gamma-herpesvirus-68 MK3 protein has an intricate interaction with the peptide loading complex that involves MK3 stabilization, a rapid degradation of MHC class I heavy chains, and a slower degradation of TAP. Here we have used tapasin chimeras to distinguish functionally the different immune evasion mechanisms of MK3. Tapasin was cloned in two alternatively spliced forms that differed by a single transmembrane valine residue. Each restored antigen presentation and MK3 function in tapasin-deficient cells. The transmembrane/cytoplasmic portion of tapasin, linked to the extracellular domain of CD8, also restored TAP stability and MK3 stability in tapasin-deficient cells. MK3 did not associate with or degrade MHC class I in these cells, which lacked the endoplasmic reticulum domain of tapasin, but degraded TAP at least as efficiently as when full-length tapasin was present. The un-degraded MHC class I consequently showed impaired maturation. The fact that MK3 required intact tapasin to degrade MHC class I but only the transmembrane/cytoplasmic portion of tapasin to degrade TAP indicated that these two immune evasion functions operate independently.     

Chaperone BAG6 is dispensable for MHC class I antigen processing and presentation

Antigen processing for direct presentation on MHC class I molecules is a multistep process requiring the concerted activity of several cellular complexes. The essential steps at the beginning of this pathway, namely protein synthesis at the ribosome and degradation via the proteasome, have been known for years. Nevertheless, there is a considerable lack of factors identified to function between protein synthesis and degradation during antigen processing. Here, we analyzed the impact of the chaperone BAG6 on MHC class I cell surface expression and presentation of virus-derived peptides. Although an essential role of BAG6 in antigen processing has been proposed previously, we found BAG6 to be dispensable in this pathway. Still, interaction of BAG6 and the model antigen tyrosinase was enhanced during proteasome inhibition pointing towards a role of BAG6 in antigen degradation. Redundant chaperone pathways potentially mask the contribution of BAG6 to antigen processing and presentation.     

MHC class II associations with autoantibody and T cell immune responses to the scleroderma autoantigen topoisomerase I

Topoisomerase I (topo I) is a major autoantigen recognized by autoantibodies in about 30% of sera from patients with systemic sclerosis (SSc). Certain HLA-DRB1 and HLA-DQB1 alleles have been reported to be associated with autoantibody and T-cell responses to topo I suggesting a T-cell dependent process. We have examined the MHC class II allele associations with anti-topo I antibodies in 16 patients with SSc compared to 250 healthy controls. Furthermore, we have studied the T cell responses to a recombinant full-length topo I molecule purified from a baculovirus expression system in eight patients with SSc and eight controls (five healthy and three with autoimmune disease). HLA-DR5 was significantly increased in patients with anti-topo I antibodies (P< 0.02). Proliferative peripheral blood mononuclear cell (PBMC) responses to soluble topo I were present in nine of 16 individuals (four of eight with SSc and five of eight controls), including the three SSc patients with anti-topo I antibodies. Homozygosity for HLA DQB1:30:Y alleles was present in five of nine responders (P< 0.03) compared to none of the non-responders. Our findings support the notion that the MHC class II background influences the ability to generate an autoimmune response to intracellular autoantigens to which the immune system may not have been tolerized. Additional factors associated with the generation of autoantibodies appear to be more intimately associated with the development of SSc.     

The MHC class I antigen presentation pathway: strategies for viral immune evasion

Presumably because of the selective pressure exerted by the immune system, many viruses have evolved proteins that interfere with antigen presentation by major histocompatibility complex (MHC) class I molecules. These viruses utilize a whole variety of ingenious strategies to inhibit the MHC class I pathway. Viral proteins have been characterized that exploit bottlenecks in the MHC class I pathway, such as peptide translocation by the transporter associated with antigen processing. Alternatively, viral proteins can cause the degradation or mislocalization of MHC class I molecules. This is often achieved by the subversion of the host cell's own protein degradation and trafficking pathways. As a consequence elucidation of how these viral proteins act to subvert host cell function will continue to give important insights not only into virus-host interactions but also the function and mechanism of cellular pathways.     

Viral evasion of the MHC class I antigen-processing machinery

In their adaptation to the immune system in vertebrates, viruses have been forced to evolve elaborate strategies for evading the hosts immune response. To ensure life-long persistence in the host, herpes viruses, adenoviruses and retroviruses have exploited multiple cellular pathways for their purpose, including the class I antigen-processing machinery. Attractive and prominent targets for viral attacks are the proteasome complex, the transporter associated with antigen processing, and MHC class I molecules. This review briefly outlines the different mechanisms of viral interference with the antigen-presentation pathway.     

Evidence for an involvement of the ubiquitin-like modifier ISG15 in MHC class I antigen presentation

The IFN stimulated gene 15 (ISG15) encodes a 15-kDa ubiquitin-like protein, that is induced by type I IFNs and is conjugated to the bulk of newly synthesized polypeptides at the ribosome. ISG15 functions as an antiviral molecule possibly by being covalently conjugated to viral proteins and disturbing virus particle assembly. Here, we have investigated the effect of ISGylation on degradation and antigen presentation of viral and cellular proteins. ISGylation did not induce proteasomal degradation of bulk ISG15 target proteins neither after overexpressing ISG15 nor after induction by IFN-β. The MHC class I cell surface expression of splenocytes derived from ISG15-deficient mice or mice lacking the catalytic activity of the major de-ISGylating enzyme USP18 was unaltered as compared to WT mice. Fusion of ubiquitin or FAT10 to the long-lived nucleoprotein (NP) of lymphocytic choriomeningitis virus accelerated the proteasomal degradation of NP while fusion to ISG15 did not detectably speed up NP degradation. Nevertheless, MHC-I restricted presentation of two epitopes of NP were markedly enhanced when it was fused to ISG15 similarly to fusion with ubiquitin or FAT10. Thus, we provide evidence that ISG15 can enhance the presentation of antigens on MHC-I most likely by promoting co-translational antigen processing.     

The role of tapasin in MHC class I antigen assembly

The discovery of tapasin has shed new light on the mechanisms of major histocompatibility complex (MHC) class I assembly in the endoplasmic reticulum (ER). Tapasin appears to play an important role in the stable assembly of class I molecules with peptide, however, the precise function of tapasin remains elusive. The pursuit of tapasin function is complicated by the observation that tapasin is not required for successful antigen presentation by all class I molecules. In addition, current data suggest that the putative role of tapasin as a bridging molecule between transporter associated with antigen presentation (TAP) and class I is only of minor importance in tapasin action, and tapasin’s major role appears to be as an active cofactor in the assembly of class I. Furthermore, it is clear that class I molecules can follow multiple pathways for successful assembly in the ER. These pathways may or may not include the interaction of class I molecules with the accessory proteins tapasin, calreticulin, ERp57, or TAP. I would like to suggest that the particular pathway utilized by a given class I molecule depends more upon the availability of appropriate peptides rather than on an intrinsic property of the class I molecule, and that tapasin may serve a peptide editing function.     

Dendritic cell/macrophage precursors capture exogenous antigen for MHC class I presentation by dendritic cells

Presentation of MHC class I antigens by professional antigen-presenting cells (APC) is an important pathway in priming cytotoxic T lymphocyte responses in vivo. This study sought to identify the nature of the professional APC responsible for indirect class I presentation by examining a special feature of professional APC, namely their ability to process exogenous forms of antigen for class I presentation. Incubation of highly purified bone marrow-derived precursor cells with chicken ovalbumin (OVA) led to the efficient presentation of the major class I-restricted OVA determinant by mature dendritic cells (DC), but not by macrophages (Mϕ) derived from the precursor population. DC as well as macrophages were, however, able to mediate class II presentation of OVA, suggesting that macrophages were deficient in class I processing but not in capturing exogenous OVA. The majority of mature DC, i.e. over 80 %, generated from the precursor cells pulsed with OVA, presented the class I OVA epitope. Upon maturation, class I presentation of OVA by DC was greatly reduced, suggesting that class I processing of exogenous antigen is modulated during DC maturation in a manner similar to class II antigen processing. This study shows that bone marrow-derived DC/Mϕ progenitors capture exogenous antigen for class I presentation, and that cells of the DC lineage can be functionally distinguished from cells of the macrophage lineage based on their ability to process exogenous antigen for class I presentation.     

Viral and bacterial minigene products are presented by MHC class I molecules with similar efficiencies

MHC class I molecules present short peptides, usually 8-10 amino acids in length, to CD8⁺ T cells. These peptides are typically generated from full-length endogenously synthesized proteins degraded by the antigen processing machinery of the target cell. However, exogenous proteins, whether originating from intracellular bacteria or parasites or via phagocytosis during cross-presentation, can also be processed for presentation by MHC class I molecules. It is currently not known whether endogenously synthesized proteins and proteins acquired from exogenous sources follow the same presentation pathway. One clue that the processing pathways followed by endogenous and exogenous proteins may not be identical is the vastly different presentation efficiencies reported for viral versus bacterial antigens. Because class I antigen processing involves multiple steps, we sought to determine where in the processing pathway these differences in efficiency occur. To accomplish this, we expressed identical minimal peptide determinants from viral and bacterial vectors using a minigene expression system and determined the rate of peptide-MHC generation per molecule of minigene product synthesized. We found that peptides expressed from either the viral or bacterial vector were presented with virtually identical efficiencies. These results suggest that differences in the processing pathways followed by endogenous versus exogenous proteins most likely occur at a point prior to where free peptide is liberated from full-length protein.     

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.     

The role of tetraspanin CD63 in antigen presentation via MHC class II

Interactions between MHC class II (MHC II)-positive APCs and CD4(+) T cells are central to adaptive immune responses. Using an Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell line (LCL) as MHC II-positive APCs and CD4(+) T-cell clones specific for two endogenously expressed EBV antigens, we found that shRNA knockdown of the tetraspanin protein CD63 in LCL cells consistently led to increased CD4(+) T-cell recognition. This effect was not due to enhanced antigen processing nor to changes in MHC II expression since CD63 knockdown did not influence the amount or dimerization of MHC II in LCL cells. We therefore investigated the possible involvement of exosomes, small MHC II- and tetraspanin-abundant vesicles which are secreted by LCL cells and which we found could themselves activate the CD4(+) T-cell clones in an MHC II-dependent manner. While equal loadings of exosomes purified from the control and CD63(low) LCLs stimulated T cells to a comparable degree, we found that exosome production significantly increased following CD63-knockdown, suggesting that this may underlie the greater T-cell stimulatory capacity of the CD63(low) LCLs. Taken together, our data reveal a new insight into the mechanisms by which tetraspanins are involved in the regulation of MHC II-dependent T-cell stimulation.     

The central repeat domain 1 of Kaposi's sarcoma-associated herpesvirus (KSHV) latency associated-nuclear antigen 1 (LANA1) prevents cis MHC class I peptide presentation

KSHV LANA1, a latent protein expressed during chronic infection to maintain a viral genome, inhibits major histocompatibility complex class I (MHC I) peptide presentation in cis as a means of immune evasion. Through deletional cloning, we localized this function to the LANA1 central repeat 1 (CR1) subregion. Other CR subregions retard LANA1 translation and proteasomal processing but do not markedly inhibit LANA1 peptide processing by MHC I. Inhibition of proteasomal processing ablates LANA1 peptide presentation. Direct expression of LANA1 within the endoplasmic reticulum (ER) overcomes CR1 inhibition suggesting that CR1 acts prior to translocation of cytoplasmic peptides into the ER. By physically separating CR1 from other subdomains, we show that LANA1 evades MHC I peptide processing by a mechanism distinct from other herpesviruses including Epstein-Barr virus (EBV). Although LANA1 and EBV EBNA1 are functionally similar, they appear to use different mechanisms to evade host cytotoxic T lymphocyte surveillance.     

Predicted MHC peptide binding promiscuity explains MHC class I ‘hotspots’ of antigen presentation defined by mass spectrometry eluted ligand data

Peptides that bind to and are presented by MHC class I and class II molecules collectively make up the immunopeptidome. In the context of vaccine development, an understanding of the immunopeptidome is essential, and much effort has been dedicated to its accurate and cost‐effective identification. Current state‐of‐the‐art methods mainly comprise in silico tools for predicting MHC binding, which is strongly correlated with peptide immunogenicity. However, only a small proportion of the peptides that bind to MHC molecules are, in fact, immunogenic, and substantial work has been dedicated to uncovering additional determinants of peptide immunogenicity. In this context, and in light of recent advancements in mass spectrometry (MS), the existence of immunological hotspots has been given new life, inciting the hypothesis that hotspots are associated with MHC class I peptide immunogenicity. We here introduce a precise terminology for defining these hotspots and carry out a systematic analysis of MS and in silico predicted hotspots. We find that hotspots defined from MS data are largely captured by peptide binding predictions, enabling their replication in silico. This leads us to conclude that hotspots, to a great degree, are simply a result of promiscuous HLA binding, which disproves the hypothesis that the identification of hotspots provides novel information in the context of immunogenic peptide prediction. Furthermore, our analyses demonstrate that the signal of ligand processing, although present in the MS data, has very low predictive power to discriminate between MS and in silico defined hotspots.     

Intracellular recycling and cross-presentation by MHC class I molecules

Cross-presentation of internalized antigens by dendritic cells requires efficient delivery of Major Histocompatibility Complex (MHC) class I molecules to peptide-loading compartments. Strong evidence suggests that such loading can occur outside of the endoplasmic reticulum; however, the trafficking pathways and sources of class I molecules involved are poorly understood. Examination of non-professional, non-phagocytic cells has revealed a clathrin-independent, Arf6-dependent recycling pathway likely traveled by internalized optimally loaded (closed) class I molecules. Some closed and all open MHC class I molecules travel to late endosomes to be degraded but might also partly be re-loaded with peptides and recycled. Studies of viral interference revealed pathways in which class I molecules are directed to degradation in lysosomes upon ubiquitination at the surface, or upon AP-1 and HIV-nef-dependent misrouting from the Golgi network to lysosomes. While many observations made in non-professional cells remain to be re-examined in dendritic cells, available evidence suggests that both recycling and neo-synthesized class I molecules can be loaded with cross-presented peptides. Recycling molecules can be recruited to phagosomes triggered by innate signals such as TLR4 ligands, and may therefore specialize in loading with phagocytosed antigens. In contrast, AP-1-dependent accumulation at, or trafficking through, a Golgi compartment of newly synthesized molecules appears to be important for cross-presentation of soluble proteins and possibly of long peptides that are processed in the so-called vacuolar pathway. However, significant cell biological work will be required to confirm this or any other model and to integrate knowledge on MHC class I biochemistry and trafficking in models of CD8⁺ T-cell priming by dendritic cells.     

MHC class II antigen presentation and immunological abnormalities due to deficiency of MHC class II and its associated genes

Antigen presentation by Major Histocompatibility Complex (MHC) class II molecules plays an important role in controlling immunity and autoimmunity. Multiple co-factors including the invariant chain (Ii), HLA-DM and HLA-DO are involved in this process. While the role for Ii and DM has been well defined, the biological function of DO remains obscure. Our data indicate that DO inhibits presentation of endogenous self-antigens and that developmentally-regulated DO expression enables antigen presenting cells to preferentially present different sources of peptide antigens at different stages of development. Disruption of this regulatory mechanism can result in not only immunodeficiency but also autoimmunity. Despite the fact that deletion of each of the three genes in experimental animals is associated with profound immunological abnormalities, no corresponding human diseases have been reported. This discrepancy suggests the possibility that primary immunodeficiencies due to a genetic defect of Ii, DM and DO in humans are under diagnosed or diagnosed as “common variable immunodeficiency”, a category of immunodeficiency of heterogeneous or undefined etiology. Clinical tests for any of these potential genetic defects are not yet available. We propose the use of multi-color flow cytometry in conjunction with intracellular staining to detect expression of Ii, DM, DO in peripheral blood B cells as a convenient reliable screening test to identify individuals with defects in antigen presentation.     

Mapping and restriction of a dominant viral CD4+ T cell core epitope by both MHC class I and MHC class II

Virus-specific CD4(+) T cells contribute to effective virus control through a multiplicity of mechanisms including direct effector functions as well as "help" for B cell and CD8(+) T cell responses. Here, we have used the lymphocytic choriomeningitis virus (LCMV) system to assess the minimal constraints of a dominant antiviral CD4(+) T cell response. We report that the core epitope derived from the LCMV glycoprotein (GP) is 11 amino acids in length and provides optimal recognition by epitope-specific CD4(+) T cells. Surprisingly, this epitope is also recognized by LCMV-specific CD8(+) T cells and thus constitutes a unique viral determinant with dual MHC class I- and II-restriction.     

Redirecting soluble antigen for MHC class I cross‐presentation during phagocytosis

Peptides presented by MHC class I molecules are mostly derived from proteins synthesized by the antigen‐presenting cell itself, while peptides presented by MHC class II molecules are predominantly from materials acquired by endocytosis. External antigens can also be presented by MHC class I molecules in a process referred to as cross‐presentation. Here, we report that mouse dendritic cell (DC) engagement to a phagocytic target alters endocytic processing and inhibits the proteolytic activities. During phagocytosis, endosome maturation is delayed, shows less progression toward the lysosome, and the endocytosed soluble antigen is targeted for MHC class I cross‐presentation. The antigen processing in these arrested endosomes is under the control of NAPDH oxidase associated ROS. We also show that cathepsin S is responsible for the generation of the MHC class I epitope. Taken together, our results suggest that in addition to solid structure uptake, DC phagocytosis simultaneously modifies the kinetics of endosomal trafficking and maturation. As a consequence, external soluble antigens are targeted into the MHC class I cross‐presentation pathway.