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.     

Polyubiquitination of lysine‐48 is an essential but indirect signal for MHC class I antigen processing

Peptides presented on major histocompatibility complex (MHC) class I molecules are generated via cytosolic proteolysis. However, the nature of the endogenous peptide precursors and the intracellular processing steps preceding protein degradation remain poorly defined. Here, we assessed whether ubiquitination is an essential signal for proteasomal cleavage of antigen substrates in human cells. Conversion into antigenic peptides occurred in the absence of any detectable N‐terminal ubiquitination of the model antigens, and did not require the presence of any of the four types, nor a minimum number of ubiquitinatable amino acids within the antigen substrate. However, the knockdown of ubiquitin, expression of a lysine 48 (K48) ubiquitin mutant, or inhibition of proteasome‐associated deubiquitinases significantly impaired antigen presentation. The results presented here are consistent with a model in which the binding of the antigen substrate by an adaptor protein leads to its K48‐polyubiquitination and the subsequent delivery of the antigen cargo for degradation by the 26S proteasome. Altogether, these findings show an important but indirect role of K48‐polyubiquitination in preproteasomal antigen sampling.     

The making of the dominant MHC class I ligand SYFPEITHI

The proteasome contributes to the generation of most of the peptide ligands of MHC class I molecules. To compare the identity of the peptides generated by the proteasome with those finally presented by MHC class I molecules, we generated a monoclonal antibody recognizing the C-terminal part of the dominant H2-K ^d ligand SYFPEITHI derived from the JAK1 tyrosine kinase. Immunoprecipitations of lysates from H2-K ^d -expressing or non-expressing cells revealed that only in the presence of H2-K ^d SYFPEITHI could be isolated. No longer potential precursor peptide containing SYFPEITHI could be detected. Surprisingly, a peptide lacking the first two amino acids, FPEITHI, was isolated independently of the presence of H2-K ^d molecules. The detection of only SYFPEITHI and FPEITHI in cell lysates corresponded with the strong generation of these two peptides in in vitro digests of elongated SYFPEITHI-containing peptides with purified 20S proteasomes. Our results indicate that MHC ligands can be generated directly by the proteasome in vivo and that at least for SYF PEITHI the expression of the corresponding MHC molecule is critical for protection of the ligand in vivo.     

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 I restricted T cell responses tolisteria monocytogenes, an intracellular bacterial pathogen

Studies of the murine immune response to infection with the intracellular bacterial pathogenListeria monocytogenes have provided a wealth of information about innate and acquired immune defenses in the setting of an infectious disease. Our studies have focused on the MHC class I restricted, CD8⁺ T cell responses of Balb/c mice toL. monocytogenes infection. Four peptides that derive from proteins thatL. monocytogenes secretes into the cytosol of infected cells are presented to cytotoxic T lymphocyte (CTL) by the H2-K^d major histocompatibility complex (MHC) class I molecule. We have found that bacterially secreted proteins are rapidly degraded in the host cell cytosol by proteasomes that utilize, at least in part, the N-end rule to determine the rate of degradation. The MHC class I antigen processing pathway is remarkably efficient at generating peptides that bind to MHC class I molecules. The magnitude of in vivo T cell responses, however, is influenced to only a small degree by the amount of antigen or the efficiency of antigen presentation. Measurements of in vivo T cell expansion followingL. monocytogenes infection indicate that differences in the sizes of peptide-specific T cell responses are more likely owing to differences in the repertoire of naive T cells than to differences in peptide presentation. This notion is supported by our additional finding that dominant T cell populations express a more diverse T cell receptor (TCR) repertoire than do subdominant T cell populations.     

A major role for tapasin as a stabilizer of the TAP peptide transporter and consequences for MHC class I expression

Tapasin is a member of the MHC class I loading complex where it bridges the TAP peptide transporter to class I molecules. The main role of tapasin is assumed to be the facilitation of peptide loading and optimization of the peptide cargo. Here, we describe another important function for tapasin. In tapasin-deficient (Tpn(-/-)) mice the absence of tapasin was found to have a dramatic effect on the stability of the TAP1/TAP2 heterodimeric peptide transporter. Steady-state expression of TAP protein was reduced more than 100-fold from about 3 x 10(4) TAP molecules per wild-type splenocyte to about 1 x 10(2) TAP per Tpn(-/-) splenocyte. Thus, a major function of murine tapasin appears to be the stabilization of TAP. The low amount of TAP moleculesin Tpn(-/-) lymphocytes is likely to contribute to the severe impairment of MHC class I expression. Surprisingly, activation of Tpn(-/-) lymphocytes yielded strongly enhanced class I expression comparable to wild-type levels, although TAP expression remained low and in the magnitude of several hundred molecules per cell. The high level of class I on activated Tpn(-/-) cells depended on peptides generated by the proteasome as indicated by blockade with the proteasome-specific inhibitor lactacystin. Lymphocyte activation induced an increase in ubiquitinated proteins that are cleaved into peptides by the proteasome. These findings suggest that in the presence of a large peptide pool in the cytosol, a small number of TAP transporters is sufficient to translocate enough peptides for high class I expression. However, these class I molecules were less stable than those of wild-type cells, indicating that tapasin is not only required for stabilization of TAP but also for optimization of the spectrum of bound peptides.     

Tapasin edits peptides on MHC class I molecules by accelerating peptide exchange

The endoplasmic reticulum (ER) protein tapasin is essential for the loading of high‐affinity peptides onto MHC class I molecules. It mediates peptide editing, i.e. the binding of peptides of successively higher affinity until class I molecules pass ER quality control and exit to the cell surface. The molecular mechanism of action of tapasin is unknown. We describe here the reconstitution of tapasin‐mediated peptide editing on class I molecules in the lumen of microsomal membranes. We find that in a competitive situation between high‐ and low‐affinity peptides, tapasin mediates the binding of the high‐affinity peptide to class I by accelerating the dissociation of the peptide from an unstable intermediate of the binding reaction.     

The phenotype of H-2M-deficient mice is dependent on the MHC class II molecules expressed

For a broader view of the role of H-2M as an accessory molecule in antigen presentation, we investigated the degree to which different MHC class II isotypes and alleles depend on H-2M to function in vivo. We generated H-2M-deficient animals expressing E^{k / b} or A^k molecules in addition to the A^b molecules already present in the mutant strain, and compared the ability of the different MHC class II molecules to present antigen at the cell surface for recognition by T cells, and contribute to positive selection of CD4⁺ T cells in the thymus. Biochemical analyses were performed to assess MHC class II maturation, and to determine the peptide content of the molecules. In the absence of H-2M, E^{k / b} molecules containd a more heterogeneous set of class II-associated invariant chain peptides (CLIP) than A^b did, which, unlike A^b -CLIP complexes, were not SDS-stable. Unlike A^b molecules, both E^{k / b} and A^k efficiently presented exogenously added peptides to T cells in the absence of H-2M. In addition, epitopes from some proteins, especially those known to be invariant chain independent, were presented by A^k molecules in the mutant animals. To our surprise, expression of E^{k / b} overcame the positive selection defect observed in H-2M-deficient mice expressing A^b alone. In contrast, A^k expression did not augment positive selection of CD4⁺ T cells in the mutant animals. Some of these findings in vivo contrast significantly with findings from in vitro studies on murine MHC class II molecules in human DM-deficient cell lines.     

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.     

Mechanistic understanding and significance of small peptides interaction with MHC class II molecules for therapeutic applications

Major histocompatibility complex (MHC) class II molecules are expressed by antigen-presenting cells and stimulate CD4⁺ T cells, which initiate humoral immune responses. Over the past decade, interest has developed to therapeutically impact the peptides to be exposed to CD4⁺ T cells. Structurally diverse small molecules have been discovered that act on the endogenous peptide exchanger HLA-DM by different mechanisms. Exogenously delivered peptides are highly susceptible to proteolytic cleavage in vivo; however, it is only when successfully incorporated into stable MHC II–peptide complexes that these peptides can induce an immune response. Many of the small molecules so far discovered have highlighted the molecular interactions mediating the formation of MHC II–peptide complexes. As potential drugs, these small molecules open new therapeutic approaches to modulate MHC II antigen presentation pathways and influence the quality and specificity of immune responses. This review briefly introduces how CD4⁺ T cells recognize antigen when displayed by MHC class II molecules, as well as MHC class II–peptide-loading pathways, structural basis of peptide binding and stabilization of the peptide–MHC complexes. We discuss the concept of MHC-loading enhancers, how they could modulate immune responses and how these molecules have been identified. Finally, we suggest mechanisms whereby MHC-loading enhancers could act upon MHC class II molecules.     

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.     

Pathways of MHC I cross-presentation of exogenous antigens

Phagocytes, particularly dendritic cells (DCs), generate peptide-major histocompatibility complex (MHC) I complexes from antigens they have collected from cells in tissues and report this information to CD8 T cells in a process called cross-presentation. This process allows CD8 T cells to detect, respond and eliminate abnormal cells, such as cancers or cells infected with viruses or intracellular microbes. In some settings, cross-presentation can help tolerize CD8 T cells to self-antigens. One of the principal ways that DCs acquire tissue antigens is by ingesting this material through phagocytosis. The resulting phagosomes are key hubs in the cross-presentation (XPT) process and in fact experimentally conferring the ability to phagocytize antigens can be sufficient to allow non-professional antigen presenting cells (APCs) to cross-present. Once in phagosomes, exogenous antigens can be cross-presented (XPTed) through three distinct pathways. There is a vacuolar pathway in which peptides are generated and then bind to MHC I molecules within the confines of the vacuole. Ingested exogenous antigens can also be exported from phagosomes to the cytosol upon vesicular rupture and/or possibly transport. Once in the cytosol, the antigen is degraded by the proteasome and the resulting oligopeptides can be transported to MHC I molecule in the endoplasmic reticulum (ER) (a phagosome-to-cytosol (P2C) pathway) or in phagosomes (a phagosome-to-cytosol-to-phagosome (P2C2P) pathway). Here we review how phagosomes acquire the necessary molecular components that support these three mechanisms and the contribution of these pathways. We describe what is known as well as the gaps in our understanding of these processes.     

Viral immune evasion: Lessons in MHC class I antigen presentation

The MHC class I antigen presentation pathway enables cells infected with intracellular pathogens to signal the presence of the invader to the immune system. Cytotoxic T lymphocytes are able to eliminate the infected cells through recognition of pathogen-derived peptides presented by MHC class I molecules at the cell surface. In the course of evolution, many viruses have acquired inhibitors that target essential stages of the MHC class I antigen presentation pathway. Studies on these immune evasion proteins reveal fascinating strategies used by viruses to elude the immune system. Viral immunoevasins also constitute great research tools that facilitate functional studies on the MHC class I antigen presentation pathway, allowing the investigation of less well understood routes, such as TAP-independent antigen presentation and cross-presentation of exogenous proteins. Viral immunoevasins have also helped to unravel more general cellular processes. For instance, basic principles of ER-associated protein degradation via the ubiquitin-proteasome pathway have been resolved using virus-induced degradation of MHC class I as a model. This review highlights how viral immunoevasins have increased our understanding of MHC class I-restricted antigen presentation.     

A Role for MHC Class II Antigen Processing in B Cell Development

For mature B cells, the encounter with foreign antigen results in the selective expansion of the cells and their differentiation into antibody secreting cells or memory B cells. The response of mature B cells to antigen requires not only antigen binding to and signaling through the B cell antigen receptor (BCR) but also the processing and presentation of the BCR bound antigen to helper T cells. Thus, in mature B cells, the ability to process and present antigen to helper T cells plays a critical role in determining the outcome of antigen encounter. In immature B cells, the binding of antigen results in negative selection of the B cell, inducing apoptosis, anergy or receptor editing. Negative selection of immature B cells requires antigen induced signaling through the BCR, analogous to the signaling function of the BCR in mature B cells. However, the role of class II antigen processing and presentation in immature B cells is less well understood. Current evidence indicates that the ability to process and present antigen bound to the BCR is a late acquisition of developing B cells, suggesting that during negative selection B cells may not present BCR bound antigen and interact with helper T cells However, the expression of class II molecules is an early acquisition of B cells and recent evidence indicates that the expression of class II molecules early in development is required for the generation of long lived mature B cells. Here we review our current understanding of the processing and presentation of antigen by mature B cells and the role for antigen processing and class II expression during B cell development.     

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.     

Endosomal compartment: Also a dock for MHC class I peptide loading

The endosomal compartment, which contains all the components required for loading peptides onto MHC class II molecules, is classically considered to be dedicated to the loading of MHC class II but not MHC class I molecules. However, a report in this issue of the European Journal of Immunology [Eur. J. Immunol. 2014. 44: 774–784], together with other recent studies, shows that the endosomal compartment also supports efficient loading of MHC class I molecules. These results bring a new perspective on the crosstalk between the MHC class II and MHC class I antigen‐processing pathways, and may inspire new ideas for the design of vaccines against viruses and tumors.     

Expression of MHC class I molecules together with antigenic peptides on filamentous phages

The availability of combinatorial T cell epitope libraries made using a phage display system would be useful for identifying the antigens recognized by T cells of unknown specificity. To this end, we have investigated here whether single chain-MHC class I molecules (scMHC-I) could be expressed together with antigenic peptides on filamentous phages. The results show that filamentous phages can express scMHC-I. Moreover, the expressed scMHC-I was able to bind antigenic peptide. These data support the use of combinatorial scMHC/T cell epitope libraries for screening potential T cell antigens.     

Inhibition of cell surface MHC class II expression by Salmonella

Peptide presentation by MHC molecules is an essential component of the adaptive immune response. To persist in a host, many pathogens have evolved strategies that interfere with MHC antigen-presentation. We show that in human cells harboring intracellular Salmonella, MHC class II cell surface expression was substantially reduced. The effect was specific for MHC class II as expression of additional surface receptors remained unchanged. We investigated the underlying mechanism and showed that class II biosynthesis and peptide loading were unaffected by the presence of Salmonella; however, infection led to an intracellular accumulation of mature molecules. The intracellular class II colocalized with lysosome-associated membrane protein-1 and HLA-DM but not with the Salmonella-containing vacuole. Using Salmonella mutants defective in different components and effectors of the Salmonella pathogenicity island-2 type-III secretion system, we traced the effect on class II to the sifA locus. SifA has been shown to be involved in recruiting membrane for the Salmonella-containing vacuoles. Our data suggest an additional role for SifA in interfering with MHC class II antigen-presentation.