Sequential cleavage by metallopeptidases and proteasomes is involved in processing HIV-1 ENV epitope for endogenous MHC class I antigen presentation

Antigenic peptides derived from viral proteins by multiple proteolytic cleavages are bound by MHC class I molecules and recognized by CTL. Processing predominantly takes place in the cytosol of infected cells by the action of proteasomes. To identify other proteases involved in the endogenous generation of viral epitopes, specifically those derived from proteins routed to the secretory pathway, we investigated presentation of the HIV-1 ENV 10-mer epitope 318RGPGRAFVTI327 (p18) to specific CTL in the presence of diverse protease inhibitors. Both metalloproteinase and proteasome inhibitors decreased CTL recognition of the p18 epitope expressed from either native gp160 or from a chimera based on the hepatitis B virus secretory core protein as carrier protein. Processing of this epitope from both native ENV and the hepatitis B virus secretory core chimeric protein appeared to proceed by a TAP-dependent pathway that involved sequential cleavage by proteasomes and metallo-endopeptidases; however, other protease activities could replace the function of the lactacystin-sensitive proteasomes. By contrast, in a second TAP-independent pathway we detected no contribution of metallopeptidases for processing the ENV epitope from the chimeric protein. These results show that, in the classical TAP-dependent MHC class I pathway, endogenous Ag processing of viral proteins to yield the p18 10-mer epitope requires metallo-endopeptidases in addition to proteasomes.     

Modulation of the phagosome proteome by interferon-gamma

Macrophages are immune cells that function in the clearance of infectious particles. This process involves the engulfment of microbes into phagosomes where these particles are lysed and degraded. In the current study, we used a large scale quantitative proteomics approach to analyze the changes in protein abundance induced on phagosomes by interferon-gamma (IFN-gamma), an inflammatory cytokine that activates macrophages. Our analysis identified 167 IFN-gamma-modulated proteins on phagosomes of which more than 90% were up-regulated. The list of phagosomal proteins regulated by IFN-gamma includes proteins expected to alter phagosome maturation, enhance microbe degradation, trigger the macrophage immune response, and promote antigen loading on major histocompatibility complex (MHC) class I molecules. A dynamic analysis of IFN-gamma-sensitive proteins by Western blot indicated that newly formed phagosomes display a delayed proteolytic activity coupled to an increased recruitment of the MHC class I peptide-loading complex. These phagosomal conditions may favor antigen presentation by MHC class I molecules on IFN-gamma-activated macrophages.     

Host immune system strikes back

Autophagy-mediated major histocompatibility complex (MHC) class I presentation can follow either the conventional MHC class I pathway or a recently described vacuolar pathway. In the vacuolar pathway, protein degradation is effected by lysosomal proteases, peptide exchange takes place with recirculating MHC complexes and the newly formed peptide-MHC complexes reach the cell surface by the endocytic pathway. This pathway is independent of the proteasome and the transporter associated with antigen processing (TAP) complex, but generates the same, or a similar, epitope as that from the conventional MHC class I pathway. Here, we discuss different mechanisms by which autophagy mediates MHC class I-restricted antigen presentation, which is crucial to its role in the control of intracellular pathogens.     

Antigen secreted from noncytosolic Listeria monocytogenes is processed by the classical MHC class I processing pathway.

Intracellular bacteria can reside in a vacuolar compartment, or they can escape the vacuole and become free living in the cytoplasm. The presentation of Ag by class I MHC molecules has been defined primarily for Ag present in the cytoplasm. It was therefore thought that Ags from bacteria that remain in a vacuole would not be presented by MHC class I molecules. Although some studies have provided data to support this idea, it is not necessarily true for all intracellular bacteria. For example, we have previously demonstrated that an epitope from the p60 protein secreted by LLO- Listeria monocytogenes, which does not reside in the cytoplasm, can be presented by MHC class I molecules to a T cell clone specific for the epitope, p60217-225. We have further examined the route by which Ag secreted by LLO- L. monocytogenes is presented by MHC class I molecules. Using pharmacological inhibitors, we demonstrate that MHC class I presentation of the p60 epitope derived from by LLO- L. monocytogenes requires phagolysosome fusion and processing by the proteasome. Lysosomal cathepsins, however, are not required for processing of the p60 epitope. Similarly, processing of the AttM epitope, secreted by LLO- L. monocytogenes and presented by H2-M3, also requires phagolysosome fusion and cleavage by the proteasome. Thus, p60 and AttM secreted by LLO- L. monocytogenes are processed via the classical class I pathway for presentation by MHC class I molecules.     

MHC class II-restricted presentation of native protein antigen by B cells is inhibitable by cycloheximide and brefeldin A

The presentation of protein Ag with MHC class II proteins involves the uptake of the protein Ag by endocytosis followed by processing, probably proteolysis, in an intracellular acidic compartment. However, there remains considerable controversy as to the precise route taken by the antigen and the MHC class II protein during this process. The unusual stability of Ag-MHC class II protein complexes has led to speculation that antigen can only associate with newly synthesized MHC class II molecules. An alternate possibility is that the MHC class II binding site can be regenerated within the cell during internalization and recycling of MHC class II proteins. To address these possibilities, three different murine B lymphoma lines were tested for their ability to process and present native protein Ag in the presence of the protein synthesis inhibitor cycloheximide or the protein synthesis inhibitor cycloheximide or the protein export inhibitor, Brefeldin A. Both agents blocked the presentation of native OVA or native hen egg lysozyme to Ag-specific T cell hybridomas. No effect was seen on peptide presentation or on presentation to allo- or autoreactive T cells. Inasmuch as Brefeldin A has been previously shown to block protein export without affecting protein internalization or protein degradation in the endocytic pathway, the simplest interpretation of these data is that antigenic fragments generated in the APC after uptake by the endocytic pathway, preferentially associate with newly synthesized rather than mature MHC class II proteins.     

Exogenous peptides delivered by ricin require processing by signal peptidase for transporter associated with antigen processing-independent MHC class I-restricted presentation

In this study we demonstrate that a disarmed version of the cytotoxin ricin can deliver exogenous CD8(+) T cell epitopes into the MHC class I-restricted pathway by a TAP-independent, signal peptidase-dependent pathway. Defined viral peptide epitopes genetically fused to the N terminus of an attenuated ricin A subunit (RTA) that was reassociated with its partner B subunit were able to reach the early secretory pathway of sensitive cells, including TAP-deficient cells. Successful processing and presentation by MHC class I proteins was not dependent on proteasome activity or on recycling of MHC class I proteins, but rather on a functional secretory pathway. Our results demonstrated a role for signal peptidase in the generation of peptide epitopes associated at the amino terminus of RTA. We showed, first, that potential signal peptide cleavage sites located toward the N terminus of RTA can be posttranslationally cleaved by signal peptidase and, second, that mutation of one of these sites led to a loss of peptide presentation. These results identify a novel MHC class I presentation pathway that exploits the ability of toxins to reach the lumen of the endoplasmic reticulum by retrograde transport, and suggest a role for endoplasmic reticulum signal peptidase in the processing and presentation of MHC class I peptides. Because TAP-negative cells can be sensitized for CTL killing following retrograde transport of toxin-linked peptides, application of these results has direct implications for the development of novel vaccination strategies.     

Induction of HPV16 capsid protein-specific human T cell responses by virus-like particles

It has been postulated that upon binding to a cell surface receptor, papilloma virus-like particles (VLPs) gain entry into the cytosol of infected cells and the capsid proteins L1 and L2 can be processed in the MHC class I presentation pathway. Vaccination of mice with human papilloma virus-like particles consisting of capsid proteins L1 and L2 induced a CD8-mediated and perforin dependent protective immune response against a tumor challenge with human papilloma virus transformed tumor cells, which express only minute amounts of L1 protein. Here we show that HPV16 capsid proteins stimulate a MHC class I restricted CTL response with human peripheral blood lymphocytes (PBL) in vitro. The vigorous response was specific for VLP-infected target cells and was MHC class I restricted. Moreover we show the presence of at least one HLA-A*0201 restricted CTL epitope within the HPV-16 capsid proteins by using a VLP-'infected' HLA-A*0201 transfected human cell line as target cells. These results demonstrated that VLPs can induce a HPV16 capsid protein-specific immune response in humans, allowing the monitoring of immune responses induced by vaccines based on chimeric VLPs carrying additional immunogenic peptides or proteins in therapeutical applications in human patients.     

Antigens and autophagy: the path less travelled?

The Epstein-Barr virus (EBV)-coded nuclear antigen (EBNA) 1, a latent cycle protein endogenously expressed in EBV-transformed B lymphoblastoid cell lines (LCLs), is reported to be processed for CD4(+) T cell recognition by an intracellular route involving antigen delivery to the endosome/lyosome (MHC class II loading) compartment via macroautophagy. In contrast we find that, in the same cell type, two other virus-coded nuclear proteins of the latent cycle, EBNA2 and EBNA3C, are processed by a different route that is unaffected by autophagy inhibition. This involves the intercellular transfer of an antigenic moiety, detectable in cell-free culture supernatants, and its uptake and processing as exogenous antigen by neighboring cells. The process is cumulative and leads over several days of LCL culture to high levels of CD4+ T cell epitope display. The presentation of certain EBV lytic cycle proteins to CD4+ T cells has also recently been found to involve a similar intercellular antigen transfer. It becomes important to know why, even in the same cell type, some antigens but not others appear to access the MHC class II presentation pathway by autophagy.     

Sequential proteolytic processing of the capsular Caf1 antigen of Yersinia pestis for major histocompatibility complex class II-restricted presentation to T lymphocytes

We studied the mechanisms of antigen presentation of CD4 T cell epitopes of the capsular Caf1 antigen of Yersinia pestis using murine bone marrow macrophages as antigen presenting cells and T cell hybridomas specific for major histocompatibility complex (MHC) class II-restricted epitopes distributed throughout the Caf1 sequence. The data revealed diversity in the pathways used and the degrees of antigen processing required depending on the structural context of epitopes within the Caf1 molecule. Two epitopes in the carboxyl-terminal globular domain were presented by newly synthesized MHC class II after low pH-dependent lysosomal processing, whereas an epitope located in a flexible amino-terminal strand was presented by mature MHC class II independent of low pH and with no detectable requirement for proteolytic processing. A fourth epitope located between the two regions of Caf1 showed intermediate behavior. The data are consistent with progressive unfolding and cleavage of rCaf1 from the amino terminus as it traverses the endosomal pathway, the availability of epitopes determining which pool of MHC class II is preferentially loaded. The Caf1 capsular protein is a component of second generation plague vaccines and an understanding of the mechanisms and pathways of MHC class II-restricted presentation of multiple epitopes from this candidate vaccine antigen should inform the choice of delivery systems and adjuvants that target vaccines successfully to appropriate intracellular locations to induce protective immune responses against as wide a T cell repertoire as possible.     

Proteasome-independent major histocompatibility complex class I cross-presentation mediated by papaya mosaic virus-like particles leads to expansion of specific human T cells

The development of versatile vaccine platforms is a priority that is recognized by health authorities worldwide; such platforms should induce both arms of the immune system, the humoral and cytotoxic-T-lymphocyte responses. In this study, we have established that a vaccine platform based on the coat protein of papaya mosaic virus (PapMV CP), previously shown to induce a humoral response, can induce major histocompatibility complex (MHC) class I cross-presentation of HLA-A*0201 epitopes from gp100, a melanoma antigen, and from influenza virus M1 matrix protein. PapMV proteins were able to assemble into stable virus-like particles (VLPs) in a crystalline and repetitive structure. When we pulsed HLA-A*0201+ antigen-presenting cells (APCs) with the recombinant PapMV FLU or gp100, we noted that antigen-specific CD8+ T cells were highly reactive to these APCs, demonstrating that the epitope from the VLPs were processed and loaded on the MHC class I complex. APCs were preincubated with two different proteasome inhibitors, which did not affect the efficiency of peptide presentation on MHC class I. Classical presentation from an endogenous antigen was abolished in the same conditions. Clearly, antigen presentation mediated by the PapMV system was proteasome independent. Finally, PapMV-pulsed APCs had the capacity to expand highly avid antigen-specific T cells against the influenza virus M1 HLA-A*0201 epitope when cocultured with autologous peripheral blood mononuclear cells. This study demonstrates the potential of PapMV for MHC class I cross-presentation and for the expansion of human antigen-specific T cells. It makes VLPs from PapMV CP a very attractive platform to trigger cellular responses for vaccine development against chronic infectious diseases and cancers.     

Pathways for lipid antigen presentation by CD1 molecules: nowhere for intracellular pathogens to hide

A crucial feature of peptide antigen presentation by major histocompatibilty complex (MHC) class I and II molecules is their differential ability to sample cytosolic and extracellular antigens. Intracellular viral infections and bacteria that are taken up in phagosomes, but then escape from the endocytic compartment efficiently, enter the class I pathway via the cytosol. In contrast, phagosome-resident bacteria yield protein antigens that are sampled deep in the endocytic compartment and presented in a vacuolar acidification-dependent pathway mediated by MHC class II molecules. Despite this potential for antigen sampling, microbes have evolved a variety of evasive mechanisms that affect peptide transport in the MHC class I pathway or blockade of endosomal acidification and inhibition of phagosome–lysosome fusion that may compromise the MHC class II pathway of antigen presentation. Thus, besides MHC class I and II, a third lineage of antigen-presenting molecules that bind lipid and glycolipid antigens rather than peptides exists and is mediated by the family of CD1 proteins. CD1 isoforms (CD1a, b, c, and d) differentially sample both recycling endosomes of the early endocytic system and late endosomes and lysosomes to which lipid antigens are differentially delivered. These CD1 pathways include vacuolar acidification-independent pathways for lipid antigen presentation. These features of presenting lipid antigens, independently monitoring various antigen-containing intracellular compartments and avoiding certain evasive techniques employed by microbes, enable CD1 molecules to provide distinct opportunities to function in host defense against the microbial world.     

Differential use of autophagy by primary dendritic cells specialized in cross-presentation

Antigen-presenting cells survey their environment and present captured antigens bound to major histocompatibility complex (MHC) molecules. Formation of MHC-antigen complexes occurs in specialized compartments where multiple protein trafficking routes, still incompletely understood, converge. Autophagy is a route that enables the presentation of cytosolic antigen by MHC class II molecules. Some reports also implicate autophagy in the presentation of extracellular, endocytosed antigen by MHC class I molecules, a pathway termed “cross-presentation.” The role of autophagy in cross-presentation is controversial. This may be due to studies using different types of antigen presenting cells for which the use of autophagy is not well defined. Here we report that active use of autophagy is evident only in DC subtypes specialized in cross-presentation. However, the contribution of autophagy to cross-presentation varied depending on the form of antigen: it was negligible in the case of cell-associated antigen or antigen delivered via receptor-mediated endocytosis, but more prominent when the antigen was a soluble protein. These findings highlight the differential use of autophagy and its machinery by primary cells equipped with specific immune function, and prompt careful reassessment of the participation of this endocytic pathway in antigen cross-presentation.     

Efficient presentation of both cytosolic and endogenous transmembrane protein antigens on MHC class II is dependent on cytoplasmic proteolysis

Peptides from extracellular proteins presented on MHC class II are mostly generated and loaded in endolysosomal compartments, but the major pathways responsible for loading peptides from APC-endogenous sources on MHC class II are as yet unclear. In this study, we show that MHC class II molecules present peptides from proteins such as OVA or conalbumin introduced into the cytoplasm by hyperosmotic pinosome lysis, with efficiencies comparable to their presentation via extracellular fluid-phase endocytosis. This cytosolic presentation pathway is sensitive to proteasomal inhibitors, whereas the presentation of exogenous Ags taken up by endocytosis is not. Inhibitors of nonproteasomal cytosolic proteases can also inhibit MHC class II-restricted presentation of cytosolically delivered protein, without inhibiting MHC class I-restricted presentation from the same protein. Cytosolic processing of a soluble fusion protein containing the peptide epitope I-Ealpha(52-68) yields an epitope that is similar to the one generated during constitutive presentation of I-Ealpha as an endogenous transmembrane protein, but is subtly different from the one generated in the exogenous pathway. Constitutive MHC class II-mediated presentation of the endogenous transmembrane protein I-Ealpha is also specifically inhibited over time by inhibitors of cytosolic proteolysis. Thus, Ag processing in the cytoplasm appears to be essential for the efficient presentation of endogenous proteins, even transmembrane ones, on MHC class II, and the proteolytic pathways involved may differ from those used for MHC class I-mediated presentation.     

Traffic of proteins and peptides across membranes for immunosurveillance by CD8(+) T lymphocytes: a topological challenge

Cytotoxic CD8(+) T lymphocytes kill infected cells that display major histocompatibility complex (MHC) class I molecules presenting peptides processed from pathogen proteins. In general, the peptides are proteolytically processed from newly made endogenous antigens in the cytosol and require translocation to the endoplasmic reticulum (ER) for MHC class I loading. This last task is performed by the transporters associated with antigen processing (TAP). Sampling of suspicious pathogen-derived proteins reaches beyond the cytosol, and MHC class I loading can occur in other secretory or endosomal compartments besides the ER. Peptides processed from exogenous antigens can also be presented by MHC class I molecules to CD8(+) T lymphocytes, in this case requiring delivery from the extracellular medium to the processing and MHC class I loading compartments. The endogenous or exogenous antigen can be processed before or after its transport to the site of MHC class I loading. Therefore, mechanisms that allow the full-length protein or processed peptides to cross several subcellular membranes are essential. This review deals with the different intracellular pathways that allow the traffic of antigens to compartments proficient in processing and loading of MHC class I molecules for presentation to CD8(+) T lymphocytes and highlights the need to molecularly identify the transporters involved.     

Cross-presentation of a CMV pp65 epitope by human dendritic cells using bee venom PLA2 as a membrane-binding vector

We have used bee venom phospholipase A2 as a vector to load human dendritic cells ex vivo with a major histocompatibility complex (MHC) class I-restricted epitope fused to its C-terminus. The fusion protein bound to human monocyte-derived dendritic cells and was internalized into early endosomes. In vitro immunization experiments showed that these dendritic cells were able to generate specific CD8 T cell lines against the epitope carried by the fusion protein. Cross-presentation did not require proteasome, transporter associated with antigen processing, or endosome proteases, but required newly synthesized MHC molecules. Comparison of the antigen presentation pathway observed in this study to that followed by other toxins used as vectors is discussed.     

The group II chaperonin TRiC protects proteolytic intermediates from degradation in the MHC class I antigen processing pathway

MHC class I molecules present precisely cleaved peptides of intracellular proteins on the cell surface. For most antigenic precursors, presentation requires transport of peptide fragments into the ER, but the nature of the cytoplasmic peptides and their chaperones is obscure. By tracking proteolytic intermediates in living cells, we show that intracellular proteolysis yields a mixture of antigenic peptides containing only N-terminal flanking residues for ER transport. Some of these peptides were bound to the group II chaperonin TRiC and were protected from degradation. Destabilization of TRiC by RNA interference inhibited the expression of peptide-loaded MHC I molecules on the cell surface. Thus, the TRiC chaperonin serves a function in protecting proteolytic intermediates in the MHC I antigen processing pathway.     

MHC class II presentation of endogenous tumor antigen by cellular vaccines depends on the endocytic pathway but not H2-M

We have developed cell-based cancer vaccines that activate anti-tumor immunity by directly presenting endogenously synthesized tumor antigens to CD4⁺ T helper lymphocytes via MHC class II molecules. The vaccines are non-conventional antigen-presenting cells because they express MHC class II, do not express invariant chain or H-2M, and preferentially present endogenous antigen. To further improve therapeutic efficacy we have studied the intracellular trafficking pathway of MHC class II molecules in the vaccines using endoplasmic reticulum-localized lysozyme as a model antigen. Experiments using endocytic and cytosolic pathway inhibitors (chloroquine, primaquine, and brefeldin A) and protease inhibitors (lactacystin, LLnL, E64, and leupeptin) indicate antigen presentation depends on the endocytic pathway, although antigen degradation is not mediated by endosomal or proteasomal proteases. Because H2-M facilitates presentation of exogenous antigen via the endocytic pathway, we investigated whether transfection of vaccine cells with H-2M could potentiate endogenous antigen presentation. In contrast to its role in conventional antigen presentation, H-2M had no effect on endogenous antigen presentation by vaccine cells or on vaccine efficacy. These results suggest that antigen/MHC class II complexes in the vaccines may follow a novel route for processing and presentation and may produce a repertoire of class II-restricted peptides different from those presented by professional APC. The therapeutic efficacy of the vaccines, therefore, may reside in their ability to present novel tumor peptides, consequently activating tumor-specific CD4⁺ T cells that would not otherwise be activated.     

A transporter associated with antigen-processing independent vacuolar pathway for the MHC class I-mediated presentation of endogenous transmembrane proteins

MHC class I molecules present peptides derived from the ectodomains of endogenous transmembrane proteins; however, the processing of these Ags is incompletely understood. As model transmembrane Ags we investigated the processing of MHC-I-derived fusion proteins containing the N-terminally extended K(b)-restricted OVA epitope SIINFEKL in the extracytoplasmic domain. In TAP-deficient, nonprofessional APCs, the epitope was cleaved out of various sequence contexts and presented to T cells. Ag presentation was inhibited by acidophilic amines and inhibitors of the vacuolar proton pump, indicating processing in endosomes. Endosomal aspartic-type cathepsins, and to some extent also the trans-Golgi network protease furin, were involved in processing. Clathrin-dependent and independent internalization from the cell surface targeted MHC-I fusion proteins to early and late endosomes, where SIINFEKL/K(b) complexes were detected by immunofluorescence microscopy. Targeting of MHC-I fusion proteins to processing compartments was independent of sequence motifs in the cytoplasmic tail. Not only TAP-deficient cells, but also TAP-competent APCs used the vacuolar pathway for processing of MHC-I fusion proteins. Thus, endosomal processing of internalized endogenous transmembrane proteins represents a novel alternate pathway for the generation of MHC-I-binding peptides.     

T Cell Detection of a B-Cell Tropic Virus Infection: Newly-Synthesised versus Mature Viral Proteins as Antigen Sources for CD4 and CD8 Epitope Display

Viruses that naturally infect cells expressing both MHC I and MHC II molecules render themselves potentially visible to both CD8⁺ and CD4⁺ T cells through the de novo expression of viral antigens. Here we use one such pathogen, the B-lymphotropic Epstein-Barr virus (EBV), to examine the kinetics of these processes in the virally-infected cell, comparing newly synthesised polypeptides versus the mature protein pool as viral antigen sources for MHC I- and MHC II-restricted presentation. EBV-transformed B cell lines were established in which the expression of two cognate EBV antigens, EBNA1 and EBNA3B, could be induced and then completely suppressed by doxycycline-regulation. These cells were used as targets for CD8⁺ and CD4⁺ T cell clones to a range of EBNA1 and EBNA3B epitopes. For both antigens, when synthesis was induced, CD8 epitope display rose quickly to near maximum within 24 h, well before steady state levels of mature protein had been reached, whereas CD4 epitope presentation was delayed by 36–48 h and rose only slowly thereafter. When antigen expression was suppressed, despite the persistence of mature protein, CD8 epitope display fell rapidly at rates similar to that seen for the MHC I/epitope half-life in peptide pulse-chase experiments. By contrast, CD4 epitope display persisted for many days and, following peptide stripping, recovered well on cells in the absence of new antigen synthesis. We infer that, in virally-infected MHC I/II-positive cells, newly-synthesised polypeptides are the dominant source of antigen feeding the MHC I pathway, whereas the MHC II pathway is fed by the mature protein pool. Hence, newly-infected cells are rapidly visible only to the CD8 response; by contrast, latent infections, in which viral gene expression has been extinguished yet viral proteins persist, will remain visible to CD4⁺ T cells.