Endosomal proteases in antigen presentation

Processing of cytoplasmic and endocytized foreign antigens were once thought to be completely separate pathways of peptide generation for MHC class I and class II peptide display. However, recent studies show actually that there is extensive overlap owing, in part, to the positioning of endosomal proteases at sites in which MHC class I and class II paths cross. The questions of how antigens access and persist at sites of endosomal processing are proving to be as important as those regarding the proteases themselves. However, individual proteases within the set of endosomal enzymes have emerged both as targets for microbial evasion from MHC-class-II-dependent immunity and as required elements in autoimmunity. Repeated implication of cathepsins in processing medically important antigens holds promise for therapeutic manipulation of proteases in diseases in which there is disordered adaptive immunity.     

Endogenous presentation of a nuclear antigen on MHC class II by autophagy in the absence of CRM1-mediated nuclear export

Accumulating evidence suggests that intracellular antigens are endogenously presented on MHC class II, but it is still unknown whether antigens within different subcellular compartments are presented with similar efficiency, and via the same or different pathways. We have previously shown that endogenous MHC class II presentation of the cytosolic bacterial antigen neomycin phosphotransferase II (NeoR) is mediated by autophagy. Here, we addressed whether secluding NeoR from this cytoplasmic pathway by directing the protein into the cell nucleus (NucNeoR) would affect antigen presentation. Unexpectedly, NucNeoR was presented at least as efficiently as the cytosolic version of the antigen. Furthermore, presentation of NucNeoR was also dependent on autophagocytosis and lysosomal processing, indicating that both antigens were presented via the same pathway. Inhibition of CRM1-mediated nuclear export did not impede antigen presentation, indicating that NucNeoR gained access to this autophagy-dependent MHC class II presentation pathway by a CRM1-independent route. Thus, this endogenous presentation pathway broadens the spectrum of intracellular antigens surveyed by CD4(+) T cells by efficiently sampling cytoplasmic as well as nuclear antigens.     

Proteases, natural protease inhibitors and activation of the machinery of antigen presentation in dendritic cells

Dendritic cells play a central role in the immune response due to their exceptionally strong capacity for presenting antigens to naive T lymphocytes. Recent evidences have demonstrated that dendritic cells (DCs) exhibit a remarkable pattern of differentiation (maturation) that is accompanied by striking changes in morphology, organization and function. The hallmark of DC maturation is the major reorganization of the MHC class II molecule intracellular transport which is in part regulated by endosomal proteases activation. The central role of the endosomal proteases in generating antigenic peptides and controlling MHC class II traffic clearly defines these enzymes as an important area of investigation.     

Late events in the intracellular sorting of major histocompatibility complex class II molecules are regulated by the 80-82 segment of the class II β chain

The molecular mechanisms that regulate sorting of major histocompatibility complex (MHC) class II molecules into the endocytic pathway are poorly understood. For many proteins, access to endosomal compartments is regulated by cytosolically expressed sequences. We present evidence that a sequence in the lumenal domain of the MHC class II molecule regulates a very late event in class II biogenesis. Class II molecules containing single amino acid changes in the highly conserved 80–82 region of the β chain were introduced into invariant chain (Ii)-negative fibroblasts with wild-type α chain, and the derived transfectants were analyzed biochemically. Using an endosomal isolation technique, we have quantified the level of class II molecules expressed in endocytic compartments and found that in the absence of Ii, approximately 15% of total cellular class II molecules can be isolated from endosomal compartments. Mutation at position 80 enhances this localization, while changes at positions 81 and 82 ablate class II expression in endosomal compartments. In addition, we have evaluated whether the induced changes in intracellular distribution of class II molecules were due to alterations in early biosynthetic events, indicative of misfolding of the molecules, or to modulation of later trafficking events more likely to be a consequence of the modulation of a specific transport event. Despite the dramatic effects on endosomal localization induced by the mutations, early bio-synthetic events and maturation of class II were unaffected by the mutations. Collectively, our data argue that late trafficking events that control the ability of the class II molecule to access antigens is regulated by the 80–82 segment of the MHC class II β chains.     

Lamp-2a facilitates MHC class II presentation of cytoplasmic antigens

Extracellular antigens are internalized and processed before binding MHC class II molecules within endosomal and lysosomal compartments of professional antigen presenting cells (APC) for subsequent presentation to T cells. Yet select cytoplasmic peptides derived from autoantigens also intersect and bind class II molecules via an unknown mechanism. In human B lymphoblasts, inhibition of the peptide transporter associated with antigen processing (TAP) failed to alter class II-restricted cytoplasmic epitope presentation. By contrast, decreased display of cytoplasmic epitopes via class II molecules was observed in cells with diminished expression of the lysosome-associated membrane protein-2 (Lamp-2). Overexpression of Lamp-2 isoform A (Lamp-2a), an established component of chaperone-mediated autophagy, enhanced cytoplasmic autoantigen presentation. Manipulating APC expression of heat shock cognate protein 70 (hsc70), a cofactor for Lamp-2a, also altered cytoplasmic class II peptide presentation. These results demonstrate a novel role for the lysosomal Lamp-2a-hsc70 complex in promoting immunological recognition and antigen presentation.     

Major histocompatibility complex class II-restricted presentation of a cytosolic antigen by autophagy

Biochemical and functional studies have demonstrated major histocompatibility complex (MHC) class II-restricted presentation of peptides derived from cytosolic proteins, but the underlying processing and presentation pathways have remained elusive. Here we show that endogenous presentation of an epitope derived from the cytosolic protein neomycin phosphotransferase II (NeoR) on MHC class II is mediated by autophagy. This presentation pathway involves the sequestration of NeoR into autophagosomes, and subsequent delivery into the lytic compartment. These results identify endosomes/lysosomes as the processing compartment for cytosolic antigens and furthermore link endogenous antigen presentation on MHC class II with the process of cellular protein turnover by autophagy.     

Excessive degradation of intracellular protein in macrophages prevents presentation in the context of major histocompatibility complex class II molecules

The endogenous major histocompatibility complex (MHC) class II presentation pathway allows biosynthesized, intracellular antigens access for presentation to MHC class II-restricted T cells. This pathway has been well documented in B cells and fibroblasts, but may not be universally available in all antigen-presenting cell types. This study compares the ability of different antigen-presenting cells, expressing endogenous C5 protein (fifth component of mouse complement) as a result of transfection, to present their biosynthesized C5 to MHC class II-restricted T cells. B cells and fibroblasts expressing C5 were able to present several epitopes of this protein with MHC class II molecules, whereas macrophages were unable to do so, but readily presented C5 from an extracellular source. However, macrophage presentation of endogenous C5 could be achieved when they were treated with low doses of the lysosomotropic agent ammonium chloride. In the presence of an inhibitor of autophagy, presentation of endogenous C5 was abrogated, indicating that biosynthesized C5 is shuttled into lysosomal compartments for degradation before making contact with MHC class II molecules. Taken together, this suggests that proteolytic activity in lysosomes of macrophages may be excessive, compared with fibroblasts and B cells, and destroys epitopes of the C5 protein before they can gain access to MHC class II molecules. Thus, there are inherent differences in presentation pathways between antigen-presenting cell types; this could reflect their specialized functions within the immune system with macrophages focussing preferentially on internalization, degradation, and presentation of extracellular material.     

Peptide loading onto recycling HLA-DR molecules occurs in early endosomes

Presentation of exogenous antigens to MHC class II-restricted T cells can follow two different processing pathways. The classical pathway requires newly synthesized MHC class II molecules, invariant chain and HLA-DM expression, whereas the alternative pathway is independent of protein synthesis, invariant chain and HLA-DM. In both cases, MHC class II molecules associate with peptides derived from exogenous antigens that have been processed in endocytic compartments. Different endosomal/prelysosomal compartments where peptide/MHC class II complexes and HLA-DM molecules accumulate have been described. We show here that the alternative pathway uses an earlier compartment than the classical pathway. Experiments with chemically cross-liniked antigen suggest that recycling MHC class II molecules present rapidly degraded antigens, leading to a rapid immune response to exogenously added influenza virus proteins.     

Cathepsins and compartmentalization in antigen presentation

Intracellular trafficking and cell surface expression of MHC class II molecules is a tightly regulated process and is to a large extent, determined by the fate of the class II chaperone, the invariant chain. Inhibition of endosomal proteases critical to invariant chain proteolysis reveals marked shunting of class II complexes to lysosomal compartments. Regulation of endosomal protease activity by expression of cystatin C directs class II cell surface expression during maturation of dendritic cells. These studies highlight the taut interactions between class-II-invariant-chain complexes and endosomal proteases during MHC class II maturation.     

Autophagy in innate and adaptive immunity against intracellular pathogens

Autophagy delivers cytoplasmic constituents for lysosomal degradation. Recent studies have demonstrated that this pathway mediates resistance to pathogens and is targeted for immune evasion by viruses and bacteria. Lysosomal degradation products, including pathogenic determinants, are then surveyed by the adaptive immune system to elicit antigen-specific T cell responses. CD4⁺ T helper cells have been shown to recognize nuclear and cytosolic antigens via presentation by major histocompatibility complex (MHC) class II molecules after autophagy. Furthermore, some sources of natural MHC class II ligands display characteristics of autophagy substrates, and autophagosomes fuse with late endosomes, in which MHC class II loading is thought to occur. Although MHC class II antigen processing via autophagy has so far mainly been described for professional antigen-presenting cells like B cells, macrophages, and dendritic cells, it might be even more important for cells with less endocytic potential, like epithelial cells, when these express MHC class II at sites of inflammation. Therefore, autophagy might contribute to immune surveillance of intracellular pathogens via MHC class II presentation of intracellular pathogen-derived peptides.     

Cross-presentation of phage particle antigen in MHC class II and endoplasmic reticulum marker-positive compartments

It has been shown that exogenous antigens can access the MHC class I pathway of professional antigen-processing cells. However, details as to how the MHC class I-peptide complex forms in the presentation pathway are still poorly understood. Here we used MHC class I-peptide-specific antibodies to investigate the formation and intracellular location of class I-peptide complexes in macrophages. We observed that the formation of class I-peptide complexes occurs within a few hours and lasts for another few hours on the cell surface of macrophages following loading with filamentous phage particles. The class I-peptide complexes in the process were co-localized with MHC class II molecules and endocytic system markers. Moreover, endosomal compartments containing class I-peptide complexes were found within intracellular organelles stained by DiOC6 and calnexin. In addition, the cross-presentation of phage particles was transporter associated with antigen processing (TAP)-dependent and sensitive to proteasome inhibitors and NH(4)Cl. These data suggest that endocytosed phage particles may be processed and cross-presented in organelles positive for phagosome and endoplasmic reticulum (ER) markers via a classical ER MHC class I loading mechanism.     

Catheptic processing of protein antigens: enzymic and molecular aspects

Processing of most exogenous protein antigens involves restricted intracellular proteolysis to yield fragments that may be bound specifically by MHC class II glycoproteins. This association is required for functional presentation to antigen-specific T lymphocytes. The proteolytic degradation takes place in antigen-presenting cells, in an acidic, endosomal compartment where antigens arrive after capture and internalization. It is catalyzed by a small number of enzymes, mainly aspartic and thiol proteases, with the cathepsins D and B--in that order--as dominant representatives. Resulting immunogenic protein fragments possess a binding site for mature MHC molecules and a recognition site for T cell receptors; topologically, these sites are interspersed, generally extending over 7-8 consecutive amino acid residues. Binding of peptides by MHC proteins of a particular genotype appears to depend mainly on specific interactions involving a common sequential motif in their primary structure in terms of non-polar and polar/charged amino acid residues and not on a general propensity to adopt a certain regular secondary structure, such as an alpha-helix.     

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.     

Autophagy proteins in antigen processing for presentation on MHC molecules

Autophagy describes catabolic pathways that deliver cytoplasmic constituents for lysosomal degradation. Since major histocompatibility complex (MHC) molecules sample protein degradation products and present them to T cells for adaptive immunity, it is maybe not too surprising that autophagy contributes to this protein antigen processing for MHC presentation. However, the recently recognized breath of pathways, by which autophagy contributes to MHC antigen processing, is exciting. Macroautophagy does not only seem to deliver intracellular but facilitates also extracellular antigen processing by lysosomal hydrolysis for MHC class II presentation. Moreover, even MHC class I molecules that usually display proteasomal products are regulated by macroautophagy, probably using a pool of these molecules outside the endoplasmic reticulum, where MHC class I molecules are loaded with peptide during canonical MHC class I antigen processing. This review aims to summarize these recent developments and point out gaps of knowledge, which should be filled by further investigation, in order to harness the different antigen-processing pathways via autophagy for vaccine improvement.     

Autophagy in innate recognition of pathogens and adaptive immunity

Autophagy is a specialized cellular pathway involved in maintaining homeostasis by degrading long-lived cellular proteins and organelles. Recent studies have demonstrated that autophagy is utilized by immune systems to protect host cells from invading pathogens and regulate uncontrolled immune responses. During pathogen recognition, induction of autophagy by pattern recognition receptors leads to the promotion or inhibition of consequent signaling pathways. Furthermore, autophagy plays a role in the delivery of pathogen signatures in order to promote the recognition thereof by pattern recognition receptors. In addition to innate recognition, autophagy has been shown to facilitate MHC class II presentation of intracellular antigens to activate CD4 T cells. In this review, we describe the roles of autophagy in innate recognition of pathogens and adaptive immunity, such as antigen presentation, as well as the clinical relevance of autophagy in the treatment of human diseases.     

Endosomal localization of MHC class II-invariant chain complexes

CD4-positive T cells recognize foreign antigens displayed on the surface of antigen-presenting cells as small peptides bound to MHC class II molecules. Thus, the ability of antigen-presenting cells to generate these class II-peptide complexes is central to the initiation and regulation of immune responses. Class II predominantly associates with peptides derived from soluble protein antigens that are internalized and degraded within endosomal compartments. It is within these endosomal compartments that class II encounters and binds antigenic peptides. A number of signals have been implicated in directing the intracellular transport of class II to endosomes. These include sequences within class II itself and within the class II-associated invariant chain (Ii).     

Presentation of antigens by MHC class II molecules: getting the most out of them.

The function of MHC class II molecules is to bind peptides derived from antigens that access the endocytic route of antigen presenting cells and display them on the plasma membrane for recognition by CD4(+) T cells. Formation of the MHC II-peptide complexes entails the confluence of the antigens and the MHC II molecules in the same compartments of the endocytic route. There, both the antigens and the MHC II molecules undergo a series of orchestrated changes that involve proteases, other hydrolases and chaperones, culminating in the generation of a wide repertoire of MHC II-peptide combinations. All the events that lead to formation of MHC II-peptide complexes show a considerable degree of flexibility; this lack of strict rules is advantageous in that it provides T cells with the maximum amount of information, ensuring that pathogens do not go undetected.     

Spatial and mechanistic separation of cross-presentation and endogenous antigen presentation

Antiviral or antitumor immunity requires activation of cytotoxic CD8+ T cells by dendritic cells, which present viral or tumor antigens on major histocompatibility complex (MHC) class I molecules. The intracellular mechanisms facilitating MHC class I-restricted presentation of extracellular antigens ('cross-presentation') are unclear. Here we demonstrate that cross-presentation of soluble antigen occurred in an early endosomal compartment distinct from the endoplasmic reticulum where endogenous antigen is loaded onto MHC class I. Efficient cross-presentation required endotoxin-induced, Toll-like receptor 4- and signaling molecule MyD88-dependent relocation of the transporter associated with antigen processing, essential for loading of MHC class I, to early endosomes. Transport of cross-presented antigen from endosomes to the cell surface was inhibited by primaquine, which blocks endosomal trafficking. Thus, cross-presentation is spatially and mechanistically separated from endogenous MHC class I-restricted antigen presentation and is biased toward antigens containing microbial molecular patterns.     

IFN-gamma facilitates release of class II-loaded intracellular pools in trophoblast cells: a novel property independent of protein synthesis.

Interferon-gamma (IFN-gamma) is an abortion-inducing factor, yet its effects in such a reaction are subject to various levels of regulation. The trophoblast cell line TROPHO-1 can be induced by IFN-gamma to express mRNA and surface class II major histocompatibility complex (MHC) proteins after 8 and 48 h of stimulation, respectively. Untreated cells, however, show an intracellular accumulation of class II antigens earlier (6 h), indicating the existence of MHC pools in the cystosol independent of any induction. On addition of IFN-y, immunofluorescence, subcellular fractionation, and ELISA experiments showed that class II antigen activity detected in the endosomal compartments of the cells could be measured in the culture supernatants. These soluble class II proteins, when isolated and purified using magnetic bead isolation techniques and tested in SDS-PAGE gel and Western blot experiments, had a molecular weight of 70 kDa. Administration of these molecules to pregnant mice as culture supernatants increased the abortion rate and decreased maternal hematocrit levels, effects that could be immunoabsorbed by anti-I-A(d) monoclonal antibodies (mAb). These results indicate that although surface class II molecules are not expressed on trophoblast cells, they accumulate in endosomal compartments and can be released from the cells on addition of IFN-gamma. This new IFN-gamma property, to mobilize intracellular pools of class II MHC antigens in trophoblast cells independent of de novo protein synthesis and induce their release to the extracellular matrix, is a mechanism that appears to be involved in the fetal rejection process, facilitating priming of the maternal organism against the fetal allograft.     

Compartmentalization of class II antigen presentation: contribution of cytoplasmic and endosomal processing

Summary: During antigen processing, peptides are generated and displayed in the context of major histocompatibility complex (MHC) class II molecules on the surface of antigen‐presenting cells (APCs) to modulate immune responses to foreign antigens and guide self‐tolerance. Exogenous and cytoplasmic antigens are processed by distinct routes within APCs to yield class II ligands. Exogenous antigens are internalized, processed, and bound to class II molecules within endosomal and lysosomal compartments of APCs. Studies reviewed here demonstrate the importance of reduction in regulating exogenous antigen presentation. The differential expression of a γ‐interferon‐inducible lysosomal thiol reductase in professional APCs and melanomas is discussed in the context of tumor immune evasion. Cytoplasmic autoantigens, by contrast, are degraded by the proteasome and other enzymes in the cytosol, with the resulting peptides translocating to endosomal and lysosomal compartments for intersection with class II molecules. Processing and editing of these antigenic peptides within endosomes and lysosomes may be critical in regulating their display via class II proteins. Multiple pathways may regulate the transit of cytosolic peptides to class II molecules. The role of lysosome‐associated membrane protein‐2a and heat‐shock cognate protein 70 in promoting cytoplasmic peptide presentation by MHC class II molecules is discussed.