Autophagy and antigen presentation

CD4(+) T cells co-ordinate adaptive immunity and are required for immunological memory establishment and maintenance. They are thought to primarily recognize extracellular antigens, which are endocytosed, processed by lysosomal proteases and then presented on major histocompatibility complex (MHC) class II. However, recent studies have demonstrated that viral, tumour and autoantigens can gain access to this antigen presentation pathway from within cells by autophagy. This review will discuss the autophagic pathways that contribute to endogenous MHC class II antigen processing. Furthermore, potential characteristics of autophagy substrates, qualifying them to access these pathways, and regulation of autophagy will be considered. Finally, I will suggest how antigen presentation after autophagy might contribute to immune surveillance of infected and transformed cells.     

Autophagy: eating for good health

A renaissance in the autophagy field has illuminated many areas of biology, and infectious disease is no exception. By identifying key components of this broadly conserved membrane traffic pathway, yeast geneticists generated tools for microbiologists and immunologists to explore whether autophagy contributes to host defenses. As a result, autophagy is now recognized to be another barrier confronted by microbes that invade eukaryotic cells. Mounting evidence also indicates that autophagy equips cells to deliver cytosolic Ags to the MHC class II pathway. By applying knowledge of the autophagy machinery and exploiting microbes as genetic probes, experimentalists can now examine in detail how this ancient membrane traffic pathway contributes to these and other mechanisms critical for infection and immunity.     

Immunoamphisomes in dendritic cells amplify TLR signaling and enhance exogenous antigen presentation on MHC-II

Autophagy, a specialized lysosomal degradation pathway, has proven to be a potent cell-autonomous defense mechanism against a range of intracellular microbes. In addition, autophagy emerged recently as a critical regulator of innate and adaptive immune responses. Links between autophagy and innate immunity are being progressively unveiled. For instance, several TLR (Toll-Like Receptor) agonists upregulate autophagy flux in immune cell types such as DC (dendritic cells) or macrophages. Conversely, and perhaps surprisingly, is the observation that TLR7-mediated responses might depend on autophagy in plasmacytoid DC, thus suggesting a more complex link between TLR-dependent responses and autophagy. Recently, the demonstration that NOD2 increases autophagy suggests that innate immune responses initiated via a broad range of pathogen recognition receptors can regulate autophagy. In addition to its involvement in innate immune responses, autophagy regulates adaptive immune responses via both MHC class I and class II molecules depending on the cellular context and the nature of the antigen.     

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.     

A modular and combinatorial view of the antigen cross-presentation pathway in dendritic cells

Major histocompatibility complex class I (MHC I) presentation of exogenous antigens (cross-presentation) by dendritic cells (DC) is essential for CD8 T-cell immunity. Most cells use MHC I molecules to present peptides derived from endogenous proteins processed in the cytosol by the proteasome. The resulting peptides are translocated into the endoplasmic reticulum for loading onto MHC I molecules, and these complexes are then transported to the cell surface. In cross-presenting DC, these steps have been proposed to occur along two major tracks. In the 'endocytic' track, exogenous antigen processing and loading occur within endosomal compartments, using MHC I molecules recycled from the plasma membrane and transported back to the surface. In the 'cytosolic' track, antigens are translocated from endosomes to the cytosol, accessing the endogenous MHC I presentation pathway. This dichotomy now appears too simplistic. Some steps may occur in locations belonging to the endosomal track and others in the cytosolic track, or in hybrid compartments combining features of both. We propose a 'modular' view of cross-presentation, whereby processing, loading and MHC I transport represent modules that can occur in one or more locations. Cross-presentation of each MHC I-peptide complex may result from combining one or more options for each of these modules.     

Binding of human cytomegalovirus US2 to major histocompatibility complex class I and II proteins is not sufficient for their degradation

Human cytomegalovirus (HCMV) glycoprotein US2 causes degradation of major histocompatibility complex (MHC) class I heavy-chain (HC), class II DR-alpha and DM-alpha proteins, and HFE, a nonclassical MHC protein. In US2-expressing cells, MHC proteins present in the endoplasmic reticulum (ER) are degraded by cytosolic proteasomes. It appears that US2 binding triggers a normal cellular pathway by which misfolded or aberrant proteins are translocated from the ER to cytoplasmic proteasomes. To better understand how US2 binds MHC proteins and causes their degradation, we constructed a panel of US2 mutants. Mutants truncated from the N terminus as far as residue 40 or from the C terminus to amino acid 140 could bind to class I and class II proteins. Nevertheless, mutants lacking just the cytosolic tail (residues 187 to 199) were unable to cause degradation of both class I and II proteins. Chimeric proteins were constructed in which US2 sequences were replaced with homologous sequences from US3, an HCMV glycoprotein that can also bind to class I and II proteins. One of these US2/US3 chimeras bound to class II but not to class I, and a second bound class I HC better than wild-type US2. Therefore, US2 residues involved in the binding to MHC class I differ subtly from those involved in binding to class II proteins. Moreover, our results demonstrate that the binding of US2 to class I and II proteins is not sufficient to cause degradation of MHC proteins. The cytosolic tail of US2 and certain US2 lumenal sequences, which are not involved in binding to MHC proteins, are required for degradation. Our results are consistent with the hypothesis that US2 couples MHC proteins to components of the ER degradation pathway, enormously increasing the rate of degradation of MHC proteins.     

Autophagy in antiviral innate immunity

Several autonomous arms of innate immunity help cells to combat viral infections. One of these is autophagy, a central cytosolic lysosomal‐dependent catabolic process constitutively competent to destroy infectious viruses as well as essential viral components that links virus detection to antiviral innate immune signals. Ongoing autophagy can be upregulated upon virus detection by pathogen receptors, including membrane bound and cytosolic pattern recognition receptors, and may further facilitate pattern recognition receptor‐dependent signalling. Autophagy or autophagy proteins also contribute to the synthesis of antiviral innate type I interferon cytokines as well as to antiviral interferon γ signalling. Additionally, autophagy may play a crucial role during viral infections in containing an excessive cellular response by regulating the intensity of the inflammatory response. As a consequence, viruses have evolved strategies to counteract antiviral innate immunity through manipulation of autophagy. This review highlights recent findings on the cross‐talk between autophagy and innate immunity during viral infections.     

Cytosol to lysosome transport of intracellular antigens during immune surveillance

The delivery of intracellular substrates such as misfolded proteins and damaged organelles from the cytosol to the lysosome for degradation is crucial for cell survival. Multiple transport pathways including bulk autophagy (microautophagy and macroautophagy) and chaperone-mediated autophagy (CMA) have been identified to efficiently facilitate this transit of macromolecules from the cytoplasm to acidic vacuolar organelles. While autophagy plays a role in the general housekeeping of cells, it also functions in more specialized processes such as development and differentiation, responses to physiological stress and immunity. The presentation of both exogenous and endogenous antigens (Ag) by major histocompatibility complex (MHC) class II molecules to CD4(+) T lymphocytes is critical for the induction of tolerance to self Ag as well as the development of immunity against intracellular pathogens and tumors. Here, we discuss the class II-mediated presentation of several endogenous Ag, dependent on either macroautophagy or CMA for their transport from the cytosol to endosomal/lysosomal compartments. Thus, the various pathways of autophagy as routes of cytoplasmic Ag delivery to lysosomes have significant implications for the MHC class II-mediated immune response to intracellular pathogens and cancer.     

Presentation of antigenic peptides by MHC class I molecules

The basis for the immune response against intracellular pathogens is the recognition by cytotoxic T lymphocytes of antigenic peptides derived from cytosolic proteins, which are presented on the cell surface by major histocompatibility complex (MHC) class I molecules. The understanding of MHC class I-restricted peptide presentation has recently improved dramatically with the elucidation of the structural basis for the specificity of peptide binding to MHC class I molecules and the identification of proteins encoded in the class II region of the MHC that are putatively involved in the production of peptides and their transport into the endoplasmic reticulum, where they assemble with class I molecules.     

MHC class Ib molecules bridge innate and acquired immunity

Our understanding of the classical MHC class I molecules (MHC class Ia molecules) has long focused on their extreme polymorphism. These molecules present peptides to T cells and are central to discrimination between self and non-self. By contrast, the functions of the non-polymorphic MHC class I molecules (MHC class Ib molecules) have been elusive, but emerging evidence reveals that, in addition to antigen presentation, MHC class Ib molecules are involved in immunoregulation. As we discuss here, the subset of MHC class Ib molecules that presents peptides to T cells bridges innate and acquired immunity, and this provides insights into the origins of acquired immunity.     

Nuclear membrane-derived autophagy,a novel process that participates in the presentation of endogenous viral antigens during HSV-1 infection

Complex membrane trafficking events are involved in the regulation of antigen processing and presentation of both endogenous and exogenous antigens. While these processes were believed to involve mainly organelles along the endo/phagocytic and the biosynthetic pathways, recent studies have shown that autophagy also participates actively in both innate and adaptive immunity. We have shown recently that, in macrophages infected with the Herpes simplex type 1 virus, autophagy plays a key role in the targeting of viral proteins to hydrolytic compartments, and their processing for presentation on MHC class I molecules. This pathway involves a novel type of autophagosomes formed by coiling of the nuclear membrane where viral proteins are highly enriched. The ability to enhance the contribution of autophagy to antigen presentation in various conditions suggests that this pathway could be used to boost the immune response against viral infection and develop new vaccines.     

Ubiquitinylation of the cytosolic domain of a type I membrane protein is not required to initiate its dislocation from the endoplasmic reticulum

Human cytomegalovirus US2 and US11 target newly synthesized class I major histocompatibility complex (MHC) heavy chains for rapid degradation by the proteasome through a process termed dislocation. The presence of US2 induces the formation of class I MHC heavy chain conjugates of increased molecular weight that are recognized by a conformation-specific monoclonal antibody, W6/32, suggesting that these class I MHC molecules retain their proper tertiary structure. These conjugates are properly folded glycosylated heavy chains modified by attachment of an estimated one, two, and three ubiquitin molecules. The folded ubiquitinated class I MHC heavy chains are not observed in control cells or in cells transfected with US11, suggesting that US2 targets class I MHC heavy chains for dislocation in a manner distinct from that used by US11. This is further supported by the fact that US2 and US11 show different requirements in terms of the conformation of the heavy chain molecule. Although ubiquitin conjugation may occur on the cytosolic tail of the class I MHC molecule, replacement of lysines in the cytosolic tail of heavy chains with arginine does not prevent their degradation by US2. In an in vitro system that recapitulates US2-mediated dislocation, heavy chains that lack these lysines still occur in an ubiquitin-modified form, but in the soluble (cytoplasmic) fraction. Such ubiquitin conjugation can only occur on the class I MHC lumenal domain and is likely to take place once class I MHC heavy chains have been discharged from the endoplasmic reticulum. We conclude that ubiquitinylation of class I MHC heavy chain is not required during the initial step of the US2-mediated dislocation reaction.     

Cross-talking between autophagy and viral infection in mammalian cells

Autophagy is a cellular process in degradation of long-lived proteins and organelles in the cytosol for maintaining cellular homeostasis, which has been linked to a wide range of human health and disease states, including viral infection. The viral infected cells exhibit a complicated cross-talking between autophagy and virus. It has been shown that autophagy interacts with both adaptive and innate immunity. For adaptive immunity, viral antigens can be processed in autophagosomes by acidic proteases before major histocompatibility complex (MHC) class II presentation. For innate immunity, autophagy may assist in the delivery of viral nucleic acids to endosomal TLRs and also functions as a part of the TLR-or-PKR-downstream responses. Autophagy was also reported to suppress the magnitude of host innate antiviral immunity in certain cases. On the other hand, viruses has evolved many strategies to combat or utilize the host autophagy for their own benefit. In this review we discussed recent advances toward clarifying the cross-talking between autophagy and viral infection in mammalian cells.     

An immunogenic antigen of murine Plasmodium yoelii 17X associates with class I MHC glycoproteins

Reticulocytes infected with the non-lethal variant of Plasmodium yoelii 17X (PY17X-NL) express elevated levels of class I, but not class II, MHC Ag when compared with non-parasitized reticulocytes. In contrast, class I Ag are not detectable on erythrocytes parasitized by the lethal variant PY17X-L. In addition, the responder status of various inbred strains of mice to PY17X-NL has been shown to positively correlate with the levels of class I MHC antigens expressed on PY17X-NL parasitized red blood cells (PRBC). MHC Ag are known to restrict, or guide, immune responses. However, earlier studies have failed to demonstrate H-2 restricted activity in the effector arm of immunity to blood-stage murine malaria. Therefore, we have examined the induction of immunity by irradiated PY17X-NL PRBC. No MHC restriction was observed in the ability of PRBC to immunize recipients. However, using irradiated PRBC bearing low, intermediate or high levels of class I Ag we found that the levels, rather than haplotype, of class I Ag expressed on irradiated PRBC greatly influenced their ability to induce immunity. Furthermore, class I-associated parasite-directed Ag were isolated as an immunogenic complex with anti-class I MHC antibody. Such complexes induced immunity in vivo in the absence of adjuvant suggesting a biologically important mechanism by which non-lethal, reticulocytic forms of malarial parasites may immunize their hosts.     

MHC class I-restricted presentation of maleylated protein binding to scavenger receptors

Pathways for loading exogenous protein-derived peptides on MHC class I are thought to be present mainly in monocyte-lineage cells and to involve phagocytosis- or macropinocytosis-mediated antigenic leakage into either cytosol or extracellular milieu to give peptide access to MHC class I. We show that maleylation of OVA enhanced its presentation to an OVA-specific MHC class I-restricted T cell line by both macrophages and B cells. This enhanced presentation involved uptake through receptors of scavenger receptor (SR)-like ligand specificity, was TAP-1-independent, and was inhibited by low levels (2 mM) of ammonium chloride. No peptide loading of bystander APCs by maleylated (maleyl) OVA-pulsed macrophages was detected. Demaleylated maleyl-OVA showed enhanced MHC class I-restricted presentation through receptor-mediated uptake and remained highly sensitive to 2 mM ammonium chloride. However, if receptor binding of maleyl-OVA was inhibited by maleylated BSA, the residual presentation was relatively resistant to 2 mM ammonium chloride. Maleyl-OVA directly introduced into the cytosol via osmotic lysis of pinosomes was poorly presented, confirming that receptor-mediated presentation of exogenous maleyl-OVA was unlikely to involve a cytosolic pathway. Demaleylated maleyl-OVA was well presented as a cytosolic Ag, consistent with the dependence of cytosolic processing on protein ubiquitination. Thus, receptor-specific delivery of exogenous protein Ags to APCs can result in enhanced MHC class I-restricted presentation, suggesting that the exogenous pathway of peptide loading for MHC class I may be a constitutive property dependent mainly on the quantity of Ag taken up by APCs.     

Cysteine proteases: destruction ability versus immunomodulation capacity in immune cells

Cysteine proteases (cathepsins) play a pivotal role in various physiological processes, as well as in several diseases. In the immune response, maturation of major histocompatibility class II (MHC II) molecules and processing of antigens for further presentation by MHC II is tightly linked to the enzymes of the endosomal/lysosomal system, of which cysteine proteases constitute a major proportion. Furthermore, the process of autophagy provides access for cytosolic antigens to proteolysis by lysosomal cathepsins and subsequent MHC II presentation. Other specific functions of proteolytic enzymes associated with the immune response, such as activation of granzymes by cathepsin C in T-lymphocytes, are introduced and covered in this review.     

Quantifying recruitment of cytosolic peptides for HLA class I presentation: impact of TAP transport

MHC class I ligands are recruited from the cytosolic peptide pool, whose size is likely to depend on the balance between peptide generation by the proteasome and peptide degradation by downstream peptidases. We asked what fraction of this pool is available for presentation, and how the size of this fraction is modulated by peptide affinity for the TAP transporters. A model epitope restricted by HLA-A2 and a series of epitope precursors with N-terminal extensions by single residues modifying TAP affinity were expressed in a system that allowed us to monitor and modulate cytosolic peptide copy numbers. We show that presentation varies strongly according to TAP affinities of the epitope precursors. The fraction of cytosolic peptides recruited for MHC presentation does not exceed 1% and is more than two logs lower for peptides with very low TAP affinities. Therefore, TAP affinity has a substantial impact on MHC class I Ag presentation.     

Thimet oligopeptidase (EC 3.4.24.15), a novel protein on the route of MHC class I antigen presentation

The initial processing of antigens leading to major histocompatibility complex (MHC) class I antigenic peptides is carried out by the proteasome. However, how the final epitopes are generated and protected from degradation by cytosolic peptidases remains unknown. Coincidentally, peptides associated with the MHC class I molecules range from 8 to 13 amino acid residues, similarly to the optimum substrate size required for the cytosolic thimet oligopeptidase. Here we have investigated the putative intracellular function of thimet oligopeptidase related to antigen presentation. Using a well-characterized antigen-presenting cell system, we were able to demonstrate either inhibition or stimulation of CD8 T cell proliferation and cytotoxicity, manipulating intracellular thimet oligopeptidase levels with its specific inhibitor cFP-Ala-Ala-Tyr-pAb or loading the enzyme itself into the antigen-presenting cells. Our results suggest that thimet oligopeptidase should take an important function in the pathway of antigen presentation via MHC class I through a mechanism yet unknown.     

MHC class I molecules and KIRs in human history, health and survival

MHC class I molecules are ligands for the killer-cell immunoglobulin-like receptors (KIRs), which are expressed by natural killer cells and T cells. The interactions between these molecules contribute to both innate and adaptive immunity. KIRs and MHC class I molecules are encoded by unlinked polymorphic gene families that distinguish all but the most related individuals. Combinations of MHC class I and KIR variants influence resistance to infections, susceptibility to autoimmune diseases and complications of pregnancy, as well as outcome after haematopoietic stem-cell transplantation. Such correlations raise the possibility that interplay between KIR and MHC class I polymorphisms has facilitated human survival in the presence of epidemic infections and has influenced both reproduction and population growth.     

Unusually high frequency MHC class I alleles in Mauritian origin cynomolgus macaques

Acute shortages of Indian origin Rhesus macaques significantly hinder HIV/AIDS research. Cellular immune responses are particularly difficult to study because only a subset of animals possess MHC class I (MHC I) alleles with defined peptide-binding specificities. To expand the pool of nonhuman primates suitable for studies of cellular immunity, we defined 66 MHC I alleles in Cynomolgus macaques (Macaca fascicularis) of Chinese, Vietnamese, and Mauritian origin. Most MHC I alleles were found only in animals from a single geographic origin, suggesting that Cynomolgus macaques from different origins are not interchangeable in studies of cellular immunity. Animals from Mauritius may be particularly valuable because >50% of these Cynomolgus macaques share the MHC class I allele combination Mafa-B*430101, Mafa-B*440101, and Mafa-B*460101. The increased MHC I allele sharing of Mauritian origin Cynomolgus macaques may dramatically reduce the overall number of animals needed to study cellular immune responses in nonhuman primates while simultaneously reducing the confounding effects of genetic heterogeneity in HIV/AIDS research.