Immune surveillance via self digestion

The adaptive immune system is orchestrated by CD4+ T cells. These cells detect peptides presented on Major Histocompatibility Complex (MHC) class II molecules, which are loaded in late endosomes with products of lysosomal proteolysis. One pathway by which proteins gain access to degradation in lysosomes is macroautophagy. We recently showed that constitutive macroautophagy can be detected in cells relevant for the immune system, including dendritic cells. In these antigen presenting cells, autophagosomes frequently fused with MHC class II antigen loading compartments and targeting of Influenza matrix protein 1 (MP1) for macroautophagy enhanced MHC class II presentation to MP1-specific CD4+ T cell clones up to 20 fold. Our findings indicate that macroautophagy is a constitutive and efficient pathway of antigen delivery for MHC class II presentation. We suggest that this pathway samples intracellular proteins for immune surveillance and induction of tolerance in CD4+ T cells, and could be targeted for improved MHC class II presentation of vaccine antigens.     

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.     

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.     

The crosstalk between autophagic and endo-/exosomal pathways in antigen processing for MHC presentation in anticancer T cell immune responses

T cells recognize antigen fragments from proteolytic products that are presented to them in the form of peptides on major histocompatibility complex (MHC) molecules, which is crucial for the T cell to identify infected or transformed cells. Autophagy, a process that delivers cytoplasmic constituents for lysosomal degradation, has been observed to provide a substantial source of intra- and extracellular antigens for MHC presentation to T cells, which will impact the tumor-specific immune response. Meanwhile, extracellular components are transported to cytoplasm for the degradation/secretion process by the endo-/exosomal pathway and are thus involved in multiple physiological and pathological processes, including immune responses. Autophagy and endo-/exosomal pathways are intertwined in a highly intricate manner and both are closely involved in antigen processing for MHC presentation; thus, we propose that they may coordinate in antigen processing and presentation in anticancer T cell immune responses. In this article, we discuss the molecular and functional crosstalk between autophagy and endo-/exosomal pathways and their contributions to antigen processing for MHC presentation in anticancer T cell immune responses.     

Immune surveillance of intracellular pathogens via autophagy

MHC class II molecules are thought to present peptides derived from extracellular proteins to CD4+ T cells, which are important mediators of adaptive immunity to infections. In contrast, autophagy delivers constitutively cytosolic material for lysosomal degradation and has so far been recognized as an efficient mechanism of innate immunity against bacteria and viruses. Recent studies, however, link these two pathways and suggest that intracellular cytosolic and nuclear antigens are processed for MHC class II presentation after autophagy.     

Autophagy-mediated antigen processing in CD4 T cell tolerance and immunity

Macroautophagy, a homeostatic process that shuttles cytoplasmic constituents into endosomal and lysosomal compartments, has recently been shown to deliver antigens for presentation on major histocompatibility complex (MHC) class II. Autophagy-mediated antigen processing in thymic epithelial cells has been suggested to be involved in the generation of a self-MHC restricted and self-tolerant CD4⁺ T cell repertoire. Furthermore, there is accumulating evidence that the up-regulation of autophagy by pattern-recognition receptor signaling represents an innate defense mechanism against intracellular pathogens. Thus, through linking pathogen breakdown with the presentation of pathogen-derived autophagy substrates on MHC class II, autophagy serves a dual function at the interface of the innate and the adaptive immune response.     

Immune Surveillance via Self Digestion

The adaptive immune system is orchestrated by CD4⁺ T cells. These cells detect peptides presented on Major Histocompatiblity Complex (MHC) class II molecules, which are loaded in late endosomes with products of lysosomal proteolysis. One pathway by which proteins gain access to degradation in lysosomes is macroautophagy. We recently showed that constitutive macroautophagy can be detected in cells relevant for the immune system, including dendritic cells. In these antigen presenting cells, autophagosomes frequently fused with MHC class II antigen loading compartments and targeting of Influenza matrix protein 1 (MP1) for macroautophagy enhanced MHC class II presentation to MP1-specific CD4⁺ T cell clones up to 20 fold. Our findings indicate that macroautophagy is a constitutive and efficient pathway of antigen delivery for MHC class II presentation. We suggest that this pathway samples intracellular proteins for immune surveillance and induction of tolerance in CD4⁺ T cells, and could be targeted for improved MHC class II presentation of vaccine antigens.

Addendum to:

MHC Class II Antigen Loading Compartments Continuously Receive Input from Autophagosomes

Dorothee Schmid, Marc Pypaert and Christian Münz

Immunity 2006; In press     

Pseudomonas exotoxin-mediated delivery of exogenous antigens to MHC class I and class II processing pathways

Peptides associated with class II MHC molecules are normally derived from exogenous proteins, whereas class I MHC molecules normally associate with peptides from endogenous proteins. We have studied the ability of Pseudomonas exotoxin A (PE) fusion proteins to deliver exogenously added antigen for presentation by both MHC class I and class II molecules. A MHC class II-restricted antigen was fused to PE; this molecule was processed in a manner typical for class II-associated antigens. However, a MHC class I-restricted peptide fused to PE was processed by a mechanism independent of proteasomes. Furthermore, we also found that the PE fusion protein was much more stable in normal human plasma than the corresponding synthetic peptide. We believe that effective delivery of an antigen to both the MHC class I and class II pathways, in addition to the increased resistance to proteolysis in plasma, will be important for immunization.     

TNF-alpha induces macroautophagy and regulates MHC class II expression in human skeletal muscle cells

Macroautophagy, a homeostatic process that shuttles cytoplasmic constituents into endosomal and lysosomal compartments, has recently been shown to deliver antigens for presentation on major histocompatibility complex (MHC) class II molecules. Skeletal muscle fibers show a high level of constitutive macroautophagy and express MHC class II molecules upon immune activation. We found that tumor necrosis factor-α (TNF-α), a monokine overexpressed in inflammatory myopathies, led to a marked up-regulation of macroautophagy in skeletal myocytes. Furthermore, TNF-α augmented surface expression of MHC class II molecules in interferon-γ (IFN-γ)-treated myoblasts. The synergistic effect of TNF-α and IFN-γ on the induction of MHC class II surface expression was not reflected by higher intracellular human leukocyte antigen (HLA)-DR levels and was reversed by macroautophagy inhibition, suggesting that TNF-α facilitates antigen processing via macroautophagy for more efficient MHC class II loading. Muscle biopsies from patients with sporadic inclusion body myositis, a well defined myopathy with chronic inflammation, showed that over 20% of fibers that contained autophagosomes costained for MHC class II molecules and that more than 40% of double-positive muscle fibers had contact with CD4(+) and CD8(+) immune cells. These findings establish a mechanism through which TNF-α regulates both macroautophagy and MHC class II expression and suggest that macroautophagy-mediated antigen presentation contributes to the immunological environment of the inflamed human skeletal muscle.     

The known unknowns of antigen processing and presentation

The principal components of both MHC class I and class II antigen processing and presentation pathways are well known. In dendritic cells, these pathways are tightly regulated by Toll-like-receptor signalling and include features, such as cross-presentation, that are not seen in other cell types. However, the exact mechanisms involved in the subcellular trafficking of antigens remain poorly understood and in some cases are controversial. Recent data suggest that diverse cellular machineries, including autophagy, participate in antigen processing and presentation, although their relative contributions remain to be fully elucidated. Here, we highlight some emerging themes of antigen processing and presentation that we think merit further attention.     

NOD2-mediated autophagy and Crohn disease

Autophagy is important in immune cells as a means of disposing of pathogens and in connecting with the antigen presentation machinery to facilitate immune priming and initiation of a correctly targeted adaptive immune response. While Toll-like receptors (TLRs) are known to regulate autophagy in this context, the extent to which other pattern recognition receptors (PRRs) are involved has been unclear. NOD2 is an intracellular PRR of the Nod-like receptor (NLR) family that is notable in that variants in the ligand recognition domain are associated with Crohn disease (CD). Our recent study shows NOD2 activates autophagy in a manner requiring ATG16L1, another CD susceptibility gene. NOD2 autophagy induction is required for bacterial handling and MHC class II antigen presentation in human dendritic cells (DCs). CD patients DCs expressing CD risk variant NOD2 or ATG16L1 display reduced autophagy induction after NOD2 triggering resulting in reduced bacterial killing and defective antigen presentation. Aberrant bacterial handling and immune priming could act as a trigger for inflammation in CD.     

Deficiency of Autophagy in Dendritic Cells Protects against Experimental Autoimmune Encephalomyelitis

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) in the immune system. DCs present antigens to CD8 and CD4 T cells in the context of class I or II MHC. Recent evidence suggests that autophagy, a conserved intracellular degradation pathway, regulates class II antigen presentation. In vitro studies have shown that deletion of autophagy-related genes reduced antigen presentation by APCs to CD4 T cells. In vivo studies confirmed these findings in the context of infectious diseases. However, the relevance of autophagy-mediated antigen presentation in autoimmunity remains to be elucidated. Here, we report that loss of autophagy-related gene 7 (Atg7) in DCs ameliorated experimental autoimmune encephalomyelitis (EAE), a CD4 T cell-mediated mouse model of multiple sclerosis, by reducing in vivo priming of T cells. In contrast, severity of hapten-induced contact hypersensitivity, in which CD8 T cells and NK cells play major roles, was unaffected. Administration of the autophagy-lysosomal inhibitor chloroquine, before EAE onset, delayed disease progression and, when administered after the onset, reduced disease severity. Our data show that autophagy is required in DCs for induction of EAE and suggest that autophagy might be a potential target for treating CD4 T cell-mediated autoimmune conditions.     

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.     

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.     

Human suppressor cell induction in vitro: preferential activation by class I MHC antigen

Human peripheral blood lymphocytes heated at 45 degrees C for 1 hr were found to continue to express all the serologically detected class II MHC antigens (HLA DR, MT, MB) but not to stimulate proliferation in primary or secondary MLR. Such cells did, however, stimulate the formation of potent suppressor cells. Three additional stimulator cell models for the presentation of either class I antigen only (purified platelets and purified T cells) or class I antigen plus nonimmunogenic class II antigen (D/DR-compatible cells) gave identical results. Supernatants from cultures stimulated with any of these cell types had significantly reduced IL 2 activity when compared to control MLR. The suppressor cells generated in such cultures were not restricted to the class I or class II MHC antigen of the original stimulator. These data are interpreted to mean that 1) the class II epitopes detected by alloantisera and the epitopes that serve as lymphocyte-activating determinants are metabolically or conformationally distinct, and 2) that presentation of class I MHC antigen alone or in conjunction with nonimmunogenic class II MHC antigen preferentially stimulates the formation of suppressor cells. It is hypothesized that the latter may be an additional mechanism that contributes to the efficacy of matching for class II determinants in human renal transplantation.     

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.     

Ubiquitin-dependent control of class II MHC localization is dispensable for antigen presentation and antibody production

Controlled localization of class II MHC molecules is essential for proper class II MHC-restricted antigen presentation and the subsequent initiation of an adaptive immune response. Ubiquitination of class II MHC molecules on cytosolic lysine (K225) of the β-chain has been shown to affect localization of the complex. We generated mice in which the endogenous β-chain locus is replaced with a GFP tagged mutant version that lacks the cytosolic lysine residue (I-A-β-K225R-EGFP). These mice have elevated levels of class II MHC as compared to I-A-β-EGFP mice, and immature bone marrow-derived dendritic cells show redistribution of class II MHC to the cell surface. Nonetheless, in these same cells efficiency of antigen presentation is unaffected in I-A-β-K225R-EGFP mice, as assayed for presentation of ovalbumin to appropriately specific T cells. The I-A-β-K225R-EGFP animals have normal CD4 T cell populations and are capable of generating antigen-specific antibody in response to model antigens and viral infection. We therefore conclude that in our experimental system modulation of trafficking by ubiquitination of residue K225 of the β-chain is not essential for the function of class II MHC products in antigen presentation or antibody production.     

Luminal antigens access late endosomes of intestinal epithelial cells enriched in MHC I and MHC II molecules: in vivo study in Crohn's ileitis

In contrast to healthy conditions, intestinal epithelial cells (IECs) stimulate proinflammatory CD4+ and CD8+ T cells during Crohn's disease (CD). The underlying regulatory mechanisms remain unknown. Here we investigated the epithelial expression of major histocompatibility complex (MHC) I and MHC II and its interference with endocytic pathways, in vivo. During ileoscopy, ovalbumin (OVA) was sprayed onto ileal mucosa of CD patients (ileitis and remission) and controls. The epithelial traffic of OVA and MHC I/II pathways were studied in biopsies using fluorescence and electron microscopy. We found MHC I and MHC II to accumulate within multivesicular late endosomes (MVLE) of IECs. Faint labeling for these molecules was seen in early endosomes and lysosomes. MVLE were entered by OVA 10 min after exposure. Exosomes carrying MHC I, MHC II, and OVA were detected in intercellular spaces of the epithelium. OVA trafficking and labeling patterns for MHC I and MHC II in IECs showed no differences between CD patients and controls. Independent of inflammatory stimuli, MHC I and MHC II pathways intersect MVLE in IECs, which were efficiently targeted by luminal antigens. Similar to MHC II-enriched compartments in professional antigen presenting cells, these MVLE might be critically involved in MHC I- and MHC II-related antigen processing in IECs and the source of epithelial-released exosomes. The access of luminal antigens to MHC I in MVLE might indicate that the presentation of exogenous antigens by IECs must not be restricted to MHC II but might also occur as "cross-presentation" via MHC I to CD8+ T cells.     

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.