• antigen presentation;
  • autophagy;
  • cross-presentation;
  • endoplasmic reticulum;
  • endosome;
  • major histocompatibility complex;
  • phagocytosis;
  • phagosome;
  • retrotranslocation;
  • toll-like receptors


  1. Top of page
  2. Abstract
  3. Processing and Presentation of Phagocytosed Antigens by MHC-I and -II
  4. Phagosomes as Antigen Presentation Compartments
  5. Innate Signaling Controls Phagosome Maturation and Antigen Processing/Presentation
  6. Autophagy and MHC Presentation from Phagosomes
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Appendix 1.: Abstract figure

Phagocytosis provides innate immune cells with a mechanism to take up and destroy pathogenic bacteria, apoptotic cells and other large particles. In some cases, however, peptide antigens from these particles are preserved for presentation in association with major histocompatibility complex (MHC) class I or class II molecules in order to stimulate antigen-specific T cells. Processing and presentation of antigens from phagosomes presents a number of distinct challenges relative to antigens internalized by other means; while bacterial antigens were among the first discovered to be presented to T cells, analyses of the cellular mechanisms by which peptides from phagocytosed antigens assemble with MHC molecules and by which these complexes are then expressed at the plasma membrane have lagged behind those of conventional model soluble antigens. In this review, we cover recent advances in our understanding of these processes, including the unique cross-presentation of phagocytosed antigens by MHC class I molecules, and in their control by signaling modalities in phagocytic cells.


Antigen presenting cells (APCs) are critical regulators of immunity that continuously survey their microenvironment, take up antigenic materials, process them and present the resulting fragments to antigen-specific T lymphocytes in the context of molecules of the major histocompatibility complex (MHC) or related proteins. APCs capture exogenous matter through different mechanisms, including receptor-mediated endocytosis, pinocytosis and phagocytosis. Phagocytosis is defined as the engulfment of large particulate antigens, such as bacterial pathogens, in a process requiring actin remodeling to form a cup around the particle that eventually closes to form a phagosome (reviewed in [1] [2]). Once formed, phagosomes mature through extensive interactions with the endocytic system into phagolysosomes, such that the enclosed particles become exposed to an increasingly harsh lumenal environment (reviewed in [1] [3], [4]). Although phagosome maturation in many ways resembles endosome maturation [4], phagosomes have properties that are distinct from those of conventional endosomes and that differ among phagocytic cell types [5]. Moreover, phagosome maturation programs can be altered in response to innate signaling pathways initiated by the enclosed particles or by effectors generated by pathogenic bacteria [1, 3, 6]. Thus, the fate of cargoes that are ingested by phagocytosis or non-phagocytic mechanisms can differ among cell types and even within the same cell type.

Initially described in invertebrates by Elie Metchnikov in 1908 [7], phagocytosis was for many years conceived as an innate immunity mechanism that served only to destroy an exogenous agent. With the study of adaptive immunity, the discovery of dendritic cells (DCs) as key APCs at the interface of innate and adaptive immune responses [8], and the description of pattern recognition receptors in APCs that can discriminate between self and non-self, [9], increased evidence shows a tight interplay between innate and adaptive immunity pathways that places the phagosome at the crossroads of different routes, one of them leading to antigen presentation. Recent studies have provided substantial insights into mechanisms of phagosome maturation, the regulation of phagosome maturation by signals from pattern recognition receptors, and the requirement for particular phagosome functions in antigen processing in ‘professional phagocytes’ (DCs, macrophages and neutrophils) and other cell types that can undergo phagocytosis under inflammatory conditions.

Here we review the cell biological aspects of antigen presentation through the eyes of phagocytes. We focus on two distinct antigen processing pathways for presentation by MHC class I (MHC-I) and MHC class II (MHC-II) molecules. Phagocytosis plays a traditional role in providing ligands for MHC-II, but recent studies suggest that phagosomes might alter the conventional pathway for MHC-II antigen processing in as yet undefined ways. MHC-I molecules are normally loaded with peptides derived from cytosolic proteolysis, but phagosomes in a sub-class of DCs are specialized for cross-presentation, an unusual process by which protein antigens on endocytosed or phagocytosed particles provide ligands for MHC-I. The mechanisms by which phagocytosed antigens access the cytosol are still unclear, but recent insights have elucidated some of the key molecular players and compartments in this process.

Throughout this review, we refer to published experiments that use different models of particulate antigens internalized by phagocytosis. These include several classes of pathogenic and non-pathogenic bacteria, live or heat killed bacteria or inert beads conjugated to model antigens and/or ligands for innate immune receptors. In some cases, the consequences of exposure to different particulate antigen models have been compared to each other and to those of soluble ligands that are internalized by other mechanisms, whereas in other cases only single models have been analyzed. It is important for readers to appreciate that both the type of particulate antigen and the mode of internalization can strongly influence the results and the models derived from them. We hope that this review makes these differences sufficiently clear and encourage others to investigate the molecular basis for them.

Processing and Presentation of Phagocytosed Antigens by MHC-I and -II

  1. Top of page
  2. Abstract
  3. Processing and Presentation of Phagocytosed Antigens by MHC-I and -II
  4. Phagosomes as Antigen Presentation Compartments
  5. Innate Signaling Controls Phagosome Maturation and Antigen Processing/Presentation
  6. Autophagy and MHC Presentation from Phagosomes
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Appendix 1.: Abstract figure

The MHC-I processing pathway is largely geared toward presentation of endogenous antigens encountered in the cytoplasm, nucleus and mitochondria of most cell types. Peptides derived by cytosolic proteolysis of such antigens are translocated into the endoplasmic reticulum (ER), where they encounter and may assemble with newly synthesized MHC-I molecules (see below). Cross-presentation by MHC-I is a distinctive process in which antigens from phagocytosed particles or soluble proteins internalized by other means are assembled with MHC-I. This process is largely restricted to specific DC subsets in vivo [10, 11], but can be replicated in other model cell types in culture. By contrast, MHC-II molecules – which are limited in expression to ‘professional’ APCs (DCs, macrophages, B lymphocytes) and to a few limited other cell types after appropriate stimulation – are specialized to present antigens internalized from the extracellular space [12]. Historically, phagosomes were well recognized to provide antigens for presentation by MHC-II molecules on phagocytic cells such as macrophages and DCs before they were considered important sources of cross-presented antigen [13-16]. Burgdorf and Kurts [17] proposed that processing of phagocytosed antigens for cross-presentation by MHC-I and for presentation by MHC-II occurs sequentially, such that the mild environment of early phagosomes would favor MHC-I-dependent cross-presentation [18] and processing for MHC-II presentation would take place on late phagosomes characterized by a decreased pH and increased proteolytic activity. Before discussing the role and regulation of phagosomes in antigen processing, we briefly summarize the general pathways of MHC-I and -II antigen presentation.

Molecular and cell biology of antigen presentation by MHC-II

MHC-II molecules are composed of two integral membrane chains, α and β, both encoded within the MHC. The peptide-binding pocket is comprised of the membrane distal domains of both chains. Association of nascent α and β chains with invariant chain (also called Ii or CD74) in the ER facilitates MHC-II assembly and ER exit, occludes the peptide binding site and prevents premature binding of peptides [19]. Ii also facilitates transport of MHC-II complexes to the endosomal system; most evidence supports transport via the cell surface [20, 21], but alternative Ii isoforms might facilitate direct transport from the Golgi to endosomes [22]. Within late endosomal compartments – which in professional APCs are often referred to as MIIC for MHC-II-containing compartments [23] – Ii is degraded by resident proteases leaving only the peptide binding region, CLIP, bound to MHC-II [24]. Simultaneously, internalized proteins are also degraded by resident proteases, generating potential antigenic and non-antigenic peptide MHC-II ligands. The MIIC-resident protein HLA-DM then facilitates peptide exchange to promote CLIP dissociation and subsequent peptide binding to the MHC-II binding pocket [25, 26]. MHC-II:peptide complexes are then delivered to the cell surface by mechanisms that are still poorly understood [27-29], where they are available to stimulate antigen-specific CD4+ T cells with cognate receptors.

Peptide loading onto MHC-II molecules can occur in multiple compartments of the endocytic pathway. Most newly synthesized MHC-II molecules are likely loaded in late endosomal MIICs in an HLA-DM-dependent mechanism, whereas internalized MHC-II αβ dimers from the cell surface can be ‘reloaded’ with peptides within the early endosomal system [30]. Late endosomal MIIC are likely to be the primary site for antigen loading for the bulk of MHC-II in B lymphocytes or DCs, because they harbor at steady state all known factors required for efficient peptide loading (acidic pH, presence of cathepsins, HLA-DM activity) [31], as recently described for a minimal in vitro reconstitution system [32]. By electron microscopy analyses in B lymphocytes and DCs, MIIC have both multivesicular and multilamellar morphologies [23, 33-35]. The current consensus suggests that these morphologies represent the extremes of different maturation states [36, 37]. Caution must be taken in inferring the sites of MHC-II: peptide complex formation based on their sites of accumulation, as recent work suggests that the pool of MHC-II in multivesicular endosomes in immature DCs does not contribute to the pool of antigen-presenting MHC-II upon DC maturation [38].

Molecular and cell biology of antigen presentation by MHC-I

MHC-I molecules are composed of two chains – the polymorphic, MHC-encoded α chain and the relatively non-polymorphic β2 microglobulin (β2m) light chain – that assemble in the ER [39]. This assembly, however, is only complete if a peptide binds in the groove of the MHC-I molecule. A sophisticated ‘quality control’ mechanism that is based on the status of the N-linked oligosaccharides on the α chain prevents the export of incompletely assembled α chain/β2m/peptide trimers from the ER (a similar mechanism operates to ensure that only complete nonameric MHC-II αβIi complexes efficiently exit the ER). The peptides that bind MHC-I molecules are usually eight to nine amino acids long. Precursors of these peptides are generated in the cytosol mainly from incomplete translation products (DRIPs) that are partially degraded by the proteasome, but can also include other misfolded proteins or retrotranslocated proteins from the ER cleaved by the proteasome or other cytosolic proteases [40]. The peptides are then translocated into the lumen of the ER by dedicated ATP-dependent TAP1/2 transporters and trimmed at their N-termini by ER proteases called ERAAPs. Transfer of the trimmed peptides from TAP onto folding MHC-I is promoted by a ‘loading complex’, composed of a dedicated chaperone, Tapasin and a series of general membrane bound and soluble chaperones (including calnexin, ERP57 and calreticulin) [39]. Once assembled, the MHC-I/β2m/peptide complexes are exported from the ER via the classical secretory pathway and are expressed at the plasma membrane. The loading of MHC-I molecules by endogenously produced peptides plays a critical role in the killing of virally infected cells, tumor cells and transplanted allogeneic cells by effector CD8+ cytotoxic T lymphocytes (CTL).

Cross-presentation by MHC-I

The endogenous pathway just described is responsible for loading all or most peptides onto MHC-I molecules in most cells in the body. During the initiation of immune responses, however, the situation turns out to be more complex. It has become clear over the last 20 years that DCs initiate most immune responses [41]. Although, some viruses can infect DCs and generate MHC-I peptide ligands by the classical pathway, in many cases – such as for responses to bacteria, parasites, tumors, allogeneic transplants and viruses that do not infect DCs – the antigens are not expressed by the DCs themselves. In many of these cases, it has been shown that DCs can phagocytose antigens from microbes or from infected or allogeneic cells, and cross-present peptides derived from these antigens on their own MHC-I. How, then, are these antigens processed for loading on MHC-I for cross-presentation?

Two main intracellular models for cross-presentation have been proposed, and are generally referred to as the ‘vacuolar’ and ‘cytosolic’ pathways [42] (Figure 1). The vacuolar pathway model proposes that internalized antigens are processed into peptides that can bind MHC-I directly in endosomes and phagosomes. The MHC-I molecules in this model likely access endocytic compartments by recycling from the plasma membrane, and would bind peptides independently of proteasomal degradation, TAP transporters or the MHC-I loading complex. The cytosolic pathway model proposes that antigens, most likely after partial degradation [43], are exported to the cytosol and processed by the proteasome as for endogenous MHC-I processing. These exogenous proteasome-processed peptides could then be loaded onto MHC-I molecules in the ER following TAP-dependent uptake. Alternatively, ER-resident proteins including TAP and the MHC-I loading machinery can be recruited to phagosomes and perhaps even to endosomes [17]; thus, peptides generated by the proteasome might be translocated back into the lumen of endocytic compartments for loading on MHC-I molecules, which could then recycle back to the plasma membrane [44]. In either case, by exploiting the proteasome and TAP, the cytosolic pathway would provide a means by which DCs can present the same peptides from internalized antigens that are generated endogenously in other cell types, and thus stimulate relevant CD8+ T cells.


Figure 1. Models of phagosomal antigen cross-presentation by MHC-I. Shown are the two main proposed models for intracellular pathways leading to cross-presentation of exogenous antigens in DCs. Left, the TAP- and proteasome-dependent cytosolic pathway. In this scenario, antigens on phagocytosed particles are exported to the cytosol by an ERAD-associated retrotranslocon, ubiquitylated and targeted to the proteasome for degradation. The generated peptides are then either translocated back into the phagosome by TAP and loaded onto MHC-I (1) or fed into the classical TAP-dependent MHC-I antigen presentation pathway in the ER (2). ERGIC-derived membranes that fuse with phagosomes are the likely source of the phagosomal retrotranslocon, TAP, and perhaps newly synthesized MHC-I. Right, the TAP- and proteasome-independent vacuolar pathway. In this scenario, peptides are generated from antigens on phagocytosed particles within phagosomes by proteolysis mediated by cathepsins. These peptides are then loaded onto itinerant MHC-I molecules within the phagosome.

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In our opinion, there are to date no definitive results that prove or exclude any of these possibilities. Most of the experimental evidence supporting presentation of a particular antigen through the vacuolar or cytosolic pathways comes from interfering with either endocytic (lysosomal proteases), cytosolic (proteasomal proteolysis) or ER-related (TAP or loading complex) functions. The problem is that the three intracellular compartments are involved in all pathways. Inhibiting the proteasome or TAP deprives newly synthesized MHC-I of peptides, causing retention of incompletely folded molecules in the ER and thereby reducing the pool of available MHC-I in endocytic compartments and the plasma membrane. Inhibiting endocytic functions can prevent generation of MHC-I binding peptides in endosomes and phagosomes, but it can also interfere with the initial partial degradation steps that are required for the export of some antigens to the cytosol. Therefore, most of the arguments in favor or against the use of the vacuolar or cytosolic pathways for presentation of particular antigens are to be taken with caution. One convincing argument supporting the use of the cytosolic pathway derives from an in vivo experiment for presentation of the male antigen Uty [45]. A peptide derived from Uty that is presented to T cells can only be generated by the immunoproteasome, which is a variant of the conventional proteasome harboring three interferon-responsive subunits; male mice lacking one of these subunits, LMP-7, cannot present the Uty epitope. Whereas endogenous MHC-I presentation of other antigens is intact in these mice, cross-presentation of Uty in vivo is also impaired in LMP-7−/− mice. This experiment provides direct evidence that the Uty epitope is processed through the cytosolic pathway even during cross-presentation.

While the relative contributions of vacuolar and cytosolic pathways to cross-presentation are murky, the molecular mechanisms underlying either process are even less well-defined. In particular, the mechanisms of antigen export to the cytosol, the intracellular compartments where peptides are loaded onto MHC-I (ER versus endocytic), the origin of the MHC-I that is loaded with cross-presented peptides (recycling or newly synthesized) and their paths for transport to the surface remain to be established.

Phagosomes as Antigen Presentation Compartments

  1. Top of page
  2. Abstract
  3. Processing and Presentation of Phagocytosed Antigens by MHC-I and -II
  4. Phagosomes as Antigen Presentation Compartments
  5. Innate Signaling Controls Phagosome Maturation and Antigen Processing/Presentation
  6. Autophagy and MHC Presentation from Phagosomes
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Appendix 1.: Abstract figure

Given the role of endosomes as antigen processing compartments and the extensive interplay between organelles of the endocytic pathway and maturing phagosomes [4], it is not surprising that antigens from phagocytosed particles are processed and presented by MHC-I and -II.

Phagosomal presentation by MHC-II

Harding and Geuze detailed the processing of phagocytosed Listeria monocytogenes and the contents of phagosomes and phagolysosomes in macrophages [14], showing that macrophages present antigen from internalized particles by MHC-II with kinetics similar to those of bacterial destruction and of arrival of MHC-II in phagosomes. Consistent with these observations, elegant studies using subcellular fractions of macrophages that had phagocytosed antigen-bound latex beads showed that phagosomes acquired MHC-II, H-2DM and Ii within 30 min following uptake, and that MHC-II:peptide complexes formed within phagosomes and were subsequently transported to the cell surface [46]. Similar observations were made with macrophages that had taken up heat-killed Mycobacterium tuberculosis [47]. These observations suggested that phagosomes themselves can serve as MHC-II processing compartments and provide an environment conducive to the assembly of MHC-II:peptide complexes, and that such complexes are delivered directly to the cell surface from maturing phagosomes (Figure 2).


Figure 2. Model of phagosomal antigen processing and presentation by MHC-II. MHC-II processing and presentation from phagosomes. (1) Phagocytosed antigen captured by APCs is unfolded and degraded in phagosomes as the phagosomes mature. Maturation is achieved by the acquisition of content (including MHC-II molecules) from early and late endosomes and lysosomes by both direct fusion (open arrows) and vesicular transport (turquoise arrows; dashed arrows indicate possible pathways for MHC-II transport that are not yet confirmed). Antigen is loaded onto MHC-II molecules predominantly in late phagosomes. Phagosome maturation is supported by autophagy (ATG; violet) proteins, which might derive from autophagosomes themselves or independently from the cytosol, and by signaling from PRRs (black arrow). Some PRRs are delivered to phagosomes from early endosomes in an AP-3-dependent manner (mauve dashed arrow). (2) From late phagosomes, peptide-loaded MHC-II molecules are delivered to the cell surface either directly (not shown) or via an intermediate tubular MHC-II storage compartment that might be similar to the compartment derived from classical non-phagosomal MIIC. Delivery to or from this compartment might be regulated by PRR signaling from the phagosome (dashed black arrows) or perhaps directly by AP-3 (dashed mauve arrow).

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In other cases, however, MHC-II:peptide complexes appear to accumulate in compartments other than phagosomes prior to cell surface appearance. For example, using subcellular fractionation of macrophages at various times following infection with Streptomyces pyogenes, von Delwig et al. found that fractions enriched in endosomes, but not phagosome-enriched fractions, could simulate pathogen-specific CD4+ T cells [48]. The time course over which these complexes formed depended on the specific peptide generated. Similarly, using immunofluorescence microscopy with an antibody that recognizes the I-Ab MHC-II molecule complexed with a peptide derived from I-Eα [49, 50], MHC-II:peptide complexes accumulated on non-endosomal/non-lysosomal vesicular compartments, but not on phagosomes, in DCs following uptake of latex beads conjugated to an Eα fusion protein [51]. These data indicate that MHC-II complexes with peptides derived from phagocytosed antigen can accumulate in distinct compartments following their formation, perhaps owing to variations on phagosome maturation that occur in macrophages during infection with certain bacteria or in DCs. Whether these complexes form in phagosomes and are then rapidly transported to post-phagosomal compartments, or form in separate MHC-II-containing compartments following antigen export from phagosomes is not yet clear. The visualization of MHC-II complexed with a peptide derived from Leishmania major within both the L. major-containing phagosome and in separate endocytic compartments in DCs [52] supports the assembly of such complexes in phagosomes with subsequent accumulation in plasma membrane-bound transport vesicles, but definitive proof of such a pathway will necessitate careful time lapse experiments.

Trafficking of MHC-II and regulators to MIIC and phagosomes

The molecular mechanisms that regulate trafficking of MHC-II from the secretory pathway to endosomes and conventional MIIC have received substantial attention, but are still unresolved. Targeting of MHC-II molecules to MIIC is facilitated by the associated Ii, and requires two dileucine-based sorting signals in the Ii cytoplasmic domain [53-55]. Similar signals in other endosomal proteins bind in vitro to the ‘trunk’ domain of members of the adaptor protein complexes, which are components of clathrin coats involved in vesicle formation and cargo sorting within the endosomal system [56]. Indeed, some evidence suggests that the Ii sorting signals bind AP-2 – the cell surface adaptor for clathrin-dependent endocytosis – and AP-1 – which functions in protein sorting at the TGN and endosomes [57]. In HeLa cells that express MHC-II and associated molecules (by virtue of expression of the CIITA transcription factor), depletion of clathrin or AP-2 delayed Ii degradation and reduced surface expression of peptide-loaded MHC-II molecules, whereas depletion of AP-1 or two other endosomal adaptors, AP-3 or AP-4, had little or no effect [21, 58]. Consistently, AP-3 – which functions in cargo sorting from early endosomes to late endosomes/lysosomes or lysosome-related organelles [59] – was dispensable for MHC-II biosynthetic transport and general peptide loading in human EBV-transformed B cells and mouse spleen cells [60, 61] and for presentation of antigen internalized by receptor-mediated endocytosis in DCs [51]. These findings demonstrate that clathrin and AP-2 participate in MHC-II molecule trafficking in vivo and that a significant pool of MHC-II molecules traffics to the endosomal-lysosomal system via the cell surface, but additional sorting events may be adaptor-independent. This was surprising given that MHC-I and -II molecules diverge at the TGN in EBV-transformed B lymphocytes [23]. Recent work has shown that non-muscle myosin II plays an important role in clustering MHC-II into centralized lysosomal compartments, perhaps via direct interaction with MHC-II/Ii complexes, and is required for subsequent formation of MHC-II:peptide complexes in B lymphocytes [62]. Whether this reflects a general role for myosin II in export from the Golgi [63, 64] or a specific role for post-Golgi MHC-II trafficking is not yet clear. MHC-II molecules accumulate on phagosomes during phagosome maturation in macrophages [14] and DCs [65], but how MHC-II molecules are transported to phagosomes is not understood. Phagosomal acquisition of MHC-II in macrophages and DCs coincides with the acquisition of lysosomal proteins such as cathepsins and the integral membrane proteins LAMP1 and LAMP2 [66, 67], suggesting that MHC-II is targeted to phagolysosomes and to traditional late endosomes and lysosomes by the same intracellular transport pathways. Alternatively, MHC-II/Ii complexes might be incorporated into phagosomal membranes during endosome/phagosome fusion [14, 68]. Recent work in macrophages has shown that the small GTPase, Arl8b, facilitates phagolysosome fusion (and lysosome maturation) by recruiting the HOPS complex – a tethering complex that facilitates SNARE-dependent fusion – to lysosomes [69]. Depletion of Arl8b impairs phagosome maturation and assembly of ligands with another antigen-presenting molecule, CD1b, and would be predicted to interfere with MHC-II peptide loading as well [69]. Resolution of the mechanism by which MHC-II and associated components (such as H-2DM) are delivered to phagosomes will likely require live-cell imaging with selective visualization of transport intermediates using photoactivatable probes. Interestingly, the rate of MHC-II transport to and retention within phagosomes can be regulated in DCs by the nature of the phagosomal cargo. For example, MHC-II is acquired more rapidly and retained longer in phagosomes that harbor TLR ligand-coated latex beads than those harboring M. tuberculosis [70].

Several studies have shown that mature MHC-II-peptide complexes can be modified by polyubiquitylation of the MHC-II β chain, and in some cases also the α chain [71], by the E3 ubiquitin ligase, MARCHI [72-75]. This modification leads to down-regulation of mature MHC-II molecules, and is at least in part responsible for the low level of surface MHC-II expression in immature DCs in which MARCHI expression is high [72, 74, 75]. The mechanism by which ubiquitylation down-regulates MHC-II is debated. While initial studies suggested that ubiquitylation influenced MHC-II:peptide endocytosis [73, 74], recent work implies instead that it functions during post-endocytic targeting of constitutively internalized MHC-II molecules for degradation in lysosomes, presumably by the multivesicular body pathway [76]. Intriguingly, ubiquitylation and down-regulation of MHC-II in mature DCs or macrophages is stimulated by at least two pathogenic intracellular bacteria, Francisella tularensis and Salmonella typhimurium [77, 78]. Nevertheless, how ubiquitylation contributes to MHC-II trafficking to or from phagosomes is not known.

Cell surface delivery of MHC-II:peptide complexes

Once they are formed in MIIC, phagosomes, or post-phagosomal compartments, MHC-II:peptide complexes must then be delivered to the plasma membrane in order to be available to stimulate antigen-specific CD4+ T lymphocytes. MHC-II cell surface delivery from conventional MIIC has been visualized in model APCs and LPS-stimulated bone marrow-derived DCs using GFP-tagged MHC-II [27-29, 79], and intermediates in transport have been visualized by electron microscopy [80-82], but the mechanisms underlying this process are poorly understood. MIIC in a model APC have been shown to move bidirectionally along microtubules through the activity of the motor proteins dynein and kinesin [83], and eventually fuse with the plasma membrane without intersecting endosomal compartments [27]. Similarly, MHC-II:peptide complexes In B lymphocytes are delivered from MIIC to the cell surface without intersecting early endosomes [84], and MIIC fusion profiles observed at the plasma membrane result in secretion of the intralumenal vesicles of multivesicular MIICs [80]. In DCs, MHC-II:peptide assembly and export from MIICs to the plasma membrane are stimulated by maturation through TLRs or other receptors [85, 86], and coincides with a transition of MIICs from vesicular to tubular structures  [28, 29, 81, 87], at least some of which lack lysosomal markers [82]. These tubules have been proposed to be the carriers to mediate cell surface delivery of MHC-II. They have not been observed in immature DCs, B cells or melanoma cells, suggesting that they represent a unique specialization of mature DCs [88]. Similar analyses have not been as thoroughly applied to phagosome-associated MHC-II:peptide complexes. Nevertheless, MHC-II-containing tubules emerging from L. monocytogenes-containing phagolysosomes in macrophages have been observed by electron microscopy [14], suggesting that delivery of phagosome-derived MHC-II:peptide complexes to the plasma membrane might also occur via tubular intermediates. The punctate compartments in which phagosome-derived MHC-II:peptide complexes accumulate following antigen-coated latex bead phagocytosis in formaldehyde-fixed DCs [51] might represent remnants of such tubules, as they lack lysosomal or early endosomal markers.

Small GTPases of the Rab and Arl family are among the few components known to regulate MHC-II trafficking following peptide loading. Rab proteins regulate membrane traffic through the selective membrane recruitment of cytosolic effector proteins, such as sorting adaptors, tethering factors, kinases, phosphatases and motors (reviewed in [89]). RAB7, a well-known regulator of late endosomal dynamics and early to late endosome maturation [90], is detected on late endosome and MIIC membranes [27, 91]. RAB7 is expressed at low levels in resting B lymphocytes but is up-regulated following stimulation, and RAB7 overexpression enhances MHC-II antigen presentation kinetics [92]. RAB7 recruits the dynein motor complex to MIIC [93, 94], allowing retrograde movement along microtubules and likely regulating MIIC release to the plasma membrane. In order to accumulate MIIC or other MHC-II-containing compartments in the periphery, dynein must be inactivated or released, which would likely require inactivation of RAB7 by a GTPase activating protein (GAP). Additional candidate regulators of late steps in MHC-II transport were recently identified in a multidimensional RNAi screen in a melanoma model APC [95]. Among the many exciting ‘hits’ in this screen was a pathway centered about the small GTPase, ARL14/ARF7. ARL14/ARF7 localized to MIIC in immature DCs, due in part to recruitment of the ARL14/ARF7 guanine nucleotide exchange factor (GEF), PSD4, by phosphoinositides generated by the phosphatidylinositide (PtdIns) kinases PIP5K1A and PIK3R2. GTP-bound ARL14/ARF7 in turn bound an effector protein, ARF7EP, which recruits the actin-based motor, MYO1E. In this way, ARL14/ARF7 may connect MIIC to the actin network via ARF7EP-MYO1E to control export of MHC-II. Consistently, silencing of ARL14/ARF7 in immature DCs resulted in a mature DC-like MHC-II distribution, as observed by confocal microscopy [95]. While a role for these pathways in MHC-II delivery from phagosomes has not yet been established, similar networks are likely to function. For example, PIP5K1A plays a critical role early in phagosome formation [96], and RAB7 has extensive interactions with phagosomes in macrophages and other phagocytes [97].

Endosomes, phagosomes and cross-presentation

By definition, the first steps of all cross-presentation pathways (vacuolar or cytosolic) occur in endocytic compartments. Models for cross-priming are mainly based on particulate antigen, as injected soluble proteins inefficiently induce cross-priming in vivo, and coupling a protein antigen to beads – which forces uptake by phagocytosis – strongly increases the efficiency of cross-presentation [98]. In macrophages, inducing macropinocytosis also stimulates cross-presentation [99]. These results suggest that the mode of internalization influences cross-presentation, but the mechanisms involved remain obscure.

Regardless of the mode of entry, the parameters that influence cross-presentation by DCs adhere to a set of rules. For example, to be cross-presented, internalized antigens need to be released in the endocytic lumen – either for lysosomal processing in the vacuolar pathway or for export to the cytosol in the cytosolic pathway. This requirement is easy to fulfill for soluble antigens that are internalized or secreted into phagosomes by living microbes, but to release non-secreted microbial antigens for cross-presentation the microbes must be killed and at least partially degraded. Furthermore, antigen release from large protein aggregates requires disaggregation and lysosomal proteolysis [43]. Therefore, the proteolytic activity of the endocytic pathway is required for cross-presentation of at least some antigens through both the vacuolar and cytosolic pathways. On the other hand, excessive endocytic proteolysis is detrimental to cross-presentation as shown using several approaches. First, the efficiency of cross-presentation is enhanced by interfering with endocytic acidification by treatment with weak bases such as chloroquine [100], and antagonized by increased acidification due to genetic defects that interfere with the alkalinizing activity or recruitment of the phagocyte NADPH oxidase (NOX2) on endosomes and phagosomes [11, 18, 101-103]. Second, cross-presentation is enhanced by targeting antigens – via antibodies or covalent modifications – to receptors that are not efficiently delivered to lysosomes, such as the mannose receptor, DEC205, DNGR-1 or CD40L [17, 102, 104]. Likewise, dead cell uptake through DGNR-1 enhances cross-presentation and retains the phagocytosed corpses in early phagocytic compartments [105, 106]. The results of these studies, however, need to be interpreted with caution. Most of these studies failed to control for the amount of antigen internalized or the kinetics of internalization under the different conditions, which can dramatically impact the availability of antigen; proper use of such a control is best exemplified in studies by the Villadangos laboratory, in which FACS sorting of cells containing single antigen-coated beads [107] excluded variations in antigen uptake. Moreover, the only reliable assay for cross-presentation is stimulation of antigen-specific T cells, which does not always have a suitable dynamic range of response and for which single antigen concentrations are often inappropriately used. Furthermore, a frequently used control for cross-presentation is antigen presentation to MHC-II-restricted T cells, for which the thresholds and sensitivity of the corresponding T cells are not always similar; thus, selective presentation to CD4+ or CD8+ T cells can often be obtained simply by using different concentrations of antigen. Finally, T cell activation is subject to many variables that are difficult to control for, such as cytoskeletal changes and content of adhesion or costimulatory molecules, complicating the use of controls.

ER-phagosome connections

How might antigens escape phagosomes to the cytosol? The best characterized system for export of secretory proteins to the cytosol is retrotranslocation of misfolded proteins from the ER by the ER-associated degradation (ERAD) machinery. If phagocytosed antigens could access this machinery, they could conceivably be translocated just like a misfolded ER protein. The idea that phagosomes can directly recruit ER resident proteins was first proposed by Desjardins and colleagues based on proteomic analyses of purified macrophage phagosomes and associated ultrastructural histochemistry that showed ER membranes in direct continuity with phagosomes or with the plasma membrane [108]. This study proposed that fusion of the ER with the plasma membrane contributed membrane for formation of the phagocytic cup during phagocytosis in macrophages. A limitation of the proteomics data is the potential for contamination of phagosome preparations with very abundant ER-derived membranes. Furthermore, subsequent work using a combination of live-cell imaging and quantitative immunoelectron microscopy challenged the microscopy data, and suggested that fusion of the ER with nascent phagosomes in macrophages was a rare event [109]. Nevertheless, several groups showed that TAP and the peptide-loading complex were recruited and functional on isolated phagosomes in DCs [110-112], making an inferential link of ER-phagosome connections to cross-presentation.

While the morphological and proteomics support for ER-phagosome fusion remain a matter of debate, several groups have provided functional evidence for a requirement for the ER in phagocytosis per se or specifically in cross-presentation. Work in Dictyostelium provided the first functional evidence that ER proteins (i.e. calnexin and calreticulin) are recruited to phagosomes and are required for phagocytosis [113], although the latter could reflect requirements for ER folding of phagosomal proteins. The Cresswell laboratory showed that a peptide bearing an N-glycosylation site and covalently linked to latex beads could be glycosylated after phagocytosis, providing direct evidence for the accessibility of the phagosome lumen to the glycosyltransferase complex of the ER [114]. Importantly, the Rothman laboratory provided evidence for a potential mechanism for ER-phagosome fusion. They identified a fusogenic SNARE complex consisting of the ER v-SNARE Sec22b and the plasma membrane t-SNAREs Syntaxin 18 and Syntaxin 4 [115]. This would potentially allow for fusion of the ER with the plasma membrane or with nascent phagosomes harboring the plasma membrane SNAREs. Several studies that investigated a possible role for ER SNAREs in ER-phagosome interactions in mammalian macrophages confusingly suggested that Syntaxin 18 and Sec22b are positive and negative regulators of phagocytosis, respectively [116-118].

Last year, two studies re-examined the issue. The first one used quantitative proteomics to evaluate the contribution of the ER (and other cellular membranes) to phagosomes in macrophages [119]. These authors found that only a subset of ER resident proteins is actually present in phagosome preparations, and that these ER residents constitute 20% of the protein in early phagosomes. The second study showed that siRNA-mediated knock down of the ER-derived vSNARE Sec22b in DCs inhibits the recruitment of ER resident proteins to phagosomes and severely impairs the efficiency of cross-presentation [120]. This was accompanied by reduced antigen export from phagosomes to the cytosol and increased degradation of phagocytosed antigen. The enrichment of Sec22b in the ER-Golgi intermediate compartment (ERGIC) suggests that in DCs, ERGIC-derived material can fuse directly with phagosomes and thus provide ER proteins required for antigen retrotranslocation and cross-presentation.

A role for ER in cross-presentation is further illustrated in immunity to Toxoplasma gondii. Goldszmid et al. showed that ER proteins are actively recruited to the parasitophorous vacuole harboring live T. gondii but not to phagosomes harboring dead parasites [121]. Concomitantly, T. gondii antigens are presented from live parasites with increased efficiency compared to dead parasites. Moreover, cross-presentation was impaired upon knock down of Sec22b, concomitant with a failure to recruit ER proteins to the parasitophorous vacuole [120]. Thus, recent evidence compellingly supports the recruitment of ER resident proteins to phagosomes and their role in cross-presentation. It will be interesting to explore the requirement for Sec22b in cross-presentation of antigens expressed by other pathogens that manipulate the recruitment of the ER to their vacuoles, such as Legionella or Brucella. Whether ER protein functions in phagosomes extend beyond cross-presentation also remains to be investigated.

Antigen export to the cytosol

Some phagocytosed bacteria, such as Listeria monocytogenes or Salmonella typhimurium, escape phagosomes to the cytosol as part of their life cycle, and can therefore provide sources of MHC-I ligands through the traditional pathway. However, antigens on non-vital bacteria, bacteria that lack such mechanisms, or even internalized proteins can also be efficiently cross-presented as well, suggesting that DCs and other cross-presenting cells must have mechanisms to export phagocytosed antigens to the cytosol.

Rock and colleagues were the first to describe processing of exogenous antigen and cross-presentation by MHC-I in cultured splenocytes [122, 123], and to show that exogenous antigens need to access the cytosol to be cross-presented [124]. Subsequent studies from several groups confirmed that exogenous proteins enter the cytosol for MHC-I presentation. Watts and colleagues reported that exogenous soluble ovalbumin was cross-presented by bone marrow-derived macrophages and DCs in a manner that required proteasome activity and TAP transporters, suggesting that antigen entry into the cytosol was a necessary step during cross-presentation [99, 125]. Finally, morphological (confocal microscopy and EM) and biochemical (subcellular fractionation) data showed that antigens from macropinosomes and phagosomes in DCs, but not macrophages, can be delivered into the cytosol [43, 126]. Export to the cytosol required proteolytic disaggregation of large complexes and was more efficient for low molecular weight cargoes [43]. Two possible mechanisms for this transport were proposed: endosome-phagosome leakage into the cytosol or selective transport through membrane transporters. A recent report provided some support for a leakage mechanism by showing that lipid body function is required during antigen cross-presentation [127]. Lipid bodies were suggested to participate in destabilizing subdomains of the ER or phagosome membrane, thereby causing leakage of their contents [87]. Our recent results, however, show no major defects in antigen export to the cytosol in mice deficient for lipid body biogenesis (Cebrian and Coll, unpublished). Other available evidence supports the existence of a transporter, most likely related to the retranslocation machinery used during ERAD.

ERAD is the main pathway for the degradation of misfolded proteins in the ER by the proteasome [128, 129]. The ERAD machinery consists of a series of E3 ubiquitin ligases and channels that include members of the DERLIN family (but not likely the SEC61 protein import channel, as originally proposed) [130]. Once engaged in the channel, misfolded ER proteins are ubiquitylated and the cytosolic AAA-ATPase VCP/p97 and/or hsp90a promote export to the cytosol. The presence of ER resident proteins in phagosomes led to the early proposal that ERAD could mediate antigen export from phagosomes to the cytosol [114, 131, 132]. Compelling support for this proposal came from the finding that: (i) ExoA, a toxin that inhibits ERAD, also inhibits cross-presentation [114]; (ii) p97 can be co-immunoprecipitated with internalized biotinylated OVA [114]; (iii) interfering with the function or expression of p97 or hsp90 in DCs inhibits cross-presentation [90, 133] and (iv) in vitro reconstituted export from phagosomes requires cytosol or recombinant p97 [134]. In a recent study, Zehner et al. showed a critical role for mannose receptor ubiquitylation in the recruitment of p97 to endosomes and in antigen export to the cytosol [132]. One criticism of these studies is the lack of a reliable quantitative assay for antigen export from endosomes or phagosomes to the cytosol. The assay used for cytosolic export relied on subcellular fractionation, but differential sensitivity of different populations of endosomes or phagosomes to mechanical rupture during cell homogenization could have significantly influenced the results. In addition, the impact of mannose receptors on cross-presentation is still a matter of debate [135-137]. The recent description of a β-lactamase-based assay for cytosolic translocation in living cells may, at least in part, help address these issues in the future [120].

Both ERAD and cytosolic delivery of internalized cargoes require protein unfolding prior to export. Indeed, elegant studies from the Creswell laboratory showed that the ER chaperones BiP, calnexin and calreticulin are present in phagosomes and may participate in protein unfolding [138, 139]. Some antigens additionally require reduction of disulfide bonds during unfolding. In some cases, this might be carried out by ER-associated protein disulfide isomerases. However, it has also been shown that GILT, a protein reductase expressed within endosomal/lysosomal compartments only in professional APCs, plays a critical role in cross-presentation of viral antigens [140, 141], as it does for MHC-II-dependent presentation of disulfide-containing antigens [142].

Innate Signaling Controls Phagosome Maturation and Antigen Processing/Presentation

  1. Top of page
  2. Abstract
  3. Processing and Presentation of Phagocytosed Antigens by MHC-I and -II
  4. Phagosomes as Antigen Presentation Compartments
  5. Innate Signaling Controls Phagosome Maturation and Antigen Processing/Presentation
  6. Autophagy and MHC Presentation from Phagosomes
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Appendix 1.: Abstract figure

Just as phagosome formation requires signaling from phagocytic receptors to induce actin and membrane remodeling [1], phagosome maturation and its associated MHC-I/II processing and presentation are also responsive to signaling. Pathogens such as M. tuberculosis interfere with such signaling to arrest phagosome maturation and block both degradative and antigen presenting functions [143]. Innate immunity to microbes relies on the detection of microbial- and danger-derived molecular signatures (collectively named microbial-associated molecular patterns or MAMPs, and danger-associated molecular patterns or DAMPs, respectively) by specific host receptors collectively termed pattern recognition receptors (PRRs) [144, 145]. PRRs include Toll-like receptors (TLRs), Nod-like receptors (NLRs) and C-type lectin receptors such as the mannose receptor, DC-SIGN, dectin-1 and DNGR-1/CLEC9A. C-type lectins detect a variety of common ligands on pathogens or tumor cells including branched oligosaccharides [146] and, for DNGR-1, F-actin released from necrotic cells [147, 148]. Phagocytic receptors for complement and immunoglobulin Fc regions do not strictly represent PRRs but can contribute to pathogen clearance through the engagement of opsonized microbes and cross-talk with PRRs [149, 150], rendering the PRR signaling network even more complex (reviewed in [151] [152]).

The best-studied family of PRRs in the context of phagocytosis and antigen presentation are the TLRs, which in mammals encodes 10–12 membrane-spanning molecules with diverse specificities for extracellular or endosomal MAMPs or DAMPs [153]. Another PRR family, the NLRs, function in the recognition of MAMPs and DAMPs in the cytosol or nuclear compartments [145, 154]. Signaling via PRRs stimulates DC maturation, and concomitantly modulates phagosomal maturation and antigen presentation. The precise mechanisms that link innate sensing, phagosome functions and antigen presentation are not yet clear, but we focus here on studies that address this link at the molecular level.

Innate sensors and antigen processing/presentation by MHC-II

LPS and other TLR ligands stimulate DCs to mature from an antigen sensing form that efficiently internalizes material from the extracellular space to an antigen presenting form that presents this material to T cells in secondary lymphoid organs [155, 156]. A pioneering study by the Mellman group dissociated antigen uptake from antigen processing/ presentation by showing that previously internalized antigens were only effectively presented by MHC-II following treatment of DCs with LPS, correlating with formation of peptide: MHC-II complexes and redistribution of MHC-II from lysosomes to post-lysosomal compartments [82]. Similarly, some activation signals also stimulated cross-presentation of previously internalized antigens by MHC-I [157]. The effect on MHC-II-associated antigen processing is due at least in part to modulation of lysosomal and phagosomal acidification by stimulating the assembly of the vacuolar ATPase complex [158].

The effects of TLR ligation on antigen processing were originally thought to be similar for antigens internalized by phagocytosis or other means. However, the presence of TLRs such as TLR2 and TLR4 on phagosomes suggested that TLR signaling from phagosomes might influence phagosome fate [159, 160]. Indeed, Blander and Medzhitov showed that TLR signaling from within a given phagosome in macrophages or DCs stimulates intrinsic phagosome maturation [66] and antigen processing [67], such that the contents of phagosomes bearing TLR ligands were preferentially presented by MHC-II over those of phagosomes containing apoptotic bodies that were devoid of TLR ligands, even when the two sorts of cargo were internalized by the same cell [67]. We recently reported similar findings regarding distinct antigen degradation and presentation properties of phagosomes in the same cell when one phagosome carried cargoes that cross-link a distinct class of phagocytic and signaling receptor family, the Fc receptor [161]. While the influence of TLR stimulation on phagosome maturation in macrophages has been disputed [162], this property in DCs likely reflects tighter regulation of TLR signaling from phagosomes than from conventional endosomes or the plasma membrane [51, 163], perhaps due to a requirement for more strict checkpoints to trigger an immune response to a live phagocytosed pathogen than to scattered soluble PAMPs [164] or a dead pathogen [165]. Interestingly, in AP-3-deficient DCs, in which recruitment of TLRs to maturing phagosomes is impaired, phagosomes matured normally and processed associated antigens for assembly of MHC-II: peptide complexes [51]. This suggests that signaling from early phagosomes might be sufficient to induce phagosome maturation, perhaps by stimulating engagement of autophagy components [163, 166]. Nevertheless, the MHC-II: peptide complexes that formed in AP-3-deficient DCs remained trapped in intracellular compartments and were not available for presentation at the plasma membrane [51]. These data support the intriguing hypothesis that proinflammatory TLR signaling from phagosomes is required to generate transport intermediates for delivery of MHC-II: peptide complexes to the plasma membrane.

TLRs such as TLR4 stimulate proinflammatory and antiviral signaling via two sets of adaptors: MyD88 and its coadaptor TIRAP/MAL initiate pro-inflammatory responses via MAPK and NFkB activation, whereas TRAM and its coadaptor TRIF stimulate a second phase of proinflammatory response and production of type I interferon via TRAF-dependent IRF3 activation [167, 168]. MyD88-deficient mice have several defects in phagocytosis and phagosome maturation [169-172], suggesting that this pathway is essential for TLR-induced phagosome maturation and downstream antigen presentation [51, 173]. Some reports suggest that MyD88 influences phagosome maturation independently of TLR signaling [162], that TLR signaling inhibits phagosome-lysosome fusion [174], and that prolonged stimulation of TLR2 by M. tuberculosis inhibits MHC-II antigen presentation, at least partially through down-regulation of CIITA activity and reduced MHC-II expression [175, 176]. Interpretation of these results is complicated by defects in inflammasome-dependent IL-1β signaling in MyD88-deficient cells, which could potentially influence phagosomal fate.

NLR engagement can also influence phagosome functions. Recent studies have shown that during infection, two members of the NLR family – Ipaf and Naip5 – restrict Legionella growth by promoting phagosome maturation and intracellular degradation, thereby restricting bacterial growth [177, 178]. Stimulation of NLRP3 has also been shown to enhance maturation of M. tuberculosis-containing phagosomes by activating IL-1β [179]. Although these reports link phagosome function with NLR activation, neither the mechanism of action nor the impact on antigen presentation has been characterized.

Innate sensors and antigen cross-presentation by MHC-I

Whereas TLR ligands clearly activate MHC-II processing and presentation on DCs, the effects of TLR or NLR stimulation on cross-presentation are less clear. Stimulation of several TLRs has been reported to either enhance or antagonize cross-presentation of soluble and particulate antigens on both DCs and macrophages, in some cases with distinct effects depending on whether stimulation occurred prior to or concomitant with Ag exposure [17, 157, 180-191]. In vivo, several reports have shown that cross-presentation can be induced by stimulation through TLRs, including TLR3 and TLR9 [192, 193]. Nevertheless, it is difficult in vivo to distinguish whether T cell priming is increased due to enhanced cross-presentation per se or to increased T cell activation due to DC maturation. More recently, NOD1 and NOD2 agonists have been reported to enhance cross-priming of soluble ovalbumin antigen by CD8+ DCs in vivo and in vitro [194]. This increase, however, might again reflect indirect effects on DC maturation rather than actual stimulation of cross-presentation. By contrast, it has been reported that a NOD2 agonist inhibits the capacity of LPS to increase cross-presentation of viral antigens [180].

Autophagy and MHC Presentation from Phagosomes

  1. Top of page
  2. Abstract
  3. Processing and Presentation of Phagocytosed Antigens by MHC-I and -II
  4. Phagosomes as Antigen Presentation Compartments
  5. Innate Signaling Controls Phagosome Maturation and Antigen Processing/Presentation
  6. Autophagy and MHC Presentation from Phagosomes
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Appendix 1.: Abstract figure

Autophagy comprises a group of processes by which cytosolic components are delivered to lysosomes for degradation [195, 196]. In particular, macroautophagy refers to the envelopment of cytoplasmic targets by nascent double-membrane compartments called autophagosomes, and subsequent degradation of autophagosome contents upon their fusion with lysosomes. Macroautophagy is thought to proceed at a low constitutive rate in all cells, but can be induced by starvation or other types of cell stress [196]. The interplay of the autophagy pathway with membrane trafficking, organelle dynamics and immunity has become the focus of intensive interest over the past few years, and has been reviewed in detail elsewhere [195-199]. Comments here will be limited to the dual role of autophagy in MHC-I and -II presentation of phagocytosed antigen.

Autophagy and class-II-mediated Ag presentation

The peptides presented on MHC-II molecules primarily derive from the degradation of exogenous antigens. However, endogenous proteins can also be presented by MHC-II molecules; indeed, analysis of peptides eluted from MHC-II molecules in B cells revealed that ∼20% of natural MHC-II ligands are derived from cytosolic and nuclear proteins, including cytoskeletal proteins, heat shock proteins and histones [200, 201]; peptides from cytosolic viral antigens, model antigens and tumor antigens have also been identified [200, 202, 203]. APCs such as B cells, DCs and activated endothelial cells constitutively form autophagosomes that eventually fuse with MIICs [200], providing a constitutive source of cytosolic antigens to the classical MHC-II processing pathway. This mechanism influences presentation of antigens from phagocytosed bacteria in two ways. First, many bacterial pathogens eject virulence factors out of the phagosome and into the host cytosol via specific type III or type IV secretion systems [204, 205]; such virulence factors are critical to bacterial survival and evasion of host cell defenses, but they also provide a source of antigen for MHC-II processing via macroautophagy. For example, infection by Yersinia pseudotuberculosis impairs antigen uptake and classical MHC-II processing by macrophages, but infected cells are still capable of stimulating CD4+ T cells specific for the outer membrane protein YopE, a substrate of the Yersinia type III secretion system [206]. MHC-II presentation of YopE is sensitive to inhibitors of autophagy and of lysosomal proteolysis, suggesting that YopE encounters MHC-II molecules in classical MIIC following autophagy. A second role for autophagy in recovering phagosomal antigens applies to bacteria such as Salmonella typhimurium and Listeria monocytogenes that escape the encapsulating phagosome into the cytosol as a pathogen strategy to avoid lysosomal degradation [207]. Macroautophagy provides a mechanism to sequester these escaped bacterial forms and protect the host from uncontrolled bacterial replication [208-211]. Similarly, group A Streptococcus that accesses the cytosol in non-phagocytic cell types is also controlled by lysosomal degradation following autophagy [212]. By delivering cytosolic bacteria to lysosomes independently of phagosome maturation, autophagy provides a ‘second chance’ for generating peptides for presentation by MHC-II. Indeed, CD4+ T cell immune responses to M. tuberculosis – a pathogen that similarly escapes phagosomes [106] – were enhanced upon stimulation of autophagy and inhibited upon interference with autophagy [213] (although this might occur by a different mechanism – see below). Hence, autophagy is an attractive pathway to counteract pathogen evasion of both innate and adaptive cellular immunity.

While macroautophagy provides a venue for MHC-II access to cytoplasmic proteins that derive from phagosomes, it also appears that autophagy regulators independently facilitate phagosome maturation to phagolysosomes. This was first recognized during infection of macrophages with mycobacteria, as M. tuberculosis has long been known to arrest phagosome maturation to evade destruction [214]. Pharmacological and physiological induction of autophagy in cells infected with the attenuated BCG strain overcomes the normal phagosome maturation arrest and results in effective phagolysosome formation [215]. This might explain the enhanced MHC-II presentation of bacterial peptides upon stimulation of autophagy in M. tuberculosis-infected macrophages and DCs [213]. A requirement for autophagy components in phagosome maturation is not limited to M. tuberculosis phagosomes. Autophagy effectors such as LC3 and Beclin 1 have been shown to be recruited to phagosomes containing bacteria, yeast, LPS-coated latex beads, apoptotic cells and entotic vacuoles [166, 216, 217], and depletion of ATG5 or ATG7 blocks phagosome acidification and content degradation [166]. Moreover, Atg5−/− DCs show impaired processing of phagocytosed antigen due to diminished phagosome maturation, leading to decreased presentation by MHC-II [163], and DCs depleted of autophagy mediators ATG5, ATG7 or ATG16L1 are impaired in controlling bacterial growth and antigen presentation upon infection with Salmonella enterica or E. coli [218]. MAMP recognition by many members of the TLR and NLR family can trigger both autophagy and LC3-associated phagosomes [216, 219], perhaps in part explaining the stimulation of phagosome maturation induced by TLRs and other PRRs. The link between PRR stimulation, autophagy and phagosome maturation is clinically important, as polymorphisms in the NLR NOD2 and the autophagy regulator ATG16L1 have been linked to Crohn's disease, an inflammatory disease in which tolerance to commensal bacteria is dysregulated [218, 220]. Together, these findings all point to a joint effort between phagocytosis and autophagy in the route to pathogen clearance and antigen processing.

Autophagy and MHC-I-restricted antigen presentation

To date, autophagy has been implicated in the classical MHC-I presentation pathway in only one report, focusing on the processing and presentation of the cytosolic HSV-1 protein gB; Desjardins and colleagues showed that upon infection by HSV-1, macrophages depleted of ATG5 have a reduced capacity compared to WT macrophages to stimulate a gB-specific CD8+ T cell response [221]. A role for autophagy in cross-presentation of phagocytosed antigens has been more controversial. One report showed that antigens conjugated to alpha-alumina nanoparticles are efficiently cross-presented by an ATG12-independent autophagy pathway [222]. They reported that siRNA knockdown of ATG6, but not ATG12, impaired cross-presentation of ovalbumin coupled to these nanoparticles. The influence of ATG6 on cross-presentation from nanoparticles, however, might reflect a property of the nanoparticles themselves rather than a general requirement for autophagy in cross-presentation from phagosomes. For example, whereas treatment of DCs with the autophagy/phosphatidylinositol(3)kinase inhibitors 3-methyladenine (3-MA; blocks early stages of autophagy and LC3-associated phagosome maturation) or wortmannin abolished cross-presentation of ovalbumin coupled to alpha-alumina nanoparticles (but not of soluble ovalbumin) [222], treatment with 3-MA had no effect on cross-presentation of HIV-1 antigens to HIV-specific CD8+ T cell clones [223]. In addition, it has been reported that Atg5-deficient DCs cross-presented both apoptotic cell-associated and soluble ovalbumin normally [163].

While a generalized role in cross-presentation is unclear, autophagy has been reported to play a role in antigen packaging in donor cells for efficient cross-presentation from dead or dying cells by DCs. In one study, cross-presentation of an expressed model antigen or an endogenous tumor antigen following coculture with DCs was enhanced when the donor cells were exposed to treatments that stimulate macroautophagy (rapamycin or starvation) and antagonized by treatment with inhibitors of macroautophagy (3-MA or wortmannin) [224]. Moreover, cross-presentation of the gp100 tumor antigen was impaired when ATG6 or ATG12 were knocked down in donor melanoma cells, and isolated autophagosomes were able to directly provide antigen to DCs. In a second study, an influenza A virus antigen was more efficiently cross-presented by DCs exposed to infected fibroblasts from Bax/Bak−/− mice, which exhibit increased features of autophagy, than from wild-type mice, and this enhancement was largely abolished by siRNA-silencing of ATG5 [225]. While these studies establish a clear role for macroautophagy in the antigenicity of dying cells for cross-presentation, the mechanisms involved remain unclear.


  1. Top of page
  2. Abstract
  3. Processing and Presentation of Phagocytosed Antigens by MHC-I and -II
  4. Phagosomes as Antigen Presentation Compartments
  5. Innate Signaling Controls Phagosome Maturation and Antigen Processing/Presentation
  6. Autophagy and MHC Presentation from Phagosomes
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Appendix 1.: Abstract figure

The last 15 years have witnessed an impressive expansion in our appreciation of phagosomes as specialized compartments for antigen processing and presentation. Once considered only a degradative compartment devoted to the killing of engulfed microbes, phagosomes have emerged as sophisticated platforms that integrate antigen processing with innate signaling, providing a molecular link between innate sensors and the generation of ligands for antigen receptors. Understanding how signaling from phagosomes affects phagosomal functions, and the consequences of phagosomal signaling on antigen processing and presentation, represents a challenge for the development of effective vaccination strategies.


  1. Top of page
  2. Abstract
  3. Processing and Presentation of Phagocytosed Antigens by MHC-I and -II
  4. Phagosomes as Antigen Presentation Compartments
  5. Innate Signaling Controls Phagosome Maturation and Antigen Processing/Presentation
  6. Autophagy and MHC Presentation from Phagosomes
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Appendix 1.: Abstract figure

This work was supported by National Institutes of Health grant AI92398 from the National Institute of Allergy and Infectious Diseases, INSERM, Institut Curie, the European Research Council (2008 Advanced Grant 233062 PhagoDC), la Ligue Nationale Contre le Cancer (LNCC) and l'Agence Nationale de la Recherche. J. G. M. received funding from Fondation Recherche Medical. The authors declare no conflict of interest.


  1. Top of page
  2. Abstract
  3. Processing and Presentation of Phagocytosed Antigens by MHC-I and -II
  4. Phagosomes as Antigen Presentation Compartments
  5. Innate Signaling Controls Phagosome Maturation and Antigen Processing/Presentation
  6. Autophagy and MHC Presentation from Phagosomes
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Appendix 1.: Abstract figure
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Appendix 1.: Abstract figure

  1. Top of page
  2. Abstract
  3. Processing and Presentation of Phagocytosed Antigens by MHC-I and -II
  4. Phagosomes as Antigen Presentation Compartments
  5. Innate Signaling Controls Phagosome Maturation and Antigen Processing/Presentation
  6. Autophagy and MHC Presentation from Phagosomes
  7. Conclusion
  8. Acknowledgements
  9. References
  10. Appendix 1.: Abstract figure

Presentation of phagocytosed antigens by MHC class I and II. In this review, we focus on the cell biological aspects of antigen presentation through the eyes of phagocytes that take up large foreign particles through the process of phagocytosis. We focus on two distinct antigen processing pathways for presentation by MHC class I (MHC-I) and MHC class II (MHCII) molecules. In this figure, we summarize the two main proposed models for intracellular pathways leading to cross-presentation of phagocytosed antigens by MHC-I in dendritic cells. Left, the TAP- and proteasome-dependent cytosolic pathway. In this scenario, antigens on phagocytosed particles are exported to the cytosol by an ERAD-associated retrotranslocon, ubiquitylated and targeted to the proteasome for degradation. The generated peptides are then either translocated back into the phagosome by TAP and loaded onto MHC-I (1) or fed into the classical TAP-dependent MHC-I antigen presentation pathway in the ER (2). ERGIC-derived membranes that fuse with phagosomes are the likely source of the phagosomal retrotranslocon, TAP, and perhaps newly synthesized MHC-I. Right, the TAP- and proteasome-independent vacuolar pathway. In this scenario, peptides are generated from antigens on phagocytosed particles within phagosomes by proteolysis mediated by cathepsins. These peptides are then loaded onto itinerant MHC-I molecules within the phagosome.