- Email: [email protected]
Topological journey of parasite-derived antigens for presentation by MHC class I molecules
Review
Topological journey of parasite-derived antigens for presentation by MHC class I molecules Nicolas Blanchard1,2 and Nilabh Shastri1 1
Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA 2 Current address: Physiopathology Research Centre of Toulouse-Purpan/INSERM U563, Department of Immunology and Infectious diseases, CHU Purpan, BP3028, 31024 Toulouse Cedex, France
Within cells of their host, many bacteria and parasites inhabit specialized compartments, such as a modified phagosome for Mycobacterium tuberculosis or a parasitophorous vacuole for Toxoplasma gondii. These locations could exclude microbial material from entry into the MHC class I surveillance pathway. Remarkably, however, under these circumstances, cells can still signal the presence of invading pathogens to circulating CD8+ T cells, which typically play a key role in protection against such intracellular organisms. Here, we review MHC I presentation pathways in various contexts, ranging from model antigens in non-infectious settings to pathogen-infected cells. We suggest that presentation of intracellular pathogens can be described as not just one, but several distinct pathways; perhaps because diverse pathogens have evolved different strategies to interact with host cells. Basic aspects of MHC I presentation of exogenous antigens CD8+ T cells are key to immune surveillance against tumors and intracellular pathogens. After recognizing their target cells, CD8+ T cells are directly cytotoxic and produce inflammatory cytokines such as interferon (IFN)-g and tumor necrosis factor (TNF)-a. The T cell antigen receptor (TCR) of CD8+ T cells recognizes short peptides presented by MHC class I molecules on the surface of transformed or infected target cells. Intracellular degradation products from self- or virus-encoded proteins constitute an important source of peptides for the MHC I presentation pathway (Figure 1). Additionally, it has long been recognized that professional antigen-presenting cells (APCs) have the capacity to internalize proteins made elsewhere and present them as peptides for ‘‘cross-priming’’, that is, to initiate naı¨ve CD8+ T cell responses [1,2]. This alternative pathway is referred to as cross-presentation (Figure 2). APCs differ in their capacity for cross-presentation and several laboratories have implicated a particular CD8a+ subset of dendritic cells (DCs) in cross-presentation of viral antigens [3–5]. The recent identification of a crucial transcription factor necessary for in vivo development of CD8a+ Corresponding authors: Blanchard, N. ([email protected]); Shastri, N. ([email protected]).
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DCs has elegantly confirmed the unique role of these cells in uptake and cross-presentation of viral and tumor antigens in vivo [6]. The ability of cells to cross-present peptide–MHC (pMHC) I appears crucial in many situations. Professional APCs, such as DCs or macrophages, are efficient in initiating naive CD8+ T cell responses, therefore, it is important that these APCs can present pMHC I even when not infected themselves [7,8]. Furthermore, infected cells (including professional APCs) are often manipulated by various pathogens to downregulate pMHC I, thereby potentially limiting or delaying the establishment of protective CD8 T cell responses [9]. In this situation, the ability of the APC to present antigens from exogenous sources becomes essential to ensure timely and efficient presentation, to allow detection and elimination of the pathogen by the host. In addition to this typical and historical view of crosspresentation, presentation of exogenous antigens can also occur in host cells infected with intracellular organisms, which synthesize their own proteins inside specialized compartments that are segregated from the cytoplasm where the classical pMHC I pathway normally begins. Despite their initial non-cytosolic location, peptides derived from these antigens can be presented on the surface of infected cells. The antigenic proteins in this case are exogenous and are not synthesized by the host protein synthesis machinery, therefore, it would be logical to consider the underlying mechanism as another manifestation of cross-presentation. However, because of the lack of consensus on the terminology, here, we define presentation of intracellular parasites in infected cells as exogenous presentation. Here, we review the studies that have led to different models for cross-presentation of non-infectious material, and then we discuss how these models apply to presentation of various intracellular pathogens. Antigens embark on an intracellular journey Presentation of exogenous material as pMHC I in normal or inflammatory contexts can steer CD8+ T cells towards tolerance or immunity. Exogenous materials can enter cells by various mechanisms. Phagocytic cells engulf apoptotic cells and debris via a variety of surface molecules such as scavenger receptors [10]. In the steady-state, cross-presentation
1471-4906/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.it.2010.08.004 Trends in Immunology, November 2010, Vol. 31, No. 11
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Figure 1. Cytosolic MHC I processing pathway. For endogenously synthesized proteins, the ‘‘direct’’ or ‘‘cytosolic’’ antigen processing pathway typically begins in the cytoplasm. Cytosolic proteases, such as the multicatalytic proteasome, fragment polypeptide substrates into smaller antigenic intermediates, which are then protected by chaperones and imported into the ER by TAP. In the ER, intermediate peptides undergo final trimming by ERAAP/ERAP1 before loading onto nascent MHC I molecules. The loading process is facilitated by several factors held together in the peptide-loading complex, including tapasin and the Erp57 thiol-disulfide oxido-reductase. MHC I molecules bound to peptides of optimal length and stability leave the ER on their way to the Golgi and the cell surface, to be ultimately scanned by circulating CD8+ T cells (reviewed in [86–88]).
of apoptotic self-antigens maintains peripheral tolerance by deletion of autoreactive CD8+ T cells. As opposed to crosspriming, this process is referred to as ‘‘cross-tolerance’’ [11]. CD8a+ DCs are also implicated in cross-tolerance [12]. When bound to antibodies, such as within immune complexes or opsonized microorganisms, antigens are internalized by Fc receptor (FcR)-triggered endocytosis or phagocytosis (see [13] for a review). In non-infectious settings, the general consequence of FcR-mediated cross-presentation is also tolerance induction, except in cases in which self-antigens are complexed with autoantibodies. In this situation, as shown in a model of mouse type II diabetes, cross-presentation of autoimmune complexes enhances activation of effector T cells and breaches tolerance [14]. An alternative mode of antigen entry into cells, potentially favoring immunity over tolerance, is when antigens are conjugated to Toll-like receptor (TLR) ligands. For example, after conjugation of TLR9 and CpG DNA receptor-mediated endocytosis enhances antigen presentation relative to presentation that occurs after normal pinocytosis [15]. Nevertheless, the same enhancement might not necessarily occur with all TLRs and their ligands [16].
Finally, soluble proteins enter cells typically via pinocytosis or macropinocytosis; two processes that are based on plasma membrane ruffling, which allow a cell to sample small or large fluid-phase volumes, respectively [17,18]. Stay on-board or hop into the cytoplasm? Once internalized in an endosomal compartment, the exogenous antigens continue their journey on the cross-presentation pathway. In the cytoplasm, the main protease that is known to produce antigenic fragments is the multisubunit proteasome. In the classical cytosolic MHC I pathway, fragments are then imported into the endoplasmic reticulum (ER) through the transporter associated with antigen processing (TAP) heterodimer. However, does exogenous antigen degradation take place in the intracellular compartment or in the cytoplasm? A large body of evidence supports a requirement for the proteasome and TAP during cross-presentation. Hence, the cytoplasm is seen as the singular location for antigen processing [18– 24]. For many antigens, the material transported from the cytosol into the ER is not the final peptide but a proteolytic intermediate that requires further processing. In fact, excessive proteolysis in the phagosome is detrimental for cross-presentation [25], which might explain why DCs, in 415
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Figure 2. Two models of MHC I cross-presentation. (a) Fusion of maturing phagosomes with the ER leads to material exchange between both compartments (brown double arrow) and results in a hybrid ER–phagosome organelle. ER–phagosomes contain the retrotranslocon factor Sec61 as well as the TAP transporter. Sec61 allows for antigen export to the cytosol where the antigen is degraded. Subsequently, TAP mediates re-import of peptides into the hybrid organelle. The pH needs to stay high enough to avoid excessive degradation of the full-length antigen. It is tightly regulated by NOX2 NADPH oxidase activity and association with lipid bodies. Intermediates might be ultimately trimmed by ERAAP and loaded onto empty MHC I molecules. (b) Alternatively, antigen processing and loading might occur entirely in the vacuole/phagosome. Processing is ensured by endosomal (IRAP) or lysosomal (cathepsin S) proteases.
particular CD8a+ DCs, are superior to macrophages in cross-presentation. It has been reported that excessive acidification of the phagolysosome in DCs is prevented through production of oxygen species by the NADPH oxidase NOX2 [26], and that this process occurs preferentially in the CD8a+ DC subset and requires the Rac2 small GTPase [27]. Phagosomal pH is also regulated by an additional mechanism [28]. Storage organelles for lipids accumulated and associated with phagosomes in a manner dependent on IGTP, an IFN-g-inducible ER-resident GTPase that is important for resistance against intracellular pathogens [29]. In a model using ovalbumin (OVA)coated latex beads as a model particulate antigen, absence of IGTP has been found to influence cross-presentation. The mechanistic details and the relevance of this pathway during infection by an intracellular pathogen remain unclear. Alternatively, in some circumstances, the final peptides can be generated in the phagosome itself (Figure 2b). This is the case when the OVA protein is incorporated into microspheres of a biodegradable polymer [poly(lactic-coglycolic) acid]. Presentation of OVA is then TAP- and proteasome-independent, but requires the lysosomal pro416
tease cathepsin S [30]. Another non-cytosolic mechanism involves the insulin-regulated aminopeptidase (IRAP), an endosomal protease found in muscle, fat, mast cells and DCs [31]. IRAP has been shown to be involved in processing OVA epitopes within DC endosomes [32]. Further investigation should clarify the potential role of IRAP in processing antigens from intracellular pathogens. In conclusion, antigens destined for cross-presentation are best preserved in compartments with low acidity. Although the final peptides are occasionally generated by local proteases, the most common route for the antigenic precursors is to enter the cytosol and serve as a substrate for the proteasome. En route to the cytosol How do antigens located in the endosome/phagosome enter the cytoplasm? This topological conundrum was first noted in macrophages, in which, after phagocytosis, a ribosomal inactivating protein inhibits protein synthesis, which suggested an exit pathway to the cytosol [19]. Further studies have proposed that, compared with macrophages, the endosome-to-cytosol transport might be more potent in DCs [33]. Using antigen-coated latex beads, certain ER proteins
Review have been observed to be recruited to the phagosome, for example, Sec61 [22,23] (Figure 2a). Sec61 is one component of the retrotranslocation channel, which exports misfolded proteins from the ER to the cytosol for degradation by the ubiquitin–proteasome system, by a process known as ERassociated degradation (ERAD). Two studies have used siRNA-mediated or chemical inhibition of retrotranslocation to confirm the functional role of the retrotranslocon in cross-presentation, and have shown that the p97 ATPase, another essential factor for ERAD, is necessary for export of antigens from purified phagosomes [34,35]. The detection of ER proteins on phagosomal membranes has prompted the idea that uptake of particulate antigens occurs through regulated ER/phagosome fusion, thereby creating a new hybrid compartment, self-sufficient for internalization of cytoplasmic antigenic fragments through TAP transport and subsequent MHC I loading [22,23] (Figure 2a). Although this model is controversial [36], it remains attractive because ultrastructural evidence supports the continuity and material exchange between the ER and the phagosome/vacuole during infection by intracellular pathogens like Leishmania [37], Toxoplasma gondii [38] and Chlamydia [39]. Does entry of soluble antigens occur in a similar fashion? By tracking the fate of soluble b2 microglobulin taken up by DCs, it has been found that this protein functionally interacts with nascent MHC I heavy chains in the ER, which suggests that retrograde transport of soluble antigens into the ER can occur [40]. Recently, an alternate model for soluble antigen processing, reminiscent of the ER–phagosome compartment, has been proposed, whereby soluble antigen-containing endosomes are equipped with TAP and MHC I loading capacity and become competent for antigen presentation [41]. Relocation of TAP to endosomes is enhanced by TLR4 ligands, which suggests that spatial separation between MHC I presentation of exogenous versus endogenous antigens is regulated by signals from the innate immune system. Putting on the final touch N-terminal trimming of antigenic intermediates by the ER aminopeptidase associated with antigen processing (ERAAP) has a major impact on the repertoire of endogenously synthesized pMHC I [42,43]. However, is ERAAP also involved in the processing of exogenous antigens? ERAAP is not involved in cross-presentation of soluble OVA by DCs [44,45], which perhaps reflects a more prominent role for the IRAP aminopeptidase in N-terminal trimming [32]. However, ERAAP is necessary for crosspresentation of immune complexes or particulate antigens by DCs [44,45]. Consistent with these results, ERAAP has been found along with other ER markers in purified latexbead-containing phagosomes [46], which suggests that it is transferred from the ER to the phagosome. Whether it actually exerts its function in the phagosomes or after antigen translocation into the ER remains to be established. In summary, these studies point to a relatively predominant pathway wherein antigenic material is first shuttled from the phagosome/endosome to the cytosol via a mechanism akin to the ERAD pathway. This material is then
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degraded by the proteasome and transported by TAP into the ER, ER–phagosome or ER–endosome hybrid compartments where subsequent trimming by ERAAP can occur. Next, we examine the various exogenous presentation mechanisms following infection by intracellular microorganisms. MHC I presentation of intracellular pathogens How does the intracellular location of the microorganism influence the presentation of the antigenic material? Here, we review well-characterized examples of intracellular pathogens with locations ranging from strictly vacuolar (Leishmania, Chlamydia or T. gondii) to cytosolic (Listeria monocytogenes or Trypanosoma) (Box 1). Notably, CD8+ T cells contribute to protective immunity to each of these pathogens; even those that do not infect professional APCs. The importance of secretion Immunodominant antigens from intracellular non-viral pathogens have consistently been found to be secreted proteins. Strong natural CD8+ responses target the circumsporozoite (CS) protein of Plasmodium berghei [47], the cysteine-rich outer membrane protein (CrpA) of Chlamydia trachomatis [48], the dense granule protein 6 (GRA6) of T. gondii [49], the lysteriolysin of L. monocytogenes [50] and the culture-filtrate protein 10 of Mycobacterium tuberculosis Box 1. Intracellular pathogens analyzed in this review At one end of the spectrum, some pathogens establish their niche in a membrane-bound compartment called vacuole (e.g. Plasmodium spp. and T. gondii), phagosome (e.g. Leishmania spp.) or inclusion (e.g. C. trachomatis) and remain segregated from the cytosol during their entire life cycle. At the other end, some intracellular organisms promptly escape to the cytosol in order to replicate (e.g. Tryp. cruzi and L. monocytogenes). Leishmania spp.: a protozoan kinetoplastid parasite that infects mammals and transmitted into skin by sandfly bite. Macrophages are infected by metacyclic promastigotes before conversion into small ovoid amastigotes. Parasites replicate in the phagosome. Tryp. cruzi: a protozoan kinetoplastid parasite that infects mammals and is transmitted by bite or feces of the blood-sucking assassin bug. Trypomastigotes force entry in vacuoles, escape into the cytosol, convert into amastigotes, and replicate in the cytosol. T. gondii: a protozoan apicomplexan parasite that infects all warmblooded animals by ingestion of contaminated food/soil, or by materno-fetal transmission. Tachyzoites actively infect any nucleated cell, and establish and replicate in a specialized nonfusogenic vacuole. Plasmodium spp.: a protozoan apicomplexan parasite that infects birds, reptiles and mammals, and is transmitted to skin/blood by mosquito bite. During the liver stage, sporozoites migrate from the bite wound to the liver, where they invade hepatocytes and replicate in vacuoles. During the blood stage, parasites reside in a vacuole in red blood cells. C. trachomatis: a Gram-negative bacterium that infects humans by sexual transmission, but mice can be used as a host model. Bacteria (elementary body) infect epithelial cells, convert into reticulate bodies and replicate in vacuoles (also called inclusion). M. tuberculosis: an acid-fast bacterium that infects humans and is transmitted by aerosol from previously infected persons. Mycobacteria infect lung alveolar macrophages, replicate in phagosomes, and potentially translocate to the cytoplasm at later stages. L. monocytogenes: a Gram-positive bacterium that infects a broad range of animals by ingestion of contaminated food. Bacteria infect epithelial or phagocytic cells, escape from vacuoles, and replicate in the cytosol.
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Review [51]; all of which are secreted molecules. This idea is supported by studies that have used genetically engineered microorganisms that express secreted or non-secreted model antigens, and is particularly striking because secreted proteins represent only a minor fraction of the entire proteome of these pathogens [52,53]. For instance, with T. gondii, only tachyzoites that secrete b-galactosidase in the vacuole induce b-galactosidase-specific CD8+ T cells in infected mice [54]. Moreover, priming of OVA-specific TCR-transgenic CD8+ T cells is optimal only with OVA-secreting Leishmania major parasites [55].
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active mechanism mediated by microorganisms and/or the host cell.
Infected versus bystander APCs Another key question about how the host mounts a protective response against intracellular pathogens is whether pathogen-derived material is presented by directly infected or bystander APCs that have ingested debris from surrounding infected cells. Once infected, any type of MHC I+ nucleated cell should be able to present antigens and become a target for killing by effector CD8+ T cells. However, only professional APCs, such as macrophages or DCs, have the capacity to internalize exogenous antigens and prime naı¨ve CD8+ T cell responses. Hence, the answer probably varies with the stage of infection (initial priming versus effector phase) and the tissue examined (peripheral site versus lymph node). For example, without being productively infected by P. berghei sporozoites, DCs can efficiently present an epitope from the CS antigen [56] and prime CS-specific CD8+ T cells in the skin-draining lymph nodes [57]. In this model, uninfected bystander DCs are believed to play a crucial role during the priming phase, whereas the later liver effector phase might be DC-independent [57]. During L. monocytogenes infection, cross-presentation by uninfected professional APCs also seems to be crucial because priming of CD8+ T cells against non-secreted antigens requires uptake of infected neutrophils [58]. Alternatively, in the T. gondii and L. major infection models, the crucial importance of protein secretion by the parasites, along with the reduced or abolished presentation of parasite antigens when dead (heat-killed) parasites are ingested by APCs, suggest that optimal T cell priming needs active infection of DCs by live parasites [59,60]. Moreover, it is likely that during the effector phase of the response to infection, exogenous MHC I presentation occurs mainly in (non-professional) APCs that are sheltering live microorganisms (Figure 3). What then are the mechanisms for exogenous presentation in these actively infected cells?
Active role for microorganisms For cytosolic pathogens that rapidly escape the phagosome, secreted proteins are directly available to the cytosolic MHC I pathway (e.g. the p60 antigen from L. monocytogenes [64]). However for vacuolar pathogens, antigens must first cross the vacuole boundary. What are the mechanisms used to accomplish this tour-de-force? Creation of pores in the vacuolar membrane constitutes one way for the pathogen to allow its antigens into the cytosol. M. tuberculosis-containing vacuoles have been shown to be permeable to fluorescent dextrans of up to 70 kDa [65]. Early secreted antigen target 6 (ESAT6) has been proposed to act as a pore-forming protein and participate in the membrane perforation of Mycobacterium marinum vacuoles [66]. However, it remains to be determined whether ESAT6 mediates access of M. tuberculosis antigens to the MHC I pathway. The vacuole membrane that surrounds T. gondii and Plasmodium falciparum also contains pores [67,68] but the relatively small opening of these ‘‘sieves’’ (<1.4 kDa) is expected to preclude polypeptides larger than 15 amino acids from diffusing, therefore pores are unlikely to be involved in the transfer of antigenic precursors to the cytosol. A second possibility for gaining access to the cytosolic MHC I pathway is by insertion within the vacuole membrane, so that all or part of the antigen is exposed to the cytoplasm, cleaved and released in the cytosol for further processing. This situation might apply to the Chlamydia epitopes from Cap1 [69] and CrpA [48], because these proteins are known transmembrane proteins that are inserted in the membrane that surrounds the inclusion. A third gateway to the cytosol is by active targeting of certain factors to the host cytoplasm. Many proteins from the Plasmodium and T. gondii apicomplexan parasites are found in the cytosol or even the nucleus of infected cells [70–72]. A conserved five-residue motif (RxLxE) called Plasmodium Export Element, or PEXEL [70], has been identified as necessary for the secretion of proteins from the P. falciparum vacuole to the cytoplasm of human erythrocytes. Notably, the CS immunodominant antigen of Plasmodium contains two PEXEL motifs that also mediate export from the parasitophorous vacuole to the cytosol in infected hepatocytes [73]. A possible translocon that mediates export of PEXEL-containing proteins to the cytosol has been recently identified [74] but whether a similar mechanism operates for T. gondii is not known.
Different pathogens, different mechanisms Generation of the pMHC I from intracellular pathogens by an exclusive vacuolar pathway (i.e. proteasome and TAPindependent) appears to be the exception more than the rule. Besides the purely phagosomal pathway used by DCs infected with OVA-expressing L. major parasites [55], data from several other infectious models (T. gondii [49,55], P. berghei [61], L. monocytogenes [24], M. tuberculosis [62] and C. trachomatis [63]) indicate that pMHC I generation requires the proteasome and TAP transport. Thus, entry of microbial material into the host cytosol requires an
Active role for the host cell Some host defense mechanisms have been shown to damage the phagosome/vacuole membrane and can therefore facilitate the release of antigens in the cytoplasm. A family of IFN-g-inducible proteins (IRGs for immunity-related GTPases, formerly known as p47 GTPases) is suspected to be involved in this process. IRGs are essential for resistance of mice to a variety of intracellular bacteria and parasites (reviewed in [75]). In the case of T. gondii, they participate in the destruction of the parasitophorous vacuole membrane [76], thereby exposing the parasite to the
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Figure 3. Exogenous MHC I presentation of intracellular pathogens. (a) In the vacuolar pathway, antigens are degraded and loaded on MHC I molecules within the phagosome/vacuole itself (e.g. L. major). (b–g) In the TAP-dependent cytosolic pathway, the antigen gains access to the cytosol where it is processed primarily by the proteasome. (b) Secreted proteins from organisms living in the cytosol (e.g. Tryp. cruzi or L. monocytogenes) directly end up in the cytosol. For organisms living in a vacuole, several exit pathways to the cytoplasm have been proposed. (c) A secreted factor might form pores in the vacuole membrane, which allow for subsequent diffusion of antigen (e.g. M. tuberculosis). (d) An active transport mechanism that potentially involves recognition of specific motifs and/or specific translocation machinery might shuttle the antigen to the cytosol (e.g. Plasmodium). (e) Host innate defense proteins such as IRGs might disrupt the vacuole membrane and release antigens into the cytosol (e.g. T. gondii). (f) In the case of a transmembrane protein, the antigen might be inserted into the vacuole membrane so that the proteasome can process the cytosolic side of the antigen (e.g. C. trachomatis). (g) Finally, fusion of the vacuole with the host ER might deliver the antigen to the Sec61-based retrotranslocation machinery that shuttles it to the cytoplasm (e.g. T. gondii). Antigenic peptide precursors are then imported into the ER for possible trimming by ERAAP and final MHC I loading.
inhospitable cytoplasmic environment. Altered levels of OVA presentation have been reported in Irgm3-deficient macrophages infected with OVA-secreting parasites [77], which implicate this IRG protein in MHC I processing. This same IRG protein has been implicated in the crosspresentation of OVA-coated latex beads via lipid bodies (see above and Figure 2a), but it is unclear if similar mechanisms are involved in both cases. Surprisingly, a presentation defect has not been observed in Irgm3-deficient DCs [77]. To date, IRGs have also been shown to participate in host resistance to M. tuberculosis [78,79], C. trachomatis [80] and Trypanosoma cruzi [81] but whether they mediate access of antigens to the MHC I pathway in these models remains to be investigated. An ER–phagosome pathway? Another pathway has been reported that is reminiscent of the ER–phagosome model already described. Sec61 has been found to have a role in MHC I presentation of OVA by T. gondii-infected DCs [38], which supports the notion that fusion between the parasitophorous vacuole and the host
ER allows antigen access to the ER, cytosol export through the retrotranslocation channel, and TAP-mediated re-import in the ER. A possible shortcoming of this model is that OVA trafficking might differ from that of natural parasite antigens. For instance, as opposed to a soluble protein like OVA, the natural T. gondii antigens GRA6 [49] and GRA4 [82] are known to associate with membranes within the vacuole, which could limit access to the ER. Hence, it is important to determine whether this appealing mechanism applies to natural T. gondii antigens as well. Concluding remarks In summary, although antigens from intravacuolar pathogens are segregated from the cytoplasm, they do enter the cytosolic MHC I pathway through a variety of ways that all have the same, paramount, downstream consequence: stimulation of a CD8+ T cell response and potential control of infection. Perhaps because each pathogen has evolved different strategies to interact with host cells, exogenous presentation of intracellular pathogens is not one but several distinct pathways. 419
Review Intriguingly, as pathogens have evolved strategies to interfere with direct, cytosolic MHC I presentation [9], pathogens also try to trick their host by dampening cross-presentation. For instance, late antigens from the vaccinia virus seem to be sequestered in virus factories, which makes them inaccessible to the cross-presentation machinery [83]. Alternatively, M. tuberculosis has evolved specific molecules to block macrophage apoptosis, thereby preventing uptake of its antigens by APCs and subsequently dampening CD8+ T cell responses [84,85]. It will be essential to delineate further how intracellular pathogens interfere with cross-presentation, which should suggest ways to counteract immune evasion mechanisms and improve vaccine efficacy.
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Acknowledgements N.B. was supported by a long-term postdoctoral fellowship from the Human Frontier Science Program Organization. N.S. was partly supported by a Lord Harris Senior Research Fellowship at the Harris Manchester College and the Weatherall Institute of Molecular Medicine at the University of Oxford, UK. Research in the N.S. laboratory is supported by grants (AI060040, AI044864, AI39548, AI065831) from the National Institutes of Health.
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Topological journey of parasite-derived antigens for presentation by MHC class I molecules Nicolas Blanchard1,2 and Nilabh Shastri1 1
Division of Immunology and Pathogenesis, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA 2 Current address: Physiopathology Research Centre of Toulouse-Purpan/INSERM U563, Department of Immunology and Infectious diseases, CHU Purpan, BP3028, 31024 Toulouse Cedex, France
Within cells of their host, many bacteria and parasites inhabit specialized compartments, such as a modified phagosome for Mycobacterium tuberculosis or a parasitophorous vacuole for Toxoplasma gondii. These locations could exclude microbial material from entry into the MHC class I surveillance pathway. Remarkably, however, under these circumstances, cells can still signal the presence of invading pathogens to circulating CD8+ T cells, which typically play a key role in protection against such intracellular organisms. Here, we review MHC I presentation pathways in various contexts, ranging from model antigens in non-infectious settings to pathogen-infected cells. We suggest that presentation of intracellular pathogens can be described as not just one, but several distinct pathways; perhaps because diverse pathogens have evolved different strategies to interact with host cells. Basic aspects of MHC I presentation of exogenous antigens CD8+ T cells are key to immune surveillance against tumors and intracellular pathogens. After recognizing their target cells, CD8+ T cells are directly cytotoxic and produce inflammatory cytokines such as interferon (IFN)-g and tumor necrosis factor (TNF)-a. The T cell antigen receptor (TCR) of CD8+ T cells recognizes short peptides presented by MHC class I molecules on the surface of transformed or infected target cells. Intracellular degradation products from self- or virus-encoded proteins constitute an important source of peptides for the MHC I presentation pathway (Figure 1). Additionally, it has long been recognized that professional antigen-presenting cells (APCs) have the capacity to internalize proteins made elsewhere and present them as peptides for ‘‘cross-priming’’, that is, to initiate naı¨ve CD8+ T cell responses [1,2]. This alternative pathway is referred to as cross-presentation (Figure 2). APCs differ in their capacity for cross-presentation and several laboratories have implicated a particular CD8a+ subset of dendritic cells (DCs) in cross-presentation of viral antigens [3–5]. The recent identification of a crucial transcription factor necessary for in vivo development of CD8a+ Corresponding authors: Blanchard, N. ([email protected]); Shastri, N. ([email protected]).
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DCs has elegantly confirmed the unique role of these cells in uptake and cross-presentation of viral and tumor antigens in vivo [6]. The ability of cells to cross-present peptide–MHC (pMHC) I appears crucial in many situations. Professional APCs, such as DCs or macrophages, are efficient in initiating naive CD8+ T cell responses, therefore, it is important that these APCs can present pMHC I even when not infected themselves [7,8]. Furthermore, infected cells (including professional APCs) are often manipulated by various pathogens to downregulate pMHC I, thereby potentially limiting or delaying the establishment of protective CD8 T cell responses [9]. In this situation, the ability of the APC to present antigens from exogenous sources becomes essential to ensure timely and efficient presentation, to allow detection and elimination of the pathogen by the host. In addition to this typical and historical view of crosspresentation, presentation of exogenous antigens can also occur in host cells infected with intracellular organisms, which synthesize their own proteins inside specialized compartments that are segregated from the cytoplasm where the classical pMHC I pathway normally begins. Despite their initial non-cytosolic location, peptides derived from these antigens can be presented on the surface of infected cells. The antigenic proteins in this case are exogenous and are not synthesized by the host protein synthesis machinery, therefore, it would be logical to consider the underlying mechanism as another manifestation of cross-presentation. However, because of the lack of consensus on the terminology, here, we define presentation of intracellular parasites in infected cells as exogenous presentation. Here, we review the studies that have led to different models for cross-presentation of non-infectious material, and then we discuss how these models apply to presentation of various intracellular pathogens. Antigens embark on an intracellular journey Presentation of exogenous material as pMHC I in normal or inflammatory contexts can steer CD8+ T cells towards tolerance or immunity. Exogenous materials can enter cells by various mechanisms. Phagocytic cells engulf apoptotic cells and debris via a variety of surface molecules such as scavenger receptors [10]. In the steady-state, cross-presentation
1471-4906/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.it.2010.08.004 Trends in Immunology, November 2010, Vol. 31, No. 11
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Figure 1. Cytosolic MHC I processing pathway. For endogenously synthesized proteins, the ‘‘direct’’ or ‘‘cytosolic’’ antigen processing pathway typically begins in the cytoplasm. Cytosolic proteases, such as the multicatalytic proteasome, fragment polypeptide substrates into smaller antigenic intermediates, which are then protected by chaperones and imported into the ER by TAP. In the ER, intermediate peptides undergo final trimming by ERAAP/ERAP1 before loading onto nascent MHC I molecules. The loading process is facilitated by several factors held together in the peptide-loading complex, including tapasin and the Erp57 thiol-disulfide oxido-reductase. MHC I molecules bound to peptides of optimal length and stability leave the ER on their way to the Golgi and the cell surface, to be ultimately scanned by circulating CD8+ T cells (reviewed in [86–88]).
of apoptotic self-antigens maintains peripheral tolerance by deletion of autoreactive CD8+ T cells. As opposed to crosspriming, this process is referred to as ‘‘cross-tolerance’’ [11]. CD8a+ DCs are also implicated in cross-tolerance [12]. When bound to antibodies, such as within immune complexes or opsonized microorganisms, antigens are internalized by Fc receptor (FcR)-triggered endocytosis or phagocytosis (see [13] for a review). In non-infectious settings, the general consequence of FcR-mediated cross-presentation is also tolerance induction, except in cases in which self-antigens are complexed with autoantibodies. In this situation, as shown in a model of mouse type II diabetes, cross-presentation of autoimmune complexes enhances activation of effector T cells and breaches tolerance [14]. An alternative mode of antigen entry into cells, potentially favoring immunity over tolerance, is when antigens are conjugated to Toll-like receptor (TLR) ligands. For example, after conjugation of TLR9 and CpG DNA receptor-mediated endocytosis enhances antigen presentation relative to presentation that occurs after normal pinocytosis [15]. Nevertheless, the same enhancement might not necessarily occur with all TLRs and their ligands [16].
Finally, soluble proteins enter cells typically via pinocytosis or macropinocytosis; two processes that are based on plasma membrane ruffling, which allow a cell to sample small or large fluid-phase volumes, respectively [17,18]. Stay on-board or hop into the cytoplasm? Once internalized in an endosomal compartment, the exogenous antigens continue their journey on the cross-presentation pathway. In the cytoplasm, the main protease that is known to produce antigenic fragments is the multisubunit proteasome. In the classical cytosolic MHC I pathway, fragments are then imported into the endoplasmic reticulum (ER) through the transporter associated with antigen processing (TAP) heterodimer. However, does exogenous antigen degradation take place in the intracellular compartment or in the cytoplasm? A large body of evidence supports a requirement for the proteasome and TAP during cross-presentation. Hence, the cytoplasm is seen as the singular location for antigen processing [18– 24]. For many antigens, the material transported from the cytosol into the ER is not the final peptide but a proteolytic intermediate that requires further processing. In fact, excessive proteolysis in the phagosome is detrimental for cross-presentation [25], which might explain why DCs, in 415
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Figure 2. Two models of MHC I cross-presentation. (a) Fusion of maturing phagosomes with the ER leads to material exchange between both compartments (brown double arrow) and results in a hybrid ER–phagosome organelle. ER–phagosomes contain the retrotranslocon factor Sec61 as well as the TAP transporter. Sec61 allows for antigen export to the cytosol where the antigen is degraded. Subsequently, TAP mediates re-import of peptides into the hybrid organelle. The pH needs to stay high enough to avoid excessive degradation of the full-length antigen. It is tightly regulated by NOX2 NADPH oxidase activity and association with lipid bodies. Intermediates might be ultimately trimmed by ERAAP and loaded onto empty MHC I molecules. (b) Alternatively, antigen processing and loading might occur entirely in the vacuole/phagosome. Processing is ensured by endosomal (IRAP) or lysosomal (cathepsin S) proteases.
particular CD8a+ DCs, are superior to macrophages in cross-presentation. It has been reported that excessive acidification of the phagolysosome in DCs is prevented through production of oxygen species by the NADPH oxidase NOX2 [26], and that this process occurs preferentially in the CD8a+ DC subset and requires the Rac2 small GTPase [27]. Phagosomal pH is also regulated by an additional mechanism [28]. Storage organelles for lipids accumulated and associated with phagosomes in a manner dependent on IGTP, an IFN-g-inducible ER-resident GTPase that is important for resistance against intracellular pathogens [29]. In a model using ovalbumin (OVA)coated latex beads as a model particulate antigen, absence of IGTP has been found to influence cross-presentation. The mechanistic details and the relevance of this pathway during infection by an intracellular pathogen remain unclear. Alternatively, in some circumstances, the final peptides can be generated in the phagosome itself (Figure 2b). This is the case when the OVA protein is incorporated into microspheres of a biodegradable polymer [poly(lactic-coglycolic) acid]. Presentation of OVA is then TAP- and proteasome-independent, but requires the lysosomal pro416
tease cathepsin S [30]. Another non-cytosolic mechanism involves the insulin-regulated aminopeptidase (IRAP), an endosomal protease found in muscle, fat, mast cells and DCs [31]. IRAP has been shown to be involved in processing OVA epitopes within DC endosomes [32]. Further investigation should clarify the potential role of IRAP in processing antigens from intracellular pathogens. In conclusion, antigens destined for cross-presentation are best preserved in compartments with low acidity. Although the final peptides are occasionally generated by local proteases, the most common route for the antigenic precursors is to enter the cytosol and serve as a substrate for the proteasome. En route to the cytosol How do antigens located in the endosome/phagosome enter the cytoplasm? This topological conundrum was first noted in macrophages, in which, after phagocytosis, a ribosomal inactivating protein inhibits protein synthesis, which suggested an exit pathway to the cytosol [19]. Further studies have proposed that, compared with macrophages, the endosome-to-cytosol transport might be more potent in DCs [33]. Using antigen-coated latex beads, certain ER proteins
Review have been observed to be recruited to the phagosome, for example, Sec61 [22,23] (Figure 2a). Sec61 is one component of the retrotranslocation channel, which exports misfolded proteins from the ER to the cytosol for degradation by the ubiquitin–proteasome system, by a process known as ERassociated degradation (ERAD). Two studies have used siRNA-mediated or chemical inhibition of retrotranslocation to confirm the functional role of the retrotranslocon in cross-presentation, and have shown that the p97 ATPase, another essential factor for ERAD, is necessary for export of antigens from purified phagosomes [34,35]. The detection of ER proteins on phagosomal membranes has prompted the idea that uptake of particulate antigens occurs through regulated ER/phagosome fusion, thereby creating a new hybrid compartment, self-sufficient for internalization of cytoplasmic antigenic fragments through TAP transport and subsequent MHC I loading [22,23] (Figure 2a). Although this model is controversial [36], it remains attractive because ultrastructural evidence supports the continuity and material exchange between the ER and the phagosome/vacuole during infection by intracellular pathogens like Leishmania [37], Toxoplasma gondii [38] and Chlamydia [39]. Does entry of soluble antigens occur in a similar fashion? By tracking the fate of soluble b2 microglobulin taken up by DCs, it has been found that this protein functionally interacts with nascent MHC I heavy chains in the ER, which suggests that retrograde transport of soluble antigens into the ER can occur [40]. Recently, an alternate model for soluble antigen processing, reminiscent of the ER–phagosome compartment, has been proposed, whereby soluble antigen-containing endosomes are equipped with TAP and MHC I loading capacity and become competent for antigen presentation [41]. Relocation of TAP to endosomes is enhanced by TLR4 ligands, which suggests that spatial separation between MHC I presentation of exogenous versus endogenous antigens is regulated by signals from the innate immune system. Putting on the final touch N-terminal trimming of antigenic intermediates by the ER aminopeptidase associated with antigen processing (ERAAP) has a major impact on the repertoire of endogenously synthesized pMHC I [42,43]. However, is ERAAP also involved in the processing of exogenous antigens? ERAAP is not involved in cross-presentation of soluble OVA by DCs [44,45], which perhaps reflects a more prominent role for the IRAP aminopeptidase in N-terminal trimming [32]. However, ERAAP is necessary for crosspresentation of immune complexes or particulate antigens by DCs [44,45]. Consistent with these results, ERAAP has been found along with other ER markers in purified latexbead-containing phagosomes [46], which suggests that it is transferred from the ER to the phagosome. Whether it actually exerts its function in the phagosomes or after antigen translocation into the ER remains to be established. In summary, these studies point to a relatively predominant pathway wherein antigenic material is first shuttled from the phagosome/endosome to the cytosol via a mechanism akin to the ERAD pathway. This material is then
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degraded by the proteasome and transported by TAP into the ER, ER–phagosome or ER–endosome hybrid compartments where subsequent trimming by ERAAP can occur. Next, we examine the various exogenous presentation mechanisms following infection by intracellular microorganisms. MHC I presentation of intracellular pathogens How does the intracellular location of the microorganism influence the presentation of the antigenic material? Here, we review well-characterized examples of intracellular pathogens with locations ranging from strictly vacuolar (Leishmania, Chlamydia or T. gondii) to cytosolic (Listeria monocytogenes or Trypanosoma) (Box 1). Notably, CD8+ T cells contribute to protective immunity to each of these pathogens; even those that do not infect professional APCs. The importance of secretion Immunodominant antigens from intracellular non-viral pathogens have consistently been found to be secreted proteins. Strong natural CD8+ responses target the circumsporozoite (CS) protein of Plasmodium berghei [47], the cysteine-rich outer membrane protein (CrpA) of Chlamydia trachomatis [48], the dense granule protein 6 (GRA6) of T. gondii [49], the lysteriolysin of L. monocytogenes [50] and the culture-filtrate protein 10 of Mycobacterium tuberculosis Box 1. Intracellular pathogens analyzed in this review At one end of the spectrum, some pathogens establish their niche in a membrane-bound compartment called vacuole (e.g. Plasmodium spp. and T. gondii), phagosome (e.g. Leishmania spp.) or inclusion (e.g. C. trachomatis) and remain segregated from the cytosol during their entire life cycle. At the other end, some intracellular organisms promptly escape to the cytosol in order to replicate (e.g. Tryp. cruzi and L. monocytogenes). Leishmania spp.: a protozoan kinetoplastid parasite that infects mammals and transmitted into skin by sandfly bite. Macrophages are infected by metacyclic promastigotes before conversion into small ovoid amastigotes. Parasites replicate in the phagosome. Tryp. cruzi: a protozoan kinetoplastid parasite that infects mammals and is transmitted by bite or feces of the blood-sucking assassin bug. Trypomastigotes force entry in vacuoles, escape into the cytosol, convert into amastigotes, and replicate in the cytosol. T. gondii: a protozoan apicomplexan parasite that infects all warmblooded animals by ingestion of contaminated food/soil, or by materno-fetal transmission. Tachyzoites actively infect any nucleated cell, and establish and replicate in a specialized nonfusogenic vacuole. Plasmodium spp.: a protozoan apicomplexan parasite that infects birds, reptiles and mammals, and is transmitted to skin/blood by mosquito bite. During the liver stage, sporozoites migrate from the bite wound to the liver, where they invade hepatocytes and replicate in vacuoles. During the blood stage, parasites reside in a vacuole in red blood cells. C. trachomatis: a Gram-negative bacterium that infects humans by sexual transmission, but mice can be used as a host model. Bacteria (elementary body) infect epithelial cells, convert into reticulate bodies and replicate in vacuoles (also called inclusion). M. tuberculosis: an acid-fast bacterium that infects humans and is transmitted by aerosol from previously infected persons. Mycobacteria infect lung alveolar macrophages, replicate in phagosomes, and potentially translocate to the cytoplasm at later stages. L. monocytogenes: a Gram-positive bacterium that infects a broad range of animals by ingestion of contaminated food. Bacteria infect epithelial or phagocytic cells, escape from vacuoles, and replicate in the cytosol.
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Review [51]; all of which are secreted molecules. This idea is supported by studies that have used genetically engineered microorganisms that express secreted or non-secreted model antigens, and is particularly striking because secreted proteins represent only a minor fraction of the entire proteome of these pathogens [52,53]. For instance, with T. gondii, only tachyzoites that secrete b-galactosidase in the vacuole induce b-galactosidase-specific CD8+ T cells in infected mice [54]. Moreover, priming of OVA-specific TCR-transgenic CD8+ T cells is optimal only with OVA-secreting Leishmania major parasites [55].
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active mechanism mediated by microorganisms and/or the host cell.
Infected versus bystander APCs Another key question about how the host mounts a protective response against intracellular pathogens is whether pathogen-derived material is presented by directly infected or bystander APCs that have ingested debris from surrounding infected cells. Once infected, any type of MHC I+ nucleated cell should be able to present antigens and become a target for killing by effector CD8+ T cells. However, only professional APCs, such as macrophages or DCs, have the capacity to internalize exogenous antigens and prime naı¨ve CD8+ T cell responses. Hence, the answer probably varies with the stage of infection (initial priming versus effector phase) and the tissue examined (peripheral site versus lymph node). For example, without being productively infected by P. berghei sporozoites, DCs can efficiently present an epitope from the CS antigen [56] and prime CS-specific CD8+ T cells in the skin-draining lymph nodes [57]. In this model, uninfected bystander DCs are believed to play a crucial role during the priming phase, whereas the later liver effector phase might be DC-independent [57]. During L. monocytogenes infection, cross-presentation by uninfected professional APCs also seems to be crucial because priming of CD8+ T cells against non-secreted antigens requires uptake of infected neutrophils [58]. Alternatively, in the T. gondii and L. major infection models, the crucial importance of protein secretion by the parasites, along with the reduced or abolished presentation of parasite antigens when dead (heat-killed) parasites are ingested by APCs, suggest that optimal T cell priming needs active infection of DCs by live parasites [59,60]. Moreover, it is likely that during the effector phase of the response to infection, exogenous MHC I presentation occurs mainly in (non-professional) APCs that are sheltering live microorganisms (Figure 3). What then are the mechanisms for exogenous presentation in these actively infected cells?
Active role for microorganisms For cytosolic pathogens that rapidly escape the phagosome, secreted proteins are directly available to the cytosolic MHC I pathway (e.g. the p60 antigen from L. monocytogenes [64]). However for vacuolar pathogens, antigens must first cross the vacuole boundary. What are the mechanisms used to accomplish this tour-de-force? Creation of pores in the vacuolar membrane constitutes one way for the pathogen to allow its antigens into the cytosol. M. tuberculosis-containing vacuoles have been shown to be permeable to fluorescent dextrans of up to 70 kDa [65]. Early secreted antigen target 6 (ESAT6) has been proposed to act as a pore-forming protein and participate in the membrane perforation of Mycobacterium marinum vacuoles [66]. However, it remains to be determined whether ESAT6 mediates access of M. tuberculosis antigens to the MHC I pathway. The vacuole membrane that surrounds T. gondii and Plasmodium falciparum also contains pores [67,68] but the relatively small opening of these ‘‘sieves’’ (<1.4 kDa) is expected to preclude polypeptides larger than 15 amino acids from diffusing, therefore pores are unlikely to be involved in the transfer of antigenic precursors to the cytosol. A second possibility for gaining access to the cytosolic MHC I pathway is by insertion within the vacuole membrane, so that all or part of the antigen is exposed to the cytoplasm, cleaved and released in the cytosol for further processing. This situation might apply to the Chlamydia epitopes from Cap1 [69] and CrpA [48], because these proteins are known transmembrane proteins that are inserted in the membrane that surrounds the inclusion. A third gateway to the cytosol is by active targeting of certain factors to the host cytoplasm. Many proteins from the Plasmodium and T. gondii apicomplexan parasites are found in the cytosol or even the nucleus of infected cells [70–72]. A conserved five-residue motif (RxLxE) called Plasmodium Export Element, or PEXEL [70], has been identified as necessary for the secretion of proteins from the P. falciparum vacuole to the cytoplasm of human erythrocytes. Notably, the CS immunodominant antigen of Plasmodium contains two PEXEL motifs that also mediate export from the parasitophorous vacuole to the cytosol in infected hepatocytes [73]. A possible translocon that mediates export of PEXEL-containing proteins to the cytosol has been recently identified [74] but whether a similar mechanism operates for T. gondii is not known.
Different pathogens, different mechanisms Generation of the pMHC I from intracellular pathogens by an exclusive vacuolar pathway (i.e. proteasome and TAPindependent) appears to be the exception more than the rule. Besides the purely phagosomal pathway used by DCs infected with OVA-expressing L. major parasites [55], data from several other infectious models (T. gondii [49,55], P. berghei [61], L. monocytogenes [24], M. tuberculosis [62] and C. trachomatis [63]) indicate that pMHC I generation requires the proteasome and TAP transport. Thus, entry of microbial material into the host cytosol requires an
Active role for the host cell Some host defense mechanisms have been shown to damage the phagosome/vacuole membrane and can therefore facilitate the release of antigens in the cytoplasm. A family of IFN-g-inducible proteins (IRGs for immunity-related GTPases, formerly known as p47 GTPases) is suspected to be involved in this process. IRGs are essential for resistance of mice to a variety of intracellular bacteria and parasites (reviewed in [75]). In the case of T. gondii, they participate in the destruction of the parasitophorous vacuole membrane [76], thereby exposing the parasite to the
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Figure 3. Exogenous MHC I presentation of intracellular pathogens. (a) In the vacuolar pathway, antigens are degraded and loaded on MHC I molecules within the phagosome/vacuole itself (e.g. L. major). (b–g) In the TAP-dependent cytosolic pathway, the antigen gains access to the cytosol where it is processed primarily by the proteasome. (b) Secreted proteins from organisms living in the cytosol (e.g. Tryp. cruzi or L. monocytogenes) directly end up in the cytosol. For organisms living in a vacuole, several exit pathways to the cytoplasm have been proposed. (c) A secreted factor might form pores in the vacuole membrane, which allow for subsequent diffusion of antigen (e.g. M. tuberculosis). (d) An active transport mechanism that potentially involves recognition of specific motifs and/or specific translocation machinery might shuttle the antigen to the cytosol (e.g. Plasmodium). (e) Host innate defense proteins such as IRGs might disrupt the vacuole membrane and release antigens into the cytosol (e.g. T. gondii). (f) In the case of a transmembrane protein, the antigen might be inserted into the vacuole membrane so that the proteasome can process the cytosolic side of the antigen (e.g. C. trachomatis). (g) Finally, fusion of the vacuole with the host ER might deliver the antigen to the Sec61-based retrotranslocation machinery that shuttles it to the cytoplasm (e.g. T. gondii). Antigenic peptide precursors are then imported into the ER for possible trimming by ERAAP and final MHC I loading.
inhospitable cytoplasmic environment. Altered levels of OVA presentation have been reported in Irgm3-deficient macrophages infected with OVA-secreting parasites [77], which implicate this IRG protein in MHC I processing. This same IRG protein has been implicated in the crosspresentation of OVA-coated latex beads via lipid bodies (see above and Figure 2a), but it is unclear if similar mechanisms are involved in both cases. Surprisingly, a presentation defect has not been observed in Irgm3-deficient DCs [77]. To date, IRGs have also been shown to participate in host resistance to M. tuberculosis [78,79], C. trachomatis [80] and Trypanosoma cruzi [81] but whether they mediate access of antigens to the MHC I pathway in these models remains to be investigated. An ER–phagosome pathway? Another pathway has been reported that is reminiscent of the ER–phagosome model already described. Sec61 has been found to have a role in MHC I presentation of OVA by T. gondii-infected DCs [38], which supports the notion that fusion between the parasitophorous vacuole and the host
ER allows antigen access to the ER, cytosol export through the retrotranslocation channel, and TAP-mediated re-import in the ER. A possible shortcoming of this model is that OVA trafficking might differ from that of natural parasite antigens. For instance, as opposed to a soluble protein like OVA, the natural T. gondii antigens GRA6 [49] and GRA4 [82] are known to associate with membranes within the vacuole, which could limit access to the ER. Hence, it is important to determine whether this appealing mechanism applies to natural T. gondii antigens as well. Concluding remarks In summary, although antigens from intravacuolar pathogens are segregated from the cytoplasm, they do enter the cytosolic MHC I pathway through a variety of ways that all have the same, paramount, downstream consequence: stimulation of a CD8+ T cell response and potential control of infection. Perhaps because each pathogen has evolved different strategies to interact with host cells, exogenous presentation of intracellular pathogens is not one but several distinct pathways. 419
Review Intriguingly, as pathogens have evolved strategies to interfere with direct, cytosolic MHC I presentation [9], pathogens also try to trick their host by dampening cross-presentation. For instance, late antigens from the vaccinia virus seem to be sequestered in virus factories, which makes them inaccessible to the cross-presentation machinery [83]. Alternatively, M. tuberculosis has evolved specific molecules to block macrophage apoptosis, thereby preventing uptake of its antigens by APCs and subsequently dampening CD8+ T cell responses [84,85]. It will be essential to delineate further how intracellular pathogens interfere with cross-presentation, which should suggest ways to counteract immune evasion mechanisms and improve vaccine efficacy.
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Acknowledgements N.B. was supported by a long-term postdoctoral fellowship from the Human Frontier Science Program Organization. N.S. was partly supported by a Lord Harris Senior Research Fellowship at the Harris Manchester College and the Weatherall Institute of Molecular Medicine at the University of Oxford, UK. Research in the N.S. laboratory is supported by grants (AI060040, AI044864, AI39548, AI065831) from the National Institutes of Health.
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