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The source of MHC class I presented peptides and its implications
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ScienceDirect The source of MHC class I presented peptides and its implications Se´bastien Apcher1, Rodrigo Prado Martins2 and Robin Fa˚hraeus2,3,4 The source of peptides that enter the major histocompatibility class I (MHCI) pathway has been intensively debated over the last two decades. The initial assumption that peptides are derived from degradation of full length proteins was challenged by a model in which alternative translation products are a source of peptides. This model has been tested and supported by scientific data. We now need new hypotheses on the physiological implications of different sources of peptides for the MHCI pathway. The aim of this overview is to give an up-todate account of the source of antigenic peptide material for the MHCI pathway and to incorporate the more recent observations of alternative mRNA translation products into existing models of the direct and cross-presentation pathways. Addresses 1 Institut Gustave Roussy, Universite´ Paris Sud, Unite´ 1015 de´partement d’immunologie, 114, rue Edouard Vaillant, 94805 Villejuif, France 2 Equipe Labellise´e la Ligue Contre le Cancer, Inserm UMR1162, Universite´ Paris 7, Institut de Ge´ne´tique Mole´culaire, 27 rue Juliette Dodu, 75010 Paris, France 3 RECAMO, Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53 Brno, Czech Republic 4 Department of Medical Biosciences, Umea˚ University, SE-90185 Umea˚, Sweden Corresponding author: Fa˚hraeus, Robin ([email protected])
Current Opinion in Immunology 2016, 40:117–122
of functional ‘‘old’’ full length proteins and instead should originate from an alternative source of peptides [1]. This theory has since been supported by a number of publications from different teams using different approaches and AP derived from cryptic and non-conventional translational products including 50 and 30 untranslated regions, intron, intron/exon junctions, have been discovered [2,3,4–6]. The question now is not so much if alternative translation products is a source for the class I pathway but how, when and for what purposes they are used? Current models on cross-presentation (cross-priming) of MHCI peptides are to a large extent based on work focused on the professional antigen presenting cells (pAPCs) and less attention has been given to the producing cells and, for that matter, to the source of peptides [7]. But if alternative sources of peptides are used for the detection of non-self antigens by CD8+ T cells via the direct presentation pathway, one could argue that the same sources of peptide material should also be used to activate the CD8+ T cells by cross-presentation. We will reconcile more recent reports on alternative sources of peptide material for the direct MHCI pathway with existing models on cross-presentation of peptides by pAPCs and how this puts the focus on the peptideproducing cells.
This review comes from a themed issue on Antigen processing Edited by Laurence C Eisenlohr and Christopher C Norbury
http://dx.doi.org/10.1016/j.coi.2016.04.002 0952-7915/# 2016 Elsevier Ltd. All rights reserved.
Introduction The detection of intron-derived peptides on MHCI molecules in the 1990s raised questions regarding the source of antigenic peptide (AP) for the MHCI pathway that the prevailing assumption at the time of full length proteins being the source of AP material could not explain. There was also the issue of the poor correlation between protein turnover rate vs antigen presentation. The latter formed a basis for Jon Yewdell’s and Jack Bennick team’s hypothesis that the source of AP for the class I pathway is unlikely to come from degradation www.sciencedirect.com
Antigen presentation is a fundamental biological process and the advancements in the immune system’s capacity to cope with parasites, or other threats to the host, have paved the way for species to climb up the evolution ladder. Hence, class I-restricted antigen presentation has become intimately linked with various intra-cellular and extra-cellular processes and it is reasonable to assume that the source of peptide material has co-evolved with other aspects of the endogenous and exogenous MHCI presentation pathways. Why the source of peptides forms a special challenge for the immune system is reflected in the fact that an estimated 105 class I molecules [8] with an approximately 24 hours turnover rate shall give a fair but sensitive picture the status of the cells. This gives a rough estimate of about 4000 new peptides can be loaded on class I molecules every hour. How is it possible to generate a sensitive system to detect infected or damaged cells under such conditions? Considering the vast amount of peptides being derived from the degradation of full length proteins or alternative translation products in the cells every hour, it implies that for both the direct and the cross-presented pathways there should be a selection of Current Opinion in Immunology 2016, 40:117–122
118 Antigen processing
Figure 1
Professional APC Effector CD8+ T-cell
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Antigenic peptides producing cell MHCI pathway mRNAs translation eIF4E
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Current Opinion in Immunology
Model on the sources of antigenic peptides for the MHCI pathway. Whereas degradation products from full-length proteins do not reach the MHCI pathway, alternative translation products such as pioneer translation products (PTPs) and defective ribosomal products (DRiPs) are selected for direct presentation [1,10]. Following proteasomal processing they are translocated into the endoplasmic reticulum (ER) via transporter (TAP) where they are further processed and loaded onto MHCI molecules and transported via the Golgi to the cell surface for presentation to CD8+ T-cells. At the same time, full length proteins and/or alternative translation products are transferred to professional antigen-presenting cells (APCs) where they are processed and presented to activate naı¨ve CD8+ T cells.
AP material in order for the immune system to detect non-self epitopes (Figure 1). The concept of alternative mRNA translation products could help to answer some basic questions regarding the immune-system’s capacity to distinguish self from non-self and it raises some hypotheses: 1. Selection of peptide substrates for the direct and crosspresented class I pathways. 2. The same source of peptides for direct and crosspresentation? 3. Cross-talk of peptide material between the class I and class II pathways. Current Opinion in Immunology 2016, 40:117–122
4. Regulation of alternative translation product synthesis. 5. Presentation of AP from non-viral pathogens.
The selection
The large discrepancy between the number of potential AP substrates derived from proteasomal degradation of peptide substrates and the number of available class I molecules suggests that the peptide material for presentation to the MHC class I pathway is selected. The fit of the peptides to the class I molecule provides one selection step [9] but it is conceivable that there are one, or more, selection steps before the peptides reach that far in www.sciencedirect.com
MHC class I peptides Apcher, Prado Martins and Fa˚hraeus 119
the class I processing pathway. The TAP transporter has a broad peptide specificity and viral evasion of the MHC class I pathway targets TAP activity in general, not a selection of peptides transported. Instead, it is possible that an important selection step lies in the source of peptides. In support of this, data from controlled expression systems, either on mRNA synthesis or full length protein degradation, support the notion that degradation products from full length proteins do not reach the direct class I pathway [10,11]. Instead, alternative sources of peptides derived from a specific mRNA translation event preceding the production of full length proteins, so-called pioneer translation products (PTPs), are efficiently selected for the direct class I pathway [10,12]. mRNAs undergo quality control before they are allowed to produce full length proteins. A well-described mechanism relates to the detection of premature stop codons that are detected by the pioneer round of translation [13,14]. However, all mRNAs, spliced, or not, undergo a pioneer translation event and transfection of mRNAs shows that MHC class I peptides are produced early and stops after a couple of hours while the production of full length proteins continues as long as the mRNAs stay intact [10]. As there is approximately one mRNA for every 3000 proteins [15] the selective use of PTP products would mean a sharp reduction in the number of potential peptide substrates that can enter the class I pathway could help address the number enigma. However, more recent works show that also intron-derived APs are equally well produced from prespliced mRNAs as compared to exon-derived products [16]. While this explains the presence of intron-derived peptides on class I molecules of tumor cells and sheds light on how the immune system generates tolerance to alternative spicing products, it also implies that the number of potential peptide products for the class I pathway remains incomprehensible high. Hence, the question of peptide selection remains unsolved but is, nevertheless, most relevant. On a more speculative note, one can make a simple thought experiment that sometime during evolution there were two predecessors, one that could distinguish mRNAs derived from self and non-self genomes and one that could not. If the first creature would selectively produce AP from non-self RNAs it would have a selective advantage when it came to fight parasites. It is tempting to speculate that the chances for this to have happened are quite high. Hence, not only the translation event that produces the AP might be of importance but also the source of the genomic material. If this is the case, it could offer a solution to the number enigma and how the MHCI pathway is sensitive to viral infection. Another potential danger signal for the host is proliferating cells, both in terms of infected and transformed cells and it will be interesting to better understand if non-self genomes and www.sciencedirect.com
cell proliferation affects the production and presentation of MHCI peptides.
The same peptide source for direct and crosspresentation?
The fact that alternative peptide substrates indeed are a source for antigen presentation puts the limelight for cross-presentation on the producing cells. If there is a selection of peptide material for the direct pathway then there should be a similar selection for cross-presented material. Treating producing cells with proteasome inhibitors increases cross-presentation and has led to the suggestion that long lived proteins is the source of peptide material for cross presentation. Similarly, it was shown that APs are better cross-presented when inserted in a stable context [17,18]. On the other hand, direct delivery of proteins to pAPCs in the form of free proteins, or protein-coated beads, is an inefficient affair [19]. Hence, if full length proteins indeed are a source for crosspresentation, then the context of how the proteins are delivered must also play a role. If one argues against full length protein being a source for direct presentation, the same arguments should also hold true for cross-presentation. If the number of potential peptide products poses a problem for the sensitivity of the direct pathway, there is little saying that this would not also be an issue for cross-presentation? If full length proteins are used for cross-presentation, the vast majority of processed peptides would come from the most abundant cellular sources such as ribosome, proteasome or chaperones. Furthermore, if PTPs helps early detection of virus-infected cells why would the immune system not use the same products for cross-presentation? If not, this would run the risk that T cells are activated towards peptides from full length proteins and not against the same peptide products used by the direct pathway. Such discrepancy would be exploited by viruses and could not be tolerated. However, if PTPs, or other translation products, are used for cross-presentation then, according to previous studies, they should be stable. PTPs for direct presentation are not stable and, hence, if PTPs are indeed used for cross-presentation, the pathways for direct and cross-presentation must diverge in the producing cells. The uptake of peptides from the cellular environment by the pAPCs shall also provide material for the MHCII pathway. The processing of peptide substrates for these two pathways is very different and it is not yet clear how the pAPCs can distinguish between full length proteins going to the class I or the class II pathways to ensure that the peptides end up in the right compartments. It shall be interesting to use an 26S proteasome inducible degradation model of full length proteins that does not result in an increase in direct MHCI presentation [10] to see what happens in terms of cross-presentation. Current Opinion in Immunology 2016, 40:117–122
120 Antigen processing
Cross-talk of peptide material for the class I and class II pathways
Having several sources of peptides for the MHCI pathway has also implications for the MHC class II pathway. While the substrates for both pathways were considered the same there was no need to address this issue. But as they are not, this aspect is also worth some thoughts. By convention, while MHCI-restricted epitopes require endogenous production and processing, it was thought that MHC class II-restricted epitopes are generated from exogenous antigens within the endocytic compartment. In the late 80s, it became apparent that non-classical pathways are also used to process MHC class II antigen presentation and that cytosolic proteins can be endogenously produced, processed and presented by the MHC class II pathway in an MHCI-like fashion [20]. Tewary et al. reported that an antigenic epitope produced from the Neuraminidase (NA) glycoprotein encoded by the A/PR/ 8/34 (PR8) influenza virus, was proteasome and TAP
dependent [21]. More recently, the same group reported that other epitopes from the same viral protein can also be endogenously produced and presented to the MHC class II pathway [22]. Hence, processing of substrates for one, or the other, pathways might not be ‘‘black and white’’, fuelling the idea that cells might have the choice in which pathway a peptide product ends up. Regulation of PTP production and delivery
Another aspect of having alternative translation events for the production of AP and full length proteins is that they could be regulated separately. The infection of a viral particle, activation of Toll-like receptors or the presence of signaling substances in the cellular environment are all events that potentially could help the immune system to detect infected cells by increasing the production of alternative translation products. The downside of coupling the production of AP substrates with signaling pathways is that it would offer viruses targets to evade
Figure 2
Bacterial invasion ? D
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Antigen presentation via MHCI during bacterial infection. Upon cell invasion, bacteria can reside in vacuoles or free in the cytosol. In vacuoles, pathogens can be processed for production of peptides that are translocated to cytoplasm via Sec61, degraded by the proteasome and subsequently follow the MHCI-classic pathway [25] (a, yellow arrows). Alternatively, bacteria can escape from these membrane-bound compartments and once in the cytosol, they can be recognized by the ubiquitination system and then be tagged for proteosomal degradation [27] (b) or be captured by autophagy (c, green arrows). In the latter case, bacteria-containing autophagosomes are fused to endosomes to form amphisomes, where antigens are preprocessed by cathepsins and transferred to the cytosol via Sec61. After being degraded by the proteasome, antigenic peptides are reimported into the amphisome by transporter associated with antigen presentation (TAP), loaded primarily on recycled MHCI and presented on the cell surface [26]. Although some pathogens like Listeria are able to replicate in the cytoplasm avoiding the described mechanisms, host cell is able to produce antigenic peptides to control infection through a pathway yet not well defined [28,29] (d). Blue and red dots denote peptides before and after proteasomal degradation, respectively. ER: endoplasmic reticulum. Red, yellow and green bacteria represent Salmonella, Chlamydia and Listeria, respectively. Current Opinion in Immunology 2016, 40:117–122
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MHC class I peptides Apcher, Prado Martins and Fa˚hraeus 121
antigen presentation. In the case of the Epstein-Barr virus-encoded EBNA1 and the LANA1 of the Kaposi sarcoma virus these proteins harbor cis-acting mechanisms to suppress the synthesis of their own mRNA and thereby keep the production of alternative translation products at a minimum while at the same time the proteins have a low turnover rate to ensure they are expressed at functional levels [23,24]. Further studies on virus-dependent evasion of the class I pathway are bound to discover cellular target factors that either prove, or disprove, the hypothesis that the cells have the capacity to regulate synthesis of alternative translation products for the class I pathway. Non-viral infection
What has been discussed so far relates to the translation machinery of the host cells. But what about pathogens that bring their own translation machinery? MHCI presentation plays a major role in immunity to intracellular bacteria like Salmonella and Chlamydia. After entry into host cells, these bacteria reside in membrane-bound compartments which do not intersect with compartments of classical MHC I pathway, denoting that alternative mechanisms are adopted to elicit cytotoxic T cells. Previous evidences indicate that processing of Salmonella antigens might involve the translocation of partial processed peptides from the Salmonella-containing vacuoles to the cytosol, followed by their proteosomal degradation and subsequent ER translocation [25]. Fiegl et al. proposed a new alternative cross-presentation model in which autophagy promotes proteasome/TAP-dependent MHC I processing of peptides from pathogens replicating in vacuoles [26]. Moreover, Salmonella can escape invasion vacuoles being released in the cytosol of infected macrophages and recognized by the ubiquitin system. This mechanism results in spatial association of proteasomes and bacteria that is expected to provide a high local concentration of substrates for degradation and could represent a link between intracellular bacteria, bacteria protein translation and antigen presentation [27]. Interestingly, a strong CD8+ T-cell response is observed upon infection by Listeria monocytogenes, in spite of the sophisticated strategies adopted by this pathogen to avoid the recognition by the ubiquitin system. In fact, processing of AP from Listeria is tightly connected to de novo bacterial protein synthesis and presentation of these peptides by MHCI is independent of the cellular half-life of the protein from which they were derived. This shares similarities to the PTP production by the host ribosome. Of note, is has been suggested that Listeria APs are generated with higher efficacy than virus-derived AP [28,29] (Figure 2). However, the identification of bacterial and host effectors involved in producing bacteria-derived AP material for the direct and cross-presentation pathways remain to be explored and could open new horizons in the studies of antigen cross-presentation in the context of non-viral infections and a better understanding of host– parasite interactions. www.sciencedirect.com
Conclusions Since the idea of alternative sources for peptides for the MHC class I pathway was first put forward, new models emerging from this concept are starting to appear that address fundamental biological questions that can help to design more effective vaccines. For example, it is long known that peptides derived from introns are presented on the class I molecules on tumor cells and we now start to understand the underlying molecular mechanisms. However, questions remain to whether/how the source of these peptide products also leads to an activation of CD8+ T cells and, hence, if they are cross-presented. Having different sources of peptides for the MHC class I pathway, produced from different events raises the question if cells have the capacity to selectively control the production of antigenic peptides material in response to stimuli such as viral infection? This would provide a selective advantage in the fight against parasites. These are just a couple of questions that hopefully will find their answers in the not too long future and that will reveal interesting cell biology as well as practical implications for better directing the immune system towards targets of our choices.
Acknowledgments This work was supported by LNCC Equipe Labellise´e, Inserm and by the project MEYS – NPS I – LO1413. AP is AVENIR/ATIP fellow.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Yewdell JW, Anton LC, Bennink JR: Defective ribosomal products (DRiPs): a major source of antigenic peptides for MHC class I molecules? J Immunol 1996, 157:1823-1826.
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Coulie PG, Lehmann F, Lethe B, Herman J, Lurquin C, Andrawiss M, Boon T: A mutated intron sequence codes for an antigenic peptide recognized by cytolytic T lymphocytes on a human melanoma. Proc Natl Acad Sci U S A 1995, 92:7976-7980. This report, along with Ref [3], demonstrate intron-derived peptides on MHC class I molecules.
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Guilloux Y, Lucas S, Brichard VG, Van Pel A, Viret C, De Plaen E, Brasseur F, Lethe B, Jotereau F, Boon T: A peptide recognized by human cytolytic T lymphocytes on HLA-A2 melanomas is encoded by an intron sequence of the Nacetylglucosaminyltransferase V gene. J Exp Med 1996, 183:1173-1183. See annotation to Ref [2].
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Malarkannan S, Horng T, Shih PP, Schwab S, Shastri N: Presentation of out-of-frame peptide/MHC class I complexes by a novel translation initiation mechanism. Immunity 1999, 10:681-690.
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Uenaka A, Ono T, Akisawa T, Wada H, Yasuda T, Nakayama E: Identification of a unique antigen peptide pRL1 on BALB/c RL male 1 leukemia recognized by cytotoxic T lymphocytes and its relation to the Akt oncogene. J Exp Med 1994, 180:15991607.
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Shastri N, Schwab S, Serwold T: Producing nature’s gene-chips: the generation of peptides for display by MHC class I molecules. Annu Rev Immunol 2002, 20:463-493.
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Yewdell JW: DRiPs solidify: progress in understanding endogenous MHC class I antigen processing. Trends Immunol 2011, 32:548-558.
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Catipovic B, Dal Porto J, Mage M, Johansen TE, Schneck JP: Major histocompatibility complex conformational epitopes are peptide specific. J Exp Med 1992, 176:1611-1618.
10. Apcher S, Daskalogianni C, Lejeune F, Manoury B, Imhoos G, Heslop L, Fahraeus R: Major source of antigenic peptides for the MHC class I pathway is produced during the pioneer round of mRNA translation. Proc Natl Acad Sci U S A 2011, 108:1157211577. 11. Khan S, de Giuli R, Schmidtke G, Bruns M, Buchmeier M, van den Broek M, Groettrup M: Cutting edge: neosynthesis is required for the presentation of a T cell epitope from a long-lived viral protein. J Immunol 2001, 167:4801-4804. 12. Apcher S, Manoury B, Fahraeus R: The role of mRNA translation in direct MHC class I antigen presentation. Curr Opin Immunol 2012, 24:71-76. 13. Ishigaki Y, Li X, Serin G, Maquat LE: Evidence for a pioneer round of mRNA translation: mRNAs subject to nonsense-mediated decay in mammalian cells are bound by CBP80 and CBP20. Cell 2001, 106:607-617. 14. Maquat LE, Tarn WY, Isken O: The pioneer round of translation: features and functions. Cell 2010, 142:368-374. 15. Schwanhausser B, Busse D, Li N, Dittmar G, Schuchhardt J, Wolf J, Chen W, Selbach M: Global quantification of mammalian gene expression control. Nature 2011, 473:337-342. 16. Apcher S, Millot G, Daskalogianni C, Scherl A, Manoury B, Fahraeus R: Translation of pre-spliced RNAs in the nuclear compartment generates peptides for the MHC class I pathway. Proc Natl Acad Sci U S A 2013, 110:17951-17956. Authors report that ribosomal scanning of prespliced mRNAs occurs in the nuclear compartment and this event produces peptide substrates for the MHC class I pathway. This finding sheds light on a unique mRNA translation event and raises the possibility that different ribosomes produce peptide with different functions. 17. Norbury CC, Basta S, Donohue KB, Tscharke DC, Princiotta MF, Berglund P, Gibbs J, Bennink JR, Yewdell JW: CD8+ T cell crosspriming via transfer of proteasome substrates. Science 2004, 304:1318-1321. 18. Wolkers MC, Brouwenstijn N, Bakker AH, Toebes M, Schumacher TN: Antigen bias in T cell cross-priming. Science 2004, 304:1314-1317. 19. Zinkernagel RM: On the role of dendritic cells versus other cells in inducing protective CD8+ T cell responses. Front Immunol 2014, 5:30. 20. Eisenlohr LC, Hackett CJ: Class II major histocompatibility complex-restricted T cells specific for a virion structural
Current Opinion in Immunology 2016, 40:117–122
protein that do not recognize exogenous influenza virus. Evidence that presentation of labile T cell determinants is favored by endogenous antigen synthesis. J Exp Med 1989, 169:921-931. 21. Tewari MK, Sinnathamby G, Rajagopal D, Eisenlohr LC: A cytosolic pathway for MHC class II-restricted antigen processing that is proteasome and TAP dependent. Nat Immunol 2005, 6:287-294. 22. Miller MA, Ganesan AP, Luckashenak N, Mendonca M, Eisenlohr LC: Endogenous antigen processing drives the primary CD4(+) T cell response to influenza. Nat Med 2015, 21:1216-1222. Using a mouse infection model, the authors show that the CD4+ T cell response to influenza is driven principally by unconventional processing of antigens synthesized within the infected antigen-presenting cell, not by classical processing of endocytosed virions or material from the infected cells. 23. Apcher S, Daskalogianni C, Manoury B, Fahraeus R: Epstein Barr virus-encoded EBNA1 interference with MHC class I antigen presentation reveals a close correlation between mRNA translation initiation and antigen presentation. PLoS Pathog 2010, 6:e1001151. 24. Kwun HJ, da Silva SR, Shah IM, Blake N, Moore PS, Chang Y: Kaposi’s sarcoma-associated herpesvirus latencyassociated nuclear antigen 1 mimics Epstein–Barr virus EBNA1 immune evasion through central repeat domain effects on protein processing. J Virol 2007, 81:8225-8235. 25. Lopez-Medina M, Perez-Lopez A, Alpuche-Aranda C, OrtizNavarrete V: Salmonella modulates B cell biology to evade CD8(+) T cell-mediated immune responses. Front Immunol 2014, 5:586. 26. Fiegl D, Kagebein D, Liebler-Tenorio EM, Weisser T, Sens M, Gutjahr M, Knittler MR: Amphisomal route of MHC class I crosspresentation in bacteria-infected dendritic cells. J Immunol 2013, 190:2791-2806. 27. Perrin AJ, Jiang X, Birmingham CL, So NS, Brumell JH: Recognition of bacteria in the cytosol of mammalian cells by the ubiquitin system. Curr Biol 2004, 14:806-811. 28. Grauling-Halama S, Schenk S, Bubert A, Geginat G: Linkage of bacterial protein synthesis and presentation of MHC class Irestricted Listeria monocytogenes-derived antigenic peptides. PLoS ONE 2012, 7:e33335. 29. Wolf BJ, Princiotta MF: Processing of recombinant Listeria monocytogenes proteins for MHC class I presentation follows a dedicated, high-efficiency pathway. J Immunol 2013, 190:2501-2509. This report demonstrates that proteins secreted from Listeria are processed for MHC class I presentation via a high efficiency processing pathway that is restricted to a subset of endogenously synthesized proteins.
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ScienceDirect The source of MHC class I presented peptides and its implications Se´bastien Apcher1, Rodrigo Prado Martins2 and Robin Fa˚hraeus2,3,4 The source of peptides that enter the major histocompatibility class I (MHCI) pathway has been intensively debated over the last two decades. The initial assumption that peptides are derived from degradation of full length proteins was challenged by a model in which alternative translation products are a source of peptides. This model has been tested and supported by scientific data. We now need new hypotheses on the physiological implications of different sources of peptides for the MHCI pathway. The aim of this overview is to give an up-todate account of the source of antigenic peptide material for the MHCI pathway and to incorporate the more recent observations of alternative mRNA translation products into existing models of the direct and cross-presentation pathways. Addresses 1 Institut Gustave Roussy, Universite´ Paris Sud, Unite´ 1015 de´partement d’immunologie, 114, rue Edouard Vaillant, 94805 Villejuif, France 2 Equipe Labellise´e la Ligue Contre le Cancer, Inserm UMR1162, Universite´ Paris 7, Institut de Ge´ne´tique Mole´culaire, 27 rue Juliette Dodu, 75010 Paris, France 3 RECAMO, Masaryk Memorial Cancer Institute, Zluty kopec 7, 656 53 Brno, Czech Republic 4 Department of Medical Biosciences, Umea˚ University, SE-90185 Umea˚, Sweden Corresponding author: Fa˚hraeus, Robin ([email protected])
Current Opinion in Immunology 2016, 40:117–122
of functional ‘‘old’’ full length proteins and instead should originate from an alternative source of peptides [1]. This theory has since been supported by a number of publications from different teams using different approaches and AP derived from cryptic and non-conventional translational products including 50 and 30 untranslated regions, intron, intron/exon junctions, have been discovered [2,3,4–6]. The question now is not so much if alternative translation products is a source for the class I pathway but how, when and for what purposes they are used? Current models on cross-presentation (cross-priming) of MHCI peptides are to a large extent based on work focused on the professional antigen presenting cells (pAPCs) and less attention has been given to the producing cells and, for that matter, to the source of peptides [7]. But if alternative sources of peptides are used for the detection of non-self antigens by CD8+ T cells via the direct presentation pathway, one could argue that the same sources of peptide material should also be used to activate the CD8+ T cells by cross-presentation. We will reconcile more recent reports on alternative sources of peptide material for the direct MHCI pathway with existing models on cross-presentation of peptides by pAPCs and how this puts the focus on the peptideproducing cells.
This review comes from a themed issue on Antigen processing Edited by Laurence C Eisenlohr and Christopher C Norbury
http://dx.doi.org/10.1016/j.coi.2016.04.002 0952-7915/# 2016 Elsevier Ltd. All rights reserved.
Introduction The detection of intron-derived peptides on MHCI molecules in the 1990s raised questions regarding the source of antigenic peptide (AP) for the MHCI pathway that the prevailing assumption at the time of full length proteins being the source of AP material could not explain. There was also the issue of the poor correlation between protein turnover rate vs antigen presentation. The latter formed a basis for Jon Yewdell’s and Jack Bennick team’s hypothesis that the source of AP for the class I pathway is unlikely to come from degradation www.sciencedirect.com
Antigen presentation is a fundamental biological process and the advancements in the immune system’s capacity to cope with parasites, or other threats to the host, have paved the way for species to climb up the evolution ladder. Hence, class I-restricted antigen presentation has become intimately linked with various intra-cellular and extra-cellular processes and it is reasonable to assume that the source of peptide material has co-evolved with other aspects of the endogenous and exogenous MHCI presentation pathways. Why the source of peptides forms a special challenge for the immune system is reflected in the fact that an estimated 105 class I molecules [8] with an approximately 24 hours turnover rate shall give a fair but sensitive picture the status of the cells. This gives a rough estimate of about 4000 new peptides can be loaded on class I molecules every hour. How is it possible to generate a sensitive system to detect infected or damaged cells under such conditions? Considering the vast amount of peptides being derived from the degradation of full length proteins or alternative translation products in the cells every hour, it implies that for both the direct and the cross-presented pathways there should be a selection of Current Opinion in Immunology 2016, 40:117–122
118 Antigen processing
Figure 1
Professional APC Effector CD8+ T-cell
Naïve CD8+ T-cell
Antigenic peptides producing cell MHCI pathway mRNAs translation eIF4E
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Proteasome
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Cytoplasm Non-spliced RNAs translation
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Current Opinion in Immunology
Model on the sources of antigenic peptides for the MHCI pathway. Whereas degradation products from full-length proteins do not reach the MHCI pathway, alternative translation products such as pioneer translation products (PTPs) and defective ribosomal products (DRiPs) are selected for direct presentation [1,10]. Following proteasomal processing they are translocated into the endoplasmic reticulum (ER) via transporter (TAP) where they are further processed and loaded onto MHCI molecules and transported via the Golgi to the cell surface for presentation to CD8+ T-cells. At the same time, full length proteins and/or alternative translation products are transferred to professional antigen-presenting cells (APCs) where they are processed and presented to activate naı¨ve CD8+ T cells.
AP material in order for the immune system to detect non-self epitopes (Figure 1). The concept of alternative mRNA translation products could help to answer some basic questions regarding the immune-system’s capacity to distinguish self from non-self and it raises some hypotheses: 1. Selection of peptide substrates for the direct and crosspresented class I pathways. 2. The same source of peptides for direct and crosspresentation? 3. Cross-talk of peptide material between the class I and class II pathways. Current Opinion in Immunology 2016, 40:117–122
4. Regulation of alternative translation product synthesis. 5. Presentation of AP from non-viral pathogens.
The selection
The large discrepancy between the number of potential AP substrates derived from proteasomal degradation of peptide substrates and the number of available class I molecules suggests that the peptide material for presentation to the MHC class I pathway is selected. The fit of the peptides to the class I molecule provides one selection step [9] but it is conceivable that there are one, or more, selection steps before the peptides reach that far in www.sciencedirect.com
MHC class I peptides Apcher, Prado Martins and Fa˚hraeus 119
the class I processing pathway. The TAP transporter has a broad peptide specificity and viral evasion of the MHC class I pathway targets TAP activity in general, not a selection of peptides transported. Instead, it is possible that an important selection step lies in the source of peptides. In support of this, data from controlled expression systems, either on mRNA synthesis or full length protein degradation, support the notion that degradation products from full length proteins do not reach the direct class I pathway [10,11]. Instead, alternative sources of peptides derived from a specific mRNA translation event preceding the production of full length proteins, so-called pioneer translation products (PTPs), are efficiently selected for the direct class I pathway [10,12]. mRNAs undergo quality control before they are allowed to produce full length proteins. A well-described mechanism relates to the detection of premature stop codons that are detected by the pioneer round of translation [13,14]. However, all mRNAs, spliced, or not, undergo a pioneer translation event and transfection of mRNAs shows that MHC class I peptides are produced early and stops after a couple of hours while the production of full length proteins continues as long as the mRNAs stay intact [10]. As there is approximately one mRNA for every 3000 proteins [15] the selective use of PTP products would mean a sharp reduction in the number of potential peptide substrates that can enter the class I pathway could help address the number enigma. However, more recent works show that also intron-derived APs are equally well produced from prespliced mRNAs as compared to exon-derived products [16]. While this explains the presence of intron-derived peptides on class I molecules of tumor cells and sheds light on how the immune system generates tolerance to alternative spicing products, it also implies that the number of potential peptide products for the class I pathway remains incomprehensible high. Hence, the question of peptide selection remains unsolved but is, nevertheless, most relevant. On a more speculative note, one can make a simple thought experiment that sometime during evolution there were two predecessors, one that could distinguish mRNAs derived from self and non-self genomes and one that could not. If the first creature would selectively produce AP from non-self RNAs it would have a selective advantage when it came to fight parasites. It is tempting to speculate that the chances for this to have happened are quite high. Hence, not only the translation event that produces the AP might be of importance but also the source of the genomic material. If this is the case, it could offer a solution to the number enigma and how the MHCI pathway is sensitive to viral infection. Another potential danger signal for the host is proliferating cells, both in terms of infected and transformed cells and it will be interesting to better understand if non-self genomes and www.sciencedirect.com
cell proliferation affects the production and presentation of MHCI peptides.
The same peptide source for direct and crosspresentation?
The fact that alternative peptide substrates indeed are a source for antigen presentation puts the limelight for cross-presentation on the producing cells. If there is a selection of peptide material for the direct pathway then there should be a similar selection for cross-presented material. Treating producing cells with proteasome inhibitors increases cross-presentation and has led to the suggestion that long lived proteins is the source of peptide material for cross presentation. Similarly, it was shown that APs are better cross-presented when inserted in a stable context [17,18]. On the other hand, direct delivery of proteins to pAPCs in the form of free proteins, or protein-coated beads, is an inefficient affair [19]. Hence, if full length proteins indeed are a source for crosspresentation, then the context of how the proteins are delivered must also play a role. If one argues against full length protein being a source for direct presentation, the same arguments should also hold true for cross-presentation. If the number of potential peptide products poses a problem for the sensitivity of the direct pathway, there is little saying that this would not also be an issue for cross-presentation? If full length proteins are used for cross-presentation, the vast majority of processed peptides would come from the most abundant cellular sources such as ribosome, proteasome or chaperones. Furthermore, if PTPs helps early detection of virus-infected cells why would the immune system not use the same products for cross-presentation? If not, this would run the risk that T cells are activated towards peptides from full length proteins and not against the same peptide products used by the direct pathway. Such discrepancy would be exploited by viruses and could not be tolerated. However, if PTPs, or other translation products, are used for cross-presentation then, according to previous studies, they should be stable. PTPs for direct presentation are not stable and, hence, if PTPs are indeed used for cross-presentation, the pathways for direct and cross-presentation must diverge in the producing cells. The uptake of peptides from the cellular environment by the pAPCs shall also provide material for the MHCII pathway. The processing of peptide substrates for these two pathways is very different and it is not yet clear how the pAPCs can distinguish between full length proteins going to the class I or the class II pathways to ensure that the peptides end up in the right compartments. It shall be interesting to use an 26S proteasome inducible degradation model of full length proteins that does not result in an increase in direct MHCI presentation [10] to see what happens in terms of cross-presentation. Current Opinion in Immunology 2016, 40:117–122
120 Antigen processing
Cross-talk of peptide material for the class I and class II pathways
Having several sources of peptides for the MHCI pathway has also implications for the MHC class II pathway. While the substrates for both pathways were considered the same there was no need to address this issue. But as they are not, this aspect is also worth some thoughts. By convention, while MHCI-restricted epitopes require endogenous production and processing, it was thought that MHC class II-restricted epitopes are generated from exogenous antigens within the endocytic compartment. In the late 80s, it became apparent that non-classical pathways are also used to process MHC class II antigen presentation and that cytosolic proteins can be endogenously produced, processed and presented by the MHC class II pathway in an MHCI-like fashion [20]. Tewary et al. reported that an antigenic epitope produced from the Neuraminidase (NA) glycoprotein encoded by the A/PR/ 8/34 (PR8) influenza virus, was proteasome and TAP
dependent [21]. More recently, the same group reported that other epitopes from the same viral protein can also be endogenously produced and presented to the MHC class II pathway [22]. Hence, processing of substrates for one, or the other, pathways might not be ‘‘black and white’’, fuelling the idea that cells might have the choice in which pathway a peptide product ends up. Regulation of PTP production and delivery
Another aspect of having alternative translation events for the production of AP and full length proteins is that they could be regulated separately. The infection of a viral particle, activation of Toll-like receptors or the presence of signaling substances in the cellular environment are all events that potentially could help the immune system to detect infected cells by increasing the production of alternative translation products. The downside of coupling the production of AP substrates with signaling pathways is that it would offer viruses targets to evade
Figure 2
Bacterial invasion ? D
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Autophagosome Endossomal recycling of MHCI
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Cytoplasm Current Opinion in Immunology
Antigen presentation via MHCI during bacterial infection. Upon cell invasion, bacteria can reside in vacuoles or free in the cytosol. In vacuoles, pathogens can be processed for production of peptides that are translocated to cytoplasm via Sec61, degraded by the proteasome and subsequently follow the MHCI-classic pathway [25] (a, yellow arrows). Alternatively, bacteria can escape from these membrane-bound compartments and once in the cytosol, they can be recognized by the ubiquitination system and then be tagged for proteosomal degradation [27] (b) or be captured by autophagy (c, green arrows). In the latter case, bacteria-containing autophagosomes are fused to endosomes to form amphisomes, where antigens are preprocessed by cathepsins and transferred to the cytosol via Sec61. After being degraded by the proteasome, antigenic peptides are reimported into the amphisome by transporter associated with antigen presentation (TAP), loaded primarily on recycled MHCI and presented on the cell surface [26]. Although some pathogens like Listeria are able to replicate in the cytoplasm avoiding the described mechanisms, host cell is able to produce antigenic peptides to control infection through a pathway yet not well defined [28,29] (d). Blue and red dots denote peptides before and after proteasomal degradation, respectively. ER: endoplasmic reticulum. Red, yellow and green bacteria represent Salmonella, Chlamydia and Listeria, respectively. Current Opinion in Immunology 2016, 40:117–122
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MHC class I peptides Apcher, Prado Martins and Fa˚hraeus 121
antigen presentation. In the case of the Epstein-Barr virus-encoded EBNA1 and the LANA1 of the Kaposi sarcoma virus these proteins harbor cis-acting mechanisms to suppress the synthesis of their own mRNA and thereby keep the production of alternative translation products at a minimum while at the same time the proteins have a low turnover rate to ensure they are expressed at functional levels [23,24]. Further studies on virus-dependent evasion of the class I pathway are bound to discover cellular target factors that either prove, or disprove, the hypothesis that the cells have the capacity to regulate synthesis of alternative translation products for the class I pathway. Non-viral infection
What has been discussed so far relates to the translation machinery of the host cells. But what about pathogens that bring their own translation machinery? MHCI presentation plays a major role in immunity to intracellular bacteria like Salmonella and Chlamydia. After entry into host cells, these bacteria reside in membrane-bound compartments which do not intersect with compartments of classical MHC I pathway, denoting that alternative mechanisms are adopted to elicit cytotoxic T cells. Previous evidences indicate that processing of Salmonella antigens might involve the translocation of partial processed peptides from the Salmonella-containing vacuoles to the cytosol, followed by their proteosomal degradation and subsequent ER translocation [25]. Fiegl et al. proposed a new alternative cross-presentation model in which autophagy promotes proteasome/TAP-dependent MHC I processing of peptides from pathogens replicating in vacuoles [26]. Moreover, Salmonella can escape invasion vacuoles being released in the cytosol of infected macrophages and recognized by the ubiquitin system. This mechanism results in spatial association of proteasomes and bacteria that is expected to provide a high local concentration of substrates for degradation and could represent a link between intracellular bacteria, bacteria protein translation and antigen presentation [27]. Interestingly, a strong CD8+ T-cell response is observed upon infection by Listeria monocytogenes, in spite of the sophisticated strategies adopted by this pathogen to avoid the recognition by the ubiquitin system. In fact, processing of AP from Listeria is tightly connected to de novo bacterial protein synthesis and presentation of these peptides by MHCI is independent of the cellular half-life of the protein from which they were derived. This shares similarities to the PTP production by the host ribosome. Of note, is has been suggested that Listeria APs are generated with higher efficacy than virus-derived AP [28,29] (Figure 2). However, the identification of bacterial and host effectors involved in producing bacteria-derived AP material for the direct and cross-presentation pathways remain to be explored and could open new horizons in the studies of antigen cross-presentation in the context of non-viral infections and a better understanding of host– parasite interactions. www.sciencedirect.com
Conclusions Since the idea of alternative sources for peptides for the MHC class I pathway was first put forward, new models emerging from this concept are starting to appear that address fundamental biological questions that can help to design more effective vaccines. For example, it is long known that peptides derived from introns are presented on the class I molecules on tumor cells and we now start to understand the underlying molecular mechanisms. However, questions remain to whether/how the source of these peptide products also leads to an activation of CD8+ T cells and, hence, if they are cross-presented. Having different sources of peptides for the MHC class I pathway, produced from different events raises the question if cells have the capacity to selectively control the production of antigenic peptides material in response to stimuli such as viral infection? This would provide a selective advantage in the fight against parasites. These are just a couple of questions that hopefully will find their answers in the not too long future and that will reveal interesting cell biology as well as practical implications for better directing the immune system towards targets of our choices.
Acknowledgments This work was supported by LNCC Equipe Labellise´e, Inserm and by the project MEYS – NPS I – LO1413. AP is AVENIR/ATIP fellow.
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