in MHC Class I Presentation and Cross-Presentation Kenneth L. Rock, Diego J. Farfán-Arribas and Lianjun Shen This information is current as J Immunol 2010; 184:9-15; ; of October 1, 2021. doi: 10.4049/jimmunol.0903399 http://www.jimmunol.org/content/184/1/9

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Proteases in MHC Class I Presentation and Cross-Presentation Kenneth L. Rock, Diego J. Farfa´n-Arribas, and Lianjun Shen Cells that have mutated their or are virally is tolerant to them. However, if cells are synthesizing mutant infected are a potential threat to a host. Consequently, or ones from viruses, then from these the immune system has evolved mechanisms for CD8 T products will also be displayed, and this allows effector CD8 lymphocytes to identify such cells and eliminate them. T cells to recognize the abnormal cells and eliminate them. The generation of CD8 T cell responses occurs in two phases, both of which critically involve the process of Ag Proteases that make the initial cleavages to generate a presented presentation. In the first phase, sentinel cells gather Ags The catabolic mechanism in cells that makes the initial cleavages present in tissues and then present them to naive CD8 to generate most presented peptides is the . Because Downloaded from T cells in ways that stimulate their maturation into the proteasome and its connection to Ag presentation has been effectors. In the second phase, these effector cells seek well described elsewhere, we will cover this subject in this paper out and eliminate the pathological cells. The abnormal relatively briefly and provide readers with references to repre- cells are identified through their presentation of immu- sentatives and/or recent papers and reviews. nogenic Ags that they are producing. The Ag presenta- The proteasome is a large barrel-shaped particle present in tion mechanisms used by the sentinel cells can be the cytosol and nucleus of all eukaryotic cells (3–5) that is http://www.jimmunol.org/ different from those in other cells. This article will re- responsible for degrading of a majority of cellular proteins (6) view these mechanisms with a focus in each case on how (Fig. 1). At its core is a 20S cylinder that is composed of two antigenic peptides are generated for presentation. The outer a rings and two inner b rings with a central channel Journal of Immunology, 2010, 184: 9–15. into which substrates enter and are cleaved (3, 6). The b ring contains three active sites, each of which is formed by a different subunit: B1, B2, and B5 (3). All three of these subunits work through the same catalytic mechanism wherein Classical (direct) pathway of MHC class I Ag presentation an N-terminal threonine residue makes a nucleophilic attack by guest on October 1, 2021 ll cells transcribe a subset of their genes and translate on a peptide bond of the substrate. However, the three active them into proteins. In parallel, they also continuously sites each have different specificities, cleaving preferentially on A degrade these proteins to regulate their levels and the carboxylic side of either hydrophobic residues (B5), basic eliminate damaged ones (1). In this process, cellular proteins residues (B1), or acidic ones (B2) (3, 6). are initially cleaved in the cytoplasm and nucleus into oligo- The strongest evidence that the proteasome plays a major peptide fragments. role in the generation of most presented peptides comes from To monitor what cells are producing, the immune system has studies using highly specific inhibitors of the proteasome’s evolved mechanisms to sample the library of peptides produced threonine-active sites. In living cells, these agents block by cellular catabolism and display them on the cell surface for completely the presentation of peptides from Ags that require scrutiny by CD8 T cells (2). In this process, a fraction of the , but have no effect on ones that do not need peptides present in the cytosol are transported into the endo- cleavage (e.g., when epitopes are expressed directly from plasmic reticulum (ER) by the TAP. In this location, peptides minigenes) and markedly limit the overall supply of peptides that are of the right length and sequence bind to newly syn- to MHC class I molecules (6). thesized MHC class I molecules, which then transport them to Whereas the immune system has used this phylogentically the cell surface. Through these mechanisms, cells display on older pathway for a source of peptides, it also evolved mod- their surface a sampling of their expressed gene products. In ifications that are thought to play a special role in Ag pre- healthy cells, all of these presented peptides come from normal sentation. One modification is an alternate set of autologous genes and are ignored because the immune system subunits (B1i/MHC-linked low molecular weight protein 2,

Department of Pathology, University of Massachusetts Medical School, Worcester, MA Abbreviations used in this paper: BH, bleomycin ; Cat, ; ER, endo- 01655 plasmic reticulum; ERAP, ER ; IRAP, insulin-regulated aminopeptidase; LAP, leucine aminopeptidase; PA28, 28-kDa proteasome activator; PA700, 700-kDa Received for publication October 20, 2009. Accepted for publication November 9, proteasome activator; PSA, puromycin-sensitive aminopeptidase; RNAi, RNA interfer- 2009. ence; TPPII, II. This work was supported by National Institutes of Health Grants 2R01AI020248-27A2 and 2R01AI043543-11A109 (to K.L.R.). Copyright Ó 2009 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 Address correspondence and reprint requests to Dr. Kenneth L. Rock, Department of Pathology, University of Massachusetts Medical School, Room S2-109, 55 Lake Avenue North, Worcester, MA 01655. E-mail address: [email protected] www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903399 10 BRIEF REVIEWS: PROTEASES IN MHC CLASS I PRESENTATION

proteasome’s active sites (3) and may do so at least in part opening the ends of the 20S particle allowing substrates to enter this particle. This change in activity can lead to altered cleavages in substrates (10) and increases or decreases in the generation of at least some MHC class I-presented peptides by purified (11, 12). Similarly, transfection of PA28 into cells enhanced the presentation of some but not all epitopes (3) and increased the surface expression of some MHC class I molecules while decreasing the levels of other 2 2 ones (12). Consistent with these findings, PA28 / mice have a defect in presenting some Ags but not others (13). So-called “hybrid” proteasomes can form with PA28 on one end and a 700-kDa proteasome activator (PA700) complex on the other. PA700 confers on the proteasome complex the ability to degrade polyubiquitinated substrates (10, 14). FIGURE 1. Schematic representation of the different proteases involved in Where examined, hybrid proteasome particles make up Ag processing. The proteasomal degradation step can lead to the generation of ∼20% of proteasomes in cells (14). Whether PA28 exerts its a mature epitope, a precursor peptide with an N-terminal extension, or the effects on Ag presentation through proteasomes with or destruction of the epitope. Precursor peptides can be further trimmed by without PA700 is presently not clear. Downloaded from to generate a mature epitope. Alternatively, amino and/or activities can lead to the destruction of the epitope. Proteasome-independent pathways of Ag processing Studies have shown that proteasome inhibitors block the as- B2i/multicatalytic endopeptidase complex-like 1, B5i/MHC- sembly of MHC class I molecules, and this is almost certainly linked low molecular weight protein 7) that, when expressed because they are stopping the production of presented peptides http://www.jimmunol.org/ preferentially, incorporate into newly assembling proteasomes that are needed for this process (6). However, the degree of in place of the “standard” B1, B2, and B5 subunits to form so- inhibition of MHC class I assembly is less marked for some called “immunoproteasomes” (3, 6). These are constitutively MHC class I molecules (15). Moreover, there are some ex- expressed in dendritic cells, lymphocytes, and thymic epi- amples of epitopes whose presentation is not blocked by thelium and are induced in all cells by proinflammatory cy- proteasome inhibitors or is actually enhanced by this treat- tokines such as IFN-g. ment (16, 17). These findings have been interpreted to in- The net effect of changing these b subunits is to alter the dicate that this proteasome inhibitor-resistant presentation is catalytic activity of the proteasome’s active sites. This changes being mediated by other proteases. This may indeed be the the cleavages made in protein substrates both quantitatively case; however, caution is warranted in interpreting these re- by guest on October 1, 2021 and qualitatively and thereby results in the production of sults because, at the concentrations used in these studies, most a different set of peptides (7). Where it has been examined for proteasome inhibitors do not block all of the proteasome’s individual Ags, immunoproteasomes sometimes make more active sites (18). Therefore, in the presence of these inhibitors, or less of a particular epitope (3, 8). It has been predicted that proteasomes may still be making cleavages that generate the immunoproteasomes generate peptides that are more favor- presented peptides. able for Ag presentation; however, how often this is the case One candidate for a that might generate presented and how important immunoproteasomes are to MHC class I peptides independently from the proteasome is tripeptidyl Ag presentation is not fully resolved (6, 7). Mice that lack one peptidase II (TPPII). TPPII was initially suggested as the major or two of the immunoproteasome subunits have defects in the mechanism by which cells chronically exposed to proteasome presentation of selected epitopes and in some cases partial inhibitors survived and generated presented peptides (19), reductions in the generation of presented peptides overall (7). although it is now thought that the proteasomes were not However, a more complete understanding of the contribution completely inhibited in these treated cells (18). However, of immunoproteasomes to Ag presentation and maybe even to purified TPPII can help generate an epitope (20), and the nonimmune functions awaits the generation of mice that lack presentation of another epitope was blocked when cells were all three inducible subunits. treated with TPPII inhibitors but not proteasome blockers Thymic medullary epithelium also constitutively expresses (16, 20). However, TPPII-null mice have been generated, and another B5 subunit (B5t) that preferentially incorporates with their cells do not show any reduction in the expression of B1i plus B2i into proteasomes called thymoproteasomes (9). MHC class I molecules (suggesting that peptide supply is Mice that lack either B2i or especially B5t show marked re- intact) (21, 22). A role for TPPII in trimming some protea- ductions in the generation of CD8 T cells and defects in the T somal products will be discussed below. cell repertoire, presumably because changes in the peptide repertoire lead to altered T cell selection in the thymus. The size of MHC class I-presented peptides and the products of Another apparent immune modification of the proteasome proteasomes is the 28-kDa proteasome activator (PA28) complex that is MHC class I-presented peptides are of a remarkable uniform constitutively expressed in dendritic cells and is induced in size, the vast majority being either 8, 9, or 10 residues, other cells by IFN-g. PA28 is a heterohexameric (a3b3) ring depending on the particular MHC class I molecule (2, 3, 6). that binds to one or both ends of the 20S proteasome. Upon This is because the ends of the Ag binding groove of the class I binding, it increases the catalytic activity of all three of the molecule are closed and have pockets that bind the N and C The Journal of Immunology 11 terminus of the presented peptide. Consequently, only pep- Cells contain many other cytosolic aminopeptidases that can tides of the exact right size stably bind to this complex. trim peptides. Three of these, leucine aminopeptidase (LAP) When purified proteasomes were incubated with Ags, the (33), bleomycin hydrolase (BH), and puromycin-sensitive majority of peptides produced were too short (,7 residues) for aminopeptidase (PSA) (34), can generate mature epitopes Ag presentation. In contrast, only a small minority (,5%) of from precursors at least in cell-free systems. However, the the peptides generated were of the correct size to bind to any contribution of these peptidases to trimming in cells is less particular MHC class I molecule, whereas a substantially larger clear. Inhibitors of BH and PSA reduced the presentation of fraction (∼20%) of products were too long for stable binding a few epitopes; however, these agents were not highly specific 2 2 2 2 2 2 (23). In the one case that has been analyzed, the peptides ones (34). On the other hand, BH / , PSA / and LAP / 2 2 2 2 produced by immunoproteasomes were on average longer mutant mice or BH / LAP / –double-deficient animals those made from standard proteasomes (24). When such long show no defect in generating and presenting mature epitopes peptides are loaded into the cytosol of cells or expressed from from N-extended precursor peptides or Ag presentation minigenes, they are trimmed and presented, indicating that generally (33, 35, 36). These negative results may indicate they can serve as precursors of presented peptides. that there is substantial redundancy among these , so What trims these long precursors into presented peptides? that trimming activity is not reduced by the loss of only one One protease involved in this process is the proteasome, which or a few peptidases. Alternatively, it is possible that there are can recleave these peptides into epitopes (25). Interestingly, other peptidases not yet identified that play a more important proteasome inhibitors block the presentation from precursors role in trimming epitopes. It is also possible that LAP, BH, that have even a single extra C-terminal residue, indicating and/or PSA are more important for the trimming of epitopes Downloaded from that these particles are the only activity in cells that can make other than the ones thus far examined. the proper C-terminal cleavage to generate an epitope (6, 25). Consistent with these findings, the cytosol of cells has little if Peptide trimming in the ER any activity (25). In contrast, precursors that It is possible to target peptides directly into the ER after they just have extra N-terminal residues are presented, but in this are synthesized by fusing a signal sequence to the N terminus of case, proteasome inhibitors do not block this process (6, 25). the peptide (6). When such constructs are expressed from http://www.jimmunol.org/ This suggested that there must be other activities in cells that minigenes in cells, the signal sequence targets the peptide into can remove N-terminal residues. It turns out that this N- the ER through the SEC61 translocon and is then removed by terminal trimming of peptides can occur in the cytosol, where the signal sequence peptidase. This delivers the peptide into they are first produced, or in the ER after they are transported the ER in a manner that is independent of the TAP. into this location. When N-extended peptides were targeted into the ER via SEC61, their extra N-terminal residues were removed and the Peptidases and trimming of peptides in the cytosol peptides were presented on MHC class I molecules (25, 27, 37). N-extended peptides that are loaded or expressed in the cytosol This indicated that aminopeptidases must exist in the lumen of by guest on October 1, 2021 of cells are trimmed and presented on MHC class I molecules the ER, and, indeed, microsomes were found to have such (6, 25). Importantly, this can occur to some extent in cells trimming activity (38, 39). These activities were purified and that are unable to cleave peptides in the ER (discussed further identified as two members of the family of ami- below) indicating that some trimming of precursors occurs in nopeptidases. Although these peptidases have been given sev- the cytosol. This trimming is mediated by aminopeptidases eral different names, they are now most commonly referred to because precursors that are resistant to hydrolysis by these as ER-associated aminopeptidase or ER aminopeptidase peptidases (due to a chemically blocked N terminus) fail to be (ERAP) 1 and 2. Mice have only ERAP1, whereas humans and trimmed and presented (26, 27). However, which specific some other mammalian species have both ERAP1 and ERAP2. aminopeptidases contribute to MHC class I Ag presentation is not well understood. ERAP1 One aminopeptidase that may play a special role in trimming Most aminopeptidases continued to hydrolyze a substrate until peptides is TPPII. TPPII is relatively unique because it trims it is converted entirely to amino acids. However, ERAP1 is longer peptides as well as shorter ones, whereas most other unique in that it trims long polypeptides rapidly until they aminopeptidases only cleave peptides ,14 aa in length. Con- reach a size of nine or eight residues and then its hydrolysis sequently, inhibition of TPPII significantly reduces the pre- stops (40, 41). Therefore, ERAP1 seems to be specialized for sentation of peptides with long N-terminal extensions but not generating peptides of the proper size for Ag presentation. shorter ones (22, 28). How important is TPPII to Ag pre- This is not to say that ERAP1 always stops trimming when it sentation? Although an initial report suggested that TPPII was has generated a mature peptide. Indeed, it trims ∼50% of 9- required for the generation of most presented peptides (29), mer peptides to 8-mers and, in so doing, will ultimately de- subsequent studies with inhibitors (30), RNA interference stroy some 9-mer epitopes (40) and limit their presentation 2 2 (RNAi)-silenced cells (28), and TPPII / mice (21, 22) found (41, 42). It has been suggested that ERAP1 might also trim no major defects in Ag presentation; these latter results are the protruding ends of long peptides when they are bound to consistent with the finding that proteasomes generate relatively MHC class I molecules (43); however, it is not known few very long peptides (see above). However, TPPII inhibitors whether this actually occurs and if ERAP1’s active site could reduce the presentation of a few individual epitopes (16), in- even access such bound peptides. crease the presentation of others (31), and do not affect re- ERAP1 plays an important role in MHC class I Ag pre- sponses to many others (32), suggesting that this peptidase may sentation. Cells in which ERAP1 is eliminated through mu- play a role in the presentation of selected sequences. tation or RNAi silencing (and also naturally lack ERAP2) are 12 BRIEF REVIEWS: PROTEASES IN MHC CLASS I PRESENTATION unable to trim N-extended peptides in the ER (41). Therefore, afew proteins that aretransported via the exocyticroute (55,56). in mouse cells and some human cells, ERAP1 is the only Because the presentation of these peptides does not require trimming in the ER, at least for these sequences. TAP, it is thought that furin may generate these peptides from ERAP1-deficient cells have reduced surface levels of MHC proteins that transit the transgolgi network. However, this is class I molecules and the peptide-MHC complexes that are unlikely to be a major source of presented peptides because made are less stable than on wild-type cells (44–46). These TAP-negative cells have very low levels of presented peptides, results suggest that ERAP1 makes an important contribution among which some are derived from signal sequences that are both to the quantity and quality of peptides available for Ag hydrolyzed in the ER, and others are not (57). Whether furin presentation. contributes to the trimming of peptides that are generated in Interestingly, loss of ERAP1 decreases the presentation of the cytosol and transported into the ER by TAP is not known. some epitopes from full-length proteins (ones presumably made as N-extended precursors), does not affect others, and Peptide destruction increases the presentation of yet others (41, 42, 46). Conse- Proteases can also cleave within epitopes, destroying them, and quently, ERAP1 deficiency markedly changes CD8 T cell this is a process that can limit Ag presentation. In fact, outside responses to various epitopes in vivo and alters im- 2 2 of their role in Ag presentation, most peptides are not useful in munodominance hierarchies (46). Remarkably, ERAP1 / cells, and their accumulation would be deleterious. As a result, T cells can recognize and respond against cells from wild-type cells have evolved robust catabolic activities to further degrade mice (44), suggesting that loss of ERAP1 may alter the overall peptides down to amino acids, which are useful to cells. repertoire markedly. Presumed polymorphisms in ERAP1 Consequently, when peptides are added to cell extracts, they Downloaded from may similarly affect responses because ERAP1 has been linked are rapidly degraded (58) and when injected into the cyto- genetically to the autoimmune disease ankylosing spondylitis plasm of living cells have a half-life of only seconds (26). (47, 48) and cancer progression (49) in humans. All of the proteases that may be involved in the generation of Based on these findings, it is clear that the Ag processing peptides can and probably do destroy some epitopes. Thus, the pathway generates many N-extended peptide precursors and presentation of some epitopes is enhanced by proteasome that trimming by ERAP1 has specificity. This specificity has inhibitors (16). Moreover, increases in the presentation of se- http://www.jimmunol.org/ not yet been fully elucidated, but studies with purified ERAP1 lected epitopes or in the overall surface expression of class I and cells expressing ERAP1 have shown that although it can molecules were seen in cells in which PSA (33), BH+LAP (35), remove all residues from the N terminus of peptides, it does so TPPII (22, 28) and ERAP1 (41, 42) were absent or inhibited. at different rates (50) and is also affected by sequences in the Another protease that has been implicated in this process is thimet epitope itself (40, 51, 52). , a cytosolic metalloprotease (59). The increased Ag presentation and higher levels of MHC class I molecules after ERAP2 inhibiting these peptidases suggest that they normally limit the ERAP2 also trims residues from the N terminus of peptides presentation of some peptides by destroying them. by guest on October 1, 2021 and appears to do so with different specificity from ERAP1, The fact that cytosol is such a hostile environment for peptides being more active in cleaving N-terminal basic residues from at has raised the question of how peptides destined for Ag pre- least some substrates (53). Therefore, the combined actions of sentation escape destruction. It may be that most are destroyed ERAP1 and ERAP2 are likely to enhance the hydrolytic ca- [it has been suggested that this is the fate of 99.9% of peptide pacity of the ER and trim a broader set of sequences. In- (60)] and that the ones that do get presented are rescued by terestingly, ERAP2 appears to form a complex with ERAP1, rapid transport through TAP and then binding to MHC class I and it has been suggested that this combination of two ami- molecules. However, it has also been proposed that there may nopeptidases may trim peptides in a cooperative manner (53). be mechanisms that protect peptides from degradation. One In contrast to ERAP1, ERAP2 does not stop trimming pep- idea is that cytosolic chaperones may bind peptides and prevent tides when they are the size of mature epitopes (52). their degradation. Indeed, peptides can be found bound to Silencing of ERAP2 with RNAi reduced the presentation of these proteins (61–63). However, whether binding to chaper- an epitope (53). However, the contribution of ERAP2 to ones is important for the presentation of most peptides is not overall ER trimming is not yet clear. Silencing of ERAP2 only clear. In fact, when thimet oligopeptidase was overexpressed in reduced overall MHC class I expression by ∼10% (53). cells, there was almost complete inhibition of the presentation Moreover, the hierarchy of specificity in removing any of the of peptides generated in the cytosol, but no effect on the ones 20 aa flanking an epitope was not different in cells expressing targeted into the ER (59). These results indicate that presented only ERAP1 or both ERAP1 and ERAP2 (50). Furthermore, peptides are susceptible to destruction in the cytosol; this ERAP2 is poorly expressed or not expressed in human brain, suggests either that protective mechanisms do not exist, or, if liver, thymus, testes, and small intestine (54), and it is not they do, then they must be in kinetic competition with the present in mice. Although additional studies are needed, these destruction pathways. The specificity of the destructive (and findings suggest that ERAP2 may play a more limited or protective) mechanisms has not been well characterized. This is specialized role in peptide trimming. an important issue because it may be yet another factor that influences what peptides ultimately get presented. Cleavage of peptides after the ER Ags that exit the ER may be cleaved by proteases in the exocytic Cross-presentation pathway. One such protease is furin, a member of the subtilisin Dendritic cells and macrophages have the unique ability to family of serine proteases that is resident in the Golgi apparatus. present on MHC class I not only peptides from their own Furin has been implicated in the generation of peptides from endogenous Ags, but also ones from Ags in their external The Journal of Immunology 13 environment through a process called cross-presentation (64– sented peptides are not generated in the phagosomes 67). After acquiring these Ags, dendritic cells carry this in- themselves. Instead, the Ags are transferred from phagosomes formation to secondary lymphoid tissues and present it to into the cytosol where they undergo cleavage. This form of naive CD8 T cells in ways that initiate immune responses cross-presentation is blocked by proteasome inhibitors, in- (64). This ability to present exogenous Ags plays a critical role dicating that these particles make the initial cleavages to gen- in immune surveillance of tissues by allowing the immune erate a cross-presented peptide (66, 67). The presentation is system to monitor tissues for the presence of cancers or viruses also dependent on TAP (66, 67) and, in at least some cases, also that do not infect dendritic cells. This pathway is also thought ERAP1 (74, 75). Thus, once the exogenous Ag is transferred to to allow the immune system to monitor the Ags in phag- the cytosol, it is thought to be processed for cross-presentation osomes of dendritic cells and macrophages and thereby allow in much the same manner as endogenous proteins. CD8 T cells to detect phagocytes that are harboring in- The peptides that are generated in the cytosol may be tracellular bacteria and eliminate them (66, 67). transported by TAP to MHC class I molecules in the ER. In Ags that are internalized by phagocytosis or macro- addition, there is evidence that many ER components, in- pinocytosis are cross-presented several thousand times more cluding TAP, somehow get incorporated into phagosomes (73, efficiently than soluble proteins (66, 67). Presumably because 76). This has suggested that some peptides generated in the of this, particulate Ags (e.g., dying cells) are a major source of cytosol might be transported by TAP to MHC class I mole- cross-presented Ags. Although still somewhat controversial, cules in phagosomes. In this location, the peptides could be there is accumulating evidence that the major source of cross- further hydrolyzed by phagosomal proteases. One vacuolar presented peptides is intact Ags (66–68) or ones that are peptidase that has been implicated in this process is insulin- Downloaded from partially cleaved (e.g., by in dying cells) (69, 70). In regulated aminopeptidase (IRAP, also known as placental this review, we will focus on the two best-characterized leucine aminopeptidase), which is a homolog of ERAP1 and mechanisms by which cross-presented peptides are generated ERAP2 (77). Like ERAP1, IRAP can remove most amino acids from proteins in phagosomes with an emphasis on what is from the N terminus of peptides; however, unlike ERAP, it can known about the proteases that generate these peptides. The continue to trim peptides to a size smaller than ,8 residues in two mechanisms we will focus on are the phagosome-to-cy- length (E. Stratikos, personal communication). http://www.jimmunol.org/ tosol and vacuolar pathways (Fig. 2). It should be noted that Dendritic cells lacking IRAP present endogenous Ags nor- there are also additional mechanisms by which cross-pre- mally, but have about a 50% decrease in their ability to cross- sentation occurs (63, 71–73), although their role in vivo is present an exogenous Ag (77). This reduction in presentation is presently unclear. of a similar magnitude to that observed in ERAP1-deficient cells. It is presently unknown whether these two peptidases are Phagosome-to-cytosol pathway working together in the phagosome or in separate compart- In the phagosome-to-cytosol pathway of cross-presentation, ments (ERAP1 in the ER and IRAP in the phagosome). blocking proteolysis in these phagosomes does not inhibit cross- by guest on October 1, 2021 presentation and may actually enhance it (66, 67). In fact, The vacuolar pathway of cross-presentation dendritic cells may promote cross-presentation by raising the In the vacuolar pathway, the generation of cross-presented pH in phagosomes and thereby reducing the proteolytic de- peptides is inhibited by inhibitors such as struction of Ags (65). Therefore, in this pathway, cross-pre- leupeptin, but is resistant to proteasome inhibitors, and their presentation is TAP-independent (66, 67). Therefore, it is thought that the cross-presented peptides are generated in the phagosome itself. One of the vacuolar cysteine proteases that generates cross- presented peptides is cathepsin (Cat) S. Cat S-deficient mice and their dendritic cells have a defect in vacuolar but not phagosome-to-cytosol cross-presentation (78, 79). Cat B-, L-, and D-deficient cells showed no defect in cross-presentation (78). In addition, IRAP might be involved in the generation of presented peptides in this compartment in DCs (77). Why Cat S-deficient plays such an important role is unclear. Perhaps it can make the appropriate cleavages to generate 8- to 9-mer peptides. In fact, recombinant Cat S was shown to generate a presented peptide in at least one case (78); however, whether it can generate other mature peptides is not known (80). Additionally, Cat S is relatively unique in being strongly FIGURE 2. Proteases in cross-presentation. Upon uptake by dendritic cells or active at neutral pH, and this may be important because MHC macrophages, exogenous Ag can be processed and presented on MHC class I class I molecules may not stably bind peptides at acidic pH. molecules by two major pathways: the phagosome-to-cytosol pathway (left) and Because Cat S-deficient mice lack the vacuolar pathway, and the vacuolar pathway (right). In the cytosolic pathway, proteasomes are the TAP-deficientanimalslackthephagosome-to-cytosolpathway,it major proteases to generate the antigenic peptide fragments. These peptides can be further trimmed by aminopeptidases in the cytosol, ER, and possibly an has been possible to use these genetic models to quantify the endosomal compartment (for details, see Fig.1). In the less well-understood contributionofthesevariouspathwaystocross-presentation.Such vacuolar pathway, Cat S plays a key role in generating class I binding peptides. It analyses have shown that both pathways contribute to the gen- is unclear whether other proteases (e.g., IRAP) are also involved. erationofCD8Tcellresponsesbutthatthephagosome-to-cytosol 14 BRIEF REVIEWS: PROTEASES IN MHC CLASS I PRESENTATION pathway contributes to a greater extent than does the vacuolar 17. Diekmann, J., E. Adamopoulou, O. Beck, G. Rauser, S. Lurati, S. Tenzer, H. Einsele, H. G. Rammensee, H. Schild, and M. S. Topp. 2009. Processing of two mechanism (66, 78). latent membrane protein 1 MHC class I epitopes requires tripeptidyl peptidase II involvement. J. Immunol. 183: 1587–1597. 18. Princiotta, M. F., U. Schubert, W. Chen, J. R. Bennink, J. Myung, C. M. Crews, Conclusions and J. W. Yewdell. 2001. Cells adapted to the proteasome inhibitor 4-hydroxy- 5- To supply MHC class I molecules with peptides, the immune iodo-3-nitrophenylacetyl-Leu-Leu-leucinal-vinyl sulfone require enzymatically ac- tive proteasomes for continued survival. Proc. Natl. Acad. Sci. USA 98: 513–518. system used, and, in some cases adapted, phylogentically older 19. Wang, E. W., B. M. Kessler, A. Borodovsky, B. F. Cravatt, M. Bogyo, H. L. Ploegh, catabolic pathways that normally break down proteins. The and R. Glas. 2000. Integration of the ubiquitin-proteasome pathway with a cyto- solic oligopeptidase activity. Proc. Natl. Acad. Sci. USA 97: 9990–9995. proteasome pathway has been used and adapted with new 20. Seifert, U., C. Maran˜o´n, A. Shmueli, J. F. Desoutter, L. Wesoloski, K. Janek, P. active sites and regulators to make the initial cleavages in Henklein, S. Diescher, M. Andrieu, H. de la Salle, et al. 2003. An essential role for proteins for direct presentation and for one of the major cross- tripeptidyl peptidase in the generation of an MHC class I epitope. Nat. Immunol. 4: 375–379. presentation pathways. Aminopeptidases in the cytosol and ER 21. Firat, E., J. Huai, L. Saveanu, S. Gaedicke, P. Aichele, K. 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