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Reading within the lines: naturally processed peptides displayed by MHC class I molecules
137
Reading within the lines: naturally processed peptides displayed by MHC class I molecules Nilabh Shastri*, Thomas Serwold and Pedro Paz A typical mammalian cell contains tens of thousands of different gene products. Snippets of this genetic information are displayed on the cell surface by MHC class I molecules as short peptides for immune surveillance by CD8 + T lymphocytes. Genetic and biochemical analysis of these peptides is revealing novel sources and mechanisms by which these peptide/MHC class I complexes arise.
Addresses Division of Immunology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA *e-mail: [email protected] Correspondence: Nilabh Shastri Current Opinion in Immunology 1998, 10:137-144
http:/Ibiomednet.com/elecref10952791501000137 © Current Biology Ltd ISSN 0952-7915
Abbreviations APC antigen-presentingcell ER endoplasmicreticulum HPLC high performance liquid chromatography ORF open reading frame TAP transporterassociated with antigen processing
Introduction
Immune surveillance by the CD8 + T cell repertoire is a mechanism for detecting and eliminating abnormal cells. These could be cells infected with viruses or bacteria, tumors, allogeneic tissues in transplantation or even self-tissues in autoimmunity. T h e antigen receptors of CD8 ÷ T cells examine the diverse repertoire of peptide/MHC complexes on the target cell surface for novel peptides that indicate expression of new or abnormal gene products. T h e peptides displayed by the MHC thus represent snippets of the cellular protein milieu. The antigen-processing pathway that extracts these internal peptides from their precursor polypeptides and leads to the expression of the informative peptide/MHC display on the surface can be considered a mechanism that 'reads' within 'lines' of amino acid sequences. In this review we focus on the antigen-processing pathway, for peptides presented by MHC class I, from the perspective of the antigen, particularly that synthesized within the antigen-presenting cell (APC) itself. Intracellular proteins undergo proteolysis to generate fragments, which are actively transported into the endoplasmic reticulum (ER) for assembly with the MHC class I molecules before being transported to the cell surface. Several key molecules involved in these steps have been subjects of excellent recent reviews [1-3]. These include the multicatalytic proteasome, the ER chaperones calnexin
and calreticulin, the peptide transporter TAP (transporter associated with antigen processing) and the recently discovered protein - - tapasin - - which bridges TAP and the MHC [4,5]. The output of the antigen-processing
pathway
The final product of the antigen-processing pathway is a short peptide bound to the MHC class I molecule on the cell surface (Figure 1). Crystal structures of several peptide/MHC class I complexes have shown that each peptide is primarily tethered to the MHC molecule by its amino- and carboxyl-termini, the backbone and the side chains of some internal residues [6]. T h e complementary pockets in the MHC molecules that interact with these features of the peptide vary due to extensive polymorphism. As a result, each MHC molecule prefers sets of peptides with distinct consensus sequences. T h e consensus sequences for MHC-bound peptides were first established by pool sequencing that allows the conserved 'anchor' residues to be distinguished from other more diverse residues [7]. T h e peptide consensus sequences have since been determined for a large number of MHC molecules [8,9]. In general the peptides bound to MHC class I are 8-10 residues in length, contain one or more conserved internal residue and share an aliphatic, aromatic or positively charged carboxyl-terminus. Knowledge of these conserved consensus motifs allows predictions of candidate antigenic peptides within protein sequences from antigens which stimulate T cells [10,11]. Exceptions to the conserved peptide sequence motifs have been found [12,13"',14"°], but what fraction of MHC-bound, naturally processed, peptides falls into this category is not yet known. T h e assignment of an amino acid sequence as the antigenic entity is demonstrated by the ability of synthetic peptides of this sequence to stimulate T cells when presented by appropriate MHC-expressing APC. In these assays, the synthetic analogue of the naturally processed peptide is usually the most active and a response by T cells is readily detectable at picomolar concentrations of peptide. Whether the active synthetic peptide actually corresponds to the naturally processed peptide is generally established by the overlap of the high performance liquid chromatography (HPLC) elution profile of the synthetic peptide with that of peptide extracts from antigen-expressing cells. This can be a technically demanding undertaking because firstly, the naturally processed peptide are expressed at low levels, secondly, the extraction efficiency varies with the chemical characteristics (e.g. hydrophobicity) of the antigenic peptide and thirdly, the HPLC set-up can be easily contaminated if a
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Immunologicaltechniques
Figure 1
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° Cell surface expression
Output Current Opinion in Immunology
Schematic view of the output, input and intervening steps in the classical MHC class I antigen processing pathway. Translated products of the mRNA serve as the input, then undergo proteolysis in the proteasome. TAP transport and potential cytoplasmic and ER trimming events occur during assembly of the peptide/MHC class I complex which is then transported to the cell surface. The peptide/MHC class I complex is offered on the APC surface as a potential ligand for the CD8 + T cells.
high concentrations of synthetic peptides are injected in order to establish their elution profiles. T h e impact of these factors can be determined by assaying HPLC fractions of mock-injections (of buffer alone) or extracts of experimental samples spiked with known amounts of synthetic peptides. Routine inclusion of these controls is critical for demonstrating the identity and abundance of naturally processed peptides and the absence of artefacts resulting from carry-over from previous samples. As an example of this analysis, consider the p2Ca peptide L S P F P F D L (in single-letter code for amino acids), which was identified as the naturally processed peptide presented by the L d MHC class I molecule to the 2C T cell clone [12,15]. T h e QL9 analogue of p2Ca, with the naturally-occurring amino-terminal glutamine (Q) flanking residue (Figure 2) is a hundred fold more active in stimulating the 2C T cells either in assays in which it is added exogenously [16], or in assays in which it is expressed endogenously in L d ceils (Figure 2). The higher activity of the QL9 DNA construct suggests that QL9 rather than p2Ca could be the naturally processed analogue. Indeed, analysis of HPLC fractionated extracts of Ld cells expressing Met-QL9 and Met-p2Ca showed that only the QL9 rather than the p2Ca peptide was detected in the cellular peptide pool. Thus, QL9, rather than the p2Ca peptide is likely to represent the major naturally processed Ld-bound ligand for the 2C T cell clone.
the MHC-bound peptide pool. In human melanoma cells, a tyrosinase peptide, YMDGTMSQV, was presented by HLA-A2.1 MHC with a post-translationally modified aspartate rather than the natural asparagine residue [17°°]. Another peptide derived from the human Y-chromosome encoded SMCY protein was presented by MHC as a cysteinylated derivative [18°°]. These precedents suggest that peptides with other post-translational modifications such as phosphorylation or glycosylation may also exist. Since these modifications are post-translational, they were not evident in the primary protein sequences and were in fact identified by innovative mass spectrometry techniques. The existence of these post-translational modifications have provided further insights into potential mechanisms for the processing of compartmentalized proteins (see below). The abundance of individual peptide/MHC complexes on the cell surface varies widely. Estimates based upon the recovery of peptides in the MHC-bound pool show that while few peptides are present at a high level, the vast majority are present at <100 copies/cell [8,19] Accordingly, several T cell stimulating peptides have been found to be present at <10-100 copies/cell [13"°,14",20]. Notably, under experimental conditions and particularly in infected cells, the expression of individual peptides can increase by several magnitudes [21,22] (P Paz, N Shastri, unpublished data).
Precursors for antigen processing Discrepancies between the predicted and observed T-cell stimulating activity or HPLC elution profiles led to the discovery of post-translationally modified peptides among
With the exception of two viral proteins no endogenously synthesized protein has so far been shown to be excluded from entry into the antigen processing
Naturally processed peptides displayed by MHC class I molecules Shastri, Serwold and Paz
139
Figure 2
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The identification of the QLg, but not p2Ca peptide among the naturally processed peptide pool in cells expressing Ld MHC and Met-p2Ca (Mp2Ca) or Met-QL9 (MQLg) precursors. Recipient COS cells were untransfected or electroporated with cDNA constructs expressing Ld MHC alone (Ld), with Ld plus Mp2Ca (Ld + Mp2Ca) or with Ld plus MQL9 (Ld + MQLg) precursors. Two days later, the untransfected or transfected cells were (a) tested directly for their ability to stimulate the lacZ activity induced in the 2CZ hybrid or (b,e) extracted with 0.20/0 (v/v) trifluoroacetic acid in water. The lacZ activity was measured as the absorbance of the cleaved product of the lacZ substrate chlorophenol red I~-galactosideat 595 nm. The extracts were fractionated by HPLC using a 10 mM triethylamine acetate, pHS.5 (Buffer A) and 10 mM triethylamine acetate, pH5.5, in acetonitrile (Buffer B) gradient. Fractions, which were collected every 3Osec while Buffer B concentration increased from 21.5% to 33°/0 in Buffer A, were dried in a vacuum centrifuge, resuspended in phosphate-buffered saline and tested for stimulating 2CZ T cells with Ld+ L cells as APC. The background response to COS cells transfected with Ld alone has been subtracted to show that the activity is due to transfected antigen genes. The arrows indicate the retention times of synthetic QL9 and p2Ca peptides determined under identical run conditions.
pathway [23,24°,25°]. These peptide/MHC complexes serve as ligands for T cell responses elicited to tumors, allogeneic tissue transplants and virally infected cells. In addition, peptide/MHC complexes can be generated from exogenous proteins that are introduced into the cytosol [26]. This process occurs naturally in cells infected with intracellular pathogens such as Listeria monocytogenes that escape into the cytosol, thus allowing their secreted proteins to gain access to the MHC class I presentation pathway [27]. Intriguingly, in certain cell types of the macrophage/dendritic lineage, antigens taken up into pinocytic or endocytic vesicles can also access the MHC class I antigen presentation pathway [28-31]. This unconventional method of accessing the MHC class I presentation pathway may be relevant to presentation of peptides from proteins synthesized in the mitochondria
and to the initiation of immune responses by cells that do not express the appropriate MHC molecules. Intriguingly, MHC class I molecules also present peptides that are encoded within regions of transcripts that are not expected to be normally translated [32-34,35°°-39°°]. These regions include 5' and 3" 'untranslated' sequences, alternate translational reading frames and translational read-through into partially spliced mRNA (Figure 3). Again, what fraction of the normal peptide pool represents such peptides is not known and would be difficult to determine given their low abundance [34, 401. Nevertheless, these cryptic translation products are capable of eliciting T cell responses in rive [35"°,36°',39°°]. Presently it is unclear whether MHC present these peptides because they are presumably labile and hence, the preferred
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Immunological techniques
substrates for the antigen processing pathway [41"'], or whether they are generated by a novel translational mechanism that feeds its products into the antigen processing pathway. Regardless of the mechanism for their generation, the existence of these peptides shows that immune surveillance may operate upon the total coding information of cellular transcripts. Accordingly, it would be more accurate to conceptualize the mRNAs, rather than their predominant protein products, as the input of the antigen processing pathway (Figure 3). Note that the origin of the processed peptide, whether it is derived from a major or minor open reading frames (ORFs) is irrelevant for immune surveillance. Site(s) of antigen
processing
No intracellular site appears exempt from providing precursors for antigen processing to the peptide/MHC class I complex. The dramar~c phenotype of TAP deficient cells, with low levels of MHC class I expression, suggests that peptides destined to bind MHC come from the cytosol [1,42]. Although exceptions to the TAP requirement exist for some proteins that are targeted into the ER [43-45], or a few membrane proteins [46,47], the bulk of MHC-bound peptides require TAP indicating that the cytosol is the primary site for antigen processing. How do proteins in membrane-bound intracellular compartments, such as the ER, become available for antigen processing in the cytoplasm? One model envisages that proteins from these compartments are retrieved for
degradation into the cytosol. The discovery of the post-translationally modified peptide with an Asp instead of the natural Asn residue (referred to above) is consistent with the view that the precursor was glycosylated within the ER and retrieved into the cytosol where enzymatic deglycosylation and conversion of the Asn to Asp occurred. Accordingly, the presentation of the peptide/MHC complex was TAP dependent [17"',48"']. Interestingly the existence of a retrieval mechanism for ER proteins was also suggested by the inhibitory effect of viral proteins on antigen presentation [49"]. Whether a similar protein retrieval/degradation pathway exists for other sub-cellular compartments, for example the nucleus or mitochondria, is not known. Alternatively, newly synthesized polypeptides that fail to reach their destination may serve as precursors for antigen processing [50,51]. Because protein synthesis occurs exclusively in the cytoplasm, this model can account for the TAP dependent presentation of any subsequently compartmentalized protein. While attractive in its elegance for explaining the source of all processed peptides, the model does not adequately explain the existence of post-translationally modified peptides (discussed above). The murky middle
steps
It is not known when endogenously synthesized proteins enter the antigen processing pathway. Unlike exogenous proteins, endogenously synthesized proteins can, in principle, be sampled for antigen processing at any stage between their biosynthesis and their turnover. Whether the sampling mechanisms involves the ubiquitin-dependent degradation pathway in antigen processing is uncertain
Figure 3
Origin of pepUdes presented by MHC class I molecules
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Current Opinion in Immunology
Schematic depiction of the origin of peptides presented by MHC class I molecules from predominant open reading frame (ORF, RFO), depicted above the mRNA shown as a straight line, as well as from other 'non-translated' regions shown below the mRNA. These include the 5' and 3' non-coding regions, as well as alternate translational reading frames, RF1 and RF2. The naturally processed peptide is depicted as a solid circle, and its polypeptide precursor sequences as wavy lines. Other mRNA features, the 5' mTG CAP,the 3' polyA tail and the Kozak context (ryyryyATGg) of the translational initiation codon (TAG), as determinants of normal translational efficiency are also indicated.
Naturally processed peptides displayed by MHC class I molecules Shastri, Serwold and Paz
J0
141
[52,53]. T h e observation that proteins may be selected at an early stage for processing is consistent with the observation that presentation of endogenously synthesized proteins is independent of their stability [54°°]. By contrast, the efficiency of presentation of exogenous proteins correlates with their relative instability [55,56]. These conflicting findings may be reconciled if ports of entry for precursors of both newly synthesized and turned-over proteins were available which lead into the antigen processing pathway.
Figure 4
Regardless of the source and the sampling criteria, and unless the precursor exactly matches the product [57], the antigenic precursors must undergo proteolysis to generate the precisely cleaved peptide/MHC complex. T h e multicatalytic proteasome is an important player in this step, because proteasome inhibitors reduce the efficiency of antigen presentation to T cells [58]. This is also evident from measurement of the level of newly assembled peptide/MHC complexes in viable cells after a quick acid-wash (which removes pre-existing peptides from the cell surface) [59]. T h e mean fluorescence intensity following staining of EL-4 cells with antibodies to Db MHC dropped from 33.8 (untreated) to 4.69 immediately after acid-wash, but increased to 22.6 after four hours in normal medium (Figure 4). This recovery of D b MHC represents newly assembled peptide/MHC complexes and this was inhibited when the proteasome inhibitors L L n L or lactacystin were included in the recovery phase (MFI=15.1 and 14.7 respectively for cells incubated for 4hr in proteasome inhibitors after acid-wash). Thus, the recovery in presence of proteasome inhibitors is partial, suggesting that other proteases may also be involved in generating antigenic peptides. T h e existence of these other yet unidentified proteases in trimming antigenic peptides has also been suggested by the lack of an effect of proteasome inhibitors and even an increase in presentation activity with some precursors in cytotoxicity assays [60].
Expressio,n of most, but not all, peptide/MHC class I complexes depends upon proteasome function. The expression of D b MHC on the surface of EL-4 cells was measured as fluorescence intensity using flow cytometry using a fluorescently conjugated antibody (B22.249). The left panel shows EL-4 cells that were either untreated, or were incubated for 3 rain at room temperature in pH3.1 citrate buffer, then were washed and were stained either immediately (acid wash; no recovery) or following incubation for 4 hr in normal medium at 37" (acid wash; 4 hr recovery). The right panel shows D b expression in acid-washed cells that were treated with the proteasome inhibitors LLnL (100p.M) or lactacystin (Lcyst, 100mM) prior to and during the 4 hr recovery phase. Both LLnL and Lcyst partially inhibit the recovery of D b MHC. Similar results were also obtained for Kb MHC expression (not shown).
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Many studies have shown that flanking sequences influence antigen presentation [61-64]. It is possible that flanking sequences alter the efficiency with which the antigenic peptides are liberated from their precursor [65-67], transported by TAP [68] or interact with MHC molecules in the ER ( T Serwold, N Shastri, unpublished data). Interestingly, proteasome inhibitors have a differential effect on cleavage of amino- versus carboxy-terminal flanking residues suggesting that the precise carboxyl- and amino-termini are generated by distinct proteolytic mechanisms [69"].
processed peptides in cell extracts are recovered only when the cells express the appropriate MHC molecules [70,71]. It is possible that the failure to detect processed peptides, that are presumably generated independently of the MHC, is due to their extreme instability, or poor extraction efficiency in the absence of the MHC molecule. Alternatively, the inability to detect these proteolytic intermediates may be due to sensitivity limits of the assays used for their detection. In contrast to the final peptide product that is active at picomolar concentrations, the presence of even a single additional residue causes a dramatic reduction in the ability to stimulate T cell activity and makes it very unlikely that peptides other than the final peptide will be detected in standard assays [71,72]. This difficulty was partially overcome by treating cell extracts with a carboxypeptidase that removed the inhibitory carboxy-terminal flanking residue, and together with HPLC analysis of cell extracts allowed detection of analogs extended at the carboxyl termini [73]. With this method, we have determined that cells generate not only the precisely cleaved final peptide product but also its analogue extended at the carboxyl terminus. This biochemically demonstrates that it is possible to generate more than one analogue of a peptide from a given precursor. Whether the longer analogue is an intermediate for the final, precisely cleaved, peptide is, however, not known.
Our current understanding of the antigen-processing pathway is primarily based upon analysis of the products and their precursors. Defining the proteolytic intermediates with or without additional flanking residues is important for furthering our understanding of this pathway. It has been evident from the earliest studies that the naturally
The ER chaperone gp96 is another source of potential processing intermediates. Several studies have shown that peptides associated with gp96 can elicit effective immune responses specific for the ceils from which they were derived [74,75,76ee]. Because the existence of at least some of these antigenic peptides depends upon TAP
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Immunological techniques
there is suggestion that gp96-bound peptides may be precursors for those eventually bound to the MHC [77"']. With the exception of one viral peptide [78°], however, the composition of the gp96-bound peptide pool and its potential role in the antigen-processing pathway remains unknown.
Conclusions T h e path from the antigenic precursor to the naturally processed peptide/MHC class I product is not straightforward. T h e clarity of the picture of the peptide/MHC complex becomes increasingly murky as one moves backwards to trace the intervening steps that account for its being. Nevertheless, answers to questions of the source of naturally processed peptides and the mechanisms that regulate their generation and abundance have revealed fundamental insights into novel translational and intracellular protein trafficking and degradation mechanisms, and more will certainly follow. In the future, we hope to learn how precursors (major or minor ORFs) are sampled for antigen processing, the identity of proteolytic intermediates and how they are delivered to TAP, as well as the extent and mechanism of ER trimming. T h e central importance of the antigen-processing pathway in generating the peptide/MHC ligands that regulate CD8 + T cell behavior from their generation in the thymus, to their eventual activation and death in the periphery, suggests that the pathway and its physiological manifestations will continue to receive intense scrutiny.
Acknowledgements We are grateful to members of our laboratory for stimulating discussions. Research in this laboratory is supported by grants from the National Institutes of Health.
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Naturally processed peptides displayed by MHC class I molecules Shastri, Serwold and Paz
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Elliott T, Willis A, Cerundolo V, Townsend A: Processing of major histocompatibility class I-restricted antigens in the endoplasmic reticulum. J Exp Med 1995, 181:1481-1491.
46.
Hammond SA, Bollinger RC, Tobery TW, Siliciano RF: Transporter-Independent Processing of HIV-1 Envelope Protein for Recognition by CD8-Positive T Cells. Nature 1993, 364:158161.
47
Lee SP, Thomas WA, Blake NW, Rickinson AB: Transporter (TAP)-independent processing of a multiple membranespanning protein, the Epstein-Barr virus latent membrane protein 2. Eur J/mmunol 1996, 26:1875-1883.
48. •,
Bacik I, Snyder HL, Anton LC, Russ G, Chen W, Bennink JR, Urge L, Otvos L, Dudkowska B, Eisenlohr L, Yewdell JW: Introduction of a glycosylation site into a secreted protein provides evidence for an alternative antigen processing pathway: Transport of precursors of major histocompatibility complex class I-restricted peptides from the endoplasmic reticulum to the cytosoL J Exp Med 1997, 186:479-487 This, together with [49"], demonstrates a novel mechanism for retrieval and degradation of endoplasmic reticulum proteins in the cytosol. These may serve as possible source of antigenic peptides derived from precursors normally present in membrane-bound subcellular compartments. 49. •
Wiertz F_JHJ,Jones TR, Sun L, Bogyo M, Geuze HJ, Ploegh HL: The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Ce//1996, 84:769-779. This, together with [48"], demonstrates a novel mechanism for retrieval and degradation of endoplasmic reticulum proteins in the cytosol. These may serve as possible source of antigenic peptides derived from precursors normally present in membrane-bound subcellular compartments. 50.
Siliciano RF, Soloski MJ: MHC class I-restricted processing of transmembrane proteins. Mechanism and biological significance. J Immunol 1995, 155:1-5.
51.
Ferris RL, Buck C, Hammond SA, Woods AS, Cotter RJ, Takiguchi M, Igarashi Y, Ichikawa Y, Siliciano RF: Class I-restricted presentation of an HIV-1 gp41 epitope containing an Nlinked glycesylation site: Implications for the mechanism of processing of viral envelope proteins. J Immunol 1996, 156:834-840.
52.
Michalek MT, Grant EP, Gramm C, Goldberg AL, Rock KL: A role for the ubiquitin-dependent proteolytic pathway in MHC class I restricted antigen presentation. Nature 1993, 363:552-554.
53.
Cox JH, Galardy P, Bennink JR, Yewdell JW: Presentation of endogenous and exogenous antigens is not affected by inactivation of E1 ubiquitin-activating enzyme in temperaturesensitive cell lines. J Immuno/1995, 154:511-519.
37. e•
Bullock TNJ, Patterson AE, Franlin LL, Notidis E, Eisenlohr LC: Initiation codon scanthrough versus termination codon readthrough demonstrates strong potential for major histocompatibility complex class I-restricted cryptic epitope expression. J Exp Med 1997, 186:1051-1058. This report, together with [35"'-37"',39"'], shows that cryptic translation products are presented by MHC class I molecules and are capable of eliciting cytotoxic T lymphocyte-mediated immune responses.
38. •e
39. ••
Mayrand SM, Schwarz DA, Green WR: An alternative translational reading frame encodes an immunodominant retroviral CTL determinant expressed by an immunodeficiencycausing retrovirus. J Immunol 1998, 160:39-50. This report, together with [35"-38"'], shows that cryptic translation products are presented by MHC class I molecules and are capable of eliciting cytotoxic T lymphocyte-mediated immune responses.
Shastri N, Nguyen V, Gonzalez F: Major histocompatibility class I molecules can present cryptic translation products to T-cells. J Biol Chem 1995, 270:1088-1091.
41. •,
36. o•
Wang R-F, Parkhurst MR, Kawakami Y, Robbins PF, Rosenberg SA: Utilization of an alternative open reading frame of a normal gene in generating a novel human cancer antigen. J Exp Med 1996, 183:1131-1140. This report, together with [35",36"',38"',39"'], shows that cryptic translation products are presented by MHC class I molecules and are capable of eliciting cytotoxic T lymphocyte-mediated immune responses.
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54. ••
Goth S, Nguyen V, Shastri N: Generation of naturally processed peptide/MHC class I complexes is independent of the stability of endogenously synthesized precursors. J/mmunof 1996, 157:1894-1904. This describes the unexpected finding that presentation of endogenously synthesized proteins does not correlate with protein stability. 55.
Grant EP, Michalek MT, GoJdberg AL, Rock KL: Rata of antigen degradation by the ubiquitin-protaasome pathway influences MHC class I presentation. J Immunol 1995, 155:3750-3758.
56.
Silts AJ, Villanueva MS, Pamer EG: CTL epitope generation is tightly linked to cellular proteolysJs of a Listeria monocytogenes antigen. J Immunol 1996, 156:1497-1503.
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Buchholz D, Scott P, Shastri N: Presentation without processing of endogenous precursors in the MHC class I presentation pathway. J Bio/Chem 1995, 270:6515-6522.
58.
Rock KL, Gramm C, Rothstein L, Clark K, Stein R, Dick L, Hwang D, Goldberg AL: Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Ceil 1994, 78:761-771. Storkus WJ, Zeh Ill HJ, Salter RD, Lotze MT: Identification of T-cell epitopes: rapid isolation of class I-presented peptides from viable cells by mild acid elution. J/mmunother 1993, 14:94-103. Vinitsky A, Anton LC, Snyder HL, Orlowski M, Bennink JR, Yewdell JW: The generation of MHC class I-associated peptides is only partially inhibited by proteasome inhibitors: involvement of nonproteasomal cytosolic proteases in antigen processing? J/mmuno/1997, 159:554-564.
59.
60.
61.
Del Val M, Schlicht H-J, Ruppert T, Reddehase MJ, Koszinowski UH: Efficient processing of an antigenic sequence for presentation by MHC class I molecules depends upon its neighboring residues in the protein. Cell 1991, 66:1145-1153.
62.
Eisenlohr LC, Yewdell JW, Bennink JR: Flanking sequences influence the presentation of an endogenously synthesized peptide to cytotoxic T lymphocytes. J Exp Med 1992, 175:481 487. Shastri N, Serwold T, Gonzalez F: Presentation of endogenous peptide-MHC class I complexes is profoundly influenced by specific C-terminal flanking residues. J Immuno/1995, 155:4339-4346. Yellen-Shaw AJ, Wherry EJ, Dubois GC, Eisenlohr LC: Point mutation flanking a CTL epitope ablates in vitro and in vivo recognition of a full-length viral protein. J/mmuno/1997, 158:3227-3234. Niedermann G, Butz S, Ihlenfeldt HG, Grimm R, Lucchiari M, Hoschutzky H, Jung G, Maier B, Eichmann K: Contribution of proteasome-mediated proteolysis to the hierarchy of epitopes presented by major histocompatibility complex class I molecules, immunity 1995, 2:289-299.
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Dick TP, Ruppert T, Groettrup M, KIoetzel PM, Kuehn L, Koszinowski UH, Stevanovic S, Schild H, Rammensee HG: Coordinated dual cleavages induced by the proteasome regulator PA28 lead to dominant MHC ligands. Ce//1996, 86:253-262. Ossendorp F, Eggers M, Neisig A, Ruppert T, Groettrup M, Slits A, Mengede E, Kloetzel PM, Neefjes J, Koszinowski LJ, Melief C: A single residue exchange within a viral CTL epitope alters proteasome-mediatad degradation resulting in lack of antigen presentation. Immunity 1996, 5:115-124. Neisig A, Roelse J, Slits AJAM, Ossendorp F, Feltkamp MCW, Kast WM, Melief C.IM, Neefjes JJ: Major differences in transporter associated with antigen presentation (TAP)-dependent
translocation of MHC class i-presentable peptides and the effect of flanking sequences. J Immuno11995, 154:1273-1279. 69. •
Craiu A, Akopian T, Goldberg A, Rock KL: Two distinct proteolytic processes in the generation of a major histocompatibility complex class I-presented peptide, Proc Nat/ Aca# Sci USA 1997, 94:10850-10855. This work leads to the suggestion that mechanism for proteolytic cleavage of the amino- or the carboxy-terminal flanking residues may differ with regard to the requirement for the proteosome. 70.
71.
Falk K, Rstzschke O, Rammensee H-G: Cellular peptide composition governed by major histocompatibility complex class I molecules. Nature 1990, 348:248-251. Malarkannan S, Goth S, Buchholz DR, Shastri N: The role of MHC class I molecules in the generation of endogenous
peptide/MHC complexes. J Immunol 1995, 154:585-598. 72.
Shastri N, Serwold T, Gonzalez F: Presentation of endogenous peptide/MHC class I complexes is profoundly influenced by specific C-terminal flanking residues. J Immuno11995, 155:4339-4346.
73.
Serwold T, Gonzalez F, Shastri N: Do MHCl molecules select peptides from a random or a nonrandom pool? J A//erg C/in /mmuno/1997, 99:$253.
74.
Arnold D, Faath S, Rammensee HG, Schild H: Cross-priming of minor histocompatibility antigen-specific cytotoxic T ceils upon immunization with the heat shock protein gp96. J Exp Med
75.
Suto R, Srivastava PK: A mechanism for the specific immunogenicity of heat shock protein-chaperoned peptides. Science 1995, 269:1585-1588.
1995, 182:885-889.
76. eo
Tamura Y, Peng P, Liu K, Daou M, Srivastava PK: Immunotherepy of tumors with autologous tumor-derived heat shock protein preparations. Science 1997, 278:117-120. This report, together with [77"'], demonstrates that gp96-associated peptides can elicit protective anti-tumor responses and that the presence of some of these peptides is transporter associated with antigen processing (TAP) dependent. 77. •*
Arnold D, Wahl C, Faath S, Rammensee HG, Schild H: Influences of transporter associated with antigen processing (TAP) on the repertoire of peptides associated with the endoplasmic reticulum-resident stress protein gp96. J Exp Med 199?, 186:461-466. This report, together with [76"], demonstrates that gp96-associated peptides can elicit protective anti-tumor responses and that the presence of some of these peptides is transporter associated with antigen processing (TAP) dependent. 78.
Nieland TJF, Agnes MC, Tan A, Monnee-Van Muijen M, Koning F, Kruisbeek AM, Van Bleek GM: Isolation of an immunodominant viral peptide that is endogenously bound to the stress protein GP96-GRP94. Proc Nat/Acad Sci USA 1996, 93:6135-6139. The first report containing biochemical evidence that gp96 is bound to a known naturally processed antigenic peptide. •
Reading within the lines: naturally processed peptides displayed by MHC class I molecules Nilabh Shastri*, Thomas Serwold and Pedro Paz A typical mammalian cell contains tens of thousands of different gene products. Snippets of this genetic information are displayed on the cell surface by MHC class I molecules as short peptides for immune surveillance by CD8 + T lymphocytes. Genetic and biochemical analysis of these peptides is revealing novel sources and mechanisms by which these peptide/MHC class I complexes arise.
Addresses Division of Immunology, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA *e-mail: [email protected] Correspondence: Nilabh Shastri Current Opinion in Immunology 1998, 10:137-144
http:/Ibiomednet.com/elecref10952791501000137 © Current Biology Ltd ISSN 0952-7915
Abbreviations APC antigen-presentingcell ER endoplasmicreticulum HPLC high performance liquid chromatography ORF open reading frame TAP transporterassociated with antigen processing
Introduction
Immune surveillance by the CD8 + T cell repertoire is a mechanism for detecting and eliminating abnormal cells. These could be cells infected with viruses or bacteria, tumors, allogeneic tissues in transplantation or even self-tissues in autoimmunity. T h e antigen receptors of CD8 ÷ T cells examine the diverse repertoire of peptide/MHC complexes on the target cell surface for novel peptides that indicate expression of new or abnormal gene products. T h e peptides displayed by the MHC thus represent snippets of the cellular protein milieu. The antigen-processing pathway that extracts these internal peptides from their precursor polypeptides and leads to the expression of the informative peptide/MHC display on the surface can be considered a mechanism that 'reads' within 'lines' of amino acid sequences. In this review we focus on the antigen-processing pathway, for peptides presented by MHC class I, from the perspective of the antigen, particularly that synthesized within the antigen-presenting cell (APC) itself. Intracellular proteins undergo proteolysis to generate fragments, which are actively transported into the endoplasmic reticulum (ER) for assembly with the MHC class I molecules before being transported to the cell surface. Several key molecules involved in these steps have been subjects of excellent recent reviews [1-3]. These include the multicatalytic proteasome, the ER chaperones calnexin
and calreticulin, the peptide transporter TAP (transporter associated with antigen processing) and the recently discovered protein - - tapasin - - which bridges TAP and the MHC [4,5]. The output of the antigen-processing
pathway
The final product of the antigen-processing pathway is a short peptide bound to the MHC class I molecule on the cell surface (Figure 1). Crystal structures of several peptide/MHC class I complexes have shown that each peptide is primarily tethered to the MHC molecule by its amino- and carboxyl-termini, the backbone and the side chains of some internal residues [6]. T h e complementary pockets in the MHC molecules that interact with these features of the peptide vary due to extensive polymorphism. As a result, each MHC molecule prefers sets of peptides with distinct consensus sequences. T h e consensus sequences for MHC-bound peptides were first established by pool sequencing that allows the conserved 'anchor' residues to be distinguished from other more diverse residues [7]. T h e peptide consensus sequences have since been determined for a large number of MHC molecules [8,9]. In general the peptides bound to MHC class I are 8-10 residues in length, contain one or more conserved internal residue and share an aliphatic, aromatic or positively charged carboxyl-terminus. Knowledge of these conserved consensus motifs allows predictions of candidate antigenic peptides within protein sequences from antigens which stimulate T cells [10,11]. Exceptions to the conserved peptide sequence motifs have been found [12,13"',14"°], but what fraction of MHC-bound, naturally processed, peptides falls into this category is not yet known. T h e assignment of an amino acid sequence as the antigenic entity is demonstrated by the ability of synthetic peptides of this sequence to stimulate T cells when presented by appropriate MHC-expressing APC. In these assays, the synthetic analogue of the naturally processed peptide is usually the most active and a response by T cells is readily detectable at picomolar concentrations of peptide. Whether the active synthetic peptide actually corresponds to the naturally processed peptide is generally established by the overlap of the high performance liquid chromatography (HPLC) elution profile of the synthetic peptide with that of peptide extracts from antigen-expressing cells. This can be a technically demanding undertaking because firstly, the naturally processed peptide are expressed at low levels, secondly, the extraction efficiency varies with the chemical characteristics (e.g. hydrophobicity) of the antigenic peptide and thirdly, the HPLC set-up can be easily contaminated if a
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Immunologicaltechniques
Figure 1
APC surface
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MHC/peptide
/~
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• Proteolysis
TCR Antigenic peptide
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• TAP transport/ • Peptide/MHC trimming assembly
Input
° Cell surface expression
Output Current Opinion in Immunology
Schematic view of the output, input and intervening steps in the classical MHC class I antigen processing pathway. Translated products of the mRNA serve as the input, then undergo proteolysis in the proteasome. TAP transport and potential cytoplasmic and ER trimming events occur during assembly of the peptide/MHC class I complex which is then transported to the cell surface. The peptide/MHC class I complex is offered on the APC surface as a potential ligand for the CD8 + T cells.
high concentrations of synthetic peptides are injected in order to establish their elution profiles. T h e impact of these factors can be determined by assaying HPLC fractions of mock-injections (of buffer alone) or extracts of experimental samples spiked with known amounts of synthetic peptides. Routine inclusion of these controls is critical for demonstrating the identity and abundance of naturally processed peptides and the absence of artefacts resulting from carry-over from previous samples. As an example of this analysis, consider the p2Ca peptide L S P F P F D L (in single-letter code for amino acids), which was identified as the naturally processed peptide presented by the L d MHC class I molecule to the 2C T cell clone [12,15]. T h e QL9 analogue of p2Ca, with the naturally-occurring amino-terminal glutamine (Q) flanking residue (Figure 2) is a hundred fold more active in stimulating the 2C T cells either in assays in which it is added exogenously [16], or in assays in which it is expressed endogenously in L d ceils (Figure 2). The higher activity of the QL9 DNA construct suggests that QL9 rather than p2Ca could be the naturally processed analogue. Indeed, analysis of HPLC fractionated extracts of Ld cells expressing Met-QL9 and Met-p2Ca showed that only the QL9 rather than the p2Ca peptide was detected in the cellular peptide pool. Thus, QL9, rather than the p2Ca peptide is likely to represent the major naturally processed Ld-bound ligand for the 2C T cell clone.
the MHC-bound peptide pool. In human melanoma cells, a tyrosinase peptide, YMDGTMSQV, was presented by HLA-A2.1 MHC with a post-translationally modified aspartate rather than the natural asparagine residue [17°°]. Another peptide derived from the human Y-chromosome encoded SMCY protein was presented by MHC as a cysteinylated derivative [18°°]. These precedents suggest that peptides with other post-translational modifications such as phosphorylation or glycosylation may also exist. Since these modifications are post-translational, they were not evident in the primary protein sequences and were in fact identified by innovative mass spectrometry techniques. The existence of these post-translational modifications have provided further insights into potential mechanisms for the processing of compartmentalized proteins (see below). The abundance of individual peptide/MHC complexes on the cell surface varies widely. Estimates based upon the recovery of peptides in the MHC-bound pool show that while few peptides are present at a high level, the vast majority are present at <100 copies/cell [8,19] Accordingly, several T cell stimulating peptides have been found to be present at <10-100 copies/cell [13"°,14",20]. Notably, under experimental conditions and particularly in infected cells, the expression of individual peptides can increase by several magnitudes [21,22] (P Paz, N Shastri, unpublished data).
Precursors for antigen processing Discrepancies between the predicted and observed T-cell stimulating activity or HPLC elution profiles led to the discovery of post-translationally modified peptides among
With the exception of two viral proteins no endogenously synthesized protein has so far been shown to be excluded from entry into the antigen processing
Naturally processed peptides displayed by MHC class I molecules Shastri, Serwold and Paz
139
Figure 2
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The identification of the QLg, but not p2Ca peptide among the naturally processed peptide pool in cells expressing Ld MHC and Met-p2Ca (Mp2Ca) or Met-QL9 (MQLg) precursors. Recipient COS cells were untransfected or electroporated with cDNA constructs expressing Ld MHC alone (Ld), with Ld plus Mp2Ca (Ld + Mp2Ca) or with Ld plus MQL9 (Ld + MQLg) precursors. Two days later, the untransfected or transfected cells were (a) tested directly for their ability to stimulate the lacZ activity induced in the 2CZ hybrid or (b,e) extracted with 0.20/0 (v/v) trifluoroacetic acid in water. The lacZ activity was measured as the absorbance of the cleaved product of the lacZ substrate chlorophenol red I~-galactosideat 595 nm. The extracts were fractionated by HPLC using a 10 mM triethylamine acetate, pHS.5 (Buffer A) and 10 mM triethylamine acetate, pH5.5, in acetonitrile (Buffer B) gradient. Fractions, which were collected every 3Osec while Buffer B concentration increased from 21.5% to 33°/0 in Buffer A, were dried in a vacuum centrifuge, resuspended in phosphate-buffered saline and tested for stimulating 2CZ T cells with Ld+ L cells as APC. The background response to COS cells transfected with Ld alone has been subtracted to show that the activity is due to transfected antigen genes. The arrows indicate the retention times of synthetic QL9 and p2Ca peptides determined under identical run conditions.
pathway [23,24°,25°]. These peptide/MHC complexes serve as ligands for T cell responses elicited to tumors, allogeneic tissue transplants and virally infected cells. In addition, peptide/MHC complexes can be generated from exogenous proteins that are introduced into the cytosol [26]. This process occurs naturally in cells infected with intracellular pathogens such as Listeria monocytogenes that escape into the cytosol, thus allowing their secreted proteins to gain access to the MHC class I presentation pathway [27]. Intriguingly, in certain cell types of the macrophage/dendritic lineage, antigens taken up into pinocytic or endocytic vesicles can also access the MHC class I antigen presentation pathway [28-31]. This unconventional method of accessing the MHC class I presentation pathway may be relevant to presentation of peptides from proteins synthesized in the mitochondria
and to the initiation of immune responses by cells that do not express the appropriate MHC molecules. Intriguingly, MHC class I molecules also present peptides that are encoded within regions of transcripts that are not expected to be normally translated [32-34,35°°-39°°]. These regions include 5' and 3" 'untranslated' sequences, alternate translational reading frames and translational read-through into partially spliced mRNA (Figure 3). Again, what fraction of the normal peptide pool represents such peptides is not known and would be difficult to determine given their low abundance [34, 401. Nevertheless, these cryptic translation products are capable of eliciting T cell responses in rive [35"°,36°',39°°]. Presently it is unclear whether MHC present these peptides because they are presumably labile and hence, the preferred
140
Immunological techniques
substrates for the antigen processing pathway [41"'], or whether they are generated by a novel translational mechanism that feeds its products into the antigen processing pathway. Regardless of the mechanism for their generation, the existence of these peptides shows that immune surveillance may operate upon the total coding information of cellular transcripts. Accordingly, it would be more accurate to conceptualize the mRNAs, rather than their predominant protein products, as the input of the antigen processing pathway (Figure 3). Note that the origin of the processed peptide, whether it is derived from a major or minor open reading frames (ORFs) is irrelevant for immune surveillance. Site(s) of antigen
processing
No intracellular site appears exempt from providing precursors for antigen processing to the peptide/MHC class I complex. The dramar~c phenotype of TAP deficient cells, with low levels of MHC class I expression, suggests that peptides destined to bind MHC come from the cytosol [1,42]. Although exceptions to the TAP requirement exist for some proteins that are targeted into the ER [43-45], or a few membrane proteins [46,47], the bulk of MHC-bound peptides require TAP indicating that the cytosol is the primary site for antigen processing. How do proteins in membrane-bound intracellular compartments, such as the ER, become available for antigen processing in the cytoplasm? One model envisages that proteins from these compartments are retrieved for
degradation into the cytosol. The discovery of the post-translationally modified peptide with an Asp instead of the natural Asn residue (referred to above) is consistent with the view that the precursor was glycosylated within the ER and retrieved into the cytosol where enzymatic deglycosylation and conversion of the Asn to Asp occurred. Accordingly, the presentation of the peptide/MHC complex was TAP dependent [17"',48"']. Interestingly the existence of a retrieval mechanism for ER proteins was also suggested by the inhibitory effect of viral proteins on antigen presentation [49"]. Whether a similar protein retrieval/degradation pathway exists for other sub-cellular compartments, for example the nucleus or mitochondria, is not known. Alternatively, newly synthesized polypeptides that fail to reach their destination may serve as precursors for antigen processing [50,51]. Because protein synthesis occurs exclusively in the cytoplasm, this model can account for the TAP dependent presentation of any subsequently compartmentalized protein. While attractive in its elegance for explaining the source of all processed peptides, the model does not adequately explain the existence of post-translationally modified peptides (discussed above). The murky middle
steps
It is not known when endogenously synthesized proteins enter the antigen processing pathway. Unlike exogenous proteins, endogenously synthesized proteins can, in principle, be sampled for antigen processing at any stage between their biosynthesis and their turnover. Whether the sampling mechanisms involves the ubiquitin-dependent degradation pathway in antigen processing is uncertain
Figure 3
Origin of pepUdes presented by MHC class I molecules
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Current Opinion in Immunology
Schematic depiction of the origin of peptides presented by MHC class I molecules from predominant open reading frame (ORF, RFO), depicted above the mRNA shown as a straight line, as well as from other 'non-translated' regions shown below the mRNA. These include the 5' and 3' non-coding regions, as well as alternate translational reading frames, RF1 and RF2. The naturally processed peptide is depicted as a solid circle, and its polypeptide precursor sequences as wavy lines. Other mRNA features, the 5' mTG CAP,the 3' polyA tail and the Kozak context (ryyryyATGg) of the translational initiation codon (TAG), as determinants of normal translational efficiency are also indicated.
Naturally processed peptides displayed by MHC class I molecules Shastri, Serwold and Paz
J0
141
[52,53]. T h e observation that proteins may be selected at an early stage for processing is consistent with the observation that presentation of endogenously synthesized proteins is independent of their stability [54°°]. By contrast, the efficiency of presentation of exogenous proteins correlates with their relative instability [55,56]. These conflicting findings may be reconciled if ports of entry for precursors of both newly synthesized and turned-over proteins were available which lead into the antigen processing pathway.
Figure 4
Regardless of the source and the sampling criteria, and unless the precursor exactly matches the product [57], the antigenic precursors must undergo proteolysis to generate the precisely cleaved peptide/MHC complex. T h e multicatalytic proteasome is an important player in this step, because proteasome inhibitors reduce the efficiency of antigen presentation to T cells [58]. This is also evident from measurement of the level of newly assembled peptide/MHC complexes in viable cells after a quick acid-wash (which removes pre-existing peptides from the cell surface) [59]. T h e mean fluorescence intensity following staining of EL-4 cells with antibodies to Db MHC dropped from 33.8 (untreated) to 4.69 immediately after acid-wash, but increased to 22.6 after four hours in normal medium (Figure 4). This recovery of D b MHC represents newly assembled peptide/MHC complexes and this was inhibited when the proteasome inhibitors L L n L or lactacystin were included in the recovery phase (MFI=15.1 and 14.7 respectively for cells incubated for 4hr in proteasome inhibitors after acid-wash). Thus, the recovery in presence of proteasome inhibitors is partial, suggesting that other proteases may also be involved in generating antigenic peptides. T h e existence of these other yet unidentified proteases in trimming antigenic peptides has also been suggested by the lack of an effect of proteasome inhibitors and even an increase in presentation activity with some precursors in cytotoxicity assays [60].
Expressio,n of most, but not all, peptide/MHC class I complexes depends upon proteasome function. The expression of D b MHC on the surface of EL-4 cells was measured as fluorescence intensity using flow cytometry using a fluorescently conjugated antibody (B22.249). The left panel shows EL-4 cells that were either untreated, or were incubated for 3 rain at room temperature in pH3.1 citrate buffer, then were washed and were stained either immediately (acid wash; no recovery) or following incubation for 4 hr in normal medium at 37" (acid wash; 4 hr recovery). The right panel shows D b expression in acid-washed cells that were treated with the proteasome inhibitors LLnL (100p.M) or lactacystin (Lcyst, 100mM) prior to and during the 4 hr recovery phase. Both LLnL and Lcyst partially inhibit the recovery of D b MHC. Similar results were also obtained for Kb MHC expression (not shown).
1O0. Acid wash /t Acidwash4hrhrecovery J no recovery ; ~ treated °oo
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;b2
Flourescence intensity
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or ++ILclan
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Many studies have shown that flanking sequences influence antigen presentation [61-64]. It is possible that flanking sequences alter the efficiency with which the antigenic peptides are liberated from their precursor [65-67], transported by TAP [68] or interact with MHC molecules in the ER ( T Serwold, N Shastri, unpublished data). Interestingly, proteasome inhibitors have a differential effect on cleavage of amino- versus carboxy-terminal flanking residues suggesting that the precise carboxyl- and amino-termini are generated by distinct proteolytic mechanisms [69"].
processed peptides in cell extracts are recovered only when the cells express the appropriate MHC molecules [70,71]. It is possible that the failure to detect processed peptides, that are presumably generated independently of the MHC, is due to their extreme instability, or poor extraction efficiency in the absence of the MHC molecule. Alternatively, the inability to detect these proteolytic intermediates may be due to sensitivity limits of the assays used for their detection. In contrast to the final peptide product that is active at picomolar concentrations, the presence of even a single additional residue causes a dramatic reduction in the ability to stimulate T cell activity and makes it very unlikely that peptides other than the final peptide will be detected in standard assays [71,72]. This difficulty was partially overcome by treating cell extracts with a carboxypeptidase that removed the inhibitory carboxy-terminal flanking residue, and together with HPLC analysis of cell extracts allowed detection of analogs extended at the carboxyl termini [73]. With this method, we have determined that cells generate not only the precisely cleaved final peptide product but also its analogue extended at the carboxyl terminus. This biochemically demonstrates that it is possible to generate more than one analogue of a peptide from a given precursor. Whether the longer analogue is an intermediate for the final, precisely cleaved, peptide is, however, not known.
Our current understanding of the antigen-processing pathway is primarily based upon analysis of the products and their precursors. Defining the proteolytic intermediates with or without additional flanking residues is important for furthering our understanding of this pathway. It has been evident from the earliest studies that the naturally
The ER chaperone gp96 is another source of potential processing intermediates. Several studies have shown that peptides associated with gp96 can elicit effective immune responses specific for the ceils from which they were derived [74,75,76ee]. Because the existence of at least some of these antigenic peptides depends upon TAP
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Immunological techniques
there is suggestion that gp96-bound peptides may be precursors for those eventually bound to the MHC [77"']. With the exception of one viral peptide [78°], however, the composition of the gp96-bound peptide pool and its potential role in the antigen-processing pathway remains unknown.
Conclusions T h e path from the antigenic precursor to the naturally processed peptide/MHC class I product is not straightforward. T h e clarity of the picture of the peptide/MHC complex becomes increasingly murky as one moves backwards to trace the intervening steps that account for its being. Nevertheless, answers to questions of the source of naturally processed peptides and the mechanisms that regulate their generation and abundance have revealed fundamental insights into novel translational and intracellular protein trafficking and degradation mechanisms, and more will certainly follow. In the future, we hope to learn how precursors (major or minor ORFs) are sampled for antigen processing, the identity of proteolytic intermediates and how they are delivered to TAP, as well as the extent and mechanism of ER trimming. T h e central importance of the antigen-processing pathway in generating the peptide/MHC ligands that regulate CD8 + T cell behavior from their generation in the thymus, to their eventual activation and death in the periphery, suggests that the pathway and its physiological manifestations will continue to receive intense scrutiny.
Acknowledgements We are grateful to members of our laboratory for stimulating discussions. Research in this laboratory is supported by grants from the National Institutes of Health.
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