Have we cut ourselves too short inmapping CTL epitopes?Scott R. Burrows1, Jamie Rossjohn2 and James McCluskey3

1Queensland Institute of Medical Research, 300 Herston Road, Brisbane, Qld 4029, Australia2The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash

University, Clayton, Vic 3800, Australia3Department of Microbiology and Immunology, University of Melbourne, Parkville, Vic 3010, Australia

MHC class I molecules generally present peptides of

eight to ten amino acids; however, peptides of 11–14

residues can also elicit dominant cytotoxic T lymphocyte

responses, sometimes at the expense of overlapping

shorter peptides. Although long-bulged epitopes are

considered to represent a barrier for T cell receptor

recognition, recent structural data reveal how these

super-bulged peptides are engaged while simul-

taneously maintaining MHC restriction. We propose

that algorithms widely used to predict class I-binding

peptides should now be broadened to include peptides

of over ten residues in length.

Mapping cytotoxic T lymphocyte epitopes

CD8C T cells recognize peptide fragments – liberated fromantigen through various proteolytic mechanisms – thatare subsequently presented on the cell surface within thebinding groove of the MHC class I molecule. A diversearray of antigenic peptides is ligated by these MHCmolecules, owing to the high degree of polymorphismwithin the six pockets of the antigen-binding cleft. Overthe past two decades, a major effort by the immunologycommunity has been directed towards defining the preciseantigenic peptides from within viral and tumor antigensthat are the targets for cytotoxic T lymphocyte (CTL)recognition, primarily for application in vaccine develop-ment and, more recently, in monitoring T cell responses.Initially, CTL epitopes were mapped using sets ofoverlapping peptides covering the entire sequence of theantigen of interest. A major breakthrough leading to moreeffective and efficient CTL epitope mapping was thediscovery that, for most MHC alleles, two of the pocketswithin the peptide-binding groove display a markedpreference for one or two amino acids at certain positionswithin the peptide [1]. Peptide binding motifs definingprimary ‘anchor’ residues responsible for making highlyconserved and energetically important MHC contactswere subsequently identified for many class I alleles.Positional scanning with random peptide libraries [2] andanalysis of natural ligands by Edman degradation [3] andmass spectrometry [4] have led to refined predictivealgorithms incorporating knowledge about secondaryanchor residues and disfavored amino acids. Peptide–

Corresponding author: Burrows, S.R. (Scott.Burrows@qimr.edu.au).Available online 16 November 2005

www.sciencedirect.com 1471-4906/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved

MHC class I predictive algorithms have become powerfultools, widely used to identify potential epitopes beforebiological validation, and listings of many motifs are freelyaccessible on the internet [5,6].

Although most currently known CTL epitopes wereidentified after computational prediction, systematicevaluation of this approach has revealed some limitations[7]. There are numerous examples of atypical ligands thateither do not conform to accepted motifs or are post-translationally modified sequences [8]. Furthermore,many predicted epitopes are nonimmunogenic as a resultof their failure to be processed and presented on antigen-presenting cells or limitations in the T cell repertoire [9].

Unusually long CTL epitopes

Historically, prediction of MHC class I ligands has beeneasier than that of their class II counterparts, owing to alength restriction resulting from structural features of theclass I peptide-binding cleft and an important set ofhydrogen bonds between the amino- and carboxy-peptidetermini and groove residues within pockets A and F, whichare highly conserved between different class I allomorphs[10]. The length of CTL epitopes is also restricted by theligand specificity of the class I antigen-processing machin-ery [11]. The proteasome complex generates fragmentswith a Gaussian-like length distribution, with a pre-ference for peptides containing eight to 11 residues [12].Peptide transport by the human transporter associatedwith antigen processing (TAP) also favors 9–11merpeptides, although much longer peptides are also boundand translocated [13]. Based on this information, web-based algorithms are currently designed to predict MHCclass I ligands limited in length to between eight and tenresidues [5,6].

Despite these restrictions, some longer peptides canalso bind to MHC class I molecules [5,14–43]. Theseinclude naturally presented self-peptides that have beeneluted from class I molecules, of which at least 5% are overten amino acids in length [42]. Six class I-bound peptidesthat are longer than ten residues have been characterizedstructurally and, in each case, the peptide adopts abulging conformation while maintaining the conservedhydrogen bonding network at the peptide amino- andcarboxy-termini [15,17,18,21,26,43] (Figure 1). Thesebulged epitopes can either be markedly rigid or display

Opinion TRENDS in Immunology Vol.27 No.1 January 2006

. doi:10.1016/j.it.2005.11.001

Figure 1. A super-bulged 13mer peptide (blue) protrudes markedly out of the

antigen-binding cleft of HLA B35 in comparison with the more standard 8mer

peptide (green) [15]. The 13mer peptide is from the EBV antigen BZLF1

(52LPEPLPQGQLTAY64) and the 8mer peptide is from HIV1 Nef (75VPLRPMTY82).

The proline residues within the longer epitope provide rigidity.

Opinion TRENDS in Immunology Vol.27 No.1 January 200612

a considerable degree of flexibility. It was suggested thatpeptides bound with a bulging conformation prevent manyT cell receptors (TCRs) from approaching the surface ofthe MHC molecule, thereby limiting the potential immu-nogenicity of unusually long class I-binding antigenicpeptides [17]. However, there are now numerous reports ofunusually long CTL epitopes of over ten amino acids inlength presented by MHC class I molecules, defined usingalternative approaches other than computer-based pre-diction algorithms (Table 1).

An extreme example is a 14mer epitope derived from analternative open reading frame of the macrophage colony-stimulating factor gene (alt.M-CSF), which is naturallypresented by HLA-B*3501 for recognition by tumor-

Table 1. Unusually long CTL epitopes

MHC Peptidea Length

H2-Db SGVENPGGYCL 11

H2-Ld IPQSLDSWWTSL 12

HLA-A*01 YSEHPTFTSQY 11

SSDYVIPIGTY 11

FLEGNEVGKTY 11

HLA-A*0201 MLMAQEALAFL 11

GLAPPQHLIRV 11

LLPENNVLSPL 11

HLA-A*03 RLRDLLLIVTR 11

HLA-A*1101 ACQGVGGPGHK 11

SVLGPISGHVLK 12

HLA-A*3101 STLPETTVVRR 11

HLA-A*6801 FVFPTKDVALR 11

HLA-B*0702 SPSVDKARAEL 11

RPHERNGFTVL 11

RPQGGSRPEFVKL 13

HLA-B*2702 RRARSLSAERY 11

HLA-B*3501 HPVGEADYFEY 11

EPLPQGQLTAY 11

LPAVVGLSPGEQEY 14

TPRLPSSADVEF 12

HLA-B*3508 LPEPLPQGQLTAY 13

CPSQEPMSIYVY 12

HLA-B*44 SELFRSGLDSY 11

HLA-B*5703 KAFSPEVIPMF 11aPrimary anchor residues shown in bold.bAbbreviations: EBNA, Epstein–Barr virus nuclear antigen; HBV, hepatitis B virus; LCM

recognized by T cells; CAMEL, CTL-recognized antigen on melanoma; HIV, human imm

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infiltrating CTLs [19,21]. Recent studies have alsoidentified a highly immunogenic 13mer epitope from theBZLF1 antigen of Epstein–Barr virus (EBV) that ispresented by HLA-B*3508 [15,22,44]. Although up to 2%of CD8C T cells from healthy, virus-exposed individualsare dedicated to recognizing this single 13mer CTLepitope, the highly restricted TCR repertoire used in thisresponse is consistent with TCR–MHC binding con-straints imposed by the highly prominent bulged peptideconformation [15,44]. A similarly restricted TCR reper-toire was reported in the very potent CTL response to anHLA-B*3501-bound 11mer epitope, also from the BZLF1antigen of EBV and overlapping with the B*3508-binding13mer [26]. CTL recognition of bulky glycopeptides thatalso project significantly above the level of the a1 and a2helices is also characterized by restricted TCR usage [45].HLA-B*3508 also presents a highly immunogenic 12merCTL epitope from the pp65 antigen of human cytomega-lovirus (HCMV), with up to 4% of CD8C cells specific forthis peptide in healthy HCMV-seropositive individuals[46]. Another 13mer epitope that binds to HLA-B*0702was recently described from the BMRF1 antigen of EBV[47] (Table 1).

Some of these unusually long epitopes are completelyoverlapping with sequences of more conventional lengththat score very well using predictive class I-bindingalgorithms but nevertheless are not reported to induceCTL responses (Table 1). For example, the HLA-A*0201-binding p53 epitope GLAPPQHLIRV (primary anchorresidues are in bold) includes the nonamer sequence(GLAPPQHLI) that binds well to HLA-A*0201 but isessentially nonimmunogenic [48]. Similarly, the highlyimmunogenic HLA-B*3501-binding 11mer epitopeHPVGEADYFEY from EBV nuclear antigen 1 is

Antigenb Refs

LCMV GP 278–286 [27]

HBV surface antigen 28–39 [28]

HCMV pp65 363–373 [29]

Tyrosinase 128–138 [30]

MART-2 381–391 [23]

CAMEL 1–11 [31]

p53 187–197 [48]

p53 25–35 [24]

HIV-1 env gp41 768–778 [32]

HIV-1 gag p24 217–227 [33]

HCMV pp65 13–24 [14]

HBV core antigen 141–151 [50]

HCMV pp65 186–196 [34]

miHAg H-Y 1041–1051 [35]

HCMV pp65 265–275 [36]

EBV BMRF1 116–128 [47]

EBV EBNA-4 243–253 [37]

EBV EBNA-1 407–417 [49]

EBV BZLF1 54–64 [22,26]

alt.M-CSF 4–17 [19,21]

Tyrosinase 309–320 [25]

EBV BZLF1 52–64 [15,22,44]

HCMV pp65 103–114 [46]

MUM-2 123–133 [38]

HIV-1 gag p24 30–40 [43]

V GP, lymphocytic choriomeningitis virus glycoprotein; MART, melanoma antigen

unodeficiency virus; miHAg, minor histocompatibility antigens.

Opinion TRENDS in Immunology Vol.27 No.1 January 2006 13

completely immunodominant over shorter peptides(HPVGEADY and HPVGEADYF) that conform to thebinding motif of this HLA allele [49]. The HLA-A*3101-binding hepatitis B virus 11mer epitope STLPETTVVRR,which is also immunogenic in the context of HLA-A*6801,is targeted by the CTL response in preference to theshorter STLPETTVVR sequence [50].

Another intriguing example of immunodominance bylonger peptides is the LPEPLPQGQLTAY 13mer epitopefrom the BZLF1 antigen of EBV that is the target of a CTLresponse in HLA-B*3508C virus carriers [15,22,44].Binding assays have shown that this peptide associateswith a higher affinity with HLA-B*3508 than with thecompletely overlapping shorter sequences EPLPQGQL-TAY and LPQGQLTAY, thus providing a possible expla-nation for the immunodominance of the 13mer peptide[22,51]. The situation with HLA-B*3501C individuals ismore complex. In these individuals, the EPLPQGQLTAY11mer dominates the EBV-specific CTL response and yetthis peptide appears to bind B*3501 with equal affinity tothe overlapping nonamer and 13mer peptides [22].Interestingly, HLA-B*3508 differs from B*3501 at just asingle amino acid position (B*3501, 156Leucine; B*3508,156Arginine), which enables the formation of a stabilizingsalt bridge between the P3-Glu of the 13mer and the Arg156 [15]; this might explain the higher-affinity bindingand immunodominance of this peptide in B*3508C

individuals. The lack of any detectible CTL response tothe LPQGQLTAY nonamer peptide could be due toconstraints in amino-terminal trimming within theendoplasmic reticulum (ER) as a result of the upstreamproline residues. Future studies should be directedtowards understanding why unusually long CTL epitopesare sometimes immunodominant over completely over-lapping shorter peptides that also bind the sameMHC allele.

Figure 2. TCR recognition of a super-bulged peptide (a) in comparison with a normal leng

TCR (a chain in red, b chain in pink) with the MHC (heavy chain in purple, light chain in

bulged peptide (orange). The determination of this structure provides insights into the

(orange) protrudes minimally from the antigen-binding cleft of MHC (heavy chain in pu

complexes involves more extensive contacts with the MHC and fewer contacts with the

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A wide range of MHC class I alleles have the structuralcapacity to present peptide ligands of over ten amino acidsin length (Table 1). However, unusually long peptideligands have not been described for allotypes such as H2-Kb, HLA-B*0801 and HLA-B*14, presumably becausethese molecules generally require a primary anchorresidue in the central part of the peptide, as well asimportant primary or secondary anchors at the extremi-ties, which is unlikely to be compatible with the loopingconformation of long peptides. MHC alleles that preferpeptide ligands with proline at position 2 are over-represented (Table 1). These MHC class I molecules,which include the very common and widely distributedHLA-B7 supertype alleles in humans and H2-Ld in mice,might have evolved the capacity to bind long peptides tocompensate for the inhibitory effects of proline peptideresidues on the class I antigen-processing pathway.Translocation of cytosolic peptides into the ER by TAP ismuch less efficient when peptides contain proline residuesnear their amino-terminus [52], implying that only longerpeptides containing proline distal to the amino-terminuswill be transported by TAP. Moreover, the unique cyclicstructure of proline restricts its susceptibility to mostpeptidases, and an aminopeptidase associated withantigen processing in the ER (ER-associated aminopepti-dase) has recently been identified as a protease with broadspecificity but which is unable to cleave the bond at theamino side of proline. This results in an accumulation ofX-Pro-Xn peptides within the ER [53]. Another probableeffect of this limitation in peptide-trimming enzymes isthat many of the X-Pro-Xn peptides that accumulatewithin the ER will be relatively long. It would therefore beadvantageous if a proportion of class I MHC moleculesthat exhibit specificity for proline at P2 could presentpeptides longer than the canonical 8–10mer length. TheHLA-B7 supertype alleles might therefore be selected

th (9mer) peptide (b). (a) This complex highlights the minimal contacts made by the

green), in particular the a1 helix. The TCR makes more extensive contacts with the

minimum requirement for MHC restriction [44]. (b) A normal length peptide [62]

rple, light chain in green). TCR (a chain in red, b chain in cyan) recognition of such

peptide.

Opinion TRENDS in Immunology Vol.27 No.1 January 200614

based upon an ability to scavenge ‘indigestible’ proline-containing peptide ligands that are sometimes longer thanusual and that are unattractive to other class I types.

Using long CTL epitopes to investigate MHC restriction

Although noncanonical, longer epitopes are clearlyimportant in MHC class I-restricted immunity, themechanism of TCR binding to these peptides has beenan enigma because they expose many more of their sidechains than usual, and their bulging from the MHC cleftposes a steric challenge to MHC-restricted ab T cellrecognition. A recently determined structure of a TCR incomplex with an HLA-B*3508-bound 13mer epitope fromEBV has shed light on how the TCR repertoire accommo-dates such ligands, as well as redefining some of the ‘rulesof engagement’ for ab T cell recognition [44] (Figure 2).The bulged peptide does act as a barrier to the TCRengaging fully with the HLA molecule, with the interfacebeing dominated by peptide-mediated interactions ratherthan by MHC-mediated contacts. The peptide contributestwice as many contacts to the TCR interface whencompared with the MHC. Whereas one to three peptideresidues from most peptide–MHC ligands interact withthe TCR [54], seven residues from the 13mer epitopeinteract with this TCR. By contrast, the TCR makes anunusually small number of contacts with HLA-B*3508,interacting only with positions 65 and 69 on the a1 helixand residues 150–158 of the a2 helix. Nonetheless, theTCR is remarkably MHC restricted and binds its cognateligand well within the normal range of affinities observedfor other TCRs. Thus, constraints imposed by the bulgedpeptide in this complex have limited the number ofMHC–TCR contacts severely, and provide clear insightsinto the minimal requirements for MHC restriction.

Interestingly, positions 65 and 69 on the a1 helix and155 on the a2 helix are involved in TCR-mediated contactsin all TCR–peptide–MHC class I structures determined sofar [55–63]. Moreover, alanine-scanning mutagenesis hasalso revealed that these three positions represent ener-getic ‘hot-spots’, providing interactions which are crucialfor TCR recognition in two different TCR–peptide–MHCsystems [64–66]. Accordingly, positions 65, 69 and 155represent a generic MHC class-I restriction element.Given this minimally conserved TCR footprint on theMHC, it seems likely that co-receptors have a guiding rolein TCR–peptide–MHC recognition [67], as evident fromtheir role within the immunological synapse and sup-ported by evidence that T cells can be triggered to respondto a single peptide–MHC ligand on the cell surface[68–70]. CD8, for example, might bind directly orindirectly to the Va chain of the TCR, thereby guidingTCR alignment to the approximate diagonal orientationthat is usually observed with class I-restricted recognition[67]. Further structural studies of TCRs specific for othernoncanonical, longer MHC class I-restricted peptideepitopes might confirm a common minimal MHC class Irestriction element or could perhaps demonstrate MHC-restricted T cell recognition without significantTCR–MHC contacts, thereby indicating that MHC restric-tion is a consequence of coreceptor interactions and

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peptide–MHC binding specificity rather than an inherentpredisposition for TCR–MHC binding.

Concluding remarks

Although the MHC class I antigen-processing andantigen-presentation pathway is biased towards 8–10mer peptides, this length restriction is clearly not asstrict for some molecules as was originally thought. Thefrequency with which CTLs recognize unusually longpeptides is difficult to estimate at this stage, because thewidely used algorithms to predict class I-binding peptidesexclude peptides of more than ten amino acids in length.Furthermore, the ‘upper limit’ of MHC class I-restrictedepitope length is unclear at present. Nevertheless, wepropose that these binding algorithms should now bebroadened for many MHC class I alleles to includepeptides of up to 14 amino acids in length.

AcknowledgementsThis work was supported by grants from the National Health and MedicalResearch Council, Australia, the Roche Organ Transplantation ResearchFund, the Juvenile Diabetes Research Foundation and the AustralianResearch Council. J.R. is a Wellcome Trust Senior Research Fellow. Wethank Anthony Purcell for helpful discussion and members of theRossjohn, McCluskey and Burrows laboratories for theirresearch contributions.

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