of January 31, 2018.This information is current as

Restricted to a HLA-B8 Moleculeagainst an Immunodominant EBV Antigen

TCR UsageβαA Structural Basis for Varied

RossjohnPurcell, Scott R. Burrows, James McCluskey and JamieHalim, Yu Chih Liu, Lars Kjer-Nielsen, Anthony W. Stephanie Gras, Pascal G. Wilmann, Zhenjun Chen, Hanim

ol.1102686http://www.jimmunol.org/content/early/2011/12/02/jimmun

published online 2 December 2011J Immunol 

MaterialSupplementary

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The Journal of Immunology

A Structural Basis for Varied ab TCR Usage against anImmunodominant EBV Antigen Restricted to a HLA-B8Molecule

Stephanie Gras,* Pascal G. Wilmann,* Zhenjun Chen,† Hanim Halim,* Yu Chih Liu,*

Lars Kjer-Nielsen,† Anthony W. Purcell,‡ Scott R. Burrows,x James McCluskey,† and

Jamie Rossjohn*

EBV is a ubiquitous and persistent human pathogen, kept in check by the cytotoxic T cell response. In this study, we investigated

how three TCRs, which differ in their T cell immunodominance hierarchies and gene usage, interact with the same EBV

determinant (FLRGRAYGL), bound to the same Ag-presenting molecule, HLA-B8. We found that the three TCRs exhibit

differing fine specificities for the viral Ag. Further, via structural and biophysical approaches, we demonstrated that the viral

Ag provides the greatest energetic contribution to the TCR–peptide-HLA interaction, while focusing on a few adjacent HLA-

based interactions to further tune fine-specificity requirements. Thus, the TCR engages the peptide-HLA with the viral Ag as the

main glue, such that neighboring TCR–MHC interactions are recruited as a supportive adhesive. Collectively, we provide

a portrait of how the host’s adaptive immune response differentially engages a common viral Ag. The Journal of Immunology,

2012, 188: 000–000.

Epstein-Barr virus is a ubiquitous human pathogen, with∼90% of the population infected; once infected, the virusis rarely cleared from the host (reviewed in Refs. 1, 2).

EBV infection, although typically asymptomatic in immunocom-petent individuals, is the causative agent of infectious mononu-cleosis and has been linked to the development of several cancers,including Burkitt’s lymphoma, immunoblastic lymphoma, andHodgkin’s disease; certain forms of T cell lymphoma; and someepithelial tumors, including nasopharyngeal carcinoma and a pro-portion of gastric cancers. Viral infection and persistence areachieved through a balance of lytic (productive) and latent (non-productive) infections that are controlled by a series of lytic and

latent proteins, respectively. Primary infection with EBV is asso-ciated with the expression of a large number of lytic proteins thatcan be classified into three temporal phases: immediate early, early,and late. The latent proteins, of which the nuclear Ags predominate(termed Epstein-Barr virus nuclear Ag [EBNAs]), enable the virusto colonize the B cell pool through latent growth-transform-ing infection and, thereby, establish a reservoir of virus genome-positive cells upon which long-term virus persistence depends.Subsequent reactivation of the B cell reservoir from latency into thelytic cycle can then initiate secondary foci of virus replication atpharyngeal sites, leading to low-level shedding of infectious virusthroughout life.EBV is contained by a vigorous cellular immune response that

is directed against a specific subset of latent epitopes. The EBV-replicative lesions are subject to direct CTL control, in which theimmediate early and early proteins are frequently the immuno-dominant targets (1). To gain a greater understanding of CTLimmunity and Ag presentation, it is important to investigate spe-cific elements relating to the protective immune response to EBV.Indeed, EBV is an important natural infection that provides anideal model for investigating the antiviral immune response inhumans because of its ubiquity in human populations and becauseit is the best-known and most widely studied herpes virus as aresult of its clinical and oncogenic importance.The cytotoxic T cell response toward viruses is directed toward

class I MHC (MHC-I) molecules complexed to viral peptide Ags(pMHC-I). These complexes are expressed on the surface ofinfected cells and are specifically recognized by clonally distrib-uted ab TCRs on CD8+ T lymphocytes, a crucial cellular com-ponent of the immune system. Typically, the antigenic peptide istethered within the Ag-binding cleft of the MHC molecule, suchthat the peptide makes a number of specificity-determining con-tacts with the MHC, whereas the “upward-pointing” amino acidsrepresent potential TCR contact points (3). Appropriately armedand activated CD8+ T cells can eliminate infected cells and pre-vent viral replication. The specificity in the cellular immune re-sponse arises from an extensive TCR repertoire, as well as from

*Protein Crystallography Unit, Department of Biochemistry and Molecular Biology,School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Aus-tralia; †Department of Microbiology and Immunology, University of Melbourne,Parkville, Victoria 3010, Australia; ‡Department of Biochemistry and Molecular Bi-ology and Bio21 Molecular Science and Biotechnology Institute, University of Mel-bourne, Parkville, Victoria 3010, Australia; and xCellular Immunology Laboratory,Queensland Institute of Medical Research and Australian Centre for Vaccine Devel-opment, Brisbane, Queensland 4029, Australia

Received for publication September 19, 2011. Accepted for publication November 2,2011.

This work was supported by the National Health and Medical Research Councilof Australia, the Roche Organ Transplantation Research Foundation, and the Aus-tralian Research Council. S.R.B. and A.W.P. are supported by the National Healthand Medical Research Council Senior Research fellowships, and J.R. is supported bya National Health and Medical Research Council Australia fellowship.

Address correspondence and reprint requests to Dr. Jamie Rossjohn or Dr. JamesMcCuskey, Protein Crystallography Unit, Department of Biochemistry and Molecu-lar Biology, School of Biomedical Sciences, Monash University, Clayton, VIC 3800,Australia (J.R.) or Department of Microbiology and Immunology, University ofMelbourne, Parkville, VIC 3010, Australia (J.M.). E-mail addresses: jamie.rossjohn@monash.edu (J.R.) and jamesm1@unimelb.edu.au (J.M.)

The online version of this article contains supplemental material.

Abbreviations used in this article: BSA, buried surface area; EBNA, Epstein-Barrvirus nuclear Ag; H-bond, hydrogen bond; MHC-I, MHC class I; PEG, polyethyleneglycol; pHLA, peptide-HLA; pMHC-I, MHC class I molecule complexed to viralpeptide Ag; r.m.s.d., root-mean-square deviation; SPR, surface plasmon resonance;vdw, van der Waals.

Copyright� 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00

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HLA polymorphism that controls the size and diversity of thepeptide repertoire presented. Indeed, the HLA locus is the most

polymorphic region of the genome, whereby single amino acid

polymorphisms between closely related HLA allomorphs can

dictate the functional outcome in viral immunity (4–6), as well as

levels of cross-reactivity and alloreactivity (7, 8). TCR diversity

is generated via gene rearrangement within the V domains of the

TCR, in which the V and J gene segments make up the Va-chain,

whereas the Vb-chain is composed of V, D, and J genes (9).

Further, there is diversity (N) at the junctions of these gene

segments. This TCR diversity is critical to accommodate the

myriad of potential peptide-MHC (pMHC) complexes compris-

ing pathogen-derived peptides. Despite the potentially vast TCR

repertoire, some immune responses, such as the CTL response to

EBV, display strong biases in TCR selection. Although our un-

derstanding of the underlying mechanisms of TCR bias is far from

complete, pMHC-I structure is considered to play a role. Some-

what surprisingly, there is relatively little information on whether

or not particular TCR bias and diversity profiles are functionally

important during infectious diseases. Defining what determines an

optimal peptide-HLA I determinant and TCR response is essential

to improve immunotherapeutic approaches for dealing with both

cancer and persistent infections that have seemingly escaped from

immune surveillance.Previously, we explored the in vivo memory CTL response to an

immunodominant HLA-B8–restricted epitope FLRGRAYGL from

the latent EBVAg EBNA 3A. In HLA-B8+ individuals, the immune

response to this peptide is characterized by biased TCR usage,

in which the archetypal TCR, LC13, is focused upon the P7-Tyr

residue of the FLRGRAYGL determinant (10–12). The LC13

TCR is alloreactive toward HLA-B44; therefore, to avoid self-

reactivity in HLA-B8–B44 heterozygotes, the public LC13 CTLs

are deleted from the mature T cell repertoire (13, 14). Nevertheless,

presumably to keep the EBV infection in check, the HLA-B8–

restricted CTL response toward FLRGRAYGL still persists but

at a slightly lower precursor frequency. These CTLs, which ex-

press different TCR gene combinations, exhibit fine specificity

shifts either toward the P1-Phe residue or the P8-Gly residue of

FLRGRAYGL (13, 15). This system, which has evolved naturally

in humans, allows us to address how TCRs specific for distinct

aspects of the same peptide-HLA (pHLA) complex apportion the

energetic contributions from the MHC H chain contacts with re-

spect to the peptide Ag, thereby providing fundamental insight into

the basis of MHC restriction. Further, the comparison of these

subdominant and immunodominant complexes will be highly in-

formative and will reveal how self-tolerance is differentially estab-

lished by shifting of fine specificity at the atomic level due to the

presence of a trans-HLA B44 allele.

Materials and MethodsCytotoxicity assay

The RL42 CTL clone was described previously (13) and was generatedusing initial stimulation of PBMCs with the FLRGRAYGL peptide, fol-lowed by biweekly restimulation with g irradiated (8000 rad) HLA-B8+

lymphoblastoid cell lines. This CTL clone was tested in duplicate in thestandard 5-h chromium-release assay for cytotoxicity against 51Cr-labeledtarget cells that had been treated for 1 h with 0.1 mM peptide. The targetcells were HLA-B8+ PHA-stimulated blasts that were raised by stimulatingPBMCs with PHA, followed by propagation in IL-2–containing mediumfor up to 8 wk. Peptides were synthesized by Mimotopes. A b scintillationcounter (Topcount Microplate; Packard Instrument) was used to measure[51Cr] levels in assay-supernatant samples. The mean spontaneous lysis fortargets in culture medium was always,20%, and variation about the meanspecific lysis was ,10%.

Protein expression, purification, and crystallization

The RL42 TCR was expressed, refolded, and purified using an engineereddisulfide linkage in the constant domains between the a- and b-chains. Boththe a- and b-chains of the RL42 TCR were expressed separately as inclusionbodies in BL21 Escherichia coli strain. Inclusion bodies were resuspended in8 M urea, 20 mM Tris-HCl (pH 8), 0.5 mM Na-EDTA, and 1 mM DTT.RL42 TCR was refolded by flash dilution in a solution containing 5 M urea,100 mM Tris (pH 8), 2 mM Na-EDTA, 400 mM L-arginine–HCl, 0.5 mMoxidized glutathione, and 5 mM reduced glutathione. The refolding solutionwas then dialyzed to eliminate the urea. The resulting protein solution wasthen purified by gel filtration and HiTrap-Q anion exchange chromatography.The LC13 and CF34 TCRs were produced, as previously described (16, 17).

Soluble class I heterodimers containing the FLRGRAYGL peptide (or thepeptide variant) were prepared, as described previously (18, 19). Briefly, thetruncated forms (amino acid residues 1–276) of the HLA-B8 H chain (ormutated H chain) and full-length b2-microglobulin were expressed in E.coli as inclusion bodies. The HLA-B8 peptide complex was refolded bydiluting the H chain and b2-microglobulin inclusion body preparations intorefolding buffer containing a molar excess of peptide ligand. The refoldedcomplexes were concentrated and purified by anion exchange chromatog-raphy. The correct fold of the pMHC and TCR proteins was assessed byconformational Abs binding W6/32 and 12H8, respectively (10).

Crystals of the unliganded RL42 TCR were obtained by hanging-drop,vapor diffusion at 20˚C with a protein concentration of 5 mg/ml. Largecube-shaped crystals grew using 13% polyethylene glycol (PEG) 3350 and0.1 M Mg formate. Crystals of the RL42–HLA-B8FLRGRAYGL complexwere grown by the hanging-drop, vapor-diffusion method at 20˚C, witha protein/reservoir drop ratio of 1:1, at a concentration of 10 mg/ml in 10mM Tris-HCl (pH 8), 150 mM NaCl. Large stick-shaped crystals grewusing 14% PEG 3350, 0.2 M ammonium tartrate, and 7% ethylene glycol.Crystals of HLA-B8–I66A in complex with the FLRGRAYGL peptide andHLA-B8 in complex with the P8-valine variant of the epitope were bothobtained by hanging-drop, vapor diffusion at 20˚C, with a protein con-centration of 10 mg/ml. Large rod-shaped crystals grew using 20% PEG4000, 0.2 M ammonium acetate, and 0.1 M trisodium citrate (pH 5.6).

Data collection and structure determination

The RL42 TCR and RL42-HLA-B8FLRGRAYGL crystals were soaked ina cryoprotectant solution containing mother liquor solution, with the PEGconcentration increased to 30% (w/v), and then flash-frozen in liquid ni-trogen. Data were collected on the protein crystallography beamlines atthe Australian Synchrotron (Clayton, Australia) using the ADSC-Quantum210 CCD on MX1 detector and an ADSC-Quantum 315r CCD on MX2detector (at 100K). Data were processed using XDS software and scaledwith XSCALE (20). The RL42 TCR crystal belonged to the space groupP1 with unit cell dimensions (Supplemental Table I), consistent with fourTCRs in the asymmetric unit.

The RL42–HLA-B8FLRGRAYGL crystal belonged to the space groupP212121 with unit cell dimensions (Supplemental Table I), consistent withfour complexes in the asymmetric unit. The structures were determined bymolecular replacement using the PHASER (21) program, with the LC13TCR as the search model for the TCR (Protein Data Bank accessionnumber, 1KGC; http://www.pdb.org/pdb/home/home.do) (22) and HLA-B8FLRGRAYGL for the HLA minus the peptide FLRGRAYGL as model(Protein Data Bank accession number, 1M05; http://www.pdb.org/pdb/home/home.do) (11). HLA-B8–I66A in complex with the FLRGRAYGLpeptide and HLA-B8 in complex with the P8-valine variant of the epitopecrystals belong to the space group P212121 with unit cell dimensions(Supplemental Table I) consistent with one complex in the asymmetricunit. The structures were determined by molecular replacement using thePHASER (21) program with HLA-B8FLRGRAYGL for the MHC modelwithout the peptide (Protein Data Bank accession number, 1M05; http://www.pdb.org/pdb/home/home.do) (11).

Manual model building was conducted using Coot software (23), fol-lowed by maximum-likelihood refinement using PHENIX (24). “Transla-tion, liberation, and screw-rotation” displacement refinement was usedto model anisotropic displacements of defined domains. The TCR wasnumbered according to the International Immunogenetics InformationSystem unique numbering system (25) whereby the CDR1 loops start atresidue number 27, the CDR2 loops start at residue number 56, and theCDR3 loops start at residue number 105. The final models were validatedusing the Protein Data Bank validation Web site, and the final refinementstatistics are summarized in Supplemental Table I. Coordinates submittedto Protein Data Bank database (http://www.rcsb.org/pdb/home/home.do),code: 3SKN (RL42 TCR), 3SJV (RL42-HLA-B8FLRGRAYGL), 3SKO(HLA-B8-A66FLRGRAYGL), 3SKM (HLA-B8FLRGRAYVL). All molecu-lar graphics representations were created using PyMol (26).

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Surface plasmon resonance measurement and analysis

All surface plasmon resonance (SPR) experiments were conducted at 25˚Con the BIAcore 3000 instrument with HEPES buffer salt buffer (10 mMHEPES [pH 7.4], 150 mM NaCl, and 0.005% surfactant P20). The HBSbuffer was supplemented with 1% BSA to prevent nonspecific binding.The human TCR-specific mAb, 12H8 (10), was coupled to research-gradeCM5 chips with standard amine coupling. For each experiment, CF34,RL42, and LC13 were passed over the flow cell until ∼200–400 responseunits were captured by the Ab. One of the four flow cells was left emptyand used as control for each experiment. HLA-B8 (or mutant), in complexwith the peptide (or variant), was injected over all four flow cells, at a rateof 20 ml/min, with a concentration range of 0.78–200 mM. The final re-sponse was calculated by subtraction of the response of the Ab alone fromthat of the Ab CF34, RL42, or LC13 complex. The Ab surface wasregenerated between each analyte injection with Actisep (Sterogene). Allexperiments were conducted in duplicate (at least). BIAevaluation Version3.1 was used for data analysis; the 1:1 Langmuir binding model, modifiedto include an additional parameter for the drifting baseline for the TCRcapture by 12H8, was used to calculate the kinetic constants.

Thermal-stability assay

A thermal-shift assay was performed to assess the effect of each mutation,either on the HLA molecule or on the peptide, on the stability of the pHLAcomplex. The fluorescent dye Sypro orange was used to monitor the proteinunfolding. The thermal-stability assay was performed in the Real TimeDetection system (Corbett RotorGene 3000), originally designed for PCR.Each pHLA complex was in 10 mM Tris-HCl (pH 8) and 150 mM NaCl, attwo concentrations (5 and 10 mM) in duplicate, and was heated from 25 to95˚C at a rate of 1˚C/min. The fluorescence intensity was measured withexcitation at 530 nm and emission at 555 nm. The thermal melt point,represents the temperature at which 50% of the protein is unfolded.

ResultsThe RL42 TCR–HLA-B8FLRGRAYGL complex

Unrelated HLA-B8+ individuals virtually all make a biased CTLresponse to the peptide FLRGRAYGL from the latent EBV AgEBNA 3A. These CTLs, of which LC13 is the archetype, all usethe same TCR a- and b-chains derived from identical Va, Ja(TRAV26-2*01, TRAJ52*01), with the same CDR3 loops se-quences and identical TCRVb, Db, Jb (TRBV7-8*03, TRBD1/D2,TRBJ2-7*01) (27) and N-region sequences (28, 29). The LC13TCR focuses upon the prominent P7-Tyr and the C-terminal end

of the HLA-B8 Ag-binding cleft (12, 13). In HLA-B8+B44+

individuals, different subdominant TCRs are deployed againstHLA-B8FLRGRAYGL, indicating that the T cell repertoire containsconsiderable redundancy in receptors with appropriate specificityfor HLA-B8FLRGRAYGL. The CF34 CTL clone (TRAV14*01-TRAJ49, TRBV11-2*03-TRBD2*01-TRBJ2-3) represents the pro-totype of the TCRs in HLA-B8+B44+ individuals who exhibitexquisite sensitivity to substitutions at P1-Phe. To account for thisP1-Phe specificity, the CF34 TCR focuses on the N-terminal re-gion of HLA-B8, in stark contrast to the C-terminal–focuseddocking mode of LC13 (16). Of interest, other HLA-B8FLRGRAYGL–restricted CTLs from HLA-B8+B44+ individuals exhibit sensitiv-ity to substitutions at both the P1 and P8 positions of the peptide(16). A more comprehensive peptide-substitution analysis con-firmed the sensitivity of one such CTL (RL42) at positions P1 andP8, as well as demonstrated that P4, P6, and P7 were criticallyimportant to the interaction (Fig. 1). The RL42 TCR exhibitedmarkedly different gene usage (TRAV12-1/TRAJ23, TRBV6-2/TRBJ2-4/TRBD1) compared with the LC13 and CF34 TCRs. Howdoes the subdominant RL42 TCR dock onto HLA-B8FLRGRAYGL

with specificity requirements for the P1, P4, P6, P7, and P8 po-sitions of the FLRGRAYGL peptide?To address this, we expressed and refolded the RL42 TCR and

determined its structure in the nonliganded state at 2.9-A resolu-tion to an Rfactor and Rfree of 19.4 and 29.1%, respectively (Sup-plemental Table I). Next, we purified the RL42 TCR–HLA-B8FLRGRAYGL complex and solved the structure at 3.1-A resolu-tion to an Rfactor and Rfree of 25.3 and 32.1%, respectively (Fig.2A, Supplemental Table I). The initial experimental phases clearlyshowed unbiased electron density for the FLRGRAYGL peptide(data not shown), and the electron density at the RL42 TCR–HLA-B8FLRGRAYGL interface was unambiguous, thereby permit-ting structural analysis to be undertaken and compared with pre-viously published LC13 TCR and CF34 TCR ternary complexes.The RL42 TCR docked at ∼65˚ across HLA-B8FLRGRAYGL,

which was similar to the LC13 TCR and CF34 TCR dockingmodes (60 and 58˚, respectively, Fig. 2). However, although theCF34 TCR and LC13 TCR docked above the N- and C-terminal

FIGURE 1. Fine-specificity analysis of

RL42CTL reactivity on peptide analogs. The

fine specificity of the RL42 CTL clone was

determined by systematically substituting

each residue in the FLRGRAYGL peptide

with the remaining 19 genetically en coded

amino acids (single letter amino acid code

shown) and then assaying their recognition

by RL42 CTL in a cytotoxicity readout.

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end of HLA-B8FLRGRAYGL, respectively, the RL42 TCR sat cen-trally over HLA-B8FLRGRAYGL, thereby providing immediate in-sight into how this TCR exhibits sensitivity toward both the P1and P8 positions of the peptide (Fig. 2B, 2C, Supplemental TableII). The total buried surface area (BSA) upon ligation was similaramong the three TCR–pMHC complexes: 2100, 2020, and 2180A2 for the RL42, LC13, and CF34 TCR complexes, respectively.Accordingly, the RL42 TCR contacted HLA-B8FLRGRAYGL toa similar extent as did the LC13 and CF34 TCRs (RL42: 116 vander Waals [vdw] contacts, 14 hydrogen bonds [H-bonds], and onesalt bridge; CF34: 127 vdw contacts and 12 H-bonds; and LC13:135 vdw contacts, 14 H-bonds, and one salt bridge). In fact, theaffinity of the RL42–HLA-B8FLRGRAYGL interaction (KD 31 mM)was lower than was the affinity of LC13 TCR and CF34 TCRtoward HLA-B8FLRGRAYGL (16 and 8 mM, respectively); there-fore, compared with LC13 TCR or CF34 TCR, the subdominantRL42 TCR did not exhibit superior docking features, despite be-ing centrally located above the HLA-B8 Ag-binding cleft.

Divergent footprints

The Va and Vb domains of the RL42 TCR contributed 64 and36%, respectively, to the BSA at the interface, indicating a biastoward the a-chain in mediating contacts with HLA-B8FLRGRAYGL,in which CDR1a, 2a, and 3a loops contributed 20, 16, and 16%BSA to the interface, respectively (Fig. 2D, Supplemental TableII). Notably, 12% BSA arose from framework residues within theVa-chain. Although the b-chain played a lesser role at the inter-face, there was a relatively even distribution of contacts among theCDR loops, with the CDR1b, 2b, and 3b loops contributing 10, 9,and 17% BSA to the interface, respectively. Thus, the relative

interplay of the CDR loops of the RL42 TCR contrasted with thatof the CF34 TCR and the LC13 TCR (Fig. 2E, 2F), in which theCDR3 loops played a dominant role in contacting the epitope (58and 45% BSA, respectively, versus 33% BSA for the RL42 TCR).RL42 TCR interacted with a stretch of the a2-helix, spanning

from Arg151 to Ala158 (Fig. 3A). The region was contacted solelyby the CDR2a loop and the surrounding Va framework residues:the Arg151 side-chain was positioned toward the CDR2a loopand contributed most of the contacts, interacting with Tyr57a(CDR2a), Ser55a, and Asn66a (Fig. 3A). The neighboring Glu154also contacted Tyr57a and Ser58a of the CDR2a loop. The in-teractions between CDR2a and the a2-helix were further sta-bilized by an intricate network of polar interactions, whichincluded an H-bond between Asn66a and Ser55a, as well as a saltbridge between Arg151 and Glu154. The RL42 TCR made moreextensive contact with the a1-helix of HLA-B8, involving five ofthe CDR loops, which contacted a large area that virtually spannedthe length of the Ag-binding cleft (from Arg62 to Arg79) (Fig.3B). Asn27a of the CDR1a loop bonded to Arg62, an HLA-B8residue that reoriented upon ligation. The three CDRb loopsinteracted with the HLA-B8 a1-helix, spanning from Thr69 toArg79 (Fig. 3C, 3D). Glu30b salt bridged to Arg79, whereasTyr31b inserted its aromatic ring between Glu76 and Thr73, H-bonded to Glu76, and formed additional contacts with Gln72 (Fig.3C). Val57 from the CDR2b loop formed vdw contacts withGln72 and H-bonded via its main chain to Arg79, whereas Gly58bmade vdw contacts with Arg79 (Fig. 3D). The only residue fromthe CDR3b loop to interact with the HLA-B8 molecule wasthe nongermline-encoded Asn110b, which interacts with Thr69,Gln72, and Thr73.

FIGURE 2. Overall docking and structural footprints of RL42, CF34, and LC13 TCRs in complex with HLA-B8FLRGRAYGL. HLA-B8 molecule (white)

bound to the FLRGRAYGL peptide (black sticks) in complex with the RL42 (A), CF34 (B), and LC13 (C) TCRs. The color scheme is the same for all

panels, with the three complexes shown in the same orientation after alignment of their a1-a2 domains of HLA-B8. The TCR a-chain is shown in pale

pink; the b-chain is shown in pale blue; the CDRa loops are shown in purple, brown, and orange; and the CDRb loops are shown in dark blue, red, and

green. Structural footprint of the RL42 (D), CF34 (E), and LC13 (F) TCRs on the surface of the HLA-B8FLRGRAYGL complex. The HLA-B8 surface is

white, and the FLRGRAYGL peptide is gray. The HLA-B8 and FLRGRAYGL peptide atoms involved in the interaction with the TCRs are colored the

same as the corresponding CDR loops. The black circles represent the mass centers for the Va and Vb domain of each TCR and specify the docking mode

of each TCR on the surface of the HLA-B8FLRGRAYGL complex.

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The RL42 footprint on HLA-B8FLRGRAYGL contrasted with thecorresponding footprints of the LC13 TCR and the CF34 TCR:although the CF34 TCR and the LC13 TCR adopted an N-terminal and C-terminal docking mode, the center of mass ofthe RL42 TCR aligned with the central region of the HLA-B8 Ag-binding cleft (Fig. 2D). Further, in contrast to the interactionsmediated by the LC13 and CF34 TCRs, the RL42 TCR exhibiteda marked bias toward interacting with the a1-helix. The con-trasting footprints were suggestive of the structural focus of theHLA-B8–restricted TCRs being dictated by the initial focus on thepeptide derived from the EBV Ag, with MHC interactions gov-erning the additional fine-specificity features.

Viral Ag-mediated contacts

The CDR1a, CDR2a, CDR3a, and CDR3b loops from the RL42TCR contacted the FLRGRAYGL EBV peptide, which contrib-uted 20% BSA of the pMHC interface in the RL42 complex, andwas similar to that observed in both the LC13 TCR and the CF34TCR ligated complexes (Fig. 2D–F). The viral Ag side-chains thatwere exposed for TCR contact were P1-Phe, P6-Ala, and P7-Tyr;

the RL42 TCR contacted all three positions, as well as the P4-Glymain chain (Fig. 4A). The extent of peptide contacts contrastedwith that of the LC13 TCR, which contacted positions P6-Ala, P7-Tyr, and P8-Gly only (Fig. 4B), and was less extensive than that ofthe CF34 TCR, which contacted P1-Phe, P4-Gly, P5-Arg (mainchain), P6-Ala, and P7-Tyr (Fig. 4C).P1-Phe, an important position for RL42 TCR recognition (Fig.

1), was only partially buried, in that it was solely contacted byAla29 of the CDR1a loop (Figs. 4A, 5A), contrasting with theCF34 TCR–P1-Phe–mediated interaction, which was shieldedfully by the CDR3a loop. Thus, the sensitivity of the RL42 TCRto substitutions at the P1 position may be more reflective of per-turbing the local environment, which included Arg62, a residuethat also contacted the CDR1a loop. P4-Gly H-bonded to the side-chain of Gln31a (Fig. 5A) and was contacted by Arg107a, a res-idue derived from the N region of the CDR3a loop (Fig. 5B). BothCDR3 loops of the RL42 TCR surrounded P6-Ala, whose smallsize permitted close interactions with Arg107a, as well as withGly109b and Asn110b from the CDR3b loop (Fig. 5B). TheRL42 TCR interacted with P7-Tyr, mostly via its CDR3b loop,

FIGURE 3. RL42 TCR interaction with

the HLA-B8 molecule. The four panels

represent the interaction of the RL42 TCR

with HLA-B8 residues. The HLA-B8

molecule is shown in white, with the residue

in stick representation. The RL42 TCR

residues interacting with HLA-B8 are also

represented as sticks and are colored ac-

cording to the CDR loops colors, CDR1-2-

3a (purple, brown, and orange) or CDR1-2-

3b (blue, red, and green). Red dashed lines

represent hydrogen bonds, and the blue

dashed lines represent the hydrophobic

interactions. Interaction of the CDR2a loop

with the tip of the HLA-B8 a2-helix (A)

and between the CDR1–3a loops and the

a1-helix (B) are shown. CDR1b interaction

with the a1-helix (C) and those between

HLA-B8 and CDR2-3b (D) are shown.

FIGURE 4. Interactions between the CDR loops of each TCR and the peptide. The peptide is represented as sticks, and the same orientation has been

conserved for all three panels; the important residues of the TCR interactions with the FLRGRAYGL peptide are also represented as sticks. A, Interaction of

the RL42 TCR with the FLRGRAYGL peptide via its CDR1a (purple), CDR3a (orange), and CDR3b (green) loops. B, CF34 TCR with its CDR3a

(orange) and CDR1-2-3b (blue, red, and green) loops interacting with the FLRGRAYGL peptide. C, Interaction between the FLRGRAYGL peptide and the

CDR1a (purple), CDR3a (orange), and CDR3b (green) loops of the LC13 TCR.

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with its aromatic ring being flanked by Gln108b and Asp112b,and was also contacted by Tyr51a of the CDR2a loop (Fig. 5C).Further, the P7-TyrOH group was stabilized by an H-bond withAsp112b (Fig. 5C). Analogous to the CF34 complex, P7-Tyr waspartially buried (∼70%) by the RL42 TCR, contrasting with theLC13 TCR, which fully encapsulates P7-Tyr (Fig. 4). Neverthe-less, although there were differences in the mode and extent ofbinding to P7-Tyr, all three TCRs formed an H-bond to the P7-TyrOH group. Interestingly, although the RL42 and CF34 TCRsdid not contact P8-Gly, the former is nevertheless sensitive tosubstitutions at this position, which contrasts with the CF34 TCR.The presence of bulky side-chains at the P8 position is likely toimpact on the conformation of Glu76 from the a1-helix of HLA-B8 (Fig. 3C). Thus, the central location resulted in the RL42 TCRexhibiting sensitivity to all of the solvent-exposed residues of theFLRGRAYGL peptide: P1-Phe, P4-Gly, P6-Ala, P7-Tyr, and P8-Gly.

Conformational plasticity

Having determined the structure of subdominant RL42 TCR andHLA-B8FLRGRAYGL in the nonliganded and liganded state, wewere able to establish the extent of plasticity required for RL42TCR-HLA-B8FLRGRAYGL ligation. Upon RL42 TCR ligation,there were very few structural changes observed in HLA-B8FLRGRAYGL, with limited movements in the peptide (root-mean-square deviation [r.m.s.d.] of 0.38 A) and in the Ag-binding cleft(r.m.s.d. of 0.69 A). Nevertheless, some side-chain movement wasobserved upon ligation, principally concerning three arginineresidues: Arg62, Arg79, and Arg151. Arg62, located at the N-terminal region of the HLA-binding cleft, was pushed away toavoid steric clashes with the CDR3a loop and H-bonded toAsn27a of the CDR1a loop, whereas Arg 79, located at the C-

terminal end of the Ag-binding cleft, flattened against the a1-helixupon RL42 TCR ligation, forming contacts with CDR1b andCDR2b loops; Arg151 reoriented 180˚ to contact Tyr57a and theVa framework residues. With respect to the RL42 TCR, there wasa 9˚ reorientation between the Va-Vb domains upon ligation (datanot shown). The major difference between the unliganded andliganded counterpart resided within the germline-encoded CDR2aloop, which shifts 2.4 A upon ligation, whereby Tyr57a exhibitedthe largest structural change (3.7 A) (Fig. 5D). The conforma-tional change of the CDR2a loop was not a result of the avoid-ance of steric clashes per se, but was attributable to specificity-governing contacts, enabling the aromatic ring of Tyr57a to packagainst P7-Tyr of the FLRGRAYGL peptide. The limited extent ofconformational changes made by the RL42 TCR in binding toHLA-B8FLRGRAYGL is in marked contrast to that of the LC13TCR, in which the CDR3a, CDR3b, CDR1a, and CDR2a loopswere remodeled upon HLA-B8FLRGRAYGL ligation (12). Accord-ingly, although the dominant LC13 TCR and subdominant RL42TCR bind to HLA-B8 complexed to the same EBV determinant,differing degrees of conformational change underpin the recog-nition event.

High specificities for the viral Ag

Previously, we demonstrated the impact of the FLRGRAYGL muta-tions at P1, P7, and P8 on both the LC13 and CF34 TCRs (16);therefore, we undertook SPR analysis with the same peptide var-iants (P1-T, P1-Y, P7-F, and P8-V) and observed their effect onRL42 TCR recognition. In addition, an alanine-scanning muta-genesis study of the peptide residues (P1, P4, P6, P7, and P8, withP6 being mutated to a Gly) contacting at least one of the three TCRswas undertaken (Fig. 6). The effects of the FLRGRAYGL residuesubstitutions were grouped into three categories: no effect, a mod-

FIGURE 5. RL42 TCR interaction with the FLRGRAYGL peptide. The FLRGRAYGL peptide is represented as gray sticks, red dashed lines represent

hydrogen bond, and blue dashed lines represent the hydrophobic interactions between the RL42 TCR and the FLRGRAYGL peptide. A, CDR1a (purple)

interacts with P1-Phe and P4-Gly of the peptide. B, RL42 TCR used both CDR3 loops (a in orange and b in green) to contact P6-Ala of the FLRGRAYGL

peptide. C, The CDR3b loop also interacted with P7-Tyr, as well as the CDR2a loop (brown) via Tyr 57. D, CDR2a movement in the RL42 TCR between

the unliganded state (yellow) and once liganded to HLA-B8FLRGRAYGL (brown). The movement of the CDR2a loop allowed Tyr57a to contact P7-Tyr of

the FLRGRAYGL peptide.

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erate effect representing a 3–5-fold loss of TCR-pMHC affinity, anda drastic effect of.5-fold loss of affinity. The peptide variants wererefolded with HLA-B8, and all were able to bind to the mAbW6/32(data not shown). The effect of the peptide mutations on HLA-B8thermal stability was assessed; with the exception of the mutation atposition P1, all of the peptide variants showed the same stability asthe wild-type HLA-B8FLRGRAYGL (Supplemental Table III). Theeffect of substitutions at the P1 position (3–6˚C) is indicative ofperturbing the architecture around the P1 pocket.The systematic substitution of the FLRGRAYGL-immunodo-

minant EBV Ag showed that the RL42 CTLs were sensitive toall solvent-exposed residues of the peptide (Fig. 1). The RL42 TCRaffinity was dramatically decreased when the P1 residue wasmutated, confirming the N-terminal sensitivity of this TCR (Fig.6A). Interestingly, the P1-Tyr substitution, which dramaticallydecreased the CF34 TCR affinity (Fig. 6B), improved the affinityof the RL42 interaction by ∼6-fold, suggesting that the additionalhydroxyl group of the P1-Tyr formed stabilizing interactions withthe RL42 TCR (Fig. 5A). The P7-Phe mutation dramatically de-creased the RL42 TCR affinity, similar to that previously observedfor the LC13 and CF34 TCRs (16). The P8-Val mutation abro-gated LC13 TCR and RL42 TCR binding; moreover, the RL42CTLs were very sensitive to most substitutions at the P8 position(Fig. 1), despite the fact that the RL42 TCR did not directlycontact this position. To address this, we solved the structure ofHLA-B8 in complex with the P8-Val variant of the FLRGRAYGLpeptide (Supplemental Table I). Although the HLA-B8 Ag-binding cleft did not change conformation appreciably (r.m.s.d.0.59 A), the conformation of the peptide itself was altered (i.e.,superposition of the wild-type peptide and the P8-Val mutantshowed an r.m.s.d. of 1.58 A, with a maximum displacement of2.6 A for the P6-Ala Ca atom and 5.8 A for the hydroxyl group ofthe P7-Tyr [Supplemental Fig. 1A]). Accordingly, the P8-Valmutation induced a structural change of the central part of thepeptide, which shifted closer to the a2-helix of HLA-B8, thereby

providing a basis for understanding the impact of P8 substitutionson the RL42 TCR interaction.Next, we undertook SPR analysis of the LC13, CF34, and RL42

TCRs against the alanine mutants of the solvent-exposed peptideresidues (Fig. 6A). The RL42 TCR affinity was moderately de-creased by the P1-Ala and P8-Ala mutations, and it was dramat-ically decreased by substitutions at P4, P6, and P7. The location ofthe key positions of the FLRGRAYGL peptide correlated verywell with the central docking of the RL42 TCR. In comparison,the CF34 TCR was extremely sensitive to substitutions at the P1,P4, P6, and P7 positions of the FLRGRAYGL peptide (Fig. 6B).The LC13 TCR was unaffected by the P1-Ala substitution, butit was adversely impacted by substitutions at P4, P6, P7, and P8(Fig. 6C). Although P4-Gly was not contacted by the LC13 TCR(12), the P4-Ala substitution likely impacted on the conformationof the closely abutting CDR3a loop, thereby affecting the LC13TCR affinity. Collectively, the substitution on the FLRGRAYGLpeptide showed a heightened peptide dependency by the threeHLA-B8–restricted TCRs, with each TCR exhibiting differingspecificities toward the same viral Ag.

The energetic hot spots on HLA-B8 are driven by the EBVdeterminant

To evaluate which HLA-B8 residues are required energetically forthe three TCR–HLA-B8FLRGRAYGL interactions, we undertookan alanine-scanning mutagenesis study across the HLA-B8 Ag-binding interface. The HLA-B8 residues targeted for substitutionwere selected based on the previously determined crystal struc-tures (12, 16) and the RL42 TCR ternary complex reported in thisarticle (i.e., alanine mutants were made for the HLA-B8 residuesthat contacted, via their side-chain, at least one of the threeTCRs). Collectively, 20 single-site HLA-B8 mutants were se-lected and assessed for binding to the three TCRs using SPR. TheW6/32 mAb was used to confirm that all of the mutant HLA-B8FLRGRAYGL complexes were correctly folded (data not shown).

FIGURE 6. SPR on the FLRGRAYGL mutants. SPR data performed on the FLRGRAYGL peptide mutants bound to the HLA-B8 molecule against the

three TCRs: RL42 (A), CF34 (B), and LC13 (C). The y-axis represents DDGeq (kcal/mol). The black line indicates a 3-fold increase when comparing the

mutant to the wild-type, and the dashed black line represents a 5-fold increase. Experiments were conducted in duplicate; error bars indicate the SD for each

duplicate.

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The effects of the HLA-B8 substitutions were grouped into threecategories: no effect, a moderate effect representing a 3–5-fold lossof affinity, and a drastic effect of .5-fold loss of affinity (Fig. 7).We also tested the effect of these mutants on the thermostability

of the pHLA complex (see Materials and Methods). As judged bythe thermal melt point, 5 of the 20 mutations affected the stabilityof the pHLA complex (i.e., Ile66, Arg79, Lys146, Trp167, and Arg170), indicating that the mutation possibly impacted on the con-formation of the HLA-B8FLRGRAYGL complex (Supplemental Ta-ble III). With the exception of Ile66, which was contacted by thethree TCRs, the mutations did not impact on the affinity of theTCRs, because they were not involved in the TCR–MHC inter-action. To assess the impact of the Ile66Ala mutation, we solvedthe structure of HLA-B8–A66 in complex with the FLRGRAYGLpeptide (Supplemental Table I). Superposing the HLA-B8 Ag-binding cleft of the wild-type and HLA-B8–A66 mutant (r.m.s.d.of 0.75 A) showed a shift in the juxtapositioning of the a1-helix inthe HLA-B8–A66 structure, which resulted in a narrower HLA-B8Ag-binding cleft. Further, the peptide moved slightly in the A66mutant, in which P4-Gly moved toward Ala66 to fill the voidcaused by the missing side-chain at position 66 of HLA-B8(Supplemental Fig. 1B). The impact of the Ile66Ala mutant canbe attributed, in part, to altering the structure of the local envi-ronment of HLA-B8FLRGRAYGL.

For the LC13 TCR–HLA-B8FLRGRAYGL interaction, 10 single-site HLA-B8 mutations had no impact, 3 had a moderate effect,and 7 positions exhibited a marked impact on the affinity of theinteraction (Fig. 7A). Of the 13 positions that had no/moderateimpact, 7 directly contacted the LC13 TCR. The mutated positionsthat had minimal impact on the affinity were clustered mostlyaround the N-terminal region of the peptide, whereas the hot spotpositions were mostly clustered around the C-terminal region ofthe Ag-binding cleft, most notably on the a1-helix that abutted theP7-Tyr residue (Fig. 7D). Previously, we conducted an alanine-scanning mutagenesis study on the LC13 TCR (10), therebyproviding us an opportunity to compare the effect of the reciprocalinterface-disrupting mutations. The majority of HLA-B8 residuesthat correspond to the hot spot area are contacted by the CDR3aand CDR3b, two loops that we previously showed to be importantin driving the energetics of the LC13 TCR–HLA-B8FLRGRAYGL

interaction (10).For the CF34 TCR–HLA-B8FLRGRAYGL interaction, 10 single-

site HLA-B8 mutations had no impact, 4 had a moderate effect, 4positions notably reduced the affinity of the interaction, and 2mutations, Gln155Ala and Glu166Ala, resulted in a 3- and 8-foldfactor improvement in affinity, respectively (Fig. 7B). Upon li-gation, Gln155 was pushed down by the CDR3a loop, and theGln155Ala mutant resulted in a slower on-rate (wild-type kon =

FIGURE 7. SPR on the HLA-B8 mutants and energetic footprints of the three HLA-B8FLRGRAYGL-specific TCRs. A–C, SPR data for the 20 HLA-B8

mutants bound to the FLRGRAYGL peptide against the three TCRs. The y-axis represents the DDGeq (kcal/mol). The orange line indicates a 3-fold

increase when comparing the mutant to the wild-type and the red line indicates a 5-fold increase. Experiments were conducted in duplicate; error bars

indicate the SD for each duplicate. Energetic footprint for the LC13 (D), CF34 (E), and RL42 (F) TCRs on the HLA-B8FLRGRAYGL complex. The surface of

HLA-B8 is white; the FLRGRAYGL peptide is pale blue. The residues of the HLA-B8 and the FLRGRAYGL peptide are colored according to the SPR

results: gray (mutation had no impact on the affinity), orange (moderate effect), red (drastic effect), and green (improved affinity due to mutation).

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6.746 1.18 104/M21s21, Gln155Ala kon = 3.936 0.11 104 M21s21)and a longer off-rate (wild-type koff = 0.266 0.01 s21, Gln155Alakoff = 0.09 6 0.01 s21) for the interaction (data not shown). All ofthe residues affecting the affinity of the interaction contacted theCF34 TCR, with the four hot spot residues (Arg62, Ile66, Thr73,and Trp167) clustering around the critical Ag residues P1-Phe, P4-Gly, and P6-Ala. Trp167 and Arg62 both surrounded the P1-Pheof the Ag (Fig. 7E); Arg62 was engulfed by the CDR3a andCDR3b loops, interacting with residues corresponding to the J andN segments, respectively. Trp167 was contacted by the CDR1aand the CDR3a via Pro30a and Asp109a (N segment), respec-tively. Thr73, in close proximity to P6-Ala, was contacted byGln57 of the CDR2b loop. Finally, Ile66, which contacted P4-Gly,a critical determinant on the FLRGRAYGL peptide, was contactedby the nongermline-encoded residue Trp110b of the CDR3b loop.Accordingly, the HLA-B8 residues that were critically importantfor the CF34 TCR–HLA-B8FLRGRAYGL interaction were engagedmostly by nongermline-encoded residues of the CF34 TCR.For the RL42 TCR–HLA-B8FLRGRAYGL interaction, 12 single-

site HLA-B8 mutations had no impact, 2 had a moderate effect,and 6 positions (Ile66, Thr69, Asn80, Lys146, Glu154, andGln155) were recognized to represent hot spots, because mutationof each notably reduced the affinity of the interaction (Fig. 7C,7F). In comparison with the LC13 TCR and CF34 TCR hot spots,the critical HLA-B8 residues for the RL42 TCR interaction weremore extensive and diffuse, located adjacent to the P1 pocket,centrally, and at the C-terminal end of the Ag-binding cleft. Thr69and Ile66, the latter of which was also a hot spot for the LC13 andCF34 TCRs, interacted with the CDR3a loop of the RL42 TCR.In the central region of the a2-helix, Glu154Ala and Gln155Alamutations had a dramatic effect on the affinity of the RL42 TCR,which is attributable to loss of contacts with the CDR2a loop.Further, the alanine-scanning mutagenesis study indicated that theC-terminal end of the binding cleft on the a1-helix was importantfor RL42 TCR recognition, with two positions having a dramaticeffect (Lys146 and Asn80) and two having a moderate effect(Glu76 and Arg79). Although the side-chains of Glu76 and Arg79were contacted by the CDR1b and CDR1/2b loops, respectively,Asn80 and Lys146 did not contact the RL42 TCR. Both of theseresidues are involved in binding to the peptide; thus, the effectsof mutating these residues to alanine were attributable to localstructural perturbations that impact on RL42 TCR recognition.Interestingly, however, the Asn80Ala and Lys146Ala mutationsdid not impact on CF34 TCR recognition, and they moderately

impacted on LC13 TCR recognition, indicating a heightenedsensitivity of RL42 TCR to this region of the Ag-binding cleft.Accordingly, our data suggested that EBV determinant drives the

energetic contribution to the TCR–Ag–HLA interaction, whereasthe energetically important HLA-based interactions colocalizedthe peptide hot spots (Figs. 7D–F, 8).

DiscussionEBV is a ubiquitous human pathogen, contained by a vigorouscellular immune response that is directed against a number ofimmunodominant EBV determinants. To gain a greater under-standing of CTL immunity and Ag presentation, we investigatedspecific elements relating to the protective immune response toEBV and have provided fundamental insight into HLA restrictionand a portrait of immunodominant versus subdominant TCR usage.With regard to MHC restriction, an appealing conceptual frame-

work suggests that the germline-encoded regions of the TCR dic-tate MHC bias. In line with this view, there is some evidence thatMHC reactivity is inherent in the germline-encoded portions of theTCR (30–34). Moreover, conserved pair-wise interactions betweenclosely related Vb8.2+ TCRs and peptide-MHC-II (pMHC-II)molecules have led to the concept of interaction motifs betweenthe CDR2b loop of a TCR and a given MHC molecule (35–38).However, other studies do not support this view in that structuraland mutagenesis studies have clearly shown a wide array of TCR-pMHC docking modes, in which the CDR3 loops often play aprominent role in interacting with the MHC, and the germline-encoded loops frequently contact the viral Ag (39, 40). A centralquestion that arises from these observations is whether the viralAg and/or the MHC H chain principally determines productiveTCR engagement. A two-step binding model proposes that theTCR binds the MHC first, then the peptide (41). Moreover, MHC-centric alloreactivity further suggests that docking orientationis governed by TCR–MHC contacts (42). However, T cell allo-reactivity can also be peptide centric, and TCR recognition ofunusually long MHC-I–restricted viral Ags demonstrates minimalMHC contacts and indicates an Ag-biased focus by the TCR (6,7, 43). Further, the mutagenesis studies reported in this articleshowed that nongermline-encoded residues of the TCRs makeimportant energetic contributions to HLA-B8 binding: the LC13TCR used two nongermline-encoded CDR3b residues (Gly97 andGln98) to interact with three HLA-B8 hot spot residues (Gln72,Ala73, and Glu76) (Fig. 8A); the nongermline-encoded residues ofthe CF34 CDR3 loops (Asp109a and Trp110b) contact the three

FIGURE 8. Nongermline-encoded TCR residue interaction with the HLA-B8 hot spot. A, The LC13 CDR3b (green) nongermline-encoded Gly97 and

Gln98 interacting with the HLA-B8 Glu76 and the Gln72 residues, both critical for the interaction (red). B, The nongermline CF34 residues from the

CDR3a (Asp109 in orange) and CDR3b (Trp110 in green) loops interacting with the energetically important residues of the HLA-B8 molecule: Arg62,

Ile66, and Trp167 (red). C, Asn110 of the RL42 CDR3b loop (green), also a nongermline–encoded residue, inserting its side-chain between two hot spots

on HLA-B8, Thr73, and Thr69 (red). Red dashed lines represent hydrogen bonds, and blue dashed lines represent the hydrophobic interactions.

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HLA-B8 hot spot residues (Arg62, Ile66, Trp167) (Fig. 8B); andthe nongermline-encoded residue Asn110b from the CDR3b loopof the RL42 TCR interacts with two HLA-B8 hot spot residues(Thr69 and Thr73) (Fig. 8C).These observations question the relative importance of the viral

determinant versus the MHC in mediating TCR contacts and, inparticular, whether the TCR has a virally encoded peptide-centricor MHC-centric view? Viral Ag peptide centricity is suggested bythe observations that displacement of the self-peptide with theforeign Ag triggers an immune response and that the viral peptidesare considered to represent structural mimics of the self-peptide;TCR–pMHC interactions are very sensitive to the peptide itself,because altered peptide ligands can markedly alter the immuneoutcome, yet they do not alter MHC docking (44); TCRs bindto superbulged pMHC complexes, such that they form a minimalfootprint on the MHC (6, 43); protective immunity can be gov-erned by a germline-encoded TCR polymorphism, which interactsexclusively with a peptide encoded by an EBV Ag (45); andalloreactivity can be governed by peptide-centric molecularmimicry (7, 46). Further, although the TCR–MHC contact is muchmore extensive compared with TCR–peptide interactions, HLA-mutagenesis studies showed that the HLA energetic hot spotcan be small and variable between various TCR–MHC systems(47). Previous studies primarily focused on TCR mutagenesis, andthe relative importance/sensitivity to alterations in peptide versusMHC H chain residues in the TCR–pMHC interaction was un-clear. Such TCR-centric mutagenesis studies include the 2C TCRsystem (48), autoreactive TCRs (49), and biased antiviral TCRs(10) (50). Two studies observed the effect of the mutation on theHLA-A2 molecule in two different systems: in the context of thetax peptide recognized by the A6 TCR and with the p1049 peptiderecognized by the AHIII TCR (47, 51). These studies revealed thatonly three HLA residues (Arg65, Lys66, and Ala69) define theenergetic hot spot for the A6 TCR (47).To address this issue of the viral Ag versus MHC H chain in

driving the TCR interaction, we examined the same Ag-presentingmolecule, bound to a common EBV determinant, and comparedrecognition against one immunodominant and two subdominantTCRs that varied in gene usage. These TCRs exhibited differingfine specificities for the viral determinant; together with the HLA-B8 mutagenesis studies, it allowed us to address the energeticlandscape of recognition (12, 19). Our data demonstrated that,although the TCRs recognize the same viral determinant, thestructural footprints vary between the three TCRs; moreover, thecorresponding energetic footprints on the MHC differ betweeneach TCR. In the case of the immunodominant LC13 and sub-dominant CF34 TCRs, the energetic footprint is clustered aroundthe site of the peptide sensitivity: for the CF34 and LC13 TCRs,the energetic hot spot is around the P1 pocket and the P7 pocket,respectively. However, for the subdominant RL42 TCR, whichadopts a central docking mode, the hot spot is more extensive anddiffuse and is reflective of the sensitivity at a number of positionswithin the peptide. Thus, our studies indicated that the energeticrequirement of HLA-B8 residues is contingent on the nature andlocalization of the broadly peptide-centric nature of the interac-tion. Thus, in contrast to the two-step TCR–pMHC binding modepreviously proposed (41), our data indicated that the viral Ag actsas the molecular glue for the TCR–pMHC interaction, whereas thenearby MHC interactions stabilize TCR ligation and modulate thefine-specificity requirements of the TCR for the viral Ag itself.Further, given that the subdominant RL42 TCR adopts a centraldocking topology on the pMHC, our data indicated that the factorsshaping dominant versus subdominant T cell repertoire selectionare not governed by favorable docking topologies. Instead, other

factors, such as affinity for the TCR–pMHC interaction, precursorfrequency, or signaling capacity, contribute to T cell immunodo-minance (52–54). Collectively, we provided insight into under-standing how varied abTCR usage against an identical HLA leadsto contrasting views of the common viral determinant.

AcknowledgmentsWe thank the staff at the MX beamlines at the Australian Synchrotron for

assistance with data collection. We also thank L.C. Sullivan, A.G. Brooks,

S.J. Turner, and J.J. Miles for input and helpful discussions.

DisclosuresThe authors have no financial conflicts of interest.

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