CTL Recognition of a Bulged Viral Peptide Involves Biased TCR Selection

John J. Miles, Diah Elhassen, Natalie A. Borg, Sharon L. Silins, Fleur E. Tynan, Jacqueline M. Burrows, Anthony W. Purcell, Lars Kjer-Nielsen, Jamie Rossjohn, Scott R. Burrows and James McCluskey
J Immunol September 15, 2005, 175 (6) 3826-3834; DOI: https://doi.org/10.4049/jimmunol.175.6.3826

Abstract

MHC class I molecules generally present peptides of 8–10 aa long, forming an extended coil in the HLA cleft. Although longer peptides can also bind to class I molecules, they tend to bulge from the cleft and it is not known whether the TCR repertoire has sufficient plasticity to recognize these determinants during the antiviral CTL response. In this study, we show that unrelated individuals infected with EBV generate a significant CTL response directed toward an HLA-B*3501-restricted, 11-mer epitope from the BZLF1 Ag. The 11-mer determinant adopts a highly bulged conformation with seven of the peptide side chains being solvent-exposed and available for TCR interaction. Such a complex potentially creates a structural challenge for TCR corecognition of both HLA-B*3501 and the peptide Ag. Surprisingly, unrelated B*3501 donors recognizing the 11-mer use identical or closely related αβ TCR sequences that share particular CDR3 motifs. Within the small number of dominant CTL clonotypes observed, each has discrete fine specificity for the exposed side chain residues of the peptide. The data show that bulged viral peptides are indeed immunogenic but suggest that the highly constrained TCR repertoire reflects a limit to TCR diversity when responding to some unusual MHC peptide ligands.

In the course of establishing protective immunity, viral clearance generally involves the recruitment of a potentially broad repertoire of T lymphocytes bearing specific αβ TCRs (1, 2, 3, 4). These receptors recognize viral Ags in the form of peptide fragments presented by MHC molecules displayed on the cell surface (5). Peptide Ags are generally 8–10 aa long, with residues involved in either MHC or TCR binding or both. Specificity for class I binding is largely conferred by two or three dominant anchor residues (6), while Ag specificity of MHC-peptide complex (MHCp)4 recognition is generally determined by the few side chains of the peptide Ag that are solvent-exposed (between one and three residues) and available for TCR contacts (5, 7). This paradigm underpins the basis of software algorithms that predict “best guess” 8–9-mer class I epitopes from protein sequences.

However, longer peptides can also bind MHC class I (MHC-I) (8) and are potential targets for CTL recognition (9, 10, 11, 12). The crystal structures of two MHC-I-bound peptides that are longer than 10 residues have been solved (13, 14), revealing that the conserved bonding networks around the N and C peptide termini are maintained, thereby displacing the center of the peptide away from the groove to an elevated position above the α helices of the MHC. There is also direct and/or indirect evidence that long class I-bound peptides can be accommodated by protruding from the binding site at the C or N terminus (15, 16, 17).

Little is known about the capacity of MHC-I-restricted T cells to recognize viral peptides of >10 aa in length and whether these ligands impose constraints on TCR repertoire and/or CDR3 length. It has been suggested that peptides bound with a bulged conformation may prevent many T cells from approaching the surface of the MHC molecule due to steric constraints, thereby severely limiting potential TCR usage toward unusually long class I-binding antigenic peptides (18). In contrast, MHC restriction results from specific TCR-MHC interactions that show considerable plasticity and appear to vary among the TCR-MHCp structures solved to date (19), despite the sharing of a broadly diagonal mode of TCR binding (7). It is therefore conceivable that bulged peptides could stimulate a particularly diverse TCR repertoire due to the availability of more exposed peptide residues and/or the potential for the peptides to shift conformation, creating multiple epitopes (13, 14). To address whether non-canonical viral peptide Ags >10 aa are immunogenic and whether they elicit a constrained TCR repertoire, we have studied the CTL response during persistent infection with the ubiquitous human herpesvirus EBV in healthy normal individuals.

In this study, we analyze a common CTL response toward an HLA-B*3501-restricted, 11-mer epitope EPLPQGQLTAY (EPLP) from the BZLF1 Ag (residues 54–64) of EBV (20). Interestingly, the natural T cell response to this unusual bulged target structure is extremely restricted in the TCR α- and β-chain sequence, with identical or closely related TCR αβ sequences dominating the response in unrelated individuals. The data show that bulged viral peptides are indeed immunogenic and sometimes elicit a highly constrained TCR repertoire that could reflect limits to the level of TCR diversity when responding to unusual MHCp ligands.

Materials and Methods

T cell cultures

CTL cultures were raised from PBMCs isolated from healthy, EBV-seropositive volunteers who were positive for HLA-B*3501, as determined by HLA sequence analysis. CTL clones were generated as previously described (20) using initial stimulation of PBMCs with the EPLP peptide, followed by biweekly restimulation with gamma irradiation (8000 rad) HLA-B*3501+ lymphoblastoid cell lines. Short-term CTL bulk cultures were also used for some TCR analyses. These were generated by culturing PBMCs (2 × 106/2-ml well) with autologous PBMCs that had been precoated with the EPLP peptide (1 μM for 1 h, responder:stimulator ratio of 2:1). Cultures were supplemented with rIL2 (20 U/ml) on day 3, were split on day 7, and were analyzed for TCR usage on day 10.

Flow cytometric analysis

PBMCs or bulk T cell cultures were incubated for 30 min at 4°C with an HLA-B*3501-EPLP, PE-labeled tetramer (ProImmune). Cells were then washed and labeled for 30 min at 4°C with Tri-color-labeled anti-human CD8 (Caltag Laboratories), allophycocyanin-labeled anti-human CD3 (BD Pharmingen), and one of the following FITC-labeled TcRβ chain-specific Abs (Serotec): Vβ1 (TRBV9), Vβ2 (TRBV20-1), Vβ3 (TRBV28), Vβ5.1 (TRBV5-1), Vβ5.2 (TRBV5-6), Vβ5.3 (TRBV5-5), Vβ6.7 (TRBV7-1), Vβ7 (TRBV4), Vβ8 (TRBV12), Vβ11 (TRBV25-1), Vβ12 (TRBV10), Vβ13.1 (TRBV6-5), Vβ13.6 (TRBV6-6), Vβ14 (TRBV27), Vβ16 (TRBV14), Vβ17 (TRBV19), Vβ18 (TRBV18), Vβ20 (TRBV30), Vβ21.3 (TRBV11-1), Vβ22 (TRBV2), or Vβ23 (TRBV13). Cells were washed and analyzed on a FACSCalibur using CellQuest software (BD Biosciences). Cell sorting was performed on a MoFlo (DakoCytomation).

Cytotoxicity assay

CTL clones were tested in duplicate in the standard 5-h chromium release assay for cytotoxicity against 51Cr-labeled target cells that had been treated for 1 h with various concentrations of peptide. The target cells were PHA-stimulated blasts that were raised by stimulating PBMCs with PHA followed by propagation in IL-2-containing medium for up to 8 wk. Percent specific lysis was calculated, and the peptide concentration required for half-maximum lysis was determined from dose-response curves. Peptides were synthesized by Mimotopes. A beta scintillation counter (Topcount Microplate; Packard Instrument) was used to measure 51Cr levels in assay supernatant samples. The mean spontaneous lysis for targets in culture medium was always <20% and variation about the mean specific lysis was <10%.

T cell repertoire analysis

T cell clones were verified for purity and peptide specificity using flow cytometry with an HLA-B*3501-EPLP tetramer before TCR analysis was performed. Total RNA was extracted from T cell clones and tetramer-sorted bulk CTL cultures using TRIzol reagent. Reverse transcription was performed with Superscript III (Invitrogen Life Technologies) and antisense TCRα and TCRβ chain primers (21, 22). PCR was performed in a 25-μl volume consisting of 200 μM dNTPs, 20 mM MgCl2, and 1.25 U of AmpliTaq Gold (Applied Biosystems) using a TCR Cα constant primer and 1 of 34 TCRVα family-specific primers, or a TCR Cβ constant primer and 1 of 26 TCR Vβ family-specific primers (22). PCR products were purified and cloned into the pGEM-T vector system (Promega) and sequenced using the ABI PRISM Big Dye Termination Reaction kit (Applied Biosystems). IMGT TCR gene nomenclature was used throughout (23) and CDR3 length was defined according to previous criteria (24).

HLA-peptide-binding assays

To assess peptide binding to HLA-B*3501, HLA stabilization studies were conducted using the mutant LCL x T lymphoblastoid hybrid cell line, 174 x CEM.T2 (referred to as T2 cells) (25), transfected with HLA-B*3501. T2.B*3501 cells were incubated in AIM V serum-free medium (Invitrogen Life Technologies) with various concentrations (0.01, 0.1, 1, 10, or 100 μM) of peptides at 26°C for 14–16 h, followed by incubation at 37°C for 2–3 h. HLA-B*3501 surface expression was then measured by flow cytometry on a FACSCalibur using a mAb to HLA-Bw6 (SFR8 Bw6). Mean fluorescence intensity (MFI) was determined, and the peptide concentration required for half-maximum MFI was calculated.

Protein expression, crystallization, and structure determination

Soluble HLA-B*3501 (residues 1–276) and full-length β2-microglobulin (residues 1–99) were expressed in Escherichia coli as inclusion bodies, refolded with the EPLPQGQLTAY peptide, and purified as previously described (26, 27). The HLA-B*3501 complex crystals were obtained by the hanging drop vapor diffusion technique. Large (0.7 × 0.5 × 0.3 mm) block-shaped crystals grew within 5 days in a condition containing 0.2 M ammonium acetate and 17% w/v polyethylene glycol 3350 (100 mM cacodylate, pH 7.6) at 4°C. Crystals were transferred directly to cryoprotectant containing 20% glycerol and flash frozen before data collection. Data were collected at the Advanced Photon Source (Argonne National Laboratory, Argonne, IL) using the 14-BM-C station. Data were processed and scaled using the HKL suite (28). The HLA-B*3501 EPLPQGQLTAY crystals belong to the space group P212121. The HLA-B*3501 structure was determined by molecular replacement using a previously determined HLA-B*3501 molecule as the search model (Protein Data Bank accession code: 1A1N) with the peptide and water molecules omitted. The model was manually built using the program “O” (29) and improved through multiple rounds of refinement using the CNS suite (30). The progress of refinement was monitored by Rfac and Rfree values. The structure has been deposited in the Protein Data Bank under accession code 1ZSD.

Results

Highly selected Vβ usage in the CTL response to EPLP-HLA-B*3501

The EPLP 11-mer epitope is an immunodominant CTL determinant in EBV-infected individuals expressing HLA-B*3501 (9, 31). To begin characterizing the TCR repertoire used against this epitope during persistent infection, TCR Vβ protein expression was examined by flow cytometry of PBMCs in unrelated individuals. Specific CD8+ T cells were characterized using an EPLP-HLA-B*3501 tetramer and 21 Vβ-specific mAbs. A significant proportion of peripheral blood CD8+ cells from four unrelated EBV-seropositive, HLA-B*3501-positive individuals were tetramer positive (1.5%, 1.0%, 2.1%, and 11.1% for donors EL, MW, DY, and GB, respectively) and, in all cases, the EPLP-specific CTL repertoire exhibited marked skewing in Vβ usage (Fig. 1⇓, a–d). The TRBV10 family (32) was observed in all donors, with frequencies ranging from 10 to 60% of EPLP-specific T cells. The response in donors EL and MW was particularly focused toward this TCR family. A second Vβ family, TRBV28, was also represented in all individuals, representing >20% of specific T cells in donors DY and GB. In contrast to the other donors, the TRBV19 family dominated the EPLP-specific response in donor DY.

FIGURE 1.
FIGURE 1.

The repertoire of TCR β-chains used against the EBV epitope EPLPQGQLTAY is restricted. PBMCs from the EBV-seropositive, HLA-B*3501+ donors EL (a), MW (b), DY (c), and GB (d), and a bulk CTL culture from donor DY, raised by in vitro stimulation with the EPLP peptide (e), were costained with an EPLPQGQLTAY-HLA-B*3501 tetramer and 1of 21 TRBV-specific Abs. A similar analysis was also done with PBMCs from donor MW (HLA-B*0801+) using an HLA-B*0801 tetramer incorporating another EBV epitope, RAKFKQLL (f). Only gated percentages of 0.02% and more were considered significant. The designation of TRBV follows IMGT TCR gene nomenclature (37 ).

Since further analysis of the EPLP-specific TCR repertoire in some donors required short-term in vitro expansion of virus-specific T cells, it was important to first verify whether such cultures accurately reflected TCR usage in vivo. A similar analysis using the panel of Vβ-specific mAbs was therefore conducted using CTL cultures from each donor that had been expanded with IL-2 for 10 days following initial stimulation with the 11-mer peptide. Although the proportion of tetramer-positive cells in these short-term CTL cultures was far greater than in PBMCs from each donor, the pattern of Vβ usage was not significantly different from those shown in Fig. 1⇑, a–d, for PBMCs (data from a representative culture raised from donor DY is shown in Fig. 1⇑e). These data indicate that short-term in vitro expansion does not induce major perturbations in the repertoire of EPLP-specific T cells compared with PBMCs.

Since donor MW expresses HLA-B*0801 as well as B*3501, it was also possible to assess TCR diversity in the response by this donor to a second highly immunogenic epitope from the BZLF1 Ag, the HLA-B*0801-binding RAKFKQLL epitope (RAK). Consistent with earlier reports, the CTL response to this short 8-mer peptide was highly diverse, with many Vβ families represented within the population of cells staining with an HLA-B8-RAK tetramer (Fig. 1⇑f).

Shared αβ TCR usage in the CTL response to EPLP-HLA-B*3501

A panel of EPLP-specific CTL clones was raised from each of the four HLA-B*3501+ donors, and their TCR V(D)J junctional sequences were determined. Surprisingly, many of the clones expressed Vα and Vβ TCR sequences that were identical or highly homologous to each other at the amino acid level, including clones isolated from unrelated individuals (Table I⇓). The most striking examples of this were the almost identical combinations of TRBV10-3/TRBJ1-5, and TRAV1-2/TRAJ6 expressed by multiple clones raised from donors MW and EL, with only one or two amino acid substitutions in each V(D)J region. Another intriguing finding was that a CTL clone from donor DY (clone DY9) expressed this same TRAV1-2/TRAJ6 TCR α-chain but in combination with a TRBV19 β-chain, distinct from the TRBV10-3 chain prominent in donors MW and EL, and yet sharing the identical TRBJ and CDR3 elements of the β-chain. Such highly homologous TCRs that select distinct but related Vβ families have not previously been described for other peptide-specific responses to our knowledge.

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Table I.

CDR3 sequences of HLA-B*3501-restricted, EPLP-specific CTL clones

The TRBV28-expressing CTL clones from donors GB and DY did not exhibit the homogeneity of the V(D)J associated with TRBV10/19-expressing clones. In contrast, the TRBV28 clones showed a preference for TRBJ2-1 and a CDR3 β-chain length of eight residues and all were associated with TRAV3 α-chains (Table I⇑).

Recurrent CDR3 sequence patterns in CTLs recognizing EPLP-HLA-B*3501

The V(D)J junctional analysis was expanded to include a pool of EPLP-HLA-B*3501 tetramer+CD8+ cells from each of the four donors. These cells were sorted using flow cytometry from PBMCs (donors MW and GB) or bulk CTL cultures raised by short-term in vitro stimulation with peptide EPLP (donors EL and DY). cDNAs corresponding to each Vβ family identified as a major expansion in the TCR mAb analysis (Fig. 1⇑) were amplified by PCR and cloned into bacteria along with TRAV1 cDNA. This method allowed a much more detailed analysis of individual clonotypes used in the EPLP-specific response. CDR3 amino acid sequences are shown for TRBV10/19, TRBV28, and TRAV1 TCRs used by each donor (Table II⇓). Consistent with the data on CTL clones, this more extensive analysis confirmed that the EPLP 11-mer epitope is targeted by CTL populations with highly focused TCR usage. Multiple sets of shared TCRs were identified in conjunction with striking conservation of certain CDR3 motifs.

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Table II.

Analysis of the HLA-B*3501-restricted, EPLP-specific CTL TCR repertoire

T cells expressing the TRBV10/19 or TRAV1 chains were detected in all donors, and the CDR3 regions displayed a high level of conservation in >120 sequences determined for each of the TCR α- and β-chains. The TRAV1 α-chain was highly conserved, being associated exclusively with TRAJ6 and a cluster of small amino acids within the CDR3 region (an SGGS motif), similar to that observed in the CTL clones. The TRBV10-3 β-chain was associated with a TGD motif within the CDR3 region and was frequently combined with TRBJ1-5 or, less commonly, with TRBJ2-7. As was observed with the CTL clones, these β-chains displayed an unusual sequence “mosaicism,” as in donor DY where TRBV10 was used in place of TRBV19 in TCRs that were highly homologous in other regions. The CDR3 length of the TRBV10/19/TRAV1 chains was restricted to nine residues in most cases.

TRBV28 TCR sequences were recovered from all donors, including MW and EL, where CTL with this β-chain were present at low frequency. These sequences exhibited significant CDR3 patterning, incorporating one of four motifs. The RPPGG motif was the most frequent TRBV28 pattern observed, usually in combination with TRBJ2-1. The LPG, LI/IG, and AGG junctional motifs occurred less frequently, but also in combination with TRBJ2-1. The TRBV28 β-chains also showed restriction in CDR3 lengths with the majority being eight or nine residues long. Taken together, the marked TCR V family preferences, V(D)J junctional patterning and CDR3 length restriction observed in EPLP-specific T cells strongly indicates stringent selection of T cell clonotypes.

Structure of the EPLP 11-mer bound to HLA-B*3501

To begin to understand the structural basis of the TCR bias toward the B*3501/11-mer complex, we solved the structure of this complex. Table III⇓ shows the x-ray data collection statistics for HLA-B*3501 in complex with the viral 11-mer peptide EPLPQGQLTAY, and Table IV⇓ shows the refinement statistics for this structure. Table V⇓ lists all of the contacts between HLA-B*3501 and the EPLPQGQLTAY peptide. Since HLA-B*3501 prefers peptide ligands with proline as a dominant anchor residue at position (P) 2, along with a large hydrophobic residue at the C terminus (33, 34, 35), it was not possible to predict the binding register of the EPLP 11-mer peptide with certainty since either of the two proline residues at P2 and P4 could serve as potential anchor residues. Moreover, HLA-B*3501 molecules are able to bind the overlapping BZLF1 9-mer (56LPQGQLTAY64) and 11-mer (54EPLPQGQLTAY64) with equal affinity, even though only the longer 11-mer peptide is immunogenic in HLA-B*3501 individuals (27). The crystal structure of the B*3501/11-mer complex was solved to 1.7 Å resolution (Rfac, 21.9%; Rfree, 24.5%; Table IV⇓) and demonstrated unequivocally that P2-Pro formed a dominant anchor residue of the EPLP peptide (Fig. 2⇓).

FIGURE 2.
FIGURE 2.

Structure of the highly bulged EPLP 11-mer complexed to HLAB*3501. a, Structure of the bulged 11-mer epitope complexed to HLA-B*3501. Electron density (2Fo–Fc) shown in mesh format, residues shown in ball-and-stick format. b, Superposition of the bulged epitope bound to HLA-B*3501 (red) compared with an 8-mer epitope (34 ) (blue).

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Table III.

X-ray data collection statistics of HLA-B3501 in complex with EPLPQGQLTAY

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Table IV.

Refinement statistics of HLA-B3501 in complex with EPLPQGQLTAY

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Table V.

Contacts between HLA-B*3501 and EPLPQGQLTAY

The bound epitope did not participate in crystal contacts and the electron density for the bound peptide was very clear. The EPLPQGQLTAY epitope bound in bulged mode with a kink at position P4-Pro, with the N and C termini anchored in the A and F pocket, respectively. Nearly all of the direct hydrogen bonds between peptide and HLA-B*3501 are located within these pockets (Table V⇑). The P1-Cα/PΩ-Cα distance is 21.8 Å in the HLA-B*3501/EPLP complex, which compares closely to the P1-Cα/PΩ-Cα distance observed in the previously determined octameric and nonameric HLA-B*3501 complexes (34, 35) (Fig. 2⇑b). In total, the 11-mer peptide makes 16 hydrogen bonds, 2 water-mediated hydrogen bonds, and 2 salt bridges. Within the A-pocket, the peptide maintains conserved hydrogen bonds with Tyr7 and Tyr159 while the P1-Glu side chain forms an additional hydrogen bond with Asn63 and salt bridges with the polymorphic Arg62. The PΩ tyrosine has nonstandard positioning in the F-pocket (34, 35). Nevertheless, the PΩ still maintains the conserved hydrogen bonds with residues in the F-pocket, including interactions with Tyr84, Thr143, and Lys146 and additional hydrogen bonds with Ser77, Asn80, and Ser116 (36, 37).

The P2-Pro binding pocket, an anchor motif for HLA-B35, is conserved between this structure and that previously described (34, 35). Notably, a network of water-mediated hydrogen bonds between HLA-B*3501 and the EPLPQGQLTAY epitope was observed that may partly assist in stabilizing this epitope within the binding groove (Table V⇑); many of these water molecules reside in the space normally occupied by the 8–9-mer peptides bound to HLA-B35.

The 11-mer epitope protrudes above the Ag-binding cleft to such an extent that the extreme tip of the bulged region of the peptide is ∼8Å higher in the peptide binding groove in comparison to the HLA-B35-octameric complex (Fig. 2⇑b). The average temperature factor of the bound 11-mer was 28.7 Å2, with the most mobile residues at the tip of the bulge (residues 5–7, temperature factors 44.1, 45.7, 49.6 Å2, respectively). Positions P4–P10 of the epitope are solvent-exposed and thus represent possible contact sites for a TCR.

Fine specificity of TCR interaction: accommodation of the 11-mer peptide bulge

To investigate how the shared B*3501-restricted TCRs interact with the bulged 11-mer peptide, the dominant CTL clonotypes were tested for their ability to tolerate single amino acid substitutions throughout the length of the EPLP peptide (Fig. 3⇓, a–e). To aid with interpretation of this data, peptide MHC-binding assays were also performed on selected peptides that were not well recognized by any of the CTL clones (Fig. 3⇓f). These binding assays measured the efficiency of stabilizing HLA-B*3501 molecules on the surface of the Ag-processing mutant T2 cell line transfected with HLA-B*3501 and pulsed with exogenously added peptide. The concentration of peptide required for half-maximum stabilization of HLA-B*3501 was then calculated (Fig. 3⇓f). Since the shared primary anchor residues at P2 and P11 were the same for all of the analogues of EPLP that were tested, it was not surprising that most of the peptides bound well to HLA-B*3501 (Fig. 3⇓). Furthermore, the impact on MHC binding of amino acid replacements at central peptide residues is likely to be marginal due to the bulged peptide conformation. The reduction in binding by the Leu→Lys replacement at P3 (Fig. 3⇓) was consistent with earlier reports defining P3 as an important secondary anchor residue for HLA-B*3501, with aliphatic hydrophobic residues preferred and basic amino acids exerting a negative impact (38).

FIGURE 3.
FIGURE 3.

The impact of single amino acid substitutions within the EPLP peptide on CTL recognition and HLA-B*3501 binding. The CTL clones ELS4 (a), ELS2 (b), GB1 (c), DY18 (d), and ELS3 (e) were tested for recognition of a panel of altered peptide ligands into which single amino acid substitutions were introduced. A range of peptide concentrations was used in these chromium release assays, and the concentration required for half-maximum lysis was calculated from this dose-response data and is shown in a–e. MHC-peptide-binding assays were also conducted by testing each peptide at a range of concentrations for its ability to stabilize HLA-B*3501 expression on the surface of the Ag-processing mutant T2 cell line. The concentration of each peptide required for half-maximum HLA-B*3501 stabilization was calculated and is shown in f.

A total of 67 analogues of the 11-mer peptide were tested for CTL recognition over a range of concentrations using chromium release assays, and the concentration of peptide required for half-maximum lysis was calculated (Fig. 3⇑, a–e). The pattern of T cell recognition of these analogues showed subtle variation between the different CTL clones. Thus, CTL clones ELS4 and ELS2 express the highly conserved TRBV10-3/TRAV1-2 shared TCR, with minor differences within the CDR3 regions of both the α- and β-chains (Table I⇑). Although the parent EPLP peptide induced half-maximal CTL lysis at concentrations between 0.1 and 1 nM, single amino acid replacements at many different positions within the peptide led to greatly reduced recognition, suggesting that this immunodominant TCR straddles most of the peptide bulge (Fig. 3⇑, a and b). There was, however, considerable flexibility in the interaction by this TCR since most of the peptide residues could be replaced with at least one other amino acid without affecting CTL recognition. The one exception was the central glycine residue that appeared most critical for recognition since substitutions at P6 invariably led to a dramatic loss of activity. Surprisingly, although P1-Glu is not well exposed (Fig. 1⇑a), it was critical for recognition by this TCR (Fig. 3⇑, a–e), and only an aspartic acid residue was tolerated as a replacement. This suggests that the P1 residue most likely controls the conformation of Arg62, a residue known to be important in TCR ligation (39). The highly prominent central P5-Gln-P6-Gly-P7-Gln residues are possible contact sites for these immunodominant TCRs since the majority of amino acid replacements led to greatly reduced CTL lysis. The P10-Ala appeared to play little role in either MHC binding or recognition by this TCR since none of the six amino acid substitutions at this position had a major impact on CTL lysis.

The CTL clones GB1 and DY18 express closely related TRAV3/TRBV28 Ag receptors, although the α-chains differ within the CDR3 and TRAJ regions (Table I⇑). Despite these sequence differences, the fine specificity of these two clones was very similar, but with GB1 being more tolerant of amino acid substitutions toward the C terminus of the peptide than DY18 (Fig. 3⇑, c and d). In comparison to the TRBV10-3-shared TCR, these TRBV28-expressing receptors displayed slightly higher peptide specificity, with peptide P4–6 (Pro-Gln-Gly) appearing to be the major focus for TCR contact. Surprisingly, the highly prominent glutamine residue at P7 could be substituted with a number of amino acids with no impact on recognition by the GB1 or DY18 CTL clones, suggesting that these TCRs avoid contact with this residue, instead focusing primarily on the N-terminal half of the peptide. As with the CTL clones expressing TRBV10-3, the TRBV28-expressing clones were highly specific for the P1-Glu. The CTL clone ELS3 expresses a subdominant TCR with no obvious homology to the others identified in this study, except for a cluster of five small amino acids (GGGGS) in the CDR3 region of the α-chain (Table II⇑). Interestingly, this clone is more sensitive to changes in the C terminus of the peptide, with all but one amino acid substitution being accommodated at P1 but very few allowed within the Gln-Gly-Gln-Leu-Thr region of the peptide (P5-P9) (Fig. 3⇑e). The bulged 11-mer peptide structure can therefore be accommodated by TCRs in more than one orientation, even though the immunodominant Ag receptors selected against the EPLP peptide all share similar footprints on the peptide-MHC complex spanning the N-terminal half of the peptide.

Discussion

The structure of HLA-B*3501 in complex with the BZLF1 peptide EPLPQGQLTAY reveals the peptide anchored into the HLA-B*3501 Ag-binding cleft at its termini, with the central residues forming a bulge somewhat analogous to that observed in the crystal structures of a rat MHC-I/13-mer (13) and a human MHC-I/14-mer (14). Although the HLA-B*3501/14-mer complex is immunogenic (11, 14), little is known about the fine specificity and diversity of the T cell response toward this ligand or toward bulging MHC-I peptides in general.

The bulge of the EPLP 11-mer protrudes further out of the Ag-binding cleft compared with the previously reported 8- and 9-mers bound to HLA-B*3501 (34, 35). Within the bulge, seven solvent-exposed residues are available for T cell recognition, (P4–P10). This significant exposure of so many protruding residues in the EPLP 11-mer epitope is likely to pose a challenging target for responding T cells and we speculated that this might constrain TCR diversity. Diversity in the TCR repertoire toward defined epitopes is believed to be advantageous to the host during protective immunity, perhaps through generation of higher avidity T cells (40, 41) and/or through avoidance of viral escape mutants that affect T cell recognition (42). Nonetheless, biased usage of V or J regions and/or conserved CDR3 lengths or sequence motifs has been observed in the CTL response to a number of viruses (12, 43, 44, 45, 46, 47).

The repertoire of HLA-B*3501-restricted, 11-mer-specific CD8+ T cells was highly restricted to combinations of TRAV1-2/TRBV10 and TRAV3/TRBV28 TCRs. TCRs with TRBV chains 10-3 and 28 showed a similar specificity for the EPLPQGQLTAY peptide analogues. Fine specificity analysis demonstrated that the selected TCRs were very sensitive to substitutions in clusters of exposed peptide residues. Thus, TRBV10-3 was highly sensitive to substitutions at P1, P5, P6, and P7, whereas TRBV28 was highly specific for P1, P4, P5, and P6. Substitution of P1-Glu with Asp was tolerated by TRBV10-3 and TRBV28, whereas other substitutions at P1 led to a significant reduction in binding. This effect is possibly attributable to the impact the substitutions have on the conformation of the polymorphic Arg62, which has been shown to be sensitive to the residues within the P1 pocket (39). Substitution of the solvent-exposed residues in the bulge of the peptide (P5, P6, and P7) was critical to the specificity of TRBV10-3, whereas TRBV28 was more affected by substitutions at P4–P6.

All EPLP-specific T cells examined from each HLA-B*3501 donor had one or another of the above TCR chain pairings containing a CDR3 sequence sharing a common, conserved motif. Notably, the CDR3 motifs were more stringently required than particular V and J region pairings, and in several donors central CDR3 motifs were conserved while V/J regions were different. For instance, in the donor DY, the majority of EPLP-reactive T cells used TRBV19 with the CDR3 motif of TRBV10 T cells. This observation is unusual but understandable in that the TRBV10 and TRBV19 genes are both related and clustered together in the Vβ phylogenetic tree (48). Another intriguing finding was that a CTL clone from donor DY (clone DY9) expressed this same TRAV1-2/TRAJ6 TCR α-chain in combination with a β-chain that differed by the use of the TRBV10-3 chain instead of the TRBV19 chain, while the TRBJ and CDR3 elements of the β-chain were conserved. The basic patterns of TCR bias or immunodominance could reflect a restricted Vα/Vβ geometry. However, the shared specificity of two different TCRs, each with the same α-chain (TRAV1-2), but paired with a different β-chain (TRBV19 or TRBV10-3) suggests that both combinations of Vα and Vβ are able to orientate the TCR correctly on top of the cognate MHCp.

In most Ag-specific T cell responses, the length and sequence of TCR CDR3α and β loops is highly variable (49), yet EPLP-specific CTL clones from unrelated HLA-B*3501+ donors had a highly restricted CDR3 length of typically eight (TRAV3/TRBV28) and nine amino acids (TRAV1-2/TRBV10–3/19) along with conservation of particular sequence motifs in both chains. Interestingly, a number of these TRBV10-3-expressing Ag receptors have previously been identified in the synovium of patients with rheumatoid arthritis, a site known to accumulate virus-specific T cells, thereby confirming the widespread distribution of this TCR in EBV-seropositive, HLA-B*3501-positive individuals (50, 51). We previously reported that TCR immunodominance in the CTL response to an EBV Ag was associated with exquisite TCR specificity and its capacity to make extravagant structural adjustments, increasing the complementarity of receptor-ligand binding (52, 53). In contrast, the highly restricted CTL response to an influenza matrix Ag appears to reflect the small target this epitope presents to T cells (54), such that only main chain atoms are available for recognition by the TCR that binds by inserting a conserved CDR3β loop into a notch on the MHCp surface (55). The precise structural role of the TCR CDR3 motifs selected in CTL responses to the EPLP-11-mer awaits the determination of the three-dimensional structure of this immunodominant TCR-MHCp complex.

The EPLP 11-mer epitope overlaps with an immunogenic 13-mer peptide that binds to HLA-B*3508 (9). Structural studies have revealed that this peptide is also bound in bulged mode, and the extreme tip of the bulged region is ∼2Å higher in the peptide binding groove in comparison to the EPLP 11-mer (56). The larger bulged region of the 13-mer peptide is more rigid than that of the 11-mer due to a central proline residue and intrapeptide water-mediated hydrogen bonds as well as water-mediated hydrogen bonds to HLA-B*3508. The TCR repertoire directed toward this 13-mer epitope is also highly restricted, primarily in α-chain usage (56).

Based on the data presented herein, it is not possible to conclude with certainty the precise mechanisms controlling TCR usage in the response to this 11-mer EBV epitope. The focused response may result from peripheral selection for the highest affinity TCRs or it could reflect a higher frequency of some TCRs in the preimmune repertoire. Alternatively, it could reflect structural constraints imposed by unusual MHCp ligands. We recently demonstrated (J.R., J.M., and A.W.P.) that a lack of prominent features in the MHCp restricts the TCR repertoire in viral immunity (57). The possibility remains at the other extreme of highly bulged, “overprominent” MHCp structures that biased TCR usage sometimes occurs as a result of a different type of structural constraint to TCR-MHCp ligation.

Acknowledgments

We thank the BioCars staff for assistance in data collection at the Advanced Photon Source, Chicago.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported by grants from the National Health and Medical Research Council, Australia; the Roche Organ Transplantation Research Fund; the Juvenile Diabetes Research Foundation, and the Australian Research Council. S.R.B. is a recipient of a National Health and Medical Research Council Career Development award, and J.R. is a Wellcome Trust Senior Research Fellow.

  • ↵2 J.J.M. and D.E. contributed equally to this work.

  • ↵3 Address correspondence and reprint requests to Dr. James McCluskey, Department of Microbiology & Immunology, University of Melbourne, Parkville 3010, Australia, E-mail address: jamesm1{at}unimelb.edu.au or Dr. Scott R. Burrows, Cellular Immunology Laboratory, Queensland Institute of Medical Research, Brisbane 4029, Australia. E-mail address: scottB{at}qimr.edu.au

  • ↵4 Abbreviations used in this paper: MHCp, MHC-peptide complex; MHC-I, MHC class I; EPLP, 11-mer EBV peptide EPLPQGQLTAY; MFI, mean fluorescence intensity; P, position.

  • Received April 6, 2005.
  • Accepted June 22, 2005.

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