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The Journal of Immunology, 2006, 177: 6804-6814.
Copyright © 2006 by The American Association of Immunologists, Inc.

TCR{alpha} Genes Direct MHC Restriction in the Potent Human T Cell Response to a Class I-Bound Viral Epitope1

John J. Miles2,*,{dagger}, Natalie A. Borg2,{ddagger}, Rebekah M. Brennan*, Fleur E. Tynan{ddagger}, Lars Kjer-Nielsen§, Sharon L. Silins*, Melissa J. Bell*, Jacqueline M. Burrows*, James McCluskey§, Jamie Rossjohn3,{ddagger} and Scott R. Burrows3,*

* Cellular Immunology Laboratory, Queensland Institute of Medical Research, Brisbane, Australia; {dagger} School of Population Health, University of Queensland, Brisbane, Australia; {ddagger} The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Australia; and § Department of Microbiology and Immunology, University of Melbourne, Parkville, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The underlying generic properties of {alpha}β TCRs that control MHC restriction remain largely unresolved. To investigate MHC restriction, we have examined the CTL response to a viral epitope that binds promiscuously to two human leukocyte Ags (HLAs) that differ by a single amino acid at position 156. Individuals expressing either HLA-B*3501 (156Leucine) or HLA-B*3508 (156Arginine) showed a potent CTL response to the 407HPVGEADYFEY417 epitope from EBV. Interestingly, the response was characterized by highly restricted TCR β-chain usage in both HLA-B*3501+ and HLA-B*3508+ individuals; however, this conserved TRBV9+ β-chain was associated with distinct TCR {alpha}-chains depending upon the HLA-B*35 allele expressed by the virus-exposed host. Functional assays confirmed that TCR {alpha}-chain usage determined the HLA restriction of the CTLs. Structural studies revealed significant differences in the mobility of the peptide when bound to HLA-B*3501 or HLA-B*3508. In HLA-B*3501, the bulged section of the peptide was disordered, whereas in HLA-B*3508 the bulged epitope adopted an ordered conformation. Collectively, these data demonstrate not only that mobile MHC-bound peptides can be highly immunogenic but can also stimulate an extremely biased TCR repertoire. In addition, TCR {alpha}-chain usage is shown to play a critical role in controlling MHC restriction between closely related allomorphs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The CTLs bearing {alpha}β TCRs are pivotal in adaptive immunity, recognizing short antigenic peptide fragments bound to class I MHC molecules (pMHC-I)4 (1, 2). MHC-I can ligate a diverse array of antigenic peptides due to the high degree of polymorphism within the six pockets of the Ag-binding cleft (3, 4). The selection of peptides that bind within the MHC-I cleft is usually determined by two pockets that specify primary anchor sites (5, 6), with one or more secondary anchor sites that fine tune the binding motifs for individual MHC-I allotypes (7). For example, the common HLA class I molecule, HLA-B*3501, preferentially binds peptide ligands with proline as a dominant anchor residue at position 2 and a large hydrophobic residue at the C terminus (8, 9, 10). Although the majority of peptides that bind to MHC-I molecules are between 8 and 10 residues in length, it is clear that some longer peptides can also bind and play an important role in anti-viral immunity (11). Several MHC-I-bound peptides that are longer than ten residues have been characterized structurally, and in each case, the peptide has been shown to adopt a centrally bulged conformation whereas maintaining the conserved hydrogen bonding network to MHC-I via the peptide N and C termini (12, 13, 14, 15, 16, 17). These bulged epitopes can either adopt a rigid conformation or display a considerable degree of flexibility; however, the impact of peptide flexibility on the T cell response to these determinants is unknown.

The interaction between {alpha}β TCRs and the pMHC has been the subject of many structural and biochemical studies (18, 19, 20, 21, 22, 23, 24, 25, 26) and some general principals have emerged. It appears that {alpha}β TCRs have inherent reactivity toward MHC molecules (22, 26) such that the TCR {alpha}-subunit is positioned near the N terminus of the peptide and the TCR β-subunit is positioned near the C terminus (23). In this manner, the germline derived CDR1 and CDR2 loops generally engage the helices of the MHC while the hypervariable CDR3 loops are considered to have a more important role in engaging the peptide (23). However, despite these general rules, the 13 independent TCR-pMHC structures determined to date have revealed a series of unique docking solutions, providing variations on the generalities of TCR/pMHC engagement (23). Consequently, since no universal set of amino acid contacts have been identified between the TCR and the MHC, the underlying "rules" governing MHC restriction remain largely unknown. Nevertheless, it appears that a so-termed "restriction triad" of MHC residues represents an important docking site for the {alpha}β TCR (25, 27).

In this study, we have investigated the impact of an MHC-I micropolymorphism on T cell repertoire selection in the CTL response to an EBV epitope. The 11-aa epitope HPVGEADYFEY (referred to as HPVG) is derived from the EBNA-1 protein (residues 407–417) (28, 29, 30) and is another example of a growing number of non-canonical (>10 aa) MHC-I-binding peptides known to be potent natural targets for CTLs (11). We found HPVG to be highly immunogenic in EBV-exposed healthy individuals expressing either HLA-B*3501 or HLA-B*3508, two alleles that differ by a single amino acid at position 156 (156Leucine vs 156Arginine, respectively). Remarkably, unrelated HLA-B*3501+ and HLA-B*3508+ individuals generated CTLs with immunodominant "public" or closely related TCR β-chain sequences, but with an alternate {alpha}-chain pairing that was dependent on the HLA-B*35 allele expression of the host. Furthermore, the major structural difference between HLA-B*3501-HPVG and HLA-B*3508-HPVG was the mobility of the peptide in each complex. These data indicate that: 1) MHC polymorphism can greatly influence the mobility of a bound peptide; 2) mobility does not necessarily disturb the immunogenicity of a peptide; 3) mobile peptides can stimulate a highly restricted TCR repertoire; and 4) the MHC restriction of a TCR can be controlled by {alpha}-chain usage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
T cell cultures

CTL cultures were raised from PBMCs isolated from healthy, EBV-seropositive volunteers who were also positive for HLA-B*3501 and/or HLA-B*3508, as determined by HLA sequence analysis. These studies have been reviewed and approved by an appropriate institutional review committee. CTL clones were generated as previously described (31) using initial stimulation of PBMCs with the HPVG peptide (0.1 µM), followed by biweekly restimulation with rIL-2 and gamma-irradiated (8000 rad) autologous lymphoblastoid cell lines. Short-term CTL bulk cultures were also used for some TCR analyses and cytotoxicity assays. These were generated by culturing PBMCs (2 x 106/2-ml well) with autologous PBMCs that had been precoated with the HPVG peptide (0.1 µM for 1 h, responder/stimulator = 2:1). Cultures were supplemented with rIL-2 (20 U/ml) on day 3, split on day 7, and 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 HPVG-HLA-B*3501 PE-labeled or an HPVG-HLA-B*3508 allophycocyanin-labeled multimer (ProImmune). Cells were then washed and labeled for 30 min at 4°C with tricolor-labeled anti-human CD8 mAb (Caltag Laboratories), allophycocyanin-labeled or PE-labeled anti-human CD3 (BD PharMingen), and one of the following FITC-labeled TCR β-chain-specific mAbs (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 (TRBV11–2), Vβ22 (TRBV2), or Vβ23 (TRBV13). Cells were washed and analyzed on a FACSCanto using FACSDiva software (BD Biosciences). Cell sorting was performed on a MoFlo (DakoCytomation).

Cytotoxicity assay

CTL clones or bulk cultures 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 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 HPVG-HLA-B*3501 or HPVG-HLA-B*3508 multimer before TCR analysis was performed. TCR {alpha}- and β-chain sequences were also determined from pools of multimer+ cells using PCR amplification with primer selection based on data presented in Fig. 1 and Table I. Thus, primers specific for TRBV9 and TRAV20 were used for HLA-B*3501+ cells and primers specific for TRBV9 and TRAV29 were used for HLA-B*3508+ cells. The subdominant TRBV10–3+ β-chains used by donors S.B. and J.W. were also sequenced using primers specific for this variable gene.


Figure 1
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  		FIGURE 1. The Vβ expression profile of TCRs specific for HPVG-HLA-B*3501 and HPVG-HLA-B*3508 is highly restricted. PBMCs or short-term HPVG-specific CTL cultures from EBV seropositive, HLA-B*3501+ or HLA-B*3508+ donors were stained with a corresponding HPVG-HLA-B*3501 or HPVG-HLA-B*3508 multimer and 1 of 21 TRBV-specific mAbs. The frequency of T cells expressing each TRBV in the HLA-B*3501+ donors T.K. (A), M.W. (B), C.S. (C), J.F. (D), E.L. (E), and the HLA-B*3508+ donors C.A. (F), S.B. (G), and J.W. (H) was calculated as a percentage of the total number of multimer+ cells. The designation of TRBV follows the international IMGT gene nomenclature.

 

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  		Table I. CDR3 analysis of TCRs expressed by HPVG-specific CD8+ T cell clones

 
Total RNA was extracted from T cell clones and multimer-sorted bulk CTL cultures using TRIzol reagent. Reverse transcription was performed with Superscript III (Invitrogen Life Technologies) and anti-sense TCR{alpha} and TCRβ chain primers (32). 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 C{alpha} constant primer and 1 of 34 TCR V{alpha} family-specific primers, or a TCR Cβ constant primer and 1 of 26 TCR Vβ family-specific primers (32). 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). The international ImMunoGeneTic database (IMGT) TCR gene nomenclature was used throughout (33) and CDR3 length was defined according to previous criteria (34).

HLA peptide-binding assays

To assess peptide binding to HLA-B*3501 and HLA-B*3508, HLA stabilization studies were conducted using the mutant lymphoblastoid cell line x T lymphoblastoid hybrid cell line, 174 x CEM.T2 (referred to as T2 cells) (35), transfected with HLA-B*3501 or HLA-B*3508. T2.B*3501 or T2.B*3508 cells were incubated in AIM V serum-free medium (Invitrogen Life Technologies) with various concentrations (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 or HLA-B*3508 surface expression was then measured by flow cytometry on a FACSCanto using a mAb to HLA-Bw6 (SFR8 Bw6). Mean fluorescence intensity was determined, and the peptide concentration required for half-maximum mean fluorescence intensity was calculated.

Protein expression, crystallization, and structure determination

Soluble HLA-B*3501 and HLA-B*3508 (aa 1–276) and full-length β2-microglobulin (residues 1–99) were expressed in Escherichia coli as inclusion bodies and refolded with the HPVG peptide as previously described (36). Purified HLA-B*3501 and HLA-B*3508 proteins were concentrated to 12 mg/ml for crystallization by the hanging drop vapor diffusion technique. HPVG-HLA-B*35 crystals were obtained at 4°C by streak seeding from a pre-existing HLA-B*35 crystal and in drops containing a 1:1 ratio of protein to reservoir. Crystals grew in drops that had been equilibrated for 2–3 days in 14–17% PEG 3350, 100 mM cacodylate (pH 7.6), and 200 mM ammonium acetate. HPVG-HLA-B*3501 and HPVG-HLA-B*3508 crystals were frozen in a stepwise manner in up to 15% glycerol, and data were collected at the Advanced Photon Source, beamline 17-ID. A 1.5 Å and 1.9 Å data set was collected for HLA-B*3501 and HLA-B*3508 in complex with peptide, respectively. The crystals belong to space group P212121, had isomorphous unit cell dimensions, and there was one molecule in each asymmetric unit. The HPVG-HLA-B*3501 and HPVG-HLA-B*3508 structures have been deposited in the Protein Data Bank under accession codes 2FYY and 2FZ3.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Highly selected TCR Vβ usage in the CTL response toward an EBV epitope presented by both HLA-B*3501 and HLA-B*3508

The HPVG epitope from the EBNA1 Ag of EBV was originally identified by the discovery of strong CTL responses to this peptide in HLA-B*3501+ virus carriers (28). Our preliminary analysis demonstrated that EBV-seropositive individuals expressing the closely related HLA-B*3508 allele also respond strongly to this peptide (data not shown). Since these two HLA alleles differ by just one residue on the {alpha}2-helix, we set out to investigate the impact of this micropolymorphism on TCR repertoire selection. TCR β-chain protein expression was examined by flow cytometry using PBMCs from unrelated HLA-B*3501+ and HLA-B*3508+ individuals. For two individuals (J.F. and J.W.), short-term HPVG-stimulated CTL cultures were used instead of PBMCs due to a relatively low frequency of specific T cells in the peripheral circulation. Peptide-specific CD8+ T cells were characterized using HPVG-HLA-B*3501 or HPVG-HLA-B*3508 multimers, and 21 mAbs specific for TRBV regions. In HLA-B*3501+ individuals, a significant portion of peripheral blood CD8+ cells stained with the multimer (0.52, 0.47, 0.40, 0.30, and 0.44% for donors T.K., M.W., C.S., J.F., and E.L., respectively) and, in all cases, the TCR repertoire exhibited a marked skewing in Vβ usage (Fig. 1, A–E). Clear focusing of the HPVG-specific response toward the TRBV9 family was observed in all HLA-B*3501+ donors, with frequencies ranging from 31% to 97% of total multimer+ cells. Three other TRBV families were also utilized (at relatively low frequency) in donors T.K., M.W., and C.S., while E.L. showed co-dominant usage of TRBV28. In HLA-B*3508+ individuals, significant levels of multimer+ cells were also found within the peripheral blood CD8+ T cell pool (0.51, 0.74, and 0.29% for donors C.A., S.B., and J.W., respectively) and, surprisingly, TRBV9+ clonotypes also dominated the HPVG-specific response, with frequencies ranging from 28% to 98% of multimer+ cells (Fig. 1, F–H).

HPVG-specific T cells that share a common TCR β-chain display differences in MHC restriction

Although both HLA-B*3501+ and HLA-B*3508+ individuals responded to the HPVG epitope with a common TRBV9 family β-chain, we were interested to know if any functional differences were displayed by these T cell populations. To determine whether the CTLs were self-MHC restricted or had the capacity to cross-recognize HPVG bound to both HLA-B*3501 and HLA-B*3508, we used peptide dose-response cytotoxicity assays (Fig. 2). Thus, HLA-B*3501+ or HLA-B*3508+ PHA blast target cells were labeled with various concentrations of the HPVG peptide and used as target cells for CTL clones or bulk cultures generated from HLA-B*3501+ donors (Fig. 2A), HLA-B*3508+ donors (Fig. 2B) or the HLA-B*3501/3508 co-expressing donor M.B. (Fig. 2C).


Figure 2
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  		FIGURE 2. HPVG-specific CTL lines display distinct HLA restriction patterns in cytotoxicity assays. HPVG-specific CTL clones and bulk T cell lines from EBV-seropositive donors who expressed HLA-B*3501 (A), HLA-B*3508 (B), or both of these HLA alleles (C) were assessed for HLA restriction using peptide dose-response cytotoxicity assays with PHA blast target cells expressing either HLA-B*3501 or HLA-B*3508 and the HPVG peptide.

 
Two CTL clones from the HLA-B*3508+ donor SB (Fig. 2B, left and middle panel), and two CTL clones from the HLA-B*3501/B*3508 co-expressing donor M.B. (Fig. 2C) were restricted exclusively through HLA-B*3508. Further analysis of these four CTL clones demonstrated strong staining with the HPVG-HLA-B*3508, but not the HPVG-HLA-B*3501, multimer (data not shown). The HPVG-stimulated CTL bulk culture from donor C.A. recognized the peptide much more efficiently in association with self-HLA-B*3508, with low level recognition of HLA-B*3501+ target cells pretreated with high peptide concentrations (Fig. 2B, right panel).

Interestingly, the HLA-B*3501+ CTL lines were able to recognize the HPVG peptide in association with either HLA-B*3501 or HLA-B*3508 (Fig. 2A). Three CTL clones from donor T.K. recognized the peptide marginally more efficiently when bound to self-HLA-B*3501, whereas CTL clones from donors C.S. and M.W., and the CTL bulk culture from donor T.K. recognized the peptide marginally more efficiently in association with non-self-HLA-B*3508. Multimer staining experiments confirmed significant staining with each multimer (data not shown).

MHC restriction of T cells specific for the HPVG epitope is controlled by TCR {alpha}-chain usage

To investigate the molecular basis for the differences in MHC restriction displayed by these HPVG-specific CTLs, a panel of nine HPVG-specific CTL clones were raised from five HLA-B*35+ individuals, and their TCR variable (V), diversity (D) and joining (J) sequences determined. Consistent with earlier data, most of the HPVG-specific clones expressed TRBV9, and remarkably, many also expressed identical or highly homologous TCR CDR3 elements (Table I). Furthermore, three unrelated HLA-B*3501+ donors (T.K., C.S., and M.W.) all produced clones with near identical {alpha}β TCR combinations of TRBV9/TRBJ2–2 and TRAV20/TRAJ58, with the highly homologous chains including only 1 or 2 aa substitutions in each V(D)J region. Notably, the minor TCR sequence variation observed between the CTL clones TK3, TK6, CS1, and MW1 was associated with small functional differences (Fig. 2A and Table I).

Three CTL clones from the HLA-B*3508+ donor SB expressed either TRBV5–1 or the common TRBV9 in combination with more diverse TRBJ, TRAV, and TRAJ elements. It was particularly interesting that two HPVG-specific CTL clones from the HLA-B*3501/3508 co-expressing donor M.B., which were restricted exclusively through HLA-B*3508 (Fig. 2C), were found to be nucleotide identical clonotypes that expressed the same "public" TRBV9/TRBJ2–2 β-chain found in HLA-B*3501+ donor T.K. However, pairing with a different TRAV29/TRAJ40 {alpha}-chain changed the specificity of the TCR, preventing cross-recognition of the HPVG peptide bound to HLA-B*3501 (Table I).

To analyze the TCR repertoire to this EBV determinant in more detail, HPVG-specific T cells were sorted using the peptide-MHC multimers from short-term bulk CTL cultures raised from five HLA-B*3501+ and three HLA-B*3508+ individuals, and donor M.B. who expressed both of these HLA alleles, and each T cell population was then subjected to mass TCR sequencing. Focused gene usage among all unrelated HLA-B*3501+ and HLA-B*3508+ donors was clear (Table II). HPVG-specific TCR β-chains from the HLA-B*3501+ donors exhibited an almost exclusive pairing of TRBV9 with TRBJ2–2, a predominant CDR3 span of 8 residues, and a predictive "ART", "VRT", or "ARS" amino acid motif within the CDR3 sequence. There appeared to be strong selection pressure for the random, N nucleotide encoded Arg at position 4 of the CDR3β sequence. The HPVG-specific TCR {alpha}-chains from the HLA-B*3501+ donors also demonstrated restricted VJ usage, with TRAV20 pairing with either TRAJ58, TRAJ39, TRAJ17, or TRAJ12. All CDR3{alpha} sequences were restricted to 10 residues in length, and there was a very strong preference for the random N nucleotide encoded Leu at position 4 of the CDR3{alpha} sequence of HLA-B*3501+ individuals.


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  		Table II. CDR3 analysis of TCR {alpha}- and β-chains expressed by HPVG-specific, multimer-sorted T cells from HLA-B*35+ individuals

 
The HPVG-specific TCR β-chains from the HLA-B*3508+ donors consisted of a combination of CDR3 elements seen in HLA-B*3501+ donors and several unique TRBV/TRBJ pairings (Table II). For instance, the TCR repertoire of donor C.A. was comprised of both the "public" TRBV9/TRBJ2–2 β-chain found in HLA-B*3501+ donors and a unique TRBV9/TRBJ2–7 combination. Interestingly, the TRAV29+ {alpha}-chains from donor C.A. were all paired to TRAJ40, with a CDR3 sequence virtually identical to that expressed by the HLA-B*3508-restricted MB1 and MB4 CTL clones (Table I and II). The subdominant TRBV10–3+ β-chains revealed in donors S.B. and J.W. using the TRBV-specific mAb (Fig. 1) had the same CDR3 length but limited sequence conservation.

Overall, the predictive V(D)J patterning observed in HPVG-specific T cells from both HLA-B*3501+ and HLA-B*3508+ individuals indicates the operation of strong immune selection. This conclusion is supported by the observation that several different nucleotide sequences were used by some of these individuals to obtain the same conserved CDR3 amino acid sequences (see footnote e in Table II). Additionally, the ability to divide distinctive TCR features among HLA-B*3501 and HLA-B*3508-restricted T cells strongly suggests that HPVG-specific TCRs are "groomed" by unique structural features within their cognate peptide-MHC ligands.

Structural differences between the HPVG-HLA-B*3501 and HPVG-HLA-B*3508 complexes

To begin to understand the structural basis underlying the MHC preferences of these HPVG-specific T cells, we determined the three-dimensional structure of both the HPVG-HLA-B*3501 and HPVG-HLA-B*3508 complexes to 1.5 Å and 1.9 Å, respectively (Fig. 3, A and B; refinement statistics shown in Table III). Both complexes crystallized under the same conditions and had isomorphous unit cell dimensions, and thus any conformational differences between the HPVG-HLA-B*3501 and HPVG-HLA-B*3508 complexes are directly attributable to the single amino acid polymorphism at position 156 (Leu156) in HLA-B*3501 vs Arg156 in HLA-B*3508, respectively).


Figure 3
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  		FIGURE 3. Structure of the bulged HPVG peptide bound to HLA-B*3501 and HLA-B*3508. The Ag-binding clefts of HLA-B*3501 (A) and HLA-B*3508 (B) in complex with the HPVGEADYFEY peptide with the {alpha}2-helix removed. The 2Fo – Fc electron density is shown in mesh format at 1.5 Å in the HLA-B*3501 complex and 1.9 Å in the HLA-B*3508 complex. C, Superposition of the Ag-binding domains of HPVG-HLA-B*3501 (shown in pink) and HPVG-HLA-B*3508 (shown in yellow). The peptide has been modeled in its entirety in the HLA-B*3508-binding cleft, but peptide residues 5–8 have been omitted from the binding cleft of HLA-B*3501.

 

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  		Table III. Data collection and refinement statistics for structural analysis

 
Peptide contacts with the H chain of both HLA-B*3501 and HLA-B*3508 are described in Table IV. In both HLA-B*3501 and HLA-B*3508 the HPVG peptide bound in an extended and bulged conformation with the N and C termini participating in a conserved network of H-bonds that anchor the peptide into the A and F pockets, respectively. The structure of HLA-B*3508 is described first, followed by a comparison to the HLA-B*3501 structure. In the HPVG-HLA-B*3508 complex, the electron density of the peptide was ordered and modeled in its entirety, with the exception of the side chain of Tyr8, which was mobile (Fig. 3B). The ordered nature of the peptide was shown by the low average temperature factor of all residues within the peptide (29.57 Å2). The HPVG peptide bulged approximately 7 Å out of the HLA-B*3508 cleft such that the side chains at positions His1, Ala6, Asp7, Tyr8, and Glu10 of the peptide were surface exposed and available for TCR interaction (Fig. 3B).


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  		Table IV. HLA contacts with the HPVGEADYFEY peptide

 
The well ordered conformation of the centrally bulged region of the epitope was attributable to a network of interactions between the peptide and HLA-B*3508, emanating from the polymorphic residue Arg156. In HLA-B*3508, Arg156 directly contacted the peptide and formed a salt bridge with Glu5-O{epsilon}1 of the HPVG peptide (Fig. 4). Furthermore, the electropositive charge of Arg156 appeared to repel the electropositive side chain of Arg97, which extended from the β-sheet floor of the Ag-binding cleft. In this position, Arg97 was able to directly contact the peptide at 3 positions, namely forming van der Waals contacts with positions 3 and 11 of the peptide and forming a salt bridge with Glu5-O{epsilon}2, which also served to stabilize the conformation of the peptide (Fig. 4).


Figure 4
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  		FIGURE 4. Local impact of the polymorphism at MHC position 156. The Ag-binding domain of HLA-B*3508 in complex with HPVGEADYFEY (yellow) (A) highlighting the stabilizing interactions between Glu5 of the peptide and Arg97 and Arg156 of the MHC. Salt bridges are shown as dashed lines and the {alpha}1-helix has been removed for clarity. Residues Arg97 and Leu156 of HLA-B*3501 are shown in magenta. Note the loss of interaction with the peptide. B, The effect of the polymorphic amino acid at position 156 on the conformation of Arg97.

 
In contrast, the electron density corresponding to the HPVG peptide bound to HLA-B*3501 was disordered, indicating a large degree of flexibility around the central positions 5–9 of the peptide. Consequently, only the terminal residues (residues 1–4 and 10–11) of the peptide could be modeled in their entirety (Fig. 3). This mobility was not attributable to the quality of the data as the structure was determined to 1.5 Å resolution, and the remainder of the structure exhibited excellent electron density.

The structures of HLA-B*3501 and HLA-B*3508 superpose extremely well (root mean square deviation of 0.2 Å) over residues 1–180 of the H chain, with no C{alpha} differences >0.4 Å apart) (Fig. 3C), and thus, the polymorphism at position 156 does not significantly affect the conformation of the H chain. Nevertheless, the differences in the mobility of the HPVG peptide in HLA-B*3501 and HLA-B*3508 are directly attributed to the polymorphic amino acid at position 156.

In HLA-B*3501, the Arg156 is non-conservatively substituted by the shorter Leu side chain and as such is no longer able to form direct contact with the peptide to maintain its stability. Furthermore, due to the shorter chain length of Leu156, the NH2 group of Arg97 was rotated relative to the position in HLA-B*3508, and Arg97 was now only able to make van der Waals contacts with position 11 of the peptide (Fig. 4). Therefore, Glu5 in the HPVG peptide had an overall net loss of two salt bridge interactions with HLA-B*3501 as a direct result of the substitution at position 156 compared with HLA-B*3508, contributing to the disordered nature of the peptide in this complex.

Fine peptide specificity of the HPVG-specific T cells

Despite its structural flexibility, the HPVG epitope is highly immunogenic in HLA-B*3501+, EBV-exposed individuals. To investigate how mobile peptides are recognized by TCRs, and determine whether the rigid terminal residues of the HPVG peptide have a more prominent role in TCR contact compared with the flexible central bulge residues, we conducted a fine peptide specificity analysis using altered peptide ligands.

Four HPVG-specific CTL clones that used distinct TCRs and/or displayed different HLA restriction patterns were tested for their ability to tolerate single amino acid substitutions throughout the length of HPVG peptide. A total of 54 single aa- substituted analogues of the HPVG peptide were tested for CTL recognition using peptide dose-response cytotoxicity assays with PHA blast target cells expressing either HLA-B*3501 (Fig. 5, A and B) or HLA-B*3508 (Fig. 5, D and E), and the concentration of peptide required for half-maximum lysis is shown in Fig. 5, A, B, D, and E. To aid with interpretation of this data, peptide-MHC binding assays were also performed on selected peptides that were not well recognized by either CTL clone in the context of HLA-B*3501/B*3508. These binding assays measured stabilization of HLA-B*3501/B*3508 molecules on the surface of the T2.B*3501 or T2.B*3508 cell lines after exogenous peptide addition, and the concentration of peptide required for half-maximum stabilization of HLA-B*3501/B*3508 was then calculated (Fig. 5, C and F). Since the primary anchor residues Pro2 and Tyr11 were maintained within all the analogues, very few amino acid substitutions at other positions within the peptide resulted in a significant reduction in binding to HLA-B*3501/B*3508.


Figure 5
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  		FIGURE 5. The impact of single amino acid substitutions within the HPVG peptide on CTL recognition and HLA-B*35 binding. The HLA-B*3501+ CTL clones CS1 (A) and TK6 (B) were used as effectors against peptide-labeled PHA blasts that also expressed HLA-B*3501. The MB4 CTL clone (D), derived from an HLA-B*3501/B*3508 co-expressing donor but which recognizes HPVG only in association with HLA-B*3508, and the HLA-B*3508+ CTL clone SB14 (E) were used as effectors against peptide-labeled PHA blasts that expressed HLA-B*3508. The peptides were altered peptide ligands of HPVG into which single amino acid substitutions were introduced at various positions (P) (shown at left of figure, single letter abbreviation used for amino acids). Amino acid replacements at primary anchor residue positions 2 and 11 were not tested. A range of peptide concentrations were used in chromium release cytotoxicity assays, and the concentration required after for half-maximum lysis was calculated from dose-response data. MHC peptide binding assays were also conducted by testing each peptide analog at a range of concentrations for its ability to stabilize HLA-B*3501 or HLA-B*3508 expression on the surface of the Ag-processing mutant T2 cell line. The concentration of each peptide required for half-maximum stabilization was calculated and is shown in C (HLA-B*3501) and F (HLA-B*3508).

 
The HLA-B*3501+ CTL clones CS1 and TK6 that used highly homologous TCRs but displayed minor functional differences (Table I and Fig. 2A) exhibited very similar fine specificity patterns for the panel of peptides (Fig. 5, A and B). Both clones were particularly affected by replacement of the highly exposed Asp7 and Tyr8 residues, indicating that these residues are critical in contacting each TCR despite their highly disordered state within the HPVG-HLA-B*3501 complex (Fig. 3A). The more rigid but less exposed His1 and Glu10 residues could be replaced by several other amino acids without reducing CTL recognition efficiency, suggesting a limited role in TCR contact.

The MB4 CTL clone shared an identical β-chain with TK6 in combination with a distinct {alpha}-chain, but unlike TK6, this clone could only recognize the HPVG peptide in association with HLA-B*3508 (Table I and Fig. 2C). This TCR {alpha}-chain difference was reflected in subtle but significant fine specificity changes compared with the TK6 CTL clone. Although the same two peptide residues (Asp7 and Tyr8) appeared to be the primary TCR contact residues for both MB4 and TK6, with most replacements at these two positions preventing recognition, MB4 displayed a reduced capacity to tolerate amino acid substitutions at position 1 but greater tolerance to replacements at positions 5, 6, 7, and 10 (Fig. 5D).

The SB14 CTL clone which is also HLA-B*3508 restricted but uses a TCR with very little sequence homology to the Ag receptors of CS1, TK6, or MB4 also displayed a critical requirement for the prominent Asp7 and Tyr8 residues. The major fine specificity differences between this clone and the others were increased tolerance for amino acid replacement at Gly4 and a more stringent requirement for Phe9, suggesting that the TCR may dock more toward the C terminus of the bound peptide.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Recent studies have suggested that germline-encoded {alpha}β TCR variable domains have an inherent predisposition to react with conserved features of MHC proteins (26); however, the solved structures of TCRs bound to their MHC peptide targets have revealed significant variation in the mode of interaction, and hence the structural principles that govern MHC restriction remain unclear (23). In this study, we have shown that MHC restriction in the CTL response to an epitope from EBV, which is naturally immunogenic across two human MHC-I alleles, can be controlled exclusively by TCR {alpha}-chain usage. TCR {alpha}-chain interactions have been shown to dominate the interface between the TCR and pMHC in many complexes (23), and recent evidence suggests that they are important in maintaining the roughly conserved docking orientation (37). In addition, V{alpha} genes have been reported to play a major role in determining whether T cells are MHC class I or class II restricted (38, 39). Our data indicate that V{alpha} genes are also critical in determining MHC restriction between different class I alleles. Interestingly, the human genome includes significantly more TCR{alpha} genes compared with TCRβ genes (2, 33), and it is conceivable that the large number of TCR{alpha} genes may have evolved to accommodate the highly polymorphic MHC locus.

Polymorphism at the MHC locus enhances immune defense across the population by ensuring wide variation in the T cell response to infecting pathogens through presentation of a broad array of target epitopes (40, 41). In addition, polymorphic MHC residues can affect peptide binding conformation as well as MHC peptide binding affinity (42, 43), and this can have a major impact on the T cell response (44). This report has demonstrated that MHC polymorphism can also greatly impact on the mobility of bound peptide, with the HPVG peptide displaying high mobility at positions 5–9 when bound to HLA-B*3501, whereas the HLA-B*3508-bound peptide is more rigid as a result of a bonding network between a central peptide residue and the single polymorphic MHC residue at position 156 that distinguishes these two HLA alleles. Although differences in real-time peptide dynamics have been described for two HLA-B*27 alleles (45), such dramatic differences in peptide mobility between MHC-I allotypes have not been observed in previous structural studies.

Compared with peptides of 8–10 residues in length, unusually long MHC-I-bound peptides have potentially greater flexibility (12, 14), and one may have predicted that highly mobile peptides are weakly immunogenic due to the disordered nature of the target epitope. However, this is clearly not the case for the 11-mer HPVG epitope since we have found peptide-specific frequencies of up to 0.52% of CD8+ cells in the peripheral circulation of healthy EBV-exposed HLA-B*3501+ individuals, and up to 2% in a patient undergoing a primary EBV infection (unpublished data), which are relatively high frequencies compared with other latent Ag epitopes from EBV (46, 47). Indeed, the HPVG peptide remained uniformly immunodominant in both HLA-B*3501+ and HLA-B*3508+ individuals, confirming that peptide mobility has little impact on immunogenicity. Moreover, we have demonstrated, via fine specificity analysis, that mobile MHC-bound peptides cannot only be strongly immunogenic, but also that TCR specificity can be critically dependent on highly mobile amino acid residues within a bound peptide. Presumably optimal binding requires a particular peptide conformation that is stabilized upon TCR ligation (48), thereby ensuring specificity for residues that are mobile before TCR docking.

It may also have been expected that the TCR repertoire used against mobile MHC-I-bound peptides would be diverse, since the various conformational isomers would create distinct multiple determinants on a single peptide. However, the T cell response to the HPVG epitope in both HLA-B*3501+ and HLA-B*3508+ individuals was comprised of a predictable assembly of highly restricted, immunodominant TCRs. This is consistent with a previous report demonstrating that CTL recognition of bulky glycopeptides that are structurally flexible is also characterized by restricted TCR usage (49). The vast majority of HPVG-specific T cells retrieved from each group of individuals expressed TRBV9 β-chains with highly homologous CDR3 sequences. Such finely tuned gene bias suggests that the specific CDR1β and CDR2β loops from the TRBV9 gene provide a selective structural advantage in HPVG recognition. Indeed, residues found only in TRBV9 include the CDR1β and CDR2β sequences Leu30-Ser31 and Tyr50-Asn51, and these regions are thought to play an important role in MHC-I anchoring (23).

Although the β-chain of HPVG-specific T cells often remained extremely conserved across both HLA-B*3501+ and HLA-B*3508+ individuals, the corresponding {alpha}-chain frequently altered in the context of these closely related HLA alleles. The dominant {alpha}β TCR pairing in this response across multiple donors appeared to be a TRBV9/TRBJ2–2 β-chain with either a TRAV20 {alpha}-chain in HLA-B*3501+ individuals, or a TRAV29 {alpha}-chain in HLA-B*3508+ individuals. The molecular basis for this TRAV gene bias probably involves preferential binding by the CDR1 and/or CDR2 loops of TRAV20 and TRAV29 to HLA-B*3501 and HLA-B*3508, respectively. However, it is impossible to predict why this might be the case by comparing the sequences of these two TRAV genes because the CDR1 and CDR2 loops of TRAV20 and TRAV29 share only 2 of 12 residues. We clearly cannot rule out other mechanisms through which the MHC dimorphism at position 156 could be influencing this peptide-specific CTL response. It is well accepted that positive and negative selection in the thymus can "warp" the TCR repertoire before Ag encounter. Such distortion could favor the appearance of different TCR{alpha} chains in genetically different individuals. However, our demonstration that the HLA-B*3501/B*3508 co-expressing individual MB uses both TRAV20 and TRAV29 in response to the HPVG peptide indicates that these TCRs escape thymic negative selection in both HLA-B*3501+ and HLA-B*3508+ individuals. Nonetheless, we cannot rule out a role for preferential thymic positive selection of these different TCRs in the context of these two closely related HLA alleles.

The results of the present study are consistent with those of a recent study of a murine T cell response in which the TCR repertoire specific for a single HVH-1 peptide was investigated across the two closely related MHC class I alleles H-2Kb and H-2Kbm8 (50). Analogous to our findings, TCRs with identical β-chains but different {alpha}-chains displayed marked differences in HLA restriction patterns. In contrast, T cells that expressed the same {alpha}-chain but different β-chains showed no difference in their ability to cross-recognize the peptide on each H-2 allele.

The fine peptide specificity analysis provides some insights into the molecular basis for the HLA restriction differences between these TCRs. It is clear from the structural studies that peptide conformation plays a critical role in controlling restriction since the Ag-binding cleft of HLA-B*3501 and HLA-B*3508 are essentially identical. However, degeneracy in HLA restriction is not simply associated with a lower specificity for peptide because CTL clones such as CS1 that are more "cross restricted" tolerated less single amino acid substitutions within HPVG compared with clones such as MB4 that are restricted exclusively through HLA-B*3508. The particular HPVG-HLA-B*3501 conformation that triggers CTL clones such as CS1 must closely resemble the more rigid and defined conformation of HPVG-HLA-B*3508. However, the HPVG-HLA-B*3508 complex must retain some unique structural features relative to the multiple conformations of the HPVG-HLA-B*3501 complex because CTLs such as MB4 fail to be triggered by the latter complex. Future studies aimed at determining the three-dimensional structures of HPVG in complex with each HLA-B*35 allotype and bound to a cross-restricted and HLA-B*3508-restricted TCR will clarify these issues.

A growing number of immunodominant or "public" TCRs are being discovered that naturally emerge against a range of viruses (15, 17, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62), but the immunological basis for their presence is unknown. One theory suggests that immunodominant TCRs are the phylogenetic answer to millions of years of co-evolution with pathogens, whereas recent evidence suggests that they may be the immunological by-product of structural constraints imposed by unconventional pMHC targets (15, 17, 25, 63, 64). Irrespective of their origins, immunodominant TCRs represent an important tool in understanding the finer points of MHC restriction in humans since they can be applied as a relatively fixed variable in the often complex TCR/pMHC recognition equation. In this study, we have examined how a highly restricted TCR repertoire accommodates a minor difference in the target pMHC ligand that results from natural MHC polymorphism, revealing novel insights into the molecular basis for MHC restriction and the impact of peptide mobility on a class I-restricted T cell response.


   Acknowledgments
 
We thank the Industrial Macromolecular Crystallography Association staff for assistance in data collection at the Advanced Photon Source.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
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 Australian National Health and Medical Research Council (NHMRC), the Roche Organ Transplantation Research Fund, the Juvenile Diabetes Research Foundation, and the Australian Research Council. N.A.B. is a recipient of an NHMRC Peter Doherty Research Fellowship, S.R.B. is a recipient of an NHMRC Career Development award, and J.R. is an Australian Research Council Federation Fellow. Back

2 J.J.M. and N.A.B. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Scott R. Burrows, Cellular Immunology Laboratory, Queensland Institute of Medical Research, Brisbane, Queensland 4029, Australia. E-mail address: Scott.Burrows{at}qimr.edu.au or Prof. Jamie Rossjohn, The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia, E-mail: Jamie.Rossjohn{at}med.monash.edu.au Back

4 Abbreviations used in this paper: pMHC-I, peptide-MHC complex class I; HPVG, 11-mer EBV peptide HPVGEADYFEY; TRBV, TCR β-chain variable. Back

Received for publication May 3, 2006. Accepted for publication July 31, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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