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Structural Aspects of the Interaction Between Heterogeneic Human Papillomavirus Type 1 E4-Specific T Cell Receptors and the Same Peptide/HLA-DQ8 Complex1 Jane C. Steele,2* Stephen P. Young,† Jane C. Goodall,3† and Phillip H. Gallimore* TCR usage has been studied in a panel of Th cell clones specific for the same peptide epitope (P N S Q D R G R P R R S D), derived from the human papillomavirus type 1 (HPV1) E4 protein, and restricted through HLA-DQ8. After identifying the V, D, and J genes used by the TCRs and sequencing across the V(D)J junctions, five different a-chain sequences and five different b-chain sequences, comprising six independent clones, were identified. A structural model of our E4 peptide/HLA-DQ8 complex predicted that the guanidinyl side chain on the arginine residue at position 6 of the peptide could exist in different orientations. An intramolecular interaction between this arginine and the glutamine residue at position four appeared to control this orientation. Interacting HPV1 E4-specific TCRs would therefore have to recognize the complex in different conformations, and molecular modeling of the TCRs suggested that this could be achieved by changing the dimensions of the central pocket formed where the CDR3 loops of the TCR a- and b-chains converge. It is known that interactions between bound peptide and amino acid residues lining the peptide-binding cleft of HLA molecules are important for determining the conformation and orientation of the peptide/ MHC complex. The suggestion here that intramolecular interactions between amino acids of close proximity on the bound peptide are also important adds a further level of complexity to the mechanism by which TCRs interact with Ag. The Journal of Immunology, 1998, 161: 4745– 4752.

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uman papillomaviruses (HPVs)4 infect cutaneous and mucosal epithelia to induce either benign or premalignant hyperplastic lesions. The chronic nature of HPV infection in an immunocompetent host suggests that the immune response during natural infection is limited. Because some HPV types are associated with malignant lesions (1–3), there is enormous interest in the development of successful vaccination strategies that could prevent HPV infection and the associated cancers or could boost the immune response and induce regression of established lesions. HPV type 1 (HPV1) induces deep cutaneous warts on the palmar and plantar surfaces of the hands and feet. These lesions are benign and are characterized by high virus content and abundant expression of the E4 protein, which can exist in tumors at levels of up to 30% of the total protein (4, 5). An association between E4 proteins and the keratin cytoskeleton of cultured epithelial cells has been reported (6 –9), but a precise function for E4 has yet to be defined. It is probable that E4 alters the normal keratinization process in the productively infected keratinocyte to favor this stage of the virus life cycle (10). We have

*Cancer Research Campaign Institute for Cancer Studies and †Department of Rheumatology, The Medical School, University of Birmingham, Edgbaston, Birmingham, United Kingdom Received for publication March 24, 1998. Accepted for publication June 22, 1998. 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 the Cancer Research Campaign (CRC) and the Medical Research Council (MRC). P.H.G. is a CRC Gibb Fellow. 2 Address correspondence and reprint requests to Dr. Jane C. Steele, CRC Institute for Cancer Studies, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TA, United Kingdom. 3 Current address: Department of Clinical Medicine, Cambridge University, Addenbrookes Hospital, Cambridge CB1 2QQ, United Kingdom. 4 Abbreviations used in this paper: HPV, human papillomavirus; HA, hemagglutinin; LCL, lymphoblastoid cell line; CDR, complementarity-determining region; APL, altered peptide ligand; GCG, Genetics Computer Group (University of Wisconsin).

Copyright © 1998 by The American Association of Immunologists

previously examined human proliferative T cell responses to the HPV1 E4 protein purified from cutaneous warts (11). Several E4specific Th cell clones were isolated, and the minimal T cell epitope and HLA restriction was defined. These investigations, together with E4 serologic studies reported by others (12–17), demonstrate the immunogenicity of the E4 protein. The fact that E4 is expressed so abundantly suggests that it could be a useful component of a vaccine, or it may represent a good target for other immunotherapeutic approaches such as adoptive transfer. A critical step toward the development of HPV-specific immunotherapy has been the identification of specific T cell epitopes presented in association with particular MHC molecules. Due to the nature of MHC restriction, protein or peptide vaccines will probably have to consist of multiple epitopes that are functional in the context of the more common HLA haplotypes among the outbred human population (18). The induction of CTLs normally requires “help” provided by CD4-expressing Th cells (19), so that an ideal HPV vaccine would therefore induce both arms of the cellular immune response. Both proliferative and CTL responses to several HPV proteins have been studied (20), and multivalent vaccines containing B cell, CTL, and Th cell epitopes have already been designed for several other infectious agents (21). To date, much of the HPV vaccine work has been confined to HPV types 16 and 18, which are commonly associated with anogenital carcinoma (1–3). The E6 and E7 early transforming proteins, or fragments derived from them, are currently the focus of vaccine trials in patients with cervical cancer (22, 23). The clinical effectiveness of such vaccines cannot yet be evaluated because the scope of the studies so far has been small, and there are several areas that require further examination. An alternative to vaccination with peptides or proteins may be to develop immunotherapy involving the adoptive transfer of autologous T cells (24). This approach is being used successfully in immunosuppressed patients at high risk of developing EBV-associated lymphoproliferative disease (25, 26). 0022-1767/98/$02.00

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Understanding the nature of the interaction between HPV-specific TCRs and the peptide/MHC complex is a necessary prerequisite to the development of this kind of immunotherapy for HPV. It may then be possible to improve the effectiveness of the T cell response to HPV Ags by manipulating the specificity of autologous T cells and/or by directing the response in the appropriate way. In other systems, it has already been possible to design Ag-specific CTLs by grafting the recognition specificites of Abs using recombinant DNA and gene transfer techniques (27–30). Previous results obtained from HPV1 E4-specific proliferative T cell clones suggested that the T cell repertoire against HPV Ags may be quite diverse, since a preliminary analysis of TCR gene rearrangements in some of these clones revealed different TCRs with the same peptide/MHC specificity (11). In this study, we have generated additional Th cell clones with the same specificity and looked in more detail at the TCRs involved in the response to HPV1 E4. In particular, we have looked at the CDR3 sequences across the V(D)J junctions, which are believed to be important for peptide recognition. We have also generated structural models for the E4 peptide/MHC complex and interacting TCRs, and we present a proposal about how different E4-specific TCRs recognize the same peptide/MHC complex.

Materials and Methods Isolation of HPV1 E4E4-specific proliferative T cell clones The isolation and characterization of Ag-specific proliferative T cell clones has already been described in detail (11). The HPV1 E4 protein was purified either from skin parings obtained from HPV1-induced verrucae or from insect cells infected with a recombinant baculovirus (31). Briefly, unfractionated mononuclear cells separated from peripheral blood were stimulated with soluble E4 (30 – 40 mg/ml), and the resulting blasts were cloned by limiting dilution on day 7 using the autologous mitomycin Ctreated lymphoblastoid cell line (LCL) and IL-2 containing supernatant from the MLA 144 cell line (32). The Ag used for cloning was either purified E4 (30 – 40 mg/ml), or the 13-mer peptide previously defined as an MHC class II-restricted T cell epitope (P N S Q D R G R P R R S D) at a concentration of 10 mM (11). Resulting clones were expanded using the same culture conditions, and their specificity determined by testing proliferative responses to purified E4 and synthetic E4 peptides presented by the autologous LCL. The MHC restriction of the clones was checked using panels of HLA class II-matched and -mismatched LCLs to present peptide to the T cell clones in proliferation assays. Proliferation assays were performed as previously described (11).

Isolation of mRNA and cDNA synthesis Cloned T-cells (1-2 3 106) were washed once in PBS by centrifugation before preparing a cell pellet. Poly(A)1 RNA was isolated from the T cells using oligo(dT)-coupled paramagnetic beads (oligo dT Dynabeads; Dynal, Oslo, Norway). Lysis buffer (0.3 ml) supplied by the manufacturer was added to the cell pellet, and after reducing the viscosity, the lysate was combined with the Dynabeads and annealed for 5 min at room temperature. The beads were collected using the magnet, and following removal of the supernatant, they were washed several times in the buffers provided. The first cDNA strand was synthesized from mRNA bound to the beads using Superscript reverse transcriptase and the supplied buffers (Life Technologies, Renfrewshire, Scotland). The total reaction volume was 50 ml containing 0.5 mM dNTPs (10 mM dNTP mix; Life Technologies, Renfrewshire, Scotland), 10 mM DTT, 50 mM Tris HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, and 500 U of reverse transcriptase. Following incubation at 37°C for 1 h, the beads were collected and the reaction mixture removed. Any remaining reverse transcriptase was inactivated by resuspending the beads in 50 ml of TB buffer (20 mM Tris HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 0.1 mg/ml BSA) and heating at 95°C for 1 min. The cDNA Dynabeads were washed and resuspended in TB buffer and stored at 4°C.

Amplification of TCR genes from cDNA Dynabeads The cDNA Dynabeads were removed from the storage buffer and resuspended in 0.5–1.0 ml of PCR buffer (10 mM Tris HCl, pH 8.8, 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100). The bead suspension was divided into aliquots so that it was possible to screen for variable (V) a and b genes. The PCR was carried out using a 59 primer from each Va or Vb

family, and a 39 primer from the respective constant (C) a or b region of the gene (50). Each reaction was carried out in a 50-ml volume containing 0.2 mM dNTPs, 2 U of Dynazyme DNA polymerase (Finnzymes Oy, Finland), and the appropriate oligonucleotide primers (Perkin Elmer, Buckinghamshire, U.K.) at a concentration of 0.25 mM in PCR buffer. The amplification was carried out in a Perkin Elmer 9600 thermal cycler; for the TCR a-chain, the conditions were 1 min of denaturation at 93°C, 1 min of annealing at 55°C, and 30 s of extension at 72°C for 32 cycles. For the TCR b-chain, the annealing temperature was increased to 60°C. In all experiments, a positive T cell cDNA control, a positive actin control, and a negative reagent control were included.

Characterization and sequencing of the PCR products The PCR products were size fractionated on a 2% agarose gel, and the Va and Vb genes, which had been rearranged by each clone, were identified. These PCR products were reamplified using the same primers to produce sufficient cDNA for sequencing. The DNA was gel purified using the QIAquick Gel Extraction Kit (Qiagen, West Sussex, U.K.) and sequenced using an Applied Biosystems 373A automated DNA sequencer. All sequencing results were confirmed in several experiments that involved repeated isolations of mRNA from the T cell clones.

Structural analysis and modeling T cell Ag receptors. Sequences derived from the TCRs of the HPV1 E4specific T cell clones were aligned with the human ab TCR specific for the Tax peptide of HTLV-1 bound to HLA-A2, recently described by Garboczi et al. (33). Alignments were made using QUANTA 4.1/Protein Workbench (Molecular Simulations, Burlington, MA). Following the addition of framework sequences, whole TCR sequences were superimposed onto the X-ray coordinates of the published TCR (kindly provided by Dr. P. Ghosh, Harvard University, Cambridge, MA), and the structures were then subjected to energy minimization (10,000 steps, Adopted Newton-Raphson algorithm) using the CHARMM program (34). Charge surface models were produced using the GRASP program (35). HPV1 E4 peptide/DQ8 (DQA1*0301/DQB1*0302) complex. Aligments between the DQA*0301 and DQB*0302 chains and the corresponding sequences from the published crystal structure of the HLA-DR1 molecule (36) were carried out using QUANTA. The DR 1-bound peptide (derived from influenza virus hemagglutinin (HA)) described by Stern et al. (36) was then aligned with the HPV1 E4 peptide (P N S Q D R G R P R R S D) described in this study. The HPV1 E4 peptide was modeled onto the DQ8 molecule using the three-dimensional coordinates for the published human class II MHC/peptide complex (kindly provided by Dr. L. Stern, Harvard University), and the side chains were regularized. The E4 peptide was used in place of the HA peptide, with the assumption that the asparagine residue at position two occupied the specificity binding pocket number 1 within the peptide-binding cleft of DQ8. The whole structure was then subjected to energy minimization as described above. Molecular dynamics. The structure generated above was solvated with water to a distance of 16 Å above the protein surface using the CHARMM program and then minimized again as previously described. Fixed constraints were then applied to water molecules .8 Å above the protein surface (to produce an unconstrained solvent environment around the protein). The model was subjected to a molecular dynamics calculation with initial warming (50 ps) and equilibration steps (50 ps) before the final simulation (500 ps) at 300 K using CHARMM. The simulation trajectory was subsequently analysed using CHARMM to calculate a variety of peptide/protein energies every 0.05 ps. This whole calculation was carried out several times.

Results Isolation of HPV1 E4-specific proliferative T cell clones We have previously isolated and characterized HPV1 E4-specific proliferative T cell clones from the peripheral blood system of a normal donor (11). Despite identical specificities, two of these clones (3F5 and 4A8) were shown by Southern blotting and PCR to have different TCR b- and g- gene rearrangements. To extend this study, we have used blood from the same donor to isolate additional Th cell clones, which were specific for the same HPV1 E4 peptide (P N S Q D R G R P R R S D) and were shown (by using a panel of LCLs of known HLA type) to be restricted through HLA-DQ8.

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FIGURE 1. Deduced amino acid sequences of TCR transcripts obtained from the HPV1 E4-specific proliferative T cell clones: A, a-chains, and B, b-chains. Assignments of the V, D, J, and C segments was made according to published sequences (37, 38, 78 – 83). Breaks in the amino acid sequence indicate the borders between the known Va/Vb and Ja/Jb sequences and the N (a-chains) or N-D-N (b-chains) region. Amino acid residues that represent N-region additions are shown in bold although, since the germline nucleic acid sequences of the Va segments are not known, the Nregion residues at the Va-N junctions in A are only predictions. The asterisk (p) indicates T cell clones using TCR chains with the same sequence.

TCR a- and b-chain rearrangements in HPV1 E4-specific T cell clones T cell clones were screened by PCR for TCR variable (V) a and b gene usage, and after reamplification of the PCR products, the aand b-chains were sequenced (summarized in Fig. 1). By comparing our findings with published sequences, it was possible to determine which joining (J) and diversity (D) genes were being used (37, 38). The position of the primers used in the PCR was such that the reaction amplified a stretch of cDNA covering the V(D)J junction, and it was therefore possible to obtain the exact sequence across the CDR3 region. Where germline sequences were available, it was also possible to identify the N-region additions. From the 12 clones under investigation, five different a-chain sequences and five different b-chain sequences were identified, although these results did not represent 5 independent T cell clones. Several of the cloning procedures yielded T cells that possessed identical a- and b-chain sequences it is probable that these clones were derived from the same progenitor T cell in vivo. Clones 2F6 and 2B7 were also found to be identical, but were obtained at different time points, suggesting that the same reactive T cells can exist in the circulation for many months. In some cases, there appeared to be preferential usage of certain a- and b-chains, because some of the clones shared the same a sequence but showed different b-chain sequences (4A8 and 2B7). Similarly, some had identical b sequences but were different at the a locus (3F5 and 2A12). Clone 4A8 was interesting because it appeared to

possess two different in-frame TCR a-chain sequences. This is not an unexpected result, since incomplete allelic exclusion of a-chains has been reported elsewhere (39, 40). Alignment of the TCR a-chain CDR3 sequences The CDR3 sequences across the V(D)J junctions of the TCR aand b-chains are important for interaction with the antigenic peptide. We were interested to look carefully at these regions in our panel of HPV1 E4-specific T cell clones and to try and explain how any differences would allow recognition of the same peptide/ MHC complex. An alignment of the five different TCR a-chain sequences obtained is shown in Figure 2. If the alignments are optimized for sequence identity on either side of the CDR3 region, it becomes apparent that these sequences are variable in length. Thus, the CDR3 sequence for clone 2B7 comprises 9 amino acids, whereas 2A12 comprises 13. Clones 2A7, 4A8, and 3C11 were intermediate in length between 2B7 and 2A12. Molecular modeling of HPV1 E4-specific TCRs To explain the CDR3 sequence differences in terms of peptide recognition, homology models of clones 2B7 and 2A12 were constructed (Fig. 3). The models were based on the recently published crystal structure of a soluble human ab TCR (33), and by comparison, it was possible to visualize the diagonal surface of the TCR believed to interface directly with the peptide/MHC complex. It was also possible to see the important central pocket where, in

FIGURE 2. Aligments of the five different TCR a-chain sequences from the HPV1 E4-specific T cells. The CDR3 sequence is taken from the conserved cysteine residue near the end of the V gene segment up to the beginning of the FG-x-GT sequence in the J gene segment. The conserved residues are all shown in bold. Sequence alignments were carried out using the GCG program (84).

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FIGURE 4. The alignment of the HLA-DR1-binding peptide (influenza virus HA) described by Stern et al. (36) with the HLA-DQ8-binding peptide derived from HPV1 E4 described in this study. A comparison was made between binding pockets 1, 4, 6/7, and 9, which lie along the peptidebinding cleft of the DR1 and DQ8 molecules, and the amino acid residues on the E4 peptide, likely to be directly involved in the interaction with these pockets within the HLA-DQ8 molecule were predicted (shown in bold type). Sequence alignments were made using the GCG program (84).

FIGURE 3. Surface models of TCRs from T-cell clones 2B7 (A) and 2A12 (B). The models were based on the crystal structure of an ab TCR specific for the Tax peptide of HTLV-1 bound to HLA-A2 published by Garboczi et al. (33). Clone 2B7 (a) had the shorter CDR3 region (Fig. 2) and appeared to possess a shallow central pocket, whereas clone 2A12 (b) was predicted to have a narrower, deeper pocket consistent with the longer CDR3 sequence (Fig. 2). Homology models were made using QUANTA, and surface models were generated using GRASP (35).

the published structure, the a- and b-chain CDR3 loops converge. The TCR models obtained from the two clones, 2B7 and 2A12 (Fig. 3), predicted that their central pockets were different in size and shape, which is presumably due to differences in their CDR3 sequence (Fig. 2). A shallow pocket was observed for clone 2B7, which is consistent with a shorter CDR3 region, whereas for clone 2A12 the central pocket appeared to be narrower and deeper, as might be expected for a longer sequence. Molecular modeling of the HPV1 E4 peptide/HLA-DQ8 complex One aim of this study was to investigate how the different TCRs could interact with the same peptide/MHC complex. It was therefore necessary to model the antigenic peptide (P N S Q D R G R P R R S D) onto the HLA-DQ8 molecule. A homology model of DQ8 was constructed based on the crystal structure of the human HLA-DR1 molecule (36), and the E4 peptide was aligned to the influenza HA peptide, which had crystallized with the DR1 protein (Fig. 4). Alignments between the DQ8 and DR1 a- and b-chains resulted in 56.3 and 66.8% identity, respectively, and required no insertions or deletions (derived from the GCG suite of sequence analysis tools). When compared with the published structure of HLA-DR1, the model of the HLA-DA8 molecule highlighted possible differences in amino acid sequence within some of the side pockets lining the

peptide-binding cleft. Together with the information available from the peptide alignments, it was possible to make predictions about what effect these side pocket changes would have on the nature of the interacting amino acids, and the residues likely to be involved in the DQ8 side pocket binding on the E4 peptide were identified (shown in bold type in Fig. 4). The asparagine residue at position 2 on the peptide was assigned the P1 anchor position, the aspartic acid at position 5 and the arginine at position 8 were the principle occupants of the next two binding pockets, and another arginine residue at position 10 appeared to be associated with the P9 pocket. All of the solved class II structures agree that bound antigenic peptides take up a similar conformation—that of a polyproline type II helix—and so it is reasonable to assume that in DQ8 a similar fold is assumed by the peptide. Thus, using the predicted alignment and the bound HA peptide coordinates, the E4 peptide was “docked” onto the DQ8 molecule and energy minimized, and a dynamics was simulation performed on the whole structure. Important structural residues involved with hydrogen bonding to the backbone of the bound peptide, such as 69N and 62N in the a-chain and 61W and 82N in the b-chain, were conserved on the DQ8 molecule, and the E4 peptide remained stably bound throughout the simulation. The molecular dynamics revealed an interesting feature of the central arginine residue at position 6 on the HPV1 E4 peptide. At different times during the calculation, it appeared that the guanidinyl side chain could either be pointing up out of the DQ8-binding cleft, or it could be lying flat (Fig. 5). Analysis of the dynamics trajectory revealed that an intramolecular interaction between this arginine and the glutamine residue at position 4 in the peptide appeared to control this orientation (Fig. 6). It was noted that the absence of this interaction coincided with the arginine being exposed (Fig. 5a), while in the presence of the glutamine interaction, the guanidinyl side chain lay closer to the peptide-binding groove (Fig. 5b). This arginine residue was not predicted to bind directly to the MHC molecule, and its position suggests that it may be more involved in interaction with the TCR. The differences in orientation suggest that interacting TCRs would have to recognize the peptide/MHC in different conformations, with the guanidinyl side chain on the central arginine residue either more or less exposed. Another interesting observation was made concerning the carboxyl end of the peptide, which appeared to extend slightly from the end of the DQ8 peptide-binding cleft. At certain time points during the energy trajectory, the aspartate residue at the C terminus (position 13 on the E4 peptide) could be seen pointing down and interacting with the arginine residue at position 79 on the DQ8 a-chain (indicated on Fig. 5).

Discussion An understanding of TCR usage in response to a given peptide/ MHC combination has implications for the treatment of human infectious diseases but has not been extensively studied. We have

The Journal of Immunology

FIGURE 5. Homology model of the HPV1 E4 peptide/HLA-DQ8 complex based on the crystal structure of the HLA-DR1 molecule containing peptide derived from influenza virus HA (36). Energy minimization procedures suggested that the guanidinyl side chain on the arginine residue at position 6 on the E4 peptide (indicated by the arrow) could exist in more than one orientation, either exposed and pointing up out of the DQ8 peptide-binding cleft (a), or lying in a flatter position (b). It is possible that clone 2A12, which was predicted to have a narrower, deeper pocket (Fig. 3b), may interact preferentially with the complex if the side chain was exposed. Conversely, clone 2B7, which appeared to possess a shallow central pocket (Fig. 3a) may interact more favorably with the E4 peptide/ HLA-DQ8 complex when the arginine side chain is lying in the flatter position. The position of an interaction predicted to occur between the aspartic acid residue at the carboxyl end of the E4 peptide and the arginine at position 79 on the DQ8 a-chain is indicated at the end of the peptidebinding cleft. Homology models were made using QUANTA.

analyzed TCR usage in a panel of 12 proliferative T cell clones specific for a particular HPV1 E4 peptide presented by HLA-DQ8. Sequence analysis of TCR a- and b-chains enabled the identification of the different TCR a and b combinations that were used to see the E4 peptide (Fig. 1). It is difficult to draw conclusions about the preferential usage of particular a or b gene segments in one donor, but the observation that some pairs of T cell clones (4A8 and 2B7; 3F5 and 2A12) used one identical and one different TCR chain indicates a certain selective pressure on some a and b rearrangements. This may reflect limited or favored TCR structures that are selected to recognize the E4 peptide in the context of HLA-DQ8. A similar situation has been reported by others working with pairs of T cells specific for the same peptide/MHC complex (41– 43). Clones 2F6 and 2B7 had been isolated from blood samples taken at different time points at least 3 yr apart and were found to be identical. This suggests that the cellular immune response to HPV may be dominated by a few expanded T cell clones that can persist in vivo for relatively long periods of time. Clone

4749 4A8 appears to be an example of a T cell that can potentially bear two Ag receptors comprising a common b-chain and either of two distinct a-chains. In these cases, there appears to be preferential association of one of the a-chains with the b-chain, and the T cell is therefore functionally monospecific (39, 44). Other studies have also attempted to define the extent of TCR diversity among T cell clones specific for a particular epitope. There are examples of both murine and human MHC class I- and II-restricted immune responses that exhibit either diverse (41, 43) or conserved (45–50) responses. In this study, the finding that 6 of the 12 T cell clones were different suggested that the class IIrestricted T cell response to HPV is quite diverse. This diversity would be beneficial for vaccine design, since it would mean that attempts to raise immune responses to HPV Ags would not be hampered by lack of repertoire. Exactly what determines the diversity of a T cell response to a given Ag is not fully understood. One theory suggests that the T cell repertoire may be influenced by the similarity of the epitope to self peptides and that a more limited repertoire is characteristic of responses to self-like Ags (51). No matches with either foreign or self Ags were found in the databases for the E4 peptide. The length and amino acid composition of the CDR3 region at the V(D)J junction of the TCR plays an important role in determining the specificity for individual peptide/MHC combinations (52, 53). It has been noted in other studies that a-chain CDR3 sequences are far less conserved in length than b-chain CDR3 regions (54); our data support this observation, since alignment of the a-chain CDR3 sequences obtained from the HPV1 E4-specific TCRs revealed that they were variable in length (Fig. 2). In particular, clones 2B7 and 2A12 were substantially different, with 2B7 possessing a relatively short CDR3 sequence of 9 amino acids and 2A12 having a longer CDR3 region of 13 residues. Molecular modeling of these two TCRs revealed interesting differences in the diagonal surface of the receptor, which interfaces with the E4 peptide/DQ8 complex (Fig. 3). The important central pocket, which lies in the centre of the TCR where the a- and b-chain CDR3 loops converge, appeared to be different in size and shape, due presumably to differences in the CDR3 sequences of the two clones. To find out more about the general features of the different TCRs identified in this study and to investigate how they interact with the same peptide/MHC complex, a structural model of our E4 peptide/HLA-DQ8 complex was constructed (Fig. 5). The peptidebinding properties of DQ molecules are not as well characterized as those of DR, upon which our model was based. This lack is due mainly to the relative paucity of DQ-restricted clones and the fact that there is great variability in DQ a-chains (55). However, the association of certain DQ alleles with insulin-dependent diabetes has led to several attempts to characterize their peptide-binding properties, although it has been difficult to define a binding motif for DQ8. The motifs that have been described have the same basic format as many HLA-DR molecules, consisting of four or five anchor residues in positions P1, P4, P6/7, and P9 from the N terminus. Based on sequence data from eluted peptides, Chicz et al. (56) suggested a positively charged amino acid at the P1 anchor position on the peptide and a small residue such as alanine or glycine at P6. In vitro peptide-binding studies by Kwok et al. (57) defined a motif, which in contrast to that described above did not allow positively charged residues at P1 but did tolerate alanine at P6. Our E4 peptide (P N S Q D R G R P R R S D) is in better agreement with studies published recently by Godkin et al. (58), because the assigned anchor residues (shown in bold) match amino acids that they have found to be enriched at the same positions using pool sequence data for HLA-DQ8. In addition, they describe a relative enrichment of arginine residues in the C-terminal half of

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FIGURE 6. Energy of interactions between peptide residues during the dynamics simulation. Electrostatic (O) and van der Waals (ª) energies were calculated and are shown. It can be seen that little interaction occurred during the early part of the simulation, but then a sustained period of interaction occurred dominated by formation of hydrogen bonds between the side chains, which caused the arginine side chain to fold downward toward the MHC. Frames from two points (no. 105 and no. 418) of the simulation are shown in Figure 5 to illustrate this interaction.

their eluted DQ8-binding peptides, which is also a feature of the E4 peptide in this study. Reasonable justification exists for constructing a model of the E4 peptide/DQ8 complex based on the structure of the influenza (HA) peptide bound to HLA-DR1 (36). There is evidence from binding studies that DQ molecules bind peptides with similar restrictions to DR (57, 59), and computer modeling using known sequences of DQ alleles in comparison with DR alleles has suggested a similar pattern of binding (60, 61). Our approach for binding the E4 peptide to the HLA-DQ8 molecule based on differences in the pockets between DR1 and DQ8 also seems reasonable. A precedence for asparagines at the P1 anchor position has already been observed (62) and fits with another model of DQ8 published by Routsias and Papadopouplos (61) who predict this pocket to be amphiphilic or hydrophilic. An interesting feature of most class II molecules is the presence of an aspartate residue at position 57 on the b-chain, which interacts with an arginine at position 79 on the a-chain forming a salt bridge at one end of the Ag-binding groove. In HLA-DQ8, however, a polymorphism at position 57 means that it possesses an alanine instead of an aspartate residue, and the potential for this salt bridge is therefore absent. In this event, there is an unopposed, positively charged arginine on the a-chain at position 79, which would interact favorably with a negatively charged amino acid at or near the end of the bound peptide. In this study, this appears to involve the aspartic acid at the carboxyl end of the E4 peptide, and when a trace of the molecular dynamics was analyzed, the interaction was clearly visible (indicated in Fig. 5). The likelihood is that in this case this interaction plays an important role in stabilizing the peptide/MHC complex. Other studies report that nega-

tively charged residues are preferred at the P9 position in DQ8binding peptides (57, 63, 64), although Gotkin et al. (58) did not find this to be essential and suggest that other P9 anchor residues could also be important. The absence of the salt bridge in DQ8 would be predicted to alter the charge properties and conformation of the molecule and would probably result in a more open structure in this region. It is therefore possible that there are less constraints on peptide binding at this end of the binding cleft, and anchor positions, particularly that of P9, may be very difficult to assign. At position 4 on the E4 peptide, the glutamine residue is largely accessible to solvent but appears to interact with the arginine at position 6, with the result that the guanidinyl side chain folds down into the groove making it transiently less accessible to the solvent and thus to the interacting TCR. It follows that interacting HPV1 E4-specific TCRs would have to recognize the complex in different conformations with the arginine side chain, either pointing up out of the DQ8-binding cleft or being less exposed and lying in a flatter position. It is tempting to speculate that the different sized and shaped pockets predicted from the models of our HPV1 E4specific TCRs may be responsible for recognizing the different conformations of the E4 peptide/DQ8 complex. It could be envisaged that the narrow, deep pocket predicted for the TCR model of clone 2A12 (with the longer CDR3 sequence) may interact with the peptide if the arginine side chain was exposed and pointing up out of the DQ8-binding cleft, whereas the shallow central pocket of clone 2B7 (with a shorter CDR3 region) would recognize the complex with the guanidinyl side chain in a flatter conformation. It is interesting to postulate what the effects of amino acid substitutions at the key positions along the E4 peptide would be. Changing the central arginine at position 6 would be unlikely to

The Journal of Immunology affect binding to the MHC but would be predicted to have an effect on T cell recognition and intramolecular interactions with neighboring peptide residues. A smaller residue than arginine may be less exposed and may therefore interact better with TCRs such as 2B7 that possess the shallower central pockets; and if the residue also possessed different charge properties to arginine, it would probably not interact as well with the glutamine at position 4. If this glutamine was substituted, there would be an effect on the intramolecular interaction with the arginine at position 6. This may well affect T cell recognition in that the arginine side chain would be predicted to exist for longer in the exposed orientation and favor TCRs such as 2A12, which have the narrower, deeper pockets. Our results support the idea that peptides can bind to the same HLA molecule in more than one conformation (65– 67), and this is the most likely explanation for the different TCRs with identical specificities identified in this study. This situation is probably more common with class II-restricted T cells, since it has been reported that there are less constraints on the binding of peptides to class II than class I MHC, and conformational differences may therefore be better tolerated (68– 70). Heterogeneity of TCRs with specificity for the same peptide/ MHC complex may arise for other reasons also. One proposal states that the complex can contain a number of different subepitopes, each capable of recognition by a different TCR (65, 71, 72), and others believe TCR heterogeneity is due to differences in the fine specificity of the peptide (41, 43, 73). In fact, subtle molecular or conformational changes in antigenic peptides produce altered peptide ligands (APLs), which are believed to have important biological effects. The indications are that APLs induce different T cell responses from those induced by the antigenic ligand and that the TCR can respond with gradations of T cell activation and effector function (74–77). Our observations imply that some peptides may have the potential to be both an epitope and an APL for the same TCR by existing in different conformations. This could be influenced by the local environment and by factors such as Ag load and the local pH at inflammatory sites. Peptide conformation could therefore have important implications both in autoimmunity and in the design of peptide therapeutics. The structural information obtained in this study has revealed important features about the interaction between HPV-specific TCRs and viral peptide that are relevant to the development of HPV-specific immunotherapy and that may be applied to other systems. In general terms, our results offer further insights into the complex inter- and intramolecular interactions that enable T cells to recognize and respond to Ag. These clones also represent a useful model system in which to study other aspects of HPV immunobiology such as the effects of APLs and the manipulation of immune responses by cytokines.

Acknowledgements We thank Darren Parkin for his technical assistance and Dr. Roger Grand for useful discussion.

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