termed anchor residues, at certain positions within the pep- tide. For example, the common class I molecule HLA- B*3501 prefers peptide ligands, with proline as a dominant anchor residue at position 2 (P2) and tyrosine (or less com- monly phenylalanine, methionine, leucine, or isoleucine) at P (the COOH terminus) (4, 5). Peptide amino acids at other secondary anchor positions can also influence allele- specific binding (6). Although immunodominance of antigenic CTL epitopes usually correlates with the abundance of peptide presented on the surface of the APC (7), there have been several re- ports where this is clearly not the case (8, 9). In these in- stances, the major factors controlling immunodominance have been proposed to be limitations and bias in the TCR repertoire imposed by thymic or peripheral selection (1, 10– 12). There has also been a suggestion that some immuno- dominant determinants may be intrinsically more immu- nogenic because of an innate propensity to interact with TCRs, perhaps through the orientation or nature of side chains available for interaction (1); however, no evidence for this theory has been presented to date. We have defined a CTL epitope (77APQPAPENAY86, referred to as APQP) from the BZLF1 or Z EBV replication activator protein of EBV that binds well to both HLA- B*3501 and HLA-B*3508, two closely related molecules that differ by a single amino acid at position 156 (156Leucine vs. 156Arginine, respectively). This epitope was found to be immunogenic in individuals expressing HLA-B*3508 but, unexpectedly, no response could be detected in HLA- B*3501 EBV-exposed individuals, indicating that factors other than the level of peptide presentation are influencing immunogenicity. Structural analysis revealed major differ- ences in the peptide conformation when bound to each HLA-B35 allotype but, surprisingly, there were no important differences in the MHC class I heavy chain conformation. These data indicate that T cell responsiveness to a foreign peptide can be influenced by its MHC-bound conformation. RESULTS The influence of a single MHC amino acid difference on the immunogenicity of a CTL epitope from BZLF1 A highly immunogenic CTL epitope (BZLF1 54–64, EPL- PQGQLTAY) that binds to HLA-B*3501 has recently been identified from the BZLF1 antigen of EBV (13, 14). A close examination of the BZLF1 protein sequence revealed a 10- mer sequence (BZLF1 77–86, APQPAPENAY) down- stream from this previously defined 11-mer epitope that also conforms to the HLA-B35 peptide-binding motif (4, 5). A peptide corresponding to this 10-mer sequence was tested for recognition by a CTL line raised from an HLA-B35 EBV-sero donor (SB) by in vitro stimulation of PBMCs with the peptide for 10 d. Our earlier studies have shown that this stimulation protocol with EBV peptides only in- duces a measurable in vitro CTL response in donors who have been previously exposed to the antigenic determinant through prior infection with EBV, and any peptide-specific 1250 CTL lysis observed in these assays reflects a substantial in vivo CTL response in these individuals (unpublished data). This CTL line recognized autologous PHA blasts (SB) only after pretreatment with the APQP peptide, thereby demon- strating that this sequence includes an EBV CTL epitope (Fig. 1 A). To determine if the HLA-B35 allele expressed by donor SB (HLA-B*3508) is responsible for presenting this Figure 1. Identification of a CTL epitope from the BZLF1 antigen of EBV that is immunogenic in HLA-B*3508 individuals. (A) Cytotoxicity by a CTL line raised against the APQP peptide of autologous PHA blast target cells (SB) and PHA blasts sharing one HLA allele with the CTLs (underlined). The target cells were tested with or without the addition of 0.1 M of the APQP peptide. E/T ratio 10:1. (B) Peptide dose-response cytotoxicity assay using a CTL line from donor SB (HLA-B*3508 ) raised against the APQP pep- tide, and PHA blast target cells also from donor SB. E/T ratio 10:1. (C) CTL recognition at a variety of E/T ratios by polyclonal T cell lines raised against the APQP peptide from PBMCs from eight HLA-B*3501 donors, three HLA-B*3501 donors, and one HLA-B*3501/-B*3508 coexpressing donor. The target cells were autologous PHA blasts that were pretreated with 0.1 M of the APQP peptide or left untreated. THE INFLUENCE OF PEPTIDE CONFORMATION ON A CYTOTOXIC T CELL RESPONSE | Tynan et al.