EBV epitope, cells expressing a range of other HLA alleles were also included as targets for this CTL line. PHA blasts sharing HLA-B*3508 with donor SB were recognized effi- ciently by the CTLs only after the addition of the APQP peptide, whereas target cells sharing other class I alleles with donor SB were not lysed, confirming that this peptide is pre- sented by HLA-B*3508 (Fig. 1 A). Because the APQP 10-mer peptide includes an 8-mer sequence that also conforms to the HLA-B35 binding mo- tif, experiments were conducted to determine the minimal length of this CTL epitope. All possible 8- and 9-mer pep- tides from within the APQP 10-mer sequence were tested for recognition by a CTL line raised from donor SB. This dose-response experiment clearly shows that the 10-mer peptide is recognized most efficiently, with high levels of ly- sis induced with peptide concentrations down to 0.001 M (Fig. 1 B), suggesting that APQPAPENAY is the naturally presented epitope. To determine if other EBV-sero HLA-B35 donors re- spond to this BZLF1 epitope, CTL lines were raised against the APQP peptide using PBMCs from a panel of such do- nors to test for reactivity with the peptide. The donors ex- pressed either HLA-B*3501 or HLA-B*3508, two allotypes of the HLA-B35 serotype that diverge by a single amino acid difference at position 156. Surprisingly, this micropolymor- phism appeared to have a major impact on responsiveness to the APQP epitope, because no evidence of a response to the peptide could be seen with these cytotoxicity assays in any of eight HLA-B*3501 donors. In contrast, three donors ex- pressing the much less common HLA-B*3508 responded well to the peptide, as did a donor positive for both HLA- B*3501 and HLA-B*3508 (Fig. 1 C). Peptide conformation, not peptide binding efficiency, controls the immunogenicity of a viral CTL epitope To determine if these dramatic differences in responsiveness to this 10-mer epitope in individuals expressing HLA-B*3501 or HLA-B*3508 are related to how well the peptide associates with each HLA-B35 subtype, MHC–peptide binding assays were conducted. These assays used the T2 cell line transfected with either HLA-B*3501 or HLA-B*3508. These antigen- presenting mutant cells express stable class I HLA molecules on their surface upon addition of exogenous HLA binding peptide (15). As positive controls for these experiments, pep- tides known to be highly immunogenic EBV epitopes in HLA-B*3501 or HLA-B*3508 individuals were used for comparison. These included the BZLF1 11-mer EPLPQGQ- LTAY (13, 14), which is immunogenic in HLA-B*3501 in- dividuals, and the unusually long 13-mer peptide LPEP- LPQGQLTAY was included for HLA-B*3508 (13, 16). An earlier study has shown that this 13-mer epitope binds to HLA-B*3508 more efficiently than the completely overlap- ping EPLPQGQLTAY 11-mer peptide. Consistent with this observation, EBV-exposed individuals expressing HLA-B*3508 show a strong CTL response to the 13-mer epitope in prefer- ence to the overlapping 11-mer peptide (13, 16). JEM VOL. 202, November 7, 2005 A R T I C L E As shown in Fig. 2 A, the APQP peptide stabilized the expression of both HLA-B*3501 and HLA-B*3508 on the surface of T2 cells, indicating efficient binding to each class I molecule. In comparison to the immunogenic 11- or 13- mer BZLF1 epitopes, the APQP 10-mer bound with similar efficiency to both HLA-B*3501 and HLA-B*3508. To investigate the possibility that the APQP peptide may bind unstably to HLA-B*3501 in comparison to an EBV epitope that is immunogenic in the context of this class I molecule, peptide–MHC dissociation rates were examined on living cells. T2.B*3501 cells were loaded with either APQP or the highly immunogenic EPLPQGQLTAY pep- tide and were then washed and assessed for B*3501 surface expression levels at various time points. The data show that the two peptides dissociate from HLA-B*3501 at essentially similar rates (Fig. 2 B), indicating that the contrasting re- sponses to the APQP epitope in individuals expressing HLA-B*3501 or HLA-B*3508 is unrelated to peptide– MHC binding stability. To determine if the T cells that respond to the APQP peptide in HLA-B*3508 donors have the capacity to recog- nize the peptide bound to HLA-B*3501, peptide-specific Figure 2. The APQP peptide binds efficiently to both HLA-B*3501 and HLA-B*3508. (A) Peptide–MHC binding assay comparing APQP with other peptides for their ability to stabilize HLA-B*3501 or HLA-B*3508 expression on T2 cells that had been transfected with these HLA genes. These other peptides included EPLPQGQLTAY, an EBV epitope immunogenic in HLA-B*3501 individuals (tested only with T2.B*3501), and LPEPLPQGQLTAY, an EBV epitope immunogenic in HLA-B*3508 individuals (tested only with T2.B*3508). As negative controls, the HLA-A24/-A23–binding EBV epitope PYLFWLAAI and a truncated version of the APQP peptide (APQPAPENA_) were also included. The T2.B*3501 data were calculated relative to the EPLPQGQLTAY peptide used at 100 M, and the T2.B*3508 data were calculated relative to the LPEPLPQGQLTAY peptide used at 100 M. (B) Peptide–MHC dissociation rates were examined for the APQP and the EPLPQGQLTAY peptides using T2.B*3501. Data were calculated relative to EPLPQGQLTAY at time 0. 1251