selects for T cells that recognize the viral peptide presented by HLA-B*3508 but not HLA-B*3501. Alternatively, cer- tain self-peptides may be presented preferentially by HLA- B*3508 that positively select T cells specific for HLA-B*3508– APQP (11, 12). In any case, the unique conformation of the APQP peptide when bound to HLA-B*3508 appears to be critical for recognition by most of the selected T cells and is, therefore, a major controlling influence over the immunoge- nicity of this EBV epitope. Another possible explanation for our data, unrelated to TCR repertoire differences between HLA-B*3501 and HLA-B*3508 individuals, is that the conformation of this EBV epitope on HLA-B*3508 is intrinsically more immu- nogenic through structural features that enable it to interact favorably with a higher frequency of TCRs (1). It is now clear that certain antigenic peptides present challenging tar- get structures for TCR recognition and are recognized by a very limited TCR repertoire (16, 28–30). It is therefore not unreasonable to expect that some peptide conformations will present docking surfaces complementary to a higher propor- tion of the TCR repertoire compared with others. Although it is impossible to decipher the exact mechanisms underlying the phenomenon, this study clearly demonstrates that pep- tide conformation can have a dramatic impact on the immu- nogenicity of an MHC–peptide complex and could there- fore play a major role in controlling determinant selection and immunodominance in other T cell responses. MATERIALS AND METHODS Cell lines. LCLs were established by exogenous transformation of periph- eral B cells with EBV and were maintained in growth medium (RPMI 1640 with 10% FCS). The mutant LCL T lymphoblastoid hybrid cell line, 174 CEM.T2 (referred to as T2 cells) (31), expressing either HLA- B*3501 (T2.B*3501) or HLA-B*3508 (T2.B*3508) was also used in this study and has been described elsewhere (13, 32). PHA blasts were generated as previously described (10). Blood donors used in this study were healthy laboratory staff selected for particular HLA alleles and prior exposure to EBV as assessed by standard virus-specific antibody tests. CTL cultures. CTL clones were generated by agar cloning as previously described (10) after initial stimulation of PBMCs for 1 h with 0.1 M of the APQP peptide. Clones were maintained with biweekly restimulation with rIL-2 and the -irradiated (8,000 rads) autologous LCLs that had been prelabeled with the APQP peptide at 0.1 M for 1 h and washed three times. Short-term CTL bulk cultures were also used as effectors in cytotox- icity assays. These were generated by culturing 2 106 PBMCs/2-ml well in growth medium with autologous PBMCs that had been precoated for 1 h with 0.1 M of the APQP peptide (responder/stimulator 2:1). Cul- tures were supplemented with 20 U/ml rIL-2 on day 3, split on day 7, and analyzed on day 10. CTL cultures were tested in duplicate or triplicate for cytotoxicity in the standard 5-h chromium release assay. In brief, CTLs were assayed against 51Cr-labeled LCL or PHA blast targets that were pre- treated with synthetic peptide (Mimotopes Ltd.) or left untreated. The mean spontaneous lysis for target cells in culture medium was always 20%, and the variation from the mean specific lysis was 10%. Short-term CTL microcultures were generated by limiting dilution as follows: PBMCs were distributed in roundbottom microtiter plates in growth medium at cell numbers ranging from 103 to 4 104 cells/well. Approximately 5 104 -irradiated (2,000 rads) autologous PBMCs that had been preincubated for 1 h with 1 M of the APQP peptide were added to each well to give a total volume of 100 l. Cultures were fed on days 4, 7, and 10 with 50 l of medium supplemented with 20 U of rIL-2 and 30% supernatant from MLA-144 cultures. On day 13, each CTL microculture was split into multiple replicates and used as effectors in a standard 5-h 51Cr- release assay against target PHA blasts that had been treated with the APQP peptide or left untreated. HLA class I peptide binding assays. To assess peptide binding to the different HLA-B35 subtypes, T2.B*3501 and T2.B*3508 cells were incu- bated in AIM V serum-free medium (Invitrogen) with various concentra- tions (0.01, 0.1, 1, 10, and 100 M) of peptides at 26 C for 14–16 h, fol- lowed by incubation at 37 C for 2–3 h and staining for HLA-B35 surface expression. To determine peptide dissociation rates, T2.B*3501 cells were pulsed with 100 M of each peptide at 26 C for 14–16 h, followed by incu- bation at 37 C for 2–3 h. Cells were then either stained immediately for HLA-B35 surface expression (0 h) or were washed three times and incubated at 37 C for 1–6 h before staining. HLA-B35 surface expression was mea- sured by a flow cytometer (FACSCalibur; Becton Dickinson) using an mAb to HLA-Bw6 (SFR8 Bw6). Data were expressed relative to the mean fluo- rescence intensity (MFI) measured using 100 M of a reference peptide that was known to bind to HLA-B*3501 or HLA-B*3508, using the following formula: ([MFI with test peptide MFI without peptide addition] 100) / (MFI with reference peptide [at 100 M] MFI without peptide addition). ELISPOT assays. IFN- ELISPOT assays were performed using cytokine capture and detection reagents according to the manufacturer’s instructions (Mabtech). In brief, anti–IFN- antibodies were coated on the wells of a 96- well nitrocellulose plate, and duplicate wells were seeded with 1,000 CD8 T cells and 50,000 target cells. Two LCLs raised from an HLA B*3501 indi- vidual were used as target cells in these experiments: one carried a WT B95.8 virus genome (BZLF1 LCL), and the other carried a B95.8 genome that had been rendered incapable of lytic cycle entry by disruption of the BZLF1 gene (BZLF1 LCL; a gift from A. Hislop and A. Rickinson, University of Bir- mingham, UK) (17). After incubation for 16 h, captured IFN- was detected with a biotinylated anti–IFN- antibody followed by development with streptavidin–horseradish peroxidase complex and chromogenic substrate, and spots were counted using an automated plate counter (AID). Expression, purification, and crystallization of HLA-B35 alleles with APQPAPENAY. Soluble HLA-B*3501 and HLA-B*3508 mole- cules (residues 1–276) and full-length 2-microglobulin (residues 1–99) were expressed, refolded with the APQP peptide, purified, and concen- trated to 10 mg/ml as previously described (33). Crystals were obtained by the hanging drop vapor diffusion technique. Block-shaped crystals grew within 4 d in a condition containing 0.2 M ammonium acetate and 16% wt/vol PEG 3350 (100 mM cacodylate, pH 7.6) at 4 C. X-ray data collection and structure determination. Crystals were soaked in reservoir solution containing increasing increments of glycerol as a cryo- protectant (5, 10, and 15%) and then flash frozen before data collection. Data were collected on an in-house radiation source and was processed and scaled using the HKL suite (34). The HLA-B35 complex structures were refined from an HLA-B*3501 structure that was previously determined in our laboratory (unpublished data). The model was manually built using the program “O” (35) and improved through multiple rounds of refinement using the CNS suite (36). The progress of refinement was monitored by Rfactor and Rfree values. Rigid-body refinement and simulated annealing were used in the first instance, but in later rounds en- ergy minimization and B-individual refinement were used to improve the quality of the model. See Table I for the final refinement and model statistics. The structures have been deposited in the Protein Data Bank under accession no. 2AXF (HLA-B*3508–APQP) and 2AXG (HLA-B*3501–APQP). Purification of HLA complexes and peptide analysis. Purification of HLA molecules was performed from 8 108 HLA-B*3501 LCLs (do- 1258 THE INFLUENCE OF PEPTIDE CONFORMATION ON A CYTOTOXIC T CELL RESPONSE | Tynan et al.