Figure 3. CTL recognition of the APQP peptide in the context of HLA-B*3508 or HLA-B*3501. (A) Peptide dose-response cytotoxicity assay using five APQP-specific CTL clones from donor SB (HLA-B*3508 ), and PHA blast target cells expressing either HLA-B*3501 or HLA-B*3508. E/T ratio 2:1. (B) Multiple CTL microcultures were established from the HLA-B*3508 donor SB and the HLA-B*3501/-B*3508 coexpressing donor MB by stimulating PBMCs at limiting dilution with the APQP peptide. On day 13, the microcultures were screened for cross-recognition of the peptide (used at 0.02 g/ml) presented on either HLA-B*3501 or HLA-B*3508 PHA blasts. The data are from CTL microcultures raised from responder PBMC concentrations from which 50% of the wells produced CTLs specific for the stimulator EBV epitope; thus, most were likely to have been generated from a single peptide-specific CTL. Data from any CTL microcultures that killed the PHA blast target cells without peptide addition was discarded, and data are only shown for microcultures that displayed considerable lysis of one or both of the peptide-coated target cells. (C) The CTL microculture 2D6 that could efficiently recognize exogenously added APQP peptide in the context of HLA B*3501 was used in an IFN- ELISPOT assay to determine if this epitope is naturally presented on EBV-infected cells expressing this HLA allele. The target cells were HLA B*3501 LCLs carrying either the WT 1252 CTL clones were raised from an HLA-B*3508 donor and screened for recognition of the epitope presented on target cells expressing either HLA-B*3501 or HLA-B*3508 (Fig. 3 A). These peptide dose-response cytotoxicity assays show that five out of five CTL clones from donor SB recognized the peptide several orders of magnitude more efficiently in the context of self–HLA-B*3508 compared with HLA-B*3501, suggesting that the peptide binds with distinct conformations on these closely related HLA molecules. To examine this same issue with a broader range of APQP-specific T cells, multiple CTL microcultures were generated from donors SB (HLA-B*3508 ) and MB (HLA-B*3501 and HLA- B*3508 ) by stimulating PBMCs at a limiting dilution with the APQP peptide. On day 13, the microcultures were screened for cross-recognition of the peptide presented by ei- ther HLA-B*3501 or HLA-B*3508 PHA blast target cells (peptide concentration 0.02 g/ml). The data shown in Fig. 3 B are from CTL microcultures raised from responder PBMC densities from which 50% of the wells produced CTL specific for the EBV epitope; thus, most were likely to have been generated from a single peptide-specific CTL. Consistent with data presented in Fig. 3 A, the vast majority of these CTLs recognized the APQP peptide much more ef- ficiently when bound to HLA-B*3508 target cells com- pared with HLA-B*3501 target cells. These data indicate that the HLA-B*3501–APQP and HLA-B*3508–APQP structures are quite distinct targets for T cell recognition. To determine if the APQP peptide is processed and pre- sented from endogenously expressed viral antigen in HLA- B*3501 cells, the single T cell microculture from donor SB (HLA-B*3508 ) that could efficiently cross-recognize the exogenously added peptide presented by HLA-B*3501 (re- ferred to as CTL 2D6; Fig. 3 B) was used as a probe for HLA-B*3501–APQP complexes. Because only a small pro- portion of cells in lymphoblastoid cell lines (LCLs) express the BZLF1 lytic antigen of EBV, it was impossible to use conventional cytotoxicity assays to test for T cell recognition of endogenously expressed antigen. Instead we used a re- cently described method (17) that measures IFN- release in an ELISPOT assay where the effector T cells were challenged with LCLs expressing HLA-B*3501 and infected with either WT EBV (BZLF1 LCLs) or a recombinant EBV in which the BZLF1 gene was deleted (BZLF1 LCLs). As shown in Fig. 3 C, the 2D6 CTLs responded strongly to the BZLF1 LCLs but not the BZLF1 LCLs, demonstrating that the APQP peptide is processed from endogenously expressed an- tigen and presented by HLA-B*3501 at sufficient levels for T cell activation. This data supports the notion that factors other than peptide presentation levels control the immunoge- nicity of this viral CTL epitope. To examine levels of the APQP peptide processed and presented from endogenously expressed viral antigen in EBV genome (BZLF1 LCL) or an EBV genome that had been rendered inca- pable of lytic cycle entry by disruption of the BZLF1 gene (BZLF1 LCL). The number of spots per well is shown. THE INFLUENCE OF PEPTIDE CONFORMATION ON A CYTOTOXIC T CELL RESPONSE | Tynan et al.