analogues, a surprising number of amino acid substitutions at other positions within the peptide reduced binding to HLA- B*3508. Consistent with the structural analysis, the glutamate at P7 appears to be a particularly important secondary anchor residue for this peptide because most substitutions at this po- sition resulted in reduced MHC binding. A total of 48 analogues of the 10-mer peptide were tested for CTL recognition over a range of concentrations using chromium release assays, and the concentration of peptide required for half-maximum lysis was calculated (Fig. 7, A–C). Each CTL clone displayed a unique pattern of fine specificity for the 48 analogues, indicating that each ex- presses a different TCR. CTL recognition by the SB8 CTL clone was particularly affected by amino acid replacements toward the COOH terminus of the peptide (Fig. 7 A), whereas the SB16 clone was focused toward central residues, with recognition less affected by substitutions at the extrem- ities of the peptide (Fig. 7 B). The SB12 CTL clone was par- ticularly tolerant of substitutions toward the COOH termi- nus, suggesting that the highly exposed peptide residues at P8 and P9 do not contact the TCR expressed by this clone (Fig. 7 C). It is notable that all three CTL clones have diffi- culty recognizing peptides with substitutions at P5 and P6, suggesting that the alanine and proline residues at these posi- tions within the EBV peptide might interact closely with the antigen receptors of these clones. Therefore, these data help explain why these clones fail to recognize the APQP peptide bound to B*3501 because the side chains of the residues at P5 and P6 are oriented quite differently when bound to B*3501 and B*3508. DISCUSSION Polymorphism at the MHC locus enhances immune defense across the population by ensuring wide variation in the T cell response to infecting pathogens through presentation of a broad array of target epitopes (22, 23). This report has demonstrated another mechanism through which MHC polymorphism can diversify the immune response to an in- fecting pathogen. Thus, polymorphic MHC residues can markedly affect peptide binding conformation as well as MHC–peptide binding affinity, and this can have a major impact on the T cell response. Although previous studies have also demonstrated peptide structural alterations induced by MHC polymorphism (24, 25), none have shown that such changes can influence a peptide-specific immune re- sponse to this extent. Our data demonstrate that a single residue polymorphism between HLA-B*3501 and HLA-B*3508 controls respon- siveness to the APQP epitope through a mechanism unrelated to peptide–MHC binding efficiency/stability (Fig. 2). Fur- thermore, HLA-B*3501 , EBV-infected lymphoblasts clearly present the APQP epitope from endogenously expressed anti- gen with similar efficiency to HLA-B*3508 LCLs (Fig. 3 C and Fig. 4), ruling out differences in epitope processing within EBV-infected cells. It should be noted, however, that the re- JEM VOL. 202, November 7, 2005 A R T I C L E sponse to this epitope could be initiated by cross-priming through dendritic cells that could process and present a differ- ent repertoire of EBV peptides compared with infected B cells. Nonetheless, direct stimulation by EBV-infected B cells is likely to have the major role in restimulating and maintain- ing these T cell populations during persistent infection. We also consider it highly unlikely that polymorphic antigen pro- cessing genes are influencing this response in our study sub- jects because our HLA-B*3501 donors were all unrelated and the HLA-B*3508 donors were from two unrelated fam- ilies from different ethnic groups. Another factor with the potential to influence the im- munogenicity of a T cell epitope is immunodomination whereby the T cell response to an immunodominant deter- minant suppresses the response to another epitope (26, 27). It was therefore feasible that HLA-B*3501, but not HLA- B*3508, presents another unknown EBV epitope that is highly immunogenic, thereby suppressing the response to APQP in HLA-B*3501 individuals. Arguing against this possibility are data presented in Fig. 1 C and Fig. 3 B show- ing that the HLA-B*3501/-B*3508–coexpressing donor MB responds to the APQP epitope. The potential role of peptide conformation in controlling the differential responsiveness to this epitope became apparent after functional assays demonstrated that APQP-specific CTL clones from an HLA-B*3508 donor were unable to recog- nize this peptide efficiently in the context of HLA-B*3501 (Fig. 3 A). Thus, although these two closely related HLA molecules present this identical peptide sequence very effi- ciently, they appear to be structurally distinct from the per- spective of many T cells. This conclusion was strengthened by our analysis of multiple CTL microcultures, most of which recognized the peptide much more efficiently when presented on HLA-B*3508 target cells compared with HLA-B*3501 targets. It is notable that one of the donors used for this analy- sis coexpressed HLA-B*3501 and HLA-B*3508, yet there was no indication that T cells had been selected for the ability to cross-recognize the peptide on each of these HLA mole- cules (Fig. 3 B). Subsequent structural analysis confirmed ma- jor conformational differences between the peptide bound to HLA-B*3501 and HLA-B*3508 resulting from direct peptide interactions with the polymorphic MHC residue (Fig. 5 and Fig. 6). Furthermore, the conformational differences were particularly striking at highly exposed peptide residues that were shown to be important TCR contact residues for three out of three APQP-specific CTL clones (Fig. 7). There are several possible mechanisms through which such peptide conformational differences could influence im- munogenicity. It is possible that thymic and postthymic se- lection influence the relative number of naive T cells with the capacity to recognize each complex. For example, there may be an abundantly presented self-peptide that binds to both HLA-B*3501 and HLA-B*3508 that negatively selects CTLs with the potential to recognize the HLA-B*3501– bound conformation of the viral peptide or that positively 1257