The essence of epitopes

D riving the rapid advance in knowledge of peptidebinding to major histocompatibility complex (MHC) molecules is the prospect of predicting which peptides from foreign proteins are likely to be antigenic, and thus effective immunogens in vivo. Despite the numerous peptides that theoretically could be generated from a foreign protein, in practice cytotoxic T lymphocytes (CTL) focus upon just a few immunodomimnt peptide epitopes presented by self MHC class I molecules (1). Underlying this selection of particular peptides, also called determinants, is the sequence polymorphism that epitomizes class I heavy chains (2). In general, differences between alleles map to the peptide-binding site of the class I molecule and serve to change the types of peptide bound. Molecular insight comes from two lines of evidence: threedimensional structures of class I molecules bound to defined peptides, and amino acid sequences of endogenously bound peptides (3). Conserved residues in the peptide-binding site enmesh the peptide termini in hydrogen bond networks, interactions that lay a foundation for high aftinity binding. Polymorphic pockets of the class 1-binding site then add sequence specificity to the interaction by selecting for particuhr amino acids at two "anchor" positions within a peptide sequence of eight to nine residues. The identity of the anchor residues and their position define minimum "allele-specific motifs" the relevance of which is demonstrated by their ability to confer class I binding when incorporated into a polyalanine peptide backbone (4, 5). This knowledge raised hope that scanning foreign protein sequences for the presence of particular motifs would facilitate prediction of CTL epitopes. Indeed in notable instances this has been achieved. The H-2K d peptide-binding motif was used to identify a protective CTL epitope from Listeria monocytogenes (6, 7), and Hill et al. (8) found a CTL epitope from Plasmodiumfaki~rum that may protect individuals who express HLA-B*5301 from severe malaria. Although most functionally defined CTL epitopes possess appropriate class I-binding motifs (9), a minority of motifcontaining peptides from a given foreign protein are actually immunogenic. For example, although ovalbumin (a favorite model antigen) contains six potential epitopes with the H-2Kb-binding motif (shown in Table 1) (10), the CTL response is dominated by just one, OVA2s7-264 (12, 13). Immunodominance is dictated by more than just the presence of an appropriate class I-binding motif, which seems hardly surprising because antigen presentation involves many steps before the binding of class I and peptide: antigenic protein undergoes proteolysis, transport to the endoplasmic reticulum, and competes there with other peptides for binding to class I molecules. Seeking an explanation for the immunodominance of OVAz57_~, Jameson and Bevan (10) showed that synthetic peptides corresponding to the six ovalbumin sequences vary widely in their ability to bind H-2K b, as measured by ability to promote stable class I assembly. Interestingly, OVA2sT-~ was the most efficient, suggesting epitope selection reflects superior binding affinity. Recent investigations evaluate the contribution of peptide affinity for class I molecules to epitope selection, with the goal of improving identification of potential CTL epitopes. One such study is reported in this issue by Chen et al. (14) who find that immunization of mice with high concentrations of ovalbumin leads to CTL against a second epitope, OVAss-62. In pursuit of the basis for dominance of the OVA2sr CTL response over that to OVAss-62 under less extreme conditions of immunization, they showed the relatively poor response to OVAss-62 could not be attributed to bias in the T cell repertoire as equivalent numbers of CTL respond to both epitopes. Thus, the onus was placed on differences in peptide generation and/or presentation. This suspicion was confirmed by comparison ofT cell hybridomas specific for OVA2sT-~ and OVAs~2, which showed the dominant epitope was processed and presented some 20-50-fold more efficiently than the sub-dominant epitope. Furthermore, a synthetic version of the dominant epitope strongly out competed T cell hybridoma recognition of the sub-dominant epitope and not vice versa. On this basis, the source of dominance was attributed to differences in binding to H-2K b. Quantitative affinity measurements were therefore undertaken using biosensor-based technology that employes surface plasmon resonance (SPR) to monitor binding of immobilized peptide analogues to a soluble form of H-2K b (Fig. 1) (15). Although several methods have been developed for studying peptide-MHC binding, they are based on measurement of affinity constants at equilibrium. SPR offers the advantage of direct and continuous monitoring of changes in concentration of soluble class I molecules at the biosensor surface, enabling association and dissociation kinetics to be measured as they occur (in real time). The association rate for the OVA2sT-264 analogue was found to be approximately 10-fold faster than that of OVAss-62, and a two to three-fold difference in the dissociation rates of the two peptides was also observed. Thus, Chen et al. (14) conclude that functional dominance of OVA2s~-~64 in vivo can be explained almost completely by its high affinity binding to H-2K b.