Michel P. M. Vierboom

Efficient MHC class I-peptide binding is required but does not ensure MHC class I-restricted immunogenicity

Cytotoxic T lymphocyte (CTL) epitopes are naturally processed peptides bound and presented by major histocompatibility (MHC) class I molecules. Since they are expressed at the cell surface in sufficient amounts to be recognized by CTL, it is generally believed, and in some cases demonstrated, that they bind efficiently to MHC class I molecules in vivo. Based on this knowledge, candidate CTL epitopes are now searched for by identifying peptides that efficiently bind to MHC class I molecules in vitro. We analysed whether this approach is valid by systematically investigating the relationship between MHC class I-peptide binding and peptide immunogenicity. Fifteen peptides that represent known CTL epitopes were tested for their MHC class I binding ability. In a comparative study with 83 peptides that bear the appropriate MHC class I allele-specific motifs but do not contain known CTL epitopes, the CTL epitope-bearing peptides showed the highest binding affinity for MHC class I. This was true for two MHC class I alleles in two different assay systems that monitor peptide-MHC class I binding. Furthermore, selected motif-bearing Kb binding peptides were used to induce peptide-specific CTL responses in mice. Only a subset of the high affinity Kb binding peptides induced reproducible peptide-specific CTL responses, whereas none of the low affinity Kb binding peptides induced a response. Taken together, these results indicate that efficient peptide-MHC class I binding is required for immunogenicity. Vice versa, immunogenicity is not guaranteed by efficient peptide-MHC class I binding, implying that additional factors are involved. Nevertheless, selection of candidate CTL epitopes on the basis of MHC class I binding seems valid. Our data indicate that, although an excess of peptides might be selected, the chance of missing immunogenic peptides is minimal.

Competition inhibition of cytotoxic T-lymphocyte (CTL) lysis, a more sensitive method to identify candidate CTL epitopes than induction of antibody-detected MHC class I stabilization

We compared the efficiency of two commonly used cellular major histocompatibility complex (MHC) class I peptide-binding assays to identify a cytotoxic T lymphocyte (CTL) epitope-containing peptide among length variants derived from the human papilloma virus type 16 (HPV 16) oncoprotein E7. Although both assays identified the same sequence (E7 49-57) as the most efficient Db-binding peptide, the efficiency by which they did so differed markedly. In a peptide competition cytotoxicity (PCC) assay, based on inhibition of CTL lysis by competition for binding to MHC class-I molecules between a known CTL epitope-containing peptide and peptide of interest, E7 49-57 bound 45-fold more efficiently to Db than the second Db-binding peptide in line. In the widely used RMA-S MHC class I peptide-binding assay, based on peptide-induced stabilization of empty MHC class-I molecules at the surface of antigen-processing defective RMA-S cells, this difference was only 3 fold. Similar differences were observed when other Db-restricted CTL clones and CTL epitope-containing peptides were used in the PCC assay. The same phenomenon was observed when peptide binding affinities for H-2Kb were analyzed in both assays. We conclude that the PCC assay discriminates more efficiently between high- and low-affinity MHC class I binding peptides than the RMA-S assay. This observation is ascribed to the fact that peptide-MHC class I dissociation is an important parameter in the PCC but not the RMA-S assay.

Fine Characterization of the HPVI6 E7 49-57 Tumor Protective Cytotoxic T Cell Epitope “Rahynivtf”

Human papillomavirus (HPV) DNA, predominantly of the HPV16 genotype, can he detected in more than 90% of the human cervical. carcinomas1. The “high risk” HPV types, including HPV16, are believed to play an important role in the pathogenesis of human cervical. cancer. The ability of HPV16 to in vitro immortalize human keratinocytes2and the dependence on HPV 16 expression for preservation of the transformed phenotype of cervical. cancer-derived cell lines3 suggests direct involvement of HPV 16 in the multi-step process of cervical. carcinogenesis. Cervical. cancer and other HPV-related cancers are more commonly seen in immunosuppressed individuals4,5. This suggests that proper immunosurveillance interferes with HPV-associated tumor development and that T cell immunity, in particular mediated by cytotoxic T lymphocytes (CTL), is important in the defense against virus induced tumors.