A Conserved E7-derived Cytotoxic T Lymphocyte Epitope Expressed on Human Papillomavirus 16-transformed HLA-A2+ Epithelial Cancers

Angelika B Riemer,Derin B Keskin,Guanglan Zhang,Maris Handley,Karen S Anderson,Vladimir Brusic,Bruce Reinhold,Ellis L Reinherz

doi:10.1074/jbc.m110.126722

Abstract

Human Papillomavirus 16 (HPV-16) has been identified as the causative agent of 50% of cervical cancers and many other HPV-associated tumors. The transforming potential/tumor maintenance capacity of this high risk HPV is mediated by two viral oncoproteins, E6 and E7, making them attractive targets for therapeutic vaccines. Of 21 E6 and E7 peptides computed to bind HLA-A*0201, 10 were confirmed through TAP-deficient T2 cell HLA stabilization assay. Those scoring positive were investigated to ascertain which were naturally processed and presented by surface HLA molecules for CTL recognition. Because IFNγ ELISpot frequencies from healthy HPV-exposed blood donors against HLA-A*0201-binding peptides were unable to identify specificities for tumor targeting, their physical presence among peptides eluted from HPV-16-transformed epithelial tumor HLA-A*0201 immunoprecipitates was analyzed by MS(3) Poisson detection mass spectrometry. Only one epitope (E7(11-19)) highly conserved among HPV-16 strains was detected. This 9-mer serves to direct cytolysis by T cell lines, whereas a related 10-mer (E7(11-20)), previously used as a vaccine candidate, was neither detected by MS(3) on HPV-transformed tumor cells nor effectively recognized by 9-mer specific CTL. These data underscore the importance of precisely defining CTL epitopes on tumor cells and offer a paradigm for T cell-based vaccine design.

Highlights

Activities and functionally inactivate the p53 and retinoblastoma (Rb) tumor suppressor proteins, respectively [3, 4]
Given that E6 and E7 are consistently expressed in human Papillomavirus (HPV)-associated cancers, these proteins themselves represent promising targets for vaccine design
We suggest that the lack of prior clinical effectiveness of targeted CTL epitope vaccination [32] is a consequence of misidentification of peptides displayed on tumor cells because of the use of indirect immunological surrogates to judge T cell epitope expression

Summary

EXPERIMENTAL PROCEDURES

Cell Lines—Three HLA-A*0201-positive, HPV-16-positive human cervical carcinoma cell lines and two cell lines transfected with HPV-16 E6 and E7 were used in this study (Table 1). In contrast to chromatographic separations, the different ionizable components are simultaneously present in the ion beam This means molecular abundance of a target can be measured by use of an added calibrant molecule at known concentration. Generation of HPV-16-specific T Cell Lines—For use as antigen-presenting cells, dendritic cells were differentiated from adherent donor monocytes in DMEM medium supplemented with 10% human serum, 1% L-glutamine, 1% penicillin/streptomycin, 100 ng/ml granulocyte-macrophage colony-stimulating factor, and 50 ng/ml IL-4 (PeproTech, Rocky Hill, NJ) for 1 week. Donor PBMC were plated at a density of 107cells/ml together with 5 ϫ 104/ml HPV-16 peptide-loaded irradiated antigen-presenting cells in 24-well culture plates in DMEM medium supplemented with 10% human serum, 1% L-glutamine, 1% penicillin/streptomycin, 0.05 mM 2-mercaptoethanol, and 10 ng/ml IL-7. Percent specific chromium release was calculated using the formula (experimental release Ϫ spontaneous release)/(maximum release in 5% Triton X-100 Ϫ spontaneous release) ϫ 100

RESULTS

HPV status

Peptide position

DISCUSSION