A conserved E7-derived cytotoxic T lymphocyte epitope expressed on human papillomavirus 16-transformed HLA-A2+ epithelial cancers

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.

Figures

FIGURE 1.

HPV-16 genome and transforming activity of E6 and E7. The left panel shows the ∼8000-bp map of this oncogenic DNA virus and its genes. In the right panel, the key functions of the early and late genes are listed (5, 85–93).

FIGURE 2.

HLA-A*0201 T2-based peptide binding assay. HLA-A*0201-positive, TAP-deficient T2 cells were pulsed with 10 μm of the respective peptides given on the graph ordinate for 6 h at 37 °C. Binding was determined with the anti-HLA-A2 antibody BB7.2 by flow cytometry and calculated relative to a known strong binder, the TAX11–19 peptide from the HTLV on the abscissa. The TAX11–19 binding was set at 1.0. The order of peptides is from predicted strongest to weakest binders, top to bottom, respectively. Shaded entries are E6-derived peptides, whereas unshaded entries are E7-derived.

FIGURE 3.

Low or undetectable memory T cells in blood of healthy individuals. Immune recognition of the 10 HLA-A*0201-binding peptides was tested in an IFNγ ELISpot assay. PBMC isolated from 6 HLA-A*0201-positive healthy donors were stimulated with 10 μm respective peptide overnight. In panel A, spots are graphed and presented as SFUs per million PBMC. SFUs of single donors are represented as dots, with a horizontal line corresponding to the mean of six donor samples. Highest SFUs are not from the same donor. In panel B, ELISpot well images from representative plates (2 × 105 cells/well) taken from several donors are shown. CEF, cytomegalovirus/Epstein-Barr-Virus/Influenza positive peptide mix. PHA, phytohemagglutinin, a mitogenic plant lectin used as another positive control.

FIGURE 4.

Methodology for immunoprecipitation of HLA-A2 molecules, elution of bound peptides, and MS3 analysis of potential CD8 T cell epitopes. MS3 analysis isolates a selected m/z window containing a target ion (e.g. m/z 555.3), fragments (by collision activation) all ions in the selected window, isolates from these fragments a second m/z window containing a target fragment (e.g. m/z 764.3), and dissociates ions in the second m/z window to form an MS3 spectrum (denoted MS3 555.3/764.4). A probabilistic measure quantifies the likelihood the MS3 spectrum generated in these steps contains a reference dissociation pattern obtained from the synthetic peptide. Details are provided under “Experimental Procedures.”

FIGURE 5.

MS3 detection analysis of E711–19, E711–20, and E629–38 HPV-16 peptides. Comparison of MS3-HLA-A2 extracts with MS3 reference patterns is shown in the left and middle columns, respectively. Poisson detection signatures are shown in the right columns. A, MS3 555.3/764.4 of HLA-A2-associated peptides extracted from 60 million CaSki cells is shown. B, MS3 555.3/764.4 of synthetic peptide YMLDLQPET corresponding to E711–19 is shown. C, shown is the Poisson detection signature of the synthetic pattern shown in B in the spectrum shown in A. The amplitude at 0 m/z shift relative to nonzero m/z shifts is a probabilistic measure of the uniqueness of the fit and is used as a marker of detection (see “Experimental Procedures” and “Results” sections). D, MS3 605.8/764.4 of HLA-A2 peptides extracted from 60 million CaSki cells is shown. E, MS3 605.8/764.4 of synthetic peptide YMLDLQPETT corresponding to E711–20 is shown. F, the Poisson detection signature of the b6 fragment (YMLDLQ-) is shown. G, MS3 578.3/935.5 of HLA-A2-associated peptides extracted from 20 million CaSki cells is shown. H, a reference spectrum of the b8 fragment (TIHDIILE)- from MS3 578.3/935.5 of the synthetic peptide TIHDIILECV is shown. I, Poisson detection signature of the b8 fragment in MS3 578.3/935.5 spectrum of peptides extracted from CaSki cells is shown.

FIGURE 6.

Poisson detection of E711–19 on all HPV-16-transformed and -transfected human epithelial cells and their HLA-A2 surface expression. Panels A–E represent Poisson detection signatures of E711–19 from 20 million C66-7 cells (A), 10 million E6/E7 transfected oral OKF6/E6E7 keratinocytes (B), 10 million E6/E7 transfected foreskin keratinocytes (N/E6E7) (C), 20 million C66-3 cells (D), and 20 million CaSki cells (E). Panel F shows mean cell fluorescence intensity values of the indicated cells reacting with the FITC-labeled anti-HLA-A2 mAb BB7.2.

FIGURE 7.

The MS2 555.3 spectra of HLA-A*0201 peptide extracts from different cell lines expressing the E7 oncoprotein and Laz 509 B cells loaded with 10 ng/ml E711–19. The common peaks at the high m/z end are fragments from different peptides sharing amino or carboxyl terminal amino acids and a molecular mass near 1108.6 Da (hence, co-selected in the m/z 555.3 window). Because the intensity of these high m/z peaks is an average of many peptides, their intensity serves as an approximate measure of the peptide background. Their amplitude relative to m/z 764.4 provides in a single spectrum a characterization of the fraction of A2-bound peptide that is E711–19 (see “Results”). The C66-7, N/E6E7, and OKF6/E6E7 samples show not just lower absolute amounts of E711–19 (Fig. 6) but also that E711–19 is a smaller relative fraction of the total peptide population.

FIGURE 8.

Quantitation of the number of E711–19 epitopes per cell on HPV epithelial transformants and peptide-pulsed lymphoid cells. To quantitate the amount of E711–19 recovered from HLA-A2 extracts, the MS3 signal abundance relative to an added control peptide is measured. A, the MS3 signals of the target E711–19 and control P (KSPWFTTK) peptides were measured at known concentrations in a mock MHC I workup. B, a known amount of the control peptide P was added to the HLA-A2 sample being analyzed, and MS3 spectra of peptide P and E711–19 were again taken. C, combining the MS3 signal ratios measured in A and B with the amount of control peptide added provided the amount of target peptide (see “Experimental Procedures”). Knowing the number of cells lysed, the target copies per cell were calculated assuming full recovery up to the point where the control peptide was added (Step IV, Fig. 4).

FIGURE 9.

Specificity of CD8 T cells elicited against E711–19 was directed against E711–19 but not E711–20. E711–19-specific T cells were generated by four weekly stimulations with autologous dendritic cells and tested for their antigen-specific proliferation by tritiated thymidine incorporation (panel A), IFNγ secretion by cytometric bead assay (panel B), IFNγ production by ELISpot (panel C), and ability to lyse autologous EBV-transformed B cells pulsed with the indicated amounts of E711–19 or an irrelevant Nef137–145 (LTFGWCFKL) HIV peptide (panel D). PHA, phytohemagglutinin.

FIGURE 10.

Conservation of E711–19 in all high risk HPV-16 strains. 15 of the 16 HPV-16 E7 sequences in the HPV data base include the E711–19 sequence. The corresponding UniProt accession number of each sequence is shown at the left of each line. Asterisk, this column contains identical amino acid residues in all sequences; colon, this column contains different but highly conserved (very similar) amino acids; no symbol indicates that this column contains dissimilar amino acids or gaps.

FIGURE 11.

E711–19 binds to the vast majority of HLA-A2 alleles. HLA binding predictions of E711–19 were performed using NetMHCpan on the 116 known HLA-A2 alleles. Panel A shows the predicted IC50 values for each allele (in nm) with strong binding (<50 nm) shaded magenta, weak binding (50–500 nm) shaded green, and no binding (>500 nm) unshaded. Panel B gives a bar graph representation of E711–19 IC50 for each allele along the x axis corresponding to those going from the left to right columns in the order defined in panel A.