Memory T cells specific for novel human papillomavirus type 16 (HPV16) E6 epitopes in women whose HPV16 infection has become undetectable

Abstract

Human papillomavirus (HPV)-specific T-cell response to the HPV type 16 (HPV16) E6 protein has been shown to be associated with successful viral clearance. The patterns of CD8 T-cell epitopes within HPV16 E6 protein were previously studied in two women with HPV16 clearance. The goal of this study was to characterize these epitopes in terms of their minimal and optimal amino acid sequences and the human leukocyte antigen (HLA) restriction molecules. The presence of the epitope-specific memory T cells after viral clearance was also examined. In subject A, the dominant epitope was characterized to be E6 75-83 (KFYSKISEY), restricted by the HLA-B62 molecule, while that of subject B was E6 133-142 (HNIRGRWTGR), restricted by the HLA-A6801 molecule. Homologous epitopes were identified in five other high-risk HPV types for both of these epitopes, but they were not recognized by respective T-cell clone cells. An enzyme-linked immunospot assay or tetramer analysis was performed on peripheral blood mononuclear cells from blood samples collected after viral clearance but prior to isolation of the T-cell clones. The presence of epitope-specific memory T cells was demonstrated. These data suggest that HPV-specific memory T cells were generated in vivo and that they may remain in circulation many months, if not years, after viral clearance. Our findings broaden the spectrum of the CD8 T-cell epitopes of the HPV16 E6 protein. The characterization of novel T-cell epitopes and long-lasting epitope-specific memory T cells may be useful for the development of a potential epitope-based vaccine.

FIG. 1.

Natural history of HPV16 infection. (a) For subject A, the HPV infection was detected at one visit. The T-cell clones isolated to define the dominant CD8 T-cell epitope were derived from a blood sample drawn 53 months after HPV clearance. PBMCs collected at 29 and 37 months after the clearance of HPV16 infection were available. Pos, positive; Neg, negative. (b) Subject B had an HPV16 infection which persisted for over 4 years. The T-cell clones isolated to define the dominant CD8 T-cell epitope were derived from a blood sample drawn 78 months after HPV16 clearance. PBMCs for tetramer staining were isolated from blood samples collected at 4, 22, 27, 48, and 74 months after the clearance of HPV16 infection.

FIG. 2.

Characterization of subject A's dominant CD8 T-cell epitope in terms of its minimal and optimal amino acid sequence and the HLA-restricting molecule. The bars represent standard errors of the means. (a) ELISPOT assay performed to determine the specificity of the screen-positive T-cell clones to one of the three 15-mer peptides contained in the HPV16 E6 61-85 region. PHA, phytohemagglutinin. (b) ELISPOT assay performed to determine whether the epitope is endogenously presented using APCs infected with E6-vac or WR. (c) ELISPOT assay performed to identify the epitope within the 15-amino-acid region using 9-mer peptides overlapping by 8 amino acids. (d) ELISPOT assay performed to determine the optimal epitope sequence using two 10-mer peptides surrounding the E6 75-83 9-mer and two 8-mer peptides within it. The E6 74-82 9-mer peptide was also tested. (e) ELISPOT assay performed using serially diluted E6 75-83 9-mer peptide and E6 74-83 10-mer peptide (10⁻⁵ M to 10⁻¹⁰ M) to determine the optimal peptide of minimum length. A representative (clone #78-A) of two T-cell clones tested is shown. (f) Chromium release assay performed to determine the restricting molecule for the HPV16 E6 75-83 epitope using peptide-pulsed autologous LCL cells or a panel of partially HLA-matched LCL cells as APCs. A representative (#78-A) of the two clones tested is shown. θ, autologous LCL cells. *, HLA type determined using one of the molecular methods.

FIG. 3.

Characterization of subject B's dominant CD8 T-cell epitope in terms of its minimal and optimal amino acid sequence and the HLA-restricting molecule. The bars represent standard errors of the means. (a) ELISPOT assay performed to determine the specificity of the screen-positive T-cell clones to one of the three 15-mer peptides contained in the HPV 16 E6 121-145 region. PHA, phytohemagglutinin. (b) ELISPOT assay performed to determine whether the epitope is endogenously presented using APCs infected with E6-vac or WR. (c) ELISPOT assay performed to identify the epitope within the 15-amino-acid region using 10-mer peptides overlapping by 9 amino acids. (d) ELISPOT assay performed to determine the optimal epitope sequence using two 11-mer peptides surrounding the E6 133-142 10-mer peptide and two 9-mer peptides within it. (e) ELISPOT assay performed using the serially diluted E6 133-142 10-mer peptide, E6 132-142 11-mer peptide, and E6 133-142 11-mer peptide (10⁻⁵ M to 10⁻¹⁰ M) to determine the optimal peptide of minimal length. (f) Chromium release assay performed to determine the restricting molecule for the HPV16 E6 133-142 epitope using peptide-pulsed autologous LCL cells or a panel of partially HLA-matched LCL cells as APCs. A representative (#87-B) of the two clones tested is shown. θ, autologous LCL cells. *, HLA type determined using one of the molecular methods.

FIG. 4.

ELISPOT assay performed to determine the frequencies of the epitope-specific T cells after clearance of HPV16 infection but prior to isolation of the T-cell clones for subject A. The bars represent standard errors of the means.

FIG. 5.

Tetramer staining performed to determine frequencies of epitope-specific T cells after clearance of HPV16 infection but prior to isolation of T-cell clones for subject B. Cells were selected using a lymphocyte gate in forward and side scatter and for being CD4/14/19 negative. (a) Tetramer staining of PBMCs from a healthy donor known to be HLA-A6801 negative mixed with HPV16 E7 133-142 T-cell clone cells (#83-B) at a 10:1 ratio (positive control). (b) Tetramer staining of PBMCs from a healthy donor known to be HLA-A6801 negative (negative control). (c) Tetramer staining of peptide-stimulated PBMC sample collected 4 months after the clearance of HPV infection. (d) Tetramer staining of peptide-stimulated PBMC sample collected 22 months after the clearance.