HLA class I binding promiscuity of the CD8 T-cell epitopes of human papillomavirus type 16 E6 protein

Abstract

One of the critical steps in the progression to cervical cancer appears to be the establishment of persistent human papillomavirus (HPV) infection. We have demonstrated that the lack of cytotoxic T-lymphocyte response to HPV type 16 (HPV 16) E6 protein was associated with persistence and that the potential presence of dominant CD8 T-cell epitopes was most frequently found (n = 4 of 23) in the E6 16-40 region by examining the pattern of CD8 T-cell epitopes within the E6 protein in women who had cleared their HPV 16 infections. The goal of this study was to define the minimal/optimal amino acid sequences and the HLA restricting molecules of these dominant CD8 T-cell epitopes as well as those of subdominant ones if present. Three dominant epitopes, E6 29-38 (TIHDIILECV; restricted by the HLA-A0201 molecule), E6 29-37 (TIHDIILEC; restricted by B48), and E6 31-38 (HDIILECV; restricted by B4002), and one subdominant epitope, E6 52-61 (FAFRDLCIVY; restricted by B35) were characterized. Taken together with a previously described dominant epitope, E6 52-61 (FAFRDLCIVY; restricted by B57), the CD8 T-cell epitopes demonstrated striking HLA class I binding promiscuity. All of these epitopes were endogenously processed, but the presence of only two of the five epitopes could have been predicted based on the known binding motifs. The HLA class I promiscuity which has been described for human immunodeficiency virus may be more common than previously recognized.

FIG. 1.

ELISPOT assays performed using E6-vac-, E7-vac-, and/or Western Reserve (WR)-infected autologous EBV LCLs as antigen-presenting cells revealed that the T-cell clones from subjects 7, 15, and 20 recognize endogenously processed E6 epitopes. The number after the hyphen indicates subject of origin. The bars represent standard errors of the means. (a) Twenty of 20 T-cell clones from the subject 7 screen positive for the E6 16-40 region were positive for an endogenously processed E6 epitope. Ten representative clones are shown. The experiment was done with an MOI of 5 in triplicates, and E6-vac and WR were tested. (b) Six of 10 T-cell clones from the subject 7 screen positive for the E6 46-70 region were positive for a endogenously processed E6 epitope. The experiment was done with an MOI of 5 in triplicates, and E6-vac and WR were tested. (c) Ten of 14 T-cell clones from the subject 15 screen positive for the E6 16-40 region were positive for a endogenously processed E6 epitope. Four representative clones are shown. The experiment was done with an MOI of 10 in duplicates, and E6-vac, E7-vac, and WR were tested. (d) Eight of eight T-cell clones from the subject 20 screen positive for the E6 16-40 region were positive for an endogenously processed E6 epitope. The experiment was done with an MOI of 5 in duplicates, and E6-vac, E7-vac, and WR were tested. (e) Six of eight T-cell clones from the subject 20 screen positive for the E6 31-55 region were positive for an endogenously processed E6 epitope. The experiment was done with an MOI of 5 in duplicates, and E6-vac, E7-vac, and WR were tested.

FIG.2.

ELISPOT assays demonstrating that the shortest and optimal peptide for subject 7's dominant epitope is E6 29-38 and that the miminal among equally effective peptides of her subdominant epitope is E6 52-61. The numbers in parentheses indicate peptide lengths in amino acids. The bars represent standard errors of the means. (a) Two of three T-cell clones tested demonstrated the highest number of spot-forming units with E6 29-38 among a number of peptides tested including two 8-mers (E6 29-36 and E6 30-37), seven overlapping 9-mers covering the E6 26-40 region, two 10-mers (E6 28-37 and E6 29-38), and one 15-mer (E6 26-40). The experiment was done in duplicates. (b) Comparison of the E6 28-38 11-mer, E6 29-39 11-mer, E6 28-37 10-mer, E6 29-38 10-mer, E6 30-39 10-mer, E6 29-37 9-mer, E6 30-38 9-mer, E6 29-36 8-mer, E6 30-37 8-mer, and E6 31-38 8-mer revealed that the optimal peptide of minimum length is likely to be the E6 29-38 10-mer. The experiment was done in triplicates. (c) Comparison of the E6 29-38 10-mer, E6 30-38 9-mer, and E6 30-37 8-mer, ranging from 10⁻⁵ M to 10⁻¹⁰ M, confirmed the optimal peptide of minimum length to be the E6 29-38 10-mer peptide. The experiment was done in duplicates, and the results of one representative clone (#59-7) out of two clones tested are shown. (d) Two of four T-cell clones tested demonstrated the most number of spot-forming units with E6 52-61 among a number of peptides tested including two 8-mers (E6 53-60 and E6 54-61), seven overlapping 9-mers covering the E6 51-65 region, two 10-mers (E6 52-61 and E6 53-62), two 11-mers (E6 51-61 and E6 52-62), and one 15-mer (E6 51-65). The experiment was done in duplicates. (e) Comparison of the E6 51-61 11-mer, E6 52-62 11-mer, and E6 52-61 10-mer, ranging from 10⁻⁵ M to 10⁻¹⁰ M, confirmed that the minimal peptide between the two optimal peptides is the E6 52-61 10-mer peptide. The experiment was done in triplicates, and the graph of one representative clone (#86-7) out of two clones tested is shown.

FIG.2.

ELISPOT assays demonstrating that the shortest and optimal peptide for subject 7's dominant epitope is E6 29-38 and that the miminal among equally effective peptides of her subdominant epitope is E6 52-61. The numbers in parentheses indicate peptide lengths in amino acids. The bars represent standard errors of the means. (a) Two of three T-cell clones tested demonstrated the highest number of spot-forming units with E6 29-38 among a number of peptides tested including two 8-mers (E6 29-36 and E6 30-37), seven overlapping 9-mers covering the E6 26-40 region, two 10-mers (E6 28-37 and E6 29-38), and one 15-mer (E6 26-40). The experiment was done in duplicates. (b) Comparison of the E6 28-38 11-mer, E6 29-39 11-mer, E6 28-37 10-mer, E6 29-38 10-mer, E6 30-39 10-mer, E6 29-37 9-mer, E6 30-38 9-mer, E6 29-36 8-mer, E6 30-37 8-mer, and E6 31-38 8-mer revealed that the optimal peptide of minimum length is likely to be the E6 29-38 10-mer. The experiment was done in triplicates. (c) Comparison of the E6 29-38 10-mer, E6 30-38 9-mer, and E6 30-37 8-mer, ranging from 10⁻⁵ M to 10⁻¹⁰ M, confirmed the optimal peptide of minimum length to be the E6 29-38 10-mer peptide. The experiment was done in duplicates, and the results of one representative clone (#59-7) out of two clones tested are shown. (d) Two of four T-cell clones tested demonstrated the most number of spot-forming units with E6 52-61 among a number of peptides tested including two 8-mers (E6 53-60 and E6 54-61), seven overlapping 9-mers covering the E6 51-65 region, two 10-mers (E6 52-61 and E6 53-62), two 11-mers (E6 51-61 and E6 52-62), and one 15-mer (E6 51-65). The experiment was done in duplicates. (e) Comparison of the E6 51-61 11-mer, E6 52-62 11-mer, and E6 52-61 10-mer, ranging from 10⁻⁵ M to 10⁻¹⁰ M, confirmed that the minimal peptide between the two optimal peptides is the E6 52-61 10-mer peptide. The experiment was done in triplicates, and the graph of one representative clone (#86-7) out of two clones tested is shown.

FIG. 3.

ELISPOT assays demonstrating that the shortest and optimal peptide for subject 15's dominant epitope is E6 29-37. The bars represent standard errors of the means. (a) Seven of seven T-cell clones tested demonstrated the highest number of spot-forming units with E6 29-38 among the seven overlapping 9-mers covering the E6 26-40 region and the 15-mer. The experiment was done in duplicates. (b) Comparison of the E6 26-40 15-mer, E6 29-38 10-mer, E6 28-37 10-mer, E6 29-37 9-mer, E6 30-37 8-mer, and E6 29-36 8-mer revealed that the optimal peptide of minimum length is likely to be the E6 29-37 9-mer. The experiment was done in triplicates. (c) Comparison of the E6 29-37 9-mer, E6 30-37 8-mer, and E6 29-36 8-mer, ranging from 10⁻⁵ M to 10⁻¹⁰ M, confirmed the optimal peptide of minimum length to be the E6 29-37 9-mer peptide. The experiment was done in triplicates, and the graph of one representative clone (#15-15) out of four clones tested is shown.

FIG. 4.

ELISPOT assays demonstrating that the shortest among equally optimal peptides for subject 20's dominant epitope present within the overlapping amino acids in the E6 26-40 and E6 31-45 regions is the E6 31-38 peptide. The bars represent standard errors of the means. (a) Clone #60-20 demonstrated large numbers of spot-forming units with E6 30-38 and E6 31-39 among the seven overlapping 9-mers covering the E6 26-40 region. The experiment was done in duplicates. (b) Clones #127-20 and #138-20 demonstrated large numbers of spot-forming units with E6 30-38 and E6 31-39 among the seven overlapping 9-mers covering the E6 31-45 region and the 15-mer. The experiment was done in duplicates. (c) Comparison of the E6 30-39 10-mer, E6 30-38 9-mer, E6 31-39 9-mer, E6 30-37 8-mer, E6 31-38 8-mer, E6 31-37 7-mer, and E6 32-38 7-mer revealed that the optimal peptide of minimum length is likely to be the E6 31-38 8-mer. The experiment was done in triplicates. (d) Comparison of the E6 30-39 10-mer, E6 30-38 9-mer, and E6 31-38 8-mer, ranging from 10⁻⁵ M to 10⁻¹⁰ M, showed that the minimal among three equally optimal peptides is the E6 31-38 8-mer peptide. The experiment was done in duplicates, and results with one representative clone (#60-20) out of two clones tested are shown.

FIG. 5.

Identifying the restricting HLA class I molecule for the CD8 T-cell epitope using chromium release assays. θ, autologous LCL; *, HLA type determined using one of the molecular methods. The bars represent standard errors of means. (a) The E6 29-38 epitope appears to be restricted by the A0201 molecule. A representative (#59-7) of two clones is shown. (b) The E6 52-61 epitope appears to be restricted by the B35 molecule. A representative (#86-7) of two clones is shown. (c) The E6 29-37 epitope appears to be restricted by the B48 molecule. A representative (#34-15) of the two clones is shown. (d) The E6 31-38 epitope appears to be restricted by the B4002 molecule. A representative (#127-20) of three clones is shown.

FIG. 6.

Diagram of the “epitope hot spots” within the HPV 16 E6 and E7 proteins, showing the clustering of the CD8 T-cell epitopes restricted by different HLA molecules.