Archives of Dermatological Research

, Volume 300, Issue 5, pp 235–242

Mapping of cytotoxic T lymphocytes epitopes in E7 antigen of human papillomavirus type 11


  • Yan Xu
    • Department of Dermatology, Sir Run Run Shaw Hospital, School of MedicineZhejiang University
  • Ke-Jian Zhu
    • Department of Dermatology, Sir Run Run Shaw Hospital, School of MedicineZhejiang University
  • Xian-Zhen Chen
    • Department of Dermatology, Sir Run Run Shaw Hospital, School of MedicineZhejiang University
  • Ke-Jia Zhao
    • Department of Dermatology, Sir Run Run Shaw Hospital, School of MedicineZhejiang University
  • Zhong-Ming Lu
    • Department of Dermatology, Sir Run Run Shaw Hospital, School of MedicineZhejiang University
    • Department of Dermatology, Sir Run Run Shaw Hospital, School of MedicineZhejiang University
Original Paper

DOI: 10.1007/s00403-008-0837-2

Cite this article as:
Xu, Y., Zhu, K., Chen, X. et al. Arch Dermatol Res (2008) 300: 235. doi:10.1007/s00403-008-0837-2


One of the critical steps in the progression to condyloma acuminatum (CA) is the establishment of a persistent human papillomavirus (HPV) infection, majority of HPV type 6 and 11. Cytotoxic T lymphocytes (CTL), which can be induced by the epitope-based peptides in vitro, are thought to be able to recognize and destroy virus-infected cells. In order to screen and identify HLA-A*0201 restricted HPV-11E7 CTL epitopes, five epitope peptides and tetramers were selected including HPV-11E7 7–15 (TLKDIVLDL), 15–23 (LQPPDPVGL), 47–55 (PLTQHYQIL), 81–89 (DLLLGTLNI) and 82–90 (LLLGTLNIV). Human monocyte-derived dendritic cells (DCs) from HLA-A*0201 healthy individuals were pulsed with these peptides to assess the expression of CD83, CD86, HLA-DR and the secretion of IL-12. The ability of peptide-loaded mature DCs to activate autologous T cells was evaluated by analyzing the frequency of specific tetramer+ CD8+ T cells using flow cytometry, and the level of IFN-γ secretion by ELISA. The ability of the epitope-specific CTLs to kill the target cells was also analysed. It was found that the immature DCs could be fully activated by all the five HPV-11E7 peptides and peptide-loaded mature DCs were able to stimulate the epitope-specific T cells in vitro. There was an increased frequency of CD8+ T cells specific for the E7 7–15 epitope when compared to other four predicted epitopes of HPV-11E7 (P < 0.05). The epitope-specific CTLs for E7 7–15 induced the strongest cytotoxicity to HPV-11E7 expressing cell line at an E:T ratio of 50:1 (P < 0.05). Taken together, these findings demonstrate that E7 7–15 (TLKDIVLDL) is an HLA-A*0201-restricted CTL epitope of HPV type 11. We propose that this epitope could be more helpful in the characterization of HPV control mechanism and be useful for the development of immunotherapeutic approaches for low-risk HPV infectious diseases such as CA.


Human papillomavirus type 11E7 antigenEpitope peptideCytotoxic T lymphocytesTetramerDendritic cells


HPV infection of the genital tract is one of the most common sexually transmitted diseases including low-risk and high-risk HPV types infection. The low-risk types of HPV 6 and 11 are shown to be responsible for the large majority of condyloma acuminatum (CA) [4]. Cell-mediated immune responses are critical in HPV attenuation, however, the antigenic epitopes involved in HPV control have not been successfully characterized [28, 18]. Previous studies have suggested that some HPV-specific CD8+ T-cell responses contribute to the control of the virus infection in cervical cancer mainly caused by high-risk types of HPV 16 or 18 [13, 29, 41]. However, less study was involved in low-risk types of HPV 6 and 11, though it is hypothesized that the weak generation of HPV-specific CTL response could be contributed to the poor treatment of CA. Therefore, identification of HPV 6/11 CTL epitopes would be helpful not only in our understanding of the protective immune response, but also in the development of immunotherapy strategies against CA.

Studies on the immune responses on the HPV early genes E6 and E7 have been the central interest in HPV research since they are required for the maintenance of cellular transformation [14]. Both E6 and E7 have immune dominant epitopes that represent ideal targets for vaccine developments against HPV [25, 19, 38]. Among those epitopes, there is a paucity of confirmed human CTL epitopes for HPV, while the majority of these are based on HLA-A*0201 [26]. Successful induction of E7-specific CTLs has been described in mice upon immunization with CTL epitope peptides of E7 [5], recombinant E7 protein [6], transfected dendritic cells [37] and recombinant plasmids expressing E7 [21, 22]. The CTL could recognize proteolysed fragments of the protein in combination with MHC class I molecules [11]. A single TCR can recognize large numbers of peptides in the context of a single MHC molecule [2]. Therefore the identification of CTL epitopes is crucial in understanding the roles of T cell activation. In this context, DCs play a crucial role in the induction of anti-viral immune responses through the recruitment and activation of T cells. Endocytosis, processing and presentation by DCs are required to induce HPV-specific CTLs [35]. However, efficient induction of CTLs is hindered by the poor immunogenicity of antigens and the poor transduction efficiency of DCs [10]. Studies have demonstrated that the HPV 16 E7-specific CTLs can be generated from in vitro vaccinated PBLs of healthy subjects [30, 42]. This observation indicates that mature dendritic cells can activate E7-specific CTLs from naive precursors in vitro [7, 36].

Previous studies have revealed that CTLs stimulated with endogenously processed HPV-11E7 antigen recognized the synthetic HLA-A2 motif-containing nonamer, HPV-11E7 4–12 and CTLs stimulated with this peptide in vitro recognized targets expressing endogenously processed E7 [32]. In the present study, we identified five HLA-A*0201-restricted CTL epitopes for HPV-11E7 using a combination of multiple computational prediction methods. Cultured mature DCs loaded with HLA-A*0201-restricted HPV-11 E7 peptides were employed to activate the antigen-specific CTLs in vitro. The presence of HPV-11 E7 epitope-specific CTLs was analysed by HLA-peptide tetrameric complexes, IFN-γ secretion and LDH release assays.

Materials and methods

Epitope selection

Epitopes were selected and analysed by a computer-assisted algorithm [23]. Consecutive overlapping nine-amino acid peptides that possessed the specific primary and secondary anchoring amino acids for HLA-A2 were identified [15, 27]. Based on the presence of favourable and unfavourable amino acids, the peptides were scored as indeterminate, low, medium, or high binding probability to HLA-A2 molecules.

Tetrameric peptide–MHC class I complexes and synthetic peptide

Tetramers of HPV-11E7, including the following HLA-A*0201-binding peptides: TLKDIVLDL, LQPPDPVGL, PLTQHYQIL, DLLLGTLNI and LLLGTLNIV were synthesized by Sanquin (Amsterdam, Netherlands). These tetramers corresponded to the peptides of HPV-11 E7 7–15, 15–23, 47–55, 81–89 and 82–90, respectively. The default panel of antibodies used for these studies was tri-colour conjugate anti-CD3 (Caltag, Burlingame, USA) and FITC-labelled anti-CD8 (Sanquin, Holland). Isotype controls were used in all flow cytometry experiments. Individual peptides were dissolved in dimethyl sulphoxide to provide stock solutions of 40–100 mg/ml.

Cell isolation

The PBMC were isolated from HLA*0201 healthy donors (young women with no sexual experience) by Lympholyte-H (Cedarlane Laboratories, ON, Canada) density gradient centrifugation as recommended by the manufacturer. T cells for one experimental setting were isolated from the same donor.

Generation of DCs and peptides pulsing

CD14 cells were isolated from PBMC by positive selection using CD14 microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) and their purity was determined by FACS with anti-CD14 Ab. DCs were generated by culturing monocytes in RPMI1640 medium containing 10% heat-inactivated fetal calf serum (FCS), 1% l-glutamine, 1% streptomycin, 1% penicillin, human recombinant interleukin-4 (rIL-4) (50 ng/ml) and human granulocyte-macrophage colony-stimulating factor (GM-CSF) (100 ng/ml) (Peprotech, ASIA) for 7 days in six-well plates at a density of 2 × 106 cells per well. Final maturation of monocyte-derived DCs was induced by exposing the cells to TNF-α (100 ng/ml) and flt-3 (50 ng/ml) (Peprotech, ASIA) for 48 h on day 5. On day 7, cells were harvested and analysed by FACS for CD1a expression. Monocytes derived DCs in the presence of GM-CSF and IL-4 without exposure to the stimulus were considered as ‘differentiated’, those induced by TNF-α and flt-3 were termed as ‘mature’ and DCs secreting cytokines named as ‘activated’ DCs.

A total of 2 × 106 differentiated DCs were incubated with each of the five HPV11E7 peptides (TLKDIVLDL, LQPPDPVGL, PLTQHYQIL, DLLLGTLNI or LLLGTLNIV) in six-well plates (20 μg/ml). After 48 h of incubation, CD1a, B7 molecule CD86, costimulatory molecule CD83 and MHC class II complexes HLA-DR expressions were analysed by flow cytometry (Beckman Coulter, Fullerton, USA) to determine the DCs maturation. IL-12 concentration in the supernatant was determined by ELISA [33, 40].

Mixed lymphocyte-DC culture and CD8+ responder T cells

Mature DCs were loaded with the aforementioned peptides at a density of 2 × 106 cells/ml by placing the DCs in a serum-free medium containing the peptides (20 μg/ml) for 1 h at 37°C. Autologous T lymphocytes were isolated from PBMC by negative selection with the pan T cell isolation kit II (Miltenyi Biotec, Bergisch-Gladbach, Germany) and mixed with the peptide-loaded mature DCs in a 24-well plates at a lymphocyte:DC ratio of 10:1. On days 7 and 14, additional DCs loaded with 20 μg/ml peptides and T lymphocytes were added to maintain the ratio of lymphocytes to DCs at 10:1 with at least 2 × 106 T cells/ml in the presence of 25 U/ml IL-2. After 3 weeks, induction of the antigen-specific T cells (suspension cells) was ready to be assessed by their capacities of IFN-γ secretion using ELISA, the tetramer staining and a LDH (lactate dehydrogenase) release assay.

Tetramers staining and flow cytometry

The antigen-specific T cells (1 × 106) were re-suspended in 50 μl of PBS and incubated with the tetramers (PE) for 20 min at room temperature. After a single wash, the tri-colour conjugate and FITC-labelled antibodies directed against CD3 and CD8 were added for 15 min. The cells were then washed with PBS again and analysed by flow cytometry (Beckman Coulter, Fullerton, USA).

CTL assay

In addition to using peptides loaded autologous DCs for assessing HPV11E7 epitope-specific CTL reactivity, a HPV-11E7 expressing cell line was established as target cell in cytolytic assay. The 293 cells were transfected with pcDNA3.1-HPV11E7-GFP by the lipofectamine kit (Invitrogen, California, USA). With a selective medium containing G418 at a concentration of 1,000 μg/ml [39], the G418-resistant positive cell clones stably expressing HPV-11E7 (HPV-11E7/293) were isolated under fluorescent microscope and identified by RT-PCR. The HPV11E7 positive 293 cells (1 × 104 per well) as target cells were incubated with the antigen-specific T cells, which served as effector cells at various effector to target cell (E/T) ratios of 10:1, 25:1, 50:1. The untransfected cells served as a negative target cell control and autologous DCs without loaded peptides were incubated with T cells and served as an effect cell control. After 6 h of co-incubation at 37°C, the supernatant was collected to assess the lactate dehydrogenase concentration using CytoTox 96 non-radioactive cytotoxicity assay kits (Promega Corp, Madison, WI, USA) with accordance to the manufacturer’s protocol. The cytotoxicity activity of T cells was assessed based on the following formula with the mean values from triplicated wells:

Percent cytotoxicity = (experimental value-effector spontaneous value-target spontaneous value)/(target maximum-target spontaneous value) × 100 [3].

Quantification of cytokines by immunoassay

Cytokine IL-12 and IFN-γ secretion levels in the culture supernatant from different experiments were determined by a direct enzyme linked immunosorbent assay (ELISA) kit according to the standard procedures (R&D, South San Francisco, USA). The ELISA plate was read with a standard ELISA reader at 450 nm. Concentration of cytokines was determined by comparing with a standard curve of serially diluted positive control samples supplied with the kit.

Statistical analysis

All data were expressed as mean ± standard deviation (SD). One-way ANOVA was used to evaluate the significance of group differences. Values of P < 0.05 were considered to be statistically significant.


Peptide selection

From the set of possible combinations of nanomer or decamer peptides in the HPV-11E7 proteins, five peptides were predicted as having a high binding ability to HLA-A2 molecules based on favourable amino acid combinations (Table 1). These HLA-A*0201-restricted HPV-11E7 peptides were selected for further immunogenicity studies.
Table 1

The novel HLA*A 0201 restricted CTL epitopes and amino acid sequences of HPV 11 E7 described by computational prediction

HPV-11E7 epitope

Amino acid sequence

Carboxy-terminus restriction site

E7 7–15


7, 9

E7 15–23


7, 9

E7 47–55


8, 9

E7 81–89


7, 9

E7 82–90


8, 9

HPV-11E7-peptides induced the maturation and activation of DCs

The purity of monocytes was determined by FACS analysis of CD14 expression on the cell surface. More than 90% of the isolated cells were CD14+. FACS analysis of differentiated DCs co-cultured with five HPV-11E7 peptides for 48 h showed high expression of HLA-DR, CD83 and CD86 on DCs with no significant difference between each group (data not shown). To determine the IL-12 secretion of peptides pulsed DCs, supernatants were obtained at 48 h from cultures. As shown in Fig. 1, IL-12 concentration in DCs cultured with the selected peptides was 30 to 60-fold higher than that of DCs cultured in medium alone (P < 0.05). IL-12 concentration in DCs cultured with E7 15–23 peptides was higher than in DCs cultured with E7 7–15 peptides (P < 0.05) and IL-12 concentration in these two groups was remarkably higher than in DCs stimulated with the other three peptides and controls (P < 0.05).
Fig. 1

IL-12 production level of DCs after incubation with different peptides. Differentiated Mo-DCs were incubated with the five peptides. As a negative control, DCs were cultured in medium alone. After 48 h of incubation, supernatants were obtained and IL-12 secretion was determined by ELISA and expressed as pg/ml ± SD of triplicate values

Peptide-loaded mature DCs promoted the activation of T cells

DCs generated from PBMC of healthy HLA-A*0201-positive donors were induced by GM-CSF, IL-4, TNF-α and flt-3 for maturation. Mature DCs were then loaded with each of the five HPV11E7 peptides and co-cultured with autologous specific T cells. As shown in Fig. 2, secretion of IFN-γ increased significantly after 3 weeks of incubation in the groups containing T cells co-cultured with peptide-loaded DCs, indicating a strong activation and stimulation of T cells. However, only a small increase of IFN-γ secretion was found both in the non-loaded DCs and in the T cells co-cultured with non-loaded DCs. Secretion of IFN-γ increased 5 to 10-fold in T cells co-cultured with peptide-loaded DCs, especially with peptides E7 7–15 (TLKDIVLDL), 15–23 (LQPPDPVGL) and 82–90 (LLLGTLNIV) (P < 0.05). T cells co-cultured with E7 7–15 peptide-loaded DCs showed the highest level of IFN-γ secretion.
Fig. 2

T cell responses against five HPV-11E7 peptides in vitro measured by ISN-γ ELISA. A total of 2 × 106 purified autologous T cells were co-cultured with 2 × 105 peptide-loaded or non-peptide-loaded mature DCs. The production of IFN-γ from each culture was measured after 21 days by ELISA. Results shown are mean ± SD of four independent experiments

HLA-A*0201-HPV-11E7 tetramers staining

Peptide-loaded DCs and T cells were co-cultured according to the above mentioned protocol. Seven days after the last round of re-stimulation, suspension cells were harvested from the cultures. Less than 0.03% tetramer+ cells detected in circulating CD8+ T cells represented the maximum staining observed in the controls (Fig. 3a). As shown in Fig. 3b–f, specific tetramer staining was observed in all cultures of autologous T cells stimulated with each of the five HPV-11E7 peptide-loaded DCs. On an average, a 100-fold increase in the frequency of specific tetramer+ CD8+ cells was observed when co-cultured with HPV-11E7 peptides-loaded DCs. Again, autologous T cells stimulated with E7 7–15 (TLKDIVLDL) peptide-loaded DCs showed the highest frequency of specific tetramer+ CD8+ cells as compared to other peptides (P < 0.05) (Fig. 3b–g).
Fig. 3

Profile of HLA-A*0201 tetramer staining for HPV-11E7 peptide-specific CTLs following multiple rounds of stimulation. T cells from donors were stimulated with DCs loaded with one of the five HPV-11E7 peptides for 3 weeks. The obtained CTLs were labelled with peptide/HLA-A*0201 tetramers for 20 min and stained with anti-CD3 and anti-CD8 antibody. The results shown are mean values of fluorescence intensity (MFI) of three representative independent experiments (af). The numbers in the upper right quadrants indicate the percentage of tetramer+ cells in the total of CD8+ cells. Cells that only stained with anti-CD3 and anti-CD8 antibodies were used as controls. a The frequency of the epitope-specific CTLs staining was calculated and the statistical difference was evaluated from three indepedent experiments. Data are presented as mean percentage of tetramer+ CD8+ cells ± SD (g)

Enhanced epitope-specific cytotoxic T lymphocyte responses

After 293 cells were transfected with pcDNA3.1/HPV11E7 plasmid, green fluorescence located in the cellular nucleus and plasma could be seen in positive clones (Fig. 4a). HPV-11E7 mRNA expression in selective cell line was also confirmed by RT-PCR (Fig. 4b). The results showed that the established cells positive for HPV-11E7 could further be used as a HPV-11E7-expressing cell line.
Fig. 4

The establishment of a HPV11E7-expressing cell line. Under the fluorescent microscope, green fluorescence could be observed in the cytoplasm and nucleus of the 293 cells 48 h after transfection with pcDNA3.1-HPV11E7-GFP (×400) (a), identification of established cell lines 293/HPV11E7 by RT-PCR (M, pUC 8 Marker; 1, positive cell line as template; 2, 293 cells without transfection with pcDNA3.1-HPV11E7-GFP; 3, ddH2O as template) (b)

Autologous T cells were co-cultured with HPV-11E7 peptide-loaded DCs as described above. Again 7 days after the last round of re-stimulation, cells from each culture were collected and analysed for the specific CTL responses. Induction of cytotoxicity in T cells in response to peptide-loaded DCs was investigated. The experiments were repeated four times with the results presented in Fig. 5. When the T lymphocytes acted as effector cells adhering to HPV11E7-expressing 293 cells at a 50:1 E/T ratio, T cells co-cultured with E7 7–15 peptide-loaded DCs had stronger cytotoxicity (91.48 ± 0.06%) than others (P < 0.05). However, this difference was not observed at the E/T ratio 25:1 or 10:1. The untransfected 293 cells did not show obvious cytolytic activity in all the groups at any E/T ratio (data not shown). These results indicate that E7 7–15 is a naturally processed CTL epitope. This was further confirmed by measuring the IFN-γ secretion of T cells and the frequency of specific tetramer+ CD8+ T cells following stimulation of E7 7–15 peptide-loaded DCs [12].
Fig. 5

The cytotoxic activities of T cells. T cells from healthy donors were stimulated with autologous mature Mo-DCs loaded with HPV-11E7 peptides 7–15, 15–23, 47–55, 81–89 or 82–90 and tested for specific lysis on day 21 by a LDH-release assay. Cytotoxicity of T cells against HPV-11E7-expressing cells was also investigated after the T cells were incubated with autologous mature DCs. Data are presented as mean percentage of cytotoxicity ± SD from four independent experiments (E, T cells isolated from PBMC by MACS co-cultured with peptides-loaded DCs; T, HPV-11E7-expressing cells)


CTL responses are considered to play an important role in viral clearance [32, 20]. The localized nature of HPV infections and resultant lesions combined with the immune evasion mechanisms of HPV, probably contributes to a weak systemic T cell response [34]. The genital HPVs are closely related to each other and the E7 proteins share significant amino acid sequence homology. Differences in the mechanisms regulating expression of the E7 proteins, rather than differences in the intrinsic biological properties of the E7 proteins, could account for the differences in the biological activities of the different HPV types [17]. Several studies have identified CTL epitopes of HPV 16/18 E6 and E7 proteins [13, 15, 19, 30, 41], while there have been only few studies on HPV-11 CTL epitopes. Identification of CTL epitopes of HPV-11E7 proteins is necessary for developing peptide-based vaccines against HPV11-associated lesions such as CA. Tarpey et al. [32] used different technologies to identify a HPV-11 E7 4–12 peptide that was able to elicit CTL response. However, we did not find information regarding the validity of using HLA allele-restricted anchor motifs or binding to HLA molecules as predictive methods for locating T-cell epitopes. In our present study, a total of five peptides selected on the basis of containing the theoretical HLA-A*0201-binding motif which were tested for the identification of HPV-11 E7 CTL epitopes. Stimulation with the peptides led to a remarkable up-regulation of IL-12 secretion in DC preparations. In vivo IL-12 produced mainly by DCs is important in the innate immune response to infection. IL-12 secretion and increased expression of two major markers for mature DCs, CD86 and CD83 costimulatory receptors which are functionally important MHC class II surface molecules, play an important role in establishing the immunological synapse with T cells. Our data show that the secretion of IL-12 in the DCs co-cultured with the peptides of E7 7–15 and E7 15–23 was higher than other peptides (P < 0.05), implying the potential role of these two peptides in inducing maturation and activation of human DCs in vitro.

It has been reported that using specific tetramers containing the relevant CTL epitope can be used to isolate HPV-specific CTLs [1, 16]. We determined the frequency of specific CD8+ T cells by PE conjugated tetramers by simultaneous multicolour flow cytometry. The frequency of HPV-11E7 7–15 (TLKDIVLDL) epitope-specific tetramer+ CD8+ T cells was higher than other epitope-specific tetramer+ CD8+ T cells (P < 0.05). Although tetramer staining provides quantitative information about the T-cell population based on its specificity, it does not provide information on its function [9]. Therefore, the functional activity of antigen-specific CD8+ T cells was examined using a standard LDH release cytotoxicity assay and an IFN-γ production assay by ELISA. The peptide-specific cytotoxicity was induced by 3 weekly rounds of stimulation with HPV-11E7 peptide-loaded mature DCs and our results showed a significant increase in IFN-γ secretion by autologous T cells. IFN-γ secretion was higher when the T cells were stimulated with the E7 7–15 (TLKDIVLDL), 15–23 (LQPPDPVGL) and 82–90 (LLLGTLNIV) . Peptide-loaded DCs as shown in Fig. 2. Of the five peptides, E7 7–15 peptide-loaded DCs seemed to be able to stimulate the T cells to secrete the highest level of IFN-γ, however, the difference of the five peptides was not statistically significant (P > 0.05). The LDH release assay used to detect the activity of CTLs against the HPV-11E7 transfected target cells showed that the specific cytolytic activity was the strongest (91.48 ± 0.06%) when the T cells were co-cultured with E7 7–15 loaded DCs at 50:1 E/T ratio (P < 0.05). Therefore, DCs loaded with HPV-11E7 7–15 (TLKDIVLDL) can induce highly effective and specific cellular immune response. These overall data of tetramer test, ELISA and LDH assay suggest that HPV-11E7 HLA-A*0201-restricted CTL epitope is a potential candidate for the development of a peptide-based vaccine, and may be used as an antigen for DC immunotherapies to treat patients with HPV-related diseases expressing this particular HLA type.

In future study, multiple tetramers comprising different CTL epitopes could be employed to allow more precise assessment of the role of CTL in HPV-associated diseases, including the possibility of HPV induced-immunological tolerance [8]. The utility of the HPV-11E7 epitope-peptides should be needed to clarify to develop new HPV vaccination strategies and evaluate the efficacy of immunotherapy by a single or combined peptide(s) of HPV-11E7.


We gratefully thank Dr. Jie He for her technical help. This work was supported by Science Foundation of Ministry of Education of China and High-level Innovation Personal Project of Health Bureau of Zhejiang Province.

Copyright information

© Springer-Verlag 2008