Mapping of cytotoxic T lymphocytes epitopes in E7 antigen of human papillomavirus type 11
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- Xu, Y., Zhu, K., Chen, X. et al. Arch Dermatol Res (2008) 300: 235. doi:10.1007/s00403-008-0837-2
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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.
KeywordsHuman 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) . 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 . 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 . Successful induction of E7-specific CTLs has been described in mice upon immunization with CTL epitope peptides of E7 , recombinant E7 protein , transfected dendritic cells  and recombinant plasmids expressing E7 [21, 22]. The CTL could recognize proteolysed fragments of the protein in combination with MHC class I molecules . A single TCR can recognize large numbers of peptides in the context of a single MHC molecule . 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 . However, efficient induction of CTLs is hindered by the poor immunogenicity of antigens and the poor transduction efficiency of DCs . 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 . 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
Epitopes were selected and analysed by a computer-assisted algorithm . 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.
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).
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 , 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 .
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.
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.
The novel HLA*A 0201 restricted CTL epitopes and amino acid sequences of HPV 11 E7 described by computational prediction
Amino acid sequence
Carboxy-terminus restriction site
HPV-11E7-peptides induced the maturation and activation of DCs
Peptide-loaded mature DCs promoted the activation of T cells
HLA-A*0201-HPV-11E7 tetramers staining
Enhanced epitope-specific cytotoxic T lymphocyte responses
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 . 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 . 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.  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 . 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 . 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.