Mapping Of Cytotoxic T Lymphocytes Epitopes In E7 Antigen Of Human Papillomavirus Type 11 And Foxp3~+CD4~+CD25~+ Regulatory T Expression In Peripheral Blood Of Patients With Condyloma Acuminatum

Condyloma acuminatum(CA)is one of the most common sexually transmitted diseases,which is difficult to cure because of its high relapse rate. The incidence of CA has a tendency of increase.Currently,we usually eliminate the neoplasm by physical therapy,including laser,microwave,liquid nitrogen,etc, followed by non-specific immunotherapy.Routine therapeutic methods can't efficiently eradicate CA in that all existing treatment options have an associated failure rate and recurrence of genital warts after treatment.Human papilloma virus(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.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.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 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 in the development of immunotherapy strategies against CA as well.Studies on the immune responses to 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. 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.The CTL could recognize proteolysed fragments of the protein in combination with MHC classâ… molecules. A single TCR can recognize large numbers of peptides in the context of a single MHC molecule,therefore,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 antiviral 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.This observation indicates that mature dendritic cells can activate E7-specific CTLs from naive precursors in vitro.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 tetramerie complexes,IFN-γsecretion and LDH release assays.Specific immune responses against viruses and cancer are controlled by a network of regulatory T cells.The naturally occurring CD4⁺CD25⁺ regulatory T cells(Tregs)is the pivotal cell type that maintains self-tolerance and exerts active immune suppression.The development and function of Tregs is controlled by the forkhead transcription factor Foxp3(forkhead boxp3).CD4⁺CD25⁺ Treg cells have been shown to suppress virus-specific CD8⁺ T cell responses and delay viral clearance.Immunized mice lacking CD4⁺CD25⁺ Tregs also showed greater resistance to viral challenge.An increased number of CD4⁺CD25⁺ cells were found in patients with autoimmunity,cancer,and chronic infection.The significantly increased frequency of CD4⁺CD25^hiFoxp3⁺ regulatory T cells was found in women who had persistent HPV16 infection.However,little is known of the frequency and distribution of regulatory T cells within PBMC of CA patients and how they are correlated with the development and recurrence of CA.The balance between type 1 and type 2 T-cell subsets in CA patients is thought to modulate antiviral immunity.A polarized Tc1/Tc2 dichotomy as well as an imbalance of Th1/Th2 are thought to be critical determinants of immune responses under certain pathologic conditions.A shift from Th1 to Th2 cytokine profile has been indicated in patients with HPV-associated cervical lesions. However,the balance of Th1/Th2 and Tc1/Tc2 cells in patients with CA is not addressed.Taken together,this project focused on the identification of HLA-A^*0201-restricted CTL epitope of HPV type 11 E7,as well as Foxp3⁺CD4⁺CD25⁺ T cells, Th1/Th2 and Tc1/Tc2 cells expression in patients with CA.It will be useful to the development of immunotherapeutic approaches for low-risk HPV infectious diseases such as CA. The project is composed of two sections.Section one:Mapping of Cytotoxic T Lymphocytes Epitopes in E7 Antigen of Human Papillomavirus Type 11One 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.Cytotoxie 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-γsecretion by ELISA.The ability of the epitope-specific CTLs to kill the target cells was also analyzed.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 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 to the development of immunotherapeutic approaches for low-risk HPV infectious diseases such as CA.1.1 Epitope selectionEpitopes were selected and analysed by a computer-assisted algorithm. Consecutive overlapping 9-amino-acid peptides that possessed the specific primary and secondary anchoring amino acids for HLA-A2 were identified. Based on the presence of favorable and unfavorable amino acids,the peptides were scored as indeterminate,low,medium,or high binding probability to HLA-A2 molecules.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 favorable amino acid combinations.These HLA-A^*0201-restricted HPV-11E7 peptides were selected for the further immunogenicity studies.1.2 Tetrameric peptide-MHC classâ… complexes and synthetic peptideTetramers of HPV-11E7,including the following HLA-A^*0201-binding peptides:TLKDIVLDL,LQPPDPVGL,PLTQI-IYQIL,DLLLGTLNI and LLLGTLNIV,were synthesized by Sanquin.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-COLOR conjugate anti-CD3 and FITC-labeled anti-CD8.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 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 hours showed high expression of HLA-DR,CD83 and CD86 on DCs with no significant difference between each group.To determine the IL-12 secretion of peptides pulsed DCs,supernatants were obtained at 48h from cultures.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).1.3 Cell isolationPBMC were isolated from HLA^*0201 healthy donors(young women with no sexual experience)by Lympholyte-H density gradient centrifugation as recommended by the manufacturer.T cells for one experimental setting were isolated from the same donor.1.4 Generation of DCs and peptides pulsingCD14 cells were isolated from PBMC by positive selection using CD14 microbeads and their purity was determined by FACS with anti-CD14 Ab.DCs were generated by culturing monocytes in RPMI1640 medium containing 10% FCS,1%L-glutamine,1%streptomycin,1%penicillin,IL-4(50ng/ml)and GM-CSF(100 ng/ml)for 7 days in 6-well plates at a density of 2×10⁶ cells per well.Final maturation of monocyte-derived DCs was induced by exposure of cells to TNF-α(100ng/ml)and flt-3(50ng/ml)for 48 hours on day 5.On day 5,2×10⁶ differentiated DCs were incubated with each of the five HPV11E7 peptides(TLKDIVLDL,LQPPDPVGL,PLTQHYQIL,DLLLGTLNI or LLLGTLNIV)in 6-well plates(20μg/ml).After 48 hours of incubation,CD1a, CD86,CD83,HLA-DR expressions were analyzed by flow cytometry to determine the DCs maturation.IL-12 concentration in the supernatant was determined by ELISA.1.5 Mixed lymphocyte-DC culture and CD8⁺ responder T cellsDCs generated from PBMC of healthy HLA-A^*0201-positive donors were induced by GM-CSF,IL-4,TNF-αand flt-3 for maturation.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(20μg/ml)for 1h at 37℃.Autologous T lymphocytes were isolated from PBMC by negative selection with the Pan T Cell Isolation Kitâ…¡and mixed with the peptide-loaded mature DCs in a 24-well plate at a lymphocyte:DC ratio of 10:1.On day 7 and 14, additional DCs loaded with 20μg/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-γsecretion using ELISA,the tetramer staining,and a LDH release assay.Secretion of IFN-γincreased significantly after 3 weeks of incubation in the groups containing T cells cocultured with peptide-loaded DCs,indicating a strong activation and stimulation of T cells.However,only a small increase of IFN-γsecretion was found both in the nonloaded DCs alone,and the T cells cocultured with nonloaded DCs.Secretion of IFN-γincreased 5 to 10-fold in T cells cocultured with peptide-loaded DCs,especially with peptides E7 7-15 (TLKDIVLDL),15-23(LQPPDPVGL)and 82-90(LLLGTLNIV)(P＜0.05).T cells cocultured with E7 7-15 peptide-loaded DCs showed the highest level of IFN-γsecretion. 1.6 Tetramers staining and flow cytometryPeptide-loaded DCs and T cells were cocultured according to the above mentioned protocol.Seven days after the last round of restimulation,suspension cells(antigen-specific T cells)were harvested from the cultures.The antigen-specific T cells(1×10⁶)were resuspended in 50μl of PBS,and incubated with the tetramers(PE)for 20 min at room temperature.After a single wash,the TRI-COLOR conjugate and FITC-labeled antibodies directed against CD3 and CD8 were added for 15 min.The cells were then washed with PBS again and analyzed by flow cytometry.Less than 0.03%tetramer⁺ cells detected in circulating CD8⁺ T cells represented the maximum staining observed in the controls.Specific tetramer staining was observed in all cultures of autologous T cells stimulated with each of the five HPV-11E7 peptide-loaded DCs.On the average,a 100-fold increase in the frequency of specific tetramer⁺ CD8⁺ cells was observed when cocultured with the 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).1.7 CTL assayIn 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.With a selective medium containing G418 at a concentration of 500μg/ml,the G418-resistant positive cell clones stably expressing HPV-11E7(HPV-11E7/293)were isolated under the 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 hours of co-incubation at 37℃,the supernatant was collected to assess the lactate dehydrogenase concentration using CytoTox 96 nonradioactive cytotoxicity assay kits 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.After the 293 cells were transfected with pcDNA3.1/HPV11E7 plasmid,green fluorescence located in the cellular nucleus and plasma could be seen in positive clones.HPV-11E7 mRNA expression in selective cell line was also confirmed by RT-PCR.The results showed that the established cells positive for HPV-11E7 could further be used as a HPV-11E7-expressing cell line.Autologous T ceils were eocultured with HPV-11E7 peptide-loaded DCs as described above.Again seven days after the last round of restimulation,cells from each culture were collected and analyzed for the specific CTL responses.Induction of cytotoxicity in T cells in response to peptide-loaded DCs was investigated. When the T lymphocytes acted as effector cells adhering to HPV11E7-expressing 293 cells at a 50:1 E/T ratio,T cells cocultured with E7 7-15 peptide-loaded DCs had a 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. Section two:Foxp3⁺CD4⁺CD25⁺ regulatory T cells expression and Th1/Th2, Tc1/Tc2 profiles in peripheral blood of patients with condyloma acuminatumCondyloma acuminatum(CA)is a disease with proliferative lesions of genital epithelium caused by human papillomavirus(HPV)infection.The balance between type 1 and type 2 T-cell subsets in CA patients is thought to modulate antiviral immunity.CD4⁺CD25⁺ Regulatory T cells(Tregs)inhibit proliferation and cytokine production by both Th1 and Th2 cells and reversibly suppress CTL-mediated immunity.A better understanding of the mechanisms of T-cell regulation in CA might help in developing more effective therapeutic strategies.2.1 PatientsA total of 30 patients(12 males and 18 females)and 20 healthy controls(9 males and 11 females)included in this study were recruited from Sir Run Run Shaw Hospital,School of Medicine,Zhejiang University,Hangzhou,People's Republic of China from October 2006 to September 2007.The clinical diagnosis of CA was made according to the criteria of China National Center for STD.None of the patients had a history of chronic systemic diseases or disorders of immunity. All of them were HIV negative at the time of the study.Fifteen patients were first onset cases and another 15 were relapsed cases of CA.Disease course ranged from 1 month to 25 months with an average of 4.7 months.The mean age of the patients was 31±3.86 years(ranged from 19 to 55 years)and controls 30.2±2.72 years (ranged from 22 to 45 years).The study was approved by the ethics committees of Sir Run Run Shaw Hospital.Informed consents were obtained from all patients.2.2 Foxp3⁺CD4⁺CD25⁺ T cells expression in peripheral blood from CA patientsIn order to identify Tregs,4 ml heparinized peripheral blood from patients or controls were obtained.PBMC were isolated by Lympholyte-H density gradient centrifugation as recommended by the manufacturer.To analyze CD4,CD25 and Foxp3 expression,PBMC were stained with anti-human CD4-PE-Cy5 and anti-human CD25-FITC monoclonal antibodies,followed by intracellular staining with anti-Foxp3 using anti-human Foxp3-PE Staining Set. Isotype-matched antibodies were used as controls.Treg cells were identified as CD25-positive and Foxp3-positive cells among CD4 cells with triple-color flow cytometry analysis.The raw data were processed with Expo32 software.The results showed that the number of Foxp3-expressing cells within the CD4⁺CD25⁺ T and CD4⁺ T lymphocyte population of CA patients were higher than health controls.The frequency of Foxp3⁺CD4⁺CD25⁺ T cells within the total CD4⁺ population was significantly increased in the blood of total CA patients (3.37±1.03)%and relapsed patients(4.68±1.17)%compared to health controls (1.18±0.53)%(p＜0.001).The frequency of Foxp3⁺CD4⁺CD25⁺ T cells was also increased in the blood of first onset CA patients(2.06±1.01)%compared to health controls but the difference was not statistically significant(P＞0.05).The frequency of Foxp3⁺CD4⁺CD25⁺ T cells was significantly increased in the blood of relapsed CA patients compared to first onset patients(p＜0.001).2.3 Type 1/ Type 2 immune response in peripheral blood from CA Patients and the health controlsHeparinized peripheral whole blood(200ul)diluted with 1:1 RPMI 1640 medium were incubated with 2μmol/L Monensin,50 ng/mL PMA and 1μmol/L Ionomycin at 37℃in 5%CO₂ for 5 h and then stained with CD3-TC and CD8-FITC MoAb for 15 min at room temperature in dark.Following two washes with PBS,the cells were resuspended and fixed with 100μl Reagent A of the Fix & Perm kit for 15 min.The cells were washed again and suspended in 100μl Reagent B of the Fix & Perm kit and incubated with IFN-γ-PE or IL-4-PE at 4℃in dark for 15 min.The cell population was detected by triple-color flow cytometry and analyzed by Expo32 software.Percentages of Th1 in PBMC of CA patients,including relapsed patients and first onset patients were all significantly lower than normal control group(P＜0.01).Percentages of Tc1 in CA patients were lower than that in normal control group,especially in relapsed patients(P＜0.05).Although percentages of Th2 and Tc2 in CA patients were lower than normal control,the difference was not significant(P＞0.05).Meanwhile,Th1 /Th2 and Tc1/Tc2 ratios of the CA patients were lower than normal control group (P＜0.05),especially Th1/Th2 ratios in relapsed patients(P＜0.01).Th1/Th2 ratio of the first onset CA patients and Tc1/Tc2 ratio all CA patients were lower than normal control group but the difference was not statistically significant(P＞0.05). Percentages of Th1 and Th1/Th2 ratio in PBMC of the relapsed CA patients were significantly lower compared to the first onset group(P＜0.05).2.4 Correlation between the frequency of Foxp3⁺CD4⁺CD25⁺ T cells and Th1/Tc1 in the peripheral blood from CA patientsCorrelations between two variables were calculated by the Spearman rank correlation test used SPSS statistical software.P＜0.05 was considered statistically significant.The frequency of Foxp3⁺CD4⁺CD25⁺ T cells was inversely correlated with Th1(r=-0.798,p＜0.05),and Tc1(r=-0.746,P＜0.05) in CA patients. Conclusion:1.From the set of possible combinations of nanomer or decamer peptides in the HPV-11E7 proteins,five peptides including HPV11E7 7-15(TLKDIVLDL), 15-23(LQPPDPVGL),47-55(PLTQHYQIL),81-89(DLLLGTLNI)and 82-90 (LLLGTLNIV)were predicted as having a high binding ability to HLA-A2 molecules based on favorable amino acid combinations.2.We have successfully established an in vitro culture system for the derivation of dendritic cells.3.We have successfully established 293 cell lines expressing HPV11E7 by transfection with pcDNA3.1-HPV11E7 plasmid,to serve as target cell models in subsequent experiments.4.IL-12 concentration in DCs cultured with the selected peptides was higher than that of DCs cultured in medium alone.DCs cultured with E7 15-23 peptides was higher than in DCs cultured with E7 7-15 peptides,and IL-12 concentration in these two groups was remarkably higher than in DCs stimulated with the other three peptides and controls.5.Secretion of IFN-γwas higher in T cells cocultured with peptide-loaded DCs.T cells cocultured with E7 7-15 peptide-loaded DCs showed the highest level of IFN-γsecretion.6.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.T cells cocultured with E7 7-15 peptide-loaded DCs had a stronger cytotoxicity than others When T lymphocytes acted as effector cells adhering to HPV11E7-expressing 293 cells. 7.E7 7-15 is a HLA-A^*0201-restricted CTL epitope CTL of HPV type 11 E7.This was confirmed by measuring the IFN-γsecretion of T cells and the frequency of specific tetramer⁺ CD8⁺ T cells following stimulation of E7 7-15 peptide-loaded DCs.8.The frequency of Foxp3⁺CD4⁺CD25⁺ T cells within the total CD4⁺ population was significantly increased in the blood of total CA patients and relapsed patients compared to health controls.The frequency of Foxp3⁺CD4⁺CD25⁺ T cells was significantly increased in the blood of relapsed CA patients compared to first onset patients.The results may indicate that Tregs be involved in the mechanisms allowing CA to occur and the relative prevalence of Tregs may be a determinant for predicting the prognosis of CA patients.9.CA patients showed a decreased proportion of Th1 and Tc1 cells,and a decreased ratio of Th1/Th2 and Tc1/Tc2.Especially remarkable decreased ratios of Th1/Th2 was found in relapsed CA patients.10.The frequency of Foxp3⁺CD4⁺CD25⁺ T cells was inversely correlated with Th1 and Tc1 in CA patients.Tregs appear to downregulate cytokine expression in both Tc1 and Th1 subsets of effector T cells,which may be responsible for antivirus responses.