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HLA-DQB1*02–Restricted HPV-16 E7 Peptide–Specific CD4⁺ T-Cell Immune Responses Correlate with Regression of HPV-16–Associated High-Grade Squamous Intraepithelial Lesions

Authors' Affiliations: Departments of ¹ Pathology, ² Obstetrics and Gynecology, ³ Molecular Microbiology and Immunology, ⁴ Oncology, and ⁵ Biostatistics, Johns Hopkins Medical Institutions, Baltimore, Maryland

Requests for reprints: T.-C. Wu, Department of Pathology, The Johns Hopkins University School of Medicine, CRBII 309, 1550 Orleans Street, Baltimore, MD 21231. Phone: 410-614-3899; Fax: 443-287-4295; E-mail: wutc{at}jhmi.edu.

	Abstract

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Purpose: The fact that up to 30% of established high-grade squamous intraepithelial lesions (HSIL) of the cervix regress spontaneously presents the opportunity to identify clinically relevant human papillomavirus (HPV) viral epitopes associated with disease outcome. Two human HPV antigens, E6 and E7, are functionally required for initiation and maintenance of cervical cancer precursor lesions and invasive cervical cancer. The identification and characterization of endogenously processed HPV antigenic epitopes in closely characterized patient cohorts will provide insight into the reasons for success or failure of therapeutic approaches.

Experimental Design: We characterized the HPV-16 E6/E7–specific T-cell epitopes using E6/E7 overlapping peptide pools with peripheral blood lymphocytes obtained from normal healthy donors. We then analyzed the difference in the HPV-16 T-cell immune responses in HPV-16+ HSIL patients with or without spontaneous regression of lesions using the statistical methods.

Results: We have identified an HPV-16 E7–specific CD4⁺ T-cell epitope [amino acids (aa) 71-85] that was restricted by HLA-DQB1*0201. Analysis of peripheral blood lymphocytes obtained from 14 HLA-DQB1*02 patients with HPV-16⁺ HSILs showed that the HPV-16+ E7 peptide (aa 71-85)–specific CD4⁺ T-cell immune response was significantly higher in the group of patients with regression compared with the patients without regression (P value <0.05).

Conclusions: The HPV-16 E7 peptide–specific CD4⁺ T-cell immune response correlates with spontaneous regression of established HPV16+ HSILs. Thus, this E7 epitope may be useful for the characterization of HPV-specific immune responses in patients infected with HPV-16 or immunized with HPV vaccines.

Therapeutic HPV vaccines targeting E6 and E7 have become an appealing approach for generating antigen-specific immunotherapy. Various forms of HPV vaccines targeting E6 and/or E7 have been explored, including viral or bacterial vector–based vaccines, peptide/protein-based vaccines, dendritic cell–based vaccines, and nucleic acid–based vaccines (for reviews, see refs. 4 and 5). We have focused on DNA vaccines because of their simplicity, stability, safety, and capacity for repeated administration (for reviews, see refs. 6–10). Several clinical trials using clinical-grade DNA vaccine targeting HPV-16 E7 are currently ongoing.

It is important to characterize HPV antigen-specific immune responses, particularly T cell–mediated immune responses, to determine the efficacy of HPV vaccines received by patients and to identify immunologic parameters that are relevant to the clinical outcomes (11–15). The identification of MHC class I–restricted CD8⁺ T cell and MHC class II–restricted CD4⁺ T cell epitopes will facilitate the development of quantitative T-cell immunologic assays for the characterization of E6/E7-specific CD8⁺/CD4⁺ T-cell immune responses in patients receiving effective HPV vaccines. Examples of these quantitative T-cell immunologic assays include intracellular cytokine staining followed by flow cytometry analysis, Enzyme-linked immunospot (ELISPOT), and MHC class I and II tetramer staining. These assays will be important for the characterization of immune responses following HPV infection and vaccination, which will allow for more efficient clinical translation of DNA vaccines against HPV-associated lesions.

Here, we describe the identification of an HPV-16 E7–specific CD4⁺ Th1 epitope that is endogenously processed and presented by HLA-DQB1*0201–restricted MHC class II molecules. We tested responses to E6 and E7 using an IFN- ELISPOT assay, in which a matrix of overlapping peptide pools spanning the length of E6 and E7 was distributed such that each individual peptide was represented in a unique combination of only two wells. We first screened samples from anonymous healthy donors and identified an HPV-16 E7–specific CD4⁺ Th1 epitope restricted to HLA-DQB1*0201. We subsequently analyzed specimens obtained from patients with HPV16+ high-grade cervical dysplasia who participated in a brief observational cohort study before standard therapeutic resection. Patients included in this analysis had at least one HLA-DQB1*02 allele. In this patient cohort, at week 15 after study entry, we observed that the HPV-16+ E7 peptide [amino acids (aa) 71-85]–specific CD4⁺ T-cell immune response was significantly higher in the group of patients with regression compared with the patients without regression (P value <0.05). Regression was determined by histologic examination for the clearance of the CIN lesions and reduction of HPV16 viral load measured by quantitative PCR. Thus, HPV-16 E7 peptide (aa 71-85)–specific CD4⁺ T-cell immune responses correlated significantly with complete regression of disease in this cohort.

	Materials and Methods

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Study subjects, HLA typing, and human papillomavirus genotyping. Buffy coats of healthy donors were obtained from the Pennsylvania Plasma Co. (North Brunswick, NJ). In addition, peripheral blood mononuclear cells (PBMC) were collected from 14 HPV-16⁺ high-grade SIL patients who had HLA-DQB1*02 alleles. These patients were part of the patients enrolled in an observational study protocol (16). All of these patients received standard therapeutic surgical resection of histologically confirmed high-grade cervical lesions as described previously (16). PBMCs were separated by density centrifugation with Ficoll-PaqueTM PLUS (Amersham Pharmacia Biotech AB, Piscataway, NJ) and cryopreserved. The human leukocyte antigen (HLA) typing was done on PBMCs through the Johns Hopkins Immunogenetics Core Laboratory. High-resolution sequence-based typing was done for HLA-A locus 1 and 2, HLA-B locus 1 and 2, and Cw locus 1 and 2. Intermediate-resolution typing was done for HLA class II loci DR and DQB. The HLA genotypes of PBMCs from the donor described in the study from which the T-cell line was generated were HLA-A*0101, 0201, HLA-B*0801, 5101, HLA-Cw*0701, 0702, 0706, 0718, 0719, 0727, HLA-DRB1*0301, 1601, HLA-DQB1*0201, and DQB1*0502. HPV genotyping was carried out on cervical specimen transport medium (STM) specimens obtained at the time of colposcopic evaluation to confirm CIN2/3 at study entry. Genotyping was carried out by pPCR by the method of Gravitt et al. (17) in the Johns Hopkins Molecular Diagnostics Core Laboratory. All of the acquisition and procedures for the handling of PBMCs from patients were approved by the Institutional Review Board (Johns Hopkins University School of Medicine IRB 99-08-27-04).

Peptides. A total of 30 HPV-16 E6 and 18 HPV-16 E7 15-mer overlapping peptides (overlapped by 10 amino acids) spanning the full length of E6 and E7 protein were synthesized by Sigma-Genosys (The Woodlands, TX). The identities of the peptides were validated by mass spectrometric analysis, and the purity of the peptides was confirmed by high-performance liquid chromatography.

Cell lines and antibodies. NIH/3T3 transfected with human CD40L was kindly provided by Dr. Hyam Levitsky (The Johns Hopkins University, Baltimore, MD) and cultured in DMEM-F12 containing 2 mmol/L glutamine, 1 mmol/L sodium pyruvate, 20 mmol/L HEPES, 0.1 mmol/L non–essential amino acids, 100 µg/mL primocin, and 10% fetal bovine serum. EBV-transformed B-cell lines (B-LCL) that are partially matched with the donor were kindly provided by Dr. Elizabeth Jaffee (The Johns Hopkins University, Baltimore, MD) and maintained in RPMI 1640 supplemented with 2 mmol/L glutamine, 1 mmol/L sodium pyruvate, 20 mmol/L HEPES, 0.1 mmol/L non–essential amino acids, 50 IU/mL penicillin, 50 µg/mL streptomycin, and 10% FBS. Antibodies against human CD3 (clone UCHT1), CD4 (clone RPA-T4), CD19 (HIB19), IFN- (clone B27), tumor necrosis factor- (TNF-; clone MAB11), interleukin 2 (IL-2; clone MQ1-17H12), IL-4 (clone MP4-25D2), and IL-10 (clone JES3-19F1) were purchased from BD PharMingen (San Diego, CA). Antibodies against HLA-ABC (clone W6/32) were purchased from eBiosciences (San Diego, CA); antibodies against HLA-DR (clone G46-6) were from BD PharMingen; antibodies against HLA-DP (clone B7/21)were from Leinco Technologies, Inc. (St. Louis, MO); and antibodies against HLA-DQ (clone SPVL3) were from Immunotech (Marseille, France).

In vitro culture of CD40-activated B cells. CD40-activated B cells were generated from PBMCs as described previously (18). Briefly, whole PBMCs were cultured at 1 x 10⁶/mL with irradiated (96 Gy) CD40L-NIH/3T3 cells in Iscove's modified Dulbecco's medium supplemented with 2 mmol/L glutamine, 1 mmol/L sodium pyruvate, 20 mmol/L HEPES, 0.1 mmol/L non–essential amino acids, 100 µg/mL Primocin, 0.2% Insulin Transferrin Ethanolamine Selenium (ITES) (Bio Whittaker, Walkersville, MD), 10% human AB serum (Gemini Bio-Products, Woodland, CA), 2 ng/mL IL-4 (PeproTech Inc., Rocky Hill, NJ), and 0.625 µg/mL cyclosporin A (Sigma, St. Louis, MO). Seven days later, cultured cells were transferred into new irradiated CD40L-NIH/3T3 cells and every 3 days after.

In vitro stimulation of PBMCs with E7 overlapping peptides and IFN- ELISPOT assay. PBMCs from healthy volunteer donors were collected and resuspended at a concentration of 2 x 10⁶/mL in RPMI 1640 supplemented with 2 mmol/L glutamine, 1 mmol/L sodium pyruvate, 20 mmol/L HEPES, 0.1 mmol/L non essential amino acids, 50 IU/mL penicillin, 50 µg/mL streptomycin, and 10% human AB serum (R10-AB). PBMCs were plated into 24-well plate at the density of 2 x 10⁶ per well. Eighteen 15-mer HPV-16 E7 overlapping peptides were divided into three pools: pool 1, including E7.1-E7.6 (E7 aa 1-40); pool 2, including E7.7-E7.12 (E7 aa 31-70); and pool 3, including E7.13-E7.18 (E7 aa 61-98), and added into PBMCs culture at the concentration of 10 µg/mL and incubated at 37°C in the presence of 5% CO₂. As a positive control, PBMCs were stimulated with CEF peptide pool containing peptides from cytomegalovirus, EBV, and Influenza (obtained through NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH). On day 3, 1 mL of R10-AB medium containing 50 IU/mL recombinant human IL-2 (PeproTech) was added into each well. On days 5 and 7, half of the culture medium was replaced with fresh R10-AB containing 50 IU/mL recombinant human IL-2. On day 9, the cells were harvested, washed, and resuspended with R10-AB medium and plated into a 24-well plate overnight. An IFN- ELISPOT assay was then done using IFN- ELISPOT assay kit from Mabtech Inc. (Cincinnati, OH). Briefly, 5 x 10⁴ to 1 x 10⁵ in vitro stimulated PBMCs were seeded in wells of 96-well polyvinylidene fluoride plates (Millipore, Bedford, MA) coated with antihuman IFN- monoclonal antibody (clone C-D1K) at a concentration of 10 µg/mL. HPV-16 E6/E7 15-mer overlapping peptide matrix pools or CEF peptide pools were added into the cells at the concentration of 10 µg/mL and incubated at 37°C in the presence of 5% CO₂ for 20 h. As a positive control, phytohemagglutinin (Remel Inc., Lenexa, KS) was added at a concentration of 2 µg/mL. Wells without any peptides and without PBMC and peptides were used as negative controls. The captured IFN- was then detected with biotin-conjugated antihuman IFN- monoclonal antibody (clone 7-B6-1) and followed by incubation with horseradish peroxidase–conjugated streptavidin. The forming spots were developed by adding avidin-enzyme complex (Vector Laboratories, Burlingame, CA) and stopped by washing with tap water. The number of spots was analyzed on an ELISPOT Analyzer 3B (Cellular Technology Ltd., Cleveland, OH).

For the characterization of the HPV-16 E7 aa 71-85 peptide-specific CD4⁺ T cell immune responses in HPV-16–positive patients with high-grade CIN lesions, PBMCs from 14 HPV-16–positive patients from our CIN2/3 observational cohort who had lesions associated with HPV16 and at least one HLADQB1*02 allele were in vitro stimulated with HPV-16 E7 overlapping peptides. E7 aa 71-85 peptide-specific T-cell responses were determined using IFN- ELISPOT assay as described above.

Generation of HPV-16 E7 peptide-specific T cells. T cells were in vitro stimulated with peptide-pulsed irradiated autologous CD40-activated B cells in the presence of IL-2 every week. After four cycles of in vitro stimulation, the HPV-16 E7 peptide specificity of the T-cell lines was validated by IFN- intracellular staining analysis.

Intracellular cytokine staining. About 1 x 10⁵ of HPV-16 E7 peptide-specific T cells were incubated with or without E7 peptide (10 µg/mL) in R10-AB medium at 37°C for 20 h in the presence of 1 µL/mL of GolgiPlug (BD PharMingen). Alternatively, B-LCLs were pulsed with 30 µg/mL of E7 aa 71-85 peptide and incubated at 37°C for 3 h. After extensive washing, the peptide-pulsed B-LCLs were cocultured with E7 peptide-specific T cells at 1:1 ratio in the presence of GolgiPlug. E7 peptide-specific T cells cocultured with unpulsed B-LCLs were used as negative controls. As positive controls, we used HiCK-1 cytokine positive control cells (for IL-2) and HiCK-2 cytokine positive control cells (for IL-4 and IL-10). Cells were surface stained with either PE-conjugated anti-CD4 or antigen-presenting cell (APC)–conjugated anti-CD8 or both. The cells were then permeabilized and fixed with Cytofic/Cytoperm (BD PharMingen) and stained for intracellular cytokines with FITC-conjugated anti–IFN-, anti–IL-2, anti–IL-4, APC-conjugated anti–IL-10, and anti–TNF-. Flow cytometry analysis was done using FACSCalibur with CELLQuest software (BD Biosciences, Mountain View, CA).

Inhibition of T-cell responses with anti-HLA class I and class II antibodies. To determine the HLA restriction element presenting the E7 aa 71-85 peptide, antibody-blocking experiments were done. The murine monoclonal antibody W6/32 that blocks peptide presentation by HLA-ABC, B7/21 that blocks HLA-DP, SPVL3 that blocks HLA-DQ, G46.6 that blocks HLA-DR, and isotype controls were added to HPV16 E7 aa 71-85–specific T cells at 5 µg/mL 90 min before the addition of E7 aa 71-85 peptides. Intracellular IFN- production was analyzed by intracellular cytokine staining as described above.

Generation of CD14⁺ monocyte-derived dendritic cells. Immature dendritic cells were generated from PBMCs by isolating CD14⁺ monocytes with CD14 magnetic beads (Miltenyi Biotech Inc., Auburn, CA) and cultured in R10-AB medium containing 100 ng/mL recombinant human granulocyte macrophage colony-stimulating factor, 50 ng/mL recombinant human IL-4 (PeproTech).

Analysis of intracellular IFN- production by E7 peptide-specific T cells stimulated with HPV-16 E7 protein-loaded autologous dendritic cells. HPV-16 E7 protein was constructed as described previously (19). HPV-16 E7 protein or bovine serum albumin (BSA; Sigma) were incubated with 25 µg/mL polymyxin B (InvivoGen, San Diego, CA) for 45 min at 37°C before adding to dendritic cells. About 50 µg/mL polymyxin B–treated HPV-16 E7 protein or BSA was added into in vitro generated immature dendritic cells. A maturation cocktail containing 10 ng/mL TNF- and IL-1ß, IL-6 (Peprotech), and 1 µg/mL of prostaglandin E₂ (Sigma) was also added into the immature dendritic cells and incubated at 37°C for 24 h. After the wash, the mature dendritic cells were cocultured with HPV-16 E7 peptide-specific T cells in a ratio of 2:1 in the presence of 1 µL/mL of GolgiPlug (BD PharMingen) and incubated at 37°C for 20 h. Production of intracellular IFN- by E7 peptide-specific T cells was analyzed using intracellular cytokine staining followed by flow cytometry analysis.

Statistical methods. All data expressed as means ± SD are representative of at least two different experiments. Data for intracellular cytokine staining with flow cytometry analysis were evaluated by ANOVA. Comparisons between individual data points were made using a Student's t test. The outcome of CIN lesions at resection was taken as a dichotomous variable, and its univariate association with T-cell immune responses against HPV-16 E7 (aa 71-85) peptide was assessed by Mann-Whitney U test using Statview 5.0 (Mac Version). A P value <0.05 is considered to be significant.

	Results

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HPV-16 E7 peptide aa 71-85 represents an HPV-16 E7-specific CD4⁺ Th epitope. To identify HPV-16 E6 and/or E7-specific CD4⁺ and/or CD8⁺ T-cell epitopes, we generated 15-mer peptides overlapping by 10 amino acids, spanning the full length of E6 (see Table 1 ) and E7 (see Table 2 ). We tested responses to peptide pools, which were configured so that any one peptide is represented in a unique combination of only two wells (Table 3 ).

View larger version (31K): [in this window] [in a new window]   Fig. 1. IFN- ELISPOT analysis to characterize HPV-16 E7 peptide-specific T-cell immune responses using PBMC from a healthy donor. PBMCs from a healthy donor were in vitro stimulated with HPV-16 E7 15-mer overlapping peptide pools at the concentrations of 10 µg/mL. Nine days later, the cells were harvested. IFN- ELISPOT analysis was done with HPV-16 E6/E7 15-mer overlapping peptide matrix pools using the procedures described in Materials and Methods. A, IFN- ELISPOT results for PBMCs stimulated with E6/E7 matrix peptide pools (50,000 cells per well). B, number of SFCs per 50,000 cells per well. Columns, mean; bars, SD.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Intracellular IFN- staining with flow cytometry analysis to determine the T-cell subset and HPV-16 E7 peptide sequence that generate E7 peptide-specific immune responses from a healthy donor. E7 peptide pool 3–stimulated PBMCs were stimulated with E6/E7 overlapping peptide matrix pools that generated E7 peptide-specific T-cell response (pools 5 and 11, see Fig. 1), peptide E7.15 (aa 71-85) and adjacent overlapping peptides aa 66-80 and aa 76-90. Cells were stained for intracellular IFN-, CD4+, and/or CD8+ T cells. A, representative figure of the flow cytometry data determining the T-cell subset activated by the E6/E7 overlapping peptide matrix pools. B, representative figure of the flow cytometry data determining the HPV-16 E7 sequence that generates E7-specific CD4+ T cell response.

HPV-16 E7 aa 71-85–specific CD4⁺ Th cells show a Th1 phenotype. To determine whether the enriched HPV-16 E7 (aa 71-85)–specific CD4⁺ Th cells had a Th1 or Th2 phenotype, we did intracellular cytokine staining followed by flow cytometry analysis. As shown in Fig. 3 , when the HPV-16 E7 (aa 71-85) peptide-specific CD4⁺ T cells were stimulated with E7.15 (aa 71-85), they secreted significant amounts of IFN- and TNF- and lesser amounts of IL-2. In contrast, no IL-4 and/or IL-10 secretion was detected, indicating that the E7 aa 71-85–specific CD4⁺ T cells had a Th1 phenotype. We used HiCK-1 cells (for IL-2) and HiCK-2 cells (for IL-4 and IL-10) as positive controls (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 3. Intracellular IFN- staining with flow cytometry analysis to determine the cytokine profiles of HPV-16 E7 (aa 71-85) peptide-specific CD4+ T cells from a healthy donor. About 1 x 105 HPV-16 E7 (aa 71-85) peptide-specific CD4+ T cell lines were stimulated with either E7 aa 71-85 peptide or no peptide in the presence of 1 µL/mL GolgiPlug. Cells were stained for intracellular IFN-, TNF-, IL-2, IL-4, and IL-10. We used HiCK-1 cells (for IL-2) and HiCK-2 cells (for IL-4 and IL-10) as positive controls for our assays (data not shown).

The HLA-DQ allele involved in presentation was determined using partial mismatch B-LCLs as APC. B-LCLs were pulsed with the E7 (aa 71-85) peptide and then used to stimulate E7 (aa 71-85)–specific T cells. IFN- production by T cells was then measured by intracellular cytokine staining. As shown in Fig. 4 , IFN- secretion was detected in T cells stimulated with peptide-pulsed B-LCLs from individuals expressing at least one DQB1*02 allele. In contrast, T cells stimulated with peptide-pulsed B-LCLs from individuals without the DQB1*02 allele (either DQB1*05,06 or DQB1*05) did not secrete IFN-, demonstrating that the HLA-DQ allele presenting E7 (aa 71-85) was HLA-DQB1*02(0201).

View larger version (22K): [in this window] [in a new window]   Fig. 4. Intracellular IFN- staining with flow cytometry analysis to determine the HLA-DQ allele responsible for presenting HPV-16 E7 (aa 71-85) peptide to CD4+ T cells. B-LCLs from individuals who have at least one HLA-DQ allele that matches that of the donor [HLA-DQB1*02 (0201) or HLA-DQB1*05(050)] were pulsed with the E7 aa 71-85 peptide and then used to stimulate E7 (aa 71-85) peptide-specific T cells. IFN- production by T cells was then analyzed by intracellular cytokine staining. A, representative figure of the flow cytometry data demonstrating the T cell IFN- production after stimulation with the peptide-pulsed B-LCLs. B, percentage of IFN-+ CD4+ T cells per total CD4+ T cells; columns, mean; bars, SD.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Intracellular IFN- staining with flow cytometry analysis to determine if HPV-16 E7 (aa 71-85) peptide represents an endogenously processed epitope. About 50 µg/mL of HPV-16 E7 protein or BSA treated with 25 µg/mL polymyxin B was loaded into autologous immature dendritic cells. The immature dendritic cells were treated with maturation cytokine cocktail and used to stimulate HPV-16 E7 (aa 71-85) peptide-specific CD4+ T cells. Cells were stained for intracellular IFN- and CD4+ T cells. A, representative figure of the flow cytometry data demonstrating the IFN- production by E7 peptide-specific T cells stimulated with HPV-16 E7 protein or BSA loaded into autologous dendritic cells. B, percentage of IFN- + CD4+ T cells per total CD4+ T cells. Columns, means; bars, SD.

View larger version (12K): [in this window] [in a new window]   Fig. 6. IFN- ELISPOT analysis to characterize E7 (aa 71-85) peptide-specific T cell responses in patients with HPV-16–associated HSIL. PBMCs from 14 HPV-16+ high-grade SIL patients with HLA-DQB1*02 were stimulated with HPV-16 E7 overlapping peptides. E7 aa 71-85 peptide-specific T cell responses were detected using IFN- ELISPOT assay as described in Materials and Methods. *, patients with lesion regression. A significant difference in the ELISPOT data between the groups of patients with or without regression was observed (P < 0.05).

	Discussion

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The HPV-16 E7 aa 71-85 CD4⁺ Th1 epitope represents a newly identified CD4⁺ Th epitope. Previous studies have identified other E7-specific CD4⁺ Th epitopes using different strategies. For instance, de Gruijl et al. (20) used IL-2 production after E7 peptide stimulation to detect CD4⁺ Th cell activity in patients with HPV-16⁺ CIN and cervical carcinoma. They found that majority of the women responded to an immunogenic region in the COOH terminus of the E7 protein (E7 aa 67-98), although women with cervical carcinoma showed a significantly reduced rate compared with the patients with CIN lesions. In another study, using PBMC cultures from HLA-typed healthy donors, Van der Burg et al. identified the central part of HPV-16 E7 (E7 aa 41-72) as a major immunogenic region and mapped three distinct Th epitopes within this region (E7 aa 50-62 restricted by HLA-DR15, E7 aa 43-77 restricted by HLA-DR3, and E7 aa 35-50 restricted by HLA-DQ2). Th1 immunity against HPV16 E7 has been detected in two of three patients diagnosed with CIN 3 and three of eight patients with cervical carcinoma (21). Warrino et al. used a predictive computer algorithm to select pan-HLA-DR candidate peptide and used a short-term in vitro T-cell sensitization assay to identify three E7-derived HLA-DR–restricted epitopes (E7 aa 1-12, E7 aa 48-62, and E7 aa 62-75). These authors found that normal donor CD4⁺ T cells failed to react against these E7 peptides, whereas patients with CIN I to III lesions displayed preferential Th1-type responses against all epitopes. Cervical cancer patients in this study showed Th1-type responses only to the E7 aa 48-62, whereas Th2-type responses to the E7 aa 1-12 and E7 aa 62-75 mainly Th2-type responses (22). In addition, Kadish et al. identified a specific HPV16 E7 peptide (aa 37-54) for which T-cell reactivity was found to correlate with regression of CIN and loss of HPV infection (23). Thus, the epitope we described in correlation with resolution of HPV-16–associated CIN 2/3 lesions has not been reported in previous studies.

In our cohort, we found that HPV16 E7 (aa 71-85)–specific T cell responses correlated significantly with the regression of HPV16-associated high-grade dysplastic cervical lesions. However, the number of subjects used in our study is limited. Thus, it is important to further characterize the E7 peptide-specific CD4⁺ T-cell immune response using a larger number of subjects in our future studies. The identification of this E7 peptide epitope will allow us to determine whether effector cells recognizing this peptide in lesions that persist and their functional phenotype. It will be of interest to characterize the HPV-16 E7 (aa 71-85)–specific CD4⁺ Th immune responses in a large population of HPV-16–positive patients with the same HLA-DQ allele as well as in patients receiving vaccines encoding HPV-16 E7 protein. This information would allow us to correlate the E7-specific CD4⁺ T cell immune responses with the natural history of HPV-associated lesions (persistent versus resolving disease). Furthermore, the antigen-specific T-cell immune responses can be correlated with vaccine effects and provides important information for the development of innovative HPV vaccines for the control of HPV-associated diseases.

	Acknowledgments

 
We gratefully acknowledge Dr. Robert Kurman for helpful discussions. We would also like to thank Ralph Hruban and David Boyd for critical review of this paper, and Archana Monie and Roanne Calizo for the preparation of the manuscript.

	Footnotes

 
Grant support: Flight Attendant Medical Research Institute Young Clinical Scientist Award and cervical cancer grants from the Specialized Programs of Research Excellence Program of the National Cancer Institute.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

Received 12/11/06; revised 1/24/07; accepted 2/ 1/07.

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