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In Vitro CD8+ T-Lymphocyte Responses of Healthy Women and Patients with Cervical Cancer to HPV-16 E7 Peptide Epitopes
Karen J. Gurski
Keerti V. Shah
John T. Schiller
Francesco M. Marincola
Michael A. Steller, MD
Section of Gynecologic Oncology, Surgery Branch, and Laboratory of Cellular Oncology National Cancer Institute
Johns Hopkins University
Key Words: HPV, peptides, CTL, vaccine, cervical cancer
Objective: Although insufficient immunologic responses have been implicated in the development of cervical neoplasia, little is known about potential difference in immune responses among healthy women and those with invasive cervical cancer. Since most women are eventually exposed to HPV, but only a small minority develops invasive cancer, we compared CD8+ T-lymphocyte (CTL) responses of normal women and patients with cervical cancer.
Methods: After screening for expression of the HLA-A*0201 allele, 6 healthy women with a recent normal Pap smear and 8 HPV-16+ cervical cancer patients underwent leukapheresis. To assess for previous or current HPV-16 infection in healthy subjects, an HPV-16 virus-like particle (VLP)–based ELISA was used to detect serum IgG antibodies against HPV-16 capsid proteins. Epitope-specific CTL were generated by in vitro stimulation using autologous dendritic cells co-incubated with the HPV-16, HLA-A*0201 restricted, synthetic peptides E711-20 or E786-93. The influenza matrix peptide M158-66 was used as a control to assess the specificity of the CTL and the ability to respond to in vitro stimulation. CTL specificity was measured by interferon-g release assay using HLA-A*0201 matched target cells.
Results: Anti–HPV-16 antibodies were not detected in the sera of any of the 6 healthy subjects. M158-66-specific CTL were induced in all subjects irrespective of disease status. E711-20-specific CTL were elicited in 3 of 6 (50%) healthy women and 0 of 7 (0%) cervical cancer patients (P < .05). E786-93-specific CTL were elicited in 2 of 5 (40%) healthy women and 3 of 8 (38%) cervical cancer patients (P < not significant).
Conclusions: That the frequency of CTL responses to E786-93 is similar for both healthy women and those with cervical cancer indicates that immunologic CTL precursors are variably present in both groups of subjects. This finding calls into question the validity of using CTL assays to assess in vivo immunologic responses to HPV antigens in vaccination protocols. Immunologic deficits other than insufficient CTL precursor frequencies in tumor-bearing patients, such as defects in T-cell signaling or differences in the mechanisms HPV-infected cells use to escape immunologic recognition, may have more important roles in the pathogenesis of HPV-associated cervical cancer.
Independent lines of evidence have emphasized the importance of immunologic responses in the pathogenesis of HPV-associated cervical neoplasia, 1,2 but the precise immunologic factors responsible for successful immunologic containment of genital HPV infections have not been fully elucidated. Several epidemiologic studies have consistently demonstrated a strong association between detection of HPV-16 DNA and the risk for cervical intraepithelial neoplasia and invasive cervical cancer. In cytologically normal women, HPV-16 also appears to be the type most commonly detected. 3 Although the natural history of HPV infection is incompletely understood, most infections in women appear to be transient and relatively harmless, 4 whereas a small subset are persistent and have the potential of inducing oncogenic progression in the cervical epithelium. 3 Recently, serologic assays have been developed using correctly assembled HPV-16 virion proteins as antigen. 5 Seroreactivity to these HPV-16 virus-like particles (VLPs) has been found to correlate with previous HPV-16 infection specifically, 5 and significantly higher seroreactivity has been found in women with persistent genital HPV-16 infection. 6 Since most sexually active women are eventually exposed to HPV, 7 but only a small minority develops high-grade dysplasia or invasive cancer, we tested the hypothesis that the ability to elicit CD8+ T-lymphocyte (CTL) responses in women with cervical cancer is different than that of healthy female counterparts. For this purpose, we compared CTL responses in healthy women with normal cervical cytology and patients with cervical cancer to two HLA-A*0201-restricted, immunodominant peptide epitopes of HPV-16 E7. We also analyzed the patients’ cervical tumors for evidence of HPV-16, while in healthy subjects, serum samples were inspected for evidence of existing or previous HPV-16 infection using an HPV-16 VLP-based assay.
MATERIALS AND METHODS
Selection of Patients and Controls
Healthy female controls were selected from a pool of normal subjects previously recruited into the Department of Transfusion Medicine donor program at the National Institutes of Health, Bethesda, Maryland. Healthy females were selected after completing a questionnaire querying for a history of abnormal cervical cytology specimens (Pap smears) and confirming having a normal cervical cytology specimen within the past 1 year. For patients with cervical cancer, members of the Surgical Pathology Section, National Cancer Institute, confirmed the diagnosis histologically in all subjects. Informed consent for blood donations was obtained from all subjects. Clinical details are summarized in Table 1.
HLA Typing and Subtyping
HLA class I type was established on PBMC as previously described, 8 and all subjects were HLA-A2 subtyped using a high-resolution nested sequence polymerase chain reaction (PCR) set to resolve the HLA-A*0201 through the HLA-A*0217 alleles.
HPV typing was performed on paraffin section samples from the patients' tumors using a PCR amplification protocol.
HPV Serologic Analysis
To assess for previous or current HPV-16 infection in healthy subjects, an HPV-16 VLP-based ELISA was used to detect serum IgG antibodies against HPV-16 capsid proteins, as previously described.9 Sera were analyzed in quadruplicate, and the optical densities (ODs) obtained for each assay were normalized relative to the mean OD obtained for a control serum tested multiple times on the same 96-well plate. The mean of the normalized ODs was used in the analysis. A preselected cut-off point equal to the reactivity of the control serum was used. This serum was previously found to have a reactivity equal to the cut-off point for seropositivity.6 Positive and negative control sera were also included in each assay.
The HPV-16 E786-93 (TLGIVCPI) and E711-20 (YMLDLQPETT) peptides and the control influenza matrix M158-66 peptide (GILGFVFTL) used for the in vitro analysis were synthesized by a solid-phase method and purified by high-pressure liquid chromatography (HPLC; >95% pure). The HPV-16 E7 peptides were selected for use in this analysis because they appear to be the immunodominant epitopes restricted by the HLA-A2 allele based on several independent investigations.8,10 All peptides were diluted from aliquots dissolved in 100% dimethyl sulfoxide and stored at -70oC.
Preparation of PBMC and Lymphocytes
We obtained 1 to 4 x 109 PBMC from all subjects by leukapheresis and separated them in Ficoll-Hypaque gradients (LSM®, Organon Teknika, Durham, NC). All PBMC preparations were frozen in human AB serum with 10% dimethyl sulfoxide (Sigma Chemical Co, St. Louis, MO) and stored in liquid nitrogen.
Preparation of Dendritic Cells
After Ficoll-Hypaque separation, 1 to 3 x 108 PBMC were processed for preparation of dendritic cells (DC) according to principles previously described.11 After physical separation of DC precursor cells, human recombinant GM-CSF (hrGM-CSF, 2000 IU/mL, Pepro Tech, Inc., Rocky Hill, NJ) and human recombinant IL-4 (hrIL-4, 2000 IU/mL), Pepro Tech, Inc.) were added every 2 to 3 days from day 0. DC were used for peptide presentation after at least 5 days of culturing following physical separation.
T2 Cell Line
T2 cells were used in cytokine release assays for the HLA-A*0201 peptide epitopes. The T2 cell line was selected as a target because it expresses only the HLA-A*0201 allele, which was the restriction element for the peptides used in this analysis.
Peptide-Pulsing of Dendritic Cells and T2 Cells
The recovered DC or T2 cells were pulsed with 20 mg/mL of E786-93 peptide, 20 mg/mL E711-20 peptide, or 1 mg/mL M158-66 peptide for 2 h in 15 mL conical tubes at 37oC at a concentration of 1 x 106 cells/mL.
In Vitro Sensitization of Peripheral Blood Lymphocytes with Dendritic Cells
CD8+ enrichment of T cells was achieved by positive selection on biomagnetic separation beads (Dynal Corp., New York, NY). In all experiments, the T-cell population was greater than 95% CD8+ and included <5% contamination with CD4+ cells by fluorescence-activated cell sorter analysis. CD8+ lymphocyte cells (4 to 5 x 106/well) were co-incubated with 1 x 106 peptide-pulsed (E786-93, E711-20, or M158-66) DC in 24-well plates and were restimulated after 1 week with 1 x 106 peptide-pulsed DC. IL-2 (300 IU/mL) was added 24 h after each stimulation and every 2 to 3 days thereafter. The effectors were tested for specificity 7 to 9 days after the restimulation.
Assessment of CTL Reactivity Using Cytokine Release Assay
Effector cells (1 x 105) were co-incubated with 1 x 105 stimulator cells for 24 h at 37oC in 200 mL of CM (5 x 105 effector cells/mL). Supernatants from these cocultures were tested for specific secretion of interferon-g (IFN-g) by human IFN-g Quantikine enzyme-linked immunosorbent assay kits (R & D Systems, Minneapolis, MN). Data are presented as picograms of IFN-g released by 5 x 105 effectors/24 h.
Specific release of IFN-g by a PBMC culture was arbitrarily defined as (1) twofold or higher difference in IFN-g production in response to relevant (T2 + E786-93 or T2 + M1 58-66) vs irrelevant (T2 alone) stimulation and (2) at least 100 pg/5 x 105 cells/24 hours production of IFN-g. Nonparametric, two-tailed Fisher’s exact test was used to compare the frequency of in vitro CTL induction between cultures from healthy donors and patients with cervical cancer. Production of IFN-g in response to M158-66 was also compared parametrically between the cervical cancer patients and the healthy female subjects using a two-tailed Student’s t test.
Patients and Controls
The clinical features of the normal donors and cervical cancer patients included in this study are summarized in Table 1. The age of the cervical cancer patients ranged form 31 to 67 years old, with a mean of 45 years of age; normal subjects ranged in age from 34 to 55 years, with a mean of 45 years of age. The histologic cell type was squamous cell carcinoma in all of the cervical cancer subjects. All of the normal donors had a normal Pap smear within the previous 1 year, and one patient described having had an abnormal Pap smear previously. All of subjects in this study expressed the HLA-A*0201.
Seroreactivity in an HPV-16 VLP ELISA
Because data on the genital HPV DNA status was not available for the healthy women, we evaluated their exposure to HPV-16 using a VLP ELISA that is a well-established and type-restricted measure of HPV-16 infection. None of the women were seropositive using a pre-assigned cut-point of 1.0 (Table 2). This cut-point was established in previous case-control studies using the same control serum.6,12
Induction of Peptide-Specific CTL
PBMC were stimulated in vitro and tested for peptide-specific CTL reactivity. CD8+ T cell reactivity was tested for IFN-g release in an HLA-A*0201-restricted assay by pulsing M158-66, E711-20, or E786-93 on T2 cells expressing HLA-A*0201 molecules (Table 3). This assay excludes non–HLA-A*0201-restricted secretion of IFN-g since the T2 cell line does not express any other HLA class I or class II alleles. Therefore, this assay is aimed at analyzing specifically HLA-A*0201-restricted secretion of IFN-g. In all subjects, CTL cultures raised against the M158-66 control peptide demonstrated specific cytokine release, indicating that the capacity to generate specific cellular immune responses is retained in both healthy women and patients with cervical cancer, even after extensive previous treatment. Anti–E786-93-specific cytokine release was noted in 2 of 5 healthy women and 3 of 8 patients with cervical cancer (P < not significant [NS]); E711-20-specific cytokine release was observed in 4 of 7 healthy women, but 0 of 7 patients with cervical cancer (P < .05).
In an effort to compare the magnitude of the CTL responses between the patients with cervical cancer and the healthy subjects, the absolute production of IFN-g among the E786-93 and the M158-66 responders were compared. For the 3 patients with cervical cancer demonstrating anti–E786-93-specific cytokine release, the mean secretion of IFN-g was 419 pg/mL/24 hours, compared with 450 pg/mL/24 hours for the two healthy subjects. For the 8 cervical cancer patients, the mean M158-66-specific response was 2086 pg/mL/24 hours, compared with 2357 pg/mL/24 hours for the 6 healthy subjects (P < NS).
We have analyzed the ability to elicit HPV-16 E7 epitope-specific reactivity in CD8+ T-lymphocyte cultures from peripheral blood samples of healthy women and those with cervical cancer using a cytokine release assay. With this assay, we were able to identify a significant proportion of both healthy subjects and those with cervical cancer with specific anti-E786-93 reactivity. Although the results using this assay correlate well with other standard measures of CTL function, such as chromium release cytotoxicity assays,13 our results are somewhat at variance with other published studies attempting to elucidate differences in cellular immune responses between healthy subjects and those with cervical cancer. For example, specific anti-M158-66 CTL responses were detected in all HLA-A*0201 cervical cancer patients tested using this assay, whereas a lack of such responses to the same peptide in a similar group of patients has been reported when chromium release assays were used.10 In addition, previous studies employing chromium release assays reported a lack of CTL reactivity against immunodominant E7 peptides or target cells expressing the E7 protein in blood samples from normal subjects,10,14-16 whereas specific anti-E786-93 CTL were generated in 40%, and anti-E711-20 CTL in 50% of our healthy subjects. Consistent with our findings, two recent studies have also described the successful generation specific anti-E786-93 CTL in healthy donors when dendritic cells were used during culture, but only after at least four total stimulations.17,18 Taken together, existing data indicates that specific anti-HPV CTL can be induced from the peripheral blood of both healthy subjects and those with cervical cancer.
The HPV-16 E7 directed CTL responses observed in 3 of the 8 cervical cancer subjects included in our study occurred exclusively against the E786-93 peptide, but not against the E711-20 peptide. This finding is also at variance with previous reports in which CTL responses using PBMC from cervical cancer patients were observed only against the E711-20 peptide.8,10,16 Among the many possible explanations, it is noteworthy that our CTL were induced differently than previous reports since we used autologous dendritic cells to present the peptides. Indeed, in one of our previous studies using a different culturing protocol, a specific CTL response to E711-20 was induced from the PBMC of one of the cancer subjects (patient 2).8 In the current study, we also used a more sensitive method of assessing CTL specificity (secretion of IFN-g into the CTL supernatant).13 Using these methods, we also found that the ability to mount HLA-restricted cellular immune responses appears to be retained in both cervical cancer patients and normal women since CTL cultures from all subjects recognized the M158-66 influenza matrix peptide. Therefore, existing data using various CTL assay techniques indicates that specific CTL directed against both HLA-A*0201-restricted HPV-16 peptides (E711-20 and E786-93) can be induced from PBMCs of HLA-A*0201 subjects, irrespective of disease status.
Although several different CTL culturing methods have been used to study differences in cellular immune responses between healthy subjects and those with cervical cancer, remarkably little attention has been focused on the gender of the healthy “controls.”10,14,17,18 Recent studies indicate that the natural history of genital HPV infection may be substantially different between men and women. For example, seroreactivity to HPV-16 VLPs is lower in high-risk men than in high-risk women,12 and detection of genital HPV infections in men appears to be more transient than that of women.19 In addition, anatomic differences between the male and female genitalia may also impact on the natural history of sexually transmitted HPV infection. For instance, the epithelial surface along the penis is predominantly cornified, whereas the female genital tract has primarily mucosal surfaces. Moreover, in studies of patients with genital warts, significantly lower seroreactivity to HPV-6 VLPs20 and to HPV-6 virions21 were found in men than in women. Therefore, immunologic responses among healthy women may differ from those of healthy men.
In the current study, we also considered current and previous exposure to genital HPV infection by using both a questionnaire and directly testing their sera for circulating anti-HPV-16 antibodies. Because the true genital HPV DNA status of the healthy women was unknown, unlike that of the cervical cancer patients, whose cancers were evaluated for HPV DNA, we evaluated these women for HPV-16 exposure using a type-restricted serologic assay based on the HPV-16 VLPs. In a previous study, over 80% of women with persistent genital tract HPV-16 DNA were seropositive.6 Therefore, it is unlikely that any of these women had a persistent HPV-16 infection. However, less than 20% of women with transient HPV-16 infections were seropositive in the previous study, so our results certainly do not preclude the possibility that the women had previously been infected with HPV-16. Transient infections could result from an effective cell-mediated immune response with the concomitant production of E7-specific memory T cells that could then be clonotypically expanded in our in vitro stimulation assays.
We have shown that HPV-specific CTL can be induced from PBMC of both patients with cervical cancer and healthy female subjects. The presence of this response in both groups suggests that specific anti-HPV CTL precursors are present irrespective of disease status or prior HPV-16 exposure. This finding calls into question the validity of using CTL assays to assess in vivo immunologic responses to HPV antigens in vaccination protocols. Immunologic deficits other than insufficient CTL precursor frequencies in tumor-bearing patients, such as defects in T-cell signaling or differences in the mechanisms HPV-infected cells use to escape immunologic recognition, may have more important roles in the pathogenesis of HPV-associated cervical cancer.
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Table 1. Clinical Characteristics of Cervical Cancer Patients and Healthy Female Subjects
*Fédération Internationale des Gynaecologistes et Obstetristes
†The genotype of both HLA alleles is presented, separated by a comma. When only one allele was typed, only one allele is presented.
Table 2: Seroreactivity to HPV-16 Virus-like Particles*
*Sera were analyzed in quadruplicate, and the optical densities (ODs) obtained for each assay were normalized relative to the mean OD obtained for a control serum tested multiple times on the same 96-well plate. Evidence of seropositivity was scored using a pre-assigned cut-point of 1.0.
Table 3. CD8+ T-lymphocyte Responses of Cervical Cancer Patients and Healthy Female Subjects*
*Effector cells (1 x 105) were co-incubated with 1 x 105 stimulator cells for 24 h, and supernatants from these cocultures were tested for specific secretion of interferon-g (IFN- g). Data are presented as picograms of IFN- g released by 5 x 105 effectors per 24 hours. Specific release of IFN- g by a PBMC culture was arbitrarily defined as (1) twofold or higher difference in IFN- g production in response to relevant (T2 + E711-20, T2 + E786-93, or T2 + M158-66) vs irrelevant (T2 alone) stimulation and (2) at least 100 pg/5 x 105 cells/24 hours production of IFN-g. Numbers in parenthesis represent nonspecific IFN- g production when CTL were reacted with irrelevant (T2 alone) target cells.
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