In Vitro CD8⁺ T-Lymphocyte Responses of Healthy Women and Patients with Cervical Cancer to HPV-16 E7 Peptide Epitopes

 

Karen J. Gurski

Masaru Murakami

Keerti V. Shah

Bryce Chackerian

John T. Schiller

Francesco M. Marincola

Michael A. Steller, MD

 

Section of Gynecologic Oncology, Surgery Branch, and Laboratory of Cellular Oncology National Cancer Institute

Bethesda, MD

and

Johns Hopkins University

Baltimore, MD

 

 

Key Words: HPV, peptides, CTL, vaccine, cervical cancer

 

ABSTRACT

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 E7_11-20 or E7_86-93. The influenza matrix peptide M1_58-66 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. M1_58-66-specific CTL were induced in all subjects irrespective of disease status. E7_11-20-specific CTL were elicited in 3 of 6 (50%) healthy women and 0 of 7 (0%) cervical cancer patients (P < .05). E7_86-93-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 E7_86-93 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.

 

INTRODUCTION

            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

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.

 

Peptide

The HPV-16 E7_86-93 (TLGIVCPI) and E7_11-20 (YMLDLQPETT) peptides and the control influenza matrix M1_58-66 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 -70^oC.

 

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 E7_86-93 peptide, 20 mg/mL E7_11-20 peptide, or 1 mg/mL M1_58-66 peptide for 2 h in 15 mL conical tubes at 37^oC 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 (E7_86-93, E7_11-20, or M1_58-66) 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 37^oC 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.

 

Statistical Analysis

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 + E7_86-93 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 M1_58-66 was also compared parametrically between the cervical cancer patients and the healthy female subjects using a two-tailed Student’s t test.

 

RESULTS

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 M1_58-66, E7_11-20, or E7_86-93 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 M1_58-66 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–E7_86-93-specific cytokine release was noted in 2 of 5 healthy women and 3 of 8 patients with cervical cancer (P < not significant [NS]); E7_11-20-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 E7_86-93 and the M1_58-66 responders were compared. For the 3 patients with cervical cancer demonstrating anti–E7_86-93-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 M1_58-66-specific response was 2086 pg/mL/24 hours, compared with 2357 pg/mL/24 hours for the 6 healthy subjects (P < NS).

 

DISCUSSION

            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-E7_86-93 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-M1_58-66 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-E7_86-93 CTL were generated in 40%, and anti-E7_11-20 CTL in 50% of our healthy subjects. Consistent with our findings, two recent studies have also described the successful generation specific anti-E7_86-93 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 E7_86-93 peptide, but not against the E7_11-20 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 E7_11-20 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 E7_11-20 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 M1_58-66 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 (E7_11-20 and E7_86-93) 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.

 

CONCLUSION

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.

 

 

REFERENCES

 

   1.   Shamanin V, Glover M, Rausch C, et al: Specific types of human papillomavirus found in benign proliferations and carcinomas of the skin in immunosuppressed patients. Cancer Res 54:4610-4613, 1994.

   2.   Nasiell K, Roger V, Nasiell M: Behavior of mild cervical dysplasia during long-term follow-up. Obstet Gynecol 67:665-669, 1986.

   3.   Schiffman MH: Recent progress in defining the epidemiology of human papillomavirus infection and cervical neoplasia. J Natl Cancer Inst 84:394-398, 1992.

   4.   Hinchliffe SA, van Velzen D, Korporaal H, et al: Transience of cervical HPV infection in sexually active, young women with normal cervicovaginal cytology. Br J Cancer 72:943-945, 1995.

   5.    Kirnbauer R, Hubbert NL, Wheeler CM, et al: A virus-like particle enzyme-linked immunosorbent assay detects serum antibodies in a majority of women infected with human papillomavirus type 16 [see comments]. J Natl Cancer Inst 86:494-499, 1994.

   6.   Wideroff L, Schiffman MH, Nonnenmacher B, et al: Evaluation of seroreactivity to human papillomavirus type 16 virus-like particles in an incident case-control study of cervical neoplasia [see comments]. J Infect Dis 172:1425-1430, 1995.

   7.   Syrjanen K, Syrjanen S: Epidemiology of human papilloma virus infections and genital neoplasia. Scand J Infect Dis 69(Suppl):7-17, 1990.

   8.    Alexander M, Salgaller ML, Celis E, et al: Generation of tumor specific cytolytic T-lymphocytes from peripheral blood of cervical cancer patients by in vitro stimulation with a synthetic HPV-16 E7 epitope. Am J Obstet Gynecol 175:1586-1593, 1996.

   9.    Nonnenmacher B, Hubbert NL, Kirnbauer R, et al: Serologic response to human papillomavirus type 16 (HPV-16) virus-like particles in HPV-16 DNA-positive invasive cervical cancer and cervical intraepithelial neoplasia grade III patients and controls from Colombia and Spain. J Infect Dis 172:19-24, 1995.

10.   Ressing ME, van Driel WJ, Celis E, et al: Occasional memory cytotoxic T-cell responses of patients with human papillomavirus type 16-positive cervical lesions against a human leukocyte antigen-A *0201-restricted E7-encoded epitope. Cancer Res 56:582-588, 1996.

11.   Sallusto F, Lanzavecchia A: Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med 179:1109-1118, 1994.

12.   Svare EI, Kjaer SK, Nonnenmacher B, et al: Seroreactivity to human papillomavirus type 16 virus-like particles is lower in high-risk men than in high-risk women. J Infect Dis 176:876-883, 1997.

13.   Cormier JN, Salgaller ML, Prevette T, et al: Enhancement of cellular immunity in melanoma patients immunized with a peptide from MART-1/Melan A [see comments]. Cancer J Sci Am 3:37-44, 1997.

14.   Evans C, Bauer S, Grubert T, et al: HLA-A2-restricted peripheral blood cytolytic T lymphocyte response to HPV type 16 proteins E6 and E7 from patients with neoplastic cervical lesions. Cancer Immunol Immunother 42:151-160, 1996.

15.   Nimako M, Fiander AN, Wilkinson GW, et al: Human papillomavirus-specific cytotoxic T lymphocytes in patients with cervical intraepithelial neoplasia grade III. Cancer Res 57:4855-4861, 1997.

16.   Evans EM, Man S, Evans AS, Borysiewicz LK: Infiltration of cervical cancer tissue with human papillomavirus-specific cytotoxic T-lymphocytes. Cancer Res 57:2943-2950, 1997.

17.   Jochmus I, Osen W, Altmann A, et al: Specificity of human cytotoxic T lymphocytes induced by a human papillomavirus type 16 E7-derived peptide. J Gen Virol 78:1689-1695, 1997.

18.   Schoell WM, Mirhashemi R, Liu B, et al: Generation of tumor-specific cytotoxic T lymphocytes by stimulation with HPV type 16 E7 peptide-pulsed dendritic cells: An approach to immunotherapy of cervical cancer [In Process Citation]. Gynecol Oncol 74:448-455, 1999.

19.   Bosch FX, Castellsague X, Munoz N, et al: Male sexual behavior and human papillomavirus DNA: Key risk factors for cervical cancer in Spain. J Natl Cancer Inst 88:1060-1067, 1996.

20.   Greer CE, Wheeler CM, Ladner MB, et al: Human papillomavirus (HPV) type distribution and serological response to HPV type 6 virus-like particles in patients with genital warts. J Clin Microbiol 33:2058-2063, 1995.

21.   Carter JJ, Wipf GC, Hagensee ME, et al: Use of human papillomavirus type 6 capsids to detect antibodies in people with genital warts. J Infect Dis 172:11-18, 1995.

 

Table 1. Clinical Characteristics of Cervical Cancer Patients and Healthy Female Subjects

Cancer Patient	Age at Diagnosis	FIGO Stage*	Histologic Type	HLA A Genotype†	HLA B Genotype†	HLA C Genotype†
1	67	IB1	Squamous Cell	0201,0201	N/A	N/A
2	31	IIB	Squamous Cell	0201,01	N/A	N/A
3	57	IB2	Squamous Cell	0201,24	07,08	07
4	60	IIB	Squamous Cell	0201,01	08,60	N/A
5	31	IIIB	Squamous Cell	0201,01	08,51	02,07
6	38	IB1	Squamous Cell	0201,11	07,44	07
7	43	IVA	Squamous Cell	0201,03	07,18	N/A
8	34	IIIA	Squamous Cell	0201,03	44,55	N/A
Healthy Female Subject	Age	H/O Abnormal PAP	Recent PAP	HLA A Genotype†	HLA B Genotype†	HLA C Genotype†
1	49	Normal	Normal	0201,24	08,44	07
2	53	Normal	Normal	0201,01	44,55	03
3	55	Normal	Normal	0201,11	35,62	03,04
4	43	Normal	Normal	0201,3402	44,35	0501,0602
5	34	Normal	Normal	0201,0205	13,44	03,07
6	33	h/o Abnormal	Normal	0201,0201	35,44	04

*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*

Cervical Cancer Subject	Tumor HPV Type		T2 + E711-20	Result E711-20		T2 + E786-93	Result  E786-93		T2 +  M158-66	Result M158-66
1	16		0	-		884 (0)	+		1804 (0)	+
2	16		0	-		34 (26)	-		648 (0)	+
3	16		12 (0)	-		0	-		748 (0)	+
4	16		26 (62)	-		0	-		2160 (126)	+
5	16		0	-		194 (2)	+		3924 (0)	+
6	16		0	-		32 (0)	-		2238 (0)	+
7	16			ND		0	-		2304 (108)	+
8	16		58 (90)	-		180 (16)	+		2862 (120)	+
TOTALS				0/7			3/8			8/8
Healthy Female Subject	Anti-HPV anitbodies		T2 + E711-20	Result E711-20		T2 + E786-93	Result  E786-93		T2 + M158-66	Result M158-66
1	neg		0	-		34 (184)	-		4428 (8)	+
2	neg		0	-		82 (78)	-		3330 (52)	+
3	neg		288 (144)	+			ND		718 (0)	+
4	neg		150 (20)	+		0	-		2156 (0)	+
5	neg		744 (0)	+		680 (0)	+		1942 (0)	+
6	neg		0	-		220 (0)	+		1566 (68)	+
TOTALS				3/6			2/5			6/6

*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 + E7_11-20, T2 + E7_86-93, 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. Numbers in parenthesis represent nonspecific IFN- g production when CTL were reacted with irrelevant (T2 alone) target cells.