Departments of Pathology1 and Obstetrics and Gynaecology2, Free University Hospital, PO Box 7057, 1007 MB Amsterdam, The Netherlands
Department of Immunology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK3
Department of Biology, YCR Cancer Research Unit, University of York, York, UK4
Author for correspondence: Jan Walboomers.Fax +31 20 4442964. e-mail jmm.walboomers{at}azvu.nl
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Abstract |
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Introduction |
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A higher incidence of HPV-associated disease in immunocompromised individuals suggests that the host immune response plays a role in controlling HPV infection and its associated lesions (Laga et al., 1992 ; Schafer et al., 1991
). Cellular immune responses against E6 and mainly E7 have been studied extensively (reviewed in Man, 1998
). While E6 and E7 are attractive targets to study in high grade dysplasia and invasive carcinoma, immune responses directed against E2 might be expected during the early stages of cervical dysplasia. E2-specific immune responses potentially play a role early on because E2 is necessary for virus replication and is therefore expected to be expressed in productive lesions, furthermore expression is expected to be reduced in more advanced lesions when virus integration may have occurred.
Serum immunoglobulin A (IgA) and IgG responses to either an E2-derived peptide or an Escherichia coli-derived fusion protein were prevalent in CIN (Reeves et al., 1990 ) and cervical carcinoma patients (Dillner et al., 1994
); however, this was not a general observation (Mann et al., 1990
; Dillner et al., 1995
). IgA responses to the native protein, produced in a baculovirus system, were associated with low grade CIN, while response rates went down in CIN III and cervical carcinoma patients (Rocha-Zavaleta et al., 1997
). Recently, it was shown that primary cytotoxic T-lymphocyte responses can be induced in healthy donors against an HLA-A2-binding peptide epitope derived from the E2 protein (Kónya et al., 1997
). However, to date nothing is known about the natural T-cell response against E2 in HPV-infected individuals. We have studied T-helper (Th) cell responses, by measuring interleukin-2 (IL-2) release, against the C-terminal and N-terminal parts of the HPV-16 E2 protein in patients participating in a non-intervention cohort study of women with abnormal cytology. In a cross-sectional analysis at the end of follow-up (FU), E2-specific IL-2 release was correlated to virus infection patterns and disease outcome.
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Methods |
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HPV genotyping.
Cervical scrapes were evaluated for the presence of HPV DNA using the general primer (Gp5+/Gp6+)-based PCR method as previously described (de Roda Husman et al., 1995 ). Positive samples were subsequently subjected to a type-specific PCR to identify 27 mucosotropic HPV types including HPV-16 (de Roda Husman et al., 1994
).
Isolation of PBMC.
PBMC were isolated from 40 ml of heparinized blood by density centrifugation with Lymphoprep (Nycomed Pharma) and cryopreserved in RPMI 1640 medium supplemented with 0·01 M HEPES buffer, 50 U/ml penicillinstreptomycin and 1·6 mM l-glutamine, containing 10% DMSO and 10% FCS.
E2 proteins.
The N-terminal and C-terminal domains of the HPV-16R E2 protein were cloned in the pET15b plasmid vector, which encodes a 20 amino acid histidine tag as an in-frame N-terminal fusion, and were expressed in E. coli. The stretch of six histidine residues allowed purification by nickel binding. Purification by nickel binding was followed by anion exchange (N-terminal domain) or heparin binding (C-terminal domain). The proteins were at least 99% pure according to the following criteria: the proteins generated a single band on SDSPAGE, a single peak on BioLogic trace and a single band on Western blot using a rabbit polyclonal antibody. Proteins were dissolved in a 20 mM HEPES buffer containing 100 mM NaCl, 20% glycerol, 0·2 mM EDTA and 5 mM DTT (pH 7·9 at 4 °C). The N-terminal protein was a 29·3 kDa protein containing a large fragment of the hinge region (bp 27563517); the C-terminal protein was a 12·5 kDa protein containing a small fragment of the hinge region (bp 35843853) (Sanders et al., 1995 ).
T-cell stimulation.
PBMC were seeded in round-bottomed 96-well culture plates (Nunclon Delta) at 2x105 per well in Iscove's modified Dulbecco's medium containing 10% human pooled serum (CLB), 50 U/ml penicillinstreptomycin, 1·6 mM l-glutamine and 0·01 mM ß-mercaptoethanol. Cells were stimulated by the addition of the C-terminal or N-terminal protein or phytohaemagglutinin (PHA) as a positive control (Murex) and cultured for 7 days at 37 °C in an incubator with a 5% CO2 humidified atmosphere. The anti-CD25 monoclonal antibody TB30 (a kind gift from R. van Lier, CLB) was added to all wells to prevent IL-2 consumption (hybridoma supernatant at a final dilution of 1:25). Optimal stimulatory concentrations for both E2 protein fragments were determined in titration experiments. Both proteins were added to the cultures at a final concentration of 4 µg/ml. PHA was used at a concentration of 30 µg/ml. PBMC cultured in medium containing the same amount of buffer as the protein supplemented conditions served as a negative control. All culture conditions were carried out in triplicate wells. After 7 days, the culture supernatants were harvested, pooled per test condition and stored at -20 °C until further use.
IL-2 bioassay.
IL-2 production in the culture supernatant was measured in a bioassay with the IL-2-dependent cell line HT2. HT2 cells were cultured at 1x104 cells per well for 24 h in Iscove's modified Dulbecco's medium supplemented with 50 U/ml penicillinstreptomycin, 1·6 mM l-glutamine, 0·01 mM ß-mercaptoethanol and 10% FCS, with the culture supernatants at final dilutions of 1:2, 1:4 and 1:8. Triplicate wells were set up per test condition and per supernatant dilution. During the last 4 h, the cells were incubated with [3H]thymidine (0·4 µCi per well; Amersham). The cells were harvested onto fibreglass filters and [3H]thymidine incorporation was determined using a flatbed liquid scintillation counter (Wallac). IL-2 titration curves were included in each assay (100, 50, 25, 12·5, 6, 3, 1·5, 0·75, 0·375, 0 IU/ml IL-2; Cetus). Counts in the E2 test wells never exceeded the linear range of the titration curves (usually between 12·5 and 0·375 IU/ml). Samples were considered positive when the mean HT2 proliferation (in c.p.m.) in the test wells exceeded proliferation in the buffer control wells by a factor of two (SIHT2 2) for at least two of the tested culture supernatant dilutions.
Statistical analysis.
Frequencies of positive Th cell responders between the different groups were compared using 2x2 table analysis and Fisher's exact test (FE). Comparisons between sets of SIHT2 were carried out using the MannWhitney U-test (MWU). Differences were considered significant when P<0·05.
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Results |
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When the responses found in CIN patients with current or past HPV-16 infections (n=37) were related to the cytopathological and histopathological status at the end of FU no clear associations with resolved lesions or low grade lesions were observed (not shown).
HPV-16 E2-specific Th cell reactivity over time in CIN patients: longitudinal analysis
Of the 59 HPV-16-positive and HPV-negative patients, 29 were available for longitudinal analysis at two to six time-points per patient. The mean number of time-points tested per patient was comparable in the different patient groups, i.e. 3·3, 3·9, 3·8 in the HPV-16 persistence, HPV clearance and HPV-negative groups respectively. Th cell reactivity was measured in relation to HPV-16 infection and disease course in seven CIN patients who had no evidence of HPV infection (mean Th FU, 18·1 months), in 11 CIN patients with cleared HPV-16 infections (mean Th FU, 25 months) and in 11 CIN patients with persistent HPV-16 infections (mean Th FU, 19·7 months). Start of FU of the clearance patients ranged from 5 months before to 43 months after the last HPV-16-positive DNA test (median, 17 months after the last HPV-16-positive DNA test). Th cell responses against the N-terminal protein were detected during FU in 4/7 HPV-negative patients, in 5/11 HPV-16 clearance patients and in 7/11 HPV-16 persistence patients (Table 1). In nine cases these responses occurred at a single time-point, while in seven cases responses were detected at two consecutive time-points. It is important to note that exposure to HPV-16 prior to FU cannot be excluded in the HPV-negative group. In the HPV-negative group, responses were found at a mean of 22 months after start of FU, while in the clearance group responses were detected at a mean of 23·6 months after the last positive HPV-16 DNA test (P=0·73). In the HPV-negative group, no responses were detected against the C-terminal protein, while in 6/11 clearance patients and in 4/11 persistence patients Th cell responses could be detected during FU (Table 1
). Responses against the C-terminal protein were all detected at a single time-point, except in one clearance patient, where both time-points analysed tested positive. In the clearance group, these responses were detected in three cases at the last positive HPV-16 DNA test; in the other cases, responses were measured at 19, 35 and 43 months after the last positive DNA test. In the latter, a positive response was also measured at 63 months after the last positive HPV-16 DNA test. Interestingly, in two patients who had initially a fluctuating infection, before in one case clearing the infection and in the other case leading to a persistent infection, Th cell responses against the C-terminal protein were detected 5 and 6 months after a positive HPV-16 DNA test. Thus, Th cell responses against the C-terminal protein were often detected shortly before or after clearance of HPV-16.
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Discussion |
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Th cell responses against the C-terminal domain were frequently detected at the last HPV-16-positive DNA test or within 6 months of the last HPV-16-positive DNA test. These findings correlate with the previous published Th cell responses against HPV-16 E7 (de Gruijl et al., 1998 ). Th cell responses against HPV-16 E7 were higher around the time of clearance than at the end of FU in a group of HPV-16 clearance patients from the same cohort study. It is not clear whether these Th cell responses are involved in virus clearance and disease regression or whether these responses result from other events important in the natural history of cervical neoplasia. In contrast, Th cell responses against the E7 protein increase as the virus persists, and 82·5% of the patients with a persistent infection had E7-specific Th cells (de Gruijl et al., 1998
; Table 1
), while Th cells against the E2 C-terminal domain are incidentally detected in patients with a persistent infection, during FU and at the and of FU at similar frequencies. While E7 expression is likely to increase as the virus persists and the CIN lesion progresses to CIN III, E2 expression is likely to decrease probably as a result of virus integration and loss of productive virus infection (Schwarz et al., 1985
; Yee et al., 1985
).
Because both capsid proteins and E2 are expressed during a productive infection we compared virus-like particle (VLP)-specific IL-2 production and IgG-specific antibodies with the Th cell responses to both E2 domains. We found no correlation between either of these different immune responses (data not shown).
Since Th cell responses directed against either the C-terminal domain or the N-terminal domain in patients with a persistent HPV-16 infection occur mainly at single time-points during FU, these may depend on E2 protein expression during the virus life-cycle. E2 is required for virus replication and the protein would therefore be expected to be expressed in productive lesions, expression levels may fluctuate during the virus life-cycle and during the natural history of CIN disease. We have recently shown that E2 expression is mainly found in koilocytosis and CIN I lesions, while expression was decreased in CIN II lesions and absent in CIN III lesions as detected with a polyclonal antibody directed against the C-terminal domain (Maitland et al., 1998 ). In concordance with these results, IgA levels to the native HPV-16 E2 protein were high in patients with low grade lesions and decreased in CIN III patients (Rocha-Zavaleta et al., 1997
). However, in the cross-sectional study Th cell responses did not correlate with disease outcome. This may be because low grade productive lesions frequently exist in close proximity to high grade lesions. Alternatively, the lack of an association with disease outcome may be due to the relatively small CIN III group or the overall relatively low response rates. The reason for the low responder frequency may be due to the fact that expression of the E2 protein is mainly found in koilocytes (Maitland et al., 1998
), which are usually located in the top layers of the squamous epithelium. This relatively remote localization could hamper the transport of E2 by migrating dendritic cells to the draining lymph nodes, which has been proposed as a crucial step in the generation of virus-specific T-cell responses (Zinkernagel, 1996
).
E2 has been suggested as a candidate protein for vaccination (Tindle, 1996 ) and has been shown to be successful in animal models (Selvakumar et al., 1995
). Although the results of the present study might suggest that, in some cases, Th cell responses against the C-terminal domain of E2 may play a role in virus clearance, the overall low response rates and the transient character of these responses would imply that E2 is less attractive than, for example E7, to which high persisting Th cell responses have been reported (see also Table 1
). However, vaccination with the E2 protein may induce T-cell responses at sufficient levels. In a prophylactic/early therapeutic vaccination setting, an E2 and VLP combined vaccination may be beneficial. While neutralizing antibodies, induced by the VLP, may prevent infection (Nardelli-Hefliger et al., 1997
), E2-specific T-cells may eliminate keratinocytes infected with HPV particles which have escaped the neutralizing antibodies.
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Acknowledgments |
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References |
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Received 10 March 1999;
accepted 19 May 1999.