1 Peter Gorer Department of Immunobiology, GKT, 3rd Floor New Guy's House, Guy's Hospital, London SE1 9RT, UK
2 Department of Obstetrics and Gynaecology, Birmingham Women's Hospital, Metchley Park Road, Edgbaston, Birmingham B15 2TG, UK
3 Department of Histopathology, St Thomas' Hospital, London SE1 7EH, UK
Correspondence
Philip Shepherd
philip.shepherd{at}kcl.ac.uk
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ABSTRACT |
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INTRODUCTION |
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A better understanding of the role of HPV-specific cell-mediated immune responses in the natural history of cervical disease requires prospective studies to be carried out whereby the immune responses of patients and controls are closely monitored over a period of time and correlated with HPV infection and disease status. This should also result in better-defined and more easily comparable patient and control groups. Relatively few such studies have been conducted to date and no clear pattern has emerged as to which T cell responses (if any) might be associated with regression or progression of disease. De Gruijl et al. (1996, 1998
) showed that patients with virus persistence and disease progression were more likely to respond to HPV-16 E7 peptides. Kadish et al. (1997)
, however, showed that responses to an N-terminal peptide of E6 and a C-terminal peptide of E7 were more strongly associated with clearance of virus infection on follow-up. A prospective study of T cell responses to L1 found no association with virus persistence or clearance (De Gruijl et al., 1999
), although the authors reported evidence of an immunogenic region at aa 311335, which is within the region previously identified by our group in cross-sectional studies (Shepherd et al., 1994
, 1996
). An association between responses to this region and the HLA DRB1*11/DQB1*0301 haplotype was also demonstrated.
We have conducted a prospective study in order to investigate further the down-regulation of HPV-16 E7-specific T cell responses, which was previously observed in women with squamous intraepithelial lesions of the cervix (SIL). The main objective of the study was to compare T cell responses of patients to HPV-16 L1 and E7 at different time points in order to determine whether T cell down-regulation might be linked to recurrence of cervical disease in patients following treatment, or progression of disease in untreated patients. The effects of treatment on both HPV-16 L1- and E7-specific T cell responses were also investigated, including whether or not effective treatment could result in a restoration of the E7 responses previously found to be absent. Other questions addressed were whether the specificity of the initial response or any changes in response observed during the follow-up period reflected the outcome of disease following treatment.
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METHODS |
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T cell culture and proliferation assays
Short-term T cell lines (STLs; 20 per antigen per patient) were established using either virus-like particles (VLPs) of HPV-16 L1 or an HPV-16 GSTE7 fusion protein as the stimulating antigen. Details of the methods involved have been described previously (Luxton et al., 1996, 1997
; Shepherd et al., 1996
). It was previously established that the majority of HPV-16-specific cell lines generated by this technique are CD4-positive (Shepherd et al., 1996
).
In standard 3-day proliferation assays, L1-specific STLs were tested for specificity against VLPs of L1, HPV-16 -galL1 fusion protein and
-galactosidase protein alone, each at 1 µg ml-1, and 15-mer peptides or pools of peptides (10 µM) representing previously identified immunogenic regions of the L1 molecule between aa 191 and 225, 281 and 295, and 311 and 345. The amino acid numbering was from the classical L1 start codon and peptides used were as described in Shepherd et al. (1996)
. E7-specific STLs were assayed against phytohaemagglutinin (1 µg ml-1) as a positive control, GSTE7 fusion protein and GST protein alone (10 µg ml-1), and three pools of 15-mer peptides overlapping by five amino acids, representing HPV-16 E7 aa 134, 3074 and 7098 (10 µM). All cell lines were tested against culture medium only as a negative control. In proliferation assays, a positive STL response was required to have a c.p.m. of >500 above the medium-only control value and a stimulation index of >2·5. In addition, a positive patient response was required to have
2/20 STLs responding to the same peptide.
HPV DNA detection in cervical biopsy tissue and cervical brush swab samples
The methods used for extraction of DNA from cervical biopsy tissue and HPV DNA typing by PCR were as previously described (Shepherd et al., 1996). The method for processing of cervical brush swabs prior to HPV DNA typing was as follows. Swabs were collected into 5 ml sterile PBS and stored at -20 °C prior to processing. The sample was thawed and vortexed vigorously for 30 s in order to free cellular material from the brush. The brush was then carefully discarded and the cell suspension transferred to a sterile centrifuge tube. The cells were pelleted by centrifugation at 1000 g for 5 min. The pellet was then resuspended in 100200 µl proteinase K buffer (0·01 M Tris/HCl, pH 7·8, 0·005 M EDTA, 0·5 % w/v SDS) containing 1 mg ml-1 proteinase K (Qiagen) and the solution was incubated at 56 °C overnight. The enzyme was inactivated by heating at 95 °C for 10 min. The digest was then centrifuged at 13 000 g for 30 s before storing at -20 °C. PCR was performed on undiluted proteinase K digests.
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RESULTS |
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HPV-16-specific T cell responses in patients and controls
In Fig. 1, the magnitude and peptide specificity of HPV-16 L1- and E7-specific T cell responses of patients with (n=42) and without (n=15) SIL on their first visit to the clinic were compared. The percentage of responders to each peptide or peptide pool is summarized in the inset figure.
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The most immunogenic region of E7 in patients with SIL was the C terminus at aa 7098 to which 15/42 (36 %) patients responded (Fig. 1a), whereas 8/42 (19 %) responded to the N-terminal region at aa 134 and 1/42 (2 %) to the central portion of the molecule at aa 3074. Fewer patients responded to any one or more E7 peptide than to L1 peptides (19/42 or 45 % compared with 28/42 or 67 %). Also, the percentage of responders to the E7 pool of aa 7098 was reduced in patients without SIL (2/10 or 20 % responders, Fig. 1b
) compared with those with SIL (15/42 or 36 %), but neither of these findings was statistically significant (P>0·05). The single patient with cervical cancer responded to the L1 regions of aa 281295 and 311345 and did not respond to E7.
HPV-16-specific T cell responses and relationship to severity of cervical lesion and infecting HPV type
The T cell responses of patients with different grades of cervical lesion were compared with those of patients who presented with no dysplasia (Fig. 1c). Patients from all histological groups responded to all peptides tested except the E7 pool of aa 3074, which failed to induce a response in patients with HPV/CIN I or CIN II lesions. Responses to most peptide pools were greater in patients with CIN III lesions than in those with HPV/CIN I or CIN II lesions, although the only statistically significant difference was between responses to the L1 peptide aa 281295 (P=0·027). On the other hand, patients responded equally well to the peptide pool of aa 311345, no matter what grade of lesion they had.
The responses detected, however, were not significantly associated with current cervical HPV-16 DNA infection (P>0·05), as 9/15 (60 %) individuals responded to L1 and 6/15 (40 %) to E7 in the HPV-16 DNA-positive group, compared with 12/18 (67 %) who responded to L1 and 10/18 (56 %) to E7 in the HPV-16 DNA-negative group. It should be noted that four individuals who responded to L1 and/or E7 were HPV31-positive, hence responses detected in the absence of HPV-16 infection may result from cross-reactivity to closely related HPV types such as HPV 31 or from previous HPV-16 infections.
HPV-16 L1- and E7-specific T cell responses in patients following treatment for SIL of the cervix
Patient follow-up, changes in histology and HPV DNA status
Thirty-three of the 58 patients who entered the study were followed up with a mean time to first follow-up of 14±11 months. Twenty-three patients were followed up after 311 months (mean±SD=7±1·9 months) and the remaining 10 patients after 1739 months (mean±SD=28±8·1 months). On their first visit to the clinic, 10 patients (30 %) had no dysplasia, 17 (52 %) had HPV/CIN I lesions, four (12 %) had CIN II lesions and two (6 %) had CIN III lesions. All patients with disease were treated on or shortly after their first visit to the clinic. Fig. 2 illustrates the outcome of treatment for each patient, comparing cervical histology at first and follow-up visits. To summarize these data, at their first follow-up visit, most patients had no dysplasia (20/33 or 61 %), 12/33 (36 %) had low-grade (HPV/CIN I) disease and one patient (3 %) had a recurrent CIN III lesion. Of those patients with no SIL at follow-up who were typed (n=7), three were HPV-16-positive (30 % of the whole group) and four were HPV-negative. Those patients with low-grade lesions that were typed (n=5) were all HPV-16-positive (29 % of the whole group) and the single CIN III lesion was HPV-negative.
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HPV-16-specific T cell responses and recurrence of cervical disease following treatment
In order to investigate the relationship between the T cell responses of individual patients and the outcome of their disease following treatment, patients with SIL at first visit were grouped according to whether or not they had recurrent disease at follow-up and those without SIL at first visit according to whether or not they had acquired disease by the time of follow-up. By analysis of HPV-16-specific T cell responses before and after treatment, we could then determine whether the peptide specificities of responses measured at the outset or any changes in the specificities of responses occurring during the period of follow-up might reflect the outcome of disease following treatment.
Of the 23 patients with SIL at first visit, nine had recurrent disease (this was recurrent high-grade disease in one case only) and 14 were disease-free at follow-up (Fig. 2). Of the 10 patients with no SIL at first visit, four had acquired low-grade lesions by the time of follow-up and six remained disease-free.
On comparison of the magnitude of responses made by patients (data not shown), 2/14 patients without recurrent lesions made particularly strong responses to the L1 region of aa 311345. However, the mean STL response to this region in patients without recurrent lesions was not significantly higher than that of patients with recurrent lesions (P>0·05). On comparison of the total percentage of patients responding to each peptide pool (Fig. 3), patients with and without recurrent lesions were found to respond equally well to all peptide pools, with the exception of the E7 pool of aa 7098 to which 67 % (6/9) of patients with recurrent disease responded compared with 14 % (2/14) of patients without recurrent disease. Therefore, T cell responses to the HPV-16 E7 region of aa 7098 detected in patients with disease at recruitment were significantly associated with recurrence of disease following treatment (P=0·017).
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Patient T cell responses were compared before and after treatment to determine whether any change (loss or gain) in response to either L1 or E7 was associated with the recurrence or absence of disease on follow-up (data not shown). However, none of the changes observed were significantly associated with recurrent disease (P>0·05).
Interestingly, on comparison of the total number of STLs per patient group responding to each L1 peptide pool before and after treatment, increases were observed in patients with recurrent disease following treatment (aa 191225, 54 %; aa 291295, 36 %; aa 311345, 48 %). In patients without recurrent disease on follow-up, there were corresponding decreases in the total numbers of L1-specific STLs of 42, 8 and 58 %, respectively.
HPV-16-specific T cell responses and acquisition of cervical disease
Fig. 4 compares the T cell responses of 10 patients who were disease-free at recruitment according to the outcome of their disease at follow-up. Three out of four (75 %) of those patients who acquired disease during the study period also gained responses to E7, including responses to the C-terminal region of aa 7098 in all cases, whereas only 1/6 (17 %) of those individuals who did not acquire disease during the study period gained responses to E7. Although this difference was not statistically significant (P=0·119) and the numbers involved were small, it suggests that responses to the E7 region of aa 7098 might be linked to the acquisition of cervical disease.
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DISCUSSION |
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On comparison of the peptide-specific responses of patients with different histological grades of lesion at first visit, the levels of response to the L1 peptide aa 281295 and E7 peptide pools of aa 134 and 7098 were clearly raised in patients with CIN III compared with those with lower-grade lesions. This difference was statistically significant for responses to peptide aa 281295 only (P=0·027). The differences observed were not directly linked to the levels of current HPV-16 positivity observed in different histological groups (P>0·05); it is therefore assumed that they resulted from recent or perhaps repeated previous exposure to HPV-16 infection.
As found in previous studies, the ability of patients with SIL to respond to any one or more HPV-16 E7 peptides differed from their ability to respond to L1 peptides. Whilst T cell responses to L1 peptides increased with increasing histological grade of lesion, responses to E7 were reduced in comparison in all grades of lesion, although none of these differences was statistically significant. It is not clear why the levels of T cell response to the L1 and E7 proteins are different. It is possible that the higher levels of E7 expression that occur in high-grade lesions might be necessary to stimulate a detectable E7-specific response, whereas in low-grade lesions, many of which involve productive virus infections, T cell responses to capsid proteins such as L1 may predominate.
HPV-16-specific T cell responses and treatment for SIL of the cervix
The HPV-16-specific T cell responses of patients with SIL before and after treatment did not change greatly, in terms of peptide specificity, the range of response observed, or the numbers of patients responding, suggesting that treatment itself had no significant positive or negative effect on these responses. Small increases in the level of T cell response to certain peptides/peptide pools were observed (the L1 peptide aa 281295, the pool of aa 311345 and the E7 pool of aa 7098), although these were not statistically significant. On comparison of patients with the same histological grade of lesion, HPV/CIN I, before and after treatment, responses to L1 peptide 281295 and the E7 pool of aa 7098 were found to be increased on follow-up. The increases in responses observed in patients with SIL at follow-up could be due to memory responses, which are boosted by reinfection with HPV-16, or the result of a new HPV-16 infection, both of which occurred in our patient group. Overall, the ability of patients with SIL to respond to any one or more E7 peptides was only slightly increased at follow-up, whereas the ability to respond to L1 peptides did not change.
Comparison of the HPV-16-specific T cell responses of patients without SIL at first and follow-up visits showed little change in the response of the group as a whole, even though 3/7 of these patients had evidence of new HPV-16 infections at follow-up by PCR (four were HPV-negative). These new infections were accompanied by the acquisition of an L1-specific response in one case and an E7-specific response in another.
HPV-16-specific T cell responses and recurrence of SIL
To investigate the relationship between HPV-16-specific T cell responses and SIL of the cervix, patients were grouped according to the outcome of their treatment, that is, whether or not they had recurrent disease, whether they remained disease-free throughout the study period or whether they acquired disease during this period. By studying the T cell responses of these groups of patients, it was found that changes in the HPV-16 L1 and E7 epitope specificity of T cells were not significantly associated with the outcome of cervical disease following treatment. Therefore, neither the absence of E7-specific responses at the outset nor the loss of any E7-specific response during the study period were associated with recurrence of disease. Conversely, successful treatment did not lead to the restoration of E7 responses in individuals where they were previously absent. Therefore, the down-regulation of HPV-16 E7-specific responses in patients with cervical disease, which was observed in this and previous studies (Luxton et al., 1997), was not associated with a worse prognosis for the patient.
Although recurrence of disease was accompanied by an increase in the frequency of HPV-16 L1-specific T cells and absence of disease on follow-up by a decrease in the frequency of L1-specific T cells, these findings were not statistically significant.
However, T cell responses to the E7 peptide pool of aa 7098, measured at the patient's first visit to the clinic, were significantly associated with the recurrence of disease following treatment (P=0·017). This does not appear to be true for healthy controls who respond to the same region of E7. The acquisition of this response also accompanied the acquisition of disease in the majority of patients (75 %) who were disease-free at the start of the study. Although the patient numbers involved were small, the data suggest that E7 aa 7098-specific responses do not protect against reinfection by HPV and recurrence of cervical disease. It is not clear why this particular T cell response should be linked with a worse prognosis for the patient at a later date; perhaps repeated or persistent infection by HPV-16 is required to achieve the levels of antigen expression necessary to stimulate E7-specific T cells and therefore these patients are those in whom disease is more likely to reoccur following treatment or progress in those who are untreated.
We propose that larger prospective studies are conducted to confirm the link between E7 aa 7098-specific T cell responses and recurrence of cervical disease. If these findings are substantiated, this T cell response might be useful in identifying a subset of patients who are at greater risk of developing recurrent or persistent disease.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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De Gruijl, T. D., Bontkes, H. J. Walboomers, J. M. M. & 9 other authors (1996). Analysis of IgG reactivity against human papillomavirus type-16 E7 in patients with cervical intraepithelial neoplasia indicates an association with clearance of viral infection: results of a prospective study. Int J Cancer 68, 731738.[CrossRef][Medline]
De Gruijl, T. D., Bontkes, H. J. Walboomers, J. M. M. & 9 other authors (1998). Differential T helper cell responses to human papillomavirus type 16 E7 related to viral clearance or persistence in patients with cervical neoplasia: a longitudinal study. Cancer Res 58, 17001706.[Abstract]
De Gruijl, T. D., Bontkes, H. J. Walboomers, J. M. M. & 10 other authors (1999). Immune responses against human papillomavirus (HPV) type 16 virus-like particles in a cohort study of women with cervical intraepithelial neoplasia I: differential T-helper and IgG responses in relation to HPV infection and disease outcome. J Gen Virol 80, 399408.[Abstract]
Gill, D. K., Bible, J. M., Biswas, C., Kell, B., Best, J. M., Punchard, N. A. & Cason, J. (1998). Proliferative T-cell responses to human papillomavirus type 16 E5 are decreased amongst women with high-grade neoplasia. J Gen Virol 79, 19711976.[Abstract]
Kadish, A. S., Romney, S. L., Ledwidge, R., Tindle, R., Fernando, G. J. P., Zee, S. Y., Van Ranst, M. A. & Burk, R. D. (1994). Cell-mediated immune responses to E7 peptides of human papillomavirus (HPV) type 16 are dependent on the HPV type infecting the cervix whereas serological reactivity is not type-specific. J Gen Virol 75, 22772284.[Abstract]
Kadish, A. S., Ho, G. Y. F., Burk, R. D., Wang, Y. X., Romney, S. L., Ledwidge, R. & Angeletti, R. H. (1997). Lymphoproliferative responses to human papillomavirus (HPV) type 16 proteins E6 and E7: outcome of HPV infection and associated neoplasia. J Natl Cancer Inst 89, 12851293.
Luxton, J. C., Rowe, A. J., Cridland, J. C., Coletart, T., Wilson, P. & Shepherd, P. S. (1996). Proliferative T cell responses to the human papillomavirus type 16 E7 protein in women with cervical dysplasia and cervical carcinoma and in healthy individuals. J Gen Virol 77, 15851593.[Abstract]
Luxton, J. C., Rose, R. C., Coletart, T., Wilson, P. & Shepherd, P. S. (1997). Serological responses to human papillomavirus type 16 L1 virus-like particles in women with cervical disease and in healthy controls. J Gen Virol 78, 917923.[Abstract]
Nakagawa, M., Stites, D. P., Farhat, S., Judd, A., Moscicki, A. B., Canchola, A. J., Hilton, J. F. & Palefsky, J. M. (1996). T-cell proliferative response to human papillomavirus type 16 peptides: relationship to cervical intraepithelial neoplasia. Clin Diagn Lab Immunol 3, 205210.[Abstract]
Shepherd, P., Rowe, A., Cridland, J., Chapman, M., Luxton, J. & Rayfield, L. (1994). An immunodominant region in HPV-16 L1 identified by T cell responses in patients with cervical dysplasias. In Immunology of Human Papillomaviruses (HPVs), pp. 233241. Edited by M. Stanley. Cambridge: Cambridge University Press.
Shepherd, P. S., Rowe, A. J., Cridland, J. C., Coletart, T., Wilson, P. & Luxton, J. C. (1996). Proliferative T cell responses to human papillomavirus type 16 L1 peptides in patients with cervical dysplasia. J Gen Virol 77, 593602.[Abstract]
Strang, G., Hickling, J. K., Angus, G., McIndoe, J., Howland, K., Wilkinson, D., Ikeda, H. & Rothbard, J. B. (1990). Human T cell responses to human papillomavirus type 16 L1 and E6 synthetic peptides: identification of T cell determinants, HLA-DR restriction and virus type specificity. J Gen Virol 71, 423431.[Abstract]
Tsukui, T., Hildesheim, A. Schiffman, M. H. & 17 other authors (1996). Interleukin 2 production in vitro by peripheral lymphocytes in response to human papillomavirus-derived peptides: correlation with cervical pathology. Cancer Res 56, 39673974.[Abstract]
Received 29 October 2002;
accepted 15 January 2003.
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