Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK1
Author for correspondence: Margaret Stanley. Fax +44 1223 333735. e-mail mas{at}mole.bio.cam.ac.uk
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Abstract |
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Introduction |
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Experimental studies on the immune response to HPVs are, however, compromised by the extreme tissue and host specificity of these agents and the absence of an in vitro culture system that supports a complete infectious cycle. The target cell for infection is the keratinocyte and, as far as is known, viral genes are expressed only in keratinocytes, with the infectious cycle being totally dependent on the implementation of the complete keratinocyte differentiation programme (Stanley, 1994 ). A mouse model that incorporates the exclusively epithelial nature of HPV has been developed to examine the host response to the HPV-16 early proteins E6 and E7. In this model, antigen is presented via a differentiated, syngeneic keratinocyte graft that expresses viral protein, a route comparable to that employed in the natural infection. Using this system, it has been shown that animals engrafted and then challenged with a recombinant vaccinia virus expressing HPV-16 E7 exhibit a delayed-type hypersensitivity (DTH) response that is E7-specific and CD4+-mediated (McLean et al., 1993
). The induction of a DTH response was measured in these studies because this type of response is associated with regression of HPV-induced genital warts (Coleman et al., 1994
) and, taken together, these studies suggest that a T helper type 1 (Th1)-type response is associated with virus clearance. Subsequent studies demonstrated the phenomenon of immune unresponsiveness in this model. Thus, animals engrafted with a low cell inoculum (low antigen dose) were unresponsive to a secondary priming challenge with antigen, whether this was delivered as a keratinocyte graft, protein or recombinant vaccinia virus. This unresponsiveness to antigen was maintained despite repeated antigen challenge, and was associated with graft persistence (Chambers et al., 1994
). There is evidence from clinical studies that progression in HPV-associated cervical disease involves a shift from a cell-mediated, Th1-type response to an antibody-associated, Th2 response (de Gruijl et al., 1996
), and this raises the possibility that, in the mouse model, different antigen loads induce either a Th1- or a Th2-type response. If this assumption is correct, the switch in the response must take place within the first days after the primary antigen encounter, and should be reflected in the cytokine profile of the lymphocytes in the nodes draining the graft. The present report presents data that examine this hypothesis.
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Methods |
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Lymphocyte isolation and culture.
Mice were sacrificed and the lymph nodes draining the graft (GDLN) from each mouse were isolated and processed individually. Single-cell suspensions were prepared by pressing the lymph node cells through a mesh (cell strainer Falcon 2340) with the help of a sterile syringe plunger. Cells were collected on complete medium (CM) containing RPMI 1640 (Gibco) supplemented with 10% heat-inactivated foetal calf serum (Gibco), 20 mM HEPES, 0·05 mM mercaptoethanol and antibiotics (penicillin, streptomycin). To induce cytokine synthesis and to facilitate its retention within the cytoplasm, a published protocol was followed (Openshaw et al., 1995 ), with slight modifications. Cells were resuspended at 1x106 cells/ml and 2 ml suspension was deposited in a well of a 24 well plate and stimulated with phorbol myristate acetate (50 ng/ml) (Sigma) plus ionomycin (500 ng/ml) (Sigma) for 4 h at 37 °C. Next, brefeldin A (Epicentre Technologies) was added at 10 µg/ml and the cells were incubated for a further 2 h. After the incubation period, cells were ready for the determination of surface markers and intracytoplasmic cytokines.
Flow cytometric simultaneous analysis of surface markers and intracellular cytokines.
After culture, cells were resuspended by pipetting and a 100 µl aliquot was deposited in each well of a V-bottomed 96 well plate (Costar). Plates were spun down and cells were resuspended in 100 µl PBS/2% BSA containing brefeldin A. Fifty µl CM containing the directly conjugated antibody (raised against mouse cell surface markers), prepared at the optimal dilution, was added to each well. Plates were incubated for 30 min at 4 °C in the dark. Plates were spun down and cells were washed twice with PBS/2% BSA. Cells were resuspended in 100 µl PBS/2% BSA/brefeldin A and 100 µl 4% paraformaldehyde, pH 7·4, was added. Cells were incubated for 20 min at room temperature. Plates were then spun down, cells were resuspended in 100 µl PBS/2% BSA/0·5% saponin and incubated for 10 min at room temperature to permit pore formation. Next, 50 µl CM, containing the directly conjugated antibody (raised against mouse cytokines) at the appropriate dilution, was added. Plates were incubated for 30 min at room temperature before being spun down. Cells were washed twice in PBS/2% BSA/0·5% saponin and once in PBS/2% BSA. Cells were resuspended in 2% paraformaldehyde, pH 7·4, and kept at 4 °C in darkness until analysed in a FACScan. Results were analysed with Lysis II software. All samples were run in the same conditions for forward and side scatter, FL1, FL2 and FL3. Lymphocytes were gated according to their scatter characteristics. In all experiments, unstained cells and cells stained separately with each fluorochrome were included to optimize compensation settings. A minimum of 10000 gated events were collected; in some experiments, 20000 or 30000 gated events were collected. Samples were regated on CD4+, CD8+ or -TCR+ cells and the percentage of cells producing cytokines within the new gate was determined. To calculate the percentage of cells producing cytokines, the cursor was set up to exclude the non-specific binding produced by the respective directly conjugated isotype control. The individual results from each mouse, from different experiments, at the same time-point were added together to calculate means and SEM.
The directly conjugated antibodies used in this study were as follows: FITCanti-mouse IFN-, FITCanti-mouse IL-2, FITCanti-mouse IL-4, FITCanti-mouse IL-10, phycoerythrin (PE)anti-mouse CD8 and FITCanti-rat IgG1 were from Pharmingen; PEanti-mouse IL-4, FITCanti-mouse IL-10, Tricoloranti-mouse CD4, Tricoloranti-mouse CD8, Tricoloranti-mouse
-TCR, FITCanti-rat IgG2a and FITCanti-rat IgG2b were from Caltag.
Statistical analysis was performed by ANOVA, considering a difference at the level of P<0·05 to be significant. Groups showing statistical differences were reanalysed by using Students two-tailed t-test; the exact level of significance is shown in each case. Data are presented as means±SEM.
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Results |
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Percentage of CD4+ GDLN cells that produced IFN-, IL-2, IL-4 and IL-10
The percentage of CD4+ cells that produced IFN-, IL-2, IL-4 and IL-10 in the GDLN was studied at days 3, 4, 5 and 7 after grafting. No significant differences were observed after comparing the experimental groups at the different time-points studied (Fig. 2
).
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Percentage of -TCR+ GDLN cells that produced IFN-
, IL-2 and IL-4
The percentage of -TCR+ GDLN cells that produced IFN-
did not differ between the experimental groups at any of the time-points studied (Fig. 4a
). The percentage of
-TCR+ GDLN cells that produced IL-2 did not differ between groups at days 3 and 5 after grafting. However, at day 4 post-grafting, IL-2 production was significantly increased in the group that received 1x107 NEK 16 cells when compared with the group that received a smaller number of NEK 16 cells [12·57±0·7 (5) vs 9·53±0·4 (5); P<0·096] (Fig. 4b
). At day 7 after grafting, the percentage of
-TCR+ GDLN cells that produced IL-2 was lower in the group that received a larger number of NEK 16 cells than in the control group [5·52±0·68 (11) vs 8·85±0·51 (7); P<0·004]. This pattern of IL-2 secretion by
T cells in the high antigen dose group is comparable to that reported to occur in trinitrochlorobenzene-induced contact sensitivity (Dieli et al., 1998
).
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Discussion |
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The data presented in Figs 2, 3
and 4
allow us differentiate the general time-dependent pattern of cytokine secretion in our experimental model from the differences amongst groups at the different time-points analysed. The cytokine profile of CD4+ cells suggested that different numbers of CD4+ cells were engaged in cytokine production at different time-points. This pattern was time-dependent and not group-dependent. Moreover, no difference was found at any time-point studied in the percentage of CD4+ cells that produced IFN-
, IL-2, IL-4 and IL-10 amongst CD4+ GDLN cells from mice that received different numbers of NEK 16 cells. These data indicate that the hypothesis that there would be a clear difference in the induction of a Th1- or Th2-type response, depending on the number of cells grafted, was not tenable.
The production of IFN- and IL-2 by CD8+ GDLN cells also failed to discriminate between groups at any time-point studied. However,
-TCR+ GDLN cells from mice that received a larger number of NEK 16 cells showed an increase in the percentage of cells that produced IL-2 associated with a decrease in IL-4 production at day 4 after grafting. The production of IL-4 and IL-10 by CD8+ cells showed striking differences at day 5 after grafting. CD8+ cells from mice that received 5x105 NEK 16 cells produced more IL-4 and IL-10 than the control group. Moreover, CD8+ cells from mice grafted with 1x107 NEK 16 cells produced more IL-10 than CD8+ cells from mice that received 5x105 NEK 16 cells or PBS. These data suggest that the essential differences in the cytokine profiles of the two experimental groups were (i) the induction, in animals that received a priming antigen dose, of a peak secretion of IL-2 by
-TCR+ cells 4 days after grafting and (ii) a peak secretion of IL-10 by CD8+ cells 5 days after grafting, together with a simultaneous up-regulation of IL-4 and IL-10 production by CD8+ cells at day 5 in animals that received a low antigen dose.
The evidence of McLean et al. (1993) showed clearly that the DTH response to E7 in this model is mediated by CD4+ cells, since treatment of animals with anti-CD4 antibodies abolished the E7-specific DTH response. Nevertheless, the results described above suggest that this is not a conventional Th1 response. This is not surprising, since several events are occurring in the grafts. The grafting procedure itself induces an inflammatory response, and this will change the cytokine milieu. Furthermore, not only is an E7-specific DTH response engendered by the priming grafts, but these grafts are then recognized as foreign and rejected by the hosts, usually by day 1416 post-grafting. The complex cytokine profile of the GDLN, with the secretion of IL-10 by CD8+ cells, reflects the complexity of these events.
Our data also suggest a complex collaboration between -TCR+ cells and CD8+ cells in the GDLN at the inductive phase of the immune response against NEK 16 cells. These data are intriguing, since a similar cross-talk and temporal relationship has been described in contact sensitivity (Dieli et al., 1998
). In this study, it was shown that, while the contact sensitivity reaction towards the hapten trinitrochlorobenzene persists for up to 21 days in the host, it can only be transferred successfully into naïve hosts at days 4 and 5 after immunization. Importantly, it was further demonstrated that IL-2-producing
-TCR+ cells obtained from the draining lymph nodes at days 4 and 5 after immunization were strictly required for a successful transfer (Dieli et al., 1998
). There is little information on the role of
T cells in the immune response to papillomavirus infections. Spontaneous regression of bovine papillomavirus-induced warts is associated with a significant
T cell infiltrate in the lesions (Knowles et al., 1996
). However, this phenomenon is not observed in spontaneous regression of HPV-6/-11-induced genital warts (Coleman et al., 1994
) or oral warts induced in the dog by the canine oral papillomavirus (P. K. Nicholls, personal communication). Further studies on
T cell function are needed in both experimental and natural papillomavirus infections.
In the original view of the Th1/Th2 dichotomy, IL-10 is secreted by Th2-type cells that inhibit cytokine secretion by Th1-type cells, switching the immune response towards a Th2 type (Howard & OGarra, 1992 ). However, IL-10 can enhance IL-2-mediated induction of CTL differentiation (Chen & Zlotnik, 1991
) and human IL-10 is a chemoattractant for CTLs (Jinquan et al., 1993
). Furthermore, IL-10 stimulates human NK cell function (Carson et al., 1995
) and IL-10 secreted by transfected tumour lines enhances anti-tumour activity by activating NK and CTL cells (Giovarelli et al., 1995
). Hence, the presence of CD8+ cells that secrete IL-10 in mice grafted with a priming dose of NEK 16 cells could favour the recruitment of E7-specific CTL and/or NK cells.
The generation of a CD8+ population that secretes IL-4 and IL-10 in the GDLN of mice grafted with 5x105 NEK cells could be associated with the induction of a conventional type 2 response mediated via CD8+ cells. There is evidence for a dichotomy amongst CD8+ cells. This phenomenon is well illustrated in lepromatous leprosy (Salgame et al., 1991 ), where CD8+ and CD4+ clones were isolated from both lesions and blood from patients. CD8+ clones produced IL-4 and suppressed the proliferation of CD4+ clones in vitro. Recently, it has been shown that low-dose tolerance to chemical allergens applied on the skin is mediated by CD8+ cells with a type 2 cytokine profile (Steinbrink et al., 1996
). These authors have shown that the induction of tolerance is strictly dependent on the amount of antigen used. This model closely resembles our system, since in both cases the access of the immune system to the antigen is very limited and in both experimental models the final outcome of the immune response depends strictly on antigen load.
In conclusion, in our experimental model, when animals were challenged locally by a high antigen dose via a graft of keratinocytes expressing HPV-16 E7, two distinctive events were observed in the lymphocytes of the GDLN; an increased secretion of IL-2 by lymphocytes at day 4 post-grafting and, at day 5, a peak secretion of IL-10 but reduced secretion of IL-4 by CD8+ cells. In contrast, in the GDLN of animals challenged with a low antigen dose, the response at day 5 was mediated by CD8+ cells (Tc2 or Ts) that secreted both IL-4 and IL-10, a scenario that could induce further unresponsiveness.
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Acknowledgments |
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References |
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Received 18 November 1999;
accepted 13 January 2000.