Affiliations of authors: Centre for Immunology and Cancer Research, University of Queensland, Princess Alexandra Hospital, Brisbane, Queensland, Australia (KM, GRL, JZ, XL, RLDK, TP, GJPF, IHF); McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine, Madison, WI (AL, PFL)
Correspondence to: I. H. Frazer, MbChB, MD, FAA, Centre for Immunology and Cancer Research, The University of Queensland, Research Wing, Building 1, Princess Alexandra Hospital, Woolloongabba, Brisbane, Queensland 4102, Australia (e-mail: ifrazer{at}cicr.uq.edu.au)
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ABSTRACT |
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INTRODUCTION |
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The cells of cervical squamous cell cancer, an epithelial cancer that is common in women worldwide, express two virally encoded nuclear oncoproteins of human papillomavirus type 16 (HPV16), E6 and E7, as do HPV16-infected epithelial cells that display various premalignant phenotypes associated with HPV16 infection (8). These proteins are therefore targets for HPV16-specific immunotherapy. To examine requirements for the elimination of HPV16-infected but non-transformed cells expressing HPV16 proteins, we established a skin graft model. In this model, skin of mice engineered to express HPV16 E6 or E7 as transgenes from the keratin 14 (K14) promoter in keratinocytes but not in professional antigen-presenting cells is grafted onto syngeneic, non-transgenic mice (9). We have previously demonstrated that K14E7 skin grafts are not rejected spontaneously, despite the generation of E7-specific humoral immunity in graft recipients. In addition, K14E7 skin grafts are resistant to active immunotherapy with E7 proteinbased immunogens that induce rejection of transgenic E7 tumors transplanted in the same mouse (10). We used this skin graft mouse model to examine the requirements for the eradication of skin cells expressing E7. Specifically, we examined whether the number of and cytokine profile of effector T cells induced following immunization determines whether E7 transgenic skin is rejected.
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MATERIALS AND METHODS |
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C57BL/6J (H-2b) mice and C57BL/6J mice transgenic for the HPV16 E7 oncoprotein driven from a K14 promoter (designated as K14E7 mice) were obtained from the Animal Resources Centre (Perth, WA, Australia). To generate H-2b K14E7 mice, H-2q K14E7 mice (hK14HPV16E6ttlE7) were backcrossed with C57BL/6J (H-2b) mice for more than 12 generations. Characterization of these mice and of transgene expression has been described (9,11). FVB (H-2q) mice (hK14HPV16E6E7ttl) transgenic for the HPV16 E6 oncoprotein driven from the K14 promoter (designated as K14E6) have been described previously (12). Lines of FVB (H-2q) mice heterozygous for a human growth hormone (hGH) transgene expressed from the K14 promoter (designated as K14hGH mice) were generated by pronuclear injection using an expression construct described by Wang et al. (13) and Zhong et al. (14). The serum hGH level in the 023 K14hGH line used in the current study is approximately 8 mIU/L, and these mice are approximately 10% larger than their sex-matched non-transgenic littermates. First generation C57BL/6JxFVB mice were recipients of first generation C57BL/6Jx FVB.K14hGH grafts.
Transgenic donors and non-transgenic syngeneic recipients aged 812 weeks were maintained under conventional conditions in specific pathogen-free holding rooms in the Princess Alexandra Hospital Biological Resources Facility (Brisbane, QLD, Australia). All animal protocols were approved by the University of Queensland Animal Ethics Committee.
Skin Grafting
Whole-thickness ear skin grafting is described elsewhere (9,10). Briefly, whole ears from donor mice (C57BL/6J.K14E7, FVB.K14E7, FVB.K14E6, C57BL/6JxFVB.K14hGH) were surgically removed, and dorsal and ventral surfaces were separated. Transgenic skin grafts were placed on the flanks of non-transgenic but otherwise syngeneic recipient mice, held in place with antibiotic-permeated Vaseline gauze (Bactigras; Smith and Nephew, London, U.K.), covered with micropore tape and elastic bandages (CoFlex; Andover, Salisbury, MA) for 7 days, and assessed as technically successful if they were adherent and vascularized on day 7. Skin grafts were observed at least three times weekly for the duration of the study. Skin graft rejection was assessed by observation and confirmed histologically by staining graft sites with hematoxylin and eosin using conventional procedures.
Adoptive Transfer of Antigen-Specific T-Cell Receptor Transgenic T Cells
HPV16 E7-specific T-cell receptor (TCR) chain transgenic (tg) C57BL/6J mice (E7TCR mice) (Leggatt GR: personal communication) demonstrated 25%40% staining of CD8+ lymphocytes with E7/H-2Db tetramers. T cells of E7TCR mice proliferate and secrete interferon gamma (IFN-
) in vitro in response to the GF001 (H-2Dbrestricted E7 cytotoxic T lymphocyte [CTL]) peptide (amino acid [aa] sequence = RAHYNIVTF). OT-1 Rag/ C57BL/6J mice (OT-1) were obtained from the Animal Resources Centre (Perth, WA, Australia). OT-1 mice express a transgenic TCR specific for H-2brestricted ovalbumin (OVA) CTL peptide (aa sequence = SIINFEKL). Splenocytes from naive E7TCR and OT-1 mice were each obtained as a single cell suspension. Red blood cells were depleted using ACK lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM EDTA, pH 7.3). After washing twice with 0.15 M phosphate-buffered saline (PBS [pH 7.4]), splenocytes were counted, diluted in PBS to the appropriate concentration, and injected intravenously into the tail vein of grafted C57BL/6J mice.
Immunization Protocol
Grafted and control mice were immunized as indicated subcutaneously at the tail base with glutathione S-transferaseHPV16 E7 fusion protein (50 µg per mouse) as previously described (15) or with ovalbumin peptide (Sigma Chemical, St. Louis, MO) (aa sequence = SIINFEKL, 50 µg per mouse). GF001 peptide (H-2Dbrestricted minimal CTL epitope of HPV16 E7 protein [aa sequence = RAHYNIVTF], 50 µg per mouse) and MT906 peptide, which includes an H-2Dbrestricted T-helper epitope and a mutant disabled form of the CTL epitope of HPV16 E7 protein (aa sequence = QAEPDRAHYNIVTKCCKCD, 50 µg per mouse) were also used for immunization. Antigens were administered with Quil A saponin (Spikoside; Iscotec AB, Stockholm, Sweden) with complete Freunds adjuvant, as indicated.
Cell Culture
To recall E7-specific memory in CTLs, splenocytes collected from C57BL/6J graft recipients and control C57BL/6J mice were restimulated with the minimal E7 CTL peptide (GF001) in vitro. Splenocytes were collected from graft recipients, red blood cells were removed with ACK lysis buffer, and the remaining splenocytes were plated at 7.5 x 106 cells per well in a 24-well plate. Syngeneic splenocytes, pulsed for 23 hours with GF001 peptide and gamma irradiated (3000 rad), were then added at 3.5 x 106 cells per well with 1 ng/mL (approximately 10 U/mL) recombinant mouse interleukin 2 (rIL-2) (BD Pharmingen, San Diego, CA) in EHAA/RPMI complete medium (50% EHAA/50% RPMI supplemented with 0.216 g/L L-glutamine, 60 mg/L benzyl penicillin [CSL, Melbourne, VIC, Australia], 100 mg/L streptomycin [CSL], 50 µmol/L 2-mercaptoethanol, and 10% fetal bovine serum [FBS; Gibco, Gaithersburg, MD]). The splenocytes were then cultured at 37 °C in a 5% CO2 incubator. After 1 week, cultured splenocytes were analyzed by flow cytometry and ELISPOT assay.
Flow Cytometric Analysis
Collected cells were resuspended in PBS/5% FBS and incubated with 1 µg of allophycocyanin- conjugated rat anti-mouse CD8+ antibody (Clone 53-6.7, BD Pharmingen) and fluorescein isothiocyanateconjugated mouse anti-mouse TCR V12+ antibody (Clone MR11-1, BD Pharmingen) for 1 hour in the dark at 4 °C. All cells were washed with PBS/5% FBS twice, resuspended in fixative solution (2% formaldehyde in PBS), and analyzed using a FACScan flow cytometer (BD Biosciences, San Jose, CA).
IFN- ELISPOT Assay
The IFN- ELISPOT assay was performed as previously described (16). Briefly, spleen cell suspensions from C57BL/6J mice were added to membrane-base 96-well plates (Millipore, Bedford, MA) that had been coated with antiIFN-
monoclonal antibody (MabTech, Stockholm, Sweden). Cells were held at 37 °C with the GF001 peptide and 50 U/mL of rIL2 (Gibco BRL) overnight. Antigen-specific IFN-
secreting cells were detected by sequential exposure of the plate to biotinylated antiIFN-
monoclonal antibody (Clone R4-6A2, MabTech), avidinhorseradish peroxidase (Sigma), and diaminobenzidine (Sigma).
Enzyme-Linked Immunosorbent Assay
Splenocytes collected from grafted and control mice and then treated with ACK lysis buffer to remove red blood cells were plated at 1 x 107 cells per well in a 24-well plate with 108 M GF001 peptide and 1 ng/mL rIL-2 (BD Pharmingen) in EHAA/RPMI complete medium. The cells were cultured at 37 °C in a 5% CO2 incubator for 72 hours. Supernatants from the cultures were tested for IFN- level by using capture enzyme-linked immunosorbent assay (ELISA). Wells of 96-well ELISA plates (Nalge Nunc, Rochester, NY) were coated with capture monoclonal antibody (100 µL per well) (Clone AN18; MabTech) at 1 µg/mL in 0.05 M Na2CO3 buffer (pH = 9.6) and incubated at 4 °C overnight. Then the wells were blocked with 200 µL of PBS containing 3% skim milk and 0.05% Tween 20 (blocking buffer) at 4 °C overnight. Supernatant sample (100 µL) at a 1 : 2 dilution in the blocking buffer was reacted with the capture antibody in the well at room temperature for 2 hours. A colorimetric assay using recombinant mouse IFN-
was used to calculate a standard curve for estimating the IFN-
concentration of the samples. Wells were washed five times with PBS containing 0.05% Tween 20 and received 100 µL per well of biotinylated detection monoclonal antibody (Clone R4-6A2; MabTech) at a concentration of 0.5 µg/mL in the blocking buffer. The wells were held at room temperature for 1 hour and washed five times with PBS containing 0.05% Tween 20. Then, 75 µL of avidinhorseradish peroxidase solution (Sigma; 1 mg/mL stock solution in PBS diluted at 1 : 400 in the blocking buffer) was added to each well, and the plates were held at room temperature for 1 hour. The plates were first washed three times with PBS containing 0.05% Tween 20, and then washed twice with PBS. Diaminobenzidine developing reagent was prepared as recommended by the manufacturer, and 200 µL was added to each well. The absorbance at 492 nm was measured after holding the plates for 15 minutes at room temperature. After 3 days of culture in vitro, supernatants were tested for IL-5 and IL-10 by using commercially available capture IL-5 and IL-10 ELISA Kits (R&D Systems, Minneapolis, MN), respectively. Antibodies to HPV16 E7 were measured by ELISA, as described previously (11).
Statistical Methods
KaplanMeier plots were used to analyze E7 skin graft survival, and a log rank test was used to examine the statistical significance of differences in the survival curves. The t test and chi-square test for trend were also used for data analysis, as indicated. All analyses were carried out using a JMP 5.01J statistics package (SAS Institute, Cary, NC). Two-tailed P values were calculated throughout and considered to be statistically significant at less than .05.
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RESULTS |
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The fate of skin from transgenic mice expressing a defined antigen from the K14 promoter and grafted onto syngeneic, non-transgenic mice was antigen-dependent (Fig. 1, A). Skin grafts expressing hGH were spontaneously rejected over 1220 days in association with a marked inflammatory cell infiltrate (data not shown), as has previously been described for grafts expressing OVA from the keratin 5 promoter (17). In contrast, skin grafts expressing the HPV16 E6 or E7 oncoprotein in a range of genetic backgrounds and demonstrating the skin phenotype of delayed cellular maturation and epithelial thickening associated with transgene expression were not rejected and appeared devoid of inflammatory immune response on histologic analysis. Placement of K14E7 H-2b skin grafts on H-2b recipients generated an HPV E7 antibody response (Fig. 1, B), as we have previously observed for H-2q recipients of K14.E7.H-2q skin (9). Thus, HPV E7 was presented by epithelial cells, and it induced a different immune response than hGH or OVA presented by the same cells.
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To determine why an immune response to E7 induced by immunization [although able to effect tumor rejection, as previously shown (9)] was inadequate to reject skin grafts expressing the same antigen, we investigated whether adoptive transfer of E7-specific CD8+ T cells to recipients of K14E7 skin grafts could facilitate rejection of the graft by increasing the precursor frequency of E7-responsive T cells. Spleen cells (5 x 106 per mouse) from mice expressing a transgenic TCR chain derived from an H-2brestricted E7 peptidespecific CTL clone (E7TCR mice) were transferred to H-2b recipients of H-2b K14E7 skin grafts (Fig. 2). Consistent with our earlier findings, H-2b K14E7 skin grafts were not rejected by untreated or by E7-immunized H-2b recipients. E7 skin graft recipients that received 5 x 106 E7TCR tg T cells rejected skin grafts infrequently. However, E7 skin grafts were rejected frequently and rapidly by mice that received E7TCR tg T cells and were immunized with E7 protein (P = .001). Although presentation of E7 antigen by keratinocytes did not adequately induce an effector T-cell response, even when considerable numbers of antigen-specific CTL precursors were present, E7 antigen presentation by cells in the graft allowed rejection if sufficient CTL effectors were otherwise induced.
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To evaluate the minimum number of precursor E7-specific T cells required to induce K14E7 graft rejection after immunization, we adoptively transferred increasing numbers of E7 TCR tg splenocytes into E7-immunized recipients of K14E7 skin grafts (Fig. 3, A). Between 5 x 103 and 5 x 104 E7 TCR tg splenocytes, including 2002000 E7/Db tetramerbinding T cells, was sufficient to cause skin graft rejection after immunization. Immunization with cell numbers above this threshold was statistically significantly associated with decreased survival of E7 skin grafts (Ptrend = .03) (Fig. 3, A).
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We have previously shown that infection with Listeria monocytogenes facilitates induction of E7-specific immunity by naturally cross-presented antigens from skin grafts and induces E7 graft rejection (9), presumably as a result of activation of innate immune responses locally in the skin. We have also shown that innate immune responses are important for determining the fate of E7- expressing transplanted tumors (18). We therefore wished to establish whether nonspecific antigenrelated mechanisms invoked by activation of large numbers of antigen-specific T cells, including release of IFN- and consequent increased susceptibility of skin cells to T-cellmediated cytotoxicity (19), might contribute to E7 transgenic skin graft rejection following adoptive transfer of specific T cells and immunization. We therefore examined survival of K14E7 skin grafts following adoptive transfer of OVA-specific TCR tg T cells together with OVA immunization (Fig. 3, B). K14E7 skin grafts were not rejected by recipients of adoptively transferred OVA-specific T cells following OVA immunization, although this treatment facilitated rapid rejection of skin grafts expressing a minimal OVA CTL epitope from the K14 promoter (20) (data not shown). Rejection of skin grafts with tumor antigenexpressing epithelial cells thus appeared to be dependent on antigen-specific CD8+ effector T cells.
Activation of Transferred CD8+ T Cells and E7 Graft Rejection
To address the possibility that spleen cells transferred from HPV E7 TCR tg mice were enhancing E7-specific immunity induced by immunization in the recipient, we immunized graft recipients with E7 protein and Quil-A saponin adjuvant 4 weeks before transferring spleen cells from E7 TCR tg mice and performing skin grafts (Fig. 3, C). Survival of skin grafts placed on previously immunized mice was similar to survival of grafts on nonimmunized animals, suggesting that direct activation of transferred T cells by E7 immunization was necessary for graft rejection. Recipients of skin grafts and E7 TCR tg T cells were then immunized with either a minimal E7 CTL epitope peptide (GF001) (21) or a mutant E7 peptide (MT906) incorporating a B-celland H-2brestricted T helper epitope of E7 in which the phenylalanine anchor residue of the CTL epitope was replaced with lysine because the T helper and CTL epitopes overlap. MT906 peptide, in contrast to GF001, cannot induce E7-specific effector CTLs but induces E7-specific antibody responses in naive C57BL/6J mice (data not shown). Immunization with the E7 CTL peptide resulted in rejection of E7 transgenic skin grafts from recipients of E7 TCR tg T cells (Fig. 3, D). Immunization with peptide was as efficient as immunization with E7 whole protein, whereas immunization with MT906 did not produce graft rejection. Without adoptive transfer of E7 TCR tg T cells, immunization with the E7 CTL peptide could not cause rejection of E7 skin grafts. Together, these findings show that activation of a sufficient number of E7-specific CD8+ T cells is necessary and sufficient to induce rejection of epithelium expressing E7 in keratinocytes in an immunocompetent recipient mouse.
Induction of Tc1-Biased Effector CD8+ T Cells Following Combined Active/Passive Immunization
To compare the immune responses associated with rejection and with failure of rejection of E7-expressing skin, we tested spleen and peripheral blood cells from E7-immunized mice for IL-10, IL-5, and IFN- secretion in vitro in response to a minimal E7 CD8+restricted epitope (GF001) at day 7 after immunization (Fig. 4, A). Recipients of adoptively transferred T cells had at least 10-fold greater E7-specific IFN-
secreting effector CD8+ T cells in both spleen and peripheral blood cells than those treated with E7 immunization alone. In contrast, lymphocytes from mice treated with adoptive transfer alone secreted IL-10 but not IFN-
in response to GF001 stimulation in short-term in vitro culture. After 3 days of GF001 stimulation in vitro, cells from mice that received adoptively transferred T cells secreted more IL-10 and IL-5 than IFN-
, whereas immunized mice secreted IFN-
and not IL-5 or IL-10. Immunized recipients of adoptively transferred T cells secreted substantially more IFN-
than immunized mice and substantially less IL-5 and IL-10 than adoptive transfer recipients (Fig. 4, BD) Thus, E7 immunization in vivo increased IFN-
production and suppressed IL-5 and IL-10 secretion by E7-specific CD8+ T cells, and subsequent skin graft rejection was associated with increased numbers of IFN-
producing cells.
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We have previously shown that animals primed to reject E7 skin grafts by grafting and administration of L. monocytogenes develop E7-specific memory such that subsequent grafts are rejected without further immune manipulation (9). To establish whether functional E7-specific memory was similarly induced by immunization in mice that received E7 TCR tg T cells, second E7 skin grafts were placed 100 days after the first graft onto immunized recipients of adoptively transferred E7 TCR tg T cells. Of 11 TCR tg T-cell recipients that were also immunized with HPV E7, seven mice rejected the first E7 skin graft and only one rejected the second E7 skin graft. To confirm that memory CTLs were established following adoptive transfer of CTLs and immunization in these mice, spleen cells from immunized recipients of V12+ TCR tg T cells, and control mice were assayed after 200 days for E7 peptidespecific IFN-
secreting T cells (Fig. 5, A) and for V
12+ CD8+ T cells (Fig. 5, B) after restimulation in vitro, and the results were compared with those obtained from cells of mice immunized 6 days previously and similarly restimulated. E7-specific IFN-
secretion by splenocytes after in vitro culture with antigen was observed in the recipients of grafts, E7 immunization, and E7 TCR tg T cells, confirming that a substantial persistent memory CTL population was present in grafted recipients.
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DISCUSSION |
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In clinical trials, specific immunotherapies for cervical cancer and precancer have induced a range of E7-specific immune responses, including E7-specific CD8+ T-cell responses (8), but evidence for therapeutic efficacy of these responses is limited. Although transfer of effector CD8+ T cells has been effective for some patients with advanced melanoma (25,26), passive immunotherapy with CD8+ T cells has not been attempted for cervical cancer. Our current data, when taken together with the outcome of these clinical studies, suggest that effective immunotherapy for HPV-associated skin cancer in humans may require a combination of active and passive immunotherapy. This observation conflicts with the finding that a range of specific immunotherapies eliminates established E7-expressing transplantable murine tumors [reviewed in (8)]; thus, skin expressing E7 may be a more demanding target for immunotherapy than a transplantable tumor expressing E7. However, B16 melanoma, a transplantable tumor that presents only self-antigen, requires not only active/passive immunotherapy but also exogenous IL-2 for successful immunotherapy (27). Thus, the requirements for effective immunotherapy of epithelial tumors expressing only self-antigen would be predicted to be even more stringent.
Findings in the murine transplant model used in this study replicate findings in cervical cancer and chronic cervical HPV infection when the E7 protein of high-risk oncogenic HPV types is presented by epithelial cells over many years to immunocompetent hosts. Immune responses to the E7 protein of HPV16 are observed in invasive malignancy (22) and in association with cervical intraepithelial neoplasia. Whether persistent infection reflects failure of an effective immune response is not yet known. Immune suppression increases the risk of persistent HPV infection and its progression to malignancy during HIV infection (28) and following transplantation (29); cell-mediated responses to HPV E7 are associated with clearance of premalignant cells (30). However, in general, persistent infection also occurs in immunocompetent individuals and, as with the mouse skin graft model, does not lead to effective presentation of E7 protein, at least as measured by antibody induction (31). As with the skin graft model, induction of immunity following immunization produces both humoral and cell-mediated immunity in patients with vulval and cervical HPV infection (8) without, however, causing disease resolution in the majority of patients. Thus the available evidence suggests that the immunobiology of cervical cancer and premalignancy more closely mimics that of E7 skin grafts than that of E7-expressing transplantable tumors, although both express E7 mRNA and protein in approximately equal amounts (10).
Why is the combination of immunization and adoptive transfer of specific CD8+ T cells necessary for effective immunotherapy of skin expressing E7 but not for that of transplantable tumors that express the same amount of E7 protein (10)? The C57BL/6J mouse mounts an effective CD8+ T-cell response to the major dominant H-2Db epitope (aa sequence = RAHYNIVTF) of HPV16 E7 protein. However, larger numbers of effector T cells may be required to kill skin cells than tumor cells, perhaps because trafficking of effector cells to skin is less effective than to tumors (3234). Certainly, more effector cells are induced by combined therapy than by immunization alone. This explanation is supported by the ability of immunization to result in tumor but not graft rejection in the same animal (10). Alternatively or additionally, E7-specific CD8+ T-cell precursors may not respond optimally to immunization in an E7 graft recipient as a consequence of presentation of E7 from the graft. However, immunization of a graft recipient before, rather than after, grafting does not render the active immunotherapy more effective (10). The results presented here thus indicate a role for activated antigen-specific CD8+ T cells in skin immunotherapy. Adoptive transfer of immunocytes might also increase capacity to deliver an innate immune response, and innate immunity can, under restricted circumstances, be sufficient to invoke rejection of HPV16 E7-expressing transplantable tumors (18). However, our current data show that E7-expressing grafts were not rejected following transfer and specific activation of OT-1 T cells, which recognize an OVA peptide. Thus, transferred innate immune response effectors, even when activated by co-administered antigen-activated T cells, are insufficient to induce graft rejectionthat is, T cells specific for an antigen expressed by graft keratinocytes are required.
The fate of grafts expressing HPV16 E6 and E7 in keratinocytes was markedly different than that of grafts similarly expressing two secreted proteinsOVA from the keratin 5 promoter (17) and hGH, from the K14 promoter; these were promptly rejected, though rejection of hGH grafts is not observed on all genetic backgrounds (14). Presentation of antigen by professional antigen-presenting cells is necessary (35,36) to induce an effective CD8+-restricted immune response but can result in functional tolerance (36,37). The amount of cross-presented antigen can determine whether immunity or tolerance is induced (38), and E6 and E7 are expressed at relatively lower levels than hGH in our model (data not shown). However, a secreted antigen is better at inducing tolerance than a non-secreted antigen (39), which is somewhat inconsistent with our findings. Systemic L. monocytogenes infection (9), induced at the time of grafting E7 skin, allows induction of specific effector responses that eliminate transgenic skin grafts, presumably by modulating cross-presentation through the induction of a wide range of proinflammatory cytokines (40,41). This result implies that the consequence of presentation of antigen is determined less by the amount of cross-presented antigen than by the environment and site. Chronic viral antigen exposure results in preferential induction of CD8+ T cells with a Tc2 phenotype (42,43). Use of an expanded antigen-specific CD8+ T-cell population in this model demonstrates that specific CD8+ T cells have a Tc2 phenotype before antigen challenge by immunization, secreting IL-10 and IL-5 but not IFN-, whereas after immunization, the phenotype is dominated by IFN-
secretion and is permissive for rejection. Thus, successful active and passive immunotherapy may be a consequence of a change of polarity of the specific immune response from Tc2 to Tc1, and the need for large numbers of precursors may reflect less efficient trafficking of effectors to skin than to other sites. The absence of second graft rejection in recipients of active and passive immunotherapy demonstrates that the newly placed grafts did not adequately cross-present E7 antigen to the circulating memory CTL population. However, mice that initially received a graft, TCR tg T cells, and immunization, when given further E7 immunization at the time of a second graft, rejected the second graft faster than they rejected the first graft (Fig. 6, B), demonstrating that the grafts themselves present antigen directly to effector T cells, once they have been induced.
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Overall, our data support a model for the consequences of peripheral antigen presentation in which a critical determinant of outcome is the amount of antigen cross-presented by professional antigen-presenting cells, as has been observed for induction of tolerance when antigen is cross-presented at different levels (44). Nonself-antigens sequestered within cells are not effectively cross-presented to naive or memory T cells, even when presented in the relatively pro-inflammatory environment of a skin graft, although stronger inflammatory signals, including infection with live L. monocytogenes, can overcome this limitation to antigen cross-presentation. Ignorance of or functional tolerance to antigens presented by skin cells, even in the face of moderate local inflammation, will thus persist. However, if antigen cross-presentation in an appropriately inflammatory environment elsewhere can induce an adequate population of effector T cells, then direct presentation of antigen, even in a less inflammatory environment, will allow epithelial cell destruction.
Interestingly, the memory effector T-cell population induced by grafting and co-administration of L. monocytogenes (9) differs substantially from that induced by adoptive transfer and immunization described in the current experiments, in that the L. monocytogenesinduced memory T-cell response could induce rejection of a second graft without further immune manipulation of the recipient mouse, whereas the memory response induced by adoptive transfer and immunization required further activation by immunization at the time of second grafting to induce graft rejection. One difference between the models is that the L. monocytogenesinvoked response is more likely to include a CD4+-restricted memory component than adoptive transfer and priming of a CD8+ TCR tg T-cell population. This difference is important because CD4+ memory is important for long-term immune effector function against tumors (45). Ongoing studies using the skin graft model may thus further clarify requirements for effective immunotherapy for human skin tumors.
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NOTES |
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Funded by grants from the National Institutes of Health (NIH), Department of Health and Human Services; the National Health and Medical Research Council of Australia; the Cancer Research Institute of New York; the Queensland Cancer Fund; and the Princess Alexandra Hospital Foundation. Tetramers were provided by the National Institute of Allergy and Infectious Diseases, NIH, tetramer production facility. K. Matsumoto was supported by a grant from the University of Tokyo.
We thank David Wiseman and Caron Maxim for their assistance with the mice used in the study.
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Manuscript received February 19, 2004; revised August 17, 2004; accepted August 31, 2004.
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