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The Journal of Immunology, 2001, 167: 6180-6187.
Copyright © 2001 by The American Association of Immunologists

Tolerance or Immunity to a Tumor Antigen Expressed in Somatic Cells Can Be Determined by Systemic Proinflammatory Signals at the Time of First Antigen Exposure1

Ian H. Frazer2,*, Rachel De Kluyver*, Graham R. Leggatt*, Hua Yang Guo3,*, Linda Dunn4,*, Olivia White*, Craig Harris*, Amy Liem{dagger} and Paul Lambert{dagger}

* Centre for Immunology and Cancer Research, University of Queensland, Brisbane, Australia; and {dagger} McArdle Institute for Cancer Research, Madison, WI 53706


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mice transgenic for the E7 tumor Ag of human papillomavirus type 16, driven from a keratin 14 promoter, express E7 in keratinocytes but not dendritic cells. Grafted E7-transgenic skin is not rejected by E7-immunized mice that reject E7-transduced transplantable tumors. Rejection of recently transplanted E7-transgenic skin grafts, but not of control nontransgenic grafts or of established E7-transgenic grafts, is induced by systemic administration of live or killed Listeria monocytogenes or of endotoxin. Graft recipients that reject an E7 graft reject a subsequent E7 graft more rapidly and without further L. monocytogenes exposure, whereas recipients of an E7 graft given without L. monocytogenes do not reject a second graft, even if given with L. monocytogenes. Thus, cross-presentation of E7 from keratinocytes to the adaptive immune system occurs with or without a proinflammatory stimulus, but proinflammatory stimuli at the time of first cross-presentation of Ag can determine the nature of the immune response to the Ag. Furthermore, immune effector mechanisms responsible for rejection of epithelium expressing a tumor Ag in keratinocytes are different from those that reject an E7-expressing transplantable tumor. These observations have implications for immunotherapy for epithelial cancers.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Somatic cell tumors frequently express tumor-specific protein Ags (TA),5 including mutated self-proteins, virus-derived proteins, and cell lineage-specific proteins. Self-Ag expressed only in somatic cells does not invoke tissue-destructive immune responses when cross-presented to cognate immunocytes by professional APCs (1). Tolerance or ignorance of cross-presented self-Ag, however induced, is presumably a mechanism for avoiding autoimmune tissue damage (2). A spontaneous TA-specific immune response would involve cross-presentation of the TA (3), and, by analogy with self-Ag, tolerance or ignorance of TA might therefore be expected. Spontaneous immune responses to TA are uncommon, although TA are generally immunogenic if delivered with appropriate adjuvants or costimuli in trials of tumor immunotherapy (4), suggesting that TA cross-presentation generally leads to ignorance rather than tolerance of TA. Induction of TA-specific immunity in humans does not generally eliminate the TA-expressing tumor (5), although there are notable exceptions (6). A similar discrepancy is reported between the immunogenicity of some viral antigenic epitopes and the effectiveness of host protective immunity induced to those epitopes (7). Discrepancies between TA immunogenicity and induction of host protective immunity could represent failure of immunization to induce an appropriate quality of immune response, perhaps on a background of partial tolerance induced by cross-presentation. Alternatively, as tumor cells often have impaired Ag presentation machinery and tumor-directed local immunosuppression prevents effector functions (8, 9), the immune response induced to TA might be appropriate, but tumor-specific factors might prevent elimination of the target tumor.

To examine the immune response necessary to eliminate tumors, and hence confirm whether immunotherapy induces an appropriate quality of immune response, models based on transplantable tumors incorporating model Ags are widely used (10). Transplantable tumors grow rapidly, so that effector responses have to be induced before or just after tumor transplantation to achieve an immune response before tumor growth requires termination of the experiment. This model therefore cannot mimic immunotherapy of a spontaneously arising tumor in humans. We have developed a model system in which a TA, the E7 protein of human papillomavirus (HPV) type 16, is expressed as a transgene in nontransformed somatic cells expressed from the keratin 14 (K14) promoter. Transgenic animals (11, 12) develop skin cancers and are partially tolerant of the E7 protein (13). To allow examination of the effects of E7-specific immunity on cells expressing E7, in an animal naive to E7, we grafted skin from K14E7-transgenic mice to nontransgenic recipients (14). Such grafts mimic HPV-associated anogenital precancers and cancers, in which the E7 proteins of oncogenic HPVs are expressed in epithelial cells (15). Previous studies with this model have shown that E7 graft rejection does not occur spontaneously, in contrast to grafts of skin exhibiting other minor transplantation Ag (MTA) differences with the graft recipient (16, 17) Allelic protein variants that can function as MTA are, unlike E7 in the K14E7 mouse, expressed in bone marrow-derived dendritic cells (BMDC), and expression in BMDC of the donor but not the recipient is held to be the determinant of whether a protein is recognized as a MTA (18). Thus, the observed failure of E7-transgenic graft recipients to reject their graft might be expected. More unexpected was the failure of immunization of graft recipients with E7 to induce graft rejection, despite induction of an E7-specific T helper and cytotoxic T cell response sufficient to prevent the growth of E7 transplantable tumors (19). These results suggested that the immune response required to eliminate an E7-expressing transplantable tumor was different from that required for elimination of skin cells expressing the same E7 Ag.

Modulation of professional APC function by bacterial or cellular products at the time of Ag presentation is a determinant of the development of a host-protective immune response (20, 21, 22, 23, 24). As Listeria monocytogenes administration has previously been demonstrated to activate autoantigen-specific T cells (25) and to overcome the ignorance of transgene-encoded Ags expressed in somatic cells (26), we therefore examined the effects of L. monocytogenes coadministration on the outcome of grafts of E7-transgenic skin to naive syngeneic recipients, to determine whether L. monocytogenes exposure could allow development of an E7-specific immune response able to effect graft rejection. We observed that L. monocytogenes exposure facilitated E7 graft rejection and induced a memory effector response enabling rejection of subsequent E7 grafts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Specific pathogen-free FVB (H-2q) mice, FVB mice homozygous for an HPV16E7 transgene expressed from the K14 promoter (K14E7) (27), and (FVB x C57BL/6J)F1 and (K14E7 x C57BL/6J)F1 mice were obtained from the Animal Resources Center (Perth, Australia). Female mice aged 4–12 wk of age were used as donors and recipients, and were maintained under conventional conditions in specific pathogen-free holding rooms in the Princess Alexandra Hospital Biological Resources Facility (Queensland, Australia). Protocols were approved by the institutional animal ethics committee.

Proteins and peptides

The sequence and synthesis of a series of overlapping peptides (GF101–109) spanning the E7 protein of HPV16, peptide BT12D, which contains three B epitopes and the universal Th epitope (DRAHYNI) of HPV16, and a control peptide (GF110), which contains a HIV-1 gp120 B epitope and a Th epitope from HPV16E7, have previously been described (12, 28). A GST HPV16E7 fusion protein was prepared in Escherichia coli as previously described (29).

Bacteria and bacterial proteins

L. monocytogenes (NCTC 11994) was routinely cultured on blood agar plates and produced {beta}-hemolytic colonies after incubation at 37°C in air. Bacteria were enumerated by limiting dilution in isotonic saline and culture on blood agar plates. "Killed" L. monocytogenes stocks were heat treated for 20 min at 121°C. Purified LPS from E. coli serotype O55:B5 was purchased from Sigma-Aldrich (L-2637; St. Louis, MO) and reconstituted with sterile isotonic saline.

Skin grafting

Whole-thickness flank skin grafting has previously been described (19). Whole-thickness ear skin grafting represents a modification of the donor tissue preparation method. Briefly, whole ears were prepared by excision of both ears from donor mice. Dorsal and ventral surfaces were then separated and underlying cartilage was removed by scraping. Grafts were assessed as technically successful if adherent and vascularized on day 8. Grafts were observed at least three times weekly for the duration of experiments.

Donor and recipient treatments

Suspensions of live (5 x 104) or killed (5 x 106) L. monocytogenes were administered into the lateral tail vein. LPS was administered i.p. (100 IU) from days 8 to 21 after grafting.

HPV16 E7-specific immunity

Ab to HPV16E7 peptides and E7 protein was measured by ELISA as previously described (30). Delayed-type hypersensitivity (DTH) to E7 protein was measured by ear challenge (31) using 10 µg of GST HPV16E7 fusion protein. Ear swelling was reported as the difference in thickness between the challenged and the control ear, 48 h after challenge. Mice were immunized with E7 protein with Quil A saponin as previously described (32) Tumor challenges with the EL4-derived C2 cell line (33) were conducted as previously described (34). E7-specific CTL responses were assayed in a standard 4-h lytic assay using C2 cells as targets and splenocytes as effectors, as previously described (11).

Presentation of E7 by dendritic cells (DC)

Briefly, BMDC were prepared from bone marrow collected from mouse femurs. RBC were removed using ACK lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM EDTA, pH 7.3). Cells were placed on plastic for 2 h, and nonadherent cells were collected and cultured with GM-CSF (10 ng/ml; Sigma-Aldrich) and IL-4 (10 ng/ml; Sigma-Aldrich) which was replenished on days 2, 5, and 7. On day 9 of culture expression, CD11c was assessed via flow cytometry using anti-CD11c. Cultured cells had DC morphology and were 50–70% CD 11c positive. Activation by BMDC of an H-2Db- restricted E7-specific CD8+ line6 specific for the major Db-restricted CTL epitope of E7(RAHYNIVTF) was assessed by IFN-{gamma} release. E7-specific cells (50,000/well) were exposed to varying numbers of BMDC for 48 h. Some BMDC were exposed to 0.01 µM RAHYNIVTF peptide for 2–3 h and washed extensively before assay. Released IFN-{gamma} was measured by ELISA. Plates were coated with 2 µg/ml anti-murine IFN-{gamma} capture Ab (R4-6A2; BD PharMingen, San Diego, CA) in bicarbonate binding buffer (pH 8.2) and blocked with 3% skim milk powder. Samples were added and held overnight at 4°C. Biotin-labeled detection Ab (XMG1.2; BD PharMingen) was added at 1 µg/ml. Plates were washed and developed with avidin-HRP followed by o-phenylenediamine substrate (Sigma-Aldrich). OD values were assessed at OD490.

Measurement of E7 protein and mRNA

HPV16 E7-specific mRNA was measured by real-time PCR of reverse transcriptase generated cDNA using the PerkinElmer Taqman system (Wellesley, MA). RNA was extracted from tissue samples stored at -70°C using TRIzol reagent according to the manufacturer’s instructions, treated with DNase (Pharmacia, Peapack, NJ), and RNA concentration established by spectrophotometry. PCR of 150 ng of mRNA was conducted with the Gene Amp RNA PCR Core kit (PerkinElmer) according to the manufacturer’s instructions using the oligo(dT) primers included in the kit, and E7-specific forward primer (5'-GAACCGGACAGAGCCCATTA-3'), reverse primer (5'-CGAATGTCTACGTGTGTGCTTTG-3'), and probe (5'-ATTGTAACCTTTTGTTGCAAGTGTGACTCTACGC-3'). Serial dilutions of plasmid pHPV16 were performed to create a series of standards ranging from 108 to 102 copies/µl, and E7 mRNA estimations were based on a standard curve derived from the results obtained with these standards. E7 protein was measured by ELISA as previously described (29).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Rejection of skin grafts expressing E7 TA in keratinocytes (KC) is enabled by L. monocytogenes infection

Epithelial grafts expressing the HPV16 E7 tumor-specific neoantigen in KC are not rejected when grafted to a naive recipient (19). To establish whether such grafts could be rendered susceptible to rejection by the delivery of a potent nonspecific inflammatory stimulus at the time of grafting, L. monocytogenes were administered to recipients of skin grafts transgenic for the E7 oncoprotein of HPV16, expressed from a K14 promoter. FVB (H-2q) and (FVB x C57BL/6J)F1 (H-2qxb) mice were infected with L. monocytogenes 8 days after grafting with E7-transgenic and control nontransgenic grafts from H-2q FVB mice. Graft outcome was observed for 100 days (Fig. 1GoA). Grafts expressing E7, but not control grafts, were rejected by animals infected with L. monocytogenes, whereas, as previously described, no E7 or control graft rejection was observed from uninfected animals. To establish whether enhancement of graft rejection required live L. monocytogenes, graft recipients were alternatively treated with killed L. monocytogenes or with E. coli endotoxin (Fig. 1GoB). A single administration of killed L. monocytogenes or repeated administration of LPS also facilitated graft rejection, and similar rejection kinetics were observed to those observed in animals treated with live L. monocytogenes. Thus, at least two distinct proinflammatory bacterial products promote rejection of skin grafts transgenic for a neoantigen expressed in KC.



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  		FIGURE 1. A, Kaplan-Meier analysis of survival of E7-transgenic skin grafts on naive otherwise syngeneic recipients. Mice with healthy grafts at day 8 were included in the survival analysis. Graft recipients were untreated ({triangleup}) or alternatively were treated with L. monocytogenes i.v. on day 8 after grafting ({circ} and {square}). Control syngeneic grafts were also assessed and survived indefinitely on all recipients. All grafts were from female donors to female recipients. FVB mice were grafted with K14E7. FVB skin ({square}) and (FVB x C57BL/6J)F1 mice were grafted with (K14E7. FVB x C57BL/6J)F1 skin({circ}). B, Kaplan-Meier analysis of survival of (K14E7 x C57BL/6J)F1-transgenic grafts on (FVB x C57BL/6J)F1 recipients treated with live ({circ}) or killed ({square}) L. monocytogenes on day 8 after grafting or with endotoxin from days 8 to 21({triangleup}).

 
TA presentation from skin grafts is indirect

As the K14 promoter is held to be active only in epithelial KC, it is likely that the presentation of E7 from skin following grafting is indirect, occurring via graft or host-derived DC. To exclude direct presentation of E7 by BMDC of graft origin, E7 mRNA was measured in BMDC from E7-transgenic FVB mice by real-time PCR (Table IGo), and no signal could be obtained after 45 cycles of amplification, corresponding to <10 copies of E7-specific mRNA per cell. In further real-time PCR experiments, background signal was consistently detected with E7 primers in samples from E7-transgenic and -nontransgenic epithelium after the same number of cycles of amplification. Confirmation that E7 was not presented by BMDC from K14E7 mice was sought using an E7-specific H-2Db-restricted CTL line. This line lyses E7-transduced EL4 thymoma cells (C2), but not EL4 cells, and is activated to secrete IFN-{gamma} by DC pulsed with 10-10 M E7 peptide (Fig. 2Go). The line was, however, not activated by BMDC from K14E7-transgenic mice. Thus, insufficient E7 is presented by DC from K14E7 mice to activate a high-affinity E7-specific CTL line.


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  		Table I. Expression of the E7 transgene in skin and tumors

 


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  		FIGURE 2. Failure of DC from K14E7-transgenic animals to present endogenous E7. BMDC from (K14E7 x C57BL/6J)F1 mice or (FVB x C57BL/6J)F1 mice were exposed at the shown E:T ratio to a CD8 T cell line specific for the dominant H-2b-restricted CTL epitope of E7 (RAHYNIVTF). In some cases ({blacksquare} and •) the DC were exposed to RA HYNIVTF peptide and washed before addition of CTL. Activation of the CTL line by presented E7 was measured as secreted IFN-{gamma}.

 
Graft rejection enabled by L. monocytogenes primes for second set rejection

Induction of an E7-specific immune response by immunization with E7 and Quil A induces rejection of an E7-transduced tumor (34), but is, in contrast, unable to promote rejection of E7-expressing skin grafts (19). L. monocytogenes infection might therefore enhance rejection of E7-transgenic skin grafts either by promoting induction of a qualitatively different E7-specific immune response from that induced by immunization with E7 and Quil A, or alternatively by rendering an E7-transgenic graft more susceptible to the effects of an E7-specific immune response. To distinguish these possibilities, mice grafted with E7-transgenic skin and exposed to live or killed L. monocytogenes were given a second graft, either with or without further killed L. monocytogenes. Mice that had previously successfully rejected an E7-expressing graft were generally able to reject a further graft (Table IIGo), with or without further L. monocytogenes exposure. Furthermore, second graft rejection occurred significantly more rapidly than first graft rejection (Kaplan-Meier analysis; p < 0.01), as might be expected in a primed animal (Fig. 3GoA). These data demonstrate that a consequence of exposure to E7 grafts and L. monocytogenes, that is not induced by E7 grafting alone or by immunization with E7 and Quil A, is induction of an E7-specific memory response of the type required for graft rejection. Furthermore, once memory is induced, no local L. monocytogenes-mediated effect is required for subsequent graft rejection.


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  		Table II. Outcome of second grafts to mice previously grafted

 


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  		FIGURE 3. A, Kaplan-Meier analysis of the survival of second grafts according to first graft outcome ({circ} and {square}: rejected; {diamond} and {triangleup}: not rejected) and treatment with second graft ({circ} and {triangleup}: L. monocytogenes; {square} and {diamond}: no L. monocytogenes). Second graft rejection in mice that had rejected a first graft was significantly more rapid (p < 0.01) than first graft rejection. B, Kaplan-Meier analysis of the survival of grafts of (K14E7 x C57BL/6J)F1-transgenic grafts on (FVB x C57BL/6J)F1 recipients treated with killed L. monocytogenes on day -4 ({triangleup}), +8 ({square}), or +15 (*) after grafting.

 
Graft rejection does not correlate with TA-specific immunity

To establish whether any particular type of E7-specific immune response could be associated with graft rejection, Ab to E7 was measured by ELISA and DTH to E7 was measured by ear challenge in mice that had rejected or failed to reject skin grafts (Table IIIGo). Significant Ab responses to a major N-terminal epitope of E7 were seen after grafting only among mice that had received live L. monocytogenes, but there was no significant correlation between the magnitude of the E7-specific immune response and the outcome of the first graft. Ab to E7 detectable after a first graft was, however, a predictor of the outcome of a second graft. Of 78 animals grafted twice, 32 rejected a second graft, and these all had low level Ab to E7 after the first graft (mean E7-specific OD, 0.192 ± 0.059; Fig. 4Go). In contrast, 46 mice failed to reject a second graft, and 12 had significant Ab to this E7 epitope (mean OD, 0.671 ± 0.143; p < 0.01), suggesting that induction of Ab to E7 was one feature of an E7-specific immune response not capable of inducing graft rejection. DTH reactions to E7 protein in all groups of E7 grafted mice were weak in comparison to the DTH induced by specific immunization with E7 and adjuvant. E7 DTH reactions were slightly but significantly greater in mice that had received live as opposed to killed L. monocytogenes, regardless of graft outcome (Table IIIGo). No correlation of E7 DTH with outcome of a first or second graft was observed (data not shown). Grafting of E7-transgenic skin and coadministration of L. monocytogenes can thus induce both humoral and cellular immunity to the E7 protein of HPV16 in the graft recipient, but induction by grafting of an E7-specific immune response does not predict the outcome of the graft.


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  		Table III. E7-specific DTH and Ab responses in recipients of E7-transgenic skin grafts

 


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  		FIGURE 4. Titer of Ab to E7 after a first E7-transgenic graft predicts outcome of a second graft. Ab to E7 was measured by ELISA at day 100 after a first K14E7-transgenic skin graft, and mice were then regrafted with K14E7-transgenic skin and followed for 100 days to determine second graft outcome.

 
L. monocytogenes enables graft rejection about the time of grafting

The presented data indicate that coadministration of L. monocytogenes to an E7-transgenic graft recipient is frequently sufficient to induce graft rejection. When L. monocytogenes was coadministered with a second graft to animals that had received a first graft along with L. monocytogenes (Table IIGo), the second E7-transgenic graft, but not a second control graft, was rejected as expected. However, animals that had not rejected the first E7-transgenic graft despite L. monocytogenes coadministration did not reject the first graft after the second administration of L. monocytogenes with a second graft, even if the second graft was rejected. These data suggest that the E7-specific immunity induced by an E7 graft and L. monocytogenes is only able to facilitate rejection of a recently placed graft. To confirm this observation, mice were grafted with E7-transgenic skin and treated with L. monocytogenes at various time points in relation to grafting. Administration of L. monocytogenes enhanced graft rejection if given 4 days before, or 8 days after, grafting (Fig. 3GoB), but no effect was seen on graft rejection when L. monocytogenes was given 21 days before or 15 days after grafting, confirming that only recently placed E7-expressing grafts were susceptible to rejection. Since it was possible that this observation might reflect diminished E7 Ag expression in mature grafts, grafts were excised at various time points after grafting and examined for E7 mRNA using quantitative real-time PCR. Expression of E7 mRNA was very similar in skin prepared for grafting and in grafts harvested between 90 and 270 days after grafting (Table IGo), confirming that a difference in E7 expression was unlikely to account for the failure of L. monocytogenes to enhance rejection of established E7-transgenic grafts.

Grafting without L. monocytogenes coadministration primes for tolerance

To establish whether exposure of mice to E7 grafts without L. monocytogenes coadministration altered their response to a further exposure to an E7 graft, mice given an E7 graft without L. monocytogenes were given a second graft after 100 days, with or without coadministered L. monocytogenes. This experiment should model immunotherapy for most human cancers in which cross-presentation of the tumor Ag to the host immune system would occur before immunotherapy. No rejection of a second E7-transgenic graft was observed among 10 FVB mice that had previously received an E7-transgenic graft without L. monocytogenes, even although these mice were given killed L. monocytogenes at day 8 after the second graft. In contrast, 16 of 19 previously ungrafted mice given an E7 graft, and given killed L. monocytogenes at day 8 after grafting, specifically rejected the E7-transgenic graft (Fisher’s exact test, p < 0.0001). Thus, prior exposure to E7 from a graft given without L. monocytogenes treatment prevents L. monocytogenes from inducing rejection of that or a subsequent E7 graft (Table IVGo).


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  		Table IV. L. monocytogenes is unable to enhance rejection of grafts placed on animals previously grafted with E7-transgenic skin

 
Rejection of grafts and tumors expressing E7 TA

Immunization of E7-grafted animals with E7 protein and Quil A induces Ab, DTH and CTL specific for E7 protein (Table VGo), and the immune response to E7 is not different from that seen in nongrafted littermate controls (19). (FVB x C57BL/6J)F1 recipients of an E7-transgenic (FVB x C57BL/6J)F1 skin graft were therefore immunized with E7 and Quil A and challenged with an E7-transduced EL4 (H-2b) tumor line(Table VGo), expressing similar amounts of E7 protein to E7-transgenic keratinocytes (Table IGo), to determine whether, in the same animal, an E7-specific immune response that could prevent growth of a transplantable tumor could result in rejection of an E7-transgenic skin graft. Of 10 E7-immunized mice that received an E7 graft and E7 tumor challenge, 8 rejected the E7-positive tumor transplant, though none rejected the graft. The rate of tumor rejection was no different from that seen previously in E7-immunized but ungrafted animals (34). Thus, the E7-specific immune response required to prevent growth of an E7-expressing transplantable tumor is different from that required to reject an E7-transgenic skin graft. Furthermore, placement of an E7 graft without coadministration of L. monocytogenes, which prevents induction by grafting and L. monocytogenes of an E7-specific immune response that can reject a further skin graft, does not prevent induction by immunization of an E7-specific response capable of rejecting an E7 tumor.


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  		Table V. E7-specific immune response, graft, and tumor rejection after immunization with E7

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The current data demonstrate that the E7 protein of HPV 16, when expressed as a transgene in keratinocytes, is only weakly immunogenic, as measured by Ab and DTH responses, when E7-transgenic skin is applied to a naive host. However, delivery to the host of a potent proinflammatory stimulus at the time of first E7 Ag exposure-induced specific rejection of an E7-transgenic graft. In contrast, immunization with E7 protein induces high-affinity E7-specific CTL and is sufficient to prevent implantation of an E7-transgenic transplantable tumor (34), but does not result in rejection of an E7-transgenic skin graft (19), suggesting that the mechanisms of E7-restricted skin graft and tumor rejection may differ. HPV16 E7 is expressed in epithelial cells in HPV-associated anogenital cancers and their precursor lesions in humans (15), and is manifestly not presented effectively to the tumor-bearing host (35). E7-specific immunotherapy of patients with cervical precancer (CIN3) induces E7-specific CTL without resolution of the disease (36, 37, 38). Thus, E7-transgenic skin grafts may model cervical cancer immunology more reliably in the mouse than E7-transplantable tumors, which are relatively easily rejected (39, 40, 41).

Skin grafts expressing a neoantigen transgene are generally rejected, probably because the viral (42, 43) or neoantigen (16) expressed in the graft is driven from a promoter active in APCs, whereas in the current model E7 mRNA expressed from the K14 promoter was, as expected, below the threshold of detection in bone marrow-derived cells as judged by real-time PCR. Minor transplantation Ags responsible for graft rejection represent a small immunogenic subset of all polymorphic proteins (17). Those that have been characterized (44, 45, 46) are expressed widely in somatic cells, including APCs and are most immunogenic when uniquely expressed on the donor rather than recipient tissue. Thus, minor transplantation Ags, like the transgenes in previous skin graft models and unlike TA or E7 in the current experiments, are presented directly to T cells by MHC-matched APC, and this appears to be crucial for their immunogenicity (46). TA and E7 protein in contrast rely on cross-presentation for immunogenicity (3). Thus, absence of an immune response to TA could be a consequence of a lack of immunogenic epitopes within the TA, which might, by analogy with the small subset of allelic variant proteins that are effective minor transplantation Ags, be a common occurrence. Alternatively, failure of effective TA cross-presentation might explain the lack of immunogenicity. This latter explanation seems more likely to explain the lack of spontaneous graft rejection in the current model as a proinflammatory stimulus such as L. monocytogenes is less likely to alter the number of immunogenic epitopes in a protein than to improve cross-presentation (47), and proinflammatory stimuli have been shown to enhance Ag cross-presentation (48).

The HPV16 E7 transgene in the current study is a well-characterized viral Ag, immunogenic in a wide variety of mouse strains, and with well-characterized B (49), Th (28), and CTL (41) epitopes, though CTL epitopes have only been identified on one murine MHC background (H-2b) and are apparently absent from at least two other backgrounds including H-2q (50). Furthermore, E7 expressed as a transgene in skin becomes immunogenic following local inflammation (12) and may be tolerogenic when expressed without inflammation (51). Thus, lack of immunogenicity of E7 expressed as a transgene in keratinocytes following grafting seems unlikely to be due to an inherent lack of antigenicity of the protein. Rejection of a primary E7 graft following exposure to L. monocytogenes could reflect a number of Ag-specific, or nonspecific, mechanisms, as previously demonstrated for other Ags at other sites using transgenic L. monocytogenes (47). Specific T cell-mediated rejection would be favored by the accelerated rejection of a second E7-transgenic graft from mice that have previously rejected an E7-transgenic graft. This observation demonstrates specific memory and thus adaptive immunity. One effect of L. monocytogenes given at the time of exposure to an E7-transgenic skin graft is therefore to facilitate effective Ag cross-presentation of E7 protein produced in keratinocytes, presumably by skin-derived Langerhans cells, as bacterial products are known to enhance Ag presentation by DC in vitro and in vivo (52, 53, 54). Effective cross-presentation might either increase the quantity or alter the quality of the E7-specific immune response. L. monocytogenes infection profoundly modulates host immune responses through IL-12 production (55) and allows cross-presentation into the class I pathway (56, 57), and hence alters the balance of specific immunity from Th2 like to Th1 like (58). As killed L. monocytogenes and chronic exposure to endotoxin achieve similar results to live L. monocytogenes in our model, induction of secretion of proinflammatory cytokines including IL-12 and IL-1 by macrophages and monocytes seem more likely to be an explanation for enhanced Ag cross-presentation than a direct effect of listerolysin. Rejection of E7-transgenic grafts was observed in donor-host pairs in which H-2q was the only MHC restriction element, and there are no MHC class I H-2q-restricted epitopes in the E7 protein (50). Further studies are underway to confirm the inference from these observations that rejection is MHC class II restricted, which would allow a hypothesis that graft rejection is enhanced by L. monocytogenes as a consequence of MHC class II-restricted cross-presentation, and the consequent induction of a Th1 type and MHC class II-restricted immune effector response to E7.

Although specific immunity to E7 apparently determines graft outcome in the current experiments, there was no correlation between the measured immune responses to E7, induced by grafting and L. monocytogenes exposure, and the outcome of the graft. This observation is paralleled in other tumor systems, both in animal models and in man (59) and has led to a search for better surrogate markers for effective tumor immunity (60, 61). Induced DTH to E7 at a low level was associated with administration of live but not killed L. monocytogenes, confirming the enhancing effect of live L. monocytogenes on development of Th1 type immune responses (58). The lack of correlation of measurable DTH with rejection confirms our previous findings that immunization capable of inducing Ab, DTH, and CTL reactivity to E7 nevertheless fails to promote graft rejection (19), although allowing tumor rejection. IgG2a Ab to E7 was no commoner in mice that successfully rejected grafts than in nonrejectors (data not shown), suggesting that a Th1 bias to the E7-specific immune response induced by L. monocytogenes was not a major contributor to L. monocytogenes-mediated effective Ag cross-presentation. Because development of Ab in the current study after grafting was associated with failure of rejection of a second graft, Ab itself may be immunomodulatory on the process of graft rejection, and this hypothesis will be tested through passive transfer experiments.

L. monocytogenes-mediated enhancement of Ag cross-presentation may be restricted to a window of opportunity around the time of grafting, as L. monocytogenes is unable to promote the rejection of established grafts, which are tolerogenic, as is E7-transgenic skin in the donor mice (11, 51, 62). Thus, newly placed grafts are more vulnerable to rejection and L. monocytogenes may act both to increase E7 presentation from a graft and to increase new graft susceptibility to an effector response. In new graft tissue, remodeling occurs and hence more apoptotic and necrotic cellular material will be available for DC uptake and cross-presentation (63, 64). Quantity of presented Ag is rate limiting for induction of immune effector function (65). Trafficking of Ag-laden DC from a newly applied graft is also likely to be enhanced during the inflammation and repair process, and this would promote cross-presentation. Lack of effective presentation of a TA in the absence of both systemic and local proinflammatory stimuli may thus limit development of tumor-specific immunity, as tumor Ag is generally presented for a long period in the absence of inflammation. Conversely, the present data support the hypothesis (26) that local inflammation, along with a profound systemic immunological upset at the time of first exposure of a naive immune system to a potential autoantigen, could play a major part in effective cross-presentation of a self-reactive Ag to generate a tissue destructive autoimmune response.


   Acknowledgments
 
The expert assistance of David Wiseman with animal care is gratefully acknowledged.


   Footnotes
 
1 This work was funded in part by grants to the authors from the National Institutes of Health (RO-1 CA 57789), 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. Back

2 Address correspondence and reprint requests to Dr. Ian Frazer, Center for Immunology and Cancer Research, Princess Alexandra Hospital, Woolloongabba, Queensland 4102, Australia. E-mail address: ifrazer{at}medicine.pa.uq.edu.au Back

3 Current address: Department of Dermatology, All Union Medical College, Beijing, China. Back

4 Current address: Queensland Institute of Medical Research, Queensland, Australia. Back

5 Abbreviations used in this paper: TA, tumor antigen; DC, dendritic cell; BMDC, bone marrow DC; DTH, delayed-type hypersensitivity; HPV, human papillomavirus; KC, keratinocyte; K14, keratin 14; MTA, minor transplantation Ag. Back

6 G. R. Leggatt, L. A. Dunn, R. L. De Kluyner, T. Stewart, and I. H. Frazer. Interferon-{gamma} enhances CTL recognition of endogenous peptide in keratinocytes without lowering the requirement for surface peptide. Submitted for publication. Back

Received for publication June 4, 2001. Accepted for publication September 24, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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