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The Journal of Immunology, 2001, 167: 4853-4860.
Copyright © 2001 by The American Association of Immunologists

Immune Responses to the HLA-A*0201-Restricted Epitopes of Tyrosinase and Glycoprotein 100 Enable Control of Melanoma Outgrowth in HLA-A*0201-Transgenic Mice1

David W. Mullins, Timothy N. J. Bullock, Teresa A. Colella, Valentina V. Robila and Victor H. Engelhard2

Department of Microbiology and Carter Immunology Center, University of Virginia, Charlottesville, VA 22908


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Many of the Ags recognized by human melanoma-reactive CTL are derived from proteins that are also expressed in melanocytes. The possibility of self-tolerance to these epitopes has led to questions about their utility for antitumor immunotherapy. To investigate the issue, we established a preclinical model based on transgenic mice expressing a recombinant HLA-A*0201 molecule and B16 melanoma transfected to express this molecule. HLA-A*0201-restricted epitopes from the melanocyte differentiation proteins (MDP) tyrosinase and gp100 are expressed in both tumor cells and melanocytes, and the former is associated with self-tolerance. However, adoptive transfer of tyrosinase or gp100-reactive CTL developed from tolerant mice delayed tumor outgrowth, as did immunization with MDP peptide-pulsed dendritic cells. Protection was enhanced by the use of peptide ligands containing conservative substitutions that were cross-reactive with the original Ags. These data establish that CTL populations reactive against MDP-derived self-Ags can be activated to mount effective antitumor immunity and strongly support their continued development for tumor immunotherapy in humans.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In light of deficits in current anticancer therapeutic approaches, tumor immunotherapy has enjoyed renewed interest. Recent studies have enhanced our understanding of anti-melanoma immune responses associated with tumor regression (reviewed in Refs. 1, 2, 3); collectively, these data suggest that activated tumor-specific CD8+ CTL are the immunological weapon of choice for potent antitumor therapy. A multitude of human and experimental tumor-associated Ags recognized by CD8+ CTL have been identified (4, 5, 6, 7, 8, 9, 10). Melanomas, in particular, have yielded a variety of immunogenic epitopes. Many melanoma-associated Ags recognized by patient-derived CTL originate from melanocyte differentiation proteins (MDP)3 expressed in normal melanocytes, including tyrosinase (11, 12, 13, 14, 15), gp100 (16, 17, 18, 19, 20), MART-1/Melan-A (21), tyrosinase-related protein (TRP)-1 (21), and TRP-2 (22).

The availability of MDP-derived Ags has shifted the focus to their use in the design of effective vaccination protocols to increase the host immune response to melanoma. However, because such responses may be complicated by preexisting self-tolerance, as well as the possibility of inducing autoimmune vitiligo (23, 24, 25), their value as immunotherapeutic agents has been questioned. In addition, although positive clinical responses have been associated with MDP-directed immunotherapies (26, 27, 28, 29, 30), evaluation of different vaccination strategies involving MDPs in clinical trials is time consuming and expensive, and variability in disease state, immune status, age, and gender often complicate data interpretation. A preclinical model that could be used to evaluate all of these issues and provide information relevant to clinical trial design is highly desirable.

Experimental systems currently available to study the immune response in tumor-bearing animals include chemically induced or spontaneous tumors, as well as a variety of transfected tumors. Insight has been gained from model tumors expressing Ags such as OVA (31, 32), {beta}-galactosidase (33), and influenza hemagglutinin (34). These models are based on highly immunogenic Ags that are overtly foreign (31, 32, 33) or are expressed in tissues and at levels that may engender different mechanisms of self-tolerance (35, 36) and may not represent optimal models for the evaluation of clinically relevant therapeutic maneuvers. More relevant models for the evaluation of the melanoma-associated Ags TRP-2, gp100, and MART-1 have been based on B16 melanoma (36, 37, 38). However, these H-2b-restricted epitopes are not presented on human melanoma and therefore are not predictive of the clinical efficacy of epitopes restricted by human MHC molecules.

We described a preclinical model using transgenic mice that express a recombinant class I MHC molecule, which consists of the peptide binding region of HLA-A*0201 linked to the CD8 binding domain of H-2Dd (AAD). The AAD molecule mediates efficient positive selection of murine T cells, and peptide epitopes presented and recognized by murine T cells in the context of AAD are similar to or identical with those recognized by HLA-A*0201-restricted human T cells (26, 39, 40). We have previously evaluated CTL responses in these mice to dendritic cells (DC) pulsed with HLA-A*0201-restricted peptide Ags from murine and human tyrosinase369–377 (peptide sequences FMDGTMSQV and YMDGTMSQV, respectively) and gp100209–217 (ITDQVPFSV in mice and humans) (41). We have also demonstrated that the expression of tyrosinase leads to substantial, although incomplete, tolerance to both human and murine tyrosinase369–377 (42). In the present study, we have developed a murine melanoma model to ask whether these epitopes can serve as tumor rejection Ags in the face of self-tolerance and to begin to determine optimal vaccination strategies for use in this situation.


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

Transgenic mice on the C57BL/6 background expressing a chimeric MHC class I composed of the {alpha}1 and {alpha}2 domains of HLA-A*0201 and AAD have been described previously (39). Nontransgenic C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice with a radiation-induced excision of the tyrosinase (c) coding locus on chromosome 7 (c38R145L/c38R145L) were obtained from Dr. L. B. Russell (Oak Ridge National Laboratories, Oak Ridge, TN) and are described elsewhere (43, 44). The c38R145L locus was crossed onto the AAD+ C57BL/6 background with three rounds of backcrossing. Transmission of the deletion was confirmed by PCR using MapPair primers (Research Genetics, Huntsville, AL) that define D7 Mit62 and D7 Mit301 (not shown). All animals were maintained in pathogen-free facilities at the University of Virginia (Charlottesville, VA). All protocols were consistent with accepted National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the University of Virginia Institutional Animal Care and Use Committee.

Peptides

Synthetic peptides were made by standard Fmoc chemistry using a model AMS422 peptide synthesizer (Gilson, Middleton, WI). All peptides were purified to >98% purity by reverse phase HPLC on a C8 column (Vydac, Hesperia, CA). Purity and identity were confirmed using a triple quadrupole mass spectrometer (Finnigan, San Jose, CA).

Cell lines

C1R-AAD is a transfectant of the human B lymphoblastoid cell line C1R with the gene for AAD (45). It was maintained under selection in RPMI 1640 supplemented with 5% FBS (Sigma, St. Louis, MO) plus SerXtend (Irvine Scientific, Santa Ana, CA) and 300 µg/ml geneticin G418 (Life Technologies, Gaithersburg, MD). HLA-A*0201+ human melanomas DM93 and DM331 were maintained in RPMI 1640 supplemented with 5% FBS-SerXtend. B16-F1 (CRL-6323) was obtained from the American Type Culture Collection (Manassas, VA), and maintained in RPMI 1640 supplemented with 5% FBS-SerXtend. B16-F1 was transfected with a plasmid containing the genes for AAD and G418 resistance (46) using LipofectAMINE PLUS (Life Technologies), and colonies were screened for stable AAD expression by flow cytometric analysis using the HLA-A*0201-specific Abs CR11.351 (47) or BB7.2 (48). A cell line that was >98% AAD positive and expressed this molecule stably for at least 3 wk in the absence of G418 was established and is referred to as B16-AAD.

DC culture and immunization

Syngeneic DC were generated as described (41). Briefly, bone marrow cells from AAD+ mice were cultured in medium containing 1000 U/ml both IL-4 and GM-CSF (generous gifts of Schering-Plough, Kenilworth, NJ) for 8 days, and DC were isolated on a StemSep column (StemCell, Vancouver, British Columbia, Canada) after incubation with a mixture of Abs for enrichment of mature DC. Activated DC were generated by overnight culture with an equal number of irradiated (3000 rad) NIH-3T3 cells transfected to express CD40 ligand (CD40L; generous gift of Dr. R. Lapoint, National Cancer Institute, Bethesda, MD). Greater than 85% of DC expressed IL-12 by intracellular cytokine staining (BD PharMingen, La Jolla, CA) after 5 h culture in the presence of 10 µg/ml brefeldin A (Sigma; not shown). For immunization, DC were pulsed with the indicated concentration of peptide for 4 h at 37°C in HBSS containing 5% FBS and 5 µg/ml human {beta}2-microglobulin (Calbiochem, La Jolla, CA), washed twice, and resuspended in physiological saline. Mice received 1 x 105 DC in 100 µl saline by s.c. injection into the scapular region.

Generation of peptide-specific HLA-A*0201-restricted CD8+ T cells

Spleens from mice primed i.v. with 1 x 107 PFU vaccinia virus (tyrosinase) or 1 x 105 peptide-pulsed DC (gp100) were harvested at least 3 wk after immunization. Responder cells (l.5 x 107) were incubated in upright 25-cm2 tissue culture flasks (Costar, Cambridge, MA) with 7 x 106 autologous irradiated (2500 rad) spleen cells that had been pulsed with the indicated concentration of peptide for 3 h at 37°C. T cell lines were derived from the initial cultures by weekly restimulations as described above, except that 5 x 105 T cells were incubated with 5 x 106 peptide-pulsed stimulators in 12-well plates (Costar); after wk 2, cultures were supplemented with 10 Cetus U (CU)/ml IL-2 (Chiron, Emeryville, CA) on the day of restimulation and then 3 days later.

Tumor induction and measurements

Subcutaneous tumors were established by injection of 4 x 105 B16-AAD in 200 µl physiological saline. Lung metastases were induced by tail vein i.v. injection of 4 x 105 B16-AAD in 200 µl physiological saline. Tumor cells were 100% viable by trypan blue exclusion and >98% AAD+ by flow cytometric analysis on the day of injection (not shown). Mice were evaluated for s.c. tumor outgrowth by palpation, and 100% of B16-AAD-challenged naive tyrosinase+ animals developed palpable tumors by day 10. Tumor growth was measured at ~72-h intervals using a vernier caliper and recorded as the product of two orthogonal diameters (a x b). The first diameter was identified as the longest surface length (a), with the second diameter being the subsequent orthogonal width (b). Repeated observations with the vernier caliper resulted in <5% SD. Lung metastases were evaluated by counting total visible lesions using a dissecting scope (x40 magnification).

Adoptive transfer of CD8+ T cells

For adoptive transfer protocols, 1 x 107 cells of the indicated CD8+ T cell line were delivered 10 days post-tumor induction, either systemically (tail vein) or directly into the tumor. T cells were coadministered with 500 CU of IL-2; subsequently, IL-2 (500 CU) was delivered daily for 3 days by i.p. injection. CD8+ T cells specific for an A2-restricted phosphorylated epitope (GLDpSYVRSL) of the MUM-2 protein were used as irrelevant control CTL (49). These CTL did not lyse B16-AAD cells or human DM93 melanoma in a 4-h 51Cr release assay (data not shown). IL-2 treatment alone did not affect B16-AAD outgrowth (data not shown).

Tumor resection and evaluation

Tumors were induced as described. When tumors achieved 250 mm2 in size, they were resected and digested in 0.5% collagenase D (Roche Diagnostics, Indianapolis, IN) for 45 min and then homogenized in a tissue grinder. A single-cell suspension was filtered through nylon; then debris and dead cells were separated on a Ficoll-Hypaque gradient (Amersham, Uppsala, Sweden). Lymphocytes were separated on a Lympholyte-M gradient (Accurate Chemical, Westbury, NY). The resulting tumor cell suspension was homogeneous on a forward scatter-side scatter flow cytometric plot and did not contain any detected CD4+ or CD8+ cells by staining with specific Abs (BD PharMingen; not shown). Cells were evaluated for AAD expression, as described.

Western analysis of tyrosinase and gp100

For Western analysis of the MDPs tyrosinase and gp100, total cellular proteins were prepared either from confluent cultures of melanoma cells or from single-cell suspension of excised melanoma cells, prepared as described. Cells were lysed in 50 mM Tris-Cl (pH 7.6) containing 10 µg/ml leupeptin and aprotinin (Sigma) and 300 mM NaCl. Membranes were pelleted by centrifugation (12,000 x g), and protein was determined by a microtiter modification of the Lowry method using Sigma reagents. Protein (15 µg) was denatured by boiling in 2-ME (5%) and separated by SDS-PAGE using GeneMates 10% Express Gels (ISC BioExpress, Kaysville, UT), transferred to Immobilon-P membrane (Millipore, Bedford, MA), and blocked using 5% nonfat milk. For tyrosinase, goat anti-mouse tyrosinase Ab (Research Diagnostics, Flanders, NJ) was used at 0.5 µg/ml, per the manufacturer’s recommendation. HRP-conjugated anti-goat IgG secondary Ab (Research Diagnostics) was used at 1/12000 dilution. For gp100, polyclonal rabbit anti-mouse gp100 antisera (generous gift of Drs. D. R. Surman and N. P. Restifo (National Cancer Institute, Bethesda, MD (50)) was used at 1/200 dilution, and HRP-conjugated anti-rabbit IgG secondary Ab (Research Diagnostics) was used at 1/12,000 dilution. Bound protein was detected on Kodak BioMax film with ECL detection reagent (Amersham Pharmacia Biotech, Piscataway, NJ).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AAD-transfected B16-F1 melanoma processes and presents HLA-A*0201-restricted MDP epitopes

To evaluate the ability of HLA-A*0201-restricted, MDP-derived epitopes to mediate tumor control, we first transfected the well-characterized and poorly immunogenic B16-F1 murine melanoma tumor with the gene for the chimeric class I HLA-A2 molecule referred to as AAD. Flow cytometric evaluation showed that a stable transfectant of B16-F1 (referred to as B16-AAD) maintained under G418 selection expressed the AAD molecule at ~20% of the level of HLA-A*0201 expressed on the human melanoma DM93 (Fig. 1GoA). Greater than 98% of cells were AAD positive, as compared with untransfected cells. AAD expression by B16-AAD remained constant after 3 wk in culture without G418 selection, the anticipated duration of in vivo tumor challenge experiments, and there was no evidence for the emergence of an AAD-negative or AAD-low subpopulation (Fig. 1GoB). Western analysis of whole cell lysates showed that B16-AAD expressed the MDPs tyrosinase and gp100 (Fig. 1GoC), consistent with the nontransfected B16-F1 line (50, 51). Most importantly, B16-AAD was recognized and efficiently lysed by murine CTL that had been elicited from tyrosinase+ mice with murine tyrosinase369–377 (abbreviated tyr369) epitope (Fig. 1GoD). These tyr369-specific CTL also efficiently recognized and lysed the human melanoma DM93, which expresses human tyrosinase369–377 (abbreviated tyr369(Y)), but not the tyrosinase-deficient human tumor DM331. Likewise, CTL from gp100+ mice raised against murine gp100209–217 (abbreviated gp100209) efficiently lysed B16-AAD but not B16-F1 (Fig. 1GoE). These data established that B16-AAD stably expressed the AAD-restricted epitopes tyr369 and gp100209, making B16-F1-based melanoma suitable for the evaluation of the antitumor efficacy of immune responses to these Ags.



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  		FIGURE 1. B16-AAD is a model tumor for evaluating HLA-A*0201-restricted melanoma epitopes. AAD+ tumor was constructed on the B16-F1 melanoma background (B16-AAD) and evaluated for use in preclinical evaluation of human melanoma Ags. A, B16-AAD tumor expression of recombinant class I molecules was measured after selection with G418 (300 µg/ml, dark histogram). AAD expression on untransfected B16-F1 (open histogram), and A2.1 expression on the human melanoma DM93 (light histogram) was also assessed. B, B16-AAD expression of AAD was measured after 3 wk culture without G418 selection (dark histogram), compared with untransfected B16-F1 (open histogram). C, Expression of the MDPs tyrosinase and gp100 was assessed in whole cell lysates of B16-F1 (lane 1), B16-AAD (lane 2), and the MDP-negative murine cell line EL4 (lane 3) by Western analysis. D and E, MDP-specific CTL recognition of B16-F1 or B16-AAD was assessed using either tyrosinase-specific (D) or gp100-specific (E) CTL by 51Cr release assay. The tyrosinase+ cell line DM93 and tyrosinase- cell line DM331 were used as controls for tyrosinase-specific CTL assays; gp100209 (10 µg/ml)-pulsed or unpulsed C1R-AAD were used as controls for gp100-specific CTL assays. All experiments were repeated at least three times; representative data are shown.

 
To evaluate the impact of AAD expression in B16 on its immunogenicity and tumorigenicity, we compared the growth of B16-F1 and B16-AAD in naive C57BL/6 or C57BL/6-AAD-transgenic mice. Expression of AAD by B16-F1 led to its rejection by normal C57BL/6 mice, consistent with its recognition as an alloantigen in this context (Fig. 2GoA). However, the growth kinetics of B16-AAD in AAD+ mice were identical with those of untransfected B16-F1 growing in either AAD+ or C57BL/6 mice (Fig. 2GoB). These results demonstrate that neither AAD nor peptide Ags presented by AAD function as effective rejection Ags in unimmunized AAD-transgenic mice. Therefore, shifts in tumor outgrowth following immunization with HLA-A*0201-restricted Ags can be interpreted as immune-mediated antitumor efficacy directed toward B16-derived MDP epitopes presented in the context of AAD. The B16-AAD melanoma is an appropriate model for the evaluation of vaccines using HLA-A*0201-restricted epitopes in a murine system.



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  		FIGURE 2. AAD chimeric HLA molecule does not induce xenogeneic rejection of B16-AAD. B16-F1 or B16-AAD tumor cells (4 x 105/animal) were delivered in 200 µl physiological saline by s.c. injection to AAD- (A) or AAD+ (B) tyrosinase+ mice. Tumor growth was measured as described. Data are mean of three separate animals ± SEM; representative data from one of three similar experiments are shown.

 
MDP-specific CTL induce vitiligo

We previously reported that adoptive transfer of tyr369-specific CTL isolated from AAD+ tyrosinase-expressing mice half-maximal stimulatory concentration (7.25 µg/ml) resulted in the rapid development of vitiligo in irradiated AAD+ recipients (42). These results established both that the tyr369 epitope was presented by normal melanocytes and that a self-reactive population of CTL capable of inducing autoimmune disease persisted in these mice. We therefore evaluated these issues with respect to the gp100209 epitope. Adoptive transfer of CTL specific for gp100209 (half maximal stimulatory concentration, 2.6 µg/ml) with IL-2 support induced vitiligo at the site of transfer in AAD+ mice (Table IGo). CTL specific for a peptide absent from melanoma fail to induce depigmentation, indicating that vitiligo is the result of specific CTL recognition of gp100209 on melanocytes. Together with previously published data (42), these results demonstrate that both tyr369 and gp100209 are naturally processed and presented on melanocytes in AAD-transgenic mice. Furthermore, the fact that the CTL causing vitiligo originate from animals that express these Ags endogenously demonstrates that CTL with avidities that are sufficient to recognize these self-Ags at the levels presented by normal host tissue persist in vivo.


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  		Table I. Induction of vitiligo in AAD mice by adoptive transfer of MDP-specific CTLs1

 
Adoptive transfer of MDP-specific CTL controls outgrowth of B16 melanoma

We next asked whether the tyr369- or gp100209-specific CTL derived from tyrosinase+ and gp100+ mice would have sufficient avidity to delay or abolish the outgrowth of B16-AAD in an adoptive transfer model. Ten days after injection of 4 x 105 tumor cells at a single s.c. site, 107 CTL were injected into the tumor. Both populations of MDP-specific CTL delayed the outgrowth of B16-AAD, whereas CTL specific for an irrelevant MUM-2 epitope had no effect (Fig. 3Go). Tyr369-specific CTL were more effective at controlling tumor than gp100209-specific CTL, in agreement with the superior in vitro recognition of B16-AAD by the tyrosinase-specific CTL used in these studies (Fig. 1Go). Similar control of B16-AAD outgrowth was achieved after adoptive transfer of MDP-specific CTL via tail vein injection (data not shown). Collectively, these data demonstrate that CTL with sufficiently high avidity to mediate partial control of tumor outgrowth can be generated from mice expressing a normal complement of MDP-derived epitopes. These data highlight the possibility that effective immune responses can be directed against shared tumor Ags, given an appropriate immunization maneuver to activate the residual MDP-reactive CTL in these animals.



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  		FIGURE 3. Adoptive transfer of MDP-specific CTL delays B16-AAD outgrowth in AAD+ mice. To assess therapeutic efficacy against melanoma, CTL (107 cells) specific for the MDPs tyrosinase or gp100 were generated from MDP+ mice, expanded in vitro, and delivered by intratumoral injection to 10-day tumors. Tumor outgrowth was measured as described. Parallel tumor-challenged animals were untreated or received irrelevant (MUM-2-specific) CTL. Data are mean of three separate animals; representative data from one of three similar experiments are shown.

 
Self-tolerance to tyrosinase limits control of melanoma outgrowth

We have demonstrated the existence of an Ag-specific repertoire of CTL capable of targeting melanomas. We were interested in determining the impact of self-tolerance to these Ags on the ability to activate an effective immune response in vivo. By taking advantage of albino mice with a complete deletion of the tyrosinase gene, we previously established that endogenous presentation of tyr369 in AAD+ mice leads to a substantial reduction in the number of Ag activatable CD8+ T cells, but a residual population of T cells specific for this epitope persists (42). In the present study, we found that B16-AAD outgrowth in AAD+ albino mice was substantially delayed compared with its outgrowth in AAD+ tyrosinase+ mice, although the tumor did eventually grow in all animals (Fig. 4Go). Interestingly, tumors in albino mice consistently became palpable 10–15 days after injection (compared with 6–10 days in tyrosinase+ animals), and the rate of tumor growth remained low for an extended period. This suggests that the initial immune response to tyrosinase-derived epitopes in AAD+ albino mice is effective in controlling tumor outgrowth but fails at later points in time. We also evaluated the development of tumor lesions in the lungs of AAD+ mice injected i.v. with B16-AAD. After 17 days, an average of almost 500 lesions were observed in tyrosinase+ mice, while an average of only 2 were detected in albino mice (Table IIGo). These results establish that self-tolerance to one or more tyrosinase-derived epitopes limits the immune response to B16-AAD.



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  		FIGURE 4. Self-tolerance limits immune-mediated rejection of B16-AAD. A, To evaluate tumor growth in the presence or absence of endogenous tyrosinase expression, 4 x 105 B16-F1 cells were delivered by s.c. injection to tyrosinase+ (Tyr+) or tyrosinase- mice. B, To evaluate the impact of HLA-A*0201 expression in this model, 4 x 105 B16-AAD were delivered by s.c. injection to tyrosinase+ or tyrosinase- mice. Tumor size was measured as described; tumor size is reported as the mean of five separate animals ± SEM; representative data from one of three similar experiments are shown.

 

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  		Table II. Lung metastases in tyrosinase+ and albino mice1

 
Importantly, the outgrowth of untransfected B16-F1 tumor was rapid and equivalent in AAD+ tyrosinase+ and AAD+ albino mice (Fig. 4Go). Similarly, there was no difference in the number of lung metastases that developed in AAD- tyrosinase+ or albino mice after injection of untransfected B16-F1 cells (Table IIGo). These data demonstrate that the tyrosinase-derived epitopes that allow enhanced control of B16-AAD outgrowth in albino mice are restricted by AAD rather than by endogenous H-2b haplotype molecules expressed by this tumor. They also suggest that no tyrosinase-derived epitopes of immunological significance for tumor control are presented by H-2Kb or Db. Collectively, these results establish that AAD-restricted tyrosinase-derived epitopes act as tumor rejection Ags, but that this ability is significantly compromised by self-tolerance.

B16 tumor outgrowth is not due to loss of Ag expression

Although tumor outgrowth was significantly delayed in albino mice, these animals failed to reject B16-AAD at the tumor burden used (4 x 105 cells/animal). There are indications that loss of MDP expression may occur in metastatic human melanomas, and it has been suggested that Ag down-regulation may represent a maneuver to evade immune-mediated destruction (52, 53, 54, 55). We therefore examined whether B16-AAD tumors that grew out in AAD+ albino mice might have down-regulated expression of MDP-associated epitopes. Excised tumors expressed both murine tyrosinase and gp100 proteins at levels similar to those of tumors that grew out in AAD+ tyrosinase+ mice, as well as cultured B16-AAD cells (Fig. 5GoA). These data demonstrate that MDP expression was maintained in situ. Interestingly, tumor cells excised from albino animals were >90% AAD+ (Fig. 5GoB), and there was no decrease in the percentage of AAD+ cells or the mean fluorescence intensity of excised tumor cells as compared with B16-AAD cultured without selection in vitro. There was also no change in AAD expression on tumors excised from tyrosinase+ animals (not shown). Thus, an active immune response to an AAD-restricted Ag does not modulate tumor cell class I expression. These data are inconsistent with the hypotheses that Ag loss or class I down-regulation allow tumor outgrowth in AAD+ albino mice.



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  		FIGURE 5. Outgrown tumors retain MDP and class I expression. A, Tyrosinase and gp100 protein in whole cell lysates from cultured B16-AAD (lane 1) or B16-AAD tumors excised from tyrosinase+ (lane 2) or albino mice (lane 3) was evaluated by Western analysis. Excised tumors were of equal size (250 mm2). B, AAD expression in outgrown tumors from albino hosts (light histogram) and B16-AAD cells cultured in vitro without G418 selection for an equal period of time (dark histogram) was assessed by FACS analysis; untransfected B16-F1 (open histogram) are shown for comparison. AAD level on tumors from tyrosinase+ animals was identical with that on tumors from albino animals (not shown). Representative data from one of three similar experiments are shown.

 
Active immunization with MDP peptide-pulsed DC delays tumor outgrowth

Because our results demonstrated that self-tolerance limited protection against melanoma, it was important to determine whether active immunization against tyr369 and gp100209 could augment this protection. We had previously established that peptide-pulsed mature DC stimulate strong CD8+ T cell responses (41). Therefore, we immunized AAD+ tyrosinase+ mice with autologous DC that had been activated overnight by coculture with irradiated NIH-3T3-CD40L cells; DC were unpulsed or pulsed with 1 µg/ml tyr369, gp100209, or M1 flu (irrelevant) peptide. Immunization using DC pulsed with either tyr369 or gp100209 peptide delayed B16-AAD outgrowth, whereas immunization with either unpulsed DC or DC pulsed with irrelevant peptide had little or no effect (Fig. 6Go). At the doses used for DC pulsing (1 µg/ml), tyr369-pulsed DC imparted protection superior to that of gp100209. This could be due to a higher density of tyr369 than gp100209 on the tumor or to differences in the average avidity or magnitude in the CTL responses to these Ags. Interestingly, tumor outgrowth in tyrosinase+ mice immunized with tyr369-pulsed DC was similar to that of unimmunized albino mice. Thus, active immunization with mature DC pulsed with MDP epitopes relevant for human cancer therapy can overcome tolerance and induce antitumor immune responses.



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  		FIGURE 6. MDP peptide-pulsed DC delay tumor outgrowth. AAD+ mice were injected with 1 x 105 CD40L-activated autologous DC that were pulsed for 3 h at 37°C with 1 µg/ml of M1 flu peptide GILGFZFTL, FMDGTMSQ, or ITDQVPFSV. Three weeks later, immunized and unimmunized mice were challenged with 4 x 105 B16-AAD cells injected s.c., and tumor growth monitored as described. Data are means of three separate animals ± SEM; representative data from one of three similar experiments are shown.

 
Previous studies have established that residual T cells reactive to tyr369 (FMDGTMSQV) in tyrosinase+ mice are difficult to activate using this peptide directly, and can be more effectively activated using the cross-reactive human homologue (YMDGTMSQV (tyr369(Y)) (41, 42). In keeping with this, immunization of tyrosinase+ mice using DC pulsed with tyr369(Y) further delayed tumor outgrowth by an additional 10 days beyond the initial delay observed in mice immunized with DC pulsed with tyr369 (Fig. 7Go). We extended these studies to evaluate a gp100209 epitope with a conservative threonine to methionine substitution at P2(IMDQCPFSV), abbreviated gp100209(M) (56, 57). Immunization using DC pulsed with gp100209(M) delayed tumor outgrowth beyond that achieved with gp100209. This demonstrates that DC pulsed with single amino acid-altered epitopes can be highly immunogenic and useful for overcoming tolerance to induce a cross-reactive and more effective antitumor response.



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  		FIGURE 7. Modified peptides enhance antitumor efficacy of MDP-targeted DC immunization. A, AAD+ mice were injected s.c. with 1 x 105 CD40L-activated autologous DC that had been pulsed for 3 h at 37°C with 1 µg/ml tyr369 or tyr369(Y). B, AAD+ mice were injected s.c. with 1 x 105 CD40L-activated autologous DC that had been pulsed for 3 h at 37°C with 1 µg/ml gp100209 or gp100209(M). Three weeks later, immunized and unimmunized mice were challenged with 4 x 105 B16-AAD cells injected s.c., and tumor growth was monitored as described. Data are means of three separate animals ± SEM; representative data from one of three similar experiments are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we report the development of a preclinical model based on human class I MHC transgenic mice and melanoma cells expressing a recombinant HLA-A*0201 molecule that can be used to quickly evaluate candidate Ags to control tumor outgrowth and immunization strategies for human use. We used this model to evaluate the significance of two Ags that had previously been identified as HLA-A*0201-restricted T cell targets on human melanoma. Tyr369 and gp100209 are important not only for their potential use in human immunotherapy but also because, as melanocyte differentiation Ags, they are subject to self-tolerance. Our data show that these epitopes are processed and presented by B16-AAD melanoma and that this expression allows the outgrowth of this tumor to be controlled in AAD-transgenic mice. We also demonstrated that self-tolerance resulting from endogenous melanocytic expression of these epitopes can be overcome by the appropriate immunization strategy. Finally, we used this model to demonstrate that use of altered peptide ligands of self-Ags and immunization directed against multiple tumor-associated epitopes can greatly augment antimelanoma immunotherapy.

As a basis for studies of antitumor immunization efficacy, we created an AAD transfectant (B16-AAD) on the B16-F1 background. These cells express tyrosinase and gp100, and AAD expression is stable for up to 10 wk in the absence of selection. The growth B16-AAD in tyrosinase+ AAD-transgenic mice is identical with that of parental B16-F1 in C57BL/6-nontransgenic mice. These data indicate that the expression of AAD in B16-F1 does not lead to the creation of significantly immunogenic Ags and are in keeping with its generally poor immunogenicity. The advantage of this is that it allows evaluation of immune responses to specific Ags with little or no "background," in terms of immune responses to the parental tumor. Conversely, the rapid growth kinetics of B16 make complete control of this tumor difficult, regardless of antigenic target or immunization strategy (31, 58, 59). Thus, we did not anticipate complete cures or absolute protection against B16 challenge. Instead, we interpret shifts in tumor outgrowth as indicators of successful immune-mediated intervention.

Importantly, B16-AAD processes and presents the murine homologues of two previously defined human HLA-A*0201-restricted peptide epitopes, tyr369 and gp100209. The gp100209 epitope is identical in mice and humans, whereas the murine tyr369 epitope differs from the human epitope by only a biochemically conservative substitution of F for Y at HLA-A*0201 binding position 1. The two tyr369 peptides bind to HLA-A*0201 with identical affinities and are cross-reactively recognized by T cells generated against either (42). Using B16-AAD, we demonstrated by two different approaches that immune responses to tyr369 or gp100209 can control tumor outgrowth. First, adoptive transfer of tyr369- or gp100209-specific CTL into established B16-AAD tumors reduced the rate of tumor growth compared with irrelevant control CTL. Secondly, we induced an active immune response using autologous, peptide-pulsed, CD40L-activated DC. Adoptive transfer of CTL with specificity for another MDP, TRP-2, has been shown to be effective in nontransgenic mice (60). In addition, protective immunity against B16 has been induced by active immunization with TRP-2180–188 (31, 37, 61), gp10025–33 (31, 59), or MART-1 (38). However, our study is the first to examine the antitumor efficacy of HLA-A*0201-restricted peptides that are highly homologous or identical with human melanoma Ags. Introduction of the chimeric HLA-A2.1 restriction element imparts B16 with the capacity to process and present epitopes homologous to human Ags, making it particularly relevant to understand the factors that may augment or limit immune responses to these Ags in patients.

A potential disadvantage in targeting immunotherapy to Ags derived from MDPs is that they are subject to self-tolerance. Indeed, we have previously shown that a robust CTL response is elicited in AAD+ albino mice using tyr369 peptide-pulsed DC or tyrosinase vaccinia virus, but this response is almost undetectable in tyrosinase+ animals (42). In keeping with this, B16-AAD outgrowth was substantially delayed in AAD+ albino mice. These results suggest that tyr369 is a strong tumor Ag in the AAD+ albino background but that self-tolerance imposes a profound limitation. In addition, B16-F1 grew out equally rapidly in both AAD- albino and tyrosinase+ mice. Thus, there do not appear to be any other tyrosinase-derived epitopes, other than those restricted by AAD, that can serve as strong tumor Ags.

Three additional observations are relevant here. First, B16-AAD tumors eventually grew in all AAD+ albino hosts, but with a significantly delayed time course. We ruled out Ag shedding or class I down-regulation as explanations for this observation. Thus, even with a strong tumor Ag and a nontolerant animal, there is an eventual loss of ability to control tumor outgrowth. The reasons for this remain to be determined. Second, despite the substantial level of self-tolerance evident in AAD+ tyrosinase+ mice, a residual repertoire of tyr369-reactive T cells remains that can induce vitiligo (42) and control tumor outgrowth after suitable vaccination. Similarly, although we cannot evaluate self-tolerance to gp100209 as rigorously, it is clear that this epitope is expressed endogenously, yet specific high avidity CTL capable of inducing vitiligo persist. Finally, we note that protective immunization was achieved in our model despite the fact that it has been difficult to measure significant primary immune responses to the same immunogens (41, 42). Collectively, these results indicate that CTL directed against MDP-derived epitopes may be effective in controlling tumor outgrowth, despite constraints imposed by self-tolerance.

A challenge remaining in the use of MDP derived Ags for immunotherapy is to overcome the constraints imposed by self-tolerance. The use of the AAD transgenic model has allowed us to evaluate the importance of this issue and ways to overcome it that are relevant in the design of human clinical trials. We found that immunization against tyr369 was enhanced by the use of DC that had been matured by culture with either TNF-{alpha} (41) or irradiated NIH-3T3-CD40L cells (66). It is possible that superior immunogenicity of peptide-pulsed DC is enhanced by maturation because peptide retention time is too short for maturation to occur in vivo. Alternatively, recent studies have implicated immature DC in the induction of T cells that negatively regulate immune responses (62, 63). In the present study, we demonstrated that MDP peptide-pulsed mature DC impart substantial protection against B16-AAD, even in the face of self-tolerance. Thus, mature DC are an effective adjuvant to overcome tolerance and induce immune responses to shared Ags. The existence of a variety of DC subsets suggests that further optimization of active immunization may come from evaluation of their function in this model system.

We previously demonstrated that immunization with peptide ligands containing conservative amino acid substitutions leads to a substantial enhancement in the magnitude of the CTL response and that a significant proportion of the CTL are cross-reactive for the native epitopes (41, 42). Indeed, we found that immunization with tyr369(Y) or the gp100209(M) altered ligands pulsed onto DC enhanced control of B16-AAD, as compared with immunization with the unmodified epitope. This is consistent with other studies that have shown that modified peptide ligands have an enhanced ability to induce antitumor responses in the face of self-tolerance to the unmodified epitope (25, 56, 57, 58). In the context of a peptide-pulsed DC immunization, some advantage may be conferred by substitutions that increase the binding affinity of the modified peptide (41, 64). However, the wild-type epitopes may also function as partial or weak agonists, leading to a suboptimal induction of MDP-specific responses, whereas the modified ligands may act as full agonists, thus affording enhanced antitumor protection through an expanded population of cross-reactive CTL (41, 42, 65).

Cancer immunotherapies continue to be refined and improved with the identification of immunogenic tumor-associated epitopes and cellular adjuvants. The AAD-transgenic mice and B16-AAD melanoma are powerful tools for the rapid evaluation and optimization of these new reagents and maneuvers in a context that would have relevance to human melanoma therapy. We have expanded this system with AAD+ albino mice, affording the possibility of comparing immunotherapy approaches in the absence or presence of self-tolerance. In the present study, we used this preclinical model to demonstrate that active immunization with the tyr369 and gp100209 epitopes in the context of DC-mediated delivery delayed melanoma outgrowth. Furthermore, we show enhanced protection with the use of peptide ligands containing conservative substitutions that were cross-reactive with the original Ags. These data suggest that active immunizations capable of overcoming tolerance may be effective in patient populations. These data strongly support the continued development of active immunization therapies for clinical intervention in human cancers.


   Acknowledgments
 
We thank Drs. Craig L. Slingluff, Jr. (Department of Surgery, University of Virginia) and Marcia J. McDuffie (Department of Microbiology, University of Virginia) for suggestions and insightful discussions.


   Footnotes
 
1 This work was supported by US Public Health Service Grants AI20963 and CA78400 (to V.H.E.). T.N.J.B. was a fellow of the Cancer Research Institute. T.A.C. was supported by U.S. Public Health Service Training Grants CA09109 and AI07496. D.W.M. was initially supported by U.S. Public Health Service Training Grant AI07496 and is a fellow of the American Cancer Society. Back

2 Address correspondence and reprint requests to Dr. Victor H. Engelhard, Carter Immunology Center, University of Virginia, Box 801386, Charlottesville, VA 22908-1386. E-mail address: vhe{at}virginia.edu Back

3 Abbreviations used in this paper: MDP, melanocyte differentiation protein; AAD, recombinant HLA-A*0201 molecule containing the murine Dd {alpha}3 region; CU, Cetus U; TRP, tyrosinase-related protein; DC, dendritic cell; CD40L, CD40 ligand. Back

Received for publication June 13, 2001. Accepted for publication August 22, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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R. Lengagne, S. Graff-Dubois, M. Garcette, L. Renia, M. Kato, J.-G. Guillet, V. H. Engelhard, M.-F. Avril, J.-P. Abastado, and A. Prevost-Blondel
Distinct Role for CD8 T Cells toward Cutaneous Tumors and Visceral Metastases
J. Immunol., January 1, 2008; 180(1): 130 - 137.
[Abstract] [Full Text] [PDF]

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L. A. Nichols, Y. Chen, T. A. Colella, C. L. Bennett, B. E. Clausen, and V. H. Engelhard
Deletional Self-Tolerance to a Melanocyte/Melanoma Antigen Derived from Tyrosinase Is Mediated by a Radio-Resistant Cell in Peripheral and Mesenteric Lymph Nodes
J. Immunol., July 15, 2007; 179(2): 993 - 1003.
[Abstract] [Full Text] [PDF]

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S. L. Sheasley-O'Neill, C. C. Brinkman, A. R. Ferguson, M. C. Dispenza, and V. H. Engelhard
Dendritic Cell Immunization Route Determines Integrin Expression and Lymphoid and Nonlymphoid Tissue Distribution of CD8 T Cells
J. Immunol., February 1, 2007; 178(3): 1512 - 1522.
[Abstract] [Full Text] [PDF]

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K. M. Hargadon, C. C. Brinkman, S. L. Sheasley-O'Neill, L. A. Nichols, T. N. J. Bullock, and V. H. Engelhard
Incomplete Differentiation of Antigen-Specific CD8 T Cells in Tumor-Draining Lymph Nodes
J. Immunol.,