Dendritic Cells Break Tolerance and Induce Protective Immunity ...

[CANCER RESEARCH 60, 6995–7001, December 15, 2000]

Dendritic Cells Break Tolerance and Induce Protective Immunity against a Melanocyte Differentiation Antigen in an Autologous Melanoma Model1 Marco W. J. Schreurs,2 Andreas A. O. Eggert, Annemiek J. de Boer, Joost L. M. Vissers, Thorbald van Hall, Rienk Offringa, Carl G. Figdor, and Gosse J. Adema3 Department of Tumor Immunology, University Medical Center St. Radboud, Philips van Leydenlaan 25, 6525 EX Nijmegen [A. A. O. E., A. J. d. B., J. L. M. V., C. G. F., G. J. A.], Department of Immunohematology and Blood Bank, Leiden University Medical Centre, 2300 RC Leiden [T. v. H., R. O.], The Netherlands

ABSTRACT Tyrosinase-related protein (TRP) 2 belongs to the melanocyte differentiation antigens and has been implicated as a target for immunotherapy of human as well as murine melanoma. In the current report, we explored the efficacy of nonmutated epitopes with differential binding affinity for MHC class I, derived from mouse TRP2 to induce CTL-mediated, tumorreactive immunity in vivo within the established B16 melanoma model of C57BL/6 mice. The use of nonmutated TRP2-derived epitopes for vaccination provides a mouse model that closely mimics human melanoma without introduction of xenogeneic or otherwise foreign antigen. The results demonstrate that vaccination with TRP2 peptide-loaded bone marrow-derived dendritic cells (DCs) results in activation of high avidity TRP2-specific CTLs, displaying lytic activity against both B16 melanoma cells and normal melanocytes in vitro. In vivo, protective antitumor immunity against a lethal s.c. B16 challenge was observed upon DC-based vaccination in this fully autologous tumor model. The level of protective immunity positively correlated with the MHC class I binding capacity of the peptides used for vaccination. In contrast, within this autologous model, vaccination with TRP2 peptide in Freund’s adjuvant or TRP2encoding plasmid DNA did not result in protective immunity against B16. Strikingly, despite the observed CTL-mediated melanocyte destruction in vitro, melanocyte destruction in vivo was sporadic and primarily restricted to minor depigmentation of the vaccination site. These results emphasize the potency of DC-based vaccines to induce immunity against autologous tumor-associated antigen and indicate that CTL-mediated antitumor immunity can proceed without development of adverse autoimmunity against normal tissue.

INTRODUCTION The cloning and characterization of tumor-associated antigens have resulted in an increased effort to develop specific antitumor immunotherapy. Among tumor-associated antigens are the differentiation antigens, nonmutated self antigens specifically expressed by normal and transformed cells of a differentiated cell type. Studies in melanoma patients have shown convincingly that melanocyte differentiation antigens, expressed by normal melanocytes and melanoma cells, can be recognized by the immune system (reviewed in Ref. 1). Melanocyte differentiation antigens include tyrosinase, TRPs,4 gp100, and MART-1 and represent well-characterized proteins involved in the biosynthesis of the pigment melanin (reviewed in Ref. 2). The recognition of these antigens by the immune system has implicated differ-

entiation antigens as attractive vaccine candidates for the induction of antitumor immunity in cancer patients. CTLs are considered as major effectors cells in the eradication of tumor cells in vivo (1). At present, different vaccination strategies for the induction of CTL-mediated antitumor immunity are available, including the use of synthetic peptides, naked plasmid DNA, and antigen-loaded DCs. These strategies have been used successfully in experimental animal models to induce tumor-reactive CTL responses in vivo, however, primarily directed against relatively immunogenic foreign or mutated antigens (3–5). In contrast, autologous nonmutated tumor antigens have proven to be poorly immunogenic, most probably because of the involvement of peripheral tolerance (6 – 8). A potential candidate to break tolerance against nonmutated antigen and activate high avidity CTLs is a DC-based vaccine. It is currently well appreciated that DCs are highly immunostimulatory antigen-presenting cells capable of activating resting T cells. Indeed, both human and animal studies have demonstrated that DCs can efficiently mediate the induction of antitumor immunity (reviewed in Refs. 9 and 10). The aim of this study was to evaluate DCs loaded with nonmutated CTL-defined epitopes derived from a melanocyte differentiation antigen as an antitumor vaccine in an autologous mouse model and compare its efficacy to other established vaccination strategies. The mouse TRP2 antigen (mTRP2), endogenously expressed by wild-type B16 melanoma as well as normal melanocytes, was chosen as a model differentiation antigen. Both human and mouse antimelanoma CTLs have been shown previously to recognize conserved nonmutated epitopes derived from TRP2 (11, 12), which renders mTRP2 an ideal model antigen to design antimelanoma vaccines in a fully autologous model for clinical application. The results demonstrate that, within this autologous tumor model, tolerance against mTRP2 can be overcome by peptide-loaded DCs but not by peptide in Freund’s adjuvant or plasmid DNA. Activation of high avidity CTLs mediating protective antitumor immunity in vivo without the development of adverse autoimmunity was observed upon DC vaccination, indicating the potency as well as the value of DC-based vaccines for immunotherapy of cancer. MATERIALS AND METHODS

Mice and Cell Lines. Male C57BL/6 (H-2b) mice were purchased from Charles River (Sulzfeld, Germany) and held under specified pathogen-free conditions in the Central Animal Laboratory (Nijmegen University, Nijmegen, Received 3/13/00; accepted 10/17/00. The Netherlands). For experimental purposes, five mice of 8 –12 weeks of age The costs of publication of this article were defrayed in part by the payment of page were used per group. charges. This article must therefore be hereby marked advertisement in accordance with The murine thymoma cell line EL4 (ATCC) was cultured in Iscove’s 18 U.S.C. Section 1734 solely to indicate this fact. 1 medium (Life Technologies, Inc., Paisley, United Kingdom), supplemented This study was supported by Grant KUN 95-911 from the Dutch Cancer Society and with 5% FCS and 50 ␮M ␤-mercaptoethanol. The murine melanoma cell line Grant ERB FMRX CT960053 from the European Community. 2 Present address: Department of Immunology, The Netherlands Cancer Institute, B16, subline F10 (13), was grown in MEM, supplemented with MEM nonesPlesmanlaan 121, 1066 CX Amsterdam, The Netherlands. sential amino acids, MEM vitamin mix, 1 mM sodium pyruvate, 0.15% sodium 3 To whom requests for reprints should be addressed, at Department of Tumor bicarbonate, and 5% heat-inactivated FCS (Life Technologies, Inc.). The Immunology, University Hospital Nijmegen St. Radboud, Philips van Leydenlaan 25, immortalized C57BL/6 melanocyte line Melan-A (14) was grown as described 6525 EX Nijmegen, The Netherlands. Phone: 31-24-3617600; Fax: 31-24-3540339; E-mail: [email protected]. for B16. Normal epidermal melanocytes were isolated from neonatal C57BL/6 4 The abbreviations used are: TRP, tyrosinase-related protein; mTRP2, mouse TRP2; mice and cultured as described (14, 15). Primary cultures of melanocytes are DC, dendritic cell; ATCC, American Type Culture Collection; IL, interleukin; LPS, further referred to as MCT. H-2Kb-expressing HeLa cells (HeLa-Kb) were lipopolysaccharide; HPLC, high-pressure liquid chromatography; CM, culture medium; generated and cultured as described (16). CMV, cytomegalovirus. 6995

DENDRITIC CELL-BASED VACCINATION AGAINST MELANOMA

For detection of mouse TRP2-reactive serum IgG, acetone-fixed cytospins of Melan-A and B16 cells were incubated for 1 h with various dilutions of serum isolated from vaccinated mice, 2 weeks after the second vaccination. Specific staining was visualized by immunoperoxidase substrate reaction as described previously (4). Polyclonal rabbit antibody ␣PEP8 (generously provided by Dr. V. Hearing, National Cancer Institute, Bethesda, MD) was used as a control for mouse TRP2-specific immunoperoxidase staining. Peptides and MHC Class I Binding Assays. Peptides were synthesized with a free COOH terminus either by f-moc peptide chemistry using a ABIMED Multiple Synthesizer or by t-boc chemistry on a Biosearch SAM2 peptide synthesizer. Peptides were ⬎90% pure as indicated by analytical HPLC. The peptides were dissolved in DMSO and stored at ⫺20°C. Peptide binding to H-2Kb was determined using the RMA/S-based MHC class I stabilization assay as described (17). Briefly, RMA/S cells (ATCC), cultured as described for EL4, were incubated at room temperature for 36 h, pulsed with peptide at the indicated concentrations for 1 h at room temperature, and subsequently incubated for 4 h at 37°C to allow turnover of “empty” MHC class I molecules. MHC class I stabilization was determined with H-2Kbspecific monoclonal antibody B8-24-3 (ATCC), followed by FITC-conjugated goat-antimouse F(ab)2 (Zymed Laboratory, South San Francisco, CA) and analyzed by flow cytometry (FACScan; Becton Dickinson, Hamburg, Germany). Peptide binding to HLA-A2.1 was determined using the JY-based MHC class I binding assay as described (18). Briefly, HLA-A2.1-presented peptides were stripped from JY cells (ATCC), cultured as described for EL4, by mild acid elution. After washing, JY cells were incubated with a mixture of fluorescein-labeled hepatitis B virus core antigen-derived reference peptide (FLPSDC[-fl]FPSV) and different concentrations of competitor peptide for 24 h at 4°C, followed by flow cytometric analysis of fluorescence intensity. Binding capacity of competitor peptides was determined as the concentration needed for 50% reference peptide binding inhibition (IC50). DC Culture and Vaccination Procedures. DCs were generated as described previously (5, 19) with minor modifications. Briefly, lymphocytedepleted bone marrow was cultured overnight in six-well plates (Costar, Badhoevedorp, The Netherlands) at 3– 4 ⫻ 106 cell/well in CM consisting of Iscove’s modified DMEM, supplemented with 10% heat inactivated FCS, 50 ␮M ␤-mercaptoethanol, and antibiotics (Life Technologies, Inc.). Nonadherent cells were harvested, resuspended in CM containing 20 ng/ml recombinant mouse granulocyte/macrophage-colony stimulating factor and IL-4 (kindly provided by Dr. G. Zurawski, DNAX, Palo Alto, CA) and cultured in six-well plates at 0.5–1 ⫻ 106 cells/well. Fresh cytokines were given on day 3. Nonadherent and loosely adherent clusters of proliferating DCs were harvested on day 6, resuspended in fresh CM containing 10 ng/ml granulocyte/macrophage-colony stimulating factor and IL-4, and cultured for 2 additional days in six-well plates. For experimental use, nonadherent DCs were harvested on day 8. Prior to vaccination, DCs were loaded in Optimem (Life Technologies, Inc.) for 1 h at 37°C, followed by 2 h at room temperature with 25 ␮M peptide in the presence of 3 ␮g/ml human ␤2-microglobulin (Symbus Bioscience, Southhampton, United Kingdom). Peptide-loaded DCs were washed twice in saline, irradiated (25 Gy), and injected s.c. in the left flank (4 ⫻ 105 in 0.2 ml saline). Construct pCMV-mTRP2 for genetic vaccination was generated by cloning the mouse TRP2 cDNA (generously provided by Dr. V. Hearing) in the coding orientation into the eukaryotic expression vector pCMV-neo (20). Mice were injected intradermally in the abdominal skin with 100 ␮g of affinity purified (Qiagen Plasmid Mega kit, Westburg, Leusden, The Netherlands) plasmid DNA in 0.1 ml of saline. Synthetic peptides (100 ␮g in 0.1 ml of saline) were emulsified with an equal volume of incomplete Freund’s adjuvant (Difco Laboratory, Detroit, MI) and injected s.c. in the left flank. All vaccinations were performed twice with a 2-week interval. Two weeks after the second vaccination, mice were challenged s.c. with 1 ⫻ 105 live B16 tumor cells in 0.1 ml of saline in the right flank. The size of growing tumors was monitored every 3 days. CTL Culture and Chromium Release Assay. Two weeks after the second DC vaccination, spleens were isolated from vaccinated mice, and 4 ⫻ 107 single-cell splenocytes were restimulated with 1 ⫻ 107 irradiated (25 Gy), peptide-loaded LPS blasts in T25 culture flasks (Costar). LPS blasts were generated from splenocytes during 3 days of culture in the presence of 25 ␮g/ml LPS from Salmonella typhosa (Sigma) and 7 ␮g/ml dextran sulfate and loaded with peptide as described above for DCs. Bulk CTLs were isolated after

6 days restimulation by density gradient centrifugation (Lympholyte-M; Cedarlane Laboratory, Sanbio, Uden, The Netherlands). CTL lines were generated by weekly restimulation of 2–5 ⫻ 105 bulk CTLs/well with 1 ⫻ 105 recombinant rat IFN-␥ (TNO, Rijswijk, The Netherlands) treated (50 units/ml for 48 h), irradiated (125 Gy) B16 cells in the presence of 1.5 ⫻ 106 irradiated (25 Gy) autologous splenocytes and 10 Cetus units/ml of recombinant human IL-2 (Cetus Corp., Emeryville, CA) in 24-well culture plates (Costar). Bulk CTLs and CTL lines were used as effectors in a chromium release assay, performed as described previously (21). Briefly, 2 ⫻ 103 Na2[51Cr]O4 (Amersham, Buckinghamshire, United Kingdom)-labeled target cells were cultured with various amounts of effector cells in triplicate wells in Ubottomed microtiter plates (Costar). After 5 h of incubation, the radioactive content of the supernatant was measured. When used in a chromium release assay, B16, Melan-A, and MCT were pretreated with IFN-␥ as described above. Analysis of Surface Expressed, Naturally Processed Peptides. Surface expressed, MHC class I-associated peptides were isolated from IFN-␥-treated B16 melanoma cells by mild acid elution as described (22). Eluted peptides were subjected to reverse-phase HPLC-mediated fractionation using a protocol described previously (23). Isolated peptide fractions were loaded onto HeLa-Kb target cells and tested for stimulation of CTL lytic activity in a chromium release assay as described above. A mixture of synthetic mTRP2derived peptides (10 ␮g each) was subjected to HPLC fractionation using identical conditions as for B16-eluted peptides.

RESULTS Differential Binding Capacity of TRP2-derived Peptides. Previously, TRP2-derived peptide VYDFFVWL (position 181–188) was identified as an epitope recognized by anti-B16 melanoma CTLs in C57BL/6 mice (11). In a subsequent report, an NH2-terminal extended peptide derived from TRP-2, SVYDFFVWL (position 180 –188) was identified as target for human HLA-A2.1-restricted antimelanoma CTLs (12). A computer-generated prediction of the binding affinity of both TRP2-derived peptides for the mouse MHC class I allele Kb and the human MHC class I allele HLA-A2.1 indicates that the NH2terminal extended peptide SVYDFFVWL may bind to both class I alleles with higher affinity as compared with the VYDFFVWL peptide.5 Analysis of the actual differential binding capacity of both peptides to Kb and HLA-A2.1 is depicted in Fig. 1. The results indeed demonstrate a significant increase in binding capacity for Kb (Fig. 1A) and HLA-A2.1 (Fig. 1B) upon NH2-terminal extension of VYDFFVWL to SVYDFFVWL. Consequently, two nonmutated CTL epitopes with differential binding capacity derived from a conserved region of the differentiation antigen TRP2 are available to explore the induction of CTL-mediated antitumor immunity with clinical relevance to the human setting. Peptide-loaded DCs Induce High Avidity TRP2-specific CTLs in Vivo. To investigate whether tolerance against an autologous differentiation antigen could be overcome and CTLs could be activated in vivo against nominal, nonmutated epitopes, mice were vaccinated with TRP2-derived epitopes VYDFFVWL and SVYDFFVWL using DCs as a vaccine. As shown in Fig. 2, A and B, after a single in vitro restimulation of immune splenocytes, bulk CTLs against either the VYDFFVWL or the SVYDFFVWL peptide specifically lyse target cells loaded with either of the TRP2-derived epitopes. Moreover, bulk CTLs also lysed B16 melanoma cells, indicating recognition of endogenously processed mTRP2-derived epitopes. Bulk CTLs generated from mice vaccinated with DCs loaded with an irrelevant peptide using similar conditions did not lyse the indicated target cells above background level (not shown). Similar results were obtained with CD3⫹CD4⫺CD8⫹TCR␣␤⫹ CTL lines LP8 (VYDFFVWL-induced)

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Internet address: http://bimas.dcrt.nih.gov/molbio/hla_bind.


Fig. 1. Binding of TRP2-derived peptides VYDFFVWL (position 181–188) and SVYDFFVWL (position 180 –188) to mouse (A) and human (B) MHC class I molecules. Stabilization of Kb MHC class I molecules expressed on RMA/S cells upon incubation with the indicated concentrations of mouse TRP2-derived peptides, positive control peptide SIINFEKL (ovalbumin position 257–264), and negative control peptide SGPSNTPPEI (adenovirus 5 E1A position 234 –243). MHC class I stabilization was determined by flow cytometric analysis of surface Kb expression (A). Binding to JYexpressed HLA-A2.1 of TRP2-derived peptides, high-affinity (positive) control peptide FLPSDCFPSV (hepatitis B virus core antigen position 18 –27), and low-affinity (negative) control peptide QLNFRATQPL (G250 RCC antigen position 372–381) is shown. Binding capacity is depicted as the peptide concentration needed to inhibit 50% binding of a fluorescein-labeled reference peptide (IC50), as determined by flow cytometry (B).

and LP9 (SVYDFFVWL-induced), generated by identical repetitive in vitro restimulation of bulk CTLs (Fig. 2, C and D, respectively). To investigate the avidity of TRP2-specific CTLs, in vitro peptide titration experiments were performed using the LP8 and LP9 CTL lines and are depicted in Fig. 3. As shown in Fig. 3A, loading of target cells with an irrelevant peptide did not result in detectable lytic activity. Recognition of the VYDFFVWL peptide started to decrease at 1 ␮M and was completely lost at 10⫺5 ␮M (Fig. 3B). In contrast, SVYDFFVWL peptide concentrations as low as 10⫺5 ␮M still resulted in stimulation of lytic activity by both CTL lines tested (Fig. 3C), in agreement with the observed differential binding capacity of both peptides depicted in Fig. 1. These data point out that DC-induced CTLs against nonmutated epitopes derived from autologous antigen can display highly efficient lytic activity. Both TRP2-derived epitopes VYDFFVWL and SVYDFFVWL Are Naturally Processed and Presented by B16 Melanoma. To demonstrate whether the VYDFFVWL, SVYDFFVWL, or both TRP2-derived epitopes are naturally processed and presented by B16 melanoma, surface-expressed peptides associated with MHC class I molecules were eluted from B16 melanoma cells and HPLC fractionated. Stimulation of lytic activity of TRP2-specific CTLs by B16presented peptide fractions is depicted in Fig. 4A. The data demonstrate that target cells loaded with peptide fractions 104 –109 are recognized by TRP-specific CTL lines LP8 and LP9. Analysis of a mixture of synthetic VYDFFVWL peptide and SVYDFFVWL peptide fractionated using identical HPLC conditions as for the B16presented epitopes indicates that the majority of VYDFFVWL peptide

peaks in fraction 105, whereas the majority of SVYDFFVWL peptide peaks in fraction 109 (Fig. 4B). These results indicate that both mTRP2-derived peptides are presented at the surface of B16 melanoma cells, suggesting that both VYDFFVWL and SVYDFFVWL are naturally processed by B16 melanoma. The observed difference in lytic activity against fractions 104 –109 in Fig. 4A remains to be investigated but probably involves differential processing as well as elution efficiency of both epitopes. Protective Immunity against Autologous Melanoma Is Induced by Peptide-loaded DCs but not Peptide or DNA Vaccination. The melanoma model presented in the current report represents a truly autologous model involving the use of nonmutated, naturally processed epitopes derived from a syngeneic tumor antigen. The availability of this autologous model that mimics the human setting allows us to investigate the induction of antitumor immunity by different established vaccination strategies. We used vaccination with peptideloaded DCs, peptide in Freund’s adjuvant and plasmid DNA, all of which have been used successfully in different mouse tumor models. Furthermore, these vaccination strategies are evaluated currently in clinical studies on tumor immunotherapy. Fig. 5 shows the induction of protective antitumor immunity in vivo by different vaccination strategies, as determined by challenge with a lethal s.c. dose of wild-type B16F10 melanoma cells. Vaccination with DCs loaded with an irrelevant H-2Kb binding peptide as a negative specificity control did not mediate protection against a B16 tumor challenge in any of the mice tested. In contrast, partial tumor protection of 20% was observed upon prophylactic vaccination with VYDFFVWL-loaded DCs (Fig. 5A). The observed tumor protection could be enhanced up to 80% when mice were vaccinated with

Fig. 2. Lytic activity of bulk CTLs (A and B) generated after a single in vitro restimulation and of CTL lines LP8 and LP9 (C and D, respectively) generated upon identical repeated in vitro restimulation of spleen-derived lymphocytes isolated after vaccination with VYDFFVWL-loaded DCs (A and C) and SVYDFFVWL-loaded DCs (B and D). Specific lysis (%) of EL4 cells loaded with 10 ␮M TRP2-derived peptides (VYDFFVWL and SVYDFFVWL) or irrelevant peptide (SIINFEKL) and of B16 tumor cells is depicted per E:T (E/T) ratio. Lytic activity of bulk CTLs generated from mice vaccinated with DCs loaded with an irrelevant peptide did not exceed background levels (not shown).

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line Melan-A as well as against normal melanocytes isolated from epidermis (MCT), whereas the nonmelanocytic cell line EL4 is lysed at background levels. The level of melanocyte lysis observed in Fig. 6 appears relatively low, despite sufficient expression levels of both MHC class I and mTRP2 of the used melanocyte target cells as determined by flow cytometric and immunocytochemical analysis (not shown). In contrast to the observed in vitro melanocyte destruction, in vivo coat depigmentation reflecting the destruction of hair bulb-residing melanocytes was detected only sporadically. Mild depigmentation developed in a minor fraction, representing ⬍10% of DC/TRP2 peptide-vaccinated mice, and was primarily restricted to the vaccination site (not shown). The use of nominal CTL epitopes for vaccination will primarily result in activation of specific CTLs, however, and may also result in a secondary antibody response. To exclude this, serum isolated from vaccinated mice was analyzed for the presence of mTRP2-reactive IgG. Immunoperoxidase staining of B16 and Melan-A failed to detect mTRP2-specific IgG in the serum of vaccinated mice, suggesting that the observed low level melanocyte destruction in vivo is CTL mediated and that peptide-loaded DC vaccination did not result in the production of potentially harmful autoantibodies (not shown). Apparently, differentiation antigenspecific CTLs can eradicate tumor cells in vivo without significantly affecting the integrity of normal tissue. DISCUSSION Differentiation antigens can effectively be recognized by the immune system in both humans and mice and may thus serve as potential tumor rejection antigens. Cloning and characterization of the murine homologues of differentiation antigens, in particular those expressed by melanocytic cells, has resulted in the availability of these antigens for preclinical studies in mice (25–27). Fig. 3. Avidity analysis of TRP2-specific CTL lines LP8 (VYDFFVWL-induced) and LP9 (SVYDFFVWL-induced). HeLa-Kb cells were loaded with the indicated titrated amounts of irrelevant (SIINFEKL; A) or relevant (VYDFFVWL and SVYDFFVWL, B and C, respectively) peptide and used as targets in a chromium release assay. Specific lysis (%) of peptide-loaded HeLa-Kb cells was determined at an E:T ratio of 10:1.

SVYDFFVWL-loaded DCs (Fig. 5A). These data indicate that the observed protective immunity depends on loading of DCs with the relevant, TRP2-derived peptides prior to vaccination. Furthermore, in line with the results obtained in vitro as depicted above in Figs. 1 and 3, the difference in binding capacity of the peptides used to load DCs prior to vaccination also appears to affect the level of protective antitumor immunity in vivo. In contrast, both peptide in Freund’s adjuvant (Fig. 5B) and plasmid DNA (Fig. 5C) as alternative vaccination strategies failed to induce significant protective immunity against B16 melanoma. Apparently, peptide in adjuvant and plasmid DNA vaccination used as such are not sufficiently effective to overcome tolerance against self antigen in the current autologous tumor model. Notably, the successful use of both vaccine procedures, as described in “Materials and Methods,” has been reported previously by us, eliminating the involvement of suboptimal vaccination procedures (4, 24). These data emphasize the potency of DCs as a vaccine adjuvant to induce tumor-reactive immunity in vivo directed against autologous tumor-associated antigen. Protective Immunity against Autologous Melanoma Can Proceed without Development of Adverse Autoimmunity. The shared expression of mTRP2 between B16 melanoma and nontransformed melanocytes may result in adverse autoimmunity when mTRP2specific CTLs are activated in vivo (8). As shown in Fig. 6, both VYDFFVWL- and SVYDFFVWL-induced CTLs (Fig. 6, A and B, respectively) display in vitro lytic activity against the melanocyte cell

Fig. 4. HPLC analysis of B16-presented, MHC class I-associated, naturally processed epitopes. Lytic activity of CTL lines LP8 and LP9 against HeLa-Kb target cells loaded with B16-eluted peptide HPLC fractions and 10 ␮M of the indicated control peptides at 10:1 E:T ratio (A) is shown. The HPLC profile in arbitrary units (AU) of fractionated mixed synthetic VYDFFVWL and SVYDFFVWL peptide (10 ␮g each; B) is shown. HPLC fractionation of the B16-eluted peptides and the mixture of synthetic VYDFFVWL and SVYDFFVWL peptide were performed using identical conditions.

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Fig. 5. Analysis of protective immunity against a lethal s.c. B16 tumor challenge induced by vaccination. Mice were vaccinated with DCs loaded with TRP2-derived peptides (VYDFFVWL and SVYDFFVWL) or with an irrelevant peptide (SIINFEKL; A), with TRP2-derived peptides (VYDFFVWL and SVYDFFVWL), or an irrelevant peptide (SIINFEKL) emulsified in incomplete Freund’s adjuvant (IFA; B) and with plasmid DNA encoding mouse TRP2 (pCMV-mTRP2) or control plasmid DNA (pCMV-neo; C). The survival (%) of mice, representing the results from three independent experiments using five mice/group, is depicted in time after tumor challenge.

Here, we present a fully autologous mouse tumor model to dissect essential requirements of effective induction of antitumor immunity against the melanocyte differentiation antigen TRP2, which is involved in mouse as well as human antimelanoma immunity. Two nonmutated TRP2-derived peptides, VYDFFVWL and the NH2terminal extended SVYDFFVWL, were used, both of which are fully conserved in mouse and human TRP2 and bind with different affinity to mouse H-2Kb and human HLA-A2.1. We used the poorly immunogenic, wild-type B16F10 mouse melanoma, which dictates a relatively stringent setting to accomplish protective antitumor immunity. It also facilitates relevant comparative analyses of established vaccination strategies. Both synthetic peptide in adjuvant and naked DNA have proven potent vaccination strategies for tumor-reactive CTL induction in mice and are currently evaluated in clinical studies. In the present autologous model, however, synthetic VYDFFVWL or SVYDFFVWL peptide in Freund’s incomplete adjuvant as well as mTRP2 encoding plasmid DNA failed to induce detectable protective immunity in vivo. Possibly, both vaccination strategies may not have reached the level of CTL activation and/or expansion required for protective immunity against B16 melanoma, although we have re-

ported previously that both vaccination approaches can be successfully applied to induce CTL-mediated immunity in mice using more immunogenic antigens (4, 24). Our results obtained in this poorly immunogenic TRP2 model are in agreement with other mouse tumor models in which tumor-protective CTL induction against an autologous antigen failed, irrespective of the use of multiple vaccination strategies including synthetic peptides and naked DNA (6, 8, 28) or recombinant adenovirus and vaccinia virus (7, 25, 29). In contrast, we show in this report that peptide-loaded DCs as a vaccine in this model did result in significant protective immunity, in line with a recent report that effective antimelanoma immunity could be induced by vaccination using DCs transduced with mTRP2-encoding adenovirus (30). The negative specificity control, DCs loaded with an irrelevant peptide, further indicates that the observed protective immunity is fully dependent of DC loading with the relevant TRP2-derived peptides. Consequently, tumor protection resulting simply from immunity induced against irrelevant antigens, including FCS components, can be excluded. Because all three vaccination modalities were used at similar levels of optimalization, these results emphasize the immunogenic potential of DCs and point out the superiority and value of DCs as vaccine adjuvant when comparatively used in a fully autologous, clinically relevant setting. Both TRP2-derived epitopes used in this study, VYDFFVWL and the NH2-terminal extended SVYDFFVWL, could be eluted from the surface of B16 melanoma cells, pointing out the truly autologous nature of the current tumor model. The observation that both epitopes are naturally processed and presented could result from differential NH2-terminal trimming of proteasome-processed TRP2. Aminopeptidases present in the endoplasmic reticulum may be responsible for generating both length variants upon transporter associated with antigen processing (TAP)-mediated translocation of a precursor peptide from the cytosol (31). Alternatively, the presence of a subdominant proteolytic cleavage site between the NH2-terminal serine and valine residues may explain the presentation of both epitopes. Elucidation of these peptide-processing mechanisms would greatly enhance future prediction of dominant epitopes. Analysis of the immune response induced by vaccination with TRP2 peptide-loaded DCs demonstrates the activation of high avidity CTLs after a single round of in vitro stimulation for both antigenic TRP2 peptides. These results indicate that the lack of endogenous immunogenicity of the TRP2 antigen can be overcome when DCs are used as vaccine adjuvant. Both epitopes mediated the induction of protective immunity against B16 melanoma in vivo when loaded onto DCs prior to vaccination. In contrast with the comparable avidity of

Fig. 6. Lytic activity of CTL lines LP8 and LP9 (A and B, respectively) generated after identical, repeated, in vitro restimulation of spleen-derived lymphocytes isolated after vaccination with VYDFFVWL-loaded DCs (A) and SVYDFFVWL-loaded DCs (B). Specific lysis (%) of EL4 thymoma cells and of melanocytes Melan-A and MCT is depicted per E:T (E/T) ratio.

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VYDFFVWL- and SVYDFFVWL-induced CTLs in vitro, the level of protective immunity in vivo was higher for the high-affinity Kb binding peptide SVYDFFVWL. This may reflect the stringent conditions of the current autologous setting, comparable as encountered in the human setting. One may hypothesize that in the current model an increase in peptide binding capacity enhances the in vivo quantity of tumor-reactive CTLs upon vaccination, rather than the quality. Possibly, the increased influx of DCs still presenting the high-affinity TRP2 peptide in the draining lymph node positively affects the magnitude of the immune response induced. In agreement with this hypothesis, phenotypic analysis of both CTL lines LP8 and LP9 did not show significant differences in expression levels of T-cell receptor, adhesion, and other accessory molecules (not shown). However, previous data have indicated that qualitative differences in mTRP2specific CTLs can be critical to their in vivo antitumor efficacy (32). Most probably an interplay between CTL quality and quantity will ultimately dictate the efficacy of antitumor immunity in vivo. The recently developed MHC tetramer technology will provide a valuable tool to investigate the relative contribution of CTL quality and quantity in antitumor immunity. The shared expression of melanocyte differentiation antigens between normal melanocytes and melanoma tumor cells may result in adverse autoimmunity mediated by differentiation antigen-directed immunity. Despite the observed in vitro lysis of normal melanocytes, albeit of relatively low level, by TRP2-specific CTLs in the current model, in vivo melanocyte destruction occurred only sporadically. In contrast to these findings, induction of immunity against mouse TRP1 (gp75) using human TRP1-encoding DNA (28) or mouse TRP1-encoding recombinant vaccinia virus (29) resulted not only in antitumor immunity but also in extensive coat depigmentation. In both studies, high titers of autoantibodies specific for mouse TRP1 were detected, and CD4⫹ but not CD8⫹ lymphocytes were shown to play an integral part in the development of TRP1-specific immunity. The results presented in this report indicate CD8⫹ CTLs as the predominant effector cells in the development of TRP2-specific immunity, appearing unable to mediate extensive autoimmunity. Recently, CTL-mediated depigmentation resulting from autoimmune destruction of normal melanocytes was reported in mice (8). In the latter study, autoimmunity depended on perforin-mediated CTL lytic activity. Alternatively, skin-homing human melanocyte-reactive CTLs were shown to require expression of the skin homing receptor cutaneous lymphocyte-associated antigen for CTL-mediated melanocyte destruction in vivo (33). The absence of extensive depigmentation in the current model may involve lack of CTL-expressed cutaneous lymphocyte-associated antigen and/or different CTL effector mechanisms. Collectively, our results demonstrate that DCs loaded with nonmutated, naturally processed epitopes derived from a melanocyte differentiation antigen enable efficient activation of high avidity CTLs and induction of antitumor immunity in vivo without extensive autoimmunity against normal tissue. Within the same autologous model, alternative well-established vaccination strategies failed to induce protective immunity. As a result, DC vaccination holds its promise. It is a superior vaccine for clinical application to treat cancer patients, despite the relatively labor-intensive procedures required to generate DC vaccines. Encouraging results have already been obtained in melanoma patients injected with peptide-loaded DCs (34, 35). The autologous tumor model described herein will contribute to further optimalization of immunotherapy using DCs as vaccine adjuvant to break peripheral CTL tolerance and allow the use of unmodified self-antigen as an antitumor vaccine.

ACKNOWLEDGMENTS We thank Jeroen van Bergen for performing HPLC analysis and Dr. Jolanda de Vries for helpful discussion.

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