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Keywords:

  • melanoma;
  • tumor antigen;
  • TRP-2;
  • T cell epitope;
  • immunotherapy

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Acknowledgements
  6. References

Tyrosinase-related protein-2 (TRP-2) is a known target antigen of spontaneous cytotoxic T cell responses in melanoma patients. Its frequent expression in metastatic tumors suggests that it might be an ideal candidate antigen for T cell-based immunotherapy. To provide knowledge about TRP-2-derived T cell epitopes useful for immunotherapy we applied a “reverse immunology strategy” based on repeated in vitro peptide stimulation of peripheral blood lymphocytes (PBL) from normal donors with predicted HLA-A*01 ligands. This led to the identification of TRP-2181–190 as the first HLA-A*01-presented TRP-2-derived epitope. T-cell lines specific for peptide TRP-2181–190 could be established from PBL of 50% of the normal HLA-A*01+ donors tested. Such T cells responded specifically to autologous dendritic cells transduced virally with TRP-2, as well as to HLA-A*01+, TRP-2+ melanoma cells, although tumor cells had to be pretreated with IFN-γ to become susceptible to T cell recognition. Interestingly, short-term in vitro peptide stimulation of PBL from HLA-A*01+ melanoma patients showed the presence of TRP-2181–190-reactive CD8+ T cells in some donors, suggesting their in vivo sensitization. Because TRP-2181–190 overlaps with the known HLA-A*0201-presented epitope TRP-2180–188, an 11mer peptide encompassing both epitopes might be of specific value for vaccination of a broad population of melanoma patients. © 2005 Wiley-Liss, Inc.

Adoptive transfer of in vitro expanded antigen-specific cytotoxic T lymphocytes (CTL) has been demonstrated recently to mediate massive destruction of metastatic tumor cells in advanced melanoma patients. The transferred T cells, either bulk or cloned CTL, exhibited specificity for HLA class I-presented epitopes derived from melanocyte/melanoma differentiation antigens (MDA).1, 2 TRP-2 is a member of the MDA-family that has been demonstrated repeatedly to be recognized by T cells isolated from tumors and the peripheral blood of melanoma patients.3, 4 Overexpression of TRP-2 has been described recently to confer a certain resistance against chemotherapeutic drugs and radiation to melanoma cells.5 This might explain why TRP-2 expression is exceptionally stable in vivo and can be detected in up to 89% of the tumor specimens tested,6 suggesting that TRP-2 might be an ideal target antigen for T cell-based active and passive immunotherapies.

Design and evaluation of such therapies is dependent on the identification of T cell epitopes derived from the target antigen. In the case of TRP-2, antigenic ligands for the HLA-A*02-, HLA-A*31/33- and HLA-Cw*08-molecule are known.3, 4, 7, 8, 9, 10, 11 Our present study provides knowledge about TRP-2-derived epitopes presented by the HLA-A*01 molecule, which is expressed in up to 26% of the Caucasian population.12 By a “reverse immunology strategy” based on epitope prediction and in vitro T cell sensitization against the predicted synthetic peptide sequences, we identified TRP-2181–190 as an HLA-A*01-presented CD8+ T cell epitope.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Acknowledgements
  6. References

Cell lines

Melanoma cell lines Ma-Mel-36 (TRP-2+, HLA-A*01+), Ma-Mel-37b (TRP-2+, HLA-A*01+) and UKRV-Mel-18a (TRP-2+, HLA-A*01) were established in our laboratory. Melanoma cells, P1.A1/hβ2m cells13 (mouse mastocytoma P1.HTR cells transfected with human β2m and HLA-A*01) and TAP-deficient, HLA-A*01+ BM36.1 cells14 (kindly provided by A. Ziegler, Charite, Berlin) were maintained in RPMI 1640/2 mM glutamine (PAA Laboratories, Cölbe, Germany) supplemented with 10% FCS (PAA Laboratories), 100 U/ml penicillin and 100 μg/ml streptomycin. Medium for maintenance of P1.A1/hβ2m cells additionally contained 800 μg/ml geneticin (Gibco-Invitrogen, Karlsruhe, Germany).

Synthetic peptides

Peptides were synthesized by Fmoc chemistry and analyzed by HPLC at the Division of Peptide Synthesis of the German Cancer Research Center, Heidelberg. Lyophilized peptides were dissolved at 50 mg/ml in DMSO and stored at −20°C.

HLA-typing of blood donors

HLA class I-type of peripheral blood mononuclear cells (PBMC) from healthy donors and melanoma patients was determined by serological typing using the commercial typing test kit Lymphotype HLA-AB 120 (Biotest, Dreieich, Germany) or by low-resolution genotyping of the HLA-A, -B loci using the PCR-SSP method (Olerup SSP AB Genovision, Vienna, Austria). PCR was carried out by using the GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA). PCR products were separated on 2% agarose gels containing ethidium bromide and visualized under ultraviolet (UV) light.

Generation of monocyte-derived dendritic cells

Frozen PBMC from normal blood donors were used as a source of monocytes and T cells, after a routine protocol.9 Monocytes were enriched from thawed PBMC by plastic adherence (Day 0) and used for the generation of dendritic cells (DC); the non-adherent cell fraction was frozen as a source of T cells for later use. Monocytes were incubated in X-VIVO15 supplemented with 10% heat inactivated human AB serum (HS) (PAA Laboratories). On Day 1 and Day 3, IL-4 (800 U/ml; R&D Systems, Wiesbaden-Nordenstadt, Germany) and GM-CSF (1,000 U/ml; Novartis, Nuremberg, Germany) was added to the culture. On Day 6, non-adherent cells were harvested as a source of immature DC and resuspended in fresh medium containing the cytokines IL-4 (800 U/ml), GM-CSF (1,000 U/ml), IL-1β (10 ng/ml; R&D Systems), IL-6 (1,000 U/ml; R&D Systems), PGE2 (1 μg/ml; Pharmacia Upjohn) and TNF-α (10 ng/ml; Boehringer Ingelheim, Germany) to induce DC maturation. After 24–48 hr, DC were harvested and used for stimulation of autologous PBL.

In vitro priming of T cells

T lymphocytes were routinely cultured in T cell medium (TCM) consisting of IMDM/25 mM HEPES (Biochrom, Berlin, Germany) supplemented with 2 mM glutamine, 10% HS, 100 U/ml penicillin and 100 μg/ml streptomycin. Cytokine-matured DC were employed for primary activation of T cells after a protocol described previously.9 In addition, a slightly modified protocol was applied. Briefly, DC were pulsed for 4 hr with synthetic TRP-2 peptides and added to autologous PBL at a ratio of 1:20 in TCM supplemented with IL-6 (1,000 U/ml) and IL-12 (1 ng/ml). At Day 10–12 of culture, autologous DC loaded with peptide (10 μg/ml) were used to restimulate lymphocytes at a ratio of 1:40 in the presence of IL-2 (20 IU/ml) and IL-7 (10 ng/ml). After the second round of stimulation, CD8+ T cells were positively selected with anti-CD8 mAb conjugated microbeads following the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). CD8+ T cells were restimulated weekly with peptide-loaded autologous monocytes.

Analysis of T cell reactivity

Reactivity of in vitro stimulated T cells was analyzed by IFN-γ ELISPOT assay. Therefore, 96-well microfiltration plates (Millipore, Eschborn, Germany) were coated with 5 μg/ml of anti-human IFN-γ mAb (1-D1K; Mabtech, Stockholm, Sweden) in PBS overnight at 4°C. Unbound antibody was removed by washing with PBS. After blocking the plates with 100 μl TCM (1 hr, 37°C), 5 × 104 target cells were seeded into the wells and peptide was added where indicated. Responder CD8+ T cells were added at the indicated numbers per well. TCM was added to a final volume of 200 μl/well. Plates were incubated at 37°C in 5% CO2 for 20 hr. For development cells were removed by washing the plates followed by the addition of biotinylated mAb anti-human IFN-γ (7-B6–1; Mabtech, Stockholm, Sweden) at 1 μg/ml in PBS plus 0.5% BSA for detection of captured cytokine. After incubation for 2 hr, plates were washed and avidin-peroxidase complex (Vectastain Elite Kit; Vector Laboratories) was added for 1 hr. Unbound complex was removed by washing. AEC substrate (Sigma, Taufkirchen, Germany) was added and incubated for 10 min. All determinations were carried out in duplicates or triplicates. Spots were imaged using the Bioreader 3000 (Bio-Sys, Karben, Germany). The data are presented as mean IFN-γ spots per the indicated amount of responder T cells.

Construction and production of recombinant MVA

Vaccinia virus strain MVA was originally obtained from Anton Mayr (University of Munich, Germany). The 1,560-nt ORF encoding human TRP-2 (sequence corresponds to GenBank accession no. S69231, except for a G to A transition at position 233 of the coding region) was cloned into vector pCMVβ (BD Bioscience Clontech, Palo Alto, CA). Three vaccinia virus early transcription termination motifs (TTTTTNT) contained in TRP-2 were altered by using the GeneEditor™ in vitro site-directed mutagenesis system (Promega, Mannheim, Germany). After mutagenesis, the entire TRP-2 gene was inserted between the PmeI and BamHI site of the pIII-ΔHR-PH5 MVA transfer vector plasmid (I.D., M.K. and G.S. unpublished results),15 placing it under the control of the vaccinia virus modified H5 early late promoter.16 Recombinant MVA viruses were propagated and titered as described recently.17, 18 To generate vaccine preparations, viruses were routinely purified by ultracentrifugation through sucrose and reconstituted in 10 mM Tris pH 7.4, 120 mM NaCl saline buffer.

Infection of DC

Before infection, MVA stock solutions were homogenized by sonication. DC (106) were infected with MVA-WT or MVA-TRP-2 at a multiplicity of infection of 5 (MOI 5) for 3 hr. Free virus particles were removed by 2 washes with fresh medium and cells were resuspended for overnight culture in 400 μl TCM. After 16 hr incubation, infected DC were used as target cells in T cell stimulation assays.

Detection of in vivo-primed T cell responses in melanoma patients

For detection of in vivo sensitized TRP-2181–190-reactive T cells, frozen PBMC from melanoma patients were thawed and seeded at 2 × 106 cells/ml per well of a 24-well plate in RPMI 1640/HEPES/2 mM glutamine supplemented with 10% human AB serum, 100 U/ml penicillin, 100 μg/ml streptomycin. Peptide (10 μg/ml), IL-2 (20 U/ml) and IL-7 (10 ng/ml) were added to the wells. After incubation for 10 days, cells were harvested and CD8+ T cells were positively selected from PBMC cultures with anti-CD8-specific mAb conjugated microbeads according to the manufacturer's instructions (Miltenyi Biotec). The indicated number of CD8+ T cells was analyzed for its reactivity toward 4 × 104 P1.A1/hβ2m13 target cells in the absence or presence of peptide TRP-2181–190 (10 μg/ml) by IFN-γ ELISPOT assay, as described above. Blood donations from patients were approved by the Institutional Review Broad and an informed consent was given by all patients.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Acknowledgements
  6. References

We applied the predictive computer algorithms of the “SYFPEITHI” database19 to obtain information about potential HLA-A*01-presented epitopes derived from the TRP-2 antigen. Of the predicted antigenic ligands, 5 were selected and synthesized as peptides, all exhibiting optimal amino acid sequences at HLA-A*01 anchor positions (aspartic acid [D] or glutamic acid [E] at position 3 and tyrosine [Y] at the C-terminal position) (Table I). These peptides were then used to sensitize T cells from HLA-A*01+ normal donors in vitro. After primary stimulation with peptide-loaded autologous DC and restimulation, specificity of bulk T cell cultures was analyzed on peptide-loaded HLA-A*01+ target cells by IFN-γ ELISPOT assay. Although each candidate peptide was analyzed for its stimulatory capacity in at least 3 different donors, we could not obtain T cell specificity for peptides TRP-265–75, TRP-2236–244 and TRP-2505–515 and only one donor responded to peptide TRP-2447–455 (data not shown). In contrast, T-cell lines specifically recognizing peptide TRP-2181–190 (VYDFFVWLHY) when pulsed on different HLA-A*01+ target cells could be generated from PBL of 6 of 11 donors (Fig. 1 and data not shown). Peptide-specificity of T cells was already detectable after 1–2 rounds of in vitro restimulation. We concentrated our further studies exclusively on peptide TRP-2181–190 and employed peptide-specific CD8+-selected T-cell lines derived from the HLA-A*01-homozygous normal donor AP for the subsequent analysis.

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Figure 1. In vitro sensitization of HLA-A*01-restricted T cells with peptide TRP-2181–190. CD8+ T cells were positively selected from in vitro TRP-2181–190-sensitized T cells of HLA-A*01-homozygous donor AP and analyzed for their specificity by co-incubation with HLA-A*01+ P1.A1/hβ2m and BM36.1 target cells, respectively, in the absence (white bars) or presence (black bars) of peptide TRP-2181–190 (10 μg/ml). (a) Stimulation of T cells was demonstrated by IFN-γ ELISPOT assay. Bars represent the mean of triplicates, error bars indicate standard deviations (SD). (b) Peptide sensitivity of CD8+ T cells from donor AP was determined by incubation with HLA-A*01+ BM36.1 target cells in the presence of titrated amounts of peptide TRP-2181–190. Determinations were carried out in duplicates. (a,b) Representative results from 1 of 2 experiments are shown.

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Table I. Predicted HLA-A*01 Epitopes Derived from TRP-2
PositionSequencePrediction scorea
  • a

    Based on predictive computer algorithms of the “SYFPEITHI” database.19

65–75RADTRPWSGPY25
181–190VYDFFVWLHY32
236–244GNESFALPY31
447–455TSDQLGYSY30
505–515LMETHLSSKRY25

To demonstrate processing of the TRP-2181–190 peptide from the TRP-2 antigen, we took advantage of a newly constructed modified vaccinia virus Ankara (MVA) recombinant for TRP-2 as an antigen delivery system. MVA is known to be capable of infecting human DC, the most important inductors of primary T cell activation. Therefore, we employed monocyte-derived DC infected with MVA-TRP-2 and wildtype MVA, respectively, as target cells for the stimulation of autologous TRP-2181–190-specific CD8+ T cells. As shown in Figure 2, T cells recognized immature and cytokine-matured DC infected with MVA-TRP-2 with high specificity compared to DC infected with wildtype MVA, verifying TRP-2181–190 as the first HLA-A*01-presented epitope processed from the TRP-2 antigen.

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Figure 2. Presentation of epitope TRP-2181–190 by MVA-TRP-2 infected DC. Stimulation of TRP-2181–190-specific CD8+ T cells from donor AP by autologous immature or mature DC infected with wildtype MVA (MVA-WT) or TRP-2-recombinant MVA (MVA-TRP-2) was analyzed by IFN-γ ELISPOT assay. Exogenous peptide TRP-2181–190 (10 μg/ml) was added where indicated. Bars = mean of triplicates, error bars = SD. Representative results from 1 of 5 experiments are shown.

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The TRP-2181–190-specific CD8+ T cells were used to analyze epitope presentation by tumor cells. Surprisingly, coincubation of T cells with the melanoma target cells Ma-Mel-36 and Ma-Mel-37b, both expressing HLA-A*01- and TRP-2, only induced background secretion of cytokine, comparable to that obtained by co-culture with the HLA-A*01, TRP-2+ control cell line UKRV-Mel-18a (Fig. 3a). The lack of specific T cell stimulation was not due to a deficiency in HLA-A*01 surface expression, because Ma-Mel-36 tumor cells were well recognized after exogenous peptide loading (Fig. 3b). Pre-treatment of Ma-Mel-36 and Ma-Mel-37b cells with IFN-γ induced effective stimulation of TRP-2181–190-specific T cells (Fig. 3a,b). This stimulation could only slightly be increased by exogenous peptide loading, indicating that reactivity of the T cell line was indeed directed toward the tumor cells presenting the TRP-2181–190 epitope processed from the endogenous TRP-2 antigen (Fig. 3b). Importantly, T cell recognition of IFN-γ-treated tumor cells was abolished in the presence of an anti-HLA class I mAb but not by an anti-HLA-BC mAb20 (Fig. 2b). Because the TRP-2181–190-specific T-cell line was established from an HLA-A*01 homozygous donor, these data suggest that recognition of the allogenic tumor cells was restricted to the HLA-A*01 allele.

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Figure 3. Stimulation of TRP-2181–190-specific T cells by tumor cells. Activation of TRP-2181–190-specific CD8+ T cells from donor AP by TRP-2+ melanoma cells was analyzed by IFN-γ ELISPOT assay. (a) Different HLA-A*01+ (Ma-Mel-36, Ma-Mel-37b) and HLA-A*01 (UKRV-Mel-18a) melanoma cells, either pre-treated with IFN-γ (500 U/ml; 48 hr) (black bars) or left untreated (white bars), were used as target cells for stimulation of T cells. Bars = mean of triplicates, error bars = SD. (b) Ma-Mel-36 cells either untreated (white bars) or pre-treated with IFN-γ (500 U/ml; 48 hr) (black bars), were used to stimulate TRP-2181–190-specific T cells. To block T cell stimulation, melanoma cells were pre-incubated with antibodies W6/32 (anti-HLA Class I) and BB1.23.2 (anti-HLA-BC) for 1 hr. Where indicated, exogenous TRP-2181–190 peptide (10 μg/ml) was added. Bars = mean of triplicates, error bars = SD. (a,b) Representative results from 1 of 2 experiments are shown.

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Analysis by FACS and RT-PCR (data not shown) demonstrated that IFN-γ treatment of Ma-Mel-36 cells did not induce a remarkable increase in HLA-A*01 surface presentation or TRP-2 antigen expression, leading to the hypothesis that the cytokine might influence the expression pattern of specific components of the cellular antigen processing machinery required for TRP-2181–190 epitope generation. We made comparable observations previously for the HLA-A*0201 epitope TRP-2360–368.21 Several melanoma cell lines had to be pretreated with IFN-γ to become capable of presenting the TRP-2360–368 antigenic ligand. It was demonstrated that IFN-γ induces the expression of the α- and β-subunits of the proteasomal activator PA28 in these cells and that PA28αβ activity is required for the efficient generation of N-terminally extended peptide precursors of the TRP-2360–368 epitope by the proteasome.21 Furthermore Schultz et al.22 demonstrated that activity of the IFN-γ inducible immunoproteasome subunit LMP-7 is required for the presentation of an epitope derived from tumor antigen MAGE-3, indicating that IFN-γ can influence epitope processing at various levels of the antigen processing machinery.

We then asked whether TRP-2181–190-reactive T cells might be detectable within the peripheral blood of HLA-A*01+ melanoma patients. To answer this, PBMC from 13 patients were stimulated only once with peptide TRP-2181–190 and analyzed for their peptide-specific reactivity after an incubation period of 10 days. This short-term in vitro peptide-stimulation has been demonstrated to recall memory T cell responses effectively without inducing activation of primary T cells,23 because it is not based on repeated T cell stimulation with peptide-loaded DC as this was done for priming of T cells from healthy individuals. Indeed, TRP-2181–190-reactive T cells could be detected in 2 of 13 donors tested. T cell reactivity was clearly restricted to the CD8+ T cell fraction (Fig. 4), suggesting that TRP-2181–190 might be useful for T cell-based immunotherapy of melanoma patients.

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Figure 4. TRP-2181–190-reactive T cells can be detected in the peripheral blood from HLA-A*01+ melanoma patients. PBMCs from 13 HLA-A*01+ melanoma patients were seeded in the presence of peptide TRP-2181–190. After incubation for 10 days, cells were harvested and peptide-reactivity of positively selected CD8+ T cells was analyzed by IFN-γ ELISPOT assay. Cytokine secretion of T cells from donors LA and KR was increased when incubated with P1.A1/hβ2m target cells loaded with TRP-2181–190 (10 μg/ml) compared to target cells alone (w/o peptide). The data are means of duplicates, error bars = SD. No specific T cell reactivity could be detected in the short-term peptide stimulation cultures of the residual 11 donors (data not shown).

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Interestingly, TRP-2181–190 (VYDFFVWLHY) overlaps by 8 amino acids with the epitope TRP-2180–188 (SVYDFFVWL) known to be presented by the HLA-A*0201 molecule.7 Consequently, an 11mer peptide encompassing both epitopes should be useful for vaccination of HLA-*01+ and HLA-A*0201+ melanoma patients, which would allow its application to a broad spectrum of patients. Because TRP-2 has been demonstrated recently to also be expressed by malignant glioma cells,24 such epitope-based vaccines might be applicable to an even larger group of tumor patients. Knowledge of TRP-2 derived T cell epitopes is not only a prerequisite for epitope-based immunotherapy, but also allows evaluation of the T cell stimulatory capacity of complex TRP-2-based vaccines, e.g., recombinant viruses. Our present study has demonstrated that MVA-TRP-2 can efficiently deliver the antigen to DC and might therefore also be used in future clinical trials for immunotherapeutic treatment of melanoma/glioma patients.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Acknowledgements
  6. References

We thank A. Sucker, C. Sterzik and A. Funk for their expert technical assistance. This work was in part supported by the EU (QLG1-CT-2002-0068, OISTER) to D.S. and by the DFG grant SFB 456/B7 to G.S. and I.D.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Acknowledgements
  6. References
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