Cytolytic T Lymphocytes Recognize an Antigen Encoded by MAGE-A10 on a Human Melanoma

Lan-Qing Huang, Francis Brasseur, Alfonso Serrano, Etienne De Plaen, Pierre van der Bruggen, Thierry Boon and Aline Van Pel
J Immunol June 1, 1999, 162 (11) 6849-6854;

Abstract

From melanoma patient LB1751, cytolytic T lymphocytes (CTL) were generated that lysed specifically autologous tumor cells. To establish whether these CTL recognized one of the Ags that had previously been defined, a CTL clone was stimulated with cells expressing various MAGE genes. It produced TNF upon stimulation with target cells expressing MAGE-A10. The Ag was found to be nonapeptide GLYDGMEHL (codons 254–262), which is presented by HLA-A2.1. This is the first report on the generation of anti-MAGE CTL by autologous mixed lymphocyte-tumor cell culture (MLTC) from a melanoma patient other than patient MZ2, from whom the first MAGE gene was identified. MAGE genes are expressed in many tumors but not by normal tissues except male germline cells and placenta, which do not express HLA molecules. Therefore, the identification of an antigenic peptide derived from MAGE-A10 adds to the repertoire of tumor-specific shared Ags available for anti-tumoral vaccination trials.

The identification of tumor Ags is essential for active immunotherapy of cancers. In recent years intensive efforts have been made in this area, which has led to the identification of a wealth of Ags recognized on human tumors by autologous T cells, i.e., T cells obtained from the same patient (1). Among them, an Ag encoded by gene MAGE-1 was initially defined by cultivating blood lymphocytes of patient MZ2 in the presence of a melanoma cell line derived from this patient. A panel of cytolytic T lymphocyte (CTL)4 clones were generated, one of which recognizes a nonapeptide presented by HLA-A1 (2, 3). It was found later that MAGE-1 belongs to a family of at least seventeen related genes, namely MAGE-1 to -12 (now named MAGE-A1 to -A12) (4), MAGE-B1 to -B4 (5, 6, 7), and MAGE-C1 (8). Genes of this family are expressed in various tumors of different histological types but are completely silent in normal tissues, with the exception of male germline cells and placenta (4, 6, 7, 8). Since male germline cells and placental trophoblast cells do not express MHC class I molecules (9), gene expression in these tissues should not lead to Ag expression. This view is supported by the observation that male mice immunized against an Ag encoded by mouse gene P1A, which has the same expression pattern as human MAGE genes, produced strong CTL responses that did not cause testicular inflammation or alteration of fertility (10). Ags encoded by MAGE genes are, therefore, highly tumor specific and are consequently suitable candidates for vaccine-based immunotherapy of cancers.

So far, however, only peptides encoded by MAGE-A1, -A3, and -A6 have been shown to be recognized by autologous CTL derived from mixed lymphocyte-tumor cell culture (MLTC), and all these CTL were generated from only a single patient, namely MZ2 (Refs. 3, 11, and 12; and P. van der Bruggen, unpublished data). We report here that CTL from another melanoma patient, LB1751, recognize an Ag that is encoded by MAGE-A10 and presented by HLA-A2.1.

Materials and Methods

Cell lines

Melanoma cell line LB1751-MEL was derived from a metastatic melanoma in axillary lymph nodes of 67-yr-old male patient LB1751 and grown by a method previously described (13). At passage 4 after the initiation of LB1751-MEL culture that grew as monolayer, aggregates of typical EBV-transformed lymphoblastoid cells appeared in the supernatant. They were collected and cultured separately to obtain B cell line LB1751-EBV. Melanoma culture LB1751-MEL was cleared of EBV-transformed B cells by limiting dilution cloning. DNA fingerprint confirmed that LB1751-MEL and LB1751-EBV originated from the same patient (data not shown).

Melanoma cell lines LB373-MEL and AVL3-MEL were derived from patients LB373 and AVL3, respectively, and were cultured in IMDM (Life Technologies, Gaithersburg, MD) containing 10% FCS. Medullary thyroid carcinoma cell line TT (ATCC No. CRL-1803) was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in DMEM supplemented with 10% FCS.

CTL clone 447A/5 was generated by MLTC as described previously with minor modifications (14). Briefly, MLTC was conducted by culturing PBMC of patient LB1751 with irradiated LB1751-MEL cells in an 8% CO2 incubator in IMDM supplemented with 10 mM HEPES buffer, l-arginine (116 μg/ml), l-asparagine (36 μg/ml), l-glutamine (216 μg/ml), 10% human serum, and 5 ng/ml of recombinant human (rh) IL-7 (Genzyme, Cambridge, MA). On day 3, rhIL-2 (Eurocetus, Amsterdam, The Netherlands) was added at a final concentration of 25 U/ml. Lymphocytes were restimulated weekly with irradiated LB1751-MEL cells in fresh medium containing 25 U/ml of rhIL-2 and 5 ng/ml of rhIL-7. On day 21, CD8+ T lymphocytes were sorted by using anti-CD8-conjugated MACS magnetic MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and cloned by limiting dilution.

Cloning of subgenic fragments of gene MAGE-A10

The 1.1-kb open reading frame (ORF) of MAGE-A10 was cloned in plasmid vector pcDNAI/Amp (Invitrogen, Oxon, U.K.). Three fragments containing the first 270, 546, and 825 nucleotides of MAGE-A10 ORF were amplified by PCR using sense primer 5′-GGAATTCATCATGCCTCGAGCTCCAAAGC-3′ and three anti-sense primers 5′-GCTCTAGAGCTTAGGCTATCTGAGCACTCTG-3′, 5′-GCTCTAGAGCTTAGCACTCGGAGGCTTCACT-3′, and 5′-GCTCTAGAGCTTACCAATCTTGGGTGAGCAG-3′, respectively. For PCR amplification Pfu DNA polymerase (Stratagene, La Jolla, CA) was used. A first denaturation step was done for 5 min at 94°C. The first cycle of amplification was performed for 1 min at 94°C followed by 1 min at 53°C and 1 min at 72°C, and then 25 additional cycles were performed as follows: 1 min at 94°C, 1 min at 65°C, and 1 min at 72°C. Cycling was concluded with a final extension step of 15 min at 72°C. The PCR products were digested with EcoRI and XbaI and unidirectionally cloned into the EcoRI and XbaI sites of plasmid pcDNAI/Amp.

Transfection of COS-7 cells

Transient transfection was performed by the DEAE-dextran-chloroquine method (15). Briefly, 2 × 104 COS-7 cells were transfected with 100 ng of plasmid pcDNAI/Amp-A2, a recombinant plasmid containing the HLA-A2.1 cDNA isolated from a CTL clone of patient SK29 (16), and 100 ng of DNA of MAGE-A cDNAs or subgenic fragments cloned in pcDNAI/Amp. Transfected COS-7 cells were tested in a CTL stimulation assay after 48 h.

CTL stimulation assay

Tumor cells and transfectants were tested for their ability to stimulate the production of TNF by the CTL as described (17). Briefly, 2 × 104 tumor cells were grown for 24 h or transfected COS-7 cells for 48 h after the transfection. The medium was discarded, and 3000 CTL were added in 100 μl of medium supplemented with 10% human serum and 25 U/ml rhIL-2. After 24 h, the supernatant was collected, and its TNF content was determined by testing its cytotoxic effect on WEHI 164 clone 13 cells (18) in a MTT colorimetric assay (17, 19). The concentration (pg/ml) of TNF in the supernatant was estimated by referring the measured OD to the standard curve obtained for known concentrations of human rTNF-β (R&D Systems, Minneapolis, MN). Inhibition with mAbs W6/32 (anti-HLA class I) (20), BB7.2 (anti-HLA-A2) (21), and B1.23.2 (anti-HLA-B and -C) (22) was performed by adding a 1/20 dilution of ascites to the test.

Antigenic peptides and CTL assay

Peptides were synthesized on solid phase using F-moc for transient NH2-terminal protection and were characterized by mass spectrometry. All peptides were >90% pure as indicated by analytical HPLC. Lyophilized peptides were dissolved at 20 mM in DMSO, diluted to 2 mM with 0.01 M PBS, and stored at −20°C. Lysis of target cells by CTL was tested by chromium release as previously described (23). In the peptide sensitization assay, target cells were 51Cr-labeled for 1 h at 37°C and then washed extensively. Target cells (1000) were incubated in 96-well microplates in the presence of various concentrations of peptides for 30 min at 37°C. CTL were added at an E:T ratio of 20. Chromium release was measured after 4 h at 37°C. Enhancement of peptide binding to the HLA-A2 molecule was achieved by incubation of target cells during 51Cr-labeling with a 1/5 dilution of hybridoma culture supernatant of mAb MA2.1 (24, 25).

PCR assay for MAGE-A10 expression

RT-PCR was performed to detect the expression of MAGE-A10 in tumor tissues. Total RNA purification and cDNA synthesis were conducted as previously described (26). One fortieth of the cDNA produced from 2 μg of total RNA was amplified using sense primer 5′-CACAGAGCAGCACTGAAGGAG-3′ and anti-sense primer 5′-CTGGGTAAAGACTCACTGTCTGG-3′, which yielded a 485-bp specific fragment of MAGE-A10. For PCR, a first denaturation step was done for 4 min at 94°C, and then 30 cycles of amplification were performed as follows: 1 min at 94°C, 1 min at 65°C, and 1 min at 72°C. Cycling was concluded with a final extension step of 15 min at 72°C.

Results

Autologous CTL clones directed against melanoma cell line LB1751-MEL

By stimulating PBL from patient LB1751 with irradiated autologous melanoma cell line LB1751-MEL, we obtained a panel of 23 CTL clones that specifically lysed LB1751-MEL cells, but not an autologous EBV-transformed B cell line LB1751-EBV or the NK-sensitive cell line K562. Among them, 5 clones were successfully maintained in long-term culture. Their lytic activities are shown by representative CTL clone 447A/5 in Fig. 1⇓.

FIGURE 1.
FIGURE 1.

Specific lysis of autologous LB1751-MEL cells by CTL 447A/5. Control targets included autologous EBV-transformed lymphoblastoid line LB1751-EBV and NK-sensitive line K562. Chromium release was measured after 4 h of incubation of chromium-labeled target cells with the CTL at different E:T ratios.

CTL clone 447A/5 produced TNF when stimulated with LB1751-MEL cells, and this production of TNF was inhibited by mAb W6/32, which recognizes all HLA class I molecules, and BB7.2, which is specific for HLA-A2, but not by mAb B1.23.2, which recognizes a common determinant of HLA-B and HLA-C molecules (data not shown), indicating that the target Ag is presented by HLA-A2.

CTL 447A/5 recognizes an Ag encoded by MAGE-A10

Because of the high expression level of almost all the MAGE-A genes in melanoma cell line LB1751-MEL (data not shown), we first tested the possibility that CTL 447A/5 recognizes an Ag encoded by one of the MAGE-A genes. We cotransfected into COS-7 cells the cDNA of each MAGE-A gene cloned in expression vector pcDNAI/Amp together with pcDNAI/Amp-A2, a construct encoding HLA-A2.1. The transfectants were tested for their ability to stimulate TNF production by CTL 447A/5. A very significant amount of TNF was produced by the CTL when stimulated with COS-7 cells transfected with MAGE-A10 (Table I⇓). Transfectants obtained with MAGE-A8 also stimulated TNF release, but to a lesser extent. No stimulation was observed with COS-7 cells transfected with HLA-A2.1 alone or with the combination of HLA-A2.1 and any of the other MAGE-A genes.

View this table:
Table I.

Stimulation of CTL 447A/5 by COS-7 cells transfected with HLA-A2.1 and MAGE-A genesa

We further examined the recognition by CTL 447A/5 of allogeneic HLA-A2+ tumor cell lines that express MAGE-A10 or MAGE-A8. The two MAGE-A10+ melanoma cell lines LB373-MEL and AVL3-MEL could stimulate the CTL to produce TNF, but MAGE-A8+ cell line TT could not (Fig. 2⇓A). In addition, LB373-MEL cells were lysed by CTL 447A/5, though less efficiently than the autologous tumor line LB1751-MEL (Fig. 2⇓B). AVL3-MEL cells showed a low level of sensitivity to lysis that could be increased by IFN-γ treatment. TT cells were not lysed by CTL 447A/5 even when pretreated with IFN-γ.

FIGURE 2.
FIGURE 2.

Recognition of allogeneic tumor cell lines by CTL 447A/5. A, LB373-MEL (MAGE-A10+), AVL3-MEL (MAGE-A10+), and TT (MAGE-A8+) cell lines derived from HLA-A2 patients were used to stimulate CTL 447A/5. Autologous tumor cell line LB1751-MEL was included as a control. After 24 h of coculture, production of TNF by the CTL was measured by testing toxicity of the supernatants to TNF-sensitive WEHI-164.13 cells. B, Lysis of LB373-MEL, AVL3-MEL and TT cell lines was assessed by the chromium release assay. Autologous tumor cell line LB1751-MEL was included as a control target. Chromium release was measured after 4 h of incubation of chromium-labeled target cells with the CTL at different E:T ratios.

Identification of the MAGE-A10 antigenic peptide

In an attempt to identify the MAGE-A10 sequence that codes for the antigenic peptide, we generated by PCR amplification fragments of different lengths starting from the initiation codon. These subgenic fragments were cloned in pcDNAI/Amp and transfected into COS-7 cells together with the construct carrying the HLA-A2 gene. CTL stimulation assay was conducted with the transfectants. As shown in Fig. 3⇓, a fragment of 825 bp rendered the transfectants capable of stimulating TNF production by CTL 447A/5, whereas a 546-bp fragment did not, indicating that the sequence coding for the antigenic peptide is located between nucleotide 522 and 825 of the MAGE-A10 ORF. In the amino acid sequence corresponding to nucleotides 522–825, we observed two nonapeptides, MLLVFGIDV (codons 183–191) and GLYDGMEHL (254–262), which conformed to the HLA-A2.1 peptide binding motif, i.e., Leu or Met at position 2 and Leu, Val, or Ile at the C terminus (27). Nonapeptide GLYDGMEHL (254–262) and, less efficiently, decapeptide GLYDGMEHLI (254–263), could sensitize the autologous lymphoblastoid cell line LB1751-EBV to lysis by CTL 447A/5 (Fig. 4⇓A). When pretreated with anti-HLA-A2 Ab MA2.1 for 1 h before peptide sensitization, LB1751-EBV cells pulsed with both peptides showed a significantly increased sensitivity to lysis by the CTL (Fig. 4⇓B). mAb MA2.1 has been reported to facilitate the binding of peptides to HLA-A2 molecules on the cell surface, thereby augmenting the sensitivity of peptide-sensitized target cells to CTL lysis (25).

FIGURE 3.
FIGURE 3.

Identification of the region coding for the antigenic peptide recognized by CTL 447A/5. PCR fragments of different lengths were cloned into pcDNAI/Amp and cotransfected into COS-7 cells with gene HLA-A2.1. Transfected cells were incubated for 24 h with CTL 447A/5, and the TNF in the supernatants was measured by its toxicity to WEHI-164.13 cells.

FIGURE 4.
FIGURE 4.

Lysis by CTL 447A/5 of peptide-sensitized LB1751-EBV cells. A, LB1751-EBV cells pulsed with peptides derived from MAGE-A10. Chromium-labeled autologous EBV-transformed lymphoblastoid cells LB1751-EBV were pulsed for 30 min with peptides as indicated at various concentrations before addition of CTL 447A/5 at an E:T ratio of 20. Chromium release was measured after 4 h. B, Enhancement by mAb MA2.1 of lysis of LB1751-EBV cells pulsed with MAGE-A10 peptides. LB1751-EBV cells were pretreated with anti-HLA-A2 Ab MA2.1 by adding the Ab during 51Cr-labeling. Peptide sensitization and chromium release assay were conducted as in A.

The fact that MAGE-A8-transfected COS-7 cells also stimulated CTL 447A/5 to produce TNF, albeit less efficiently (Table I⇑), prompted us to test peptide GLYDGREHS encoded by the homologous region of MAGE-A8. This peptide did not sensitize LB1751-EBV cells to lysis by CTL 447A/5 unless the peptide was used at 100 μM, a concentration that is too high for a peptide to be considered as a specific Ag in physiological conditions. Even after pretreatment with Ab MA2.1, LB1751-EBV cells were not lysed unless the peptide was used at a concentration as high as 10 μM. The overlapping decapeptide GLYDGREHSV also failed to sensitize LB1751-EBV cells to lysis. The ability of COS-7 cells transfected with MAGE-A8 to stimulate CTL 447A/5 to produce TNF is presumably due to the considerable replication of the transfected gene, resulting in very high levels of MAGE-A8 peptide GLYDGREHS presented on the surface by HLA-A2.1 molecules.

MAGE-A10 is expressed in a variety of tumors

The expression of MAGE-A10 has been studied previously only in a small number of tumors. To obtain a better evaluation of the frequency of tumors expressing this gene, we tested a series of 314 samples of tumors of various histological types by RT-PCR with primers ensuring specificity for gene MAGE-A10. As shown in Table II⇓, it was expressed in a high proportion of lung carcinomas, bladder carcinomas, head and neck and esophageal carcinomas, and melanomas. Of the 71 tumor samples expressing MAGE-A10, all but two expressed simultaneously at least one of genes MAGE-A1, -A2, -A3, -A4 or -A6 (data not shown).

View this table:
Table II.

Expression of MAGE-A10 in tumors

Discussion

The MAGE, BAGE, and GAGE gene families have been identified on the basis of anti-tumoral CTL clones obtained following autologous MLTC conducted with a single melanoma patient, namely patient MZ2 (2, 3, 11, 12, 28, 29). Six different Ags encoded by these genes and presented by HLA-A1, Cw16, and Cw6 were identified. Curiously, when we later analyzed the CTL responses of several other patients bearing melanoma tumors expressing various MAGE-type genes, we obtained no CTL directed against MAGE, BAGE or GAGE Ags. The CTL were directed either against differentiation Ags such as tyrosinase or Melan-A/Mart-1 or against Ags encoded by mutated or overexpressed genes (30, 31, 32, 33, 34, 35). However, autologous CTL were also obtained recently that were directed against Ags encoded by MAGE-type gene NY-ESO-1 (36, 37). CTL clone 447A/5 reported here is the first anti-MAGE CTL obtained by autologous MLTC with a patient other than MZ2. This CTL recognizes the MAGE-A10-encoded nonapeptide GLYDGMEHL, which is presented by HLA-A2. The CTL recognized not only the autologous melanoma cells but also other HLA-A2 tumor cells expressing MAGE-A10. We have confirmed that MAGE-A10 is expressed in a variety of tumor types including melanomas, lung cancers, head and neck and esophageal carcinomas, and bladder carcinomas, where the gene is expressed in more than 30% of the samples.

In a previous report, we suggested, on the basis of semiquantitative RT-PCR assays, that all the tumors expressing MAGE-A10 expressed the gene at a very low level (4). The level we observed was deemed unlikely to be sufficient for the production of enough antigenic peptides to allow recognition by CTL (38). However, it was later observed with a mAb that a tumor with a high level of MAGE-A1 expression contained similar amount of MAGE-A1 and MAGE-A10 protein (39). This led us to reconsider our MAGE-A10 assay, and we have now reached by two different methods the conclusion that the tumors expressing MAGE-A10 express this gene at levels that are similar to those observed for MAGE-A1. In melanoma MZ2-MEL, for instance, the frequency of the MAGE-A1 message is 1/5500 and that of the MAGE-A10 message is 1/6600 (our unpublished data). We conclude that there is no discrepancy between the level of expression of MAGE-A10 and the fact that an Ag encoded by MAGE-A10 can be recognized by a CTL.

An immunotherapy trial of metastatic melanoma patients with detectable disease, which involves immunization with MAGE-A3 peptide presented by HLA-A1, has recently been completed (40, 41). Of the 25 patients who received the complete treatment, i.e., three monthly injections of peptide in skin location distant from the tumor, seven patients showed partial or complete tumor regressions. Surprisingly, no CTL response against the MAGE-A3 Ag could be documented with lymphocytes present in the blood. In trials involving peptide-pulsed dendritic cells, it was reported that some patients developed delayed-type hypersensitivity or CTL responses (42, 43, 44). The identification of a MAGE-A10 Ag presented by HLA-A2 will make it possible to extend such immunotherapy trials to a wider group of patients.

Acknowledgments

We thank Madeleine Swinarska, Anne Gustin, and Maria Panagiotakopoulos for technical assistance. We are grateful to Vincent Stroobant and Anne Authom for the synthesis of peptides.

Footnotes

  • ↵1 L.-Q. H. was supported by a postdoctoral fellowship from the International Institute of Cellular Pathology, Brussels, Belgium.

  • ↵2 Current address: Serv. Analisis Clinicos (Inmunologia), Hospital Virgen de las Nieves, 18014 Granada, Spain.

  • ↵3 Address correspondence and reprint requests to Dr. Aline Van Pel, Ludwig Institute for Cancer Research, Brussels Branch, 74 Avenue Hippocrate, UCL 7459, B-1200 Brussels, Belgium. E-mail address: vanpel{at}licr.ucl.ac.be

  • ↵4 Abbreviations used in this paper: CTL, cytolytic T lymphocyte; MLTC, mixed lymphocyte-tumor cell culture; ORF, open reading frame; rh, recombinant human.

  • Received December 21, 1998.
  • Accepted March 9, 1999.

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The Journal of Immunology: 162 (11)
The Journal of Immunology
Vol. 162, Issue 11
1 Jun 1999
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