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The Journal of Immunology, 2000, 164: 6041-6045.
Copyright © 2000 by The American Association of Immunologists

Cytolytic T Lymphocytes Raised Against a Human Bladder Carcinoma Recognize an Antigen Encoded by Gene MAGE-A121

Leonora Heidecker, Francis Brasseur, Michael Probst-Kepper2, Maryse Guéguen3, Thierry Boon and Benoît J. Van den Eynde4

Ludwig Institute for Cancer Research, Brussels Branch, and Cellular Genetics Unit, Université Catholique de Louvain, Brussels, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human bladder carcinoma line LB831-BLC expresses several distinct Ags that are recognized by different autologous CTL. Here, we show that one of these Ags is presented by HLA-Cw7 and encoded by gene MAGE-A12. This is the first time that CTL directed against a MAGE-encoded Ag have been derived from the lymphocytes of a patient with cancer other than melanoma. This new Ag was found to be nonapeptide VRIGHLYIL, corresponding to position 170–178 of the MAGE-A12 protein. Gene MAGE-A12 is silent in normal tissues except in male germline cells, which do not express HLA molecules. It is expressed in 26–62% of melanomas, infiltrating bladder carcinomas, lung carcinomas, esophageal carcinomas, and head and neck carcinomas. Because HLA-Cw7 is present in 43% of Caucasians, this new Ag is shared by many tumors and should be a useful target for cancer immunotherapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Avariety of genes that are expressed in many tumors but not in most normal tissues encode tumor-specific peptides that can be recognized by CD8+ T cells (1). Genes MAGE, GAGE, and BAGE were initially identified because they encode Ags recognized by CTL derived from the lymphocytes of a melanoma patient that were stimulated in vitro with autologous tumor cells (2, 3, 4, 5, 6, 7). This patient enjoys an extraordinarily favorable clinical course 13 years after abdominal metastases were treated by surgery, chemotherapy, and vaccination with irradiated autologous tumor cells. MAGE-specific CTL were also obtained later, either by stimulating lymphocytes of a few other melanoma patients with autologous tumor cells or by stimulating lymphocytes from non-cancer patients with APC pulsed with MAGE peptides or infected with MAGE-containing recombinant viruses (8, 9, 10, 11, 12).

Because isolating CTL against tumors other than melanoma appears to be more difficult, less is known about the Ags that can be recognized by T cells on these tumors. We previously reported the isolation of autologous tumor-specific CTL from bladder carcinoma patient LB831. These CTL clones recognized three distinct Ags. One of them could be identified and was found to result from a point mutation in a gene with ubiquitous expression and unknown function (13). Using the same bladder carcinoma line, LB831-BLC, we have established another series of MHC class I-restricted CTL clones showing specific lysis of the autologous tumor cells, and we show here that one of them is directed against an Ag encoded by gene MAGE-A12 and presented by HLA-Cw7.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

Bladder carcinoma cell line LB831-BLC was derived from the primary invasive bladder tumor (pT3,G3) of a 67-yr-old male patient, LB831 (HLA-A*2403, -A3, -B*4403, -B*4901, -Cw*0401, and -Cw*07011). The karyotype of the cell line performed at passage 7 showed that the number of chromosomes varied from 56 to 144, confirming that the LB831 cell line was a tumor line. MI13443-MEL is a melanoma cell line and LE9211-RCC is a renal carcinoma cell line. Both were derived from HLA-Cw7 patients. LB373-MEL is a melanoma line derived from an HLA-Cw7-negative patient. The tumor cells were cultured in Iscove’s medium (Life Technologies, Gaithersburg, MD) containing 10% FCS (Life Technologies). Lymphoblastoid cell line LB831-EBV was derived from PBLs of patient LB831 with 1 µg/ml cyclosporin A (Sandoz, Basel, Switzerland) and 20% of supernatant of EBV-transformed B95-8 cells using standard techniques. This cell line was grown in RPMI 1640 (Life Technologies) containing 10% FCS. 293-EBNA cells are human embryonic kidney cells expressing constitutively EBV nuclear Ag 1 (EBNA-1).5 They were obtained from Invitrogen (Carlsbad, CA) and grown in DMEM containing 10% FCS. All media were supplemented with L-arginine (116 µg/ml), L-asparagine (36 µg/ml) and L-glutamine (216 µg/ml).

Antitumor CTL clones

An autologous mixed lymphocyte-tumor cell culture (MLTC) was performed by mixing irradiated CD80-transfected LB831-BLC cells cultured in human serum and CD8+ T lymphocytes, as described previously (13). Irradiated non-CD8+ cells were added to the mixed culture during the first stimulation. On day 3, IL-2 25 U/ml was added. After 1 wk, 5 x 105 lymphocytes were restimulated with irradiated CD80-transfected tumor cells and 25 U/ml IL-2. On day 28, lymphocytes from the culture were cloned by limiting dilution in Iscove’s medium supplemented with IL-2 (50 U/ml).

CTL stimulation assay

A total of 3000 CTLs were added to microwells containing 10,000 stimulator cells in 100 µl Iscove’s medium supplemented with 10% human serum and 25 U/ml IL-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 (14) in a MTT colorimetric assay (15). Inhibition with mAbs W6/32 (anti-HLA class I), B1.23.2 (anti-HLA-B and -C) was performed by adding a 1/20 dilution of ascites to the test.

Antigenic peptides and CTL assay

All peptides were synthesized in house on solid phase using Fmoc for transient NH2 terminal protection (16). Peptides were characterized by mass spectrometry, lyophilized, and stored at 20 mg/ml in DMSO at -20°C.

Lysis of target cells by CTL was tested by chromium release as previously described (17). Briefly, in 96-well conical microplates, 1000 51Cr-labeled target cells were incubated for 30 min at 37°C with various concentrations of peptides. CTLs were then added at different E:T ratios. The supernatants were collected after 4 h of incubation at 37°C for the measurement of the chromium released from lysed cells.

Transient transfections

Transient transfections were performed with the Lipofectamine reagent (Life Technologies). Briefly, 5 x 104 293-EBNA cells were transfected in a flat-bottom 96-well plate with 100 ng DNA of the MAGE-A12 cDNA or subgenic fragments cloned into pcDNA3 (Invitrogen), 50 ng plasmid pcDNA3 containing the HLA-Cw7 cDNA, and 1.5 µl Lipofectamine. LB373-MEL cells (10,000) were transfected with 150 ng of the HLA-Cw7 construct and 1 µl Lipofectamine. Transfected cells were tested in a CTL stimulation assay after 24 h. Cloning of the HLA-Cw7 cDNA from LB831-BLC was performed as described previously (13). Its sequence was identical with the reported sequence of allelic subtype Cw*07011, except at position 1087 of the coding sequence where G was found instead of A. This nucleotide change causes the replacement of a threonine by an alanine in the intracytoplasmic domain of the molecule. We then cloned and sequenced the Cw7 allele from cell line LCL721 (European Collection of Cell Cultures), which is the reference line for allele Cw*07011, and we also found a G at position 1087. We conclude that the reported Cw*07011 sequence is erroneous and shoud have G at this position.

Cloning of subgenic fragments of gene MAGE-A12

A MAGE-A12 cDNA containing the entire open reading frame (ORF) of 945 bp was used as template for PCR amplification (18). Eight fragments containing the first 195, 342, 525, 540, 591, 651, 683, and 816 nucleotides of the MAGE-A12 ORF were amplified using the forward primer 5'-CCTACCTGCTGCCCTGACCA-3' (LHE7) and reverse primers 5'-CCTAAGGACTGTGGGGAGGA-3' (LHE2), 5'-CCAACTAAGCCATCTTCCTA-3' (LHE3), 5'-GTGACAAGGATCTACAAGTG-3' (LHE4), 5'-CCAGTCAGGTGACAAGGATG-3' (LHE10), 5'-CCTGTCTAGGGCACGATCTG-3' (LHE8), 5'-CTCCTAAGGGGCACAGTCGC-3' (LHE9), 5'-TCAGATGCCTACAACACACT-3' (LHE5), and 5'-GGACCCTACAGGAACTCGTA-3' (LHE6), respectively. Taq DNA polymerase (TaKaRa Taq) was used for PCR amplification. A first denaturation step was performed for 5 min at 94°C, and then 25 cycles of amplification were performed as follows: 1 min at 94°C for all primers; 2 min at 62°C for primers LHE3, LHE4, LHE5 or 2 min at 64°C for primers LHE2, LHE6, LHE8, LHE9, LHE10; and 3 min at 72°C for all primers. Cycling was concluded with a final elongation step of 10 min at 72°C. The PCR products were cloned into vector pcDNA3 using the Bidirectional Eukaryotic TOPO TA Cloning Kit (Invitrogen).

PCR assay for MAGE-A12 expression

RT-PCR was performed to detect the expression of MAGE-A12 in tumor tissues. Total RNA purification and cDNA synthesis were conducted as previously described (19). One-fortieth of the cDNA produced from 2 µg total RNA was amplified using sense primer 5'-CGTTGGAGGTCAGAGAACAG-3' and antisense primer 5'-GCCCTCCACTGATCTTTAGCAA-3'. For PCR, a first denaturation step was done for 4 min at 94°C, and then 32 cycles of amplification were performed as follows: 1 min at 94°C; 2 min at 62°C; and 3 min at 72°C. Cycling was concluded with a final extension step of 15 min at 72°C.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Autologous CTL clones recognizing bladder carcinoma line LB831-BLC

By stimulating blood lymphocytes from bladder carcinoma patient LB831 with irradiated autologous tumor cells transfected with the cDNA of the costimulatory molecule CD80 (B7-1), we previously obtained a panel of HLA class I-restricted CD8+ T cell clones showing specific lysis of the autologous tumor cells. On the basis of inhibition with anti-HLA-Abs and recognition of allogeneic tumor cells, the CTL clones were distributed into three groups that recognized three distinct Ags, LB831-A, -B, and -C (13). By repeating the same type of MLTC using CD80-transfected tumor cells, we established another series of LB831-specific CTLs. Some of them, such as CTL 501D/19, recognized an Ag distinct from the first three, which we called LB831-D (Fig. 1Go).



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  		FIGURE 1. Recognition of autologous and allogeneic tumor cell lines by CTL 501D/19. A, The following target cells were tested in a standard 4-h chromium release assay: LB831-BLC, the autologous bladder carcinoma line; LB831-EBV, autologous B cells transformed with the EBV; LE9211-RCC, an allogeneic HLA-Cw4 and HLA-Cw7-positive renal carcinoma line; MI13443-MEL, an allogeneic HLA-Cw4 and HLA-Cw7-positive melanoma line; K562, NK target. The allogeneic and the autologous tumor lines were pretreated with IFN-{gamma} for 1 or 5 days, respectively, before being used as targets. B, Stimulation of CTL 501D/19 with the same tumor cell lines as in A, and with LB373-MEL, a melanoma cell line derived from an HLA-Cw7-negative patient. LB373-MEL was transiently transfected with an HLA-Cw7 plasmid construct. Three thousand CTL were added to 10,000 stimulator cells, and the production of TNF by the CTL was measured after 24 h.

 
To determine the MHC restriction of these CTL clones, we studied the inhibitory effect of anti-HLA mAbs on their stimulation. CTL clone 501D/19 produced TNF when stimulated with LB831-BLC cells, and this production was completely blocked in the presence of anti-HLA class I mAb W6/32, or in the presence of mAb B1.23.2, which is directed against a common determinant of HLA-B and -C molecules (data not shown). Considering the HLA type of the patient, this indicated that the presenting molecule was HLA-B44, B49, Cw4, or Cw7. CTL 501D/19 also lysed two allogeneic tumor lines: melanoma line MI13443-MEL; and renal carcinoma line LE9211-RCC (Fig. 1GoA). Both cell lines shared only the Cw4 and the Cw7 specificity with LB831-BLC. A number of allogeneic tumor lines were also recognized by the CTL after transient transfection of HLA-Cw7, indicating that Ag LB831-D is presented by the HLA-Cw7 molecule (Fig. 1GoB).

Ag LB831-D is encoded by gene MAGE-A12

We transfected 293-EBNA cells with the HLA-Cw7 cDNA and with the cDNA of a series of genes including the MAGE, GAGE, BAGE, and LAGENY-ESO1 genes, which were expressed at a high level in the bladder tumor sample of patient LB831 (data not shown). The transfectants were tested for their ability to stimulate TNF production by CTL 501D/19. TNF was only produced by the CTL when stimulated with 293-EBNA cells transfected with HLA-Cw7 and gene MAGE-A12 (Fig. 2Go). No stimulation was observed with 293-EBNA cells transfected with HLA-Cw7 alone or with the combination of HLA-Cw7 and any other gene.



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  		FIGURE 2. CTL 501D/19 recognizes an Ag encoded by MAGE-A12 and presented by HLA-Cw7. 293-EBNA cells were cotransfected with the indicated coding sequences cloned into expression vectors. CTL 501D/19 was added, and TNF production was measured after 24 h. LB831-BLC cells were used as a positive control.

 
By RT-PCR, we ascertained that all the tumor lines that were recognized by CTL 501D/19 expressed MAGE-A12, confirming that the Ag is encoded by this gene (data not shown). We also examined other HLA-Cw7 restricted CTL clones generated in the same MLTC, and we found 6 other CTL clones that recognized 293-EBNA cells transfected with gene MAGE-A12 and HLA-Cw7. Because CTL 501D/19 could be maintained easily in long term culture, we continued our analysis with this CTL clone.

Identification of the antigenic peptide encoded by gene MAGE-A12

To identify the MAGE-A12 sequence coding for the antigenic peptide, we generated nested fragments by PCR (Fig. 3Go). These subgenic fragments were cloned into pcDNA3 and transfected into 293-EBNA cells together with the HLA-Cw7 construct. Cells transfected with fragments of 540 bp or more were capable of stimulating CTL 501D/19, whereas those transfected with shorter fragments were not (Fig. 3Go). This indicated that the end of the sequence coding for the antigenic peptide was located between nucleotides 525 and 540 of the MAGE-A12 ORF. In this region, we found two overlapping peptides, EVVRIGHLY (codons 168–176) and VRIGHLYIL (codons 170–178), which conformed to the HLA-Cw7 peptide binding motif, i.e., tyrosine or leucine at the C terminus (20). Nonapeptide VRIGHLYIL sensitized autologous EBV-transformed B cells to lysis by CTL 501D/19, whereas peptide EVVRIGHLY did not (Fig. 4Go). Octapeptide RIGHLYIL was also recognized, but less efficiently. Half-maximal lysis was obtained with a remarkably low concentration of nonapeptide VRIGHLYIL, i.e., 100 pM, indicating that this peptide is recognized very efficiently by CTL 501D/19.



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  		FIGURE 3. Identification of the MAGE-A12 region coding for the antigenic peptide recognized by CTL 501D/19. PCR fragments of different lengths were cloned into expression vector pcDNA3 and cotransfected into 293-EBNA cells together with the HLA-Cw7 construct. Transfected cells were incubated for 24 h with CTL 501D/19, and TNF production was measured. The numbering corresponds to the nucleotides of the coding region.

 


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  		FIGURE 4. Lysis of LB831-EBV cells pulsed with MAGE-A12 peptide VRIGHLYIL (170–178). Chromium-labeled LB831-EBV cells were pulsed for 30 min with various concentrations of nonapeptide VRIGHLYIL or octapeptide RIGHLYIL before the addition of CTL 501D/19 at an E: T ratio of 10. Chromium release was measured after 4 h. Peptides lacking the C-terminal leucine were also tested and were not recognized.

 
We tested the presentation of the MAGE-A12 peptide by different allelic subtypes of HLA-Cw7 and observed that cells bearing either Cw*07011 or Cw*0702 were recognized by CTL 501D/19 with the same efficiency when pulsed with peptide VRIGHLYIL. These two allelic subtypes are the most frequent and together account for nearly 100% of Cw7-positive Caucasians (21). Two much rarer alleles, namely Cw*0704 and Cw*0711, were not able to present the peptide.

Expression of gene MAGE-A12 in tumor samples

To evaluate the frequency of tumors expressing gene MAGE-A12, a series of 1005 tumor samples of various histological types were tested by RT-PCR with primers specific for gene MAGE-A12. As shown in Table IGo, MAGE-A12 is expressed in a high proportion of melanomas, infiltrating bladder carcinomas, lung carcinomas, esophageal carcinomas, and head and neck carcinomas.


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  		Table I. Expression of gene MAGE-A12 in tumor samples

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To our knowledge, this report is the first to describe MAGE-specific CTL isolated from the lymphocytes of a patient with cancer other than melanoma. The MAGE-A gene family comprises 12 highly homologous genes that are completely silent in normal tissues, with the exception of male germline cells, which do not have HLA molecules on their surface (18, 22, 23). At least seven MAGE-A genes are frequently expressed in human tumors and code for peptides that are presented to tumor-specific CTL by HLA class I molecules (2, 3, 4, 7, 8, 9, 10, 11, 12, 24). Interestingly, the peptide described here is located in a region of the MAGE-A12 protein that overlaps the MAGE-A1 and MAGE-A3 region containing the peptides presented by HLA-A1 and B44 (3, 4, 24). The 20 other MAGE-A antigenic peptides that have been identified thus far are scattered along the MAGE protein sequences, which are ~320 residues long, but are remarkably absent from the first 60 amino acids. Besides MAGE-A, two other families of closely related genes have been described (25, 26). They have an expression profile similar to that of the MAGE-A family and were named MAGE-B and MAGE-C. Another family of more distantly related genes, named MAGE-D, are widely expressed in normal tissues (27, 28). However, none of the MAGE antigenic peptides identified thus far is conserved in the MAGE-D sequences. This is also the case for the MAGE-A12 peptide described here. Therefore, the expression of MAGE-D in normal tissues does not jeopardize the tumor specificity of these antigenic peptides.

Bladder carcinoma is a tumor type that appears to be sensitive to immunological control, as suggested by the frequent success of adjuvant treatments based on intravesical instillation of bacillus of Calmette-Guérin (BCG) in superficial tumors (29, 30, 31). After local tumor resection, intravesical BCG instillation has become the standard adjuvant treatment for high risk patients with superficial bladder cancer, providing a 10-year disease-specific survival of 75% instead of 55% with local surgery alone (30). Although the exact mechanism of the antitumor effect of BCG is unclear, it induces a strong local inflammation that may result in a better presentation of tumor Ags and a better recruitment of tumor-reactive lymphocytes.

The MAGE-A genes are expressed in a high fraction of bladder tumors: 10 to 33% of superficial and 32 to 57% of infiltrating bladder carcinomas, depending on the MAGE-A gene considered (32, 33, 34). Given their apparent sensitivity to immune attack and their frequent expression of the MAGE-A genes, bladder cancers appear to be good candidates for specific immunotherapy based on MAGE Ags. Clinical trials of cancer vaccines based on defined Ags are currently pursued in melanoma. We recently reported significant tumor regressions in 7 of 25 metastatic melanoma patients who received 3 monthly injections of a MAGE-A3 peptide presented by HLA-A1 (35). Three regressions were complete and two of these led to a disease-free state that persisted for >3 yr after the beginning of treatment. In another trial, six patients with advanced melanoma were injected with autologous dendritic cells pulsed with MAGE peptides, and a partial tumor response was observed (36). These observations in small pilot studies should motivate larger trials in patients with more limited disease and also with other types of cancer, such as bladder carcinoma.

The identification of new antigenic peptides derived from tumor proteins and presented by frequent HLA molecules such as HLA-Cw7 (43% of Caucasians) increases the number of patients who are eligible for peptide vaccination trials and allows the design of multipeptide vaccines, which should decrease the risk of tumor escape by Ag loss. Furthermore, to optimize the immunization procedure, it is essential to analyze the immune response of vaccinated patients and to compare CTL induction by the various immunization modalities. Most of the immunological tests available to measure CTL responses require the use of peptides. This is true for CTL assays, ELISPOT, tetrameric HLA-peptide stainings and IFN-{gamma} assays (37, 38, 39, 40). The identification of many peptide epitopes is therefore also essential for the immunological monitoring of vaccination trials based not only on peptides but also on proteins or full length recombinant vectors.


   Acknowledgments
 
We thank L. Pilotte for technical assistance, A. Authom and V. Stroobant for the peptide synthesis, S. Depelchin and S. Mapp for editorial assistance, and P. van der Bruggen for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by the Belgian Program on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister’s Office, Science Policy Programming, and by grants from the Association Contre le Cancer, Brussels, Belgium, from the Fonds J. Maisin, Belgium, from Caisse Générale d’Epargne et de Retraite-Assurances, and VIVA, Brussels, Belgium. L.H. was supported by a postdoctoral fellowship of the Deutsche Krebshilfe. M.P.-K. was supported by a postdoctoral fellowship of the Deutsche Forschungsgemeinschaft and by the Schering Foundation, Germany. Back

2 Current address: Molecular Immunology Group, Gesellschaft für Biotechnologishe Forschung-National Research Center for Biotechnology, Braunschweig, Germany. Back

3 Current address: SmithKline Beecham Biologicals, 1330 Rixensart, Belgium. Back

4 Address correspondence and reprint requests to Dr. Benoît J. Van den Eynde, Ludwig Institute for Cancer Research, Avenue Hippocrate 74, UCL 7459, B-1200 Brussels, Belgium. Back

5 Abbreviations used in this paper: EBNA, EBV nuclear Ag; MLTC, mixed lymphocyte-tumor cell culture; ORF, open reading frame; BCG, bacillus of Calmette-Guérin. Back

Received for publication November 24, 1999. Accepted for publication March 13, 2000.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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