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. 2005 Dec;54(12):1214-20.
doi: 10.1007/s00262-005-0705-2. Epub 2005 Jul 16.

A MAGE-1 antigenic peptide recognized by human cytolytic T lymphocytes on HLA-A2 tumor cells

Sabrina Ottaviani  1 , Yi Zhang, Thierry Boon, Pierre van der Bruggen
Affiliations

A MAGE-1 antigenic peptide recognized by human cytolytic T lymphocytes on HLA-A2 tumor cells

Sabrina Ottaviani et al. Cancer Immunol Immunother. 2005 Dec.

Abstract

"Cancer-germline" genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in normal tissues. They encode shared tumor-specific antigens that have been used in therapeutic vaccination trials of cancer patients. It was previously demonstrated that MAGE-1 peptide KVLEYVIKV was presented by HLA-A 0201 molecules on the surface of a human breast carcinoma cell line, but no human specific CTL had been isolated so far. Here, we have used HLA-A2/MAGE-1 fluorescent multimers to isolate from blood cells three human CTL clones that recognized the MAGE-1 peptide. These clones killed efficiently HLA-A2 tumor cells expressing MAGE-1, whether or not they were treated with IFN-gamma, suggesting that the MAGE-1 antigen is processed efficiently by both the standard proteasome and the immunoproteasome. These results indicate that the MAGE-1.A2 peptide can be used for antitumoral vaccination.

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Figures

Fig. 1
Fig. 1
Overview of the procedure used to isolate anti-MAGE-1 CTL clones (A) and examples of positive microcultures (B). The control multimer is an HLA-A2 multimer containing an EBV peptide. The cells represented in the figure (B) are CD4− cells.
Fig. 2
Fig. 2
Effector functions of the anti-MAGE-1 CTL clones. (A) Labeling of T cell clones with A2/MAGE-1 multimers. The VβJβ usage and the amino-acid sequence of the CDR3 region is indicated for each clone. Cells were labeled for 15 min at room temperature with the A2/MAGE-1 multimer (20 nM) conjugated to PE and the control multimer (5 nM) conjugated to APC. Anti-CD8 antibodies were then added for 15 min. (B) Recognition of cells transiently transfected with MAGE-1. COS-7 cells were transiently transfected using lipofectamine with a MAGE-1 and an HLA-A*0201 coding sequence. One day after transfection, 5,000 CTL were added to the transfected cells. IFN-γ production was measured by ELISA after overnight co-culture. (C) Lysis of HLA-A2 targets loaded with MAGE-1 peptide or transduced with a retrovirus containing the coding sequence of MAGE-1. Target cells were 51 Cr-labeled and, as indicated, pulsed or not for 15 min with 1 μg/ml of peptide KVLEYVIKV. (D) Lysis of HLA-A2 tumor cell lines expressing MAGE-1. Cell line K562 is a target for natural killer cells. Target cells were 51Cr-labeled and incubated for 4 h with CTL at the indicated effector-to-target ratios. Chromium release was measured after 4 h. As indicated, cells were incubated or not with 1 μM of peptide before being put into contact with CTL. (E) Titration of the MAGE-1 peptide. Cells were 51Cr-labeled and incubated for 15 min with threefold dilutions of the synthetic peptide. CTL were subsequently added at an effector-to-target ratio of 10:1. Chromium release was measured after 4 h. The concentrations indicated in the figure correspond to the concentrations during the 4-h incubation.
Fig. 3
Fig. 3
Loss of recognition by CTL clone 89 of a tumor cell line treated by lactacystin. HLA-A2 tumor cell line CP50-MEL expresses MAGE-1. Cells were first treated at pH3 for 10 sec to remove peptides bound to HLA molecules on the surface. Cells were then incubated for 1 h with 10 μM of lactacystin, followed by overnight incubation with 1 μM of lactacystin. In total, 30,000 tumor cells were co-cultured for 8 h with 5,000 CTL of the clone 89. To establish the viability of the cells, they were also pulsed with 2 μg/ml of peptide and tested for recognition by the CTL.
Fig. 4
Fig. 4
Lysis by CTL clone 89 of a tumor cell line treated with IFN-γ. The tumor cell line LB1017-SCCHN is a squamous cell carcinoma of the head and neck. The cell line was treated with 50 U/ml of IFN-γ for either 2 or 7 days. Targets were 51Cr-labeled and chromium release was measured after 4 h.
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