Transfection and expression of a gene coding for a human melanoma antigen recognized by autologous cytolytic T lymphocytes

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Abstract
Human melanoma line MZ2-MEL expresses several antigens recognized by autologous cytolytic T lymphocytes (CTL). As a first step towards the cloning of the gene coding for one of these antigens, we tried to obtain transfectants expressing the antigen. The DNA recipient cell was a variant of MZ2-MEL which had been selected with a CTL clone for the loss of antigen E. It was cotransfected with genomic DNA of the original melanoma line and with selective plasmid pSVtkneoβ. Geneticin-resistant transfectants were obtained at a frequency of 2 × 10−4. These transfectants were then screened for their ability to stimulate the production of tumor necrosis factor by the anti-E CTL clone. One transfectant expressing antigen E was identified among 70 000 drug-resistant transfectants. Its sensitivity to lysis by the anti-E CTL was equal to that of the original melanoma cell line. When this transfectant was submitted to immunoselection with the anti-E CTL clone, the resulting antigen-loss variants were found to have lost several of the transfected pSVtkneoβ sequences. This indicated that the gene coding for the antigen had been integrated in the vicinity of pSVtkneoβ sequences, as expected for cotransfected DNA.