GENE-TRANSFER OF THE CD80 COSTIMULATORY MOLECULE INTO OCULAR MELANOMA-CELLS USING A NOVEL EPISOMAL VECTOR

Purpose. The CD80 (B7.1) molecule, which is a necessary costimulatory signal for T-cell activation and proliferation, is a powerful inducer of antitumor immunity. In this study, primary human ocular melanoma ce lls were transfected with a novel vector (B45-Neo episomal vector) con taining the complementary DNA (cDNA) for human CD80 to determine if th is vector system is useful for stimulating CD8(+) T cells. Methods. Oc ular melanoma cells were transfected with the B45-Neo episomal vector containing the cDNA for human CD80 and were positively selected in med ium containing geneticin. The transcription of plasmid cDNA, plasmid c opy number, and cell surface expression were determined on transfected tumor cell lines, and cloned tumor cells were obtained by limiting di lution techniques. The stability of CD80 expressed on tumor cells was determined after prolonged culture without geneticin and on irradiated cells. Autologous lymphocytes were restimulated with CD80(+) tumor ce lls in the presence of recombinant interleukin-2 to determine whether CD8(+) T cells were stimulated. Results. CD80 was expressed on tumor c ells transfected with the B45-Neo vector containing the cDNA for CD80. The level of CD80 expressed on different transfected tumor cell lines was heterogeneous and dependent on the plasmid copy number. High CD80 expression was observed on cloned tumor cells that possessed more tha n 520 plasmids per cell; intermediate levels were observed on tumor ce lls with approximately 240 to 520 plasmids. CD80(+) ocular melanoma ce lls maintained a stable CD80 expression even after prolonged culture w ithout geneticin, and on irradiated tumor cells, CD80 expressed on tum or cells was biologically functional and stimulated autologous CD8(+) cells. Conclusions. The B45-Neo episomal vector induces stable express ion of the CD80 costimulatory molecule on ocular melanoma cells. Our r esults indicate that this vector is suitable for experiments designed to genetically engineer ocular melanoma cells to stimulate CD8(+) T ce lls.