Gene transfer of the CD80 costimulatory molecule into ocular melanoma cells using a novel episomal vector.
OBJECTIVE
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 cells were transfected with a novel vector (B45-Neo episomal vector) containing the complementary DNA (cDNA) for human CD80 to determine if this vector system is useful for stimulating CD8+ T cells.


METHODS
Ocular melanoma cells were transfected with the B45-Neo episomal vector containing the cDNA for human CD80 and were positively selected in medium containing geneticin. The transcription of plasmid cDNA, plasmid copy number, and cell surface expression were determined on transfected tumor cell lines, and cloned tumor cells were obtained by limiting dilution 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 cells in the presence of recombinant interleukin-2 to determine whether CD8+ T cells were stimulated.


RESULTS
CD80 was expressed on tumor cells 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 than 520 plasmids per cell; intermediate levels were observed on tumor cells with approximately 240 to 520 plasmids. CD80+ ocular melanoma cells maintained a stable CD80 expression even after prolonged culture without geneticin, and on irradiated tumor cells. CD80 expressed on tumor cells was biologically functional and stimulated autologous CD8+ cells.


CONCLUSIONS
The B45-Neo episomal vector induces stable expression of the CD80 costimulatory molecule on ocular melanoma cells. Our results indicate that this vector is suitable for experiments designed to genetically engineer ocular melanoma cells to stimulate CD8+ T cells.
Uno
T
T
Schepens Eye Research Institute, Harvard Medical School, Boston, MA 02114, USA.
Chen
P W
PW
Murray
T G
TG
Podack
E R
ER
Ksander
B R
BR
D018122:B7-1 Antigen / Q000096:biosynthesis / Q000235:genetics*; D052688:Bovine papillomavirus 1 / Q000235:genetics; D018414:CD8-Positive T-Lymphocytes / Q000502:physiology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects / Q000502:physiology; D008545:Melanoma / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D014162:Transfection; D014407:Tumor Cells, Cultured; D014604:Uveal Neoplasms / Q000235:genetics / Q000378:metabolism*
D018122:B7-1 Antigen; D018076:DNA, Complementary; D007376:Interleukin-2