Gene transfer of the B7.1 costimulatory molecule into human ocular melanoma cells

T. Uno, T. G. Murray, P. W. Chen, E. R. Podack, B. R. Ksander

Research output: Contribution to journalArticle

Abstract

Purpose Since the B7.1 cosiimulalory molecule is known to activate specific T cells thai eliminate tumor cells, the following experiments were performed to determine if sustained high levels of the B7.1 costimulatory molecule could be expressed on primary ocular melanoma cells, and whether B7.1 positive tumor ceils stimulate autologous CD8-T cells. Methods Primary ocular melanoma cells were transfected with the B45Nco episomal vector containing the cDNA for human B7.1. Transcription and surface expression of B7.1 cDNA was determined by specific RT-PCR and flow cytometry. respectively. The vector copy number was determined by semi-quantitative analysis of plasmid DNA. Tumor cell clones were established by limiting dilution techniquev Autologous lymphocytes were stimulated with transfecled tumor cells in mixed-lymphocyte-tumor cell culiures containing exogenous IL-2. Results Untransfected ocular melanoma cells failed to express B7.1. By contrast, transfected tumor ceils expressed B7.1, although the level of expression was heterogeneous and varied considerably. Clones of transfected tumor cells were established to obtain tumor cells that expressed stable and high levels of B7.1. The intensity of B7.1 on the clones corresponded with ihe relative amount of B7.1 mRNA and plasmid copy number. Tumor cells with a low copy number (<50/cell) possessed less B7.1 mRNA and weak surface expression, whereas tumor cells with a copy number greater than 200/cell uniformly expressed high levels of B7.1. To determine if transfected tumor cells stimulate CD8+ T cells, auiologous peripheral blood lymphocytes were stimulated with either B7.1 positive, or negative tumor cells in the presence of exogenous IL-2. Only B7.1 positive lumor cells successfully stimulated CD8+ T cells. Conclusions The B45Neo episomal vector can be used to induce stable expression of high levels of the B7.1 costimulatory molecule in primary ocular melanomas. B7.1 positive tumor cells successfully stimulated autologous CD8+ T cells, implying that lumor cells genetically engineered to express B7.1 can stimulate tumor-specific T cells.

Original languageEnglish (US)
Pages (from-to)S469
JournalInvestigative Ophthalmology and Visual Science
Volume38
Issue number4
StatePublished - Dec 1 1997

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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    Uno, T., Murray, T. G., Chen, P. W., Podack, E. R., & Ksander, B. R. (1997). Gene transfer of the B7.1 costimulatory molecule into human ocular melanoma cells. Investigative Ophthalmology and Visual Science, 38(4), S469.