Abstract

Epstein-Barr Virus (EBV)-transformed lymphoblastoid B cell lines, BLCL, which expresse antigens, are potential antigen-presenting cells (APCs) for the induction of CTL in vitro. However, transfection of BLCLs with subsequent selection by antibiotics is notoriously difficult because plating efficiencies of BLCLs are reported to be 1% or less. To generate stable transfectants of BLCLs, we produced high titers of retroviruses encoding pp65 antigen of human cytomegalovirus as foreign antigens and transduced them to BLCLs. The pp65 gene was cloned into the retroviral vector pLXSN. The recombinant retroviral vector was transfected to ecotropic packaging cell line, GP&E86, and this polyclonal recombinant retrovirus was transduced to PA317 that is amphotropic packaging cell line. The titers of cloned PA317 amphotropic retroviruses ranged from 5 to 7 X 10^6 colony forming units (CFU) per ml (CFU/ml). We performed three rounds of consecutive transductions to BLCLs in order to improve the cloning efficiencies. The expression of recombinant HCMV-pp65 antigen was more than 20% after the final transduction. The third-transduced BLCLs were easily selected in optimal concentration of G418. BLCLs expressing foreign antigens could be used as target cells for CTL assay and/or as APCs for induction of in vitro CTL responses specific for viral and tumor antigens.

• Keywords: EBV-transformed lymphoblastoid B cell lines (BLCL); antigen-presenting cells (APCs); Recombinant ret