Establishment and characterization of the T-cell line, EBT-8 latently infected with Epstein-Barr virus from large granular lymphocyte leukemia.

An Epstein-Barr virus (EBV) genome-positive T-cell line, designated EBT-8, was established from peripheral blood of a patient with EBV genome-positive large granular lymphocyte leukemia of T-cell origin. The cells have been cultured continuously in RPMI-1640 medium supplemented with 10% fetal calf serum and 40 U/ml interleukin-2 for more than 18 months. Analysis of T-cell receptor gene rearrangement demonstrated similar rearrangement between the fresh leukemic cells and EBT-8 cell line. The cell line has several azurophilic granules in its cytoplasm and activated cytotoxic/suppressor T-cell surface antigens (CD2, CD3, CD8, HLA-DR and T-cell receptor alpha/beta). Karyotypic analysis of the cell line showed several chromosomal abnormalities. EBV DNA was demonstrated in the cells by Southern blot hybridization and about five copies of covalently closed circular DNA per cell were detected by Gardella gel analysis. Clonotypic episomal EBV DNA was observed in the cells by Southern blot hybridization with EBV-terminal fragment probe. EBV-encoded small RNA, EBER1 were demonstrated in all cells by in situ hybridization. EBV-encoded proteins, EBNA and LMP1 were demonstrated by immunofluorescence technique. EBV activation was observed after 12-O-tetradecanoylphorbol-13- acetate treatment of the cells. These results demonstrated the establishment of a T-cell line with latent EBV genomes and suggested the involvement of EBV to the large granular lymphocyte leukemia of T cells.