Establishment of human T lymphocyte clones highly cytotoxic for an EBV-transformed B cell line in serum-free medium: isolation of clones that differ in phenotype and specificity.

Abstract A human T cell culture (HLA A2; w23 B40; w44 Cw4) was established by repetitive stimulation with an Epstein Barr virus-transformed B cell line JY in a serum-free culture medium. This T cell culture was highly cytotoxic for JY but had negligible nonspecific natural killer celllike activity against the myeloid cell line K562. Cloning by limiting dilution procedures in serum-free medium resulted in a high plating efficiency and the establishment of a large number of cytotoxic clones. Three of these clones (31, 38, and 77) were selected. Phenotypic characterization by using a series of monoclonal antibodies and fluorescein isothiocyanate-labeled goat anti-mouse immunoglobulin revealed that clone 31 was OKT 3+, 4−, T8+, Ia+, OKM 1−, whereas clone 38 was OKT 3+, 4+, T8−, la+, and OKM 1−, indicating that cytotoxic T cells are not restricted to the T8+ subset and can reside in the OKT 4+ “inducer/helper” T cell subset. Because the cytotoxic reaction of clone 31 was inhibited by a monoclonal antiserum that recognizes all HLA A, B, and C specificities, whereas that of clone 38 was not blocked, it was concluded that in contrast to clone 38, clone 31 recognized HLA A, B, or C antigens. A preliminary analysis indicated that clone 31 reacted with HLA B7. The specificity of clone 38 remained unclear. The cytotoxic reaction of the T8+ clone 31 was blocked by anti-T8, a monoclonal antibody specific for human cytotoxic/suppressor T cells, suggesting that the T8 determinant plays a role in the cytotoxic process. The cytotoxic reaction of the T8− clone 38 was not blocked by anti-T8. The third clone (77), which was OKT 3+, 4−, T8+, Ia+, and OKM 1−, was cytotoxic only in the presence of minimal concentrations of lectin. Our results indicate that stable cytotoxic T cell clones can be established successfully in serum-free medium.