Growth of human B-lymphocyte colonies in vitro

Abstract

RECENTLY developed in vitro culture systems that support clonal formation of lymphoid cells have promoted remarkable advances in our understanding of lymphocyte proliferation and the nature of the proliferating cells. Human T lymphocytes from venous blood, bone marrow and lymphoid organs and murine T and B lymphocytes from lymphoid tissues have been grown into colonies of T and B cells in semisolid culture. In these systems, T-lymphocyte colony formation requires that the seeded cells undergo T-mitogen stimulation, that is, by phytohaemagglutinin (PHA) or concanavalin A, and B-lymphocyte colony growth necessitates sensitisation by mercaptoethanol, a humoral thymic factor, or a polyclonal B-cell activator, such as lipopolysaccharide, purified protein derivative or dextran sulphate^1–7,17. Previous attempts to achieve colony formation of human B cells were unsuccessful, even in the presence of known polyclonal B-cell activators. However, in certain experimental conditions, the plant lectins, pokeweed mitogen (PWM) and PHA, stimulate both human B and T lymphocytes into transformation and proliferation in cell culture with a synergistic effect between the sensitised T and B cells^8–13. We report here that, based on this information and earlier techniques for clonal formation of human lymphocytes in vitro, we have evolved a two-layer soft agar culture technique in which the mitogens, PHA and PWM, were able to generate the appropriate signal for triggering B-lymphocyte precursors of venous and umbilical cord blood, tonsil, adenoid and chronic lymphatic leukaemia (CLL) origin into colony-forming and immunoglobulin-producing cells.