Colony growth of T lymphocytes in vitro. Control and regulation of clonal formation

Human lymphocytes stimulated with PHA in liquid phase (step 1) and then seeded in a two-layer soft agar system (step 2) grew and developed into T cell colonies. Colony formation was enhanced when the agar culture was supplemented with culture fluid obtained from phytohemagglutin-treated lymphocytes (Ly-CF). Untreated lymphocytes plated directly in the soft agar system also developed into colonies if the culture medium contained Ly-CF. Mitogen-sensitized T lymphocytes produced a lymphocyte colony enhancing factor in the culture fluid which stimulated lymphocytes into colony formation. The best plating efficiency (1:250) was attained when blood mononuclear cells were seeded. When spleen cell culture fluid or a highly concentrated blood-adherent cell population was added to the soft agar, colony development was strongly inhibited. Monocytes-macrophages secrete a lymphocyte colony inhibiting factor in the culture medium. These lymphokines exert a competitive influence on T cells and thus control and regulate clonal proliferation.