Highly purified human T-lymphocytes do not respond to mitogens--including calcium ionophore and phorbol ester

The proliferative, and other, in vitro responses of human lymphocytes are dependent upon interactions with accessory cells. Studies of the nature of these required interactions are dependent upon the ability to isolate functionally pure populations of lymphocytes and accessory cells. We describe here a four-step separation procedure using plastic adherence, passage through columns of nylon wool and Sephadex G-10, and treatment with L-leucine methyl ester which produced T-lymphocyte populations which were functionally free of accessory cells, and accessory cell populations which were highly enriched for macrophages. T-lymphocytes were 91-98% Leu-4-positive, contained fewer than 0.05% Mo1- or Mo2-positive cells and fewer than 0.1% nonspecific esterase-positive cells, showed relatively little change in the T4/T8 ratio, and gave no proliferative response to a panel of mitogens. The accessory cell populations were 72-98% nonspecific esterase-positive, 91-99% OKM-1-positive and only 0-4% Leu-4-positive, and could fully restore the proliferative responses of the mitogen-treated lymphocytes. Using these purified cell populations, we have found that, contrary to a number of published observations, both the calcium ionophore A23187 and the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate were dependent on the presence of accessory cells to produce a mitogenic response by human lymphocytes.