EFFECTS OF CYCLOPHOSPHAMIDE ON IN VITRO HUMAN LYMPHOCYTE CULTURE AND MITOGENIC STIMULATION

Cyclophosphamide (CY) has been reported to be inactive in vitro under certain conditions. In the present study, CY was tested for its ability to inhibit human lymphocyte proliferation and to modulate lymphocyte response to mitogens in vitro. The inhibition of or the increase in 3H-thymidine incorporation in mitogen-stimulated and unstimulated lymphocytes by CY was used as a measure of CY activity in vitro. The results demonstrate that lymphocytes from 10 different persons had a mean decrease of 74% in 3H-thymidine incorporation in the presence of CY (P less than 0.005). The effect was maximal at a concentration of 160 micrograms/ml. A mean inhibition of 35 and 55% was caused by 10 and 40 micrograms/ml concentrations of CY, respectively. CY also was able to reduce the number of viable cells during 5 days in culture and had a profound effect on mitogen stimulation of lymphocytes. In all cases, CY modulated the stimulation of lymphocytes by phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM) either by augmenting or suppressing the responses. At low concentrations (10 micrograms/ml) it augmented mitogenic stimulation by 46 to 281%. At higher concentrations (20 to 160 micrograms/ml), CY had a suppressive effect with a maximum suppression of 99%. The CY-induced immunomodulation is perhaps caused by its action on the regulatory T cells. When tested in vitro, CY had inhibitory activity on T cells.