Immunosuppression Derived from Human B-Lymphoblastoid and Melanoma Cell Lines

Abstract

Previous work conducted by the authors, using a murine model, suggested that soluble factors secreted by tumor cells suppress lymphocyte responses. To apply this premise to human tumors, the effects of UC729-6 (lymphoblastoid B-cell) and M21-HPB (malignant melanoma) conditioned media (CM) on normal lymphocyte proliferation, as well as on tumor cell growth in autologous CM was studied. The CM was collected at 2-5 day intervals from cultures of UC729-6 and M21-HPB cells in serum-free media. Phytohemagglutinin- and concanavalin A-stimulated mononuclear peripheral blood cells from healthy human donors showed decreased pHJthymidine ([³H]Tdr) uptake in the presence of each CM when compared with controls. In assays using 100% CM, mitogen stimulation was 68-85% less than that of controls and 40-50% less using 50% CM. The suppression was more pronounced with UC729-6 CM than with M21-HPB CM. In mixed lymphocyte cultures (MLC), addition of 50% CM from either tumor cell line resulted in 40-50% reduction in pHJTdr uptake by lymphocytes. Incubation of UC729-6 cells in 5% to 100% ofUC729-6 fCM (filter-concentrated) produced a decrease in pHJTdr uptake which was directly proportional to the amount of f CM present. In contrast, M21-HPB cell growth in autologous f CM was dependent on cell number, as well as on the amount offCM used. Treatment of the UC729-6fCM using acid (pH4.5), trypsin (100fig/ml), and heat (56^oC) did not restore mitogen-stimulated lymphoproliferation. However, the inhibition observed with UC729-6fCM was partially reversed after dialysis with membranes having M_r limits of 2.5 X 10⁴, 1.5 X 10⁴, or 1 × 10⁴.