In vitro lymphocyte immunization to cultured human tumor cells: parameters for generation of cytotoxic lymphocytes

Conditions for optimal immunization of allogeneic lymphocytes against human tumor cells were studied. Maximal cell-mediated cytotoxic responses were generated usually with 2-4 X 10(4) tumor cells and 1 X 10(6) lymphocytes after a 5-day incubation. A further increase in the number of stimulating cells usually suppressed the immunization of lymphocytes. Viable untreated tumor cells were generally more immunogenic than were the mitomycin C-treated tumor cells. Lymphocytes (1 X )0(6)) sensitized in small volumes (0.5-1 ml) of medium were invariably transformed into cytotoxic effector cells, whereas the same number of lymphocytes sensitized in relatively larger volumes (4 ml) of medium had little or no cytotoxic activity. Occasional shaking of mixed culture during the sensitization provided better immunization than that in similar stationary culture. The replacement of culture fluids with fresh medium during incubation decreased the cytotoxicity level of sensitized cells.