Lectin-Dependent Cell-Mediated Cytotoxicity Following In Vitro Culture of Normal Lymphocytes in Medium Containing 2-Mercaptoethanol

Abstract

Cell-mediated cytotoxic reactivity resulting from the in vitro incubation of normal lymphocytes was assessed using nonspecific lectin-dependent cell-mediated cytotoxicity (LDCC) as a measure of overall reactivity. Spleen cells from nonimmune C57BL/6 mice were incubated in vitro in RPM1-1640 supplemented with 10% fetal calf serum and 2-mercaptoethanol (2ME). Cytotoxicity was assayed against syngeneic Cr51-labeled EL-4 cells in the presence of Con A or PHA. Optimal LDCC was observed after 8 days of culture in the presence of 5 × 10^-5 M 2ME. Cytotoxicity was mediated by an activated T-lymphocyte population whose development did not appear to require macrophages. Usually LDCC in the presence of PHA was significantly greater than that obtained in the presence of Con A. The presence of 2ME during the initial phase of culture was crucial for the development of cytotoxicity, since early removal of 2ME after 1 or 3 days of culture did not alter the subsequent development of cytotoxicity, whereas delayed addition of 2ME on day 1 or 3 failed to produce cytotoxic reactivity. This rapid conversion from a 2ME sensitive state to a 2ME insensitive state may be related to a rapid loss of accessory cell viability during the early phase of culture. Together the results indicate that this system may provide a useful model for the investigation of the events leading to the development of CTL in vitro.