The cytotoxic activity of spleen and peritoneal cells from C57Bl/6 mice immunized with irradiated syngeneic EL4 leukemia cells was tested in vitro on 51Cr labelled target cells following various fractionation and purification procedures. At the peak of the response, immune spleen cells showed very low cytotoxic activities. Fractionation on a simple BSA density gradient yielded a low-density (less than 1.08 g/cm3) spleen-cell population with a five- to 10-fold increased cytotoxic activity. A similar increase was observed after preincubation of immune spleen cells for 24 h at 37° C, confirming results of Ortiz de Landazuri and Herberman (1972). A successive application of preincubation followed by BSA gradient separation resulted in a cumulative 30- to 100-fold increase in cytotoxicity. Removal of high-density small lymphocytes before preincubation led to populations with considerably decreased activity, suggesting the formation of low-density cytotoxic lymphocytes from high-density precursors during preincubation. Immune peritoneal cells showed a three- to five-fold increase in cytotoxicity after (1) removal of phagocytic cells, (2) 24 h preincubation followed by removal of adherent cells, and (3) removal of phagocytic cells followed by preincubation. This indicated that adherent cells were not involved in but appeared to inhibit the cytotoxic activity of immune peritoneal cells. Treatment of preincubated or gradient-separated immune spleen and peritoneal cells with anti-θ serum and complement and passage through Sephadex lg-anti-Ig columns demonstrated the T-cell nature of the cytotoxic effector cells. In vitro studies also showed an enhanced protective effect of pretreated immune spleen cells when compared to the original population.
Dr. Damodaran Vasudevan, K Brunner, T., and Cerottini, J. - C., “Detection of cytotoxic T lymphocytes in the EL4 mouse leukemia system: increased activity of immune spleen and peritoneal cells following preincubation and cell fractionation procedures”, International Journal of Cancer, vol. 14, pp. 301–313, 1974.