Immunological Studies on Human Malignant Tumor VIII Cytotoxicity Assay of Normal Human Lymphocytes Against 51Cr Labelled Target Cells

Abstract

Cytotoxicity occurring in vitro when lymphocytic cells from a normal individual are added to the target cells has been termed spontaneous cell mediated cytotoxicity (SCMS). The present studies were undertaken to identify the effector cells in human peripheral blood mediating SCMC. The target cell was an in vitro cultured cell line derived from maxillary cancer of a epidermoid carcinoma characteristic. The target cells were labelled with 51CrNa2O4 under an optimal condition. The mononuclear cells were separated by centrifugation on Ficoll-Hypaque gradients. The suspension of mononuclear cells containing a high percentage of lymphocytes was then incubated at 37°C for 1hr in a plastic dish to remove adherent cells. The separation of T cell and non-T cell populations from the suspension of non-adherent cells was a final preparation. The rosetting procedure with sheep red cell (E) was empolyed. T cells forming rosettes with E were obtained by Ficoll Hypaque gradients and the attached E were lysed with treatment of 0.75% ammonium chloride. Each lymphocyte suspension was used for 51Cr release assay according to Hersey's methods.The Cytotoxicity of effector cells was expressed as a percentage of 51Cr release, when the effector cells were added to the culture of 51Cr labelled target cells. The cytotoxicity of effector cells was measured at different ratios to the target cells. Mononuclear cells showed a cytotoxicity with a percent lysis ranging from 6.7 to 11.2. An additional experiment revealed that the cytotoxicity of non-adherent cells was higher than that of mononuclear cells. The cytotoxicities of T cells and non-T cells separated from the non-adherent cells greatly decreased. The exact mechanisms involved in SCMC are now being studied.