Quantitative In Vitro Assay of Cytotoxic Cellular Immunity

Abstract

Studies of immune lymphoid cell cytotoxicity in ^{in vitro} systems were undertaken to establish a rapid, sensitive, and quantitative assay that truly measured cellular cytotoxicity. The technique of Brunner, Mauel, Cerottini, and Chapuis (Immunology 14: 181–196, 1968), based on the release of ⁵¹Cr from labeled target cells, was modified by the introduction of gentle rocking of the incubation mixtures. The sensitivity of the assay allowed detection of target cell damage at 1% above background and 30 minutes after the cells were mixed. Studies of the rates of target cell damage indicated that the actual immune cell-damaging process occurred rapidly after contact of the immune and target cells. Peak activity was evident within 2 hours of the start of the assay, when spontaneous death of immune and target cells was still minimal. Many observations indicated that this assay reflected a cell-mediated immune process, without detectable contribution by humoral antibody or complement. Added complement (fresh guinea pig serum) failed to enhance, and added complement inhibitors failed to inhibit the cytotoxic effect. Added isoantibody (against target cell antigens) inhibited the cytotoxic effect. Antisera reacting with all known heavy- and light-chain mouse immunoglobulin classes only minimally reduced the cytotoxicity. Nonspecific cytotoxic factors in cytotoxic systems requiring long incubation periods were not detected. Presumably, they have no significance outside the immediate microenvironment of the immune cell.