Cytoplasmic granules from cloned CTL were prepared by Percoll gradient centrifugation of homogenates prepared by nitrogen cavitation. In addition to the cytolysin these granules contain a DNAse activity capable of releasing 125I-UdR from nuclei. This nuclease activity is found in far smaller amounts in granules of non-cytotoxic lymphocytes, and the level of activity in cloned CTL is greater that that seen in LGL tumor cells. This enzyme is active at neutral pH, and was shown to cleave high molecular weight DNA. Thus it appears that the reports of rapid target cell DNA cleavage during CTL-mediated cytotoxicity can ba accounted for by CTL granule DNAse without postulating endogenous DNAses in the target cell. In order to test the granule exocytosis model for lymphocyte toxicity on lymphocytes which are neither LGL nor classical CTL, cytoplasmic granules were prepared from mouse LAK cells, spleen lymphocytes cultures with recombinant IL-2 which develop cytotoxic activity. It was found that cytolytic activity in the granules appears on about day three of culture and continues to increase for the next 6 days. This pattern roughly follows LAK cytotoxic activity but begins later and does not plateau with time. IL-2 titration shows similar amounts of lymphokine are needed to generate granule lytic activity and LAK. The LAK cytolysin shows a similar spectrum of target cell killing and a similar dependence on calcium and rapid linetics as do the cytolysisns from LGL and CTL. The LAK cytolysin is also neutralized by anti-LGL granule antibodies. These results support the granule exocytosis model for LAK-mediated lysis.
