In order to test the granule exocytosis model for lymphocyte cytotoxicity, we have examined the function of a non-cytotoxic cell which possesses a well-defined regulated secretory system after transfection for the granule cytolysin of cytotoxic T lymphocytes. The cells chosen for this are RBL, a rat mucosal mast cell tumor line, which degranulates in response to cross- linking its IgE Fc receptor. In order to allow transfection, a cDNA cloned for the cytolysin was inserted into the mammalian cell expression vector PSVL. This construct was co-transfected with the neo-containing pSV3 using calcium phosphate, and RBLs grown in G418 to select for transfectants. Northern blots revealed a low but positive level of cytolysin expression in the surviving cells, which were then cloned by limiting dilution. Clones were individually screened by Northerns and those with the highest expression were selected for further study (RBL-cy). Homogenates of RBL and RBL-cy were fractionated on Percoll gradients, and the dense granule fractions examined for cytolysin activity. The calcium dependent hemolytic activity characteristic of cytolysin was found in RBL-cy but not RBL granules. Rbl-cy were found to have a potent cytolytic activity against TNP-RBC in the presence of an IgE anti-DNP, giving 50% lysis at an E/T of about 1. Under these conditions, cytotoxic activity was undetectable in RBL, as was the activity of RBL-cy in the absence of IgE. RBL-cy cytotoxic activity was completely abolished in the presence of EGTA, as expected from the granule exocytosis model. Bystander RBC labeled with 51 Cr but not TNP-modified were not lysed by RBL-cy in the presence of an excess of co-pelleted unlabelled TNP-RBC, thus reproducing the classic sparing of bystanders by CTL. TNP modified tumor targets were lysed only to a marginal level by RBL-cy under conditions giving excellent lysis of TNP-RBC.
