THE ROLE OF THE FAS LYTIC PATHWAY IN A PERFORIN-LESS CTL HYBRIDOMA

The murine CTL hybridoma PMMI has been shown by the most sensitive tec hniques to be devoid of perforin. We thus used PMMI activated with PMA and ionomycin, to investigate possible alternate lytic pathways in CT Ls in the absence of perforin. We found that PMMI is equipped with mem brane TNF-alpha as a potential lytic mechanism, but TNF-alpha is unlik ely to be involved in acute (4 h) lytic reactions. On the other hand, PMMI readily lyses target cells expressing the gene for the Fas Ag, bu t does not lyse target cells expressing las antisense DNA. The generat ion of fas-dependent lysis required protein synthesis in PMMI, but tar get cell protein synthesis was not required for lysis. Lysis of Fas-po sitive target cells by PMMI was accompanied by DNA fragmentation, and both lysis and DNA fragmentation were blocked by inhibition of protein synthesis in the effector cell. We find the relative extent and kinet ics of fas-dependent lysis and DNA fragmentation indistinguishable fro m that seen in ''classical'' CTL lytic assays. Both fas- and perforin- dependent lysis were blocked by inhibitors of poly(ADP) ribosylation. We found very little difference in the sequence of events in target ce lls lysed by the las pathway compared with the classical (probably per forin) lytic pathway. Given the widespread distribution of las, partic ularly in hematopoietic target cells, caution may be required in inter preting the relationship between parameters such as DNA fragmentation and Cr-51-release solely on the basis of the granule exocytosis model.