Summary The reason(s) why individual cytotoxic T lymphocytes (CTL) possess a fast‐acting, perforin/granzyme‐mediated, as well as a much slower, Fas ligand (FasL) ‐driven killing mechanism is not clear, nor is the basis for wide variations in killing activity exhibited by individual CTL, ranging from minutes to hours. We show that perforin expression among individual, conjugated CTL varies widely, which can account for the heterogeneity in killing speeds exhibited by individual CTL. Despite a 2‐hr lag in FasL‐based killing, CTL lytic action is enhanced when the two mechanisms operate in concert. This is explained by finding that the two pathways in fact are jump‐started simultaneously with the lag in FasL lytic action reflecting pre‐lytic caspase‐8 activation and BH3‐interacting domain (BID) cleavage. The complementary action of the two lytic pathways, co‐expressed at varying levels among individual CTL, facilitates the lytic action of late‐stage poor perforin‐expressing CTL, ensuring optimal cytocidal action throughout the CTL response.

Summary Although CD8+ cytotoxic T lymphocytes (CTL) exhibit both Fas ligand (FasL) ‐based and perforin‐based lytic activities, the accepted hallmark of a fully active CTL remains its perforin killing machinery. Yet the origin, rationale for possessing both a slow‐acting (FasL) and a fast‐acting (perforin) killing mechanism has remained enigmatic. Here we have investigated perforin expression in CTL directly involved in acute tumour (i.e. leukaemias EL4 and L1210) allograft rejection occurring within the peritoneal cavity. We show that at the height of the immune response, the majority of conjugate‐forming CD8+ CTL express high levels of perforin messenger RNA and protein, and kill essentially via perforin. Later however, coinciding with complete rejection, fully cytocidal CTL emerge which exhibit a stark decrease in perforin and now kill preferentially via constitutively expressed FasL. Although late in emergence, and persistent, these powerful CTL are neither effector‐memory nor memory CTL. This finding has implications for the monitoring of anti‐transplant responses in clinical settings, based on assessing perforin expression in graft infiltrating CD8+ T cells. The results show that as the immune response progresses in vivo, targeted cellular suicide mainly prunes high perforin‐expressing CD8+ cells, resulting in the gradual switch in effector CTL, from mostly perforin‐based to largely Fas/FasL‐based killers. Hence, two kinds of CD8+ CTL have two killing strategies.