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How many ways remain to be discovered to account for how cytotoxic T lymphocytes (CTLs) kill tumor cells? Here our efforts are directed towards the roles of CTL lipases, including an IL-4 inducible lipase, pancreatic lipase-related protein 2 (PLRP2) that is normally secreted into the gut to digest dietary fats. We compared tumor cell death mediated by freshly activated CTLs generated from splenocytes of Balb/c wild type (WT) and Balb/c PLRP2-/- (KO) littermate mice. Concanavalin-A activated splenic T cells were cultured with IL-2 or with IL-4 (500 u/ml) for 6 days. We assayed cytotoxicity by anti-CD3 antibody redirected lysis of Fc-IgG-receptor-bearing P815 mastocytoma cells, monitoring 51Cr release from the dead cells. We also monitored the frequency of Grz B+ CD8+ CTLs and their levels of Grz B by flow cytometry. PLRP2 was detected by RT-PCR for gene expression and by immunoblots of the CTL proteins developed with cross-reacting chicken antibodies to human PLRP2. Lipase activity was monitored by pre-labeling the target P815 cell lipids with 3H-oleic acid or by enzymatically monitoring lipase released from the CTL upon stimulation with phorbol myristic acid and calcium ionophore. We found that the WT CTLs were consistently 2-4 fold more cytotoxic than the PLRP2-/- CTLs, with greater differences following culture with IL-4. The frequency and mean fluorescent intensity of the signal of Grz B+ CTLs was similar for WT and KO CTLs cultured with identical cytokines. The similarities indicate that the differences responsible for the increased toxicity of the WT cells were unbiased by CTL frequency and independent of Grz B expression. There was extensive 3H released into the supernatant from the oleic-acid labeled P815s which could reach 30% of the total label of the P815s. The release correlated with the effector CTL to target cell ratios and was absent without anti-CD3 when lysis failed to occur. It was blocked by tetrahydrolipstatin, an inhibitor of triglyceride lipases. The 3H release, thought to be associated with lipid products, appeared regardless of the WT or KO status of the CTL, suggesting that the lipase(s) mediating most of the target cell lipid degradation are independent of PLRP2. Upon stimulation, the WT CTLs induced with IL-4 released activity that degraded the triolein substrate that is selectively hydrolysed by lipases like PLRP2 while the IL-4 cultured PLRP2-/- CTLs lacked this activity. From these date, we conclude that PLRP2 is a lipase of CTLs that is important for CTL activity and probably released during T cell mediated killing. A different lipase is responsible for the extensive degradation of target cell lipids. Overall, the data indicate that lipases appear to be a new means to account for how cytotoxic T lymphocytes (CTLs) kill tumor cells. Supported in part by NIH R01 CA38942 & T32 CA09563 and the Reno Cancer Foundation.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA