The lipases of T cells and their function in cytotoxicity
AuthorAlves, Bryce Nicholas
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Cytotoxic T lymphocytes (CTLs) eliminate virally infected cells and tumor cells through the use of cytotoxic proteins and death-inducing ligands. Through decades of research, scientists have identified and characterized many of these cytotoxic proteins but spent little time on phospholipases and lipases as cytotoxic enzymes released during CTL mediated killing. Pancreatic lipase related protein 2 (PLRP2) is an interesting candidate lipase found in CTLs. This dissertation has three parts: (1) determination of the role of T cell-derived lipases in tumor cell membrane degradation, (2) characterization of the induction of PLRP2 and determination as to how PLRP2 affects CTL mediated killing, and (3) investigation if CTL-derived lipases can mediate indirect toxicity toward tumor cells. During part (1), using tumor cells with radioactive lipids in their membranes, I found no evidence for gross membrane damage mediated directly by CTL lipases. In part (2), using quantitative RT-PCR I found that PLRP2 is consistently induced in CTLs by IL-4. With consistent induction of this lipase in wild type (WT) CTLs, it was possible to compare their activity with PLRP2-/- ("knock out") CTLs that completely lack PLRP2. Cytotoxicity of the WT CTLs was higher than the PLRP2-/- CTLs. Attempts to match the elevated cytotoxicity with PLRP2 lipase activity left the issue unanswered. In fact, the results even suggest that PLRP2, as a nutritional enzyme in early mouse development, may affect differentiation of CD8 CTLs. During part (3), when I monitored cytotoxicity mediated directly by pure PLRP2 or indirectly by PLRP2 plus lipid substrate, there was cytotoxicity only indirectly, when lipid was present. Live CTLs expressing PLRP2 lacked lipid-dependent killing. There was a notable exception in which live CTLs could mediate lipid-dependent cytotoxicity. This exception was for CTLs repeatedly induced with a unique lot of IL-4. Despite my many efforts, the critical properties of this lot are still unknown. In conclusion, the possibility of PLRP2 performing a direct role in T cell mediated cytotoxicity has been eliminated. My studies into indirect lipase toxicity revealed a novel lipid-dependent cytotoxic pathway induced by a yet to be identified factor. At the end of this project, an intriguing new cytotoxic mechanism awaits future investigation.