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Title: Expression of human perforin in a mouse cytotoxic T lymphocyte cell line: evidence for perturbation of granule-mediated cytotoxicity.
Austin Authors: Thia, K Y;Smyth, Mark J;Trapani, Joseph A
Affiliation: Cellular Cytotoxicity Laboratory, Austin Research Institute, Austin Hospital, Heidelberg, Victoria, Australia
Issue Date: 1-Dec-1993
Publication information: Journal of Leukocyte Biology; 54(6): 528-33
Abstract: Expression of the pore-forming protein perforin is normally restricted to the cytolytic granules of cytotoxic T lymphocytes and natural killer cells. Perforin, which causes cell death by osmotic lysis, has the ability to form transmembrane channels in target cell membranes. This function makes perforin crucial in the granule-exocytosis model of T cell-mediated cytotoxicity. In the present study, variants of the mouse cytotoxic T lymphocyte cell line CTLL-R8 have been produced which express human perforin. A full-length cDNA clone (HP-10) encoding human perforin was inserted in the sense orientation into the expression plasmid pCMV5neo. The resultant construct, designated pCMV5neoHP-10, was used to transfect CTLL-R8 cells. Of eight G418-resistant clones studied, four clones expressed human perforin mRNA by Northern analysis and three of these clones also expressed human perforin protein by Western blotting. The expression of human perforin protein was associated with a pronounced (55-74%) and consistent reduction in the killing of three target cell lines, P815, YAC-1, and EL4, compared with parental CTLL-R8 cells. The reduction in target cell lysis could not be attributed to nonspecific effects of the transfection, as clones transfected with neo alone showed no reduction in killing in comparison with parental CTLL-R8 cells. Clones expressing human perforin showed very similar growth characteristics, surface phenotype, and N-alpha-benzyloxycarbonyl-l-thiobenzyl-esterase release compared with untransfected CTLL-R8 cells. The mechanism of reduction of cytolysis is unclear but may involve competition by human perforin in the handling or packaging of endogenous granule constituents (including mouse perforin) or assembly of human perforin into mouse polyperforin channels in target cell membranes. The expression of human perforin in mouse cytotoxic T cells provides a potential model for studying how cytotoxic T cells process, package, utilize, and protect themselves against the perforin molecules they produce.
Gov't Doc #: 8245705
Type: Journal Article
Subjects: Animals
Cell Line
Clone Cells.chemistry
Cytoplasmic Granules.physiology
Membrane Glycoproteins.genetics.physiology
Mice, Inbred BALB C
Mice, Inbred C57BL
Pore Forming Cytotoxic Proteins
T-Lymphocytes, Cytotoxic.chemistry.ultrastructure
Transfection.drug effects
Appears in Collections:Journal articles

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