Study of diverse mechanisms of cell-mediated cytotoxicity in gene-targeted mice using flow cytometric cytotoxicity assay.
Cytotoxic lymphocytes kill tumor or virus-infected target cells utilizing two mechanisms-(1) release of lytic granules (containing perforin and granzymes), and (2) Fas ligand (FasL)/Fas or TNF-initiated apoptosis. We have examined mechanisms of target cell lysis by effector T cells from gene-targeted and mutant mice using a new Flow Cytometric Cytotoxicity Assay (FC Assay). Target cells were labeled with PKH67 dye. Cell death was estimated by 7-AAD inclusion and annexin V-PE binding. A direct correlation has been found between the percentage of dead target cells in FC Assay and the results of 111In release cytotoxicity assay when effector T cells from either Pfp -/- (perforin knockout) or gld (non-functional Fas Ligand) mice were used. As shown by the 4 h FC assay, the granule-mediated mechanism was utilized by T cells from gld mice. In contrast, T cells from Pfp -/- mice used death receptor-mediated lysis. Therefore, cytotoxic cells from gene-targeted and mutant mice can serve as valuable tools for studying different mechanisms of cell-mediated cytotoxicity, and the FC assay could be applied irrespective of which cytotoxic effector pathway is involved.
Malyguine
Anatoli
A
SAIC Frederick, Inc. NCI at Frederick, P.O. Box B, Frederick, MD 21702-1201, USA. amalyguine@mail.ncifcrf.gov
Derby
Eric
E
Brooks
Alan
A
Reddy
Vasavi
V
Baseler
Michael
M
Sayers
Thomas
T
D000818:Animals; D003601:Cytotoxicity Tests, Immunologic; D003602:Cytotoxicity, Immunologic; D053222:Fas Ligand Protein; D005434:Flow Cytometry; D018390:Gene Targeting; D007111:Immunity, Cellular; D008562:Membrane Glycoproteins / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D013601:T-Lymphocytes; D014407:Tumor Cells, Cultured
D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin