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Cloning and in vitro antiapoptotic effects of pig FLIPs
1Division of Organ Transplantation, Department of Molecular Therapeutics, Osaka University Graduate School of Medicine, Osaka, Japan.
Introduction:
Cellular FLICE-like protein (cFLIP) inhibits death receptor-mediated apoptosis signal transduction, such as that induced by Fas and TNFR. The present study examined the role of antiapoptotic molecules to protect pig cells from human natural killer (NK) cells in vitro, as a model of delayed-type xenograft rejection.
Methods:
Pig FLIPs were cloned using the TBLASTIN program to search for cDNA fragments of pig FLIPs. The sequence was identified using the dideoxy chain termination method and an ABI PRISM3100 genetic analyzer. The cDNA of pig FLIPs was inserted into the cloning site of the chicken beta-actin promoter (pCXN2). The cDNA was then transfected into pig endothelial cells (PEC), to establish several stable PEC clones containing the cDNA. Expression of the pig FLIP gene was evaluated by reverse-transcriptase polymerase chain reaction, and NK cell-mediated cytolysis assessed, using YT cells (an NK-like cell line).
Results:
The full-length pig FLIP encoding sequence, total 5'-region to 3'-region, was defined for the first time. PEC transfectants with the FLIP showed moderate expression of FLIPs. Transfection of PEC with plasmids encoding FLIPs inhibited NK cell-mediated PEC lysis. While approximately half of parental PEC were injured by the human NK-like YT cells, the injury rate was relatively lower in the transfectants.
Conclusion:
Overexpression of the antiapoptotic molecules, pig FLIPs, has the potential for use in protecting graft cells from human NK cells.
Insights
Overexpressing pig cellular FLICE-like protein (cFLIP) in pig endothelial cells (PECs) protected them from human natural killer (NK) cell attack. This offers a potential strategy to prevent delayed-type xenograft rejection.
Area of Science:
- Immunology
- Cell Biology
- Xenotransplantation
Background:
- Cellular FLICE-like protein (cFLIP) is an antiapoptotic molecule that inhibits death receptor-mediated apoptosis.
- Natural killer (NK) cells mediate xenograft rejection, posing a challenge for xenotransplantation.
- Understanding mechanisms to protect donor cells from NK cell-mediated lysis is crucial for xenograft survival.
Purpose of the Study:
- To investigate the role of pig cFLIP (pig FLIPs) in protecting pig endothelial cells (PECs) from human NK cell-mediated lysis.
- To establish a model for assessing xenograft rejection and potential protective strategies in vitro.
Main Methods:
- Pig FLIPs cDNA was cloned and sequenced.
- Pig FLIPs cDNA was transfected into PECs to create stable transfectants.
- NK cell-mediated cytolysis was assessed using human NK-like YT cells and parental/transfected PECs.
Main Results:
- The full-length pig FLIP encoding sequence was determined.
- Transfected PECs expressed moderate levels of pig FLIPs.
- PECs transfected with pig FLIPs exhibited significantly reduced lysis by human NK-like YT cells compared to parental PECs.
Conclusions:
- Overexpression of pig FLIPs confers resistance to human NK cell-mediated cytotoxicity.
- Pig FLIPs represent a potential therapeutic target for preventing delayed-type xenograft rejection.
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