Significant inhibition of human CD8(+) cytotoxic T lymphocyte-mediated xenocytotoxicity by overexpression of the human decoy Fas antigen.

BACKGROUND

Human CD8(+) CTL-mediated killing may be important for xenograft rejection. The purpose of this study was to explore the preventing methods for CTL-mediated xenocytotoxicity by overexpression of human decoy Fas, which lacks a death domain in its cytoplasmic region, by binding competition with endogenous pig Fas. Moreover, the cytoprotective effect of this CTL-killing of membrane-bound human FasL, which is resistant to metalloproteolytic cleavage, was also assessed.

METHODS

Human CTL were generated by the stimulation of human PBMC with swine endothelial cells (SEC) and human IL-2, subsequently a CD8(+) population were selected by magnetic beads and employed as the effector cells. Stable SEC transfectants expressing either decoy Fas or membrane-bound FasL were established. Double-transfectants were also created. The amelioration of cytotoxicity to these transfectants was examined with Cr release assay. RESULTS.: Human CD8(+) CTL were highly detrimental against parental SEC. This CTL-killing was strongly inhibited by anti-FasL mAb treatment, however partial suppression was observed by Concanamycin A treatment. The overexpression of either decoy Fas or membrane-bound FasL in SEC markedly inhibited CTL-xenocytotoxicity. The double expressions of these molecules also significantly reduced this xenocytotoxicity despite the low levels of expression of either decoy Fas or membrane-bound FasL.

CONCLUSION

These findings indicate that the strong xenocytotoxicity of human CD8(+) CTL is mediated mainly by the Fas/FasL pathway. The overexpression of either decoy Fas or membrane-bound FasL were quite effective in preventing CTL-killing. Furthermore, the combined expression of both molecules in pig cells may create a window of opportunity for prolonging xenograft survival.