PREVENTING HUMAN CD8+ CYTOTOXIC T LYMPHOCYTES (CTL)-MEDIATED CYTOTOXICITY AGAINST SWINE ENDOTHELIAL CELLS BY OVEREXPRESSION OF HUMAN DECOY FAS ANTIGEN

Abstract

P1060 Aims: Human CD8+ cytotoxic T lymphocytes (CTL)-mediated killing in xenograft recipients may be a new immunological obstacle for long-term graft survival. The objectives in this study are to assess the mechanism of human CTL-mediated xenocytotoxicity and to explore the method to prevent this killing with overexpression of human decoy Fas antigen by binding competition with endogenous Fas antigen on swine cells for common ligand, human FasL. Furthermore, we generate a membrane-bound form of human FasL that can not be cleaved with metalloproteinase and assess the usefulness of this molecule in inhibition of CTL cytotoxicity. Methods: Generation of human CD8+ CTL: PBMC freshly obtained from healthy volunteers were separated by centrifugation onto a cushion of Histopaque-1077. The fresh lymphocytes were co-cultured for 14 days with irradiated swine endothelial cells (SEC) and human IL-2 (100 U/ml). We employed cultured lymphocytes as effector cells in cytotoxicity assay. Cytotoxicity assay and blockade of lymphocytes killing in the cytotoxicity assay: The Cytotoxicity of human CTL incubated under various effector-to-target ratios was assessed by Terasaki plates. Parental SEC and SEC transfectants were plated as target cells in those plates. Furthermore, the blocking experiments were performed using Concanamycin A to inhibit perforin-dependent lysis and an anti-human FasL mAb to block Fas/FasL pathway. Gene: cDNAs of human decoy Fas which does not contain the death domain in its cytoplasmic tail and FasL carrying the deletion at the cleavage site with metalloproteinase, were subcloned into pEF-BOS expression vector (promoter of human elongation factor 1α), respectively. Expression of plasmids: These plasmids were transfected into SEC by lipofection, respectively. The expression of these molecules on SEC transfectants were assessed with FACS analysis using PE-conjugated mAbs. Results: Mechanism of human CTL cytotoxicity: The human CTL killing was dramatically inhibited by anti-FasL mAb, whereas the killing with them was partially suppressed by Concanamycin A. These results are averages of 3∼6 experiments in the table.FigureConclusions: The Fas/FasL pathway is a main contributor to human CTL killing against xenograft cells. SEC transfectants with either human decoy Fas antigen or membrane-bound FasL gene were highly resistant to human CTL-mediated lysis. Our data demonstrate that these novel molecules may represent a step forward toward preventing cellular xenograft rejection and that the combinatorial expression of both molecules may be more beneficial to protect xenograft cells.