In vitro prevention of cell-mediated xenograft rejection via the Fas/FasL-pathway in CrmA-transducted porcine kidney cells

Cell-mediated cytotoxicity may be involved in delayed and/or chronic xenogr aft rejection in which apoptosis is induced in the grafted cells via the Fa s/Fas-ligand (FasL) and perforin/granzyme pathways. One barrier to the pote ntial use of xeonogenic grafts for humans may be Fas/ Fast-mediated apoptos is, which would be blocked by the gene expression of cytokine response modi fier A (CrmA), a cowpox virus gene product. The purpose of this study is to explore whether crmA is an effective candidate gene for inhibiting apoptos is in an in vitro model of xenograft rejection, using Fas-expressing non-pr imate cells cultured with a soluble recombinant human Fast (sFasL). A recom binant adenovirus vector expressing CrmA (AxCALNLCrmA) was successfully gen erated with a Cre-mediated switching system. PK15 cells, derived from a por cine kidney and infected with AxCALNLCrmA and/or AxCANCre at a multiplicity of infection (MOI) ranging from 0.1 to 100, were cultured with human sFasL derived from KFL74.18, a human FasL-overexpressed cell line. The geneexpre ssion level of the PK15 cells was confirmed by CrmA-immune staining. Approx imately 70% of the control PK15 cells showed induced apoptosis when culture d with sFasL. In contrast, the apoptosis was dramatically reduced in cr crm A-gene-transduced PK15 cells. The inhibitory effect of apoptosis increased with an increase in the infection dose of AxCANCre. In addition, the activi ty of caspases 3 and 8 was significantly inhibited in the a crmA-transduced cells. These results indicate that CrmA is an effective gene product for i nhibiting Fas/FasL-mediated apoptosis, which suggests the potential therape utic use of its gene transduction to protect against graft damage due to de layed and/or chronic xenograft rejection.