CD4+ CELLS PLAY A MAJOR ROLE IN XENOGENEIC HUMAN ANTI-PIG CYTOTOXICITY THROUGH THE FAS/FAS LIGAND LYTIC PATHWAY1,2

In this study, the role of cell-mediated cytotoxicity by human leukocytes against pig endothelial cells was examined in vitro. The aim was to determine which cell subsets were responsible for this phenomenon and which pathways were involved in cell lysis.Primed human peripheral blood mononuclear cells (PBMC) or purified CD4+ or CD8+ T cells were used in a cell-mediated cytotoxicity assay in which cytotoxicity of an SV40 transformed porcine endothelial cell (EC) line (SVAP) was determined by Annexin V binding.Human PBMC demonstrated specific lysis of porcine EC that was proportional to the effector: target ratio. CD4+ T cells accounted for >60% of this lysis, whereas CD8+ T cells accounted for <20%. CD4+ T cell-mediated lysis depended on direct recognition of porcine major histocompatibility complex class II molecules as inhibition of swine leukocyte antigen class II on porcine EC-inhibited CD4+ T cell cytotoxicity. This lysis was mediated through the Fas/FasL pathway as addition of anti-Fas and/or anti-FasL antibody profoundly inhibited antiporcine lysis. In addition, FasL gene expression was detected in primed PBMC and CD4+ T cells by RT-PCR, whereas granzyme B gene expression was not. Primed CD4+ T cells demonstrated high level FasL protein by Western blotting and two-color FACS analysis, whereas NK cells and CD8+ T cells did not. Finally, recombinant human FasL induced apoptosis in Fas expressing porcine EC cells, demonstrating that human FasL interacted with and activated Fas on porcine EC cells.In conclusion, human to pig cell-mediated cytotoxicity was mediated predominantly by CD4+ T cells through the Fas/FasL pathway of apoptosis. These results suggest that direct cytotoxicity by xenoreactive CD4+ T cells may be one of several effector mechanisms involved in cellular xenograft rejection.