CD4+ cells play a major role in xenogeneic human anti-pig cytotoxicity through the Fas/Fas ligand lytic pathway

Background. 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 mere responsible for this phenomenon and w hich pathways were involved in cell lysis, Methods. Primed human peripheral blood mononuclear cells (PBMC) or purified CD4+ or CD8+ T cells were used in a cell-mediated cytotoxicity assay in wh ich cytotoxicity of an SV40 transformed porcine endothelial cell (EC) line (SVAP) was determined by Annexin V binding. Results. Human PBMC demonstrated specific lysis of porcine EC that was prop ortional 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 l ysis depended on direct recognition of porcine major histocompatibility com plex class II molecules as inhibition of swine leukocyte antigen class II o n porcine EC-inhibited CD4+ T cell cytotoxicity. This lysis was mediated th rough the Fas/FasL pathway as addition of anti-Fas and/or anti-Fast antibod y profoundly inhibited antiporcine lysis. In addition, Fast 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 Fast protein by Western blotting. and two-color FAGS analysis, whereas NK cells and CD8+ T cells did not. Finally, recombinant human Fast induced apoptosis in Fas expressing porcine EC cells, demonstrating that human Fast interact ed with and activated Fas on porcine EC cells, Conclusions. In conclusion, human to pig cell-mediated cytotoxicity was med iated predominantly by CD4+ T cells through the Fas/FasL pathway of apoptos is. These results suggest that direct cytotoxicity by xenoreactive CD4+ T c ells may be one of several effector mechanisms involved in cellular xenogra ft rejection.