Xenogeneic human anti-pig cytotoxicity mediated by activated natural killer cells

Even if hyperacute rejection, which is mediated by human natural antibodies (nAb) and complement, could be prevented, xenoreactive human anti-pig cellular responses may lead to delayed and/or chronic xenograft rejection. Among the cell populations participating in such rejection, Mt cells have been proposed as an important component. In this study we report the in vitro cytotoxic activity of natural killer (NK) cells obtained from healthy human donors against porcine target cells. Freshly isolated peripheral blood mononuclear cells (PBMC) and purified NK cells (CD16+/CD56+, CD3-, CD20-, CD33-) exhibited little or no cytotoxic activity when tested on porcine phytohemagglutinin (PHA)-stimulated lymphoblasts or bone marrow- or aortic-derived endothelial cell lines in the presence of serum-free medium. Killing was considerably higher in the presence of human decomplemented plasma, containing xenoreactive nAb, or purified Gal(alpha 1,3)Gal-reactive antibodies, suggesting that antibody dependent cell-mediated cytotoxicity (ADCC) mediated by NK cells is an important mechanism involved in xenogeneic cytotoxicity. After incubation of human PBMC for 6 days in the presence of irradiated xenogeneic porcine or allogeneic stimulator cells, or in the presence of exogenous interleukin 2 (IL-2), the cytotoxic activity of the bulk cultures as well as that of isolated NK cells (separated from stimulated bulk cultures) against xenogeneic targets increased considerably, and corresponded to an increased NK-specific lysis of K562 target cells. Cell surface staining and flow cytometry showed that CD16+/CD56+, CD3- NK cells composed ca. 25% of short-term (6 days) xenogeneic, allogeneic, or IL-2 stimulated bulk cultures. In summary, these data suggest that, in contrast to allogeneic cell-mediated killing, xenogeneic human anti-porcine cytotoxicity includes an important contribution from NK cells.