Inactivation of the al-3 Galactosyltransferase Gene Protects Murine Hearts from Xenoantibody-dependent Human NK Cell / Monocyte Mediated Injury.

Abstract

174 NK cells and monocytes are the predominant cell type in rejecting vascularized discordant xenografts in which hyperacute rejection has been prevented by complement inhibition i.e. in delayed xenograft rejection. a-Gal is the major porcine xenoantigen and anti-Gal Fab fragments inhibit human NK cell adhesion and cytotoxicity to porcine endothelial cells in vitro. In an attempt to further define the relationship between endothelial expression of a-Gal and NK cell-mediated cardiac rejection we studied a-Gal deficient mice in an ex-vivo perfusion model. A modified Langendorff apparatus was used to study hearts from mice with a targeted deletion in the Gal transferase gene (Gal KO) and size matched non-transgenic control mice. Cardiac work was determined by a pressure transducer affixed to the ventricle. Freshly isolated human PBMC (peripheral blood mononuclear cells) were obtained from healthy volunteers by density gradient sedimentation of whole blood. Hearts were perfused with buffer alone, human IgG, PBMC or the combination of human IgG and PBMC on the same day. A minimum of 4 perfusions were performed in each treatment group. After 70 minutes perfusion, hearts were snap frozen for histological analysis. Perfusion of control hearts with PBMC or IgG did not significantly alter the cardiac work over 70 minutes compared to buffer alone. When control hearts were perfused with PBMC+IgG there was a prompt and significant fall in cardiac function within 20 minutes which continued to decline throughout the perfusion period. In contrast to control hearts, there was no significant difference in performance between Gal KO hearts, perfused with human IgG alone or PBMC+IgG. Histological examination of control hearts perfused with PBMC+IgG showed marked tissue disruption of non-transgenic hearts whereas identically perfused Gal KO mouse hearts showed minimal damage. This study shows that anti-Gal xenoantibody facilitates PBMC mediated cardiac dysfunction and morphological damage in this ex vivo model and that removal of Gal epitope by gene targeting abrogates the influence of IgG xenoantibody. Thus genetic manipulations which reduce or remove the a-Gal epitope may in addition to inhibiting hyperacute rejection, have an important role in inhibiting the cell-mediated component of delayed xenograft rejection.