Demonstration of the functional importance of the Gal epitope in an ex vivo model of xenotransplantation

Abstract

The galactose a 1‐3 galactose terminal disaccharide (Gal epitope) has been identified as the major porcine xenoantigen recognised by xenoantibody in human plasma. Elimination or suppression of the epitope or antibody will be a major factor in overcoming hyperacute rejection. Inhibition of the antibody by depletion or elimination of the epitope by gene knockout may reveal the importance of other xenoantibodies, and in addition elimination of the epitope may unmask or produce other xenoantibody combinations. This study aims to determine the relative importance of anti‐Gal antibody and Gal epitope elimination in a functional model of xenotransplantation, ex vivo perfusion of mouse hearts with human plasma on a Langendorff apparatus. Perfusion of mouse hearts with human plasma depleted of anti‐Gal antibody demonstrates a protective effect compared to hearts perfused with undepleted plasma with prolongation of survival time from 24.1 to 44.5 min. Similarly, elimination of the epitope is also protective. Hearts from Gal knockout mice, which were generated by gene targeting of the al,3 galactosyltransferase gene, and hearts from appropriate control mice were perfused with human plasma. Gal knockout mice hearts demonstrated an increase in survival time from 10.2 to 33.8 min compared to control hearts. This was accompanied by a decrease in C3c and IgM, but little change in IgG deposition. The protective effect is incomplete, probably due to the effect of antibodies against non‐Gal xenoantigens. There was no functional evidence for generation of neo‐antigens in the Gal KO mice that were I recognised by naturally occurring human xenoantibodies.

Naturally acquired anti‐αGal antibodies in a murine allograft model similar to delayed xenograft rejection

“…Like humans, Gal KO mice lack the αGal epitope [11]. Hearts from Gal KO mice function longer than control hearts when perfused ex vivo with human plasma [12], confirming along with other in vitro analyses [13, 14] the importance of αGal in HAR. However, a suitable in vivo model is necessary to study the role of anti‐αGal antibodies in DXR.…”

α(1,2)‐Fucosylation prevents sialyl Lewis x expression and E‐selectin‐mediated adhesion of fucosyltransferase VII‐transfected cells

“…The aim of this study was to evaluate the effect of expressing HT or ST6Gal‐I on Sle x synthesis and E‐selectin adhesion. The strategy described here was drawn from xenotransplantation studies in which it has been shown by transfection or transgenic techniques, that HT reduces graft rejection between pig and man, by replacing the porcine xenoantigen Galα(1,3)Gal structures with the H(O) blood group Fucα(1,2)Gal [41–43]. Likewise, we addressed here the possibility that the synthesis of functional E‐selectin ligand sLe x , which requires the presence of α(2,3)‐linked sialic acid to be further fucosylated on its GlcNAc residue(s) by FucT‐VII [25], may be impaired after HT‐transfection.…”

Xenoantigens and Xenoantibodies: Their Modification

“…In the absence of ␣Gal these strains develop low levels of anti-␣Gal antibody (which can be boosted to higher levels by exposure to ␣Gal-containing antigens) and are therefore proving invaluable in the investigation of this antigen-antibody system. The organs from ␣Gal-knockout mice, however, still appear to be susceptible to antibody-mediated rejection by ex vivo perfusion with human serum [23], suggesting that previously cryptic antigens have been exposed, against which humans also have natural antibody. The relevance of this observation with regard to potential ␣Gal-knockout pigs remains unknown.…”