High-level endothelial expression of human CD59 prolongs heart function in an ex vivo model of xenograft rejection.

BACKGROUND: Hyperacute rejection of discordant xenografts is dependent on activation of the complement system of the recipient. Transgenic expression of recipient complement regulatory factors in donor tissue has proved to be a promising approach to dealing with hyperacute rejection, although the relationship between the level of complement regulatory factor expression and the degree of protection is not well established. Here, we examine this relationship using CD59 transgenic mouse hearts in an ex vivo model of xenograft rejection. METHODS: The level of expression of CD59 in two lines of transgenic mice, in which CD59 is expressed under the control of either the murine H2Kb (MHC class I) promoter (line CA-17) or the endothelium-specific human intercellular adhesion molecule-2 promoter (line 237-7), was compared by immunohistochemistry and flow cytometry. Hearts from both groups and wild-type controls were perfused ex vivo with human plasma, and mean heart work for each group was compared over a 60-min period. RESULTS: CD59 expression on cardiac endothelial cells isolated from homozygous CA-17 mice was 25- to 30-fold lower than that on cardiac endothelial cells from heterozygous 237-7 mice. CA-17 hearts perfused with 6% human plasma exhibited a reduction in deposition of the membrane attack complex, but not a prolongation of function, compared with nontransgenic mouse hearts. In contrast, 237-7 hearts showed significantly prolonged function during perfusion with 20% plasma. CONCLUSIONS: High-level endothelial-specific expression of CD59 was effective in prolonging the function of mouse hearts perfused with 20% human plasma, whereas low-level, broader expression did not provide protection from 6% plasma.

Combination of decay-accelerating factor expression and alpha1,3-galactosyltransferase knockout affords added protection from human complement-mediated injury.

BACKGROUND: Hyperacute rejection (HAR) currently prevents the use of pigs as organ donors for humans. It is now generally accepted that the key instigators of HAR are naturally occurring xenoantibodies against the terminal disaccharide galactose alpha1,3-galactose (Gal), and the species incompatibility between human complement and porcine complement regulatory molecules. Using two in vitro models and an ex vivo mouse heart perfusion model, we have shown previously that cells and tissues from Gal knockout (Gal KO) and transgenic mice expressing the human cell surface complement regulator decay-accelerating factor (DAF/CD55) are partially, but not completely, protected from human complement-mediated injury. METHODS: In the present study, Gal KO mice were crossed with DAF transgenic mice and bred to homozygosity (DAF/Gal KO). Isolated splenocytes were incubated with human serum, and the protective effect of DAF and Gal KO was assessed by measuring complement deposition and cell lysis. Hearts perfused ex vivo with human plasma were examined for human antibody and complement deposition, and assessed functionally by measuring work performed by the heart. RESULTS: Splenocytes from DAF/Gal KO mice were found to be more resistant to complement-mediated injury than cells from either DAF transgenic or Gal KO mice. In addition, hearts from DAF/Gal KO mice, when perfused with human plasma, displayed prolonged survival compared with hearts from Gal KO mice. This was associated with a reduction in the extent of endothelial deposition of IgG, IgM, and complement C3b. CONCLUSIONS: These findings demonstrate that expression of human DAF in association with elimination of the Gal epitope provides added protection from complement-mediated injury in these models of HAR.

The alpha-1,3-galactosyltransferase knockout mouse. Implications for xenotransplantation.

Organ xenografts in discordant combinations such as pig-to-man undergo hyperacute rejection due to the presence of naturally occurring human anti-pig xenoantibodies. The galactose alpha(1,3)-galactose epitope on glycolipids and glycoproteins is the major porcine xenoantigen recognized by these xenoantibodies. This epitope is formed by alpha(1,3)-galactosyltransferase, which is present in all mammals except man, apes, and Old World monkeys. We have generated mice lacking this major xenoantigen by inactivating the alpha(1,3)-galactosyltransferase gene. These mice are viable and have normal organs but develop cataracts. Substantially less xenoantibody from human serum binds to cells and tissues of these mice compared with normal mice. Similarly, there is less activation of human complement on cells from mice lacking the galactose alpha(1,3)-galactose epitope. These mice confirm the importance of the galactose alpha(1,3)-galactose epitope in human xenoreactivity and the logic of continuing efforts to generate pigs that lack this epitope as a source of donor organs.