Selective igm depletion prolongs organ survival in an ex vivo model of pig-to-human xenotransplantation

In the pig-to-primate model, xenograft hyperacute rejection (HAR) is mediated by antibody and complement. Previous studies have implicated xenoreactive IgM natural antibody (nAb) as the predominant immunoglobulin involved in HAR. To further evaluate the role of IgM, we selectively reduced IgM levels in human blood, without changing IgG and IgA levels, and then used this blood to perfuse porcine hearts ex vivo. Specific IgM depletion was accomplished with an immunoabsorption column containing sheep anti-human IgM (mu-chain specific) conjugated to Sepharose beads. Human blood was separated into plasma and cellular components. For control experiments, those components were unmodified and recombined in the perfusion system. For experiments with IgM reduced blood, the plasma was passed through the IgM column. Immunoabsorption resulted in similar to 90% reduction in xenoreactive IgM levels, as measured by ELISA. Porcine hearts perfused with unmodified human blood survived 25+/-5.6 min (n=5). Porcine hearts perfused with human blood containing reduced levels of IgM survived 229+/-45.2 min (n=4; P<0.01). Organ survival was negatively associated with xenoreactive IgM nAb levels measured immediately before perfusion (r=-0.83; P=0.01), and not with IgG nAb levels (r=-0.21; P=0.62). The ability of plasma from IgM-depleted blood to elicit complement activation, measured by iC3b binding to porcine aortic endothelial cells in vitro, was also strongly associated with IgM xenoreactive nAb levels (r=0.92; P<0.0001), control hearts perfused with unmodified human blood showed typical widespread histologic features of HAR, while porcine hearts perfused with IgM-reduced blood demonstrated milder and less uniform changes. Immunopathological analysis of heart tissues obtained at the completion of each study showed similar deposition of IgG; between groups but markedly less IgM, C3, C4, and C9 in the IgM reduction group, These results suggest that selective IgM reduction delays HAR with prolongation of survival and that xenoreactive IgM may be the predominant immunoglobulin involved in HAR in the pig-to-human combination.