Characterization of porcine tumor necrosis factor receptor 2: Implications for xenotransplantation

Clinical solid organ xenotransplantation is precluded by the strong immune response that results in rejection of pig xenografts in primate models. Innate immunity seems to play a major role in this process. In particular, tumor necrosis factor (TNF), produced by natural killer cells and macrophages, contributes to xenograft rejection by promoting endothelial cell activation and the recruitment of inflammatory cells. To further elucidate its molecular mechanism, we cloned the full-length cDNA of porcine TNF-Receptor 2 (pTNFR2, p75) by reverse transcriptase polymerase chain reaction (PCR) of total RNA isolated from porcine peripheral blood mononuclear cells. To this end, we used degenerate primers based on the sequences of the mouse, rat, and human homologues. Two PCR fragments were obtained that contained the pTNFR2 sequence, but differed in size. The shorter clones lacked the sequence corresponding to exon 4 by homology but identical for the rest, suggesting there is an alternative spliced mRNA variant of the porcine receptor. The predicted protein sequence (461 amino acids, containing exon 4) exhibited 72.5% identity to the human TNFR2 and 58.7% to the mouse molecule. By predicted protein sequence analysis, we determined that it comprised the four TNFR cysteine-rich repeats conserved between species. However, the molecule missing exon 4 lacks one cysteine-rich repeat. To assess function, we produced two recombinant proteins containing the extracellular domain of each pTNFR2 variant fused to the Fc portion of human IgG1. Next, we examined their ability to inhibit human TNF-mediated activation of porcine aortic endothelial cells. The addition of the whole pTNFR2 fusion protein to the TNF treatment blocked the up-regulation of activation markers. However, the fusion protein lacking exon 4 failed to effectively counteract TNF effects. These two pTNFR2 isoforms may play differential roles in the process of xenograft rejection.