AUTHOR=Lee Haneulnari , Park Eun Mi , Ko Nayoung , Choi Kimyung , Oh Keon Bong , Kang Hee Jung 

TITLE=Effect of Factor H on Complement Alternative Pathway Activation in Human Serum Remains on Porcine Cells Lacking N-Glycolylneuraminic Acid

JOURNAL=Frontiers in Immunology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2022.859261

DOI=10.3389/fimmu.2022.859261

ISSN=1664-3224

ABSTRACT=<sec><title>Background</title><p>Triple knockout (TKO) donor pigs lacking alpha-1,3-galactose (Gal), <italic>N</italic>-glycolylneuraminic acid (Neu5Gc), and Sd(a) expressions were developed to improve the clinical success of xenotransplantation. Neu5Gc, a sialic acid expressed on cell surfaces, recruits factor H to protect cells from attack by the complement system. Lack of Neu5Gc expression may cause unwanted complement activation, abrogating the potential benefit of gene-modified donor pigs. To investigate whether TKO porcine cells display increased susceptibility to complement activation in human serum, pathway-specific complement activation, apoptosis, and human platelet aggregation by porcine cells were compared between <italic>alpha-1,3-galactosyltransferase</italic> gene-knockout (<italic>GT</italic>KO) and TKO porcine cells.</p></sec><sec><title>Methods</title><p>Primary porcine peripheral blood mononuclear cells (pPBMCs) and endothelial cells (pECs) from <italic>GT</italic>KO and TKO pigs were used. Cells were incubated in human serum diluted in gelatin veronal buffer (GVB<sup>++</sup>) or Mg<sup>++</sup>-EGTA GVB, and C3 deposition and apoptotic changes in these cells were measured by flow cytometry. C3 deposition levels were also measured after incubating these cells in 10% human serum supplemented with human factor H. Platelet aggregation in human platelet-rich plasma containing <italic>GT</italic>KO or TKO pECs was analyzed.</p></sec><sec><title>Results</title><p>The C3 deposition level in <italic>GT</italic>KO pPBMCs or pECs in GVB<sup>++</sup> was significantly higher than that of TKO pPBMCs or pECs, respectively, but C3 deposition levels in Mg<sup>++</sup>-EGTA-GVB were comparable between them. The addition of factor H into the porcine cell suspension in 10% serum in Mg<sup>++</sup> -EGTA-GVB inhibited C3 deposition in a dose-dependent manner, and the extent of inhibition by factor H was similar between <italic>GT</italic>KO and TKO porcine cells. The percentage of late apoptotic cells in porcine cell suspension in GVB<sup>++</sup> increased with the addition of human serum, of which the net increase was significantly less in TKO pPBMCs than in <italic>GT</italic>KO pPBMCs. Finally, the lag time of platelet aggregation in recalcified human plasma was significantly prolonged in the presence of TKO pECs compared to that in the presence of <italic>GT</italic>KO pECs.</p></sec><sec><title>Conclusion</title><p>TKO genetic modification protects porcine cells from serum-induced complement activation and apoptotic changes, and delays recalcification-induced human platelet aggregation. It does not hamper factor H recruitment on cell surfaces, allowing the suppression of alternative complement pathway activation.</p></sec>