Human CD55 Expression and C1 Inhibition Partially Protect Gene-Edited Pig Red Blood Cells From Human Complement-Mediated Hemolysis In Vitro.

Abstract

Background: In recent years, gene-edited pigs have become sources of organs for clinical xenotransplantation. They have the potential to be sustainable sources of red blood cells (pRBCs). We investigated in vitro the effect of human complement inhibition by using (i) human CD55-expressing pRBCs from pigs with 10 gene-edits (10GE) and (ii) a C1-esterase inhibitor (C1-INH).

Methods: RBCs were collected from pigs (triple-knockout [TKO] with or without expression of "protective" human transgenes [10GE] on peripheral blood mononuclear cells [PBMCs], including two complement-regulatory proteins, hCD46 and hCD55). hCD46 and hCD55 expression, anti-pRBC antibody binding, and C3b/iC3b deposition were measured by flow cytometry. Hemolysis by complement-dependent cytotoxicity (CDC) was measured by a plate reader. A C1-INH was added to the hemolysis assay.

Results: HCD46 was not expressed on either TKO or 10GE pRBCs. hCD55 was expressed at low levels on 10GE pRBCs. Hemolysis induced by human complement and anti-pRBC antibodies was less when pRBCs were from 10GE pigs than from TKO pigs (57.3% ± 2.2% vs. 26.2% ± 3.8%, p < 0.01). C3b/iC3b deposition of 10GE pRBCs under nonhemolytic conditions was also lower. C1-INH decreased hemolysis (No C1-INH = 18.6% ± 2.3% vs. 2.5U/mL C1-INH = 7.0% ± 1.1%, p < 0.05). C3b/iC3b deposition on pRBCs was also decreased (gMFI: No C1-INH = 2680 ± 82 vs. 2.5 U/mL C1-INH = 719 ± 57, p < 0.01).

Conclusions: Even low expression of hCD55 contributes to the protection of pRBCs from hemolysis by CDC, but the possibility of phagocytosis still remains. However, C1-INH partially protects pRBCs from hemolysis and C3b/iC3b deposition. Therefore, higher hCD55 expression and the administration of a complement inhibitor are likely to prolong pRBC survival after clinical xenotransfusion.