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Background: ABO-incompatible (ABOi) organ transplantation is performed owing to unremitting donor shortages. Defining mechanisms of antibody-mediated rejection, accommodation, and tolerance of ABOi grafts is limited by lack of a suitable animal model. We report generation and characterization of a murine model to enable study of immunobiology in the setting of ABOi transplantation., Methods: Transgenesis of a construct containing human A1- and H-transferases under control of the ICAM-2 promoter was performed in C57BL/6 (B6) mice. A-transgenic (A-Tg) mice were assessed for A-antigen expression by histology and flow cytometry. B6 wild-type (WT) mice were sensitized with blood group A-human erythrocytes; others received passive anti-A monoclonal antibody and complement after heart transplant. Serum anti-A antibodies were assessed by hemagglutination. "A-into-O" transplantation (major histocompatibility complex syngeneic) was modeled by transplanting hearts from A-Tg mice into sensitized or nonsensitized WT mice. Antibody-mediated rejection was assessed by morphology/immunohistochemistry., Results: A-Tg mice expressed A-antigen on vascular endothelium and other cells including erythrocytes. Antibody-mediated rejection was evident in 15/17 A-Tg grafts in sensitized WT recipients (median titer, 1:512), with 2 showing hyperacute rejection and rapid cessation of graft pulsation. Hyperacute rejection was observed in 8/8 A-Tg grafts after passive transfer of anti-A antibody and complement into nonsensitized recipients. Antibody-mediated rejection was not observed in A-Tg grafts transplanted into nonsensitized mice., Conclusions: A-Tg heart grafts transplanted into WT mice with abundant anti-A antibody manifests characteristic features of antibody-mediated rejection. These findings demonstrate an effective murine model to facilitate study of immunologic features of ABOi transplantation and to improve potential diagnostic and therapeutic strategies.

The ABO histo-blood group system presents a major barrier in organ transplantation that limits organ availability. A reduction in donor A/B antigens may represent a novel approach to allow safe ABOi transplantation. The utility of FpGalNAc deacetylase and FpGalactosaminidase (Azymes) to convert blood group A-antigen to H-antigen in an ex vivo human lung perfusion model was recently demonstrated. However, as A-glycosyltransferases are constitutively expressed, the duration of A-antigen removal remains unclear. A-transgenic (Tg) mice developed by our group constitutively express A-antigen on vascular endothelium and erythrocytes and have been used to model ABOi Tx. Using this model, we assessed A-antigen removal and re-expression following Azyme administration in vitro and in vivo. Azyme function was studied using A-Tg BALB/c (male n=5, female n=3) and A-Tg C57BL/6 (male n=2, female n=3) mice (10-44 wk). In vitro : Red blood cells (RBC) were assessed for A- and H-antigen expression by hemagglutination at various times (0.5-4 hr) post-Azyme treatment (2-50 µg/mL) of whole blood. In vivo : A-Tg mice were injected iv with Azyme (0.4-0.8 mg/kg); blood was sampled at various times and RBC were tested for A-antigen by flow cytometry and for A- and H-antigens by hemagglutination. Heart and lung tissue were harvested at various times (1-96 hr) and assessed for expression of A- and H- antigens by immunohistochemistry. In vitro , Azyme cleaved A-antigen from A-Tg mouse RBC, resulting in H-antigen. In vivo , Azyme treatment resulted in the absence of A-antigen and the appearance of H-antigen on RBC up to 2 hr post-injection, with detection of A-antigen reappearing after 4 hr. Heart and lung tissue showed a reduction in A-antigen and the appearance of H-antigen up to 8-hr post-injection. No significant differences between males and females were observed. Our preliminary data confirm that Azymes effectively remove A-antigen from RBC and heart and lung tissues in a mouse model. Future studies will assess the efficacy of higher doses of Azymes and the resulting timing of A-antigen re-expression. Clinical application of Azyme technology has the potential to expand ABOi organ transplantation, allowing life-saving transplants in individuals for whom compatible organs cannot be found, and use of donated organs otherwise discarded due to lack of compatible recipients. [ABSTRACT FROM AUTHOR]