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Study of complement activation in a life supporting pig to primate xenotransplantation model using GAL KO pigs

Marta Vadori¹, Moh Daha², Ngaisah Klar², Federica Besenzon¹, Nicola Baldan³, Laura Cavicchioli⁴, Giulia Maria De Benedictis⁵, Massimo Boldrin¹, Valentina Innocente¹, Fabio Fante¹, Fiorella Calabrese⁶, Gilles Blancho⁷, Pierre Giannello⁸, Cesare Galli⁹, Mark B. Nottle¹⁰, Peter Cowan¹¹, Anthony J.F. D’Apice¹¹, Emanuele Cozzi^1,12

¹Immunology Laboratory, CORIT (Consortium for Research on Organ Transplantation), Padua, Italy; ²Department of Nephrology, Leiden University Medical Center, Leiden, Netherlands; ³Department of Surgical and Gastrointestinal Sciences, Clinica Chirurgica I, University of Padua, Padua, Italy; ⁴Department of Public Health, Comparative Pathology and Veterinary Hygiene, University of Padua, Padua, Italy; ⁵Clinical Veterinary Medicine, University of Padua, Padua, Italy; ⁶Department of Pathological Anatomy, University of Padua, Padua, Italy; ⁷INSERM U643, CHU Hotel Dieu, Nantes, France; ⁸Institut de recherche expérimentale et clinique, University of Louvain, Bruxelles, Belgium; ⁹Centro riproduzione animale, Avantea, Cremona, Italy; ¹⁰Discipline of Obstetrics and Gynaecology, University of Adelaide, Adelaide, Australia; ¹¹Immunology Research Centre, St Vincent’s Hospital, Melbourne, Victoria, Australia; ¹²Direzione Sanitaria, Padua General Hospital, Padua, Italy

Background: Generation of α1,3-galactosyltransferase gene-knockout (GTKO) pigs that are also transgenic for human complement regulatory proteins (hCRP) may improve xenograft survival in the pig-to-primate model. In this study, complement activation was evaluated in primate recipients of a GTKO pig kidney expressing hCRP in a life supporting model. The impact of two different induction treatments on humoral responses and classical and alternative complement cascade was also evaluated.

Methods: Ten bilaterally nephrectomized cynomolgus monkeys received a kidney from GTKO pigs transgenic for human CD39, CD55, CD59 and fucosyltransferase (FT). Animals in group 1 (n=6) received cyclophosphamide, cyclosporin A, mycophenolate sodium and steroids. Animals in Group 2 (n=4) received Rituximab instead of cyclophosphamide. IgM and IgG anti- donor porcine aortic endothelial cells (PAEC) were measured by flow cytometry pre- and post-transplantation. Classical and alternative complement pathway and properdin, C1q, C3, C4, complement fragments were also measured.

Results: Median survival was 16 and 12 days in Group 1 and 2, respectively. Except for two animals, all primates were euthanized in the presence of kidney failure. In Group 1, anti-PAEC IgM and IgG increased in the first week and persisted throughout the post operative period. In Group 2, a limited amount of elicited anti pig antibodies was observed. A progressive activation of the classical and alternative pathway complement cascades was detected in both groups, with consumption of C1q, C4 and C3. In Group 2, a delay in classical pathway activation was observed. Additionally we detected a decrease in circulating properdin in agreement with alternative pathway involvement.

Conclusion: In our model, rituximab therapy is useful in controlling anti-donor antibody production and delay the activation of the complement pathways. These studies implicate that a combination of inhibitory antibodies together with downmodulation of complement activation may be useful to control the rejection of xenotransplants using GTKO pig organs.