Cardiac xenografts show reduced survival in the absence of transgenic human thrombomodulin expression in donor pigs

Abstract

A combination of genetic manipulations of donor organs and target-specific immunosuppression is instrumental in achieving long-term cardiac xenograft survival. Recently, results from our preclinical pig-to-baboon heterotopic cardiac xenotransplantation model suggest that a three-pronged approach is successful in extending xenograft survival: (a) α-1,3-galactosyl transferase (Gal) gene knockout in donor pigs (GTKO) to prevent Gal-specific antibody-mediated rejection; (b) transgenic expression of human complement regulatory proteins (hCRP; hCD46) and human thromboregulatory protein thrombomodulin (hTBM) to avoid complement activation and coagulation dysregulation; and (c) effective induction and maintenance of immunomodulation, particularly through co-stimulation blockade of CD40-CD40L pathways with anti-CD40 (2C10R4) monoclonal antibody (mAb). Using this combination of manipulations, we reported significant improvement in cardiac xenograft survival. In this study, we are reporting the survival of cardiac xenotransplantation recipients (n = 3) receiving xenografts from pigs without the expression of hTBM (GTKO.CD46). We observed that all grafts underwent rejection at an early time point (median 70 days) despite utilization of our previously reported successful immunosuppression regimen and effective control of non-Gal antibody response. These results support our hypothesis that transgenic expression of human thrombomodulin in donor pigs confers an independent protective effect for xenograft survival in the setting of a co-stimulation blockade-based immunomodulatory regimen.

Figure 1.. Hematological Parameters;

(A). White blood Counts (WBC), (B). Hematocrit (HCT) and (C). Platelets of cardiac xenograft recipient receiving from non-hTBM donor pig (i.e. GTKO.CD46)

Figure 2.. Cardiac xenograft survivals and histopathological examination;

(A) Cardiac xenograft survivals of both GTKO.CD46 and GTKO.CD46.hTBM xenograft in recipient baboons. (B) Hematoxylin and eosin staining of biopsy of xenograft expressing GTKO.CD46.hTBM gene (i (20X) & ii (400X)) without any sign of rejection or thrombotic microangiopathy; and, explanted GTKO.CD46 xenograft without hTBM expression (iii, iv & v (20X)) indicating myocardial destruction and widespread fibrin micro-thrombi. (Arrows).

Figure 3.. Coagulation, Troponin release and Ultrasonography:

(A).Coagulation profile (e.g. (i) PT, (ii) PTT & (iii) Fibrinogen levels) of recipient baboon receiving non TBM and TBM expressing donor xenoheart;(pvalue=*<0.5; **<0.05) (B) Troponin Release, and, (C) Ultrasonography of GTKO.CD46 xenograft (i.e. (i) a representative ultrasound screenshot showing an empty cavity of transplanted xenoheart just after the xenotransplantation and (ii) with clot filled in transplanted heart during the rejection) from recipient baboon receiving non TBM donor pig.

Figure 4.. Non-Gal IgG and IgM levels;

Anti pig non-Gal (A) IgG and (B) IgM antibodies levels in mean fluorescence intensity (MFI) was measured in cardiac xenograft grafts baboon recipient’s serum by flow cytometry using GTKO porcine endothelial cells.