Early graft failure of GalTKO pig organs in baboons is reduced by expression of a human complement pathway-regulatory protein

Abstract

We describe the incidence of early graft failure (EGF, defined as loss of function from any cause within 3 days after transplant) in a large cohort of GalTKO pig organs transplanted into baboons in three centers, and the effect of additional expression of a human complement pathway-regulatory protein, CD46 or CD55 (GalTKO.hCPRP). Baboon recipients of life-supporting GalTKO kidney (n = 7) or heterotopic heart (n = 14) grafts received either no immunosuppression (n = 4), or one of several partial or full immunosuppressive regimens (n = 17). Fourteen additional baboons received a GalTKO.hCPRP kidney (n = 5) or heart (n = 9) and similar treatment regimens. Immunologic, pathologic, and coagulation parameters were measured at frequent intervals. EGF of GalTKO organs occurred in 9/21 baboons (43%). hCPRP expression reduced the GalTKO EGF incidence to 7% (1/14; P < 0.01 vs. GalTKO alone). At 30 mins, complement deposits were more intense in organs in which EGF developed (P < 0.005). The intensity of peri-transplant platelet activation (as β-thromboglobulin release) correlated with EGF, as did the cumulative coagulation score (P < 0.01). We conclude that (i) the transgenic expression of a hCPRP on the vascular endothelium of a GalTKO pig reduces the incidence of EGF and reduces complement deposition, (ii) complement deposition and platelet activation correlate with early GalTKO organ failure, and (iii) the expression of a hCPRP reduces EGF but does not prevent systemic coagulation activation. Additional strategies will be required to control coagulation activation.

Keywords: baboon; complement-regulatory protein; pig; xenograft rejection; xenotransplantation; α1, 3-galactosyltransferase gene-knockout.

Figure 1. Macroscopic and microscopic analysis of xenografts

(A) Macroscopic appearances of rejected pig organs [left], and microscopic appearances (trichrome staining [middle] and hematoxylin and eosin staining [right]) are shown for representative heart and kidney xenografts with EGF, and hematoxylin and eosin staining for representative heart and kidney 30min biopsies with DXR [far right]. (B) Immunofluorescence for tissue deposition of IgM, IgG, C3, C5b-9, CD41 and fibrin from representative biopsies for each group.

Figure 1. Macroscopic and microscopic analysis of xenografts

(A) Macroscopic appearances of rejected pig organs [left], and microscopic appearances (trichrome staining [middle] and hematoxylin and eosin staining [right]) are shown for representative heart and kidney xenografts with EGF, and hematoxylin and eosin staining for representative heart and kidney 30min biopsies with DXR [far right]. (B) Immunofluorescence for tissue deposition of IgM, IgG, C3, C5b-9, CD41 and fibrin from representative biopsies for each group.

Figure 2. Role of antibody and complement

(A) Baboon anti-nonGal IgM levels were measured by flow cytometry on pig aortic endothelial cells and data expressed as median fluorescence intensity (MFI) (n = 35). Pre-transplant anti-nonGal antibody levels did not predict graft outcome. Anti-nonGal antibody deposition was associated with both EGF and DXR. The dotted box represents anti-nonGal IgM levels in a pool of normal human serum. (B) Complement activation in the graft was quantified using a semi-quantitative score as defined in Methods (n = 22). Complement deposition (at 30 min) was significantly associated with EGF. Each dot represents an individual xenograft; the horizontal line represents the median.

Figure 2. Role of antibody and complement

(A) Baboon anti-nonGal IgM levels were measured by flow cytometry on pig aortic endothelial cells and data expressed as median fluorescence intensity (MFI) (n = 35). Pre-transplant anti-nonGal antibody levels did not predict graft outcome. Anti-nonGal antibody deposition was associated with both EGF and DXR. The dotted box represents anti-nonGal IgM levels in a pool of normal human serum. (B) Complement activation in the graft was quantified using a semi-quantitative score as defined in Methods (n = 22). Complement deposition (at 30 min) was significantly associated with EGF. Each dot represents an individual xenograft; the horizontal line represents the median.

Figure 3. Activation of platelets and coagulation

(A) Platelet activation (determined by plasma levels of baboon β–thromboglobulin) was significantly increased when EGF developed (n = 30). (B) Coagulation activation in the graft and blood was quantified using a semi-quantitative score as defined in Methods (n = 32). EGF of GalTKO pig xenografts was associated with a higher cumulative coagulation score, but coagulation was not decreased by expression of hCPRP. Each dot represents an individual xenograft; the horizontal line represents the median.

Figure 3. Activation of platelets and coagulation

(A) Platelet activation (determined by plasma levels of baboon β–thromboglobulin) was significantly increased when EGF developed (n = 30). (B) Coagulation activation in the graft and blood was quantified using a semi-quantitative score as defined in Methods (n = 32). EGF of GalTKO pig xenografts was associated with a higher cumulative coagulation score, but coagulation was not decreased by expression of hCPRP. Each dot represents an individual xenograft; the horizontal line represents the median.