The innate immune response and activation of coagulation in alpha1,3-galactosyltransferase gene-knockout xenograft recipients

Abstract

Background: The role of the innate immune system in the development of thrombotic microangiopathy (TM) after alpha1,3-galactosyltransferase gene-knockout (GTKO) pig organ transplantation in primates is uncertain.

Methods: Twelve organs (nine hearts, three kidneys) from GTKO pigs were transplanted into baboons that received no immunosuppressive therapy, partial regimens, or a full regimen based on costimulation blockade. After graft failure, histologic and immunohistologic examinations were carried out.

Results: Graft survival of less than 1 day was prolonged to 2 to 12 days with partial regimens (acute humoral xenograft rejection) and to 5 and 8 weeks with the full regimen (TM). Clinical or laboratory features of consumptive coagulopathy occurred in 7 of 12 baboons. Immunohistochemistry demonstrated IgM, IgG, and complement deposition in most cases. Histopathology demonstrated neutrophil and macrophage infiltrates, intravascular fibrin deposition, and platelet aggregation (TM). Grafts showed expression of primate tissue factor (TF), with increased mRNA levels, and TF was also expressed on baboon macrophages/monocytes infiltrating the graft.

Conclusions: Our data suggest that (1) irrespective of the presence or absence of the adaptive immune response, early or late xenograft rejection is associated with activation of the innate immune system; and (2) porcine endothelial cell activation and primate TF expression by recipient innate immune cells may both contribute to the development of TM.

Figure 1

Histopathologic features of failed heart grafts. Graft failure occurred after 24 hours in Exp 2, showing (A) hemorrhage, thrombosis and infarction (H&E, x400), (B) fibrin deposition (MSB, x400), and (C) platelet accumulation (IHC, x400). IgM (D), IgG (E), and complement C3 (F) deposition (all indicated by brown staining - arrows) were present in the heart of Exp 3 that underwent failure at 7 days from AHXR (x400). Graft failure occurred on day 6 in Exp 6, showing significant neutrophil (H&E, x200) (G) and macrophage (H) infiltration, with relatively less T (I) and B (J) cell infiltration (all IHC, x200, stained brown).

Figure 2

Histopathologic features of the heart graft that failed after 8 weeks (Exp 12), showing (A) thrombosis and infarction (H&E, x200), (B) fibrin deposition (MSB x400), and (C) massive platelet accumulation (IHC, x400). A neutrophil infiltrate can be seen in the interstitium (IHC, x400) (D); these cells are also present in the vessels of the graft (E), but there are relatively few infiltrating macrophages (IHC,200) (F), and very few T (G) and B (H) lymphocytes (stained brown) (magnification x200).

Figure 3

Staining for primate TF in heart grafts that rejected at day 12 (A; Exp 5) and at 8 weeks (B; Exp 12), showing strong staining in the thrombosed vessels and less staining in the interstitium (arrows) (x600). Co-localization of primate TF (red stain) and macrophages (stained for CD68, brown) in heart grafts excised on day 12 (C, Exp 5) and at 8 weeks (D, Exp 12) is indicated by arrows (x600). Biopsies from 3 other rejected xenografts showed similar results. Primate TF mRNA levels in a rejected porcine heart xenograft by qRT-PCR (E); the heart failed from TM at 8 weeks (Exp 12) and showed high levels of primate TF. Tissue from a normal baboon heart was used as a positive control, while a porcine heart was used as a negative control to ensure the specificity of the primate TF primer. Real-time PCR data were plotted as the ΔRn fluorescence signal versus the cycle number. The expression of each gene was normalized to actin mRNA content and calculated relative to control using the comparative CT method.