The role of human CD46 in early xenoislet engraftment in a dual transplant model

Abstract

Background: Membrane cofactor protein CD46 attenuates the complement cascade by facilitating cleavage of C3b and C4b. In solid organ xenotransplantation, organs expressing CD46 have been shown to resist hyperacute rejection. However, the incremental value of human CD46 expression for islet xenotransplantation remains poorly defined.

Methods: This study attempted to delineate the role of CD46 in early neonatal porcine islet engraftment by comparing Gal-knocked out (GKO) and hCD46-transgenic (GKO/CD46) islets in a dual transplant model. Seven rhesus macaques underwent dual transplant and were sacrificed at 1 hour (n = 4) or 24 hours (n = 3). Both hemilivers were recovered and fixed for immunohistochemistry (CD46, insulin, neutrophil elastase, platelet, IgM, IgG, C3d, C4d, CD68, Caspase 3). Quantitative immunohistochemical analysis was performed using the Aperio Imagescope.

Results: Within 1 hour of intraportal infusion of xenografts, no differences were observed between the two types of islets in terms of platelet, antibody, or complement deposition. Cellular infiltration and islet apoptotic activity were also similar at 1 hour. At 24 hours, GKO/CD46 islets demonstrated significantly less platelet deposition (P = 0.01) and neutrophil infiltration (P = 0.01) compared to GKO islets. In contrast, C3d (P = 0.38) and C4d (P = 0.45) deposition was equal between the two genotypes.

Conclusions: Our findings suggest that expression of hCD46 on NPIs potentially provides a measurable incremental survival advantage in vivo by reducing early thrombo-inflammatory events associated with instant blood-mediated inflammatory reaction (IBMIR) following intraportal islet infusion.

Keywords: CD46; instant blood-mediated inflammatory reaction; islet transplantation; xenotransplantation.

Figure 1.

Weight of pancreas harvested (A) and yield of islet cells per gram of pancreas tissue (B) between the two genotypes and wild type piglets were not significantly different. (C) Technical success for islet segregation by contralateral hemiliver was confirmed by CD46 staining at both 1hr and 24hrs (p<0.01). (D) Genotype of NPIs was confirmed by sequencing (data not shown, by Revivicor Inc.) and flow cytometry using piglet PBMCs. Representative hCD46 IHC slides of the hemilivers where GKO islets were delivered (E) and where GKO/CD46 islets were delivered (F) at 1 hr.

Figure 2.

Development of instant blood mediated inflammatory response (IBMIR) observed in vivo over time in GKO/CD46 NPIs. Representative neutrophil IHC slides of hemilivers (GKO/CD46 side) procured at 1-hour (A), 24-hour (B) and 7-day (C). Representative platelet immunohistochemistry slides at various time points shown in (D-F). Neutrophil infiltration (G) and platelets deposition (H) were heavily featured early in IBMIR but their involvement diminished over time. (I) Other components of IBMIR observed at various early time points following portal infusion of islets.

Figure 3.

The role of hCD46 in islet xenotransplantation. Significantly less neutrophil infiltration (p=0.01) and platelet deposition (p=0.01) were observed with GKO/CD46 islets compared to GKO islets at 24 hours following intraportal infusion, even though no differences was seen at 1 hour (A, D). Representative immunohistochemistry images on both L and R hemilivers precured from the same animal at 24 hours stained for neutrophil elastase (B-C) and platelet (E-F).

Figure 4.

Other measured variables, including T cell and macrophage infiltration, C3d, C4d, IgM and IgG deposition were comparable between GKO and GKO/CD46 islets at both 1hour (A) and 24 hours (B).