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Complement is not activated at the graft site in non-immunosuppressed immunocompetent mice transplanted with GTKO/hCD46 pig pancreatic tissue

Suzanne Bertera^1,2, Rita Bottino^1,2, Jing He¹, Dirk van der Windt², David Ayares³, David KC Cooper², Massimo Trucco¹

¹Children’s Hospital of Pittsburgh, University of Pittsburgh, Pittsburgh, PA; ²Thomas E Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA; ³Revivicor Inc., Blacksburg, VA; United States

Purpose: Our group has demonstrated that islets from pigs homozygous for alpha1,3-galactosyltransferase gene-knockout (GTKO) additionally transgenic for the human complement-regulatory protein, hCD46, transplanted into immunosuppressed diabetic cynomolgus monkeys achieve and maintain normalization of blood glucose. However, the mechanisms behind successful engraftment need further elucidation. Testing the immunogenicity of these genetically-modified pig islets in a small animal model may provide valuable information that will contribute towards clinical application. We transplanted pancreatic digest from GTKO/hCD46 pigs into immunocompetent non-diabetic mice and examined complement activation as well as cell infiltration at the graft site, using wild-type (genetically-unmodified) pig tissue as control.

Methods: Approximately 30-60ml of pancreatic tissue containing 30% islets from DKO/hCD46 or wild-type pigs was transplanted under the kidney capsule of non-immunosuppressed immunocompetent B6 mice. Mice were euthanized at one of four time-points post-transplant (24 hours, 4, 8, and 12 days) and sections of the graft-bearing kidney were fixed for histological examination. Sections were stained for CD4⁺ and CD8⁺ cells, macrophages (F4/80 antigen⁺), and complement activation marker C4d.

Results: In vivocomplement activity was evident at the graft site of wild-type pig tissue as early as 24 hours after tissue transplantation, while complement activation was not detected at any time-point in the grafts of GTKO/hCD46 pig tissue. Relatively numerous CD4⁺ and CD8⁺ T cells and macrophages were already present in the wild-type islet grafts by day 4 post-transplantation. Although macrophages were seen in the GTKO/hCD46 islet grafts on day 4, CD4⁺ cells were not seen until the next time-point (day 8). A few CD8⁺ cells were also seen on day 8, but were not abundant until day 12.

Conclusions: The genetic modification, GTKO/hCD46, works to protect pig islet grafts from complement injury after xenotransplantation in a pig-to-mouse model. There is also a delay in cell infiltration of grafts from GTKO/hCD46 pigs when compared with those from wild-type pigs. This study demonstrated significant advantages of the transplantation of islets from GTKO/hCD46 pigs.