Mechanistic insights from transcript analysis of long-term pig to non-human primate kidney xenografts

Porcine xenografts are a potential future organ source. Limited short-term clinical studies have suggested different mechanisms in xenografts than allografts. Here, we sought further insights from transcript analysis of xenografts in non-human primates that achieved up to two years of survival.

Transcript analysis was performed on 58 samples from eGenesis pig kidney xenografts in Cynomolgus monkeys, including donor, post-perfusion, protocol, and terminal samples, using the Banff Human Organ Transplant panel, supplemented with 35 pig specific probes. Glomerular regions of interest in donor and terminal samples were analyzed using the GeoMx Immune Pathways Panel and 10 custom probes. Antibodies that distinguish donor and recipient molecules were used to confirm protein expression.

Protocol biopsies with no histologic evidence of rejection had a marked rise in CD45 transcripts compared with donor samples, similar to CD45 levels in stable human allografts that did not portend rejection. Terminal samples showed increases in antibody and T cell mediated rejection gene sets and prominence of alternatively activated macrophages. Reduction of VEGFA transcripts correlated with transplant glomerulopathy and glomerular thrombotic microangiopathy and was localized to glomeruli by spatial transcriptomics. Loss of donor and gain of recipient endothelial transcripts were detected in the graft, with widespread expression of recipient PECAM1, vWF, and MHC class I and II proteins in xenograft endothelium.

Marked transcript elevation occurred in stable grafts probably due to repopulation by recipient leukocytes. VEGFA loss in podocytes preceded and potentially contributed to thrombotic microangiopathy. Loss of donor endothelial transcripts contrasts with antibody mediated rejection in allografts. Unexpected gain of recipient endothelial transcripts and proteins was shown, a novel process detected in the xenograft.