Core B: Pathology Mechanistic Core

ABSTRACT This Core provides comprehensive pathology and molecular analysis for each of the three projects. The Core will provide expert pathological and immunopathological interpretation of each multi-GE xenograft in the three Projects, critical in the evaluation of rejection and mechanisms of graft injury. The pathology techniques include standard scoring based on Banff, ISHLT and custom schemata, immunohistochemistry and immunofluorescence for cell identification, in situ hybridization and whole slide digital imaging. Bulk transcript analysis of protocol biopsies and terminal xenograft samples will be done using the Nanostring nCounter platform to evaluate the nature of the cellular infiltrate, cytokine, chemokine and other mediators, and effects on parenchymal and endothelial cells. The 770 gene set used in human allografts (Banff-Human Organ Transplant panel), has been adapted and supplemented with 35 pig specific probes, and used successfully in our NHP and human renal pig xenograft studies. Spatial transcriptomics will be applied to xenografts using the Nanostring CosMx Spatial Molecular Imager (SMI) platform. The goal is to fully characterize the interactions and mediators of the infiltrating cells, the responses of the endothelium and the expression of the human transgenes. We have used this technique to reveal new mechanisms of antibody mediated rejection in human renal allografts. We will modify the 1000-plex Human Universal Cell Characterization probe set to select those probes that are highly homologous to pig, cynomolgus and baboon genes. These will be supplemented with 50 pig specific probes for endothelium and parenchymal cells, human transgenes and probes to distinguish pig and primate cells.