Presence of graft-infiltrating regulatory T cells are associated with long term cardiac xenograft survival in non-human primate
Avneesh Singh1, Corbin E. Goerlich1, Gheorghe Braileanu1, Alena Hershfeld1, Tianshu Zhang1, Ivan Tatarov1, Billeta Lewis1, Faith Sentz1, David Ayares2, David Kaczorowski1, Bartley P. Griffith1, Muhammad Mohiuddin1.
1Surgery, SOM, University of Maryland Baltimore, Baltimore, MD, United States; 2Revivicor Inc., Blacksburg, VA, United States
Introduction: Regulatory T (CD4+CD25HiFoxP3+) cells (Treg) are a very small subset of CD4+ T cells that have been shown to play an important role in immune regulation and induce tolerance in transplantation. Previously we have reported the increased number of Treg cells in peripheral blood of long term cardiac xenograft survival recipients. However, in both kidney and liver transplantation in humans, FoxP3+ Tregs have also been associated with clinical rejection. Therefore, the role and function of graft infiltrating Tregs have been of great interest. In this study, we have examined the FoxP3+ Tregs in graft-infiltrating lymphocytes (GILS) from cardiac xenograft.
Materials and Methods: Genetically engineered (GE) donor pig heart was heterotopically transplanted in 3-4-year-old specific pathogen-free baboon (n=6). These GE pigs were alpha 1-3 galactosidase knockout and had multiple combinations of transgenic expression of human complement inhibitory protein (CD46), thrombomodulin (TBM), endothelial protein C receptor (EPCR), tissue factor pathway inhibitor (TFPI), and decay-accelerating factor (DAF) and protective gene against phagocytosis (CD47). Immunosuppression consisted of targeted T and B cell depletion and conventional anti-rejection agents. Immuno-phenotyping on GILS from explanted xenograft was performed to analyze the percentage of Treg cells with anti-human CD3, CD4, CD25, CD127, FoxP3, and Helios monoclonal antibodies.
Results and Discussion: Cardiac xenotransplantation were performed without complication. Baboons were extubated immediately following surgery and were active, eating, and generally well soon after. WBC and lymphocytes counts were low in recipients after the xenotransplantation due to immunosuppression but recovered in a few days to a normal level. Three of six cardiac xenografts were explanted after 115 days of cardiac xenotransplantation and GILS were examined. A small population of lymphocytes was seen in all the explanted xenograft and CD4+CD25HiFoxP3+ Tregs were examined.
Conclusion: We found that long term survivors had an increased percentage of FoxP3+ Treg cells compared to the GILS in cardiac xenografts with earlier graft failure. Tregs were not identified in GILS from recipients with early graft failure. The presence of Tregs was associated with improved graft function. This suggests a TReg-dependent mechanism of survival for xenografts and supports future experiments investigating this mechanism to bring xenotransplantation to clinical reality.
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