*Purpose: 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 among the graft-infiltrating lymphocytes (GILs) in the cardiac xenograft.

*Methods: Genetically engineered (GE) donor pig heart was transplanted in the abdomen (heterotopically, i.e., HHXTx; (n=6) and in the chest (life-supporting orthotopically, i.e. OHXTx; (n=6)) in 3-4-year-old baboons. These GE pigs were depleted of carbohydrate antigen (GTKO, B4KO, CMAH) and GHR along with expression of human CD46, TBM, EPCR, TFPI, DAF, CD47, and HO1 genes in different combinations. Previously described immunosuppression was used which consisted of targeted T and B cell depletion, conventional anti-rejection agents like MMF and steroids, and anti-CD-40 mAb. Immuno-phenotyping on GILs from explanted xenograft was performed to analyze the percentage of Treg cells with anti-human CD3, CD4, CD25, CD127, and FoxP3 mAbs.

*Results: The recipient with HHXTX was euthanized after 120 days with a functional xenograft. Recipients with OHXTX survived up to 264 days and grafts were explanted at different timepoints. The WBC and lymphocyte counts were low in recipients after the xenotransplantation due to immunosuppression but recovered in a few days to a normal level. GILs from the explanted hearts from both HHXTx and OHXTX were examined. A small population of lymphocytes was seen in all the explanted xenografts and the percentage of CD4+CD25HiFoxP3+ Tregs was examined. We found an increased percentage of FoxP3+ Treg cells in GILs from long-term survivors who were electively euthanized. These Treg cells were also increased recipients who did not have xenograft rejection, as compared to recipients with xenograft failure/dysfunction (66.97%+8.7 vs 31.88+4.6; p = 0.0006).

*Conclusions: These results suggest that Tregs play a role in preventing xenograft rejection and help in prolonging cardiac xenograft survival. The exact mechanism of Treg help is currently under investigation in our laboratory.

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