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 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 used immunosuppression was used which consisted of targeted T and B cell depletion and conventional anti-rejection agents along with 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, FoxP3, and Helios monoclonal antibodies.

Results and Discussion: Heterotopic and life-supporting orthotopic cardiac xenotransplantation were performed without complication. Baboons were extubated immediately following surgery and were active, eating, and generally well soon after and survived up to 264 days (HHTx). WBC and lymphocyte 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 from HHXTx and one of six from OHXTx were euthanized after 115 days and 180 days, respectively. 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 CD4+CD25HiFoxP3+ Tregs were examined. We found that increased percentage of FoxP3+ Treg cells in GILs of 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). These results suggest that Tregs prevent xenograft rejection and help in prolonging cardiac xenograft.