Background: Peripheral blood derived Treg can be cultured and trained as xenoantigen specific Treg which is a promising way for xenotransplantation. However an abnormal quantity and/or quality of regulatory T cells were reported in some autoimmune diseases such as T1D. Thus using an alternative source and selecting Treg-friendly drugs may supply a efficacy immunosuppressive regimen for islet xenotransplantation. This study aims to give experimental evidence regarding the influence of current using immunosuppressive drugs on xenoantigen-stimulated cord blood derived Treg. 
Methods: Human cord blood derived CD4+CD25+CD127low Treg were cultured with IL-2, rapamycin and irradiated porcine PBMC as xenoantigen stimulation for 2 weeks. The effects of major ISDs as tacrolimus (TAC), mycophenolate (MPA) and new ISD beletacept (BEL) and the mixture of three ISDs (ISDs mix) on xenospecific Treg were test to analysis the cell viability, phenotype, proliferation, function, stability and metabolism.
Results: After two weeks induction, xenoantigen stimulated cord blood derived-Tregs (Xn-Treg) have enhanced suppressive capacity in xeno MLR and higher expression of chemokine receptor CCR4, CCR6, CXCR3 and CCR7 compared to polyclonal Treg (Pc-Treg). Further the expression of function molecular CD39, CD73, ICOS, GITR, CTLA-4 and IDO was also upregulated compared to Pc-Treg which confirmed the function of Xn-Treg. Mycophenolate have a negative impact on viability and proliferative capacity of Xn-Treg in a dose-dependent manner. however, BEL and TAC did not affect the viability and proliferative capacity of xenospecific Treg severely. No matter Treg were treated with ISD alone or the mixture of ISDs, the immunosuppressive treatments have no effect on the expression of Treg functional molecules but affected chemokine receptors. From the OCR assay, MPA and ISDs mix significantly increased the maximal respiratory capacity and mitochondrial mass of Xn-Treg compared to the control group. While TAC and BEL did not have any change. For xeno-MLR assays, TAC and MPA did not have any negative effect on the suppressive capacity of Xn-Treg but rather BEL strengthened it at the ratio of responder :Treg 64:1. The results of CBA assay showed when activated CD25-responder cells co-cultured with Treg and ISDs, BEL and Treg have synergetic effect to reduction of IL6, TNF-α, IL17A and IFN-γ which explained why BEL could strengthen the suppressive ability of Treg.
Conclusion: Our results suggested xenoantigen-stimulated UCB-Treg show enhanced suppressive capacity in the pig-human MLR and different immune modulatory drugs have different influence on Xn-Treg. We speculate that a low dose combination of ISDs would be better able to support Treg while adequately preventing rejection while minimizing toxicity.