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Life-long immunosuppression is required to prevent xenograft rejection. Induction of antigen specific tolerance by cell therapy with regulatory T-cells (Tregs) represents an attractive alternative approach. T regulatory type 1 (Tr1) cells can be generated from naive T cells in the presence of interleukin-10 (IL-10) and represent one promising therapeutic choice. This study aimed to expand xenoantigen-specific human Tr1 cells for immunotherapy in xenotransplantation.Human naïve CD4+ T cells isolated from peripheral blood mononuclear cells (PBMC) were stimulated with anti-CD3/CD28 beads and IL-2 in the presence of rhIL-15 and rhIL-10 for 16 days as polyclonal expansion Tr1 cells (PC-1). Naïve CD4+ T cells were expanded with two cycles (7 days for each cycle) of xenoantigen stimulation with irradiated porcine PBMC, anti-CD3/CD28 beads and IL-2 in the presence of rhIL-15 and rhIL-10 (XN-1). Tr1 cells phenotype and their suppressive capacity were assessed after two cycles of stimulation.After two cycles of stimulation, Tr1 cells both from polyclonal and xenoantigen stimulation upregulated expression of CTLA-4, HLA-DR, CD49b, LAG-3 and CD39 but did not express Foxp3. Interestingly, Tr1 cells stimulated with polyclonal or xenoantigen both showed memory/effector Treg phenotype markers (CD45RO+CCR7-). In the subset of CD45RO+CCR7- Tr1 cells, xenoantigen stimulation led to expression more CD39 than polyclonal stimulation (Figure 1).Moreover, xenoantigen stimulation led to upregulate the expression of IL-10, IL-5, and GITR, showed 2.51-fold, 2.53-fold and 1.63-fold more than polyclonally expanded Tr1 cells, respectively. An 1.41-fold, 1.96-fold and 3.06-fold increased expression in TCR Vβ2, Vβ9 and Vβ13 in xenoantigen stimulation Tr1 cells compared to polyclonal stimulation (Figure 2).In addition, xenoantigen stimulated Tr1 cells secreted more IL-10 with enhanced suppressive capacity in xeno-stimulated MLR (Figure 3).This study shows a feasible strategy to obtain human functional xenoantigen-stimulated Tr1 cells for immunomodulation in xenotransplantation by minimizing systemic immunnosuppression.National Natural Science Foundation of China (No. 81501602).