In Vitro Xenoantigen Stimulated Human Tr1 Cells Enhanced Suppression of the Xenogeneic T Cell Response with Upregulated Expression of CD39

Abstract

Introduction 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. Method 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. Results 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). Conclusion 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).