Mesenchymal Stem Cells Generate De Novo Functional CD4+CD25+CD127- Regulatory T Cells with Highly Methylated FOXP3 DNA

Abstract

Introduction: The ability of mesenchymal stem cells (MSC) to apply immunosuppression makes them interesting candidates for cellular therapy in solid organ transplant recipients. MSC exert their function through the inhibition of effector T cell proliferation. In animal models, MSC treatment led to an increase of CD4+CD25+CD127-FOXP3+ regulatory T cells (Treg). The present study aimed to elucidate the cell population of origin for Treg induction by MSC. Methods: MSC were isolated from perirenal fat tissue of kidney donors. PBMC were derived from blood bank donors; CD25- cells and natural Treg (nTreg) were obtained by cell separation. Induction of Treg was achieved through allogeneic stimulation of CD25- effector cells in the presence of MSC. CD4+CD25- T cells and CD4+CD25+CD127- T cells were sorted and analyzed; the functionality was tested in secondary mixed lymphocyte reactions (MLR) and the methylation status of the FOXP3 gene's Treg-specific demethylated region (TSDR) was determined. Results: In the presence of MSC a significant induction of CD25+CD127- FOXP3+ cells within the CD4+ T cell population originating from CD25- effector cells was observed (p=0.001). These de novo CD4+CD25+CD127-FOXP3+ T cells inhibited effector T cell proliferation as effectively as nTreg (62% [p=0.007] and 48%, [p=0.003], respectively); the CD4+CD25- T cells had no suppressive effect on T cell proliferation. Both CD4+CD25+CD127-FOXP3+ T cells and CD4+CD25- T cells had a highly methylated TSDR (93% and 99%, respectively). This indicates that the newly induced CD4+CD25+CD127-FOXP3+ T cells are of CD25- origin and distinct from nTreg, which have a highly demethylated TSDR. Conclusion: This study demonstrates that MSC contribute to the generation of an immunosuppressive environment not solely through the inhibition of allo-activated effector T cells, but also through the induction of de novo CD4+CD25+CD127-FOXP3+ Treg from CD25- cells which by themselves possess repressive capacities. This apparent diversity of MSC function emphasizes the potential of MSC immunotherapy in solid organ transplantation.