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Abstract
BackgroundMesenchymal stem cells (MSCs) are known for their ability to induce the conversion of conventional T cells (Tconvs) into induced regulatory T cells (iTregs) in specific inflammatory contexts. Stable Foxp3 expression plays a major role in the phenotypic and functional stability of iTregs. However, how MSCs induce stable Foxp3 expression remains unknown.MethodsWe first investigated the role of cell–cell contact and cytokine secretion by bone marrow-derived MSCs (BM-MSCs) on the induction, stability, and suppressive functions of Tregs under various experimental conditions that lead to Foxp3 generation by flow cytometry and ELISA respectively. Second, we studied the effect of MSCs on TRAF6, GRAIL, USP7, STUB1, and UBC13 mRNA expression in CD4+ T cells in correlation with the suppressive function of iTregs by real-time PCR; also, we investigated Foxp3 Treg-specific demethylated region (TSDR) methylation in correlation with Foxp3 stability by the high-resolution melting technique. Third, we studied the effect of ex-vivo-expanded BM-MSCs on the induction of transplant tolerance in a model of fully allogeneic skin transplantation. We further analyzed the cytokine secretion patterns in grafted mice as well as the mRNA expression of ubiquitination genes in CD4+ T cells collected from the spleens of protected mice.ResultsWe found that in-vitro MSC-induced Tregs express high mRNA levels of ubiquitination genes such as TRAF6, GRAIL, and USP7 and low levels of STUB1. Moreover, they have enhanced TSDR demethylation. Infusion of MSCs in a murine model of allogeneic skin transplantation prolonged allograft survival. When CD4+ T cells were harvested from the spleens of grafted mice, we observed that mRNA expression of the Foxp3 gene was elevated. Furthermore, Foxp3 mRNA expression was associated with increased TRAF6, GRAIL, UBC13, and USP7 and decreased STUB1 mRNA levels compared with the levels observed in vitro.ConclusionsOur data suggest a possible ubiquitination mechanism by which MSCs convert Tconvs to suppressive and stable iTregs.
Highlights
Mesenchymal stem cells (MSCs) are known for their ability to induce the conversion of conventional T cells (Tconvs) into induced regulatory T cells in specific inflammatory contexts
The Foxp3 mRNA levels in the cells were measured by RT-PCR at 6 h, 12 h, 24 h, 48 h, 72 h, and 5 days of coculture and compared with the levels of these mRNAs in Induced regulatory T cell (iTreg) obtained by classical in-vitro T-cell activation in the presence of TGF-β and IL-2 for 5 days and with those of CD4+CD25− T cells isolated from allogeneic mixed lymphocyte reaction (MLR) after 5 days of culture
We show for the first time that MSC-iTreg generation is associated with changes in the mRNA expression of ubiquitination genes (TRAF6, Gene related to anergy in lymphocytes (GRAIL), Ubiquitin-specific protease 7 (USP7), Ubiquitin-conjugating enzyme 13 (UBC13), and STIP1 homology and U-box-containing protein 1 (STUB1)) that are involved in the suppressive phenotype of Tregs obtained from CD4+CD25− conventional T cells
Summary
Mesenchymal stem cells (MSCs) are known for their ability to induce the conversion of conventional T cells (Tconvs) into induced regulatory T cells (iTregs) in specific inflammatory contexts. The maturation, function, and differentiation of DCs are affected by MSCs through the reduction in the surface membrane expression of MHC class II costimulatory molecules and reduced secretion of IL-12 and tumor necrosis factor alpha (TNF-α) [19]. We and others reported that MSC-induced DCs express ILT3, ILT4, PDL-1, and IDO, molecules that enhance the differentiation of suppressive Tregs [18, 38,39,40]. These tDCs can induce or enhance the suppressive function of existing Tregs and convert activated T cells into induced Tregs [36]
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