Nitric Oxide from Mesenchymal Stem Cells Regulated T-Cells through Inhibiting Expression of the T-bet.

INTRODUCTION: More recently, experimental evidence and preliminary clinical studies have demonstrated that mesenchymal stem cells (MSCs) have an important immune modulatory function. Many reports have shown that MSCs regulate functions of T-cells and suppress its proliferation, but the molecular mechanisms are poorly understood. Here We found that T-bet expression in T cells is suppressed in the presence of MSCs, and nitric oxide (NO) is involved in the regulation of T-cell status and T-bet expression.

METHODS: Adult C57BL/6 mouse bone marrow MSCs were characterized by surface molecules, the typical spindle-shaped morphology, and the ability of differentiation. T-lymphocytes were isolated from the spleen of rats by using mouse CD4, CD8, and CD19 MACS beads, of which the purity determined by flow cytometry was more than 85%. Responding recipient cells (1×10 ⁵) and an equal number of irradiated (3000 cGy) donor stimulating cells or MSCs were cocultured. Cell proliferation was assessed using enzyme-linked immunosorbent assay (ELISA), in accordance with the BrdU user’s manual.

RESULTS: Flow cytometry showed that expression of the activation markers CD25 and CD69 on CD4 or CD8 T cells increased significantly in the presence of stimulating cells, and the production of IFN-and expression of its T-bet in T cells in response to allogeneic stimulation increased to 18 times and 15times, respectively. However, after 24 hours in the presence of MSCs, IFN-γ in T cells was diminished, expression of the T-bet was reduced more than 10 times, T cell proliferation was suppressed by 73–92% in response to allogeneic stimulation or in response to ConA activation. Reverse transcriptase-polymerase chain reaction and Western blot analysis showed that the expression of iNOS in MSCs cocultured with activated splenocytes increased significantly, but not in MSCs alone, or splenocytes alone. The immunofluorescence studies showed that iNOS was exclusively expressed by large adherent CD45⁻ cells, which correspond to MSCs. To further determine a functional immunosuppressive role for NO expressed by MSCs, N-nitro-L-arginine methyl ester, the NO-specific inhibitor was used. As a result, it reversed the suppression of T-cell proliferation and the T-bet expression.

CONCLUSION: Taken into account of T-bet’s functions in T cells, these results suggest that NO produced by MSCs is one of the major mediators of T-cell suppression by MSCs.