Ex vivo–induced bone marrow-derived myeloid suppressor cells prevent corneal allograft rejection in mice

Abstract

PURPOSE. To investigate the effects of ex vivo–induced bone marrow myeloid-derived suppressor cells (BM-MDSCs) on allogeneic immune responses in corneal transplantation. METHODS. Bone marrow cells from C57BL/6J (B6) mice were cultured with IL-6 and GM-CSF for four days. The ex vivo induction of the BM-MDSCs was assessed using flow cytometry, inducible nitric oxide synthase (iNOS) mRNA expression using reverse transcription–quantitative polymerase chain reaction, and nitric oxide (NO) production in allogeneic stimulation. T-cell proliferation and regulatory T-cell (Treg) expansion were investigated on allogeneic stimulation in the presence of ex vivo–induced BM-MDSCs. IFN-γ , IL-2, IL-10, and TGF-β1 protein levels were measured using enzyme-linked immunosorbent assays. After subconjunctival injection of ex vivo–induced BM-MDSCs, the migration of the BM-MDSCs into corneal grafts, allogeneic corneal graft survival, neovascularization, and lymphangiogenesis were assessed using flow cytometry, slit-lamp microscopy, and immunohistochemistry. RESULTS. The combination of GM-CSF and IL-6 significantly induced BM-MDSCs with increased iNos mRNA expression. The ex vivo–induced BM-MDSCs promoted NO release in allogeneic stimulation in vitro. The ex vivo–induced BM-MDSCs inhibited T-cell proliferation and promoted Treg expansion. Decreased IFN-γ and increased IL-2, IL-10, and TGF-β1 production was observed in coculture of ex vivo–induced BM-MDSCs. Injected ex vivo–induced BM-MDSCs were confirmed to migrate into the grafts. The injected BM-MDSCs also prolonged corneal graft survival and prevented angiogenesis and lymphangiogenesis. CONCLUSIONS. The ex vivo–induced BM-MDSCs have suppressive effects on allogeneic immune responses and prolong corneal allograft survival via the iNOS pathway, indicating that they may be a potential therapeutic tool for corneal transplantation.