Mesenchymal stromal cells exert rapid immunoregulation functions on pediatric steriod-resistant aGVHD patients through TCR-Ca2+signalling

Abstract

Background & Aim Severe acute graft-versus-host disease (GVHD) after transplantation is associated with a high mortality rate and is a major threat to a successful outcome. Mesenchymal stromal cells (MSC) has been reported recently as a promising treatment for severe steriod-resistant acute graft-versus-host disease. MSCs have shown great promise in preclinical and clinical therapies for severe acute condition within hours, such as septic shock and acute lung injury. Here, we demonstrated that MSCs could able to rapidly reduce the expressions of TNF-α and IFN-γ in activated T cells in vitro and in vivo, mainly by a rapid suppression on their TCR signaling. Methods, Results & Conclusion Methods 1.Isolation of BMSCs from donors and cultured for the functional experiments. 2.Processing of peripheral blood cells and plasma from healthy donors and pediatric aGVHD patients, which were used for Real-Time qRT-PCR and Elisa assays to measure the levels of TNFα and IFN-γ. 3.Western blotting Analysis and Phospho-epitope Staining for Flow Cytometry were used to test the levels of TCR. 4.Measurements of Ca2+concentrations in responding T cells throgh Microscope and Flow Cytometry by using a Ca2+ indicator- Fluo-4 AM. Results MSCs rapidly suppressed the expressions of TNF-α and IFN-γ, TCR-proximal signaling and Ca2+ signaling of T cells in vitro. For aGVHD patients, we observed, after MSC infusion, TNF-α and IFN-γ at mRNA levels of T cells were decreased in 6 of 10 (more than half) patients, 2 patients without significant change and 2 patients increased a little. These matched up the Ca2+ level of T cells and TCR-proximal signaling reductions of these patients. Conclusion Using T/MSCs co-culture system, we found that MSCs were able to downregulate the TNF-α and IFN-γ of activated T cells within 1 hour. Also, MSCs were observed to rapidly suppress the TCR signaling. The alterations from aGVHD patient, which received MSCs infusion, further confirmed these MSCs-mediated short-term effects on T cells in vivo. This might make it more understandable for the feasibility of MSCs to control the severe acute immune disorders, such as septic shock and acute lung injury.