Effect And Mechanism Of Novel Recombinant Human Interferon-γ And Receptor 1 Lentivirus Transgenic Mesenchymal Stem Cells On GVHD Post HSCT

Objective:Human interferon-γ(hIFN-γ)can enhance the immunosuppressive properties of mesenchymal stem cells(MSCs),mainly through specific binding to its receptor 1.In order to enable human umbilical cord stem cells(HUMSCs)to sustainably and stably enhance their immunosuppressive capacity by activating IFN-γ signaling pathway,we constructed a novel recombinant lentiviral vector containing hIFN-γand its receptor 1(hIFN-γ R1)and infected HUMSCs to explore the biological properties of transgenic HUMSCs with the membrane chimeric hIFN-γ-R1(IFN-γ-R1-HUMSCs).And to investigate the immunomodulatory effect of hIFN-γ-R1-HUMSCs on T lymphocytes in vitro and the efficacy of preventing graft-versus-host disease(GVHD)in a mouse model of hematopoietic stem cell transplantation(HSCT).Materials and Methods:1.Construction of a novel human IFN-γ-R1 lentiviral vector and identification of biological characterization of hIFN-γ-R1 transgenic HUMSCs.HIFN-γ-R1 lentiviral vector was constructed and HUMSCs overexpression hIFN-γ-R1(hIFN-γ-R1-HUMSCs)were formed by infecting HUMSCs with hIFN-γ-R1 lentivirus vectoe.The expression of IFN-γ,IFN-γ-R1 and PD-L1 on membrane of HUMSCs was detected by flow cytometry.The expression of indoleamine 2,3-dioxygenase(IDO)and PD-L1 in cells was detected by q RT-PCR.The expression of JAK1/p-JAK1,STAT1/p-STAT1 and IDO protein in JAK-STAT signaling pathway was determined by WB,and the espression of STAT3/p-STAT3,IRF1 and PD-L1 protein was detected by WB.2.Investigation of the immunosuppressive mechanism of hIFN-γ-R1-HUMSCs on T cells in vitro.(1).To investigate the effect of hIFN-γ-R1-HUMSCs on T cells proliferation and differentiation of Treg and Th17 cells in vitro.After co-culture of human or mouse T cells with hIFN-γ-R1-HUMSCs in vitro,the inhibition rates of proliferation of human or mouse T cells were detected by CCK8.The effects on the differentiation of T cells into Treg and Th17 cells were determined by flow cytometry under co-culture.(2).To explore the effects of hIFN-γ-R1-HUMSCs on human or mouse T cells PD-1 through PD-1/PD-L1 pathway in vitro.Flow cytometric detection of T cellsPD-1 expression after contact co-culture of hIFN-γ-R1-HUMSCs with human and mouse T cells.3.To investigate the preventive effects of hIFN-γ-R1-HUMSCs on a GVHD of allo-HSCT mouse model.Allo-HSCT mouse model was established using 8-10week-old male C57BL/6 mice as donors and 8-10 week-old male BALB/C mice as recipients to observe the effects of hIFN-γ-R1-HUMSCs on hematopoietic and immune functional reconstruction,a GVHD score and survival of mice after transplantation.Results:1.A novel recombinant lentivirus vector with hIFN-γ and its receptor 1 gene was successfully constructed.After infected with the IFN-γ-R1 lentivirus vector,the expression of IFN-γ,IFN-γ R1 and PD-L1 on hIFN-γ-R1-HUMSCs membrane was significantly enhanced compared with that of normal HUMSCs or HUMSCs infected with p WPXLD no-load lentivirus.This indicated that the lentivirus could successfully infect HUMSCs and possessed biological activity.The IDO and PD-L1 gene expression level was obviously higher in hIFN-γ-R1-HUMSCs group than that in control group and vector group.The protein expression levels of IFN-γ,IFN-γ-R1,JAK1/p-JAK1,STAT1/ p-STAT1,IRF-1,IDO and PD-L1 in hIFN-γ-R1-HUMSCs group were significantly higher than those in control group and vector group by WB detection.It was confirmed that hIFN-γ-R1-HUMSCs could up-regulate the gene and protein levels of IDO and PD-L1 throughIFN-γ-JAK-STAT signaling pathway.2.Study on the immunosuppressive mechanism of hIFN-γ-R1-HUMSCs in vitro.(1).Through the overexpression of IDO,hIFN-γ-R1-HUMSCs could significantly inhibit the proliferation of T cells and promote the differentiation of T cells to Treg cells,while inhibit the differentiation of T cells to Th17 cells.(2).HIFN-γ-R1-HUMSCs up-regulated the expression of its PD-L1 gene and protein through the activation of IFN-γ-JAK-STAT-IRF1 signaling pathway.In vitro experiments confirmed that hIFN-γ-R1-HUMSCs could enhance the expression of PD-1 in activated T cells through the PD-1/PD-L1 pathway and promote the immune fatigue phenotype of T cells.3.The allo-HSCT mouse model was successfully established.There was no significant diference in the engraftment of leukocyte and platelet of recipient mouses in hIFN-γ-R1-HUMSCs group.However,the percentage of peripheral blood Treg cells in mice and the overall survival rate was obviously improved,while the clinical manifestation of GVHD and GVHD score was reduced of the recipient mice in the hIFN-γ-R1-HUMSCs group compared to the control group after allo-HSCT.Conclusion:In this study,we first successfully constructed a recombinant lentivirus vector with human IFN-γ and its receptor 1 which can effectively infect HUMSCs with biological characteristics.HUMSCs transgenic with membrane chimeric hIFN-γ-R1(hIFN-γ-R1-HUMSCs)can significantly up-regulate the expression of PD-L1 and PD-1 in T lymphocytes,which may exert immunosuppressive effects through the PD-1/PD-L1 pathway.The hIFN-γ-R1-HUMSCs,through overexpression of IDO,were shown to up-regulate Treg cells and perform immunosuppressive effects in vivo and in vitro,increasing the survival rate of allogeneic transplantation recipient mice and reducing the aGVHD response.