PD-L1 Overexpression Enhances The Immunosuppressive Effect Of Mesenchymal Stem Cells On T Lymphocytes And Its Regulatory Mechanisms

BackgroundAcute graft-versus-host disease(aGVHD)is one of the severe complications post allogeneic stem cell transplantation(allo-HSCT).It usually causes the morbidity and mortality of the receipients.Published results of clinical trials indicated mesenchymal stem cells(MSC)might play an important role in preventing and treating aGVHD.However,the clinical use of MSC is still under debate.New methods are needed to improve the immunomodulatory capacity of MSC in aGVHD.ObjectiveThe aim of this study is to investigate whether programmed death-ligand 1(PD-L1)gene manipulation can enhance the immunosuppressive effect of MSC on immune effector cells and to provide basic research evidence for further clinical application in aGVHD prevention and treatment.Methods and results1.MSC culturing and identification.The human umbilical cord MSC we used in this study is spindle-shaped adherent cells that arranged in swirl during ex vivo culture;flow cytometry analysis showed this less than 2%of the cells express CD11b?CD19?CD34?CD45 or HLA-DR,more than 99%of the cells express CD73?CD90 and CD 105;adipogenesis potential of this MSC is confirmed by Oil Red O staining,osteogenesis potential is confirmed by quantative PCR of osteogenic markers RUNX2 and Osteocalcin(OC)?2.The influence of PD-L1 overexpression on MSC immunoregulatory function.PD-L1 gene is manipulated in MSC by using adenovirus,an overexpression of PD-L1 is confirmed by flow cytometry analysis;cell couting kit-8 and flow cytometry analysis revealed the overexpression of PD-L1 do not alter the cell morphology?proliferation?apoptosis level and cell surface markers of MSC;phytohemagglutinin(PHA)was used to stimulate human peripheral blood mononuclear cells(PBMC),MSC and PBMC were cocultured,T lymphocyte proliferation was analysed by CFSE staining assey and T lymphocyte activating markers CD25?CD69 were evaluated by flow cytometry,we found?when the ratio of MSC and PBMC was 1:5 or 1:10,in PD-L1 overexpression MSC coculture group,the proliferation of CD3+T lymphocyte and CD3+CD8+T lymphocyte were reduced significantly(1:5,CD3+T lymphocyte number 486 vs 310 p=0.007,CD3+CD8+T lymphocyte number 249 vs 102 p=0.001,CD3+T lymphocyte proliferation index 1.72 vs 1.10 p=0.005,CD3+CD8+T lymphocyte proliferation index 2.37 vs 1.26 p=0.005;1:10,CD3+T lymphocyte number 1015 vs 671 p=0.014,CD3+CD8+T lymphocyte number 521 vs 245 p=0.002,CD3+T lymphocyte proliferation index 1.16 vs 1.05 p=0.005,CD3+CD8+T lymphocyte proliferation index 1.95 vs 1.26 p=0.002)?when the ratio of MSC and PBMC was 1:10,in PD-L1 overexpression MSC coculture group,CD3+CD8+T lymphocyte activation was reduced significantly(CD25+CD3+CD8+T lymphocyte number 13 vs 4,p=0.019,percent of CD25 positive cells in CD3+CD8+T lymphocyte 4.07%vs 1.13%,p=0.039).3.Expression regulating mechanism of PD-L1 in human umbilical cord MSC.qPCR revealed a significant expression increase of PD-L1 after IFN y preconditioning,IRF1?STAT3n NF?B and A20 expression were upregulated simultaneously,among them IRF1 changed the most(more than 70 folds);to investigate whether IRF1 participate in regulating the expression of PD-L1 in MSC,IRF1 were knocked down by siRNA,qPCR revealed a significant simultaneous decrease of PD-L1 and it decreased the most under the condition of 150nM siRNA and 30ng/ml IFN ? for 20h(IRF11 vs 0.68 p<0.001,PD-L11 vs 0.53 p=0.001);on the other hand,we overexpressed IRF1 by using lentivirus,qPCR revealed a significant simultaneous up regulation of PD-L1(IRF1 1 vs 30.80 p=0.018,PD-L1 1 vs 10.87 p=0.009).Conclusions1.The human umbilical cord MSC used in this study is well cultured and identified according to ISCT criteria;2.PD-L1 gene manipulation enhanced the immunosuppressive effect of MSC on T lymphocyte activation and proliferation;3.IRF1 participate in regulating the expression of PD-L1 in human umbilical cord MSC.