Interferon-? Promotes Immunomodulatory Of Adipose Tissue-derived Mesenchymal Stem Cells On Peripheral Blood Lymphocytes

Objective:Mastering the extraction and culture methods of autologous adipose tissue-derivedmesenchymalstemcells(ADSCs).Toinvestigatethe immunomodulatory effects and the mechanism of interferon-?(IFN-?)-pretreated adult autologous ADSCs on peripheral blood lymphocytes.Methods:In this study,ADSCs were collected from healthy volunteers and cultured to the third generation,observed the morphology of the cells and identified the cell surface differentiation antigens by flow cytometry,adipogenic and osteoblast differentiation experiments were carried out to identified ADSCs.Isolated peripheral blood mononuclear cells(PBMCs)and co-cultured with ADSCs for 5 days in presence of concomitant phytohemagglutinin(PHA)/interleukin(IL)-2 stimulation.This study was divided into three groups.IFN-?-pretreated group was IFN-?-induced ADSCs group,IFN-?-unpretreated group was without IFN-?-induced ADSCs,IDO blocking group was added 1-methyltryptophan(1-MT)to ADSCs induced by IFN-?,In the control group,PHA and IL-2 were used to stimulate T cells.Each group was cultured with autologous peripheral blood mononuclear cells(PBMCs)at different concentration ADSCs-to-PBMCs ratios,and then measured the absorbance of T cell proliferation by MTT,thus the inhibition rate of T cell proliferation was calculated.Using flow cytometry detected the percentage of CD4~+CD25~+regulatory T cells(Treg).Detected the changes of IDO gene and protein levels after IFN-?-induced ADSCs.Results:The morphology of ADSCs was typical fusiform and adherent growth after 3generations.The positive rate of CD73,CD90 and CD105 was more than 95%and the positive rate of CD45 was low.Flow cytometry identification of surface markers conforms to international standards.ADSCs had the ability to differentiate into adipogenic and osteoblast.The proportion of ADSCs and lymphocyte at1:50,1:10,1:5,the T cell proliferation inhibition rate respectively were(20.25±3.10)%,(52.75±4.85)%,(65.75±5.12)%.In the IFN-?-pretreated group,the proportion of ADSCs and lymphocyte at 1:50,1:10,1:5,the T cell proliferation inhibition rate respectively were(20.25±3.10)%,(52.75±4.85)%,(65.75±5.12)%.There was a dose-dependent relationship between the inhibition rate of T cell proliferation and the proportion of ADSCs and lymphocyte.IFN-?-pretreated group significantly increased the proliferation inhibition rate of activated T cells,the percentage of CD4~+CD25~+Treg,and the expression levels of IDO mRNA and protein as compared with IFN-?-unpretreated group.However,the IDO-blocker,1-methyl-tryptophan(1-MT),significantly increased activated T cells proliferation ability as compared with IFN-?-pretreated group(P<0.01).Conclusion:In a dose-dependent manner,the inhibition rate of ADSCs on the proliferation of activated T cells was significantly increased with the decrease of co-culture ratio of ADSCs and T cells.After pretreatment with IFN-?,it was found that IFN-?stimulation significantly enhanced the negative regulatory effect of ADSCs.The ability of ADSCs to induce the transformation of activated T cells into CD4~+CD25~+Treg subsets was enhanced after ADSCs was pretreated with IFN-?.The results showed that the expression of IDO mRNA was affected by IFN-?and IFN-?could promote the expression of IDO mRNA.IFN-?also promoted the expression of IDO protein at protein level.1-MT can inhibit the immunosuppression mediated by ADSCs to a great extent.In conclusion,IFN-?promotes the immunosuppressive ability of ADSCs on activated T cells.IDO plays an important role in ADSCs mediated immunosuppression.