Effects Of IL-27 And IFN-? On HPMSCs Immunoregulatory Function To Induce The Generation Of Different Regulatory T Cell Subsets

Objective:To elucidate the whether human placenta-derived mesenchymal stem cells(hPMSCs)could induce the generation of CD4+IL-10+IFN-?+T cells and balance the serum levels of IL-27,IFN-? and IL-10 in graft-versus-host disease(GVHD)mice model.Then we further studied the effects of IL-27 on hPMSCs ability to induce the generation of CD4+IL-10+IFN-?+T cells.Meanwhile,we investigate the effects of interferon(IFN)-? on hPMSCs,in particular,their adhesion,proliferation and migration and modulatory effects on the CD4+CXCR5+Foxp3+T cell subset.And we compared hPMSCs ability to induce the generation of different regulatory T cell(Treg)subsets in response to treatment with IFN-?.Methods:1.To induce humanized murine models of GVHD,non-obese diabetic/severe combined immunodeficient(NOD/SCID)mice were been used.Then hPMSCs were transfused intravenously to treat the humanized murine models of GVHD.The mice were sacrificed after hPMSCs treatment for 7 days and 14 days respectively.2.Flow cytometry(FCM)was used to detect the changes of CD4+IL-10+IFN-?+T cells in spleen and liver.3.Hematoxylin and eosin(HE)staining was used to observe the morphological changes of lung and kidney in GVHD mice before and after treatment by hPMSCs.4.The concentration of human IFN-?,IL-10 and IL-27 were determined in mice serum by ELISA.5.And FCM was used to detect ? the expression of IL-27 receptor(IL-27R)in hPMSCs,? the percentages of CD4+IL-10+IFN-?+T cells in stimulated PBMC cocultured with hPMSCs with or without IL-27 pretreated and the expression of PDL2 on hPMSCs,? the CD4+CXCR5+Foxp3+ and other Treg subsets in PBMC from hPMSCs pretreated by IFN-y or not and PBMC coculture system.6.The protein levels of p-STAT1,STAT1,p-STAT3 and STAT3 in hPMSCs were determined by Western blot.7.The adhesion,proliferation and migration of hPMSCs with IFN-y was measured by Real-Time Cell DP Analyzer in real time.Results:1.hPMSCs could induce the generation of CD4+IL-10+IFN-?+T cell subset and alleviate the symptoms of humanized murine models of GVHD(1)The results showed that the percentages of CD4+IL-10+IFN-?+T cells in PH A activated peripheral blood mononuclear cell(PBMC)significantly increased in the presence of hPMSCs.(2)Humanized murine models of GVHD was successfully constructed.(3)Besides,in humanized xenogeneic GVHD the elevation of CD4+IL-10+IFN-?+T cells in liver were greater than that in spleen with hPMSCs treatment compared with PBS group(P<0.01).(4)The levels of IL-10 in serum was increased with hPMSCs treatment for 7 days comapred with PBS control group(P<0.01).However,the levels of IL-27 and IFN-? were decreased(P<0.01).After treating by hPMSCs for 14 days,the levels of these cytokines were futher changed(P<0.01).(5)Histologically,the morphological changes of liver and kidney in hPMSCs treatment group were improved compared with PBS control group.(6)Compared PBS control group,in hPMSCs treatment group the survival time was remarkably prolonged(P<0.001),the clinical score was significantly reduced(P<0.001),and the weight of mice was gradually restored(P<0.001).2.hPMSCs ability to induce the generation of CD4+IL-10+IFN-?+T cell subset could be enhanced by IL-27(1)The FCM results demonstrated that IL-27R was expressed in hPMSCs.(2)Compared with hPMSCs group,the percent of CD4+IL-10+IFN-?+T cells from PBMC was significant increased in IL-27 pretreated hPMSCs group.(3)IL-27 increased PDL2 expression levels in hPMSCs via the JAK/STAT pathway.3.Effects of IFN-? on hPMSCs adhesion,proliferation,migration and their ability to induce different Treg subsets(1)The cell index of proliferation and migration for hPMSCs was significantly decreased after 64 and 20 hours respectively in presence of IFN-?(10 ng/ml,20 ng/ml or 30 ng/ml)while the adhesion of hPMSCs was not influenced by IFN-y.(3)And percentages of CD4+CXCR5+Foxp3+Treg subsets from activated PBMC were increased in presence of hPMSCs and the increase could be enhanced by IFN-?.(4)Besides,the percentages of CD4+CXCR5+Foxp3+Treg subset increased fastest from activated PBMC in presence of hPMSCs preteated by IFN-y compared with CD4+CD25+Foxp3+,CD8+CD25+Foxp3+,CD4+IL-10+ and CD8+IL-10+ Treg subsets.Conclusion:1.The symptoms in humanized xenogeneic GVHD(xeno-GVHD)NOD/SCID model could be alleviated by hPMSCs through upregulating CD4+IL-10+IFN-?+T cells in the spleen and liver and balancing the serum levels of IL-10,IFN-y and IL-27.2.IL-27 increased PDL2 expression levels in hPMSCs via the JAK/STAT pathway,which then enhanced the ability of hPMSCs to induce the generation of CD4+IL-10+IFN-?+T cells from stimulated PBMC.3.IFN-y suppressed the proliferation and migration of hPMSCs.Besides hPMSCs ability to induce the generation of different Treg subsets could be enhanced by IFN-y.And maximal effectiveness of IFN-y treated hPMSCs upon inducing the generation of Treg subsets was for CD4+CXCR5+Foxp3+Treg subset as compared with that of CD4+CD25+Foxp3+,CD8+CD25+Foxp3+,CD4+IL-10+ and CD8+IL-10+Treg subsets.