Preliminary Study On The Mechanisms Of Defective Formation Of Induced Regulatory T Cells Of Systemic Lupus Erythematosus Patients

Background and objective:Systemic lupus erythematosus(SLE)is an autoimmune disease which can cause multiple tissues and organs damage.The immune regulation of SLE patients is dysfunctional.Regulatory T cells(Treg)play an important role in maintaining immune tolerance and regulating the immune response.Some researches reported that both of the function and the quantity of Treg in SLE patients are abnormal.IL-2 and TGF-?are key cytokines in promoting the formation of induced Treg(iTreg).It has been confirmed that the abnormal expression of IL-2and its receptor is closely related to the pathogenesis of SLE.The thymus-derived natural Treg(nTreg)has been widely studied in SLE.However,the factors which influence on the development of iTreg in peripheral blood of SLE patients are poorly elucidated.The aim of the present study is to assess the expressions of IL-2 receptor?(CD122)and?_c(CD132)chains on CD4~+T cells in peripheral blood of SLE patients,and to analyze the effect of IL-2 receptor deficiency on the induction of Foxp3~+iTreg,for a better understanding for the molecular mechanisms of peripheral immune dysfunction in SLE.Method:(1)Peripheral blood mononuclear cells(PBMCs)were isolated from health control and SLE or rheumatoid arthritis(RA)patients,and then further sorted into CD4~+CD25~-T cells by MACS separator;(2)CD4~+CD25~-T cells isolated from PBMCs were induced into CD25~+Foxp3~+iTreg through culture with TGF-?and IL-2in vitro,the ratio of CD25~+Foxp3~+/CD4~+was analyzed by flow cytometry(FCM);(3)PBMCs were analyzed for the proportion of CD25~-CD122(IL-2R?chain)~+and CD25~-CD132(?_c chain)~+T cells in CD4~+T cells by FCM;The relative abundance of CD122 and CD132 mRNA in CD4~+CD25~-T cells were analyzed by fluorescence quantitative PCR.Then,detected the expressions of CD122 and CD132 on the surface of CD4~+CD25~+induced regulatory T cells by FCM;(4)CD4~+CD25~+iTreg derived from CD4~+CD25~-T cells by culture in vitro were detected for the phosphorylation levels of Stat5 and the relative abundance of SMAD3 and SOCS3mRNA;(5)The?_c gene of CD4~+CD25~-T cells from health control was inhibited by siRNA,and then the expressions of Foxp3 and pStat5 were detected after culture with TGF-?and IL-2 for inducing CD4~+CD25~+T cells.Results:(1)The proportion of CD25~+Foxp3~+T cells in the cells which had been induced from CD4~+CD25~-T cells by culture with TGF-?and IL-2 was significantly lower in SLE patients than that in the control group or in RA patients,and negatively correlated with SLEDAI.There was no abnormality in CD25~+Foxp3~+T cell induction in RA patients,compared with health control;(2)In SLE patients,compared with the health control,the ratio of CD25~-CD132~+/CD4~+was lower and negatively correlated with SLEDAI,although there was no significant change in the expression of CD122on CD4~+CD25~-T cells;Furthermore,the relative abundance of CD132 mRNA was lower.After culture with TGF-?and IL-2 for inducing iTreg,the ratios of CD25~+CD122~+/CD4~+and CD25~+CD132~+/CD4~+of induced cells in SLE patients were lower than those in control group and RA patients,but only the latter was negatively correlated with SLEDAI and positively correlated with the expression of Foxp3;(3)The expression of phosphated Stat5(pStat5)in CD25~+T cells which were induced from the CD4~+CD25~-T cells was lower than that in the control group;The ratio of pStat5~+/CD4~+CD25~+was negatively correlated with SLEDAI and positively correlated with the ratio of CD25~+Foxp3~+/CD4~+.There were no differences in the relative abundance of SMAD3 and SOCS3 mRNA in the induced CD4~+CD25~+T cells between SLE and control groups;(4)The expression of CD132 on CD4~+CD25~-T cells was reduced significantly after it was functioned by siRNA,the expression of pStat5 in the induced CD4~+CD25~+T cells was also significantly reduced and the proportion of induced CD4~+CD25~+Foxp3~+iTreg was also lower than that in the control group.Conclusion:The expression of CD132(?_c chain)on CD4~+CD25~-T cells in SLE patients was significantly defective,resulting in the impairment of intracellular IL-2receptor signaling pathway and the reduction of Stat5 phosphorylation levels,furthermore leading to the defective expression of Foxp3,affecting the conversion from CD4~+CD25~-T cells into Foxp3~+iTreg.This immunological abnormity is closely related with the pathogenesis of SLE,while no related with RA.