A Preliminary Study On The Abnormal Phenotypies Of CD4~+CD25~+ Foxp3~+ In Vitro Induced Treg Cells Of Patients With Systemic Lupus Erythematosus

Background and objective:Systemic lupus erythematosus(SLE)is a common autoimmune disease which causes multiple tissue and organ damages.The autoimmune response mediated by T lymphocytes plays a very important role in the pathogenesis of SLE.CD4~+T lymphocytes differentiate into different subsets in different conditions or environments.Among these T subsets,regulatory T cells(Treg)may play an important role in SLE.The deficiencies of Treg in numbers and functions have been found in SLE patients and animal models,which were correlated with disease activity.The induction and function of Treg involve a series of complex mechanisms,including many cytokines and membrane surface molecules.In the previous study,our laboratory found that CD4~+CD25~+Foxp3~+iTreg induced in vitro from CD4~+CD25-T cells of SLE patients were reduced.But,it is unknown whether they have some important changes in their phenotype,including the expressions of cytokines and membrane molecules related to their functions.In the present study,our objective is to finding the differences of cytokines and membrane molecule expressed by CD4~+CD25~+Foxp3~+iTreg induced in vitro from CD4~+CD25-T cells between SLE patients and healthy controls,and to get a better understanding of the deficiency of SLE Treg.Methords:(1)Peripheral blood mononuclear cells(PBMCs)were isolated from healthy donors and SLE patients respectively,and then CD4~+CD25-T cells were further isolated by MACS separator.These CD4~+CD25-T cells were induced to differentiate into CD4~+CD25~+Foxp3~+iTreg through culturing with antibodies to CD3 and CD28,TGF-? and IL-2 in vitro.(2)ELISA was employed to examine the concentrations of IL-4 and IFN-y of the supernatants of the cell cultures.(3)Fluorescence quantitative RT-PCR was used to determine the mRNA levels of IL-2,IL-4,IL-10,TGF-? and IFN-y in these cells.(4)Flow cytometry(FCM)was applied to analyse the expressions of cytoplasmic and membrane IL-10(cIL-10 and mIL-10),membrane TGF-?(mTGF-?),LAP,PD-1,PD-L1,CTLA-4 and CD39 by CD4~+CD25~+T subset and CD4~+CD25~+Foxp3~+iTreg induced from CD4~+CD25-T cells.Results:(1)CD4~+CD25~+T subsets and CD4~+CD25~+Foxp3~+iTreg induced from CD4~+CD25-T cells of both healthy controls and SLE patients highly expressed PD-1,the differences between healthy controls and SLE patients were no significant in statistics;while the expressions of PD-L1 by CD4~+CD25~+T subset and CD4~+CD25~+Foxp3~+iTreg induced from CD4~+CD25-T cells of SLE patients were significantly lower than those of healthy controls,which were negatively correlated with SLEDAI.(2)The expressions of CTLA-4 by CD4~+CD25~+T subset and CD4~+CD25~+Foxp3~+iTreg induced from CD4~+CD25-T cells of SLE patients were also significantly reduced as compared with those of healthy controls,which were negatively correlated with SLEDAI yet.(3)The expression of TGF-? on the membrane surface(mTGF-?)of CD4~+CD25~+T subset and CD4~+CD25~+Foxp3~+iTreg induced from CD4~+CD25-T cells of SLE patients was also reduced as compared with that of healthy controls,but there was no significant difference between active and non-active patients.While,the expressions of LAP and CD39 between SLE patients and healthy controls did not show significant differences.(4)The expressions of cIL-10 by CD4~+CD25~+T subset and CD4~+CD25~+Foxp3~+iTreg induced from CD4~+CD25-T cells of SLE patients were significantly increased as compared with those of healthy controls,but they were no correlated with SLEDAI,and their expressions of mIL-10 did not show significant difference between healthy controls and SLE patients.(5)The mRNA levels of IL-2,IL-4,IL-10,TGF-? and IFN-?,and the concentrations of IL-4 and IFN-y of the supernatants of these cells also did not show any differences between healthy controls and SLE patients in statistics.Conclution:CD4~+CD25~+Foxp3~+iTreg induced in vitro from CD4~+CD25"T cells of SLE patients might have some deficiencies in the expressions of PD-L1 and CTLA-4,which might relate with the defective function of Treg and take part in the pathological process of SLE.