Expression And Function Of Peripheral Blood Tfr And Tfh Cells In Patients With Rheumatoid Arthritis

Background:Rheumatoid arthritis(RA)is a chronic,systemic autoimmune disease characterized by the inflammation of the synovial membrane,which eventually can lead to the destruction of articular cartilage and bone.It has been proved that the imbalance and overactivation of T/B lymphocytes and the production of a large number of autoantibodies are important stages in the development of RA.Follicular helper T cells(Tfh)and Follicular regulatory T cells(Tfr),two new subsets of CD4~+T cells founded in the latest researches,can participate in formation of germinal centers(GCs)and regulate B cell proliferation and differentiation,thus playing important roles in maintaining immune homeostasis.Tfh cells can promote B cells to differentiate into plasma cells to produce high affinity antibodies,while Tfr cells play an opposite regulatory role and can inhibit the function of Tfh.The imbalance between the Tfr and Tfh cells may be an vital mechanism for the production of abnormal autoantibodies such as rheumatoid factor(RF)and anti cyclic citrullinated peptide antibody(Anti CCP)in RA.At present,there are few studies about Tfr and Tfh in peripheral blood of RA.This study intends to explore the expression and function of Tfr,Tfh cells and their subsets in peripheral blood of RA patients,so as to explore the mechanism of antibody production in RA and provide new therapeutic directions for it.Objective:In order to explore the expression of Tfr,Tfh and their subsets in peripheral blood between RA patients and healthy controls,and analyze the correlation between them with indicators of disease activity,abnormal autoantibodies.And we examined the expression of IL-21,Bcl-6,Blimp-1mRNA which as the related function molecules.Methods:1.47 RA patients including 17 new RA patients and 30 treated RA patients,and 18healthy controls were enrolled in our study.The disease activity of RA patients were evaluated according to DAS28,and ESR,CRP,anti-CCP antibodies et al indicators were collected.And the peripheral blood of RA patients was collected,and the percentage of Tfr,Tfh and their subsets in CD4~+T cells was detected by flow cytometry.Tfr is thought to be a subset of FOXP3~+Treg(CD4~+CD25~+FOXP3~+)cells,and CD45RA acts as a marker to distinguish native and memory T cells,CD45RA~-cells represent memory cells.So in our study,(1)CD3~+CD4~+CD25~+CXCXR5~+FOXP3~+labeled Tfr cells,CD3~+CD4~+CD25~+CXCR5~+CD45RA~-FOXP3~+labeled memory Tfr cells.(2)CD4~+CXCR5~+cells are recognized as the phenotype of circulating Tfh cells,so in our study CD3~+CD4~+CXCR5~+CD45RA~-descrised Tfh cells.Moreover,PD-1 was labeled on Tfh cells in peripheral blood,and PD-1~+Tfh cells represent the effector memory Tfh(Tfh-EM)cells which was a functional subset of Tfh cells.In addition,according to the expression of CXCR3 and CCR6,Tfh were divided some major subsets:CXCR3~+CCR6-(Tfh1),CXCR3~-CCR6~-(Tfh2),CXCR3~-CCR6~+(Tfh17)and CXCR3~+CCR6~+cells.2.The expression of IL-21,Bcl-6 and Blimp-1mRNA were detected by RT-PCR in10 new RA patients and 7 healthy controls.Results:1.Compared to the healthy controls,the percentage of Tfr,memory Tfr cells were significantly decreased in RA group,while the percentage of Tfh,PD-1~+Tfh(Tfh-EM),Tfh17 and CXCR3~+CCR6~+cells in RA group were significantly increased(P<0.05).And the ratio of Tfr/Tfh in RA group was reduced(P<0.05).2.(1)Compared to the healthy controls,the percentage of memory Tfr,PD-1~+Tfh,Tfh17 and CXCR3~+CCR6~+cells in the new RA group was decreased(P<0.05).And the ratio of Tfr/Tfh was decreased(P<0.05).(2)Compared to the healthy controls,the percentage of Tfr and memory Tfr cells in the treated RA group was decreased(P<0.05),the percentage of PD-1~+Tfh and Tfh17 cells was increased(P<0.05).And the ratio of Tfr/Tfh in treated RA group was decreased(P<0.05).(3)Compared to the treated RA group,the percentage of Tfr,Tfh,PD-1~+Tfh,Tfh1 and CXCR3~+CCR6~+cells was increased(P<0.05).3.The level of Tfh(r=0.326,P=0.025),Tfh1(r=0.346,P=0.017)and CXCR3~+CCR6~+(r=0.363,P=0.012)cells were positively correlated with ANA.4.The level of Blimp-1 mRNA in the periphral blood of RA patients was lower than the healthy controls,the level of IL-21 and Bcl-6mRNA had no difference between two groups.Conclusion:Decreased Tfr cells and increased Tfh cells in RA lead to the imbalance of Tfr/Tfh,which may be an important pathogenesis of RA.In addition,reduced memory Tfr and increased PD-1~+Tfh cells were involved in the RA pathogenesis.Tfh subsets in the different stages of disease express differently.Moreover,Tfh17 and CXCR3~+CCR6~+subsets cells which currently has not been reported in the RA may play key pathogenic roles.The Blimp-1 can regulate the expression of Tfr and Tfh.Our results indicated Blimp-1 may play a positive regulatory role in the Tfr differentiation and a negative regulatory role in the Tfh differentiation.