The Effect Of PD-1/PD-L1 On CTfh And CTfr In Rheumatoid Arthritis And Its Mechanism

Objective:To study the expression of circulating follicular helper T cells,circulating follicular regulatory T cells,B cells and related molecules(s PD-1,s PD-L1,s PD-L2)in rheumatoid arthritis(RA)and the effect of PD-L1 on circulating follicular helper T cells and circulating follicular regulatory T cells and its possible mechanism.Methods:Fresh peripheral blood,serum and clinical data of patients with rheumatoid arthritis were collected,and gender and age-matched healthy persons were included as controls.Flow cytometry was used to study the frequency of follicular helper T cells(cTfh),follicular regulatory T cells(cTfr),CD3~-CD19~+B cells and CD3~-CD20~+B cells in peripheral blood of patients with rheumatoid arthritis(RA)and healthy persons.The serum levels of soluble programmed death-1(s PD-1),soluble programmed death ligand-1(s PD-L1)and soluble programmed death ligand-2(s PD-L2)in patients with rheumatoid arthritis and healthy subjects were detected by ELISA.Furthermore,CD4~+CXCR5~+T cells were selected from peripheral blood mononuclear cells of RA patients and healthy people by magnetic bead sorting method for in vitro culture,and exogenously added recombinant human PD-L1 protein to stimulate,through CCK-8,Flow cytometry,ELISA,RT-PCR to detect CD4~+CXCR5~+T cell proliferation inhibition rate,cTfh,cTfr frequency,cytokine IL-21 expression level,PI3K,AKT,Bcl-6,Blimp-1m RNA Expression level,to explore the effect of PD-1/PD-L1 on cTfh and cTfr immune disorders in RA patients and possible mechanisms.Results:1.The percentage of cTfh in CD4~+T cells in peripheral blood of RA patients was significantly increased(4.09±3.04 vs 0.55±0.51,P<0.001),and the percentage of cTfr in CD4~+T cells was also significantly increased(1.93±1.12 vs 0.50±0.28,P<0.001),the ratio of cTfr to cTfh in peripheral blood of RA patients was lower than that of HC(0.58±0.35 vs 1.31±1.15,P<0.001).2.The percentage of cTfh cells in peripheral blood CD4~+T cells of RA patients was positively correlated with the serum levels of CRP,ESR,RF,Anti-CCP,Ig G and DAS28-CRP index(r=0.470?P=0.021,r=0.528?P=0.008,r=0.681?P<0.001,r=0.522?P=0.009,r=0.418?P=0.042,r=0.452?P=0.027),while the ratio of cTfr/cTfh was negatively correlated with the serum levels of CRP,ESR,RF,Anti-CCP,Ig G and DAS28-CRP index(r=-0.456?P=0.025,r=-0.412?P=0.048,r=-0.419?P=0.042,r=-0.629?P=0.001,r=-0.410?P=0.047,r=-0.519?P=0.009).3.The expression level of serum s PD-1 in RA patients was significantly higher than that in HC patients(68.65±14.81 pg/ml vs 12.11±1.68 pg/ml,P<0.001),which was statistically significant.The serum concentrations of s PD-L1 and s PD-L2 in RA patients were not statistically different from those in HC.Moreover,the expression level of serum s PD-1 in RA patients was positively correlated with cTfh,disease activity indexes such as ESR and CRP(r=0.667?P<0.05,r=0.544?P=0.006,r=0.44?P=0.03).4.The proportion of CD3~-CD19~+B cells in peripheral blood lymphocytes of RA patients was significantly higher than that of HC,with a statistical difference(6.3±2.18 vs 3.86±1.12,P<0.05).The proportion of CD3~-CD20~+B cells in peripheral blood lymphocytes of RA patients was also significantly higher than that of HC,with a statistical difference(6.1±2.05 vs 3.62±1.21,P<0.05).5.The level of cTfh cells in peripheral blood of RA patients was positively correlated with the level of CD3~-CD19~+B cells in peripheral blood(r=0.72,P<0.05),and was positively correlated with the level of CD3~-CD20~+B cells in peripheral blood(r=0.71,P<0.05).6.Recombinant human PD-L1 protein significantly inhibited the proliferation of CD4~+CXCR5~+T cells in peripheral blood during the active phase of RA,and the inhibition increased with the increase of dose.However,the inhibitory effect of recombinant human PD-L1 on the proliferation of CD4~+CXCR5~+T cells in peripheral blood of healthy people was weak.7.Recombinant human PD-L1protein reduced the frequency of cTfh in CD4~+CXCR5~+T cultured cells in peripheral blood during the active phase of RA,while the frequency of cTfr did not change significantly.The increase of cTfr/cTfh ratio had no significant effect on the frequency of cTfh and cTfr CD4~+CXCR5~+T cultured cells in peripheral blood in healthy.8.Recombinant human PD-L1 protein reduced the level of cytokine IL-21 in superfluid of CD4~+CXCR5~+T cell culture in peripheral blood of RA at the active stage.9.In active RA peripheral blood CD4~+CXCR5~+T cell culture,the expression of PI3K,AKT and Bcl-6m RNA in the recombinant human PD-L1 protein group was decreased.There was a statistical difference.However,the change of Blimp-1 m RNA was not significant,showing no statistical difference.Conclusion:The immune imbalance of cTfr/cTfh in RA peripheral blood,and the abnormal distribution of effector molecules of cTfh cells or cTfr cells may contribute to the immune pathogenesis of rheumatoid arthritis.The ratio of cTfr/cTfh in peripheral blood of RA patients may be used as a biomarker to assess the severity of disease in RA patients.PD-1/PD-L1 may inhibit the proliferation,differentiation and activation of cTfh through the PI3K/AKT signaling pathway,regulate the immune balance between it and cTfr,and regulate the interaction between PD-1/PD-L1 may be a kind of RA Promising treatment.