| Objective:(1)To investigate whether Tfh cells and Tfr cells participate in the immune disorder of IMN.(2)To investigate whether the immune-regulatory function of Tfh cells or Tfr cells is abnormal.And to explore the possible causes of the abnormal immune-regulatory function.(3)To investigate whether the overexpression of IL-21 in Tfh cells of IMN patients result in the excessive promotional function of Tfh cells in IMN patients,which leads to the over activation of humoral immunity and participates in the immune disorder of IMN.Methods:(1)41 patients with new-onset idiopathic membranous nephropathy from the nephrology department of the people’s Hospital of Xinjiang Uygur Autonomous Region during January 2020 to December 2020 were recruited as the IMN group,and 30 healthy volunteers from medical examination center of the people’s Hospital of Xinjiang Uygur Autonomous Region were recruited as healthy control group.The frequency of CD4~+CXCR5~+Foxp3~+Tfr cells,Tfr cell subsets,CD4~+CXCR5~+Foxp3~-Tfh cells were tested together with the concentrations of serum proinflammatory cytokines:IL-6,IL-17A,IL-21,serum Ig G4,24-hour total urinary protein,serum creatinine,urea nitrogen and other laboratory indexes,to explore the relationship between Tfh cells,Tfr cells and immune disorder of IMN.(2)In vitro cell coculture was used to simulate the interaction between Tfh cells and B cells in the secondary lymphoid structure in order to test the immune promotional function of Tfh cells of IMN patients.B cells of healthy donors were cocultured with the peripheral blood Tfh cells of healthy donors(group A)and IMN patients(group B),At the same time,the B cells of healthy donors(group C)and the B cells of IMN patients(group D)were cultured separately as the control group.The concentrations of Ig G4,lactic acid,IL-6,IL-17A and IL-21 in the cultured system were analyzed to assess the immune promotional function of Tfh cells of IMN patients.In vitro cell coculture was used to simulate the interaction between Tfr cells,Tfh cells and B cells in the secondary lymphoid structure in order to test the immune-suppressive function.of Tfr cells of IMN patients.Tfr cells from peripheral blood of healthy donors(group E)and Tfr cells from peripheral blood of patients with IMN(group F)were added to the coculture system composed of Tfh cells and B cells from peripheral blood of patients with IMN respectively.At the same time,a blank control(group G)was set up,in which the system only contained Tfh cells and B cells from peripheral blood of patients with IMN.The concentrations of Ig G4,lactic acid,IL-6,IL-17A and IL-21 in the cultured system were analyzed to assess the immune-suppressive function of Tfr cells of IMN patients.Further,the expression of IL-21 and PD-1 in Tfh cells and PD-1,CTLA-4and IL-2R in Tfr cells of IMN patients were analyzed by RT-PCR,in order to explore the possible causes of abnormal function of Tfh cells and Tfr cells in IMN patients.(3)In vitro cell co culture was used to explore whether the overexpression of IL-21 in Tfh cells of IMN patients led to the immune promotional hyperfunction of Tfh cells and the over activation of humoral immunity.A system composed of TFH cells of IMN patients,Tfr cells of healthy donors and B cells of IMN patients was set up as the control group(group J).The intervention measures were applied on the basis of group J:1.adding exogenous IL-21(group H),2.adding exogenous IL-21 and blocking IL-21R on B cells of IMN patients at the same time(group I).The concentrations of Ig G4,lactic acid,IL-6,IL-17A and IL-21 in the system after coculture were tested to assess whether the increased expression of IL-21 in Tfh cells of IMN patients leads to the immune promotional hyperfunction of Tfh cells in IMN patients,which leads to the over activation of humoral immunity and participates in the immune disorder of IMN.Results:(1)the frequency of Tfh cells(P<0.001)and Tfr cells(P<0.001)and the ratio of Tfr/Tfh cells(P<0.001)in peripheral blood of patients with IMN were significantly higher than those of healthy donors;Serum IL-6,IL-17A and IL-21 were significantly increased in patients with IMN(P<0.001).Serum IL-21 was positively correlated with the frequency of Tfh cells in peripheral blood(r=0.829,P<0.001);The frequency of Tfh cells in peripheral blood of patients with IMN was positively correlated with 24-hour total urinary protein(r=0.798,P<0.001);In IMN patients,the frequency of CTLA-4~+Tfr cells decreased(P<0.001)while the frequency of PD-1~+Tfr cells increased(P=0.022),and the ratio of CTLA-4~+Tfr/PD-1~+Tfr decreased(P<0.001);The ratio of CTLA-4~+Tfr/PD-1~+Tfr was negatively correlated with the concentration of serum Ig G4 in patients with IMN(r=-0.924,P<0.001).(2)The concentrations of lactic acid,Ig G4,IL-6,IL-17A and IL-21 in group B were significantly higher than those in group A,C and D(P<0.001);The concentrations of lactic acid,Ig G4,IL-6,IL-17A and IL-21 in group F were significantly higher than those in Group E(P<0.001),but lower than those in group G(P<0.001).There was a higher expression of IL-21(P<0.01)and a lower expression of PD-1(P<0.01)of Tfh cells in IMN patients.A lower expression of CTLA-4(P<0.001),IL-2R(P<0.05)and higher expression PD-1(P<0.05)were detected in Tfr cells of IMN patients.(3)The concentrations of lactate,Ig G4,IL-6 and IL-17A in group H were significantly higher than those in group I and group J(P<0.001).The concentrations of IL-6 and IL-17A in group I were higher than those in Group J(P<0.05),but the concentrations of lactate and Ig G4were lower than those in Group J(P<0.05).Conclusion:(1)Tfh cells and Tfr cells are involved in the immune disorder of IMN.(2)The immune promotional function of Tfh in IMN patients was hyperactive,which may be caused by higher expression of IL-21 or lower expression of PD-1.The immunosuppressive function of Tfr cells was impaired in patients with IMN,which may be caused by the increase of expression of PD-1 and the decrease of expression CTLA-4 or IL-2R.(3)The higher expression of IL-21 in Tfh cells of patients with IMN result in immune promotional hyperfunction of Tfh cells,which mediated Tfh cells participating in the immune disorder of IMN. |
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