T Follicular Helper Cells Enhance IgA Class Switching And Gd-IgA1Generation In Children With IgAN

Part Ⅰ. Tfh and Expression of Tfh-related Cytokines of IL-21, TGFβ1and mIgA in Peripheral Blood in IgAN PatientsObjective To investigate Tfh and the expression of Tfh-related cytokines of IL-21and TGF β1and mIgA from peripheral blood in IgAN patients. Methods Tfh in CD4+lymphocytes and mIgA in CD19+lymphocytes from peripheral blood were quantitated by flow cytometry in immunosuppressive agent-treated or untreated IgAN children and healthy controls. Meanwhile, expressions of CHal, CHa2(IgA gene) were determined by RT-PCR and expression of Tfh-related cytokines including IL-21and TGFβ1were measured in plasma of immunosuppressive agent-untreated IgAN children and healthy controls by Elisa. Results Tfh proportion were (19.87±1.00)%in20untreated IgAN,(13.87±0.83)%in34immunosuppressive agent-treated IgAN and (10.28±0.68)%in22controls and all p values between each two groups<0.001. mlgA ratio in CD19+B cells was (13.21±0.85)%in IgAN but (8.75±0.32)%, p<0.0001. Tfh-related cytokines IL-21and TGFβ1were (102.2±9.5) pg/ml and (80392±2274) pg/ml in immunosuppressive agent-untreated IgAN children, both of them were significantly higher than controls with (74.38±2.6) pg/ml and (29384±1828) pg/ml, P values<0.05. Conclusion It was found that the expression of mIgA, Tfh and Tfh-related cytokines IL-21and TGF β1from peripheral blood significantly upregulated in children with IgAN which indicating enhanced IgA class switching may occur in IgAN patients and immunosuppressive agent treatment showed their effects on lowering Tfh quantitation in IgAN.Part II. T Follicular Helper Cells Enhance IgA Class Switching of B cells in Children with IgANObjective To investigate if T follicular helper cells(Tfh) enhance IgA class switching in B cells of children with IgAN. Methods Magnetic bead was performed to sort Tfh cells. and naive B cells from untreated IgAN and healthy children and then co-cultured Tfh cells with naive B cells in each group. Genes expression of TGF-β1、IL-21Bcl-6. PRDM-1, AID. Ir3Cr3, Iα1Cα1, Ia2Ca2were determined by real-time RT-PCR and IgA class switching products of CHαl、 CHα2genes were detected by conventional RT-PCR and secretory expression of IL-21, TGF-β1, IgA were measured in co-cultured supernatants by Elisa. The isotype changes of immunoglobulins on CD19+B cells were detected by flow cytometry and confocal microscopy was used to determine IgA expression in Tfh and Naive B co-cultured cells and isolated Naive B cells cultured with cytokines of IL-21or TGF-β1or IL-4. Results CHal and CHa2expression were most significant at the6th hour, accompanied with similar trends in expression of Ir3Cr3, IalCαl, Ia2Ca2, TGF-β1, IL-21, Bcl-6, PRDM-1and AID genes, all of them declined over time thereafter. At the9th day, the proteins expressions of mIgA, IL-21, TGF-β1and IgA were significantly increased with declined mIgM in IgAN than in control (all P values<0.05). Confocal microscope showed IgA expressions were higher in Tfh and Naive-B co-cultured cells as well as naive B cells treated with TGF-β1or IL-21or IL-4than cytokine-untreated naive B cells. Conclusions This study first demonstrated that Tfh cells enhance IgA class switching in B cells of children with IgAN, probably through upregulated expression of IL-21and TGFβ1. Part Ⅲ. Tfh Cells and Tfh Related Cytokines Promote the Secretion of Galactose-deficient IgAl from B cells in Children with IgANObjective To explore the galactose-deficient IgAl(Gd-IgAl) secretion from B cells induced by Tfh or Tfh related-cytokines in children with IgA nephropathy Methods Magnetic bead was performed to sort peripheral blood Naive B cells (CD27"IgD+) and follicular T helper cells(CD4+CXCR5+) in IgA nephropathy children and healthy controls and co-cultured them. Meanwhile, Naive B cells were cultured respectively with IL-21, TGF-β1or IL-4. Supernatant was collected to detect the excretion of IgA and galactose-deficient IgAl(Gd-IgAl) by elisa. Results Excretion of IgA and level of Gd-IgAl were2881±611(ug/ml),2573±540.2(U/ml) in children with IgA, and2337±428(ug/ml),1021±132.5(U/ml) in control group, only Gd-IgAl level showed significant difference between IgA children and control group(p=0.03). The level of IgA in supernatant of co-cultured Tfh and NB cells from children with IgA were much higher than those of NB with IL-21or NB with TGF-β1or NB with IL-4or control group[4.82±0.39(ug/ml)vs.2.28±0.26(ug/ml),2.4±0.27(ug/ml),2.02±0.32(ug/ml), all P values＜0.001. In addition, the ratios of Gd-IgAl to IgA in co-cultured Tfh and NB cells from IgA nephropathy children was4.04±0.32(U/ug), which was much lower than NB with IL-21of7.73±1.31(U/ug), NB with TGF-β1of7.56±1.13(U/ug) and NB with IL-4of8.64±1.23(U/ug), all P values＜0.05. Results Tfh cells and Tfh-related cytokines may play important roles in secretionof Gd-IgAl, and participate in the pathogenesis of IgA nephropathy.