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Background: Owing to individual heterogeneity, patients with idiopathic membranous nephropathy (IMN) exhibit varying sensitivities to immunotherapy. This study aimed to establish and validate a model incorporating pathological and clinical features using deep learning training to evaluate the response of patients with IMN to immunosuppressive therapy. Methods: The 291 patients were randomly categorized into training (n = 219) and validation (n = 72) cohorts. Patch-level convolutional neural network training in a weakly supervised manner was utilized to analyze whole-slide histopathological features. We developed a machine-learning model to assess the predictive value of pathological signatures compared to clinical factors. The performance levels of the models were evaluated using the area under the receiver operating characteristic curve (AUC) on the training and validation tests, and the prediction accuracies of the models for immunotherapy response were compared. Results: Multivariate analysis indicated that diabetes and smoking were independent risk factors affecting the response to immunotherapy in IMN patients. The model integrating pathologic features had a favorable predictive value for determining the response to immunotherapy in IMN patients, with AUCs of 0.85 and 0.77 when employed in the training and test cohorts, respectively. However, when incorporating clinical features into the model, the predictive efficacy diminishes, as evidenced by lower AUC values of 0.75 and 0.62 on the training and testing cohorts, respectively. Conclusions: The model incorporating pathological signatures demonstrated a superior predictive ability for determining the response to immunosuppressive therapy in IMN patients compared to the integration of clinical factors.
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Aprendizado Profundo , Glomerulonefrite Membranosa , Humanos , Glomerulonefrite Membranosa/tratamento farmacológico , Rim/patologia , Análise Multivariada , ImunoterapiaRESUMO
Excessive levels of cadmium (Cd) in soil exert serious negative impacts on soil ecosystems. Microorganisms are a common component of soil and show great potential for mitigating soil Cd. This review summarizes the application and remediation mechanisms of microorganisms, microbial-plants, and microbial-biochar in Cd-contaminated soil. Microorganisms such as Bacillus, Acinetobacter, Pseudomonas, and arbuscular mycorrhizal fungi (AMF) can change the biological validity of Cd through adsorption, mineralization, precipitation and dissolution. Different factors such as pH, temperature, biomass, concentration, and duration have significant effects on Cd bioavailability by microorganisms. Pseudomonas, Burkholderia, and Flavobacterium can promote the uptake of Cd2+ by hyperaccumulator through promotion and activation. Biochar, a soil amendment, possesses unique physicochemical properties and could act as a shelter for microorganisms in agriculture. The use of combined microbial-biochar can further stabilize Cd compared to using biochar alone.
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Cádmio , Poluentes do Solo , Ecossistema , Carvão Vegetal/química , Solo/químicaRESUMO
The objective of this study is to detect the number of different subsets of TFH and B cells in renal transplant recipients (RTR) with antibody-mediated acute rejection (AMR), acute rejection (AR), chronic rejection (CR), or transplant stable (TS). The present study was a prospective study. The numbers of ICOS +, PD-1+ and IL-21+ TFH, CD86+, CD38+, CD27+, and IgD- B cells in 21 patients with end-stage renal disease (ESRD) and post-transplant times were measured by flow cytometry. The level of serum IL-21 was detected by ELISA. The numbers of circulating CD4+CXCR5+, CD4+CXCR5+ICOS+, CD4+CXCR5+PD-1+, CD4+CXCR5+IL-21+ TFH, CD19+CD86+, and CD19 +CD86+CD38+ B cells as well as the level of serum IL-21 in the AMR, AR, and CR groups at post-transplantation were significantly higher than those at pre-transplantation. In contrast, the number of circulating CD19+CD27+IgD B cells was significantly increased in the TS groups in respect to the other groups. Moreover, the numbers of circulating CD4+CXCR5+IL-21+ TFH cells, CD19+CD86+CD38+ B cells as well as the level of serum IL-21 were positive related to the level of serum Cr while showing negative correlated with the values of eGFR in the AMR groups at post-transplantation for 4 and 12 weeks. Circulating TFH cells may be a biomarker in RTR with AMR, which can promote the differentiation of B cells into plasma cells by activating B cells, thereby promoting disease progression.
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Linfócitos B/imunologia , Rejeição de Enxerto/sangue , Transplante de Rim , Células T Auxiliares Foliculares/imunologia , ADP-Ribosil Ciclase 1/imunologia , Doença Aguda , Adulto , Antígeno B7-2/imunologia , Biomarcadores/sangue , Feminino , Rejeição de Enxerto/imunologia , Humanos , Terapia de Imunossupressão , Interleucinas/imunologia , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , TransplantadosRESUMO
BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is a kidney-specific autoimmune disease, but its pathogenesis is not fully known. The present study detected the frequencies of circulating memory B cells and plasmablasts and other clinical parameters in FSGS. METHODS: We monitored 16 primary FSGS patients and 23 healthy controls (HC). Flow cytometry was used to analyze circulating memory B cell and plasmablastspercentages. Serum IgG levels were detected using a cytometric bead array (CBA). RESULTS: The proportions of CD27 + IgD- class-switched memory B cells (P = 0.0002), CD27 + IgD-IgG + class-switched memory B cells (P < 0.0001), CD27hiCD38hi plasmablasts (P < 0.0001) and CD138 + plasma cells (P < 0.0001) were markedlyelevated in FSGS patients, and the frequency of CD38 + IgG + plasmablasts (P < 0.0001) and serum IgG levels (P < 0.0001) were lower compared to HC. In the FSGS patients, the frequency of CD27 + IgD-IgG + class-switched memory B cells negatively correlated with CD38 + IgG + plasmablasts (P = 0.0183, R = -0.3375), serum IgG levels (P = 0.0061, R = -0.4263) and estimated glomerular filtration rate (eGFR) (P = 0.0074, R = -0.4114) but positively correlated with 24-h urinary protein levels (P = 0.0077, R = 0.4085). The proportion of CD38 + IgG + plasmablasts positively correlated with serum IgG levels (P = 0.0151, R = 0.3538). CONCLUSIONS: We speculate that alterations in the frequencies of CD27 + IgD-IgG + class-switched memory B cells and plasmablasts may be responsible for the etiopathogenesis of FSGS.
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Linfócitos B/imunologia , Glomerulosclerose Segmentar e Focal/imunologia , Imunoglobulina G/sangue , Plasmócitos/imunologia , ADP-Ribosil Ciclase 1/metabolismo , Adulto , Idoso , Feminino , Citometria de Fluxo , Taxa de Filtração Glomerular , Glomerulosclerose Segmentar e Focal/diagnóstico , Humanos , Switching de Imunoglobulina , Imunoglobulina D/metabolismo , Memória Imunológica , Imunofenotipagem , Masculino , Pessoa de Meia-Idade , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Regulação para CimaRESUMO
Recent research has identified a population of PD-1hiCXCR5- 'peripheral helper' T (Tph) cells that simulate plasma cell differentiation by interactions between IL-21 and SLAMF5. However, the alteration of circulating Tph and CD138+ B in IgA nephropathy (IgAN) remains poorly understood. Flow cytometry analysis was used to measure the frequency of circulating PD-1hiCXCR5- T cells and CD138+ B cells in 37 patients with IgAN and 23 healthy controls (HCs). Estimated glomerular filtration rate (eGFR), 24 h urinary protein and serum cytokine concentrations were measured. The percentage of different subsets of circulating PD-1hiCXCR5- T cells and CD138+ B cells were significantly higher in patients with IgAN compared to HCs. Pretreatment, the percentage of different subsets of circulating PD-1hiCXCR5- T cells and CD138+ B cells were negatively correlated with eGFR, the percentage of circulating CD138+ B cells was positively correlated with 24-h urinary protein concentration, and the percentage of circulating PD-1hiCXCR5-, CD28+ and ICOS+ T cells. Posttreatment, the percentage of different subsets of circulating PD-1hiCXCR5- T cells and CD138+ B cells and serum IL-21 concentration were significantly reduced. Different subsets of circulating PD-1hiCXCR5- T cells contribute to the progression and pathogenesis of IgAN by regulating the differentiation of CD138+ B cells through a combination of surface molecules.
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Linfócitos B/imunologia , Glomerulonefrite por IGA/tratamento farmacológico , Receptor de Morte Celular Programada 1/metabolismo , Receptores CXCR5/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Adolescente , Adulto , Idoso , Benzazepinas/administração & dosagem , Benzazepinas/uso terapêutico , Estudos de Casos e Controles , Feminino , Glomerulonefrite por IGA/metabolismo , Humanos , Interleucinas/sangue , Masculino , Pessoa de Meia-Idade , Prednisona/administração & dosagem , Prednisona/uso terapêutico , Sindecana-1/metabolismo , Valsartana/administração & dosagem , Valsartana/uso terapêutico , Adulto JovemRESUMO
Background: Circulating B cells are crucial for the pathogenesis of IgA nephropathy (IgAN). This study aimed at investigating the relationship between frequency of different subsets of circulating B cells and clinical measures in IgAN patients.Methods: The percentages of different subsets of circulating B cells in 23 IgAN patients and 14 healthy controls (HC) were determined by flow cytometry. Their serum IL-6 levels were measured by Cytometric Bead Array (CBA). Clinical parameters in five patients were measured before and after treatment for 8-12 weeks. The potential relationship between variants was analyzed.Results: In comparison with the HC, the frequency of CD3-CD19+ CD27+ IgD+IgM+ non-switched memory B cells (P = .0038) and CD3-CD19+ CD27hiCD38hi plasmablasts (P = .0467) and serum IL-6 (P = .0392) levels significantly increased in IgAN patients. The percentages of non-switched memory B cells were positively correlated with plasmablasts (R = 0.5781, P = .0039) and serum IL-6 levels (R = 0.6663, P = .0005) in the patients. The percentages of non-switched memory B cells (R = 0.8399, P < .0001), plasmablasts (R = 0.4486, P = .0318) and the levels of serum IL-6 (R = 0.5461, P = .0070) were positively correlated with the values of 24-h urine proteins in IgAN patients. The serum levels of IL-6 were negatively correlated with the eGFR values. Following standard treatment, the frequency of non-switched memory B cells and plasmablasts and the levels of 24-h urine proteins (P = .0317, P = .0079, P < .05) significantly decreased in IgAN patients.Conclusions: Abnormally higher frequency of non-switched memory B cells and plasmablasts may contribute to the pathogenesis of IgAN and be potential biomarkers for IgAN.
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Subpopulações de Linfócitos B/imunologia , Glomerulonefrite por IGA/imunologia , Interleucina-6/sangue , Plasmócitos/imunologia , Adolescente , Adulto , Idoso , Feminino , Glomerulonefrite por IGA/sangue , Glomerulonefrite por IGA/patologia , Humanos , Memória Imunológica/imunologia , Interleucina-6/imunologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Regulatory B cells participate in the pathogenesis of autoimmune disease. This study aimed to examine the putative contribution of regulatory B cells to the pathogenesis of DN. The number of circulating CD19+CD24hiCD38hi B cells, CD19+CD24hiCD38hiCD5+ B cells, and CD19+CD24hiCD38hiIL-10+ B cells were significantly lower in DN patients (pâ¯<â¯0.05) than the control group. The number of circulating CD19+CD24hiCD38hi B cells was positively correlated with the levels of eGFR and serum IL-10 levels, but negatively correlated with urinary protein levels in DN patients. Treatment significantly increased the number of CD19+CD24hiCD38hi B cells, CD19+CD24hiCD38hiCD5+ B cells, CD19+CD24hiCD38hiIL-10+ B cells, and the levels of serum IL-10 (pâ¯<â¯0.05). We conclude that regulatory B cells may present new targets for intervention of DN.
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ADP-Ribosil Ciclase 1/imunologia , Antígenos CD19/imunologia , Linfócitos B Reguladores/imunologia , Antígeno CD24/imunologia , Nefropatias Diabéticas/imunologia , Glicoproteínas de Membrana/imunologia , Adulto , Idoso , Contagem de Células/métodos , Feminino , Humanos , Interleucina-10/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
The interleukin (IL)33/suppression of tumorigenicity 2 (ST2) axis regulates Th2 reactivity, and ST2 is the receptor for IL33. In this study, the roles of IL33 and soluble ST2 (sST2) in the pathogenesis of membranous nephropathy (MN), and their association with disease severity were evaluated. Serum levels of IL33 and sST2 in 93 patients, and 34 healthy controls (HCs) were measured by enzymelinked immunosorbent assays. Clinical characteristics were recorded and the estimated glomerular filtration rates (eGFRs) were computed. In addition, the association between serum IL33 and sST2 levels, and clinical measurements in patients with MN was analyzed. No difference in the serum levels of IL33 was identified between the patients with MN and HCs. However, the serum levels of sST2 were considerably higher in the MN patients compared with in the HCs at every stage. Higher concentrations of serum IL2, IL4, IL10, IL17A, and IFNγ were measured in the MN patients compared with in the HCs. Serum sST2 concentrations were negatively correlated with IL4 concentrations in the patients with MN. Furthermore, serum sST2 levels were negatively correlated with the eGFRs and serum calcium levels. Serum sST2 levels, but not IL33 levels, were positively correlated with the 24h urine protein and serum phosphorus levels. Following treatment, serum sST2 levels were considerably reduced, whereas serum IL4 and IL10 levels were significantly increased. These data suggest that sST2 and IL4, but not IL33, contribute to the pathogenesis of MN.
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Glomerulonefrite Membranosa/sangue , Glomerulonefrite Membranosa/diagnóstico , Proteína 1 Semelhante a Receptor de Interleucina-1/sangue , Interleucina-4/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Estudos de Casos e Controles , Citocinas/sangue , Feminino , Seguimentos , Glomerulonefrite Membranosa/terapia , Humanos , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Resultado do Tratamento , Adulto JovemRESUMO
Inflammation is a complex response to diverse pathological conditions, resulting in negative rather than protective effects when uncontrolled. Orientin (Ori), a flavonoid component isolated from natural plants, possesses abundant properties. Thus, we aimed to discover the potential therapeutic effects of orientin on lipopolysaccharide- (LPS-) induced inflammation in RAW 264.7 cells and the underlying mechanisms. In our studies, we evaluated the effects of Ori on proinflammatory mediator production stimulated by LPS, including tumor necrosis factor- (TNF-) α, interleukin- (IL-) 6, IL-18, and IL-1ß, along with prostaglandin E2 (PGE2) and NO. Our data indicated that orientin dramatically inhibited the levels of these mediators. Consistent with these results, the expression levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were also reduced. Further study demonstrated that such inhibitory effects of Ori were due to suppression of the nuclear factor-kappa B (NF-κB) pathway and nucleotide-binding domain- (NOD-) like receptor protein 3 (NLRP3) inflammasome activation, which may contribute to its anti-inflammatory effects. Together, these findings show that Ori may be an effective candidate for ameliorating LPS-induced inflammatory responses.
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BACKGROUND: T follicular helper (Tfh) cells are known to regulate humoral immune response. In this study we examined the correlation of different subsets of peripheral blood Tfh cells in patients with diabetic nephropathy (DN). METHODS: A total of 23 DN patients and 15 healthy controls (HC) were investigated for various subsets of Tfh cells by flow cytometry. The molecules ICOS+, PD-1+, CD28+, CD154+, IL-21+, IFN-γ+, IL-4+, IL-17+ Tfh cells were examined. The subsets of B cells were investigated by flow cytometry. The levels of 24 h urinary protein and estimated glomerular filtration rate (eGFR) were calculated. A potential correlation between the number of different subsets of Tfh cells, B cells and DN, was assessed. RESULTS: The circulating CD4+CXCR5+PD-1+, PD-1+CD154+, PD-1+CD28+, PD-1+IL-21+, PD-1+IL-4+, PD-1+-IL-17+-Tfh cell counts, CD38+CD19+, CD38+CD19+CD40+ B cells and plasma levels of IL-21 were significantly increased in DN patients (p < 0.05), as compared to that in the HC group. Furthermore, the circulating CD4+CXCR5+PD-1+ Tfh cell counts negatively correlated with eGFR; Tfh cell counts positively correlated with 24 h urinary protein concentration in DN patients. Post-treatment, there was a significant reduction in the CD4+CXCR5+PD-1+ Tfh cell counts and its subsets, with a corresponding decrease in plasma levels of IL-6 and IL-17A (p < 0.05) in DN patients, as compared to the HCs. CONCLUSION: An increased number of CD4+CXCR5+PD-1+ Tfh cells were observed in DN patients, which may be new targets for intervention in DN.
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Nefropatias Diabéticas/sangue , Nefropatias Diabéticas/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biomarcadores , Citocinas/sangue , Nefropatias Diabéticas/tratamento farmacológico , Feminino , Humanos , Imunofenotipagem , Contagem de Linfócitos , Masculino , Receptores CXCR5/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
BACKGROUND: Henoch-Schoenlein purpura is the one of most common types of systemic vasculitis that involves impaired renal function and Henoch-Schoenlein purpura nephritis (HSPN). The diagnosis of this condition is largely based on immunohistologic detection of immunoglobulin A1-containing immune complex in the glomerular deposits of mesangium. Despite clinical advances, the etiopathogenesis of HSPN is still largely unknown. METHODS: In this study, we enrolled 25 newly diagnosed HSPN patients and 14 healthy controls. Then, fractions of B cell subtypes were determined in venous blood using flow cytometry. The serum interleukin (IL)-10 concentration was determined by enzyme-linked immunosorbent assay. RESULTS: Compared to those in healthy controls, the numbers of CD38+CD19+, CD86+CD19+, CD38+CD86+CD19+, and CD95+CD19+ B cells per microliter of blood were significantly higher in HSPN patients. In contrast, the numbers of CD5+CD19+, IL-10+CD19+, CD5+CD1d+CD19+, and IL-10+CD5+CD1d+CD19+ B cells per microliter of blood and the serum IL-10 concentration were significantly lower in HSPN patients. Following treatment, the numbers of CD38+CD19+ and CD86+CD19+ B cells per microliter of blood were significantly reduced in HSPN patients. However, the numbers of CD5+CD1d+CD19+, CD5+CD1d+IL-10+CD19+, and IL-10+CD19+ B cells per microliter of blood and the serum IL-10 concentration were significantly increased in HSPN patients following treatment. The estimated glomerular filtration rate (eGFR) was negatively correlated with the number of CD38+CD19+ B cells but positively correlated with the numbers of IL-10+CD19+, CD1d+CD5+CD19+, and IL-10+CD1d+CD5+CD19+B cells per microliter of blood and the serum IL-10 concentration. The 24-h urinary protein concentration was positively correlated with the number of CD38+CD19+B cells but negatively correlated with the numbers of IL-10+CD19+, CD1d+CD5+CD19+, and IL-10+CD1d+CD5+CD19+B cells per microliter of blood and the serum IL-10 concentration. CONCLUSION: Our results suggest that CD38+CD19+ and CD1d+CD5+CD19+ B cells (Bregs) contribute to the pathogenesis of HSPN.
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Linfócitos B Reguladores/imunologia , Vasculite por IgA/imunologia , Nefrite/imunologia , ADP-Ribosil Ciclase 1/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD19/metabolismo , Linfócitos B Reguladores/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Feminino , Humanos , Vasculite por IgA/sangue , Vasculite por IgA/complicações , Imunoglobulina A/sangue , Interleucina-10/sangue , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Nefrite/etiologia , Adulto JovemRESUMO
BACKGROUND: T follicular helper (TFH) cells are involved in the humoral immune responses. This study is aimed at examining the frequencies of different subsets of CD4(+)CXCR5(+) TFH cells in adult patients with minimal change disease (MCD). METHODS: A total of 27 patients and 14 healthy controls (HC) were characterized for the levels of sera cytokines, inducible T-cell costimulator (ICOS), and programmed death 1 (PD-1) of positive TFH cells by flow cytometry. The level of sera IL-21 was examined; 24 h urinary protein and eGFR were calculated. The potential correlation between the frequency of different subsets of TFH cells and the values of clinical measures in MCD patients were analyzed. RESULTS: The frequency of circulating CD4(+)CXCR5(+), CD4(+)CXCR5(+)ICOS(+), and CD4(+)CXCR5(+)PD-1(+) TFH cells and the levels of sera IL-17A, IFN-γ, IL-2, IL-10, IL-4, and IL-21 were significantly higher in MCD patients (P < 0.05) than that in the HC group. Furthermore, the percentages of circulating CD4(+)CXCR5(+) TFH cells were negatively correlated with the values of eGFR (r = -0.4849, P < 0.05) and the percentages of CD4(+)CXCR5(+)PD-1(+) TFH cells were correlated positively with the levels of serum IL-21 (r = 0.6137, P < 0.05) and 24 h urinary protein (r = 0.1410, P < 0.05) in those patients. Also, the percentages of CD4(+)CXCR5(+)ICOS(+) TFH cells were correlated positively with the levels of serum IL-21 (r = 0.6201, P < 0.05) and 24 h urinary protein (r = 0.7519, P < 0.05). Following standard therapies, the percentages of circulating CD4(+)CXCR5(+), CD4(+)CXCR5(+)PD-1(+), and CD4(+)CXCR5(+)ICOS(+) TFH cells and the levels of serum IL-21 were significantly reduced, but the levels of serum IL-4 and IL-10 were increased (P < 0.05). CONCLUSIONS: A higher frequency of CD4(+)CXCR5(+) TFH cells that existed in adult patients with MCD could be new target for intervention of MCD.
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Antígenos CD4/sangue , Nefrose Lipoide/sangue , Nefrose Lipoide/epidemiologia , Receptores CXCR5/sangue , Linfócitos T Auxiliares-Indutores/química , Adolescente , Adulto , Idoso , Anti-Inflamatórios/uso terapêutico , Linfócitos B/química , Linfócitos B/citologia , Estudos de Casos e Controles , Citocinas/sangue , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Nefrose Lipoide/tratamento farmacológico , Prednisolona/uso terapêutico , Linfócitos T Auxiliares-Indutores/citologia , Adulto JovemRESUMO
The frequency of different subsets of CD4(+)CXCR5(+) TFH cells and serum cytokine levels were analyzed in a total of 14 patients with newly diagnosed hepatitis B virus-associated membranous nephropathy (HBV-MN), 12 individuals with immune-tolerant HBV infection (HBV-IT) and 12 healthy controls (HC). Serum cytokine levels were measured before and 10-12 weeks after treatment. Significantly higher frequency of CD4(+)CXCR5(+), CD4(+)CXCR5(+)ICOS(+) and CD4(+)CXCR5(+)PD-1(+) TFH cells, and higher serum levels of IL-17A, IFN-γ, IL-2, IL-10, IL-4 and IL-21 were detected in HBV-MN patients compared to the HC. The percentage of CD4(+)CXCR5(+) TFH cells and serum IL-21 level in HBV-MN patients were also higher than the HBV-IT. The percentage of CD4(+)CXCR5(+) TFH cell was negatively correlated with the value of eGFR, and the percentage of CD4(+)CXCR5(+)ICOS(+) TFH cells was positively correlated with the 24-h urinary protein concentration. Notably, the percentage of CD4(+)CXCR5(+)PD-1(+) TFH cells was positively correlated with serum IL-21 level and 24-h urinary protein concentration. Treatment with prednisone or/and immunosuppressive drugs significantly reduced the frequency of CD4(+)CXCR5(+), CD4(+)CXCR5(+)PD-1(+) TFH cells and serum IL-21 level, but increased IL-4 and IL-10 levels in the patients. CD4(+)CXCR5(+) TFH cells, especially CD4(+)CXCR5(+)PD-1(+) TFH cells may participate in the pathogenesis of HBV-MN.