Application of a reagent for detecting Tph cell levels in hepatitis B cirrhosis

Abstract

This invention provides the application of a reagent for detecting Tph cell levels in hepatitis B cirrhosis. The reagent for detecting Tph cell levels is used to prepare a kit for detecting hepatitis B cirrhosis. The reagent for detecting Tph cell levels includes CD4 antibody, PD-1 antibody, and CXCR5 antibody, and also includes a reagent for detecting Tph cell levels using flow cytometry. This invention detects hepatitis B cirrhosis based on the level of Tph cells in peripheral blood. Tph cells have high specificity and sensitivity in the detection of hepatitis B cirrhosis and have good detection value.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to the application of a reagent for detecting Tph cell levels in hepatitis B cirrhosis. Background Technology

[0002] Hepatitis B cirrhosis (HBC) is one of the adverse progressions of chronic hepatitis B (CHB) caused by HBV infection. In 2019, the WHO reported that approximately 820,000 people worldwide died from liver failure, cirrhosis, or liver cancer caused by HBV infection. Currently, HBV infection accounts for 60% of cirrhosis cases in my country, with an annual incidence of CHB progressing to cirrhosis of 2%–10%, while the diagnosis and treatment rates for CHB are only 22% and 15%, respectively. If HBV infection is not cleared in time, it leads to persistent viral replication, repeated intrahepatic inflammation, and subsequently, fibrosis, lobular destruction, and pseudolobular formation, gradually progressing to cirrhosis. During this process, the body's sustained immune response produces antigen-antibody immune complexes, and extensive hepatocyte necrosis leads to liver dysfunction. Therefore, exploring new diagnostic biomarkers is crucial for improving the clinical diagnosis and treatment of hepatitis B cirrhosis.

[0003] Both peripheral helper T cells (Tph) and follicular helper T cells (Tfh) can promote antibody production by B cells. However, Tph cells do not express chemokine receptor type 5 (CX-Cmotif chemokine receptor type 5, CXCR5) on their surface, and thus interact with memory B cells in peripheral inflammatory tissues to produce antibodies. Studies have reported that B cells are involved in viral clearance during HBV infection; reversing excessive B cell activation and liver damage can clear HBV to achieve HBsAg seroconversion, suggesting the important role of humoral immune response in HBV infection. Dynamic changes in Tfh cell frequency can serve as a marker for predicting HBeAg seroconversion. Furthermore, Tfh cells are enriched in the peripheral blood of CHB patients, indicating their involvement in the pathogenesis of CHB. Current research mainly focuses on the CHB stage, but the dynamic changes, clinical significance, and specific association with liver function impairment of Tph cells during the progression of CHB to cirrhosis remain unclear. Summary of the Invention

[0004] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a reagent for detecting Tph cell levels in hepatitis B cirrhosis. The AUC of Tph cells as a marker for detecting hepatitis B cirrhosis reaches 0.968, with a sensitivity and specificity of 95.83%, which is superior to existing markers. Moreover, it is significantly correlated with liver injury indicators and can be used for disease activity assessment. It has the advantages of high sensitivity, high specificity, and non-invasive detection.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: the application of a reagent for detecting Tph cell levels in hepatitis B cirrhosis, wherein the reagent for detecting Tph cell levels is used to prepare a kit for detecting hepatitis B cirrhosis; the reagent for detecting Tph cell levels includes: CD4 antibody, PD-1 antibody and CXCR5 antibody.

[0006] Preferably, the reagents for detecting Tph cell levels also include reagents for detecting Tph cell levels using flow cytometry.

[0007] Preferably, the surface marker of the Tph cells is CD4. + PD-1 + and CXCR5 - .

[0008] Preferably, the Tph cell level refers to the Tph cell level in peripheral blood.

[0009] Compared with the prior art, the present invention has the following advantages: 1. The Tph cells of the present invention have good value in detecting hepatitis B cirrhosis and can be used as biomarkers for detecting hepatitis B cirrhosis and indicators for monitoring disease activity.

[0010] 2. The Tph level in peripheral blood samples in this invention can also effectively distinguish between hepatitis B cirrhosis and chronic hepatitis B.

[0011] 3. In this invention, the Tph level in peripheral blood samples is associated with the disease activity of hepatitis B cirrhosis.

[0012] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0013] Figure 1 In Example 2 of this invention, flow cytometry analysis was performed on CD4+ levels in peripheral blood of the HC group, HBC group, and CHB group. + PD-1 + Diagram showing the proportions of T, Tfh, and Tph cells.

[0014] Figure 2 CD4 in Embodiment 3 of the present invention + PD-1+ ROC curves of T, Tfh, and Tph cells distinguishing between patients with hepatitis B cirrhosis and chronic hepatitis B.

[0015] Figure 3 This is a correlation analysis diagram of Tph cells in peripheral blood of hepatitis B cirrhosis patients with ALT, AST, GGT, TP and GLO in Example 4 of the present invention; wherein, Figure A is ALT; Figure B is AST; Figure C is GGT; Figure D is TP; and Figure E is GLO.

[0016] Figure 4 In Example 4 of this invention, CD4 in peripheral blood of patients with hepatitis B-related cirrhosis... + PD-1 + Correlation analysis diagrams of T cells with ALT, GGT, and GLO; where Figure A represents ALT; Figure B represents GGT; and Figure C represents GLO. Detailed Implementation

[0017] Example 1

[0018] This example involves the collection of data from the control group, patients with chronic hepatitis B, and patients with hepatitis B cirrhosis.

[0019] Twenty-four patients with hepatitis B cirrhosis (HBC) and 24 patients with chronic hepatitis B (CHB) admitted to Changshu Clinical College of Xuzhou Medical University from July 2024 to April 2026 were included in this study. In addition, 24 healthy individuals who underwent physical examinations during the same period and were matched for sex and age were included as the control group.

[0020] Demographic characteristics and laboratory parameters of all study subjects were collected from the hospital information system (HIS), including age, sex, alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), total protein (TP), ALB, and globulin (GLO), as shown in Table 1.

[0021] Patient inclusion criteria: According to the diagnostic criteria for hepatitis B cirrhosis and CHB in the "Guidelines for the Prevention and Treatment of Chronic Hepatitis B (2022 Edition)," patients with hepatitis B cirrhosis must meet either of the following ① or ②: ①Those who are HBsAg positive, or HBsAg negative, anti-HBc positive and have a clear history of chronic HBV infection (HBsAg positive for more than 6 months), and whose liver biopsy pathology is consistent with the diagnosis of cirrhosis and other causes have been excluded; ② Excluding those with non-cirrhotic portal hypertension, and meeting two or more of the following five criteria: a. Imaging examinations show signs of cirrhosis and / or portal hypertension; b. Endoscopic findings show esophageal and gastric varices; c. Liver stiffness measurements (LSM) are consistent with cirrhosis (LSM ≥ 12.0 kPa when ALT < 1 × ULN; LSM ≥ 17.0 kPa when 1 × ULN < ALT < 5 × ULN). d. Blood biochemistry tests showed decreased albumin (ALB) levels (<35g / L) and / or prolonged prothrombin time (PT) (>3s longer than control). e. A complete blood count shows a platelet count <100×10⁻⁶. 9 / L etc.

[0022] Exclusion criteria for patients: ① non-alcoholic fatty liver disease; ② alcoholic liver disease; ③ drug-induced liver injury; ④ autoimmune liver disease; ⑤ coexisting viral hepatitis; ⑥ hepatocellular carcinoma.

[0023] Inclusion criteria for healthy controls: ① Negative for markers such as HBsAg, HBeAg, and HBV-DNA; ② Normal liver function; ③ No history of liver disease and previously healthy.

[0024] Exclusion criteria for healthy controls: positive HBsAg or HBeAg or HBV-DNA markers, abnormal liver function, or a history of liver disease.

[0025] The results are shown in Table 1. There were no statistically significant differences in gender and age between patients with hepatitis B cirrhosis (HBC group) and the control group (HC group) (P > 0.05). Serum ALT, AST, ALP, GGT, TP, and GLO levels were statistically significant among the control group, patients with hepatitis B cirrhosis, and patients with chronic hepatitis B (CHB group) (P < 0.05), while serum ALB levels showed no statistically significant difference among the three groups (P > 0.05). Peripheral blood CD4 levels in the HC, CHB, and HBC groups were also statistically significant. + PD-1 + The differences among T, Tfh, and Tph cells were all statistically significant (P < 0.05); the general information of the control group, patients with hepatitis B cirrhosis, and patients with chronic hepatitis B are shown in Table 1.

[0026] Table 1 General information of the control group, CHB group and HBC group Note: *P<0.05 compared with the control group.

[0027] Example 2

[0028] This embodiment describes the use of flow cytometry to detect CD4 levels in the peripheral blood of patients with hepatitis B-related cirrhosis. + PD-1 + The proportions of T, Tfh, and Tph cells.

[0029] The experimental reagents and instruments used in this embodiment were as follows: BV421-labeled anti-CD4 antibody (CD4-BV421), BV510-labeled anti-CD45 antibody (CD45-BV510), PerCP-Cy5.5-labeled anti-CXCR5 antibody (CXCR5-PerCP-Cy5.5), PE-labeled anti-CD279 (PD-1) antibody (PD-1-PE), and Fixed Viability Stain 700 were all purchased from BDBiosciences; FITC-labeled anti-CD3 antibody (CD3-FITC), PE-Cy7-labeled anti-CD19 antibody (CD19-PE-Cy7), and APC-Cy7-labeled anti-CD8 antibody (CD8-APC-Cy7) were all purchased from Biolegend, Inc.; erythrocyte lysis buffer was purchased from Beijing Tongsheng Times Biotechnology Co., Ltd.; PBS was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; and the flow cytometer was purchased from Beckman Coulter, Inc.

[0030] Collect 1 mL of EDTA-anticoagulated whole blood from all subjects. Transfer 50 μL of fresh whole blood to a flow cytometry tube. Stain cells with FixedViability Stain 700 to identify viable cells. Simultaneously, add monoclonal antibodies labeled with the following fluorescent dyes and incubate at room temperature in the dark for 20 min: CD4-BV421, CD45-BV510, CXCR5-PerCP-Cy5.5, CD279(PD-1)-PE, CD3-FITC, CD19-PE-Cy7, and CD8-APC-Cy7. Then, add 1 mL of 1× erythrocyte lysis buffer, mix well, and incubate at room temperature in the dark for 10 min. Centrifuge at 500×g for 5 min and discard the supernatant. Wash cells with 1 mL of 1× PBS, mix well, centrifuge at 500×g for 5 min, discard the supernatant, resuspend cells in 300 μL of PBS, and collect cells using CytExpert forDXFLEX software on a flow cytometer. Analyze the cells using FlowJo... v10.8.1 software is used for data analysis.

[0031] The results are as follows Figure 1 As shown, CD4 in peripheral blood + PD-1 + T, CD4 + PD-1 + CXCR5 + Tfh and CD4 + PD-1 + CXCR5 - Flow cytometry gate strategy of Tph cells; compared with the HC group, the HBC group had CD4 in peripheral blood. + PD-1 + The proportions of T (P < 0.0001), Tfh (P < 0.0001), and Tph (P < 0.0001) cells were significantly increased in the peripheral blood of the CHB group. + PD-1 + The proportions of T (P=0.014) and Tfh (P<0.0001) cells were significantly higher in the HBC group than in the control group, while the proportion of Tph cells was not statistically significant between the two groups (P=0.140). Furthermore, the peripheral blood CD4 count in the HBC group was significantly higher. + PD-1 + The proportions of T (P<0.001) and Tph (P<0.0001) cells were significantly higher in the T group than in the CHB group.

[0032] Example 3

[0033] This embodiment is CD4. + PD-1 + Value analysis of T, Tfh, and Tph cells in detecting hepatitis B cirrhosis.

[0034] Tfh, Tph, and CD4 were evaluated using indicators such as ROC curves, sensitivity, and specificity. + PD-1 + The value of T cells in detecting hepatitis B cirrhosis and chronic hepatitis B.

[0035] The results are as follows Figure 2 As shown in Table 2, ROC curve analysis revealed that the AUC of Tph cells for detecting hepatitis B-related cirrhosis was 0.968 (P < 0.0001), with a cut-off value of 9.55%, a sensitivity of 95.83%, and a specificity of 95.83%; the AUC of Tfh cells for detecting hepatitis B-related cirrhosis was 0.560 (P = 0.477), with a cut-off value of 2.76%, a sensitivity of 70.83%, and a specificity of 66.67%; CD4 + PD-1 + The AUC of T cells in detecting hepatitis B-related cirrhosis was 0.906 (P<0.0001), with a cut-off value of 12.56%, a sensitivity of 87.50%, and a specificity of 75.00%. These results indicate that the AUC, sensitivity, and specificity of Tph cells were all higher than those of Tfh and CD4. + PD-1 + T cells can effectively distinguish between hepatitis B cirrhosis and chronic hepatitis B.

[0036] Table 2 Tfh, Tph and CD4 + PD-1 + Detection efficacy of T cells in hepatitis B cirrhosis Example 4

[0037] This example illustrates the CD4+ levels in the peripheral blood of patients with hepatitis B-related cirrhosis. + PD-1 + The relationship between T, Tfh, and Tph cells and disease activity.

[0038] Further analysis of peripheral blood CD4 levels in patients with hepatitis B cirrhosis + PD-1 + Correlation analysis was performed on T, Tfh, and Tph cells with various clinical indicators to assess their relationship with disease activity.

[0039] The results are shown in Table 3. Figure 3 and Figure 4As shown, correlation analysis revealed that the proportion of peripheral blood Tph cells in patients with hepatitis B cirrhosis was positively correlated with serum ALT (r=0.486, P=0.016), AST (r=0.463, P=0.023), and GGT (r=0.539, P=0.007) levels; conversely, the proportion of Tph cells was negatively correlated with serum TP (r=-0.416, P=0.043) and GLO (r=-0.494, P=0.014) levels. Additionally, CD4+ levels in the peripheral blood of patients with hepatitis B cirrhosis were significantly higher than those in peripheral blood. + PD-1 + The proportion of T cells was positively correlated with serum ALT (r=0.419, P=0.042) and GGT (r=0.421, P=0.041) levels, and negatively correlated with serum GLO (r=-0.475, P=0.019) levels. The proportion of peripheral blood Tfh cells in patients with hepatitis B cirrhosis was not correlated with serum ALT, AST, ALP, GGT, TP, ALB, and GLO levels, and the differences were not statistically significant. This suggests that peripheral blood Tph cells may be associated with disease activity in hepatitis B cirrhosis.

[0040] Table 3. Peripheral blood Tfh, Tph, and CD4 levels in patients with hepatitis B-related cirrhosis. + PD-1 + Correlation between T cells and clinical indicators In summary, this study analyzed peripheral blood Tfh, Tph, and CD4 levels in patients with hepatitis B-related cirrhosis. + PD-1 + This study investigates changes in T cell levels, explores their relationship with liver injury indicators, and examines their clinical significance. Tph cells have good clinical diagnostic value and are closely related to disease activity, making them a potential diagnostic marker and disease activity monitoring indicator for hepatitis B-related cirrhosis.

[0041] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. The application of a reagent for detecting Tph cell levels in hepatitis B cirrhosis, characterized in that, The reagent used to detect Tph cell levels is used to prepare a kit for detecting hepatitis B cirrhosis. Reagents for detecting Tph cell levels include: CD4 antibody, PD-1 antibody, and CXCR5 antibody.

2. The application of the reagent for detecting Tph cell levels according to claim 1 in hepatitis B cirrhosis, characterized in that, Reagents for detecting Tph cell levels also include those that use flow cytometry to detect Tph cell levels.

3. The application of the reagent for detecting Tph cell levels according to claim 1 in hepatitis B cirrhosis, characterized in that, The surface marker of the Tph cells is CD4. + PD-1 + and CXCR5 - .

4. The application of the reagent for detecting Tph cell levels according to claim 1 in hepatitis B cirrhosis, characterized in that, The Tph cell level refers to the Tph cell level in peripheral blood.

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