Application of liver gamma delta T cell as biliary atresia biomarker and therapeutic target

Abstract

The invention discloses application of liver gamma delta T cells as biliary atresia biomarkers and therapeutic targets. Research finds that on the contrary to typical autoimmune diseases, late BA is mainly based on gamma delta Treg amplification, gamma delta Th17 is up-regulated, and the proportion of gamma delta Treg cells in the liver of a BA patient is obviously increased; contrast with reduced gamma delta Treg cell levels observed in other autoimmune diseases is formed. The result shows that the gamma delta T cells can play a role in protecting lymphocytes under the background of BA. The significant increase of gamma delta Treg cells prompts a compensatory protection mechanism, and gamma delta T cells are located as key regulatory factors for BA progression. The invention provides a theoretical basis for gamma delta T cell targeted therapy of biliary atresia, and provides a new direction and treatment strategy for treatment of biliary atresia.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of hepatic γδT cells as biomarkers and therapeutic targets for biliary atresia. Background Art

[0002] Biliary atresia (BA) is a biliary disease that can affect both the extrahepatic and intrahepatic bile ducts and is characterized by progressive hepatic fibrosis—inflammatory damage, cholestasis, and fibrotic obstruction of the extrahepatic bile ducts. Currently, the diagnosis of BA relies primarily on serum biochemical markers, imaging, and liver histopathology. However, these methods have limitations such as insufficient sensitivity, high invasiveness, and the inability to dynamically monitor the patient's liver immune status, hindering timely clinical medication guidance. Currently, treatment for BA is limited to a surgical procedure called KPE, which still fails to improve the condition in nearly 50% of patients and leaves the intrahepatic bile duct lesions unresolved. Furthermore, in many cases, BA leads to fibrosis, portal hypertension, and liver failure, ultimately requiring liver transplantation, which necessitates lifelong immunosuppression and compromises the quality of life of BA patients. Therefore, identifying new diagnostic and therapeutic targets is crucial for improving clinical diagnosis and treatment.

[0003] Various immune mechanisms have been proposed, including immune dysregulation, autoimmunity, and the susceptibility of the immature neonatal immune system. Although the exact interplay between these processes is not fully understood, extensive research in the field has identified key components of the early and late innate and adaptive immune responses in BA. The innate immune response plays a key role in early pathogen defense through PRRs, particularly TLRs. TLRs are upregulated in BA and recognize PAMPs or stimuli released by apoptotic / necrotic cells, known as DAMPs. Following TLR activation, type I interferons are released, initiating a complex immune signaling cascade through TNF-α, IL-1, IL-6, IL-8, and IL-157. Cholangiocytes, macrophages, and dendritic cells play a major role in the early immune response, leading to neutrophil recruitment and activation of adaptive immunity.

[0004] γδT cells, a unique subset of innate lymphocytes, perform innate immune functions. Their TCRs are composed of γ and δ chains. γδT cells are immune cells that can kill cancer cells and tumor stem cells while also recognizing cancer antigens. γδT cells can differentiate into at least three distinct subpopulations: IFN-γ-producing γδT cells, IL-17-producing γδT cells, and Foxp3-producing γδT cells. Functional subpopulations of γδT cells can be distinguished based on surface markers.

[0005] Current research on BA has largely focused on the potential role of viral infection and immune responses in its pathogenesis. Given the complex process of bile duct damage in BA, some studies have proposed that BA may be an autoimmune disease. Imbalance in the Th17 / Treg axis has been widely reported in other autoimmune diseases, such as experimental colitis and allergic asthma. γδ T cells possess the unique ability to differentiate into both the γδ Th17 and γδ Treg subtypes. However, there are currently no reports on whether a similar immune imbalance exists in γδ T cells during BA. Summary of the Invention

[0006] The purpose of the present invention is to address the above-mentioned problems and provide an application of hepatic γδT cells as a biomarker and therapeutic target for biliary atresia.

[0007] In order to achieve its purpose, the present invention adopts the following technical solutions:

[0008] A first aspect of the present invention provides use of liver γδ T cells as a target for developing or screening drugs for treating biliary atresia.

[0009] In the above-mentioned technical scheme of use, the liver γδT cells, among which the γδTh17 cells that produce IL-17 play a pathogenic role, and the γδTreg cells play an immune homeostatic role.

[0010] In the above-mentioned technical solution of use, the drug promotes the differentiation of liver γδT cells into γδTreg cells.

[0011] A second aspect of the present invention provides use of an agent that promotes the differentiation of hepatic γδT cells into γδTreg cells in the preparation of a medicament for treating biliary atresia.

[0012] A third aspect of the present invention provides the use of a substance for detecting a biomarker in the preparation of a product for diagnosing biliary atresia, wherein the biomarker is hepatic γδT cells, and the product includes a detection reagent, a kit, or a detection system.

[0013] In patients with biliary atresia, hepatic γδT cells increased, including γδTh17 subsets, γδTreg subsets, and CD4 - CD8 - γδT cells, CD3 + γδ T cells and CCR6 + The proportion of γδT cells increased.

[0014] A fourth aspect of the present invention provides the use of a substance for detecting a biomarker in the preparation of a product for evaluating the progression of biliary atresia, wherein the biomarker is liver γδT cells, and the product includes a detection reagent, a kit or a detection system.

[0015] Compared with early-stage patients, the ratio of γδTreg / γδTh17 was increased in patients with advanced biliary atresia.

[0016] A fifth aspect of the present invention provides a system for assessing the progression of biliary atresia, the system comprising:

[0017] (1) an analysis unit, the analysis unit comprising: a detection substance for determining the proportion of liver γδ T cells as a biomarker in a test sample of a subject;

[0018] (2) An evaluation unit, comprising: determining the progression of biliary atresia in the subject based on the biomarker ratio determined in (1).

[0019] In the above-mentioned system technical solution for evaluating the progression of biliary atresia, the liver γδT cell ratio refers to the cell ratio of the γδTreg subpopulation and the γδTh17 subpopulation. Compared with early patients, the γδTreg / γδTh17 ratio of subjects with late-stage biliary atresia is increased.

[0020] The beneficial effects of the present invention are:

[0021] This study used qPCR, immunofluorescence, and flow cytometry to analyze liver tissue from 100 patients with BA and 30 non-cholestatic controls to assess their clinical correlation with liver function parameters. The results showed significant increases in the proportion of γδT cells (P < 0.05), CCR6+ γδT cells (P < 0.05), and the γδTh17 subset (P < 0.05) in BA livers. Furthermore, γδTreg cells showed a more pronounced increase (P < 0.05). Patients with higher levels of hepatic γδT cells had better liver function in advanced BA.

[0022] The research presented in this paper suggests that γδT cells may play a protective lymphocyte role in the setting of biliary atresia. The significant increase in γδTreg cells suggests a compensatory protective mechanism, positioning γδT cells as key regulators of BA progression. This invention provides new directions and therapeutic strategies for the early detection and treatment of biliary atresia. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1Figures: A: qPCR analysis of lymphocytes isolated from liver tissue revealed increased relative expression levels of γδT cell phenotypic receptor genes (CDG), IL-17, CCR 6, and Foxp 3; B: The left figure shows HE staining of liver tissue sections from the control group and BA group, and the right figure shows Sirius red staining of liver tissue sections from the control group and BA group; C: Immunofluorescence images of γδT and CCR 6 in liver tissue sections from the control group and BA group; D: The left figure shows a bar graph of γδT cell fluorescence intensity analysis between the control group and BA group, indicating a significant increase in γδT cell infiltration in BA tissue; the right figure shows a bar graph of CCR6 fluorescence intensity analysis between the control group and BA, indicating increased CCR6 expression in BA tissue; β-actin (β-Actin) was used as a housekeeping gene, and the ΔΔCt method was used to detect the relative expression levels of the above four genes, *P < .05; **P < .01; *P < .001; *P < .0001.

[0024] Figure 2 Figure 3: CD3+γδT (total γδT cells in T cells) flow cytometry scheme; B: Statistical bar graph of the proportion of CD3+γδT cells in T cells; C: CD4-CD8-γδT cell flow cytometry gating scheme; D: Statistical bar graph of the proportion of CD4-CD8-γδT cells to CD4-CD8-T cells; *P<0.0001.

[0025] Figure 3 Figure 3: A: Flow cytometry gating scheme for CCR6+γδT cells; B: Statistical bar graph of the ratio of CCR6+γδT cells to CD3+γδT cells; *P<0.0001.

[0026] Figure 4 Figure 3: A: Flow cytometric gating strategy for γδTh17 and γδTreg cells; B: Histogram of Foxp3 in γδT cells obtained by flow cytometry; C: Bar graph showing the proportion of γδTh17 cells in CD3+γδT cells; D: Bar graph showing the proportion of γδTreg cells in CD3+γδT cells; E: Bar graph showing the MFI of Foxp3 in CD3+γδT cells in the control and BA groups; F: Bar graph showing the ratio of γδTreg / γδTh17 cells in the control and BA groups; *P < .05; **P < .01; *P < .001; *P < .0001.

[0027] Figure 5Figure 3 Flow cytometric analysis of hilar lymph node lymphocytes: A: Statistical bar graph showing the proportion of CD3+γδT cells to T cells; B: Statistical bar graph showing the proportion of CD4-CD8-γδT cells to CD4-CD8-T cells; C: Statistical bar graph showing the proportion of CCR6+γδT cells to CD3+γδT cells; D: The left panel shows the proportion of γδTh17 cells to CD3+γδT cells, and the right panel shows the proportion of γδTreg cells to CD3+γδT cells; E: Bar graph showing the MFI of Foxp3 in CD3+γδT cells; F: Bar graph showing the ratio of γδTreg / γδTh17 cells; *P < .05, *P < .001, ns indicates no statistical significance.

[0028] Figure 6 AH: Correlation simulation curves of the eight indicators with statistical differences in the correlation analysis; I: Since the best-fit curve of PAB is a logarithmic fit and the unit on the x-axis changes accordingly, a scatter plot is created for PAB.

[0029] Figure 7 To analyze the differences and mechanisms of differentiation of γδTh17 / γδTreg cells in liver tissue of advanced BA and normal liver tissue. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to the embodiments, but the present invention is not limited thereto.

[0031] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0032] Example 1

[0033] 1 Materials and Methods

[0034] 1.1 Study Design and Patients

[0035] The specimens for this study were collected from patients who visited the Department of Hepatobiliary Surgery, Chongqing Medical University Children's Hospital between April 2021 and April 2024. Among them, 100 were children with BA who required liver transplantation due to cholestatic cirrhosis, and 30 were children with non-cholestatic cirrhosis. Among the cases of non-cholestatic cirrhosis, 16 were diagnosed with hepatoblastoma based on preoperative liver biopsy, and 14 were diagnosed with non-cholestatic cirrhosis diseases such as hepatic hemangioma, glycogen storage disease, and portal vein stenosis. Liver specimens from BA patients during transplantation were used as the experimental group (BA group); liver specimens from patients with non-cholestatic cirrhosis collected during surgery were used as the control group (non-tumor tissue more than 1 cm away from the hepatoblastoma was considered normal tissue), marked as the Ctrl group. All clinical samples from patients were approved by the Ethics Committee of Chongqing Medical University for clinical research.

[0036] 1.2 Cell separation and purification

[0037] Improved collagenase digestion and Ficoll density gradient centrifugation were used to separate and extract liver lymphocytes. Conventional methods were used to separate liver lymphocytes.

[0038] 1.3 RNA extraction and RT-qPCR

[0039] Total RNA was extracted from isolated cell populations using nucleic acid extraction or purification reagents (TT-BD-RNA-48, TETE MED, China). Complementary DNA was prepared for qPCR using ABScript Neo RTMaster Mix with gDNA removal reagent (RK20433, ABclonal, China). All other transcripts were detected using 2X Universal SYBR Green Fast qPCR Mix (RK21204, ABclonal, China) on an Applied CFX Opus 96 Real-Time PCR System (Bio-RAD). β-actin (β-actin) was used as a housekeeping gene, and relative gene expression values were normalized using the 2-ΔΔCt method.

[0040] 1.4 Histology

[0041] After surgery, liver tissue specimens were fixed with 4% paraformaldehyde. Subsequently, liver tissue sections were mounted on adhesive slides and subjected to hematoxylin and eosin (HE) staining, Congo red staining, and immunofluorescence staining.

[0042] Liver paraffin sections were subjected to immunofluorescence using the following primary antibodies: TCR GAMMA / DELTA (1:100, TCR1061, Thermo), Anti-Macrophage Inflammatony Protein 3α antibody (1:100, ab9829-100ug, Abcam).

[0043] 1.5 Flow cytometry

[0044] Dead cells were excluded using 7-AAD (1:100, PerCP, 420404, biolgend). For T cell phenotype, surface staining was performed using the following antibodies in the presence of TCRγδ (1:100, APC, 331212, biolgend), CD3 (1:100, APC-cy7, 557832, BD), CD4 (1:100, BV650, 317436, biolgend), CD8 (1:100, BV711, 583877, BD), and CCR6 (1:100, BV510, BD).

[0045] To measure cytokine production (IL-17A and Foxp3), cells were restimulated with 50 ng / ml phorbol 12-myristate 13-acetate (PMA, Merck Millipore) and 500 ng / ml ionomycin (Sigma-Aldrich) for 4 to 6 hours (5% CO2, 37°C). For intracellular staining, cells were fixed for 20 minutes at room temperature using Fixation Buffer (1:3, Cat. No. 2921581, eBioscience). Cell membranes were permeabilized using Permeabilization Buffer (1:10, Cat. No. 2256153, eBioscience) for 5 minutes at room temperature. Antibodies to IL-17A (1:100, BV421, Cat. No. 512322, Biolgend) and Foxp3 (1:100, FITC, Cat. No. 320106, Biolgend) were then incubated for 30 minutes at room temperature. Samples were acquired using a FACS Canto II flow cytometer (BD) and analyzed using FlowJo software (BD).

[0046] 1.6 Statistical analysis

[0047] Data with homogeneous variance were statistically analyzed using paired t-tests and expressed as mean ± standard deviation. Data with unequal variance were analyzed using Student's two-tailed t-tests and expressed as median (range). Laboratory index data were tested for normality using the Shapiro-Wilk test. Normally distributed data were analyzed using Pearson correlations, while those with non-normal distributions were analyzed using Spearman correlations.

[0048] Statistical software used in this study included GraphPad Prism 8.1, FlowJo 10.9.0, ImageJ 1.52, and R software 4.4.2. Statistical results were plotted using tools including GraphPad Prism 8.1, R software 4.4.2, Figdraw, and Adobe Illustrator 2024. R packages used included ggplot2, tidyverse, and ggpmisc.

[0049] 1.7 Abbreviations

[0050] The biochemical indices and their abbreviations used in this study are as follows:

[0051] Total bilirubin (TBIL), direct bilirubin (DBIL), indirect bilirubin (IBIL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), albumin (ALB), globulin (GLB), serum total protein (STP), cholinesterase (CHE), prealbumin (PAB), total bile acid (TBA), amylase (AMY), creatinine (Cr), uric acid (UA), total cholesterol (TC), triglycerides (TG), G), high-density lipoprotein (HDL), platelets (PLT), white blood cells (WBC), low-density lipoprotein (LDL), red blood cells (RBC), hemoglobin (HB), lymphocytes (L), neutrophils (N), monocytes (MNC), eosinophils (Eos%), basophils (Bas%), prothrombin, prothrombin time (PT), fibrinogen (FIB), international normalized ratio (INR), activated partial thromboplastin time (APTT), thrombin time (TT), lactic acid (LAC), ammonia (NH3).

[0052] 2 Results

[0053] 2.1 BA liver RT-qPCR and histopathological analysis

[0054] Previous studies have identified that CCR6+γδT cells in the liver of BA patients secrete IL-17, which has been identified as a driver of inflammatory and autoimmune processes in BA. Furthermore, studies have found that Treg cells secrete a variety of anti-inflammatory cytokines and inhibit the activation of other lymphocytes.

[0055] We performed RT-qPCR analysis on lymphocytes extracted from liver tissues of the BA group and the control group (Ctrl) and found that the transcriptional levels of γδT cell phenotype-related genes CDG (p = 0.002), CCR6 (p = 0.018), IL-17 (p = 0.019), and Foxp3 (p = 0.027) were upregulated ( Figure 1 A).

[0056] Histological examination of liver sections stained with hematoxylin and eosin (HE) showed that compared with the Ctrl group, the liver lobule structure in the BA group was significantly irregular, bile acid deposition in some cells, and immune cell infiltration in the portal vein area ( Figure 1 B). Sirius red staining (Sirius red) showed that compared with the Ctrl group, the BA group had extensive portal fibrosis and a large amount of collagen fiber deposition ( Figure 1 B). In addition, immunofluorescence analysis of liver sections showed that in the BA group, there were obvious CCR6+ T cells and γδ T cells clustered around the portal vein area and also distributed in the liver lobules ( Figure 1CD).

[0057] 2.2 CD3 in BA liver tissue + γδ T cells and CD4 - CD8 - Increased proportion of γδ T cells

[0058] In our preliminary study, we performed surface staining analysis of lymphocytes extracted from liver tissues of BA patients collected from April 2021 to April 2023 (a total of 130 cases) using flow cytometry and found that CD3 + The proportion of γδT cells increased (total γδT cells, P < 0.001) ( Figure 2 AB). Among them, CD4 - CD8 - γδT cells are a subset of γδT cells. + or CD8 + Compared with γδT cells, this subset usually has stronger tissue infiltration ability and can secrete large amounts of IFN-γ and IL-17, participating in the regulation of inflammation and liver fibrosis. In our study, we found that the proportion of γδT cells in this subset was also increased in BA liver tissue (P<0.001) ( Figure 2 CD).

[0059] 2.3BA CCR6 in liver + Increased proportion of γδT cells

[0060] CCR6 is a chemokine receptor that binds to its ligand CCL20 (MIP-3α). In BA, the expression of CCL20 in damaged bile ducts, bile duct cells and liver tissues is significantly increased, promoting the activation of CCR6. + γδT cells are recruited to the site of inflammation. Related studies have shown that CCR6 + γδT cells are one of the main sources of IL-17. In our study, flow cytometric analysis of lymphocytes from BA liver tissue showed that CCR6 + The proportion of γδT cells increased (P<0.001) ( Figure 3 ).

[0061] 2.4BA liver γδT cell subset immune imbalance

[0062] γδT cells can be classified according to their cytokine secretion profiles, including γδTh1, γδTh2, γδTh17, and γδTreg subpopulations. It is worth noting that γδTh17 and γδTreg cells account for the majority of these subpopulations. Flow cytometry analysis of lymphocytes extracted from liver tissues of 32 BA patients collected from April 2023 to April 2024 showed an increase in the proportion of γδTh17 cells (P<0.001) and the proportion of γδTreg cells (P<0.001) after intracellular staining ( Figure 4 A, C, D).

[0063] In our study, flow cytometry analysis showed that the mean fluorescence intensity of Foxp3 in the CD3+γδT cell population in the BA group showed an increased trend compared with the Ctrl group (P=0.002) ( Figure 4 B,E).

[0064] In addition, analysis of the ratio of γδTreg to γδTh17 cells (P = 0.018) showed that the differentiation of γδT cells in the BA group was more unbalanced than that in the Ctrl group ( Figure 4 F), indicating an immune imbalance between γδ Th17 and γδ Treg cells within the γδ T cell population; however, this imbalance differs from the classical definition.

[0065] The proportion of γδT cells in the portal vein lymph nodes of patients with 2.5BA is significantly increased

[0066] We often observe enlarged lymph nodes in the portal vein region during liver transplantation for BA in children. Therefore, we performed flow cytometric analysis of lymphocytes from these enlarged lymph nodes (n=11). Compared with the control group (n=6), we found that CD3 + γδT (P = 0.024), CD4 - CD8 - γδT (P = 0.043) and CCR6 + The proportion of γδT cells increased (P<0.001) Figure 5 AC). In addition, we analyzed the subtypes of γδT cells and found that the proportions of γδTh17 cells and γδTreg cells increased (P<0.001) ( Figure 5 DE).

[0067] Based on these findings, we wanted to know whether there is also an immune imbalance of γδT cells in the portal vein lymph nodes of children with BA. We found that although the MFI of Foxp3 was increased in the γδT cell population (P = 0.0002), Figure 5 E), but the ratio of γδTreg / γδTh17 (P=0.5205) was not statistically significant ( Figure 5 F) These results suggest that although inflammatory responses may be present in the hepatic portal lymph nodes of patients with BA, the immune imbalance of γδ T cells is limited to the liver.

[0068] Correlation and linear regression analysis between laboratory parameters and γδT cell ratio in the 2.6BA group

[0069] We conducted a correlation analysis between the γδT cell ratios obtained by flow cytometry and corresponding laboratory parameters in the BA group. This analysis showed that the γδT cell ratio was positively correlated with GLB (P = 0.021) and STP (P = 0.034). There was a negative correlation with AST (P = 0.007), PAB (P = 0.035), CHE (P = 0.007), UA (P = 0.035), LDL (P < 0.001), and L (P = 0.038), as shown in Table 1.

[0070] Table 1 Overall situation and correlation analysis of laboratory indicators in BA patients

[0071]

[0072]

[0073] In order to intuitively illustrate the relationship between the proportion of γδT cells and the corresponding laboratory indicators, based on the analysis results, we drew the best fitting curves of laboratory indicators with statistical differences (fitting types include: linear fitting, polynomial fitting, polynomial fitting, logarithmic fitting, logistic fitting) ( Figure 6 ).

[0074] 3 Analysis

[0075] Despite extensive research, a unified understanding of the pathogenesis of BA remains elusive. Previous studies have shown that CCL20 expression is significantly increased in human and animal liver tissues with liver fibrosis. It has been reported that CCL20 can induce CCR6 + γδT cells produce IL-17, which stimulates the expression of α-smooth muscle actin in hepatic stellate cells (HSCs). This leads to the production of TGF-β, actin, IL-6, and collagen, which together promote the progression of liver fibrosis. Consistent with these findings, our study also showed that the proportion of CCR6+γδT cells in BA increased ( Figure 3 ), while the proportion of γδTh17 cells increased ( Figure 4 Furthermore, the MFI of IL-17 on γδ T cells in BA livers was increased, indicating increased IL-17 secretion ( Figure 4 ).

[0076] CD4+ regulatory T cells (Tregs) are one of the most extensively studied immunoregulatory T cell subtypes and are defined by the expression of Foxp3. Foxp3 can also be transiently induced in non-Treg cells, including γδT cells. The main function of Tregs is to inhibit the activation and function of other leukocytes, thereby maintaining immune homeostasis. In addition, Tregs are cytotoxic and inhibit effector cells by inducing apoptosis. Previous studies in mouse BA models have reported a decrease in the number and function of Treg cells. Therefore, we hypothesized that the proportion of γδTreg cells in BA would also be reduced. However, contrary to our expectations, we observed an increase in the proportion of γδTreg cells ( Figure 4 The MFI of Foxp3 in γδ T cells also showed an increasing trend, challenging our initial hypothesis. These findings suggest that γδ T cells in BA may have dual functions, with IL-17-producing γδ Th17 cells playing a pathogenic role and γδ Treg cells playing a role in immune homeostasis.

[0077] Some studies have proposed that BA is an autoimmune disease. Immune imbalance of the Th17 / Treg axis has been reported in other autoimmune diseases, such as experimental colitis and uveoretinitis. Similarly, an imbalance of γδT cells in the γδTh17 / γδTreg ratio has been found in experimental allergic asthma. Our study revealed a similar γδTh17 / γδTreg imbalance in BA. However, unlike the common imbalance (increased γδTh17 ratio and decreased γδTreg ratio), we observed an increased ratio of γδTh17 and γδTreg cells, as well as an increased MFI of Foxp3 in BA ( Figure 4 The ratio of γδTreg / γδTh17 in the BA group was higher than that in the control group ( Figure 4 This may indicate that although γδT cells in the BA group differentiate into γδTh17 and γδTreg subtypes, differentiation into γδTreg cells is more obvious. Based on our findings and previous studies, we analyzed the differentiation of γδT cells in the liver of late BA and the potential underlying mechanisms ( Figure 7 Furthermore, this discrepancy may reflect differences between early and late stages of BA. In mouse BA models, an increase in the ratio of γδTh17 to γδTreg cells may occur during the early stages of inflammation. However, in late-stage BA, as seen in pediatric patients undergoing liver transplantation, inflammatory triggers may no longer be present, and γδTreg cells may assume a dominant role in maintaining immune balance. This could explain the higher γδTreg differentiation observed during this stage.

[0078] To further explore the impact of γδT cells in patients with BA, we performed correlation analyses between the γδT cell ratio and laboratory parameters. Although several markers (GLB, AST, CHE, PAB, UA, LDL, L, and STP) were statistically significant in the correlation analyses, we currently do not believe that γδT cells influence these laboratory parameters, as GLB, UA, and LDL are within normal ranges in patients with BA. Serum total protein is composed of multiple proteins, primarily ALB and GLB. Elevated serum total protein may reflect accelerated protein synthesis in the liver in response to immune challenges and during repair. In BA, liver damage and immune system activation lead to an increase in the γδT cell ratio, which may also promote the synthesis of certain proteins, including immunoglobulins. Therefore, a positive correlation was observed between serum total protein and the γδT cell ratio.

[0079] AST is primarily released from hepatocytes, reflecting hepatocyte damage, and increases during the progression of liver fibrosis. This negative correlation suggests that γδT cells (with an increased proportion of γδTreg cells) may play a protective role in late-stage BA-associated liver fibrosis. However, the persistent secretion of IL-17 by γδT cells highlights their conflicting roles. It is possible that γδT cells play a pathogenic role in early BA and a protective role in late BA.

[0080] CHE is an enzyme synthesized by the liver, and its level is closely related to liver synthetic function. PAB is a protein synthesized by the liver that primarily functions as a transporter and plays an important role in plasma as an indicator of nutritional status. CHE, PAB, and γδ T cells are negatively correlated, suggesting that liver function may gradually decline with increasing γδ T cell levels. Combined with the previous discussion on γδ T cells and AST, we propose that although γδ T cells exhibit some protective effects, the expansion of protective γδ Treg cells may occur too late in advanced BA to offset established fibrosis or IL-17-driven inflammation.

[0081] In conclusion, our study highlights a unique pattern of γδ T cell immune imbalance in advanced BA, which differs from other autoimmune diseases. Our discovery of a potential protective role for γδ T cells in advanced BA highlights the importance of γδ Treg cells in maintaining immune homeostasis. These results provide a foundation for γδ T cell-targeted therapies in BA.

Claims

1. Use of liver γδ T cells as a target for the development or screening of drugs for the treatment of biliary atresia.

2. The use according to claim 1, characterized in that: Among the liver γδT cells, γδTh17 cells that produce IL-17 play a pathogenic role, and γδTreg cells play an immune homeostatic role.

3. The use according to claim 2, characterized in that: The drug promotes the differentiation of liver γδT cells into γδTreg cells.

4. Use of an agent that promotes the differentiation of hepatic γδT cells into γδTreg cells in the preparation of a drug for treating biliary atresia.

5. Use of a substance for detecting a biomarker in the preparation of a product for diagnosing biliary atresia, characterized in that: The biomarker is liver γδT cells, and the product includes a detection reagent, a kit or a detection system.

6. The use according to claim 5, characterized in that: In patients with biliary atresia, hepatic γδT cells increased, including γδTh17 subsets, γδTreg subsets, and CD4 - CD8 - γδT cells, CD3 + γδ T cells and CCR6 + The proportion of γδT cells increased.

7. Use of a substance for detecting a biomarker in the preparation of a product for assessing the progression of biliary atresia, characterized in that: The biomarker is liver γδT cells, and the product includes a detection reagent, a kit or a detection system.

8. The use according to claim 7, characterized in that: Compared with early-stage patients, the ratio of γδTreg / γδTh17 was increased in patients with advanced biliary atresia.

9. A system for assessing the progression of biliary atresia, characterized in that The system comprises: (1) an analysis unit, the analysis unit comprising: a detection substance for determining the proportion of liver γδ T cells as a biomarker in a test sample of a subject; (2) An evaluation unit, comprising: determining the progression of biliary atresia in the subject based on the biomarker ratio determined in (1).

10. The system according to claim 9, characterized in that: The liver γδT cell ratio refers to the cell ratio of the γδTreg subpopulation to the γδTh17 subpopulation. Compared with early-stage patients, the γδTreg / γδTh17 ratio is increased in subjects with late-stage biliary atresia.

Images