Application of Gal-9 + Th cell and / or Gal-9 + Treg as AIH detection target

Abstract

The invention discloses an application of a Gal-9 + Th cell and / or Gal-9 + Treg as an AIH detection target spot. Researches find that compared with a healthy control group, the peripheral blood Gal-9 + Treg cell proportion and Gal-9 + Th cell proportion of an AIH patient baseline group are remarkably increased, and after ALT of a patient in a treatment group returns to normal, the Gal-9 + Treg cell proportion and the Gal-9 + Th cell proportion are both decreased compared with those before treatment, and it is indicated that the Gal-9 + Treg cell proportion and the Gal-9 + Th cell proportion are effective indexes for monitoring disease improvement. However, the AIH is a chronic disease process, even if the ALT level returns to normal, Gal-9 + Treg cells and Gal-9 + Th cells still do not return to the level of a healthy control group, the Gal-9 + Th cells are taken as detection targets, the progress of the AIH disease course can be monitored more accurately, and disease repetition and biochemical rebounding caused by premature drug withdrawal are prevented.

Description

Technical Field

[0001] The present invention relates to the application of Gal-9+Th cells and / or Gal-9+Treg as AIH detection targets. Background Art

[0002] Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease characterized by elevated serum transaminases, circulating autoantibodies, hypergammaglobulinemia, characteristic liver histological changes (interface hepatitis, portal lymphoplasmacytic infiltration, and rosetting), and a poor response to immunosuppressive therapy. AIH is a global disease that can occur at any age, with an insidious onset and progression to cirrhosis. A recent meta-analysis showed that the annual incidence of primary hepatocellular carcinoma (HCC) in AIH patients was 3.06‰, while the annual incidence of HCC in AIH patients with cirrhosis was 10.07‰. Notably, in this analysis, 92 of the 93 HCC patients were found to have cirrhosis at the time of diagnosis.

[0003] Therefore, timely patient assessment is crucial, necessitating a clear understanding of the disease's pathogenesis and therapeutic targets. However, the exact etiology and pathogenesis of AIH remain incompletely understood. Several studies examining the role of Treg cells in AIH have yielded inconsistent and even conflicting results. Longhi et al. suggest that the number of peripheral blood Tregs is significantly reduced at the onset of AIH compared with drug remission. Unlike healthy controls, Tregs are unable to regulate CD8 T cell expansion and the production of cytokines such as IFN-γ. However, some Treg cell function can be restored during remission. Peiseler et al., in their study, found no significant difference in the number or function of CD4+CD25+CD127-FOXP3+ Treg cells between AIH patients and healthy controls. In fact, Treg cells were increased in patients with active AIH compared with those in remission. In liver biopsy specimens, the number of Treg cells in the liver was higher in AIH patients than in patients with nonalcoholic steatohepatitis (NASH), suggesting that Treg cell number may be positively correlated with the severity of the inflammatory response.

[0004] Therefore, it is very important to further study the role of Treg cells in AIH patients.

[0005] Galectin-9 (Gal-9) is a novel eosinophil chemoattractant and a member of the galectin family. Highly expressed in the liver, it maintains hepatic homeostasis by exerting biological functions in innate and adaptive immunity. It exhibits important pleiotropic immunomodulatory properties, including involvement in cell differentiation, maturation, adhesion, aggregation, chemotaxis, activation, and apoptosis. It is currently the only confirmed ligand for the Tim-3 protein. Observations have shown a negative correlation between Gal-9+ Tregs and immunoglobulin levels and autoantibody titers, both of which are considered serological hallmarks of the disease. This study further treated Tregs in each group with Gal-9 interfering RNA to downregulate Gal-9 gene expression. The study found that the suppressive function of T lymphocytes on Tregs was significantly reduced in all groups, further supporting the idea that Gal9+ Tregs may be a key target for disease control in AIH.

[0006] In co-culture experiments, Gal-9+ Th cells can regulate Th17 / Treg development. These regulatory effects are sensitive to Gal-9 antagonists, inhibiting IL-17A production, but insensitive to IL-10 and TGF-β blockers. This suggests that Gal-9+ Th cells play an independent and important role in the diagnosis and treatment of immune diseases. However, unlike Gal-9+CD4+CD25+ Treg cells, Gal-9+ Th cells rarely co-express Tim-3. The frequency of IL-10+ CD4+ T cells is significantly lower in Gal-9 knockout mice than in wild-type mice. In vitro, Gal-9+ Th cells in the peripheral blood of healthy individuals secrete larger amounts of Gal-9 and higher levels of IL-10 and TGF-β mRNA than Gal-9-deficient Th cells. There is no difference in IL-2 and IFN-γ expression, while IL-4 and IL-17 levels are significantly reduced.

[0007] Th cells are a functional subset that assists T cell responses, while Treg cells are a subset with immunosuppressive functions. It is necessary to study Gal-9+ Th cells and Gal-9+ Tregs to observe the impact of the Tim-3 signaling pathway and its role in the pathogenesis of AIH. Summary of the Invention

[0008] The purpose of the present invention is to provide the use of Gal-9+Th cells and / or Gal-9+Treg as AIH detection targets. By detecting the ratio of Gal-9+Th cells and / or Gal-9+Treg, the progression of AIH can be more accurately reflected.

[0009] The technical solution adopted in the present invention is: Application of Gal-9+Th cells and / or Gal-9+Treg as AIH detection targets.

[0010] The AIH is autoimmune hepatitis.

[0011] Preferably, Gal-9+Th cells are used as targets for AIH detection.

[0012] The present study showed that the AIH patient baseline group had significantly increased peripheral blood Gal-9+ Treg cell and Gal-9+ Th cell ratios compared to the healthy control group, indicating that AIH patients have related immune dysfunction. The peripheral blood Gal-9+ Treg cell ratio and the Gal-9+ Th cell ratio are significantly positively correlated. Given the significant positive correlation between the peripheral blood Gal-9+ Treg cell ratio and the Gal-9+ Th cell ratio, it is possible to detect only Gal-9+ Th cells as the detection target for AIH without simultaneously detecting Gal-9+ Treg cells. This simplifies the detection process and effectively reflects the relevant disease status.

[0013] Furthermore, Gal-9+Th cells and / or Gal-9+Tregs can be used as detection targets during the recovery period of AIH treatment.

[0014] The present invention also provides the use of Gal-9+Th cells and / or Gal-9+Treg as detection targets in the preparation of AIH detection reagents.

[0015] Furthermore, it is preferred to prepare AIH detection reagents using Gal-9+Th cells as detection targets.

[0016] The present invention also provides an AIH detection reagent, which includes a reagent for detecting the ratio of Gal-9+ Th cells and / or Gal-9+ Treg cells. Preferably, the AIH detection reagent includes a reagent for detecting the ratio of Gal-9+ Th cells.

[0017] The AIH detection reagent is preferably used to monitor the progression of AIH during the recovery period of AIH treatment.

[0018] AIH detection reagents can quantitatively measure the proportion of Gal-9+ Th cells and / or Gal-9+ Treg cells in peripheral blood, thereby assisting in the diagnosis of the activity and severity of autoimmune hepatitis (AIH). This test reagent can provide clinicians with an objective biomarker to help assess disease progression, monitor treatment efficacy, and predict the risk of disease relapse. It provides an important basis for the development of personalized treatment plans and improves the precision and effectiveness of treatment.

[0019] Reagents for detecting the proportion of Gal-9+ Th cells are generally based on flow cytometry (FACS) technology. Specific antibodies are used to label cell surface molecules such as Galectin-9 (Gal-9) and CD3, CD4, CD25, and CD127, thereby achieving quantitative analysis of Gal-9+ Th cells and indicating the AIH condition.

[0020] The present invention has the following beneficial effects: During the treatment of AIH, biochemical indicators such as ALT levels reaching normal values ​​are typically used as a criterion for evaluating treatment efficacy. However, the present invention's study found that the percentage of peripheral blood Gal-9+ Treg cells and Gal-9+ Th cells was significantly elevated in AIH patients at baseline compared to the healthy control group, indicating related immune dysfunction in AIH patients. In contrast, after ALT levels returned to normal in the treatment group, both the percentage of Gal-9+ Treg cells and the percentage of Gal-9+ Th cells decreased compared to pre-treatment, demonstrating that both are effective indicators for monitoring disease progression. However, these levels remained elevated relative to the healthy control group. Furthermore, there was no correlation between Gal-9 Treg cells and Gal-9+ Th cells and peripheral blood ALT levels. This suggests that AIH is a chronic disease process. Even after ALT levels return to normal, Gal-9 Treg cells and Gal-9+ Th cells may not return to healthy control group levels. Therefore, immune recovery in AIH is a chronic process. Therefore, normal peripheral biochemical indicators do not indicate immune homeostasis, requiring long-term medication observation. Using Gal-9+Th cells as detection targets can more accurately monitor the progression of AIH and prevent premature discontinuation of medication leading to disease recurrence and biochemical rebound. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure is a bar graph comparing the proportion of Gal-9+Treg cells in peripheral blood among the three groups.

[0022] Figure 2 The figure is a bar graph comparing the ratio of Gal-9+Th cells in peripheral blood among the three groups.

[0023] Figure 3 This is a bar graph comparing the peripheral blood IL10 levels among the three groups.

[0024] Figure 4 The figure is a bar graph comparing the peripheral blood ALT levels among the three groups.

[0025] Figure 5 This is a correlation analysis chart between peripheral blood Gal-9Treg and Gal-9+Th. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

[0027] Example 1 1 Research subjects Twenty patients with autoimmune hepatitis who were outpatients and inpatients in Hangzhou First People's Hospital from April 2018 to December 2020 were selected, including 17 females and 3 males, aged 28 to 80 years, with an average age of 58.6±16.5 years. At the same time, 20 healthy people of the same age group who underwent physical examination in our hospital and had normal physical examination indicators were selected, including 16 females and 4 males, aged 35 to 70 years, with an average age of 53.0±10.7 years. There was no significant difference in gender and age between the two groups (P>0.05).

[0028] 1.1 Inclusion criteria Diagnostic Criteria: Patients with autoimmune hepatitis must meet the clinical diagnosis of AIH (according to the AIH diagnostic scoring system updated by the International Autoimmune Hepatitis Group (IAIHG) in 1999). All patients must not have used glucocorticoids or other medications that affect immune function within the six months prior to enrollment.

[0029] 1.2 Exclusion criteria 1) Exclude patients with concurrent hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis G, HIV and other infectious diseases; 2) Exclude other liver diseases such as fatty liver, schistosomiasis-induced liver fibrosis, and drug-induced hepatitis; 3) Exclude serious heart, brain, kidney, tumor and other diseases; 4) Pregnant or breastfeeding women were excluded. 1.3 Grouping criteria According to the 2015 consensus on the diagnosis and treatment of autoimmune hepatitis in my country, 20 AIH patients without treatment were divided into the baseline group, those with ALT levels within the normal range after treatment were divided into the treatment group, and 20 healthy controls were divided into the control group.

[0030] 1.4 Treatment plan: Standard regimen group: Hormones alone or in combination with immunosuppressants (drugs and dosages are based on the 2015 consensus treatment plan for the diagnosis and treatment of autoimmune hepatitis in my country) are used to ultimately achieve normal peripheral blood ALT levels.

[0031] Oral prednisone (sole) 30 mg / d for 1 week, 20 mg / d for 2 weeks, and 15 mg / d for 4 weeks. When the prednisone (sole) dose is lower than 15 mg / d, it should be gradually reduced by 2.5 mg / d to the maintenance dose (5-10 mg / d). After one week of oral prednisolone, add azathioprine 50 mg / d.

[0032] 2 Experimental methods 2.1 Patients signed informed consent forms. Baseline data were collected from the patients. Peripheral blood was drawn to detect the levels of Gal-9+Th cells, Gal-9+Treg cells, related cytokines such as IL-10, liver function, autoantibody titers, IgG levels, etc. Liver puncture biopsy was performed for pathological examination.

[0033] 2.2 After the peripheral blood ALT level reached the normal range after treatment, the treatment group was tested for peripheral blood Gal-9+Th cells, Gal-9+Treg cell levels, related cytokines such as IL-10 and liver function ALT.

[0034] 2.3 The levels of peripheral blood Gal-9+Th cells, Gal-9+Treg cells, cytokines such as IL-10, and liver function ALT were detected in the healthy control group.

[0035] 2.4 Data Analysis Statistical analysis was performed with SPSS Statistics 16.0, using the t-test. One-way analysis of variance was used for comparisons between groups. Correlation and regression analysis were used for data correlations, and nonparametric tests were used for skewed distributions. P < 0.05 was considered significant.

[0036] 3 Detection methods ① Peripheral mononuclear cell isolation Peripheral blood mononuclear cells (PBMCs) were isolated using lymphocyte separation medium.

[0037] ② Detection of Gal-9+Th and Gal-9+Treg cell numbers Flow cytometry was used to detect the number of Gal-9+Th and Gal-9+Treg cells using fluorescently labeled antibodies.

[0038] ③ IL-10 was detected by flow cytometry; serum liver function, IgG, autoantibody titer, etc. were measured using a fully automatic biochemical analyzer and related instruments.

[0039] In step ②, the method for detecting peripheral blood CD3+CD4+CD25+CD127+anti-galectin9-Treg (Gal-9+Treg) and CD3+CD4+CD25+ anti-galectin9 (Gal-9+Th) cells is as follows: a. Number the flow cytometry tubes and add 10 μL FITC-labeled mouse anti-human CD4 monoclonal antibody, 10 μL FITC-labeled mouse anti-human CD3 monoclonal antibody, 3 μL PE-labeled mouse anti-human CD25 monoclonal antibody, 3 μL PE-Cy7-labeled mouse anti-human CD127 monoclonal antibody, and 3 μL PE-labeled mouse anti-human Anti-Galectin9 monoclonal antibody to the tubes in sequence; b. Carefully add 25 μL of the mixed EDTA anticoagulated blood sample to the bottom of the test tube, swirl thoroughly at low speed for 3 seconds, and incubate at room temperature (20-25°C) in the dark for 15 minutes (Note: Avoid direct sunlight exposure during the incubation process, and avoid the sample from sticking to the wall of the test tube during the addition process); c. Add 1 mL of 10-fold diluted FACS hemolytic reagent to each tube, immediately vortex thoroughly at low speed for 3 seconds, and incubate at room temperature in the dark for 10 minutes. d. Add 2 mL of PBS buffer to each tube, vortex thoroughly at low speed for 3 seconds, centrifuge at 1200 rpm for 5 minutes, and discard the supernatant. e. Add 3 mL of PBS to each tube to resuspend the cells, vortex thoroughly at low speed for 3 seconds, centrifuge at 1200 rpm for 5 minutes, and discard the supernatant; f. Add 0.25 mL of PBS to each tube to resuspend the cells to create the desired single-cell suspension. h. Flow cytometry The prepared single-cell suspension was tested using a Calibur flow cytometer from BD (BioNTech). After startup, the instrument's sensitivity was tested using CaliBRITE 4 fluorescent microspheres and FACSComp software. Experimental acquisition conditions, including photomultiplier tube voltage and fluorescence compensation, were automatically set. CellQuest software was then used to further optimize the experimental acquisition conditions using samples. Each group of sample tubes was tested, acquiring 10,000 cells. Finally, CellQuest software was used to analyze the various CD molecules on the cell surface.

[0040] 4 Test results 4.1 Patient Baseline Characteristics There were 20 AIH patients and 20 healthy subjects. There was no significant difference in gender and age between the two groups ( P >0.05), the baseline peripheral blood Gal-9+Treg, Gal-9+Th, and ALT levels in AIH patients were significantly higher than those in the healthy group, and the differences were statistically significant ( P <0.01), and there was no significant difference in peripheral blood IL-10 between the two groups ( P >0.05). The comparison of baseline indicators of 20 AIH patients and healthy controls is shown in Table 1.

[0041] Table 1 Comparison of baseline indicators of 20 AIH patients and healthy controls 4.2 Flow cytometry detection of the ratio of Gal-9+Treg and Gal-9+Th cells in peripheral blood 4.2.1 Gal-9+ Tregs were almost undetectable in healthy control samples (0.00±0.00)%. Compared with the healthy control group, the proportion of Gal-9+ Tregs in AIH baseline samples was (18.84±17.46)%, which was significantly increased ( P <0.01), the proportion of Gal-9+Treg in the samples of the treatment group was (12.43±13.44)%, which was significantly higher than that in the healthy control group (P<0.01), but there was no significant difference compared with the baseline group ( P =0.35). The comparison of the ratio of Gal-9+Treg cells in peripheral blood among the three groups is shown in the bar graph. Figure 1 shown.

[0042] 4.2.2 The proportion of Gal-9+Th in healthy control samples was (0.20±0.18)%. Compared with the healthy control group, the proportion of Gal-9+Th in AIH baseline samples was significantly increased (8.26±9.18)%. P <0.01), the ratio of Gal-9+Th in the samples of the treatment group was (4.41±5.91)%, which was higher than that in the healthy control group, but there was no statistical difference ( P =0.109), and there was no significant difference compared with the baseline group ( P =0.124). The comparison of the ratio of Gal-9+Th cells in peripheral blood among the three groups is shown in the bar graph. Figure 2 shown.

[0043] 4.3 Flow cytometry detection of peripheral blood IL-10 content The peripheral blood IL-10 level in the healthy control group was (1.79±0.58) pg / ml; the peripheral blood IL-10 level in the AIH baseline group was (1.98±2.13) pg / ml; and the peripheral blood IL-10 level in the AIH treatment group was (2.19±0.45) pg / ml. There was no significant difference between the three groups (F=0.473, P =0.625), the comparison of peripheral blood IL10 levels among the three groups is shown in the bar graph Figure 3 .

[0044] 4.4 Detection of ALT levels in peripheral blood by fully automatic biochemical analyzer The peripheral blood ALT level of the healthy control group was (18.80±3.86) U / L; the peripheral blood ALT level of the AIH baseline group was (69.00±62.96) U / L. Compared with the healthy group, the AIH baseline group was significantly higher, with significant differences ( P <0.01), the peripheral blood ALT level in the AIH treatment group was (27.15±9.65) U / L, which was significantly lower than that in the baseline group ( P <0.01), and there was no significant difference compared with the healthy group ( P =1.0), the comparison of peripheral blood ALT levels among the three groups is shown in the bar graph Figure 4 .

[0045] 4.5 Bivariate correlation analysis of peripheral blood ALT level and IL-10 content with the peripheral blood Gal-9+Treg cell ratio and Gal-9+Th cell ratio showed no correlation.

[0046] 4.6 Correlation between the Peripheral Blood Gal-9 Treg Cell Proportion and Gal-9+ Th The bivariate correlation analysis of the ratio of Gal-9 Treg cells (10.42±14.78)% and the ratio of Gal-9+ Th cells (4.37±7.00)% in the three groups showed a significant positive correlation (r=0.791, P <0.01). Correlation analysis between peripheral blood Gal-9Treg and Gal-9+Th is shown in the figure Figure 5 .

[0047] 5 Research results First, the proportion of Gal-9+Treg cells and Gal-9+Th cells in the peripheral blood of the AIH patient baseline group was significantly increased compared with the healthy control group, indicating that AIH patients have related immune dysfunction.

[0048] Second, there was a significant positive correlation between the proportion of Gal-9+Treg cells and the proportion of Gal-9+Th cells in peripheral blood, and peripheral blood Gal-9+Th cells may play an independent role in the diagnosis and treatment of AIH.

[0049] Third, the proportion of Gal-9+Treg cells and Gal-9+Th cells in the peripheral blood of the AIH treatment group (biochemical response) decreased compared with before treatment, but there was no statistical significance, which may indicate that AIH immune recovery is a chronic process and requires long-term medication observation.

[0050] The present invention observes the changes in relevant immune cells in AIH patients by detecting and comparing the ratios of Gal-9+ Treg cells and Gal-9+ Th cells in the peripheral blood of AIH patients before and after treatment. After AIH reaches a biochemical response, the peripheral blood Gal-9+ Treg cells and Gal-9+ Th cells still do not reach the levels of the healthy control group, indicating that AIH is a chronic disease that requires long-term treatment; peripheral blood Gal-9+ Th cells may play an independent role in the diagnosis and treatment of AIH.

[0051] Before the present study, it was expected that the levels of Gal-9+ Th cells and Gal-9+ Treg cells in the peripheral blood of AIH patients would be lower before treatment (disease phase) compared with the healthy control group; after treatment (drug remission phase), the levels would show no significant difference from the healthy control group, but would be significantly higher than before treatment. However, during the study, flow cytometry revealed that when cells were permeabilized to detect Gal-9+ Treg and Gal-9+ Th cells, both were elevated in the healthy control group and the baseline group, reaching almost 100%, making the comparison insignificant. Without permeabilization, significant differences were found between the two groups. In healthy control samples, Gal-9+ Tregs were almost undetectable, while the proportion of Gal-9+ Tregs in AIH baseline samples was significantly increased compared with the healthy control group, demonstrating a significant difference. In post-treatment follow-up observations of samples from the treated group (where ALT levels normalized), the proportion of Gal-9+ Tregs was significantly increased compared with the healthy control group, but not significantly different from the baseline group. Similarly, the proportion of Gal-9+ Th cells in AIH baseline samples was significantly higher than that in the healthy control group, demonstrating a statistically significant difference. In samples from patients in the treatment group, the proportion of Gal-9+ Th cells increased compared with the healthy control group and decreased compared with the baseline group, but neither difference was significant. Bivariate correlation analysis revealed a significant positive correlation between Gal-9+ Treg cells and Gal-9+ Th cells. However, neither correlated with peripheral blood ALT levels or IL-10 levels, respectively. Therefore, this study reveals that AIH is a chronic disease process that requires long-term medication to achieve immune homeostasis. Normal peripheral biochemical parameters do not indicate immune homeostasis, which is one reason why many AIH patients are prone to disease progression and biochemical rebound after drug discontinuation. The finding that both Gal-9+ Treg and Gal-9+ Th cells were significantly elevated in AIH patients compared with healthy controls and decreased with biochemical response to treatment suggests that both are effective indicators for monitoring disease progression. Their significant positive correlation suggests that monitoring Gal-9+ Th cells may be the optimal indicator for detecting AIH.

Claims

1. Application of Gal-9+Th cells and / or Gal-9+Treg as targets for AIH detection.

2. The use according to claim 1, characterized in that Application of Gal-9+Th cells as targets for AIH detection.

3. Application of Gal-9+Th cells and / or Gal-9+Treg as detection targets in the preparation of AIH detection reagents.

4. The use according to claim 3, characterized in that AIH detection reagent was prepared using Gal-9+Th cells as detection targets.

5. An AIH detection reagent, comprising a reagent for detecting the ratio of Gal-9+ Th cells and / or Gal-9+ Treg cells.

6. The AIH detection reagent according to claim 5, characterized in that The AIH detection reagent includes a reagent for detecting the ratio of Gal-9+Th cells.

Images