Application of T cells as biomarkers in monitoring or predicting the efficacy of anti-HBV treatment or predicting the risk of relapse

Abstract

The present invention discloses the application of T cells as biomarkers in the preparation of products for monitoring or predicting the efficacy of anti-HBV treatment, wherein the T cells include Treg cells, CD4+T cells, and CD8+T cells; and also discloses the application of T cells as biomarkers in the preparation of products for predicting the risk of relapse after achieving clinical functional cure in anti-HBV treatment, wherein the T cells include CD8+ subgroup CXCR5+CD8+T cells and CD4+ subgroup CXCR5+CD4+T cells. The present invention can be applied to guide interferon antiviral treatment of chronic hepatitis B, provide new immune targets for exploring antiviral treatment of chronic hepatitis B, improve cure rate, and reduce relapse rate.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of T cells as biomarkers in monitoring or predicting the efficacy of anti-HBV treatment or predicting the risk of recurrence. Background Art

[0002] Chronic hepatitis B (CHB) is a type of infectious disease caused by the hepatitis B virus (HBV) that seriously endangers the lives of the public. The treatment of CHB aims to reduce the occurrence of cirrhosis, liver failure and HCC, and improve the quality of life of patients and prolong their survival. "Actual functional cure" is the goal pursued by medical workers now. "Functional cure" is different from "inactive HBV carriers after drug withdrawal". It means that HBsAg turns negative, anti-HBs is negative or positive, HBeAg is negative, serum HBVDNA is below the detection limit, and the risk of HCC decreases over time; it is different from "complete and thorough cure" in that the cccDNA in the liver is still detectable but inactive. As a first-line drug for achieving clinical cure, pegylated interferon (Peg-IFN) has a dual mechanism of direct antiviral and immunomodulatory action, which can significantly increase the HBsAg loss rate of chronic hepatitis B patients. In terms of antiviral effect, it can block the formation of core particles, regulate cccDNA epigenetics, and ISGs-mediated antiviral defense in infected and non-infected cells, thereby reducing HBVDNA and HBV antigen (HBsAg, HBcAg, HBeAg) expression; in terms of immunomodulatory effect, it can increase NK cell TRAIL expression, activate CD56brihgtNK cells, increase CD8+T cell response in small amounts, and regulate CD4+T cell response, thereby enhancing the production of direct antiviral IFN-γ, promoting T cell response, and finally achieving clinical cure. The study by WuD et al. believes that the future of chronic hepatitis B patients is to combine multi-target intervention with immunomodulation on the basis of sustained viral control and reshape the body's antiviral immune efficacy. In recent years, reports on immunomodulatory research have received increasing attention from scholars. XiaY et al. believed that IFN-γ and TNF-α produced by T cells can reduce HBV cccDNA by inducing deamination and subsequent cccDNA decay; Wang et al. believed that the interaction mechanism between Foxp3 (surface marker of Treg) and IL-17 / IL-23 can become a new breakthrough point for HBV clearance, and Foxp3 is positively correlated with HBV DNA and HBsAg.

[0003] In CHB, the activation of the CXCL13 and IL-21 axis is significantly correlated with the occurrence of functional cure. Shen Z et al. demonstrated through animal models that IL-21 and IL-33 are closely related to the clearance of HBV. CXC chemokine receptor 5 (CXCR5) and its cognate receptor CXCL13 are members of the CXC subtype of the chemokine superfamily, which can chemotactic mature B cells and T follicular helper cells to migrate to B cell follicles and germinal centers, secreting memory B cells and plasma cells. The subset of CD8+T cells, CXCR5+CD8+T cell follicular cytotoxic T cells, play an important role in controlling chronic viral infection. Professor Ren Hong's research group found that the proportion of γδT cells was positively correlated with the replication of HBV and negatively correlated with the degree of liver inflammation. The lower the proportion of its effector memory subpopulation, the better the efficacy of Peg-IFNα-2a combined with TDF in the treatment of CHB.

[0004] In summary, many studies have confirmed the importance of enhancing host immune function in Peg-IFN combined with TDF anti-HBV treatment, which is one of the key factors for us to improve the clinical cure rate of chronic hepatitis B. However, after achieving clinical functional cure, some patients, about 9.6%, relapsed, that is, hepatitis B surface antigen was positive or HBV-DNA was positive. Therefore, studying the differences between relapse and non-relapse is of great significance for guiding interferon antiviral treatment of chronic hepatitis B, providing new immune targets for exploring antiviral treatment of chronic hepatitis B, improving cure rate, and reducing relapse rate. Summary of the invention

[0005] One of the purposes of the present invention is to provide the use of T cells as biomarkers in the preparation of products for monitoring or predicting the efficacy of anti-HBV treatment, wherein the T cells include Treg cells, CD4+T cells, and CD8+T cells.

[0006] In the above technical scheme, changes in CD4+T cell content, Treg cell content and CD4 / CD8 ratio are used to predict or monitor the efficacy of anti-HBV treatment; compared with the reference, the subject's CD4+T cell content is significantly decreased, the CD4 / CD8 ratio shows a significant downward trend, and the Treg cell content is significantly decreased, indicating that the drug is effective, and vice versa; preferably, the reference is the cell content of a normal group and / or the subject before medication.

[0007] Another object of the present invention is to provide the use of T cells as biomarkers in the preparation of products for predicting the risk of relapse after achieving clinical functional cure in anti-HBV treatment, wherein the T cells include CD8+ subpopulation CXCR5+CD8+T cells and CD4+ subpopulation CXCR5+CD4+T cells.

[0008] In the above technical scheme, compared with the reference group of the non-relapsed group, a significant decrease in the subjects' CXCR5+CD8+T cells and CXCR5+CD4+T cells indicates a high risk of HBsAg re-positivity. Conversely, a high level of CXCR5+CD8+T cells and CXCR5+CD4+T cells in T cells indicates no or low risk of re-positivity.

[0009] Another object of the present invention is to provide a method for detecting the amount of Treg cells, CD4+T cells, and CD8+T cells for use in preparing a product for monitoring or predicting the efficacy of anti-HBV treatment.

[0010] In the above technical scheme, changes in CD4+T cell content, Treg cell content and CD4 / CD8 ratio are used to predict or monitor the efficacy of anti-HBV treatment; compared with the reference, the subject's CD4+T cell content is significantly decreased, the CD4 / CD8 ratio shows a significant downward trend, and the Treg cell content is significantly decreased, indicating that the drug is effective, and vice versa.

[0011] Another object of the present invention is to provide a method for detecting the amount of CD8+ subpopulation CXCR5+CD8+T cells and CD4+ subpopulation CXCR5+CD4+T cells in the preparation of a product for predicting the risk of relapse after achieving clinical functional cure in anti-HBV treatment.

[0012] In the above technical scheme, compared with the reference group of the non-relapsed group, a significant decrease in the subjects' CXCR5+CD8+T cells and CXCR5+CD4+T cells indicates a high risk of HBsAg re-positivity. Conversely, a high level of CXCR5+CD8+T cells and CXCR5+CD4+T cells in T cells indicates no or low risk of re-positivity.

[0013] In the above-mentioned application technology scheme, the T cells refer to T cells in peripheral blood; the anti-HBV treatment refers to Peg-IFN treatment, preferably a combination of Peg-IFN and tenofovir.

[0014] The substance is selected from antibodies and chips.

[0015] The beneficial effects of the present invention are:

[0016] The present invention analyzes the effects of various immune cell functions on clinical recurrence after Peg-IFNα combined with TDF treatment of chronic hepatitis B achieves clinical functional cure, as well as the differences between recurrence and non-recurrence, and observes the expression of various immune cells in whole blood specimens after interferon treatment achieves functional cure. Through the above research results, we can guide the interferon antiviral treatment of chronic hepatitis B, provide new immune targets for exploring antiviral treatment of chronic hepatitis B, improve the cure rate, and reduce the recurrence rate.

[0017] The present invention has been found through research, and through experimental results and clinical data analysis, it can be seen that the distribution and dynamic changes of peripheral blood T lymphocytes of Peg-IFN combined with TDF after anti-HBV achieves clinical cure, among which the total proportion of CD4+T cells and Treg cells shows a downward trend, and the total proportion of CD8+T cells has no obvious change. The changes in CD4+T cells, Treg cells and CD4 / CD8 ratio can be used to monitor and predict the efficacy of Peg-IFNα-2b combined with tenofovir in anti-HBV treatment; CXCR5 plays an important role in the process of clinical cure and relapse after Peg-IFN combined with tenofovir in anti-HBV treatment. The total proportion of Tfh and Tfc in the Cure group is higher than that in the Re group. Similarly, the Re group is also lower than the CL group and the HC group. Therefore, when surface antigen clearance is achieved, Tfh and Tfc are maintained at a higher concentration, and their rapid decline is likely to indicate the re-positivity of HBsAg. Maintaining a high level of proportion is more conducive to the clearance of HBsAg and achieving clinical functional cure. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a flow cytometry analysis of CD3, CD4, CD8 and Treg cells in PBMC.

[0019] Figure 2 is the distribution of CD4+T cells in each group.

[0020] Figure 3 is the distribution of CD8+T cells in each group.

[0021] Figure 4 It is the distribution of the ratio of CD4+T cells to CD8+T cells in each group.

[0022] Figure 5 is the distribution of Treg cells in each group.

[0023] Figure 6 This is a flow cytometry analysis of Tfh cells and Tfc cells in PMBC.

[0024] Figure 7 is the distribution of CXCR5+CD4+T cells in each group.

[0025] Figure 8 is the distribution of CXCR5+CD8+T cells in each group. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0027] The experimental methods in the following examples are conventional methods unless otherwise specified; the reagents used are conventional reagents in the art and can be obtained commercially unless otherwise specified.

[0028] Example 1

[0029] 1 Materials and methods

[0030] 1.1 Research subjects

[0031] A total of 35 CHB patients who visited the First Affiliated Hospital of Chongqing Medical University from October 2019 to October 2021 were included as subjects.

[0032] Inclusion criteria: ① Adult (age >18 years) chronic hepatitis B patients, but younger than 60 years old; ② Agree to use Peg-IFNα-2b combined with tenofovir (TDF) for treatment and can adhere to outpatient follow-up; ③ Have understood the risks of this study and treatment and signed the informed consent form.

[0033] Exclusion criteria: ①Combined with other viral infections, such as hepatitis A, hepatitis C, hepatitis E and HIV infection; ②Contraindications to IFN treatment: such as severe infection, schizophrenia, severe end-stage liver disease, pregnant women, etc.; ③Combined with serious chronic diseases, such as coronary heart disease, alcoholic liver disease, end-stage renal disease and severe lupus erythematosus; ④Some serious adverse reactions occurred when using Peg-IFNα-2b combined with TDF.

[0034] Discontinuation criteria: normal liver function; HBsAg <0.04IU / mL (undetectable); HBV DNA <100IU / mL.

[0035] The personal and clinical information of the enrolled patients was recorded, including age, gender, treatment cycle, etc. The follow-up of the patients during treatment was maintained, including blood routine, liver function, hepatitis B two-to-half, HBV DNA content, AFP and other indicators. This study has been approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (2020-409).

[0036] 1.2 Experimental reagents and instruments

[0037] 1.2.1 Main experimental reagents

[0038]

[0039]

[0040] 1.2.2 Main experimental instruments and equipment

[0041]

[0042] 1.3 Experimental methods

[0043] 1.3.1 Specimen collection

[0044] 10 ml of peripheral blood was collected from healthy volunteers and patients included in the study in the outpatient clinic using vacuum blood collection tubes and the specimens were processed within two hours.

[0045] 1.3.2 PBMC extraction

[0046] (1) Centrifuge blood sample at 1200r / min for 10min

[0047] (2) Pipette 1 ml of serum into a 1.5 ml EP tube

[0048] (3) Add the remaining plasma into a 15 ml centrifuge tube containing 4 ml PBS and mix well.

[0049] (4) Slowly add 4 ml of eluent (slowly add at a 45° angle)

[0050] (5) Place in a centrifuge at 400G for 40 minutes and slowly rise and fall

[0051] (6) Place the serum in a centrifuge at 12000r for 10 minutes and then transfer it to a new EP tube.

[0052] (7) Use a capillary tube to aspirate the PBMCs suspended in the centrifuge tube

[0053] (8) Place in centrifuge and centrifuge at 300G for 8 minutes

[0054] (9) Aspirate the upper liquid

[0055] (10) Cell counting

[0056] (11) Place in centrifuge and centrifuge at 300G for 5 minutes

[0057] (12) Aspirate the upper liquid

[0058] (13) Add RPMI 1640 (800ul or 1.2ml) to the centrifuge tube

[0059] (14) Slowly add an equal amount of cell freezing solution

[0060] (15) Mix well and separate into 1.5 ml cryotubes (2 or 3 tubes)

[0061] (16) After gradient freezing, place the cryovials in liquid nitrogen for storage

[0062] 1.3.3 Cell recovery

[0063] (1) Prepare about 5 ml of RPMI1640 + 10% FBS as culture medium

[0064] (2) Take the PBMC out of the liquid nitrogen tank and place it in a 37°C water bath. When the ice is almost melted, transfer the PBMC to the culture medium.

[0065] (3) Centrifugation: 300 g, 5 min, room temperature, discard the supernatant;

[0066] (4) PBS counting: aspirate 20ul of PBMC and place it into a cell counter. Ensure that the number is no less than 2×10 under the microscope. 6 ;

[0067] (5) Centrifugation: 300 g, 5 min, room temperature, discard the supernatant and divide them into three tubes of 50 μl × 3 tubes, tube 1 blank tube, tube 2 sample tube, tube 3 isotype control.

[0068] 1.3.4 Flow cytometric analysis of peripheral blood mononuclear cells (PBMC)

[0069] (1) Fc blocking: add 2.5 μl of blocking agent to 50 μl of cell system, protect from light for 8 min, and do not wash;

[0070] (2) Add antibodies to each tube:

[0071] Tube 1 (blank): None

[0072] Tube 2 (sample tube):

[0073] PE MouseAnti-Human CD3: 0.5μl

[0074] FITC MouseAnti-Human CD8: 2μl

[0075] PerCP-Cy5.5 MouseAnti-Human CD4: 1μl

[0076] BV650 MouseAnti-Human CD25: 1μl

[0077] BV421 Rat Anti-Human CXCR5(CD185): 0.5μl

[0078] Alexa 647Mouse Anti-Human CD127: 2μl

[0079] Tube 3 (isotype control):

[0080] PE Mouse Anti-Human CD3: 0.5μl

[0081] FITC Mouse Anti-Human CD8: 2μl

[0082] PerCP-Cy5.5 Mouse Anti-Human CD4: 1μl

[0083] BV650 Mouse IgG1,κIsotype Control: 0.5μl

[0084] Alexa 647Mouse IgG1,κIsotype Control:0.5μl

[0085] BV421 Mouse IgG2b,κIsotype Control:0.5ul

[0086] (3) Add 1 ml of PBS and keep the tube in a dark place for 20 min.

[0087] (4) Centrifugation: 300 g, 5 min, room temperature, discard the supernatant;

[0088] (5) Add 400 μl of PBS again and centrifuge at 300 g for 5 min at room temperature to resuspend;

[0089] (6) Move through the filter membrane into the flow tube;

[0090] (7) Preparation of single positive tube: BD TM CompBeads Anti-Mouse Ig, Kappa and BD TM CompBeads

[0091] Negative Control was mixed into 6 tubes at a ratio of 1:1, with 50ul in each tube, and added to PE Mouse Anti-Human CD3, FITC Mouse Anti-Human CD8, PerCP-Cy5.5 Mouse Anti-Human CD4, BV650 Mouse IgG1,κIsotype Control, Alexa 647Mouse IgG1,κIsotype Control, BV421 MouseIgG2b,κIsotype Control in several tubes.

[0092] (6) Repeat the steps (3) and (6);

[0093] (7) Debug the instrument and conduct on-machine testing.

[0094] (8) Data analysis was performed using FlowJo.

[0095] 1.4 Statistical analysis

[0096] GraphPadPrism 8.0 was used for drawing and analysis. For measurement data, t-test was used when normality and variance homogeneity were met; Welch's corrected t-test was used when variance was unequal; Mann-Whitney rank sum test was used when normality was not met. Fisher's exact test was used for enumeration data. When P < 0.05, the difference between the groups was statistically significant.

[0097] 2 Results

[0098] 2.1 The distribution and dynamic changes of peripheral blood T lymphocytes were obvious after clinical cure of HBV by Peg-IFN combined with TDF

[0099] We divided PBMC into several groups: Healthy control (HC, n = 10), HBsAg clearance group (CL, n = 10), cure group (Cure, n = 10), and relapse group (Recrudescence, RE, n = 15). The healthy control group consisted of samples from healthy people with negative HBsAg and positive HBsAb. The HBsAg clearance group consisted of samples from patients who were HBsAg negative in the first test after Peg-IFN combined with TDF treatment. The cure group consisted of samples from patients whose HBsAg turned negative after Peg-IFN combined with TDF treatment and remained negative for half a year or more. The relapse group consisted of samples from patients whose HBsAg turned negative after Peg-IFN combined with TDF treatment and who became positive again after outpatient follow-up. CD3, CD4, CD8, and Treg cells in their PBMCs ( Figure 1 ) for flow cytometry analysis.

[0100] 2.1.1 Distribution of CD4+ T cells

[0101] The percentage of CD4+T cells in T cells (abbreviated as CD4+T total percentage) was significantly different between the Re group and the HC group, and between the Re group and the CL group. The HC group was higher than the Re group (P=0.0029), and the CL group was higher than the Re group (P<0.001). There was no significant difference between the Re group and the Cure group (P>0.05). Figure 2 .

[0102] 2.1.2 Distribution of CD8+ T cells

[0103] In the flow cytometry analysis, there were no significant differences in the percentage of CD8+T cells in T cells (abbreviated as CD8+T total percentage) between the Re group and the HC group, the Re group and the Cure group, and the Re group and the CL group (P>0.05). Figure 3 .

[0104] 2.1.3 Ratio of CD4+ T cells to CD8+ T cells

[0105] The ratio of CD4+T cells to CD8+T cells (abbreviated as CD4 / CD8 ratio) was significantly different between the Re group and the CL group, and the CL group was higher than the Re group (P=0.010). There was no significant difference between the Re group and the HC group, and between the Re group and the Cure group (P>0.05). Figure 4 .

[0106] 2.1.4 Distribution of Treg cells

[0107] The proportion of Treg cells in T cells (abbreviated as Treg proportion) was significantly different between the Re group and the CL group, and between the Re group and the HC group. The CL group was higher than the Re group (P=0.003), and the HC group was higher than the Re group (P=0.03). There was no significant difference between the Re group and the Cure group (P>0.05). Figure 5 .

[0108] In summary, it can be found that there are significant differences in the distribution of CD4+ and Treg cells in peripheral blood PBMCs in patients with CHB treated with Peg-IFNα-2b combined with TDF after hepatitis B surface antigen clearance, clinical cure, and relapse. It also shows that the dynamic changes of these immune cells are related to the efficacy of Peg-IFNα-2b. Among them, the changes in CD4+T cells are the most obvious. From the hepatitis B surface antigen clearance period to clinical cure or relapse, CD4+T cells are showing a downward trend. In comparison, the change trend of CD8+T cells is not obvious; and the CD4 / CD8 ratio has certain differences between the CL group and the Re group and the CL group and the Cure group. The CD4 / CD8 ratio shows a downward trend from the CL stage to the Re stage, while the difference in the CD4 / CD8 ratio from the CL stage to the Cure stage is not obvious. Changes in CD4+T cells, Treg cells and CD4 / CD8 ratio can be used to predict or monitor the efficacy of Peg-IFNα-2b combined with TDF.

[0109] 2.2CXCR5 plays an important role in whether HBV relapses after clinical cure with Peg-IFNα-2b combined with TDF anti-HBV treatment

[0110] Similarly, we studied two subpopulations of cells: CXCR5 follicular helper T cells (CXCR5+CD4+T cells) and follicular cytotoxic T cells (CXCR5+CD8+T cells). Figure 6 ) were used for flow cytometry analysis to investigate the differences among the HBsAg clearance group (CL group), cured group (Cure group), relapse group (Re group) and healthy control group (HC group).

[0111] 2.2.1 Distribution of CXCR5+CD4+T cells

[0112] The proportion of CXCR5+CD4+T in T cells (abbreviated as TFH proportion) was significantly different between the Re group and the CL group, the Re group and the HC group, and the Re group and the Cure group. The CL group was higher than the Re group (P<0.001), the HC group was higher than the Re group (P=0.0097), and the Cure group was higher than the Re group (P=0.031). Figure 7 .

[0113] 2.2.2 Distribution of CXCR5+CD8+T cells

[0114] The proportion of CXCR5+CD8+T in T cells (abbreviated as TFC proportion) was significantly different between Re and CL, Re and HC, and Re and Cure groups. The proportion of CXCR5+CD8+T in T cells was higher in CL group than in Re group (P=0.0006), higher in HC group than in Re group (P=0.0021), and higher in Cure group than in Re group (P=0.023). Figure 8 .

[0115] The results of flow cytometry experiments showed that there were significant differences in the distribution of TFH cells and TFC cells in patients with CHB treated with Peg-IFNα-2b combined with TDF after hepatitis B surface antigen clearance, clinical cure, and relapse. From the hepatitis B surface antigen clearance period to clinical cure or relapse, TFH cells and TFC cells showed a downward trend. In the early stage of hepatitis B surface antigen clearance, the TFH cells and TFC cells in the patient's peripheral blood were in a high expression state higher than that of the healthy group. As the treatment progressed, when the patient achieved functional cure, Tfh cells and Tfc cells showed a downward trend lower than that of the healthy group. In the relapse group, the patient's Tfh cells and Tfc cells showed an expression trend lower than that of the cured group. It can be seen that the exhaustion of Tfh cells and Tfc cells is closely related to the efficacy of Peg-IFNα-2b combined with TDF. The detection of such subcell populations can be used to predict the anti-HBV efficacy of Peg-IFNα-2b combined with TDF or to find new immune targets to reduce the relapse rate and improve the cure rate.

[0116] 3 Analysis and Conclusion

[0117] Our study found that the total percentage of CD4+T cells in both the Cure and Re groups was lower than that in the HC and CL groups. Our previous study found that the total percentage of CD4+T cells in CHB patients was higher than that in the HC group, and the total percentage of CD8+T cells was lower than that in the HC group. The CD4 / CD8 ratio also showed that the degree of T cell dysfunction and exhaustion in the Cure and Re groups was restored compared with that in CHB patients. The characteristic of CD8+T cells is that they can eliminate abnormal factors or resist the invasion of foreign factors by amplifying in large numbers and differentiating into cytotoxic effector cells. The exhaustion of CD8+T cells is related to the gradual impairment of their functions, especially in terms of amplification capacity, cytokine secretion, and antigen elimination. We collected clinical data and found that compared with CHB patients, HBV-DNA in the Cure group and the Re group was below the detection limit, although the HBV-DNA in the Re group would increase over time; in terms of the distribution of Treg cells, the Re group was significantly lower than the HC group and the CL group. Treg cells are a subpopulation of CD4+T cells that have a negative immune regulatory effect, can inhibit the auxiliary effect of CD4+T cells on CD8+T cells, and can non-specifically inhibit the activation and proliferation of CD4+T cells and CD8+T cells by secreting inhibitory cytokines. In the Re group, Treg cells are more active than those in the Cure group, and the negative feedback mechanism can reflect that the clearance of HBsAg in the Re group is weaker than that in the Cure group. Therefore, for the efficacy of achieving functional cure and recurrence, Treg cells and CD8+T cells are good monitoring indicators. Of course, at the same time, the detection of other indicators such as HBV DNA, AST, ALT, etc. should also be analyzed together. The control of the dynamic changes of each indicator can better predict the efficacy.

[0118] Follicular helper T cells (Tfh, CXCR5+CD4+T cells) and follicular cytotoxic T cells (Tfc, CXCR5+CD8+T cells) have attracted more and more attention and attention from experts and scholars in the treatment of CHB in recent years. Their iconic surface molecules CXCR5 and its ligand CXCL13 have also been included in extensive research.

[0119] The coordination role of Tfh cell subsets in the humoral immunity of adaptive immune response and the auxiliary role in the production of various antibodies are our main research directions. Mature Tfh cells are located in the follicles of secondary lymphoid organs (SLOs), and they assist in activating B cells located in the germinal center (GC) to differentiate into memory B cells and plasma cells and produce corresponding mature antibodies. Not only that, Tfh cells play a very important role in cancer prevention and treatment, anti-microbial pathogen infection, and prevention of excessive autoimmunity as a reservoir of HIV. Of course, Tfh cell subsets are no exception in anti-HBV. In the present study, it was found that the total proportion of Tfh in the Re and Cure groups was lower than that in the CL and HC groups, and the Re group was also lower than the Cure group. This shows that the total proportion of Tfh is directly related to the clearance of HBsAg, and has a good immunological value for predicting the efficacy of Peg-IFN-α combined with TDF treatment.

[0120] CXCR5+CD8+T cells are a special type of CD8+ subpopulation, which has only been discovered and studied in recent years. Im Se Jin et al. conducted a detailed characterization study of this subpopulation of cells at the transcriptome level and found that this subpopulation is an adaptive response to exhaustion of PD-1+CD8+T cells. During chronic infection of chronic hepatitis B, they have stem cell-like characteristics, that is, they can differentiate into terminal CXCR5+CD8+T cells while self-renewing to exert antiviral efficacy. Transcription factor TCF1 is essential for the generation of this subpopulation, and PD-1 blockade can cause the rapid expansion of this subpopulation and differentiation to the terminal stage. CXCR5+CD8+T cells play a key role in controlling viral replication. These CXCR5+CD8+T cells that migrate to B cell follicles under the influence of CXCL13 chemotaxis have fewer inhibitory receptors and show stronger cytotoxicity than CXCR5- subsets; they also show stronger therapeutic potential in HIV. This study found that the total TFC proportion in the Re and Cure groups was lower than that in the CL and HC groups, and the Re group was also lower than the Cure group. This shows that TFC cells showed better antiviral efficacy in the early stage of CL, while in the Re group, HBsAg re-positivity occurred due to the exhaustion of CD8+T cells. The total Tfc proportion in the Cure group was higher than that in the Re group, which shows that Tfc plays a key role in controlling the replication of HBV virus, indicating that CXC5+CD8+T cells can be used as a breakthrough point for improving the cure rate and reducing the recurrence rate of anti-HBV treatment.

[0121] In summary, through experimental results and clinical data analysis, the present invention shows that the distribution and dynamic changes of peripheral blood T lymphocytes are obvious after Peg-IFN combined with TDF achieves clinical cure in anti-HBV, among which the total proportion of CD4+T cells and Treg cells shows a downward trend, and the total proportion of CD8+T cells does not change significantly. The changes in CD4+T cells, Treg cells and CD4 / CD8 ratio can be used to monitor and predict the efficacy of Peg-IFNα-2b combined with tenofovir in anti-HBV treatment.

[0122] CXCR5 plays an important role in the process of clinical cure and relapse after Peg-IFNα-2b combined with tenofovir in anti-HBV treatment. The total proportion of Tfh and Tfc in the Cure group was higher than that in the Re group. Similarly, the Re group was lower than that in the CL group and the HC group. Therefore, when surface antigen clearance is achieved, Tfh and Tfc are maintained at a high concentration, and their rapid decline is likely to indicate the re-positivity of HBsAg. Maintaining a high level of proportion is more conducive to the clearance of HBsAg and achieving clinical functional cure. CXCR5 / CXCR13 is expected to become a new immune target for achieving functional cure.

Claims

1. The use of T cells as a biomarker in the preparation of a product for predicting the risk of relapse after achieving clinical functional cure in anti-HBV treatment, characterized in that: The T cells include CD8+ subpopulation CXCR5+CD8+T cells and CD4+ subpopulation CXCR5+CD4+T cells; the anti-HBV treatment is a combination treatment of Peg-IFN and tenofovir.

2. The use according to claim 1, characterized in that: Compared with the controls in the non-relapse group, a significant decrease in the subjects' CXCR5+CD8+T cells and CXCR5+CD4+T cells indicated a high risk of HBsAg re-positivity. Conversely, a high level of CXCR5+CD8+T cells and CXCR5+CD4+T cells in T cells indicated no or low risk of re-positivity.

3. Use of a substance for detecting the amount of CD8+ subpopulation CXCR5+CD8+T cells and CD4+ subpopulation CXCR5+CD4+T cells in the preparation of a product for predicting the risk of relapse after achieving clinical functional cure in anti-HBV treatment, wherein the anti-HBV treatment refers to a combination of Peg-IFN and tenofovir.

4. The use according to claim 3, characterized in that: Compared with the controls in the non-relapse group, a significant decrease in the subjects' CXCR5+CD8+T cells and CXCR5+CD4+T cells indicated a high risk of HBsAg re-positivity. Conversely, a high level of CXCR5+CD8+T cells and CXCR5+CD4+T cells in T cells indicated no or low risk of re-positivity.

5. The use according to claim 1 or 3, characterized in that: The T cells refer to T cells in peripheral blood.

6. The use according to claim 3 or 4, characterized in that: The substance is selected from antibodies and chips.

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