Research method of NK cell subset change in interferon treatment process

Abstract

The invention discloses a research method of NK cell subset change in an interferon treatment process, and relates to the technical field of interferon treatment. According to the research method for the NK cell subgroup change in the interferon treatment process, research objects are grouped according to an inclusion standard and an exclusion standard, and an initial treatment group, a platform group and an oral medicine group are included; respectively carrying out sample collection on the initial treatment group, the platform group and the oral medicine group by taking the starting time of the IFN-a as a baseline; performing peripheral blood NK cell phenotype analysis on the basis of the collected sample; clinical indexes are detected, wherein the clinical indexes comprise HBsAg, HBeAg, HBV DNA, ALT and AST; statistical analysis is carried out on all the obtained data, the mechanism of HBsAg disappearance of patients with surface antigen level decline and stagnation in the plateau stage through intermittent treatment can be obtained, and therefore the problem of how to research NK cell phenotype changes and diversity in the intermittent treatment process is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of interferon therapy, and specifically to a research method for changes in NK cell subsets during interferon therapy. Background Art

[0002] Currently, approximately 257 million people are infected with HBV. According to a survey in 2015, 887,000 people died from HBV-related diseases, including liver cirrhosis and hepatocellular carcinoma (HCC), accounting for approximately 52% and 38% respectively. Due to the huge threat and damage brought by CHB patients to this disease, it is very necessary to receive treatment in a timely manner. The elimination of hepatitis B surface antigen (HBsAg) is an important indicator for detecting the long-term prognosis of CHB patients and is also a key result of functional cure. Yang et al. found in a recent meta-analysis that in CHB patients without any antiviral treatment, the annual disappearance rate of HBsAg was only 1.02%. Based on the above research, it is therefore considered necessary for CHB patients to receive antiviral treatment.

[0003] In CHB patients treated with PEG-IFN combination therapy or extended therapy, the disappearance rate of HBsAg can be maintained at 15%, which is better than using NAs alone. However, during the process of HBsAg elimination, it is not easy to achieve through a one-time combination therapy or extended therapy, but rather through multiple stages, which is called intermittent therapy. When receiving long-term IFN therapy, the decline level of HBsAg in some patients may tend to reach a plateau, that is, compared with the previous detection, the HBsAg level decreases by <0.5 lg IU / ml. Even if the previous IFN therapy is continued during the plateau phase, it is not easy for patients to obtain a sustained virological response. At the same time, the guidelines also recommend that if the decline in HBsAg after 24 weeks of IFN therapy is <1 lg IU / ml, NAs should be used to replace antiviral therapy. Therefore, for patients who are stagnant in the plateau phase, intermittent therapy is carried out on them. Approximately 20% of the patients achieved the disappearance of HBsAg through this mode, and the disappearance of HBsAg did not occur in patients who stopped interferon during the plateau phase and did not receive interferon retreatment. This indicates that interferon intermittent therapy has an ideal prospect of improving the HBsAg conversion rate in CHB patients, but its molecular mechanism is worthy of study. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention provides a research method for changes in NK cell subsets during interferon therapy, and solves the problem of how to study the changes and diversity of NK cell phenotypes during intermittent therapy.

[0005] To achieve the above object, the present invention is realized through the following technical solutions: A research method for the changes of NK cell subsets during interferon treatment, comprising the following steps: grouping the research objects according to the inclusion criteria and exclusion criteria, including the initial treatment group, the plateau group, and the oral drug group; taking the start time of IFN-a as the baseline, and collecting samples from the initial treatment group, the plateau group, and the oral drug group respectively; performing peripheral blood NK cell phenotype analysis based on the collected samples; performing clinical index detection, and the clinical indexes include HBsAg, HBeAg, HBV DNA, ALT, and AST; statistically analyzing all the obtained data by linear regression, determining the dependent variable and the independent variable, establishing a regression model, and using the root mean square error to measure the accuracy, wherein the dependent variable is the change of NK cell subsets, and the independent variable is the clinical index.

[0006] Further, the inclusion criteria are as follows: patients who have never received any treatment before and start using IFN for the first time are the initial treatment group; patients who have been taking antiviral drugs orally for more than 6 months are the oral drug group; after using IFN for a period of time, those with a decrease in HBsAg level of <0.5 lg IU / ml are the plateau group.

[0007] Further, the exclusion criteria include: co-infection with hepatitis C virus, hepatitis D virus, and hepatitis E virus; long-term use of immunosuppressive drugs or drugs that are likely to cause liver damage; suffering from liver-related alcoholic hepatitis, autoimmune hepatitis, metabolic liver disease, cirrhosis, and liver tumors; mental diseases; severe cardiovascular and cerebrovascular diseases; and systemic diseases with greater harm.

[0008] Further, before collecting samples from the initial treatment group, the plateau group, and the oral drug group respectively, the initial treatment group is subcutaneously injected with PEG-IFN-a 180 mg per week, combined with first-line antiviral drugs, and the first-line antiviral drugs include one or more of entecavir 0.5 mg / d, tenofovir 300 mg / d, and tenofovir alafenamide fumarate 25 mg / d. According to their clinical characteristics and personal wishes, taking the start time of IFN-a as the baseline, 4 weeks and 12 - 24 weeks are taken as the follow-up time points; when the plateau group stops using interferon due to reaching the plateau phase, first-line antiviral drugs are used for maintenance treatment, and the first-line antiviral drugs include one or more of entecavir 0.5 mg / d, tenofovir 300 mg / d, and tenofovir alafenamide fumarate 25 mg / d, and follow-up is performed at 12 to 24 weeks after discontinuation and 12 to 24 weeks of re-treatment with interferon; the oral drug group takes the baseline as the enrollment time and does not require follow-up.

[0009] Further, the steps of collecting samples from the initial treatment group, the platform group, and the oral drug group are as follows: At baseline and each follow-up time point, we collected peripheral venous blood using two 9-ml EDTA purple tubes respectively, and detected clinical indicators of HBsAg, HBeAg, HBV DNA, and liver function. Within 4 hours after collecting peripheral blood, peripheral blood mononuclear cells were extracted for determination, namely PBMC determination, followed by staining and flow cytometry.

[0010] Further, the process of performing peripheral blood NK cell phenotype analysis based on the collected samples is as follows: Extract PBMC and resuspend it with phosphate-buffered saline; Add anti-human antibodies to 100 ml of PBMC cell suspension; After complete vortex mixing, let the sample stain in the dark at room temperature for 15 - 20 minutes; After staining, add 2 ml of PBS, centrifuge at 1200 g for 5 minutes, discard the supernatant, add 200 ml of PBS to homogenize the cell mass at the bottom of the tube; Detection was performed using flow cytometry. The voltage and fluorescence compensation were adjusted to the optimal state, cells were collected, and data was obtained using Flowjo software for gating analysis.

[0011] Further, the anti-human antibodies include CD3 / CD19-fluorescein isothiocyanate (FITC) anti-human antibody, CD14-phycoerythrin (PE) anti-human antibody, CD16-pericp chlorophyll protein (PERCP) anti-human antibody, CD56-bright violet 510 (BV510) anti-human antibody, CD57-BV421 anti-human antibody, CD69-allophycocyanin (APC-Fire750) anti-human antibody, TIGIT-PE anti-human antibody, PD-1-APC anti-human antibody.

[0012] Further, the method for detecting clinical indicators is as follows: HBsAg and HBeAg were detected by chemiluminescent particle method; The concentration of HBV DNA in serum was detected by real-time quantitative polymerase chain reaction; ALT and AST were detected by blood test.

[0013] Further, if the concentration of HBsAg is greater than 250 U / ml, the sample is diluted to 1:500, and the specific concentration is calculated.

[0014] Further, the steps of statistically analyzing all the obtained data are as follows: All data are analyzed using SPSS 25.0; the measurement data are first subjected to a normality test using the Shapiro-Wilk test; those that meet the normality test are expressed as mean ± standard deviation, while those that do not conform to the normal distribution are expressed as the median; the comparison of data between groups is performed using the t-test, Wilcoxon test, or Mann-Whitney test; a linear regression analysis is used to analyze the relationship between NK cell subsets and clinical indicators, and if P < 0.05, it is considered that the difference is statistically significant; for repeated measurements, the split method is used, and P < 0.017 is set as statistically significant.

[0015] The present invention has the following beneficial effects:

[0016] The research objects are grouped according to the inclusion criteria and exclusion criteria, including the initial treatment group, the plateau group, and the oral drug group; taking the start time of IFN-a as the baseline, sample collection is performed on the initial treatment group, the plateau group, and the oral drug group respectively; peripheral blood NK cell phenotype analysis is performed based on the collected samples; clinical indicator detection is performed, and the clinical indicators include HBsAg, HBeAg, HBV DNA, ALT, and AST; statistically analyzing all the obtained data can obtain the mechanism by which intermittent treatment achieves the disappearance of HBsAg in patients in the plateau stage, thus solving the problem of how to study the changes and diversity of NK cell phenotypes during intermittent treatment.

[0017] Of course, it is not necessary for any product implementing the present invention to simultaneously achieve all the above-mentioned advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a flow chart of the research method for the changes of NK cell subgroups during the interferon treatment process of the present invention.

[0019] Figure 2 It is an analysis result diagram of the gating analysis obtained by using Flowjo software to obtain data in the present invention.

[0020] Figure 3 It is a dynamic change diagram of NK cell subsets in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0022] In the description of the present invention, it should be understood that the terms "open hole", "upper", "lower", "thickness", "top", "middle", "length", "inner", "periphery", etc. indicating orientation or positional relationship are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the components or elements referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as a limitation to the present invention.

[0023] In CHB patients treated with PEG-IFN combination therapy or extended therapy, the disappearance rate of HBsAg can be maintained at 15%, which is better than that of NAs alone. However, during the process of HBsAg elimination, it is not easy to achieve through a single combination therapy or extended therapy, but through multiple stages, which is called intermittent therapy. When receiving long-term IFN therapy, the decline level of HBsAg in some patients may tend to a plateau, that is, compared with the previous detection, the HBsAg level has decreased by <0.5 lg IU / ml. Even if the previous IFN therapy is continued during the plateau phase, it is not easy for patients to obtain a sustained virological response. At the same time, the guidelines also recommend that if the HBsAg level decreases by <1 lg IU / ml after 24 weeks of IFN therapy, NAs should be used for alternative antiviral therapy. Therefore, for patients who are stagnant in the plateau phase, we conducted intermittent therapy on them. Approximately 20% of the patients achieved HBsAg disappearance through this mode, and HBsAg disappearance did not occur in patients who stopped interferon during the plateau phase and did not receive interferon retreatment, indicating that interferon intermittent therapy has an ideal prospect of improving the conversion rate of HBsAg in CHB patients.

[0024] Please refer to Figure 1 , the embodiment of the present invention provides a technical solution: a research method for changes in NK cell subsets during interferon therapy, including the following steps: grouping the research objects according to the inclusion criteria and exclusion criteria, including the initial treatment group, the plateau group, and the oral drug group; taking samples from the initial treatment group, the plateau group, and the oral drug group respectively with the start time of IFN-a as the baseline; performing peripheral blood NK cell phenotype analysis based on the collected samples; performing clinical index detection, and the clinical indexes include HBsAg, HBeAg, HBV DNA, ALT, and AST; statistically analyzing all the obtained data by linear regression, determining the dependent variable and the independent variable, establishing a regression model, and using the root mean square error to measure the accuracy, where the dependent variable is the change in NK cell subsets and the independent variable is the clinical index.

[0025] Specifically, the inclusion criteria are as follows: patients who have never received any treatment before and start using IFN for the first time are in the initial treatment group; patients who have been taking oral drugs for more than 6 months are in the oral drug group; patients whose HBsAg level has decreased by <0.5 lg IU / ml after using IFN for a period of time are in the plateau group.

[0026] The exclusion criteria include: co-infection with hepatitis C virus, hepatitis D virus, and hepatitis E virus; long-term use of immunosuppressive drugs or drugs prone to liver damage; having liver-related alcoholic hepatitis, autoimmune hepatitis, metabolic liver disease, cirrhosis, liver tumors; mental diseases; severe cardiovascular and cerebrovascular diseases; and systemic diseases with greater harm.

[0027] In this implementation plan, patients diagnosed with CHB at the Liver Disease Center of Beijing Ditan Hospital from November 2021 to August 2022 were selected as the research subjects and divided into an initial treatment group, a plateau group, and an oral drug group. CHB is defined as HBsAg being positive for ≥24 weeks, with or without hepatitis B antigen (HBeAg) being positive, and ALT being abnormal (>120 U / L) for more than 12 weeks. Patients who have never received any treatment before and start using IFN for the first time are in the initial treatment group; patients who have taken first-line oral medications continuously for more than 6 months are in the oral drug group; after using IFN for a period of time, those with an HBsAg level reduction of <0.5 lg IU / ml are in the plateau group. Exclusion criteria: 1) Co-infection with other viruses, such as hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus, etc.; 2) Long-term use of immunosuppressive drugs or drugs prone to liver damage; 3) Other liver-related diseases, such as alcoholic hepatitis, autoimmune hepatitis, metabolic liver disease, cirrhosis, liver tumors, etc.; 4) Mental diseases, such as depression; 5) Severe cardiovascular and cerebrovascular diseases or other systemic diseases with greater harm.

[0028] An oral drug group was established and compared cross-sectionally with the initial treatment group and the plateau group, aiming to explore the regulatory effects of oral drugs and PEG-IFN a treatment on NK cells. At the same time, the results also showed that compared with the oral drug treatment group, the percentages of NK cell regulatory subsets and killing subsets did not change significantly. To further study the regulation of oral drugs on NK cell subsets, regular follow-up should be conducted. However, this study mainly discussed the changes in NK cell subgroups during interferon treatment. Therefore, the dynamic changes during the follow-up period of the oral drug group were not included in this study. Establishing a corresponding control group (continuing IFN treatment after the HBsAg plateau phase) can better reflect the advantages of intermittent treatment and the relationship between NK cells and treatment strategies. However, according to the Guidelines for the Prevention and Treatment of Chronic Hepatitis B in China (2019 Edition), when the decline in HBsAg is <1 lg IU / ml, IFN should be stopped after 24 weeks of PEGIFN treatment. Therefore, in this study, we did not establish such a control group.

[0029] Specifically, before collecting samples from the initial treatment group, platform group and oral medication group, the initial treatment group was subcutaneously injected with PEG-IFN-a 180 mg per week, combined with first-line antiviral drugs, the first-line antiviral drugs included one or more of entecavir 0.5 mg / d, tenofovir 300 mg / d, and tenofovir alafenamide fumarate 25 mg / d. According to their clinical characteristics and personal wishes, the start time of IFN-a was taken as the baseline, and 4 weeks and 12-24 weeks were taken as follow-up time points; when the platform group stopped using interferon due to reaching the plateau period, first-line antiviral drugs were used for maintenance treatment, the first-line antiviral drugs included one or more of entecavir 0.5 mg / d, tenofovir 300 mg / d, and tenofovir alafenamide fumarate 25 mg / d, and follow-up was carried out 12 to 24 weeks after stopping interferon and 12 to 24 weeks after re-enabling interferon; the oral medication group used the baseline as the entry time and no follow-up was required.

[0030] Specifically, the steps for collecting samples for the initial treatment group, platform group and oral medication group were as follows: at baseline and each follow-up time point, we used two 9 ml EDTA purple tubes to collect peripheral venous blood, tested HBsAg, HBeAg, HBV DNA and clinical indicators of liver function, and extracted peripheral blood mononuclear cells within 4 hours after peripheral blood collection, i.e., PBMC assay, followed by staining and low cytometry.

[0031] Specifically, the process of performing peripheral blood NK cell phenotype analysis based on the collected samples is as follows: extract PBMC and resuspend with phosphate buffered saline; add anti-human antibody to 100 ml PBMC cell suspension; after complete vortex mixing, let the sample be stained at room temperature away from light for 15-20 minutes; after staining, add 2 ml PBS, centrifuge at 1200g× for 5 minutes, discard the supernatant, add 200 ml PBS, and homogenize the cell mass at the bottom of the test tube; use flow cytometry for detection, adjust the voltage and fluorescence compensation to the optimal state, collect cells, and use Flowjo software to obtain data for gate analysis. The analysis results are as follows: Figure 2 shown.

[0032] Specifically, the anti-human antibodies include CD3 / CD19-fluorescein isothiocyanate (FITC) anti-human antibodies, CD14-phycoerythrin (PE) anti-human antibodies, CD16-peripherin green protein (PERCP) anti-human antibodies, CD56-brilliant violet 510 (BV510) anti-human antibodies, CD57-BV421 anti-human antibodies, CD69-allophycocyanin (APC-Fire750) anti-human antibodies, TIGIT-PE anti-human antibodies, and PD-1-APC anti-human antibodies.

[0033] Specifically, the method for detecting clinical indicators is as follows: HBsAg and HBeAg are detected by chemiluminescent particle method; the concentration of HBV DNA in serum is detected by real-time quantitative polymerase chain reaction; ALT and AST are detected by blood test. If the concentration of HBsAg is greater than 250 U / ml, the sample is diluted to 1:500, and the specific concentration is calculated.

[0034] Specifically, the steps for statistically analyzing all the obtained data are as follows: all data are analyzed using SPSS 25.0; measurement data are first subjected to normality test using Shapiro-Wilk test; data that conform to the normality test are expressed as mean ± standard deviation, while those that do not conform to the normal distribution are expressed as median; the comparison of data between groups is performed using t-test, Wilcoxon test or Mann-Whitney test; linear regression analysis is used to analyze the relationship between NK cell subsets and clinical indicators. If P < 0.05, the difference is considered statistically significant; for repeated measurements, the split method is used, and P < 0.017 is set as statistically significant.

[0035] A total of 176 CHB patients were included, including 51 cases in the initial treatment group, 50 cases in the plateau group, and 75 cases in the oral drug group. The average age of the initial treatment group was 34.24 years (31.22, 41.16), the average age of the plateau group was 34.24 years (31.22, 41.16), and the average age of the oral drug group was 48.00 years (41.13, 58.01).

[0036] Baseline clinical characteristics and NK cell patient phenotypes

[0037] Between the initial treatment group and the oral drug group, there were no significant differences in the CD56dimD16+ subgroup [80.90 (69.41, 86.40) vs 79.30 (66.70, 84.82), Z = -1.03, P = 0.305] and the CD56brightCD16- subgroup [3.58 (1.94, 5.60) vs 2.37 (1.70, 4.30), Z = 11.78, P = 0.075)), but there were significant differences compared with the platform group [CD56dimCD16+: 80.90 (69.41, 86.40) vs 46.51 ± 18.51, Z = -7.08, P < 0.001 (initial treatment group vs platform group); 79.30 (66.70, 84.82) vs 46.51 ± 18.51, Z = -6.44, P < 0.001 (oral drug group vs platform group); CD56brightCD16-.001: 3.58 (1.94, 5.60) vs 11.64 (6.05, 19.61), Z = -6.35, P < 0.001 (initial treatment group vs platform group); 2.37 (1.70, 4.30) vs 11.64 (6.05, 19.61), Z = -7.74, P < 0.001 (oral drug group vs platform group)).

[0038] When comparing the platform group, the initial treatment group, and the oral drug group of CD69+CD56dim, the former was significantly higher than the other two groups [10.49 (5.27, 19.07) vs 5.03 (3.67, 8.58), Z = -3.11, P = 0.002; 10.49 (5.27, 19.07) vs 4.04 (1.90, 7.26), Z = -5.30, P < 0.001], while CD57+CD56dim was significantly lower than the initial treatment group and the oral drug group [68.42 ± 10.37 vs 55.85 ± 12.87, t = 5.84, P < 0.001; 76.38 ± 9.49 vs 55.85 ± 12.87, t = -9.65, P < 0.001]. The CD56brightCD16- platform group was significantly higher than the initial treatment group and the oral drug group [11.64 (6.05, 19.61) vs 3.58 (1.94, 5.60), Z = -6.35, P < 0.001; 11.64 (6.05, 19.61) vs 2.37 (1.70, 4.30), Z = -7.74, P < 0.001]. The baseline characteristics are shown in Table 1.

[0039]

[0040] Changes in NK cells - Phenotype during treatment

[0041] In the initial treatment group, in the CD56dimCD16+ subgroup, for CD57+CD56dim and TIGIT+CD56dim, the differences among all patients gradually decreased with the prolongation of treatment time. Compared with the baseline, the differences were statistically significant [80.90(69.41, 86.40) vs 51.18(34.34, 60.00), Z=-3.81, P<0.001; 68.42±10.37 vs 57.14±10.92, t=3.74, P=0.001; 49.94±21.19 vs 30.04±14.22, t=3.29, P=0.004]. In the CD56brightCD16- subgroup, it increased with the prolongation of treatment time, and the difference was statistically significant compared with the baseline group [3.58(1.94, 5.60) vs 10.70±5.95, Z=-3.17, P=0.002].

[0042] In the platform group, for CD57+CD56dim, compared with the baseline level, after stopping interferon for 12 - 24 weeks, its expression level increased significantly (55.85±12.87 vs 65.95±12.94, t=-2.78, P=0.011). There was no significant difference between the baseline group and the untreated group (65.95±12.94 vs 68.42±10.37, t=1.12, P=0.266). The dynamic changes of NK cell subsets are shown in Table 2 and Figure 3 .

[0043]

[0044] Study on the correlation between NK phenotype and clinical indicators

[0045] In the initial treatment group, HBV DNA was positively correlated with CD69+CD56dim (b=-0.35, t=-2.27, P=0.029). On the other hand, it was negatively correlated with CD56brightCD16- (b=0.43, t=2.15, P=0.038), as shown in Tables 3 to 5.

[0046] Table 3

[0047]

[0048] Table 4

[0049]

[0050] Table 5

[0051]

[0052] During the antiviral treatment of CHB, its therapeutic effect is closely related to the virus type, treatment strategy, and immune status. During the use of IFN, our previous study found that the disappearance of 75% of HBsAg occurred after 48 weeks, and extended treatment helped the disappearance of HBsAg and the maintenance of antiviral effect. However, not all patients can achieve ideal results through extended treatment, and the decline of HBsAg in some patients stagnates with the extension of treatment time. Based on this phenomenon, we conducted a clinical study and found that about 20% of patients achieved the disappearance of surface antigen through the intermittent treatment mode of initial-intermittent-retreatment, demonstrating the effectiveness of this strategy. However, the molecular mechanism behind intermittent treatment and the reasons for the plateau phase of the stagnant decline of HBsAg level remain to be explored. This study explored the molecular mechanism of the plateau phase by observing the changes of NK cell-related phenotypes at different times in patients at different treatment stages, providing a theoretical basis for intermittent treatment.

[0053] NK cells are a component of innate immune cells, which are of great significance in the human body and play a role in eliminating viruses by inducing inflammatory responses through cytotoxicity or cytokine secretion. Chronic HBV infection leads to a decrease in the count and function of NK cells. As an antiviral drug recommended by the guidelines, IFN induces ISG transcription through the JAK / STAT pathway and interferes with different stages of virus replication, including virus invasion, gene recombination, and assembly and release, playing an antiviral role in virus infection. It has been reported that interferon can significantly expand CD56brightCD16- cells and promote the activation of CD56dimCD16+. CD56dimCD16+ and CD56brightCD16- are the main subsets of NK cells. CD56dimCD16+ plays a key role in killing, accounting for about 90% of NK cells, and exerts cytotoxic effects by secreting granzymes and perforins. CD56brightCD16- mainly has regulatory functions, accounting for about 10% of the total number of NK cells, and secretes cytokines such as TNF-a and IFN-Y. In this study, in addition to detecting the frequencies of the two subsets of CD56brightCD16- and CD56dimCD16-, we also detected the expressions of CD69, CD57, TIGIT, and PD1 in these two subgroups respectively. CD69 is an activation molecule and killing receptor of NK cells. CD57 is expressed in mature and differentiated NK cells, marking the maturity of NK cells. Both TIGIT and PD-1 are receptors specifically expressed on the surface of NK cells and T cells with co-inhibitory effects, playing a co-inhibitory role. There are research reports that TIGIT acts on hepatocyte cytotoxic T lymphocytes (CTLs), leading to immune tolerance and making it difficult to clear HBV. Blocking the TIGIT pathway or inhibiting TIGIT expression can restore immune function. In animal experiments, PD-1-deficient mice have an advantage in clearing adenovirus from the liver, but they are also prone to immune damage, presumably due to the excessive autoimmune response caused by PD-1 deficiency. Therefore, the moderate expression of TIGIT and PD1 is also extremely important in innate immunity.

[0054] Our research found that in patients who had received IFN treatment for a period of time, there were significant differences in the percentage of CD56dimCD16+. The frequencies of CD69+CD56dim and CD57+CD56dim, compared with the baseline, showed that in patients receiving IFN treatment, the number of mature cells in CD56dimCD16+ decreased, indicating that after receiving IFN treatment for a period of time, the number of subsets with killing function weakened and the number of mature cells decreased. The increase in the frequency of CD69+CD56dim indicated that in patients receiving IFN treatment, although the killing subset decreased, the NK cell activity in the killing subset increased. Research has shown that the increase in transaminase in the interferon trial is related to the upregulation of NKCD69. This study further confirmed that in patients receiving IFN treatment, the killing subset CD69+CD56dim was significantly upregulated, which enhanced the killing activity and may have led to abnormal transaminases. During the follow-up of patients initially receiving IFN treatment, CD56dimCD16+ gradually decreased over time with the prolongation of IFN use, indicating that with the application of IFN, the killing function of NK cells gradually weakened, which may have led to the appearance of a plateau phase. At the same time, the frequency of CD56brightCD16- gradually increased because the differentiation direction of NK cells is from the regulatory subset to the killing subset, that is, from CD56brightCD16- to CD56dimCD16+, until finally exhausted. Therefore, it can be considered that the change in the frequency of CD56brightCD16- is a compensatory increase caused by the decrease in CD56dim.

[0055] Research has shown that after 12 weeks of PEG-IFN treatment, HBsAg decreased by less than 0.5 lgIU / ml compared with the baseline, indicating that it is difficult to achieve sustained virological remission after stopping treatment. This indicates that if there is no significant and sustained decrease in HBsAg during treatment, it is difficult to obtain good clinical outcomes. Therefore, we used a decrease in HBsAg range ≤ 0.5 lgIU / ml as the criterion for the plateau phase.

[0056] In the plateau phase group, with the prolongation of the IFN withdrawal period, the frequencies of NK cell-related subsets gradually recovered. The frequency of CD57+CD56dim, compared with the baseline, increased significantly after 12 - 24 weeks of IFN treatment, indicating the maturation of the killing subset. Compared with CHB patients who had not received any antiviral treatment (the baseline of the initial treatment group), the frequency of CD57+CD56dim recovered to the ideal level 12 - 24 weeks after stopping IFN in the plateau phase.

[0057] Studies have shown that reducing the viral load through antiviral therapy can partially reshape the function of NK cells. Reducing the viral load is beneficial to improving the function of NK cells. In pregnant women infected with HBV, antiviral therapy can significantly increase the occurrence frequency of NK cells. Through multiple linear regression analysis of baseline clinical indicators and NK subsets in this study, it was found that the HBVDNA level in the initial treatment group was negatively correlated with the frequency of CD69+CD56dim, indicating that the viral load may inhibit the activation of NK cells.

[0058] In summary, this study increased the differences in the expression of NK cell subsets in patients who had not received any treatment and IFN treatment, providing a partial theoretical basis for the emergence of the plateau phase in IFN treatment. From the perspective of NK cells, it provides theoretical support for the intermittent treatment of interferon. The results showed that during IFN treatment, the killing subsets of NK cells gradually decreased, the compensatory subsets increased, and the activity of the killing subsets increased. After stopping interferon, the frequency and function of NK cell subsets gradually recovered. Higher virus levels can affect the function of NK cells and the activation of NK cells.

[0059] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or device.

[0060] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor limit the invention to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of this specification. These embodiments are selected and specifically described in this specification to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and utilize the present invention well. The present invention is only limited by the claims and their full scope and equivalents.

Claims

1. Research methods for changes in NK cell subsets during interferon treatment, It is characterized in that The following steps are involved: The subjects were divided into groups according to the inclusion and exclusion criteria, including the initial treatment group, platform group, and oral medication group; Taking the start time of IFN-a as the baseline, samples were collected from the initial treatment group, platform group, and oral medication group; Perform peripheral blood NK cell phenotype analysis based on collected samples; Conducting clinical index testing, wherein the clinical indexes include HBsAg, HBeAg, HBV DNA, ALT and AST; All the obtained data were statistically analyzed by linear regression to determine the dependent variable and independent variable, establish a regression model, and use the root mean square error to measure the accuracy. The dependent variable was the changes in NK cell subgroups, and the independent variable was the clinical index.

2. The method for studying changes in NK cell subsets during interferon treatment according to claim 1, Features: The inclusion criteria are as follows: patients who have never received any treatment and are starting to use IFN for the first time are the initial treatment group; patients who have been taking oral antiviral drugs for more than 6 months are the oral drug group; after using IFN for a period of time, the HBsAg level decreases by <0.5lg IU / ml is the platform group.

3. The method for studying changes in NK cell subgroups during interferon treatment according to claim 2, It is characterized in that The exclusion criteria include: Co-infection with hepatitis C virus, hepatitis D virus, and hepatitis E virus; Long-term use of immunosuppressive drugs or drugs that may cause liver damage; Suffering from liver-related alcoholic hepatitis, autoimmune hepatitis, metabolic liver disease, cirrhosis, liver tumors; Mental illness; Severe cardiovascular and cerebrovascular diseases; A systemic disease with serious consequences.

4. The method for studying changes in NK cell subsets during interferon treatment according to any one of claims 2 to 3, Features: Before the samples were collected from the initial treatment group, platform group and oral medication group, the initial treatment group was subcutaneously injected with PEG-IFN-a 180 mg per week, combined with first-line antiviral drugs, which included one or more of entecavir 0.5 mg / d, tenofovir 300 mg / d, and tenofovir alafenamide fumarate 25 mg / d. According to their clinical characteristics and personal wishes, the start time of IFN-a was used as the baseline, and 4 weeks and 12-24 weeks were taken as the follow-up time points; When the platform group stopped interferon due to reaching the plateau period, first-line antiviral drugs were used for maintenance treatment, including one or more of entecavir 0.5 mg / d, tenofovir 300 mg / d, and tenofovir alafenamide fumarate 25 mg / d, and follow-up was performed 12 to 24 weeks after discontinuation of interferon and 12 to 24 weeks after re-enabling interferon; The baseline of the oral medication group was set as the enrollment time, and no follow-up was required.

5. The method for studying changes in NK cell subsets during interferon treatment according to claim 4, It is characterized in that The steps of collecting samples from the initial treatment group, platform group and oral medication group are as follows: At the baseline and each follow-up time point, we collected peripheral venous blood using two 9-ml EDTA purple tubes respectively, and detected HBsAg, HBeAg, HBV DNA and clinical indicators of liver function. Peripheral blood mononuclear cells were extracted within 4 hours after peripheral blood collection for determination, namely PBMC determination, followed by staining and flow cytometry.

6. The research method for the change of NK cell subsets during interferon treatment according to claim 5, characterized in that, the process of performing peripheral blood NK cell phenotype analysis based on the collected samples is as follows: Extract PBMC and resuspend it with phosphate buffered saline; Add anti-human antibodies to 100 ml of PBMC cell suspension; After complete vortex mixing, let the sample stain for 15 - 20 min at room temperature in the dark; After staining, add 2 ml of PBS, centrifuge at 1200 g × for 5 min, discard the supernatant, add 200 ml of PBS to homogenize the cell mass at the bottom of the tube; Perform detection by flow cytometry, adjust the voltage and fluorescence compensation to the optimal state, collect cells, and use Flowjo software to obtain data for gating analysis.

7. The research method for the change of NK cell subsets during interferon treatment according to claim 6, characterized in that: The anti-human antibodies include CD3 / CD19-fluorescein isothiocyanate anti-human antibody (FITC), CD14-phycoerythrin (PE) anti-human antibody, CD16-pericentrin green protein anti-human antibody, CD56-bright violet 510 anti-human antibody, CD57-BV421 anti-human antibody, CD69-allophycocyanin anti-human antibody, TIGIT-PE anti-human antibody, PD-1-APC anti-human antibody.

8. The research method for the change of NK cell subsets during interferon treatment according to claim 6, characterized in that, the method for performing clinical index detection is: HBsAg and HBeAg are detected by chemiluminescent particle method; The concentration of HBV DNA in serum is detected by real-time quantitative polymerase chain reaction; ALT and AST are detected by blood test.

9. The research method for the change of NK cell subsets during interferon treatment according to claim 8, characterized in that: If the concentration of HBsAg is greater than 250 U / ml, the sample is diluted to 1:500 and the specific concentration is calculated.

10. The research method for the change of NK cell subsets during interferon treatment according to claim 8, characterized in that, the steps for performing statistical analysis on all the obtained data are as follows: All data are analyzed using SPSS 25.0; Measurement data are first subjected to normality test using Shapiro-Wilk test; Those that meet the normality test are expressed as mean ± standard deviation, while those that do not conform to the normal distribution are expressed as median; The comparison of data between groups uses t-test, Wilcoxon test or Mann-Whitney test; Linear regression analysis is used to analyze the relationship between NK cell subsets and clinical indicators. If P < 0.05, it is considered that the difference is statistically significant; For repeated measurements, the split method is used, and P < 0.017 is set as statistically significant.

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