Method for increasing percentage of HLA-DR+CD38dim subpopulation on T cell

Abstract

The invention discloses a method for increasing the percentage of HLA-DR + CD38dim subgroups on T cells, and relates to the technical field of IFN treatment. The method for increasing the percentage of the HLA-DR + CD38dim subgroups on the T cells comprises the following steps: bringing a patient who has been treated by PEG-IFN alpha 180 [mu] g / week but enters a plateau phase due to the fact that hepatitis B surface antigens cannot be continuously reduced into a plateau phase group; pEG-IFN alpha is stopped from being used for patients in the plateau group, IFN treatment is recovered after 12-24 weeks, and only first-line NAs drug treatment is accepted during the intermittent period. Through intermittent treatment of the IFN, the percentage of HLA-DR + CD38dim subgroups is remarkably increased, and the antiviral curative effect of the IFN can be played again during retreatment of the IFN.

Description

Technical Field

[0001] The present invention relates to the technical field of IFN treatment, specifically a method for increasing the percentage of HLA-DR + CD38 dim subgroups on T cells. Background Art

[0002] Chronic hepatitis B virus (HBV) infection is a major public health problem in China and even globally. In China, there are approximately 86 million chronic HBV infected patients, among which about 30 million are chronic hepatitis B (CHB) patients, and there are also approximately 400,000 HBV-related death cases every year. In addition, among patients with liver cirrhosis and liver cancer in China, those caused by HBV infection account for 77% and 84% respectively. (Chinese Society of Infectious Diseases, Chinese Society of Hepatology. Guidelines for the Prevention and Treatment of Chronic Hepatitis B (2019 Edition)). Antiviral treatment is the most important measure to delay the disease progression of such patients and improve their long-term prognosis.

[0003] Interferon (IFN) and nucleoside (nucleotide) analogs (NAs) are two effective antiviral drugs for the current treatment of CHB. Compared with NAs, IFN has the dual functions of direct antiviral and immune regulation, and plays an increasingly important role in antiviral treatment. However, although numerous studies have shown that long-term antiviral treatment with IFN can increase the clearance rate of hepatitis B surface antigen (HBsAg), which helps to achieve durable immune control and clinical cure. But there are still quite a number of patients who cannot achieve the clearance of HBsAg from long-term IFN treatment. When the HBsAg level of patients stagnates during IFN treatment, that is, reaches the plateau phase, at this time, even if IFN continues to be used for antiviral treatment, the HBsAg level cannot be further reduced.

[0004] The clearance of HBV depends on the immune response, in which the activation of T cells plays the most important role. The expression of transmembrane glycoprotein molecules HLA-DR and CD38 on the surface of T cells, especially the co-expression of the two, can better reflect the activation state of T cells. According to the expression of CD38, the HLA-DR + CD38 + co-expression subgroup on T cells can be further divided into two heterogeneous subgroups: HLA-DR + CD38 dim and HLA-DR + CD38 hiCompared with HLA-DR in an overactivated or exhausted immune disorder state with impaired cell killing function + CD38 hi Subgroup, HLA-DR + CD38 dim The subpopulation expresses low levels of inhibitory checkpoints and has strong cell-killing potential. + CD38 dim The percentage of subgroups, thereby breaking the deadlock of IFN treatment of CHB entering the plateau period of HBsAg decline, is a problem that needs to be solved. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for increasing the expression of HLA-DR on T cells. + CD38 dim The subset percentage method solves the problem of how to increase the HLA-DR on T cells + CD38 dim The percentage of subpopulations can break the deadlock of IFN treatment of CHB entering the plateau stage where HBsAg decrease stagnates.

[0006] To achieve the above objectives, the present invention is implemented by the following technical solutions: increasing the HLA-DR + CD38 dim The method of subgroup percentage includes the following steps: patients who have received polyethylene glycol (PEG)-IFNα180 ug / week treatment but entered the plateau phase due to the inability to continuously decrease HBsAg are enrolled in the plateau phase group; CHB patients who have not been treated with IFN are treated with PEG-IFNα after enrolment, and the CHB patients who have not been treated with IFN include patients enrolled in the naive group and the NA-treated group; blood samples are collected from patients in the plateau phase group, the naive group and the NA-treated group at each follow-up time point; HBV virological and serological indicators and biochemical indicators are measured in the blood samples, and T cell-related phenotypes are detected by flow cytometry, and HLA-DR on T cells are analyzed. + CD38 dim Whether the subpopulation percentage has increased.

[0007] Furthermore, the treatment-naive group consists of CHB patients who have never received antiviral treatment, and the NA-treated group consists of CHB patients who have only received first-line oral antiviral drug treatment.

[0008] Furthermore, for the patients in the plateau group, PEG-IFNα was discontinued, and IFN treatment was resumed after an interval of 12-24 weeks. Only first-line NAs treatment was received during the IFN interval; the first-line oral antiviral drugs included one or more of ETV 0.5 mg / d, TDF 300 mg / d, and TAF 25 mg / d; for the patients in the treatment-naive group, subcutaneous injection of PEG-IFNα-2a 180 μg / week was started, and first-line oral antiviral drugs were selected for combination treatment based on their clinical data and personal wishes; for the NA-treated group, subcutaneous injection of PEG-IFNα-2a 180 μg / week was started in combination with the original oral antiviral drugs.

[0009] Furthermore, the inclusion criteria for patients in the plateau group, the treatment-naive group and the NA-treated group are: age 18-65 years old; hepatitis B surface antigen positivity (HBsAg ≥ 0.05 IU / mL) for > 6 months; the treatment-naive group had never received antiviral treatment before; the NA-treated group had only taken first-line oral antiviral drugs for > 6 months; the plateau group had IFN antiviral treatment for ≥ 6 months and then had a plateau of hepatitis B surface antigen level decrease; the plateau period was when the hepatitis B surface antigen level decreased by < 0.5 lg IU / mL compared with the previous detection time point, and the interval compared with the previous detection time point was ≥ 3 months.

[0010] Furthermore, the exclusion criteria for patients in the plateau group, the treatment-naive group and the NA-treated group were: previously known viral hepatitis excluding hepatitis B; infection with cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus and non-hepatitis virus; concurrent liver disease, including alcoholic hepatitis, autoimmune hepatitis, metabolic-related fatty liver disease, hepatolenticular degeneration, decompensated cirrhosis and liver cancer; long-term alcoholism and / or use of drugs that damage the liver; mental illness; severe heart, brain, lung and kidney system diseases; use of hormones and / or immunosuppressants or liver protectants.

[0011] Furthermore, the steps of collecting blood samples from patients in the plateau group, the naive group and the NA-treated group are as follows: blood samples are collected from patients in the naive group and the NA-treated group at baseline before IFN treatment, in the first month of IFN treatment and after 3-6 months of IFN treatment, respectively; blood samples are collected from patients in the plateau group at baseline of the plateau period, after 3-6 months of intermittent IFN treatment and after 3-6 months of resuming IFN treatment, respectively, and the plateau baseline is when IFN is discontinued.

[0012] Furthermore, the blood samples were tested for HBV virological and serological indicators, biochemical indicators, and T cell-related phenotypes were detected by flow cytometry to analyze HLA-DR on T cells.+ CD38 dim The steps for whether the subgroup percentage is increased are as follows: Obtain peripheral blood mononuclear cells (PBMCs) from the collected blood samples, prepare the samples before detection on the instrument using PBMCs, collect data using a FACSCanto flow cytometer, and perform data analysis using FlowJo software; conduct clinical index detections, where the clinical indexes include HBsAg, hepatitis B e antigen (HBeAg), HBV DNA, alanine aminotransferase (ALT), and aspartate transaminase (AST), and perform statistical analysis on all the obtained data.

[0013] Further, the steps for obtaining PBMCs from the collected blood samples and preparing the samples before detection on the instrument using PBMCs are as follows: Place the collected peripheral venous blood of the patient into an EDTA anticoagulation tube, and separate PBMCs by density gradient centrifugation using lymphocyte separation medium; Incubate PBMCs with directly conjugated antibodies in the dark at 4°C for 20 minutes; Add 2 mL of 1×PBS to wash the cells, spin at 1200 rpm for 5 minutes, and discard the supernatant; Add 200 μL of 1×PBS to prepare the samples before detection on the instrument.

[0014] Further, the directly conjugated antibodies include: anti-human CD4-APC-Fire750 (Clone SK3), CD8-BV510 (Clone SK1), HLA-DR-PE-Cy7 (Clone L243), CD38-APC (Clone HB-7).

[0015] Furthermore, for the clinical index detection, the clinical indexes include HBsAg, HBeAg, HBV DNA, ALT and AST. The steps for statistically analyzing all the obtained data are as follows: SPSS 26.0 software is used for statistical analysis. Categorical variables are expressed as frequency and percentage, and chi-square test is used to compare the differences between groups. The normal distribution of continuous variables is evaluated by Shapiro-Wilk test. Continuous variables conforming to the normal distribution are expressed as mean ± standard deviation, and continuous variables not conforming to the normal distribution are expressed as median and interquartile range. For baseline data, according to whether it conforms to normality, One-way ANOVA test and Kruskal-Wallis test are respectively used to compare among the plateau group, naïve group and NA-treated group. Subsequently, Tukey’s multiplicity test and Dunn’s multiplicity test are respectively used for pairwise comparison between groups. The linear mixed effect model is used to analyze the changes of each parameter at different time points within the plateau group, naïve group and NA-treated group, and Bonferroni method is used to adjust the P value for multiplicity.

[0016] The present invention has the following beneficial effects: The method for increasing the percentage of HLA-DR + CD38 dim subpopulation on T cells divides the HLA-DR + CD38 + T cell population into two subpopulations with different characteristics: HLA-DR + CD38 dim that plays a role in virus clearance and HLA-DR + CD38 hi that represents overactivation or senescence. During the long-term use of IFN, the percentages of HLA-DR + CD38 + subpopulation on CD4 + CD38 dim T cells and CD8 + CD38 hi T cells first increase significantly and then gradually decrease, while the percentage of HLA-DR + CD38 hi subpopulation increases significantly. During the long-term use of IFN, the HBsAg level is significantly negatively correlated with the percentage of HLA-DR + CD38 hi subpopulation. The continuously increasing percentage of HLA-DR + CD38 hi subpopulation is related to the emergence of the IFN plateau phase. After the intermittent treatment with IFN, the percentage of HLA-DR + CD38 hi subpopulation decreases significantly, and the percentage of HLA-DR + CD38dim The percentage of the subgroup increased significantly. After resuming IFN treatment, the HBsAg level began to decline again. Meanwhile, HLA-DR + CD38 hi The percentage of the subgroup increased significantly, but was still lower than the level at the baseline of the IFN plateau phase. During the long-term IFN treatment, the co-expression subgroup spectrum of HLA-DR and CD38 on T cells changed. The appearance of the IFN plateau phase was related to the continuously high percentage of the HLA-DR + CD38 hi subgroup. Intermittent treatment could significantly reduce the percentage of this subgroup and increase the percentage of the HLA-DR + CD38 dim subgroup, thus helping to restore the antiviral efficacy of IFN during re-treatment with IFN.

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

[0018] Figure 1 It is a flowchart of the method for increasing the percentage of the HLA-DR + CD38 dim subgroup on T cells according to the present invention. EMBODIMENTS

[0019] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with 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.

[0020] In the description of the present invention, it should be understood that the terms "openings", "upper", "lower", "thickness", "top", "middle", "length", "inner", "periphery", etc. indicating the 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 of the present invention.

[0021] A total of 150 CHB patients were included in this study, including 53 patients in the naïve group, 51 patients in the NA-treated group, and 36 patients in the plateau phase group. Subsequently, according to the patients' wishes, a total of 37 patients in the naïve group were willing to start IFN treatment, that is, interferon treatment, and a total of 93 samples were collected at 3 follow-up time points for subsequent detection and analysis; a total of 13 patients in the NA-treated group were willing to add IFN on the basis of the original NA treatment, and a total of 33 samples were collected at 3 follow-up time points for subsequent detection and analysis; 13 patients in the plateau phase group were unable to complete the subsequent follow-up examinations in our hospital, so a total of 23 patients collected 56 samples at 3 follow-up time points for subsequent detection and analysis.

[0022] Please refer to Figure 1 , an embodiment of the present invention provides a technical solution: a method for increasing the percentage of HLA-DR + CD38 dim subsets of T cells, comprising the following steps: Patients who have received PEG-IFNα 180 μg / week treatment but enter the plateau phase due to the inability of HBsAg to continuously decline are included in the plateau phase group; CHB patients who have not received IFN treatment are treated with PEG-IFNα after enrollment, and the CHB patients who have not received IFN treatment include patients included in the naïve group and the NA-treated group; for the plateau phase group, the naïve group and the NA-treated group, blood samples are collected at the corresponding follow-up time points; for the blood samples, HBV virological and serological indicators, biochemical indicators are measured, and T cell-related phenotypes are detected by flow cytometry to analyze whether the percentage of HLA-DR + CD38 dim subsets of T cells increases.

[0023] Specifically, the naïve group is CHB patients who have never received antiviral treatment, and the NA-treated group is CHB patients who have only received first-line oral antiviral drugs before.

[0024] According to the patients' treatment conditions, they are divided into the following three groups: CHB patients who have never received antiviral treatment are included in the naïve group; CHB patients who have only received first-line oral antiviral drugs before are included in the NA-treated group; patients who have received PEG-IFNα 180 μg / week treatment before but enter the plateau phase due to the inability of HBsAg to continuously decline are included in the plateau phase group.

[0025] Specifically, for the patients in the plateau phase group, PEG-IFNα was discontinued, and IFN treatment was resumed after an interval of 12-24 weeks, during which only first-line NAs treatment was received; the first-line oral antiviral drugs included one or more of ETV 0.5 mg / day, TDF 300 mg / day, and TAF 25 mg / day; for the patients in the treatment-naive group, subcutaneous injection of PEG-IFNα-2a 180 μg / week was started, and first-line oral antiviral drugs were selected for combination treatment based on their clinical data and personal wishes; for the NA-treated group, subcutaneous injection of PEG-IFNα-2a 180 μg / week was started in addition to the original oral antiviral drugs.

[0026] The steps of collecting blood samples from patients in the plateau group, the naive group and the NA-treated group are as follows: blood samples are collected from patients in the naive group and the NA-treated group at baseline before IFN treatment, in the first month of IFN treatment and after 3-6 months of IFN treatment; blood samples are collected from patients in the plateau group at baseline of the plateau, after 3-6 months of intermittent treatment and after 3-6 months of resuming IFN treatment, and the plateau baseline is when IFN is discontinued.

[0027] In the treatment-naive group, patients started to receive subcutaneous injection of PEG-IFNα-2a 180 μg / week, and selected first-line oral antiviral drugs such as ETV 0.5 mg / day or TDF 300 mg / day or TAF 25 mg / day for combination therapy based on their clinical data and personal wishes. Patients in the NA-treated group started subcutaneous injection of PEG-IFNα-2a 180 μg / week on the basis of taking the original oral antiviral drugs. Blood samples were collected from patients in the treatment-naive group and the NA-treated group at baseline before IFN treatment, in the first month of IFN treatment, and after 3-6 months of IFN treatment to measure HBV virological and serological indicators, biochemical indicators, and T cell-related phenotypes.

[0028] For patients in the plateau group, when HBsAg decreased to the plateau, IFN treatment was suspended, and then intermittent treatment for 3-6 months was started. During this period, only first-line oral antiviral drugs such as ETV 0.5 mg / day or TDF 300 mg / day or TAF 25 mg / day were used to maintain the antiviral efficacy. After the intermittent period, PEG-IFNα-2a was added to NA for IFN retreatment. Blood samples were collected from patients in the plateau group at the baseline of the plateau, after 3-6 months of intermittent treatment, and after the 3rd to 6th month of resuming IFN treatment, to measure HBV virological and serological indicators, biochemical indicators and T cell-related phenotypes. In addition, each group underwent liver ultrasound examination every 3-6 months at baseline and during follow-up.

[0029] Specifically, the inclusion criteria for patients in the plateau group, the first-treatment group and the NA-treated group are: age 18-65 years old; hepatitis B surface antigen positivity (HBsAg ≥ 0.05 IU / mL) for > 6 months; the first-treatment group had never received antiviral treatment before; the NA-treated group had only taken first-line oral antiviral drugs for > 6 months; the plateau group had IFN antiviral treatment for ≥ 6 months and then experienced a plateau of HBsAg level decrease; the plateau period was when the HBsAg level decreased by < 0.5 lg IU / mL compared with the previous detection time point, and the interval from the previous detection time point was ≥ 3 months.

[0030] Specifically, the exclusion criteria for patients in the plateau group, the treatment-naive group and the NA-treated group are: previously known viral hepatitis excluding hepatitis B; cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus and non-hepatitis virus infection; combined liver disease, including alcoholic hepatitis, autoimmune hepatitis, metabolic-related fatty liver disease, hepatolenticular degeneration, decompensated cirrhosis and liver cancer; long-term alcoholism and / or use of drugs that damage the liver; mental illness; severe heart, brain, lung, and kidney system diseases; use of hormones and / or immunosuppressants or liver protectants.

[0031] Specifically, the blood samples are tested for HBV virological and serological indicators, biochemical indicators, and T cell-related phenotypes are detected using flow cytometry to analyze HLA-DR on T cells. + CD38 dim The steps to determine whether the percentage of subpopulations is increased are as follows: PBMCs are obtained from the collected blood samples, and PBMCs are used to prepare samples before machine testing, data are collected using a FACSCanto flow cytometer, and data are analyzed using FlowJo software; clinical indexes are tested, including HBsAg, HBeAg, HBV DNA, ALT and AST, and all the data obtained are statistically analyzed.

[0032] Specifically, the steps of obtaining PBMCs from the collected blood sample, PBMCs are a type of white blood cells found in peripheral blood, including lymphocytes and monocytes, and using PBMCs to prepare samples before machine testing are as follows: the collected peripheral venous blood of the patient is placed in an EDTA anticoagulant tube, and PBMCs are separated by density gradient centrifugation using lymphocyte separation fluid; PBMCs are incubated with directly coupled antibodies in a dark environment at 4°C for 20 minutes; 2 mL of 1×PBS is added to wash the cells, and the supernatant is discarded after rotating at 1200 rpm for 5 minutes; 200 mL of 1×PBS is added to prepare samples before machine testing.

[0033] Specifically, the direct conjugate antibodies include: anti-human CD4-APC-Fire750 (Clone SK3), CD8-BV510 (Clone SK1), HLA-DR-PE-Cy7 (Clone L243), CD38-APC (Clone HB-7).

[0034] Specifically, for the detection of clinical indicators, the clinical indicators include HBsAg, HBeAg, HBV DNA, ALT, and AST. The steps for statistical analysis of all the obtained data are as follows: Statistical analysis is performed using SPSS 26.0 software. Categorical variables are expressed as frequency and percentage, and the chi-square test is used to compare the differences between groups. The normal distribution of continuous variables is evaluated using the Shapiro-Wilk test. Continuous variables that conform to the normal distribution are expressed as mean ± standard deviation, and continuous variables that do not conform to the normal distribution are expressed as median and interquartile range. For baseline data, according to whether they conform to normality, the One-way ANOVA test and the Kruskal-Wallis test are respectively used to compare the plateau group, naïve group, and NA-treated group. Subsequently, Tukey's multiplicity test and Dunn's multiplicity test are respectively used for pairwise comparisons between groups. A linear mixed-effects model is used to analyze the changes in each parameter at different time points within the plateau group, naïve group, and NA-treated group. The Bonferroni method is used to adjust the P value for multiplicity. All meaningful analyses are two-tailed analyses, and a P value < 0.05 is considered statistically significant.

[0035] The serum HBV DNA load is detected using a Roche Cobas TaqMan 96 fully automated real-time fluorescence quantitative PCR detector (Roche, Pleasanton, CA, USA), the serum HBsAg and HBeAg levels are detected using Abbott Architect i2000 kits (Abbott Diagnostics, Abbott Park, IL, USA), and the liver function indicators are detected using a Hitachi 7600 fully automated biochemical analyzer (Wako Pure Chemical Industries, Ltd., Japan).

[0036] Statistical analysis was performed using SPSS 26.0 (IBM Corporation, New York, NY, USA) and GraphPad 5 (GraphPad Software, La Jolla, CA, USA) software. Categorical variables were expressed as frequency and percentage, and the chi-square test was used to compare differences between groups. The Shapiro-Wilk test was used to evaluate the normal distribution of continuous variables. Continuous variables with a normal distribution were expressed as mean ± standard deviation, and continuous variables without a normal distribution were expressed as median and interquartile range.

[0037] For baseline data, according to whether they conformed to normality, One-way ANOVA test and Kruskal-Wallis test were used for comparison among multiple groups, and then Tukey’s multiple comparisons test and Dunn’s multiple comparisons test were used for pairwise comparison between groups. The linear mixed-effects model (restricted maximum likelihood estimation [REML]) was used to analyze the changes of each parameter at different time points within each group, and the Bonferroni method was used to adjust the P value for multiplicity.

[0038] The patient was used as a random effect, and the group and measurement time point were used as fixed effects. The likelihood ratio test was used to select the standard model. There was no violation of the standard model assumptions. The adjusted model used age and gender as covariates to exclude potential confounding factors. The Spearman correlation test was used to obtain the P and correlation coefficient values. All meaningful analyses were two-tailed analyses, and P < 0.05 was considered statistically significant.

[0039] In the naïve group, NA-treated group, and plateau group, 32, 25, and 17 HBeAg-positive patients were included respectively. There was no statistical difference among the three groups (60.38% [32 / 53] vs. 49.02% [25 / 51] vs. 47.22% [17 / 36], P = 0.3747). The HBsAg level in the plateau group was significantly lower than that in the naïve group and the NA-treated group (P < 0.0001; P = 0.0122). The HBsAg level in the NA-treated group was slightly lower than that in the naïve group, but there was no statistical difference (P = 0.0680). The HBV DNA level in the naïve group was significantly higher than that in the NA-treated group and the plateau group (P < 0.0001; P < 0.0001), while there was no significant difference in the HBV DNA level between the NA-treated group and the plateau group (P = 0.9019). The ALT and AST levels in the NA-treated group were significantly lower than those in the naïve group and the plateau group (ALT: P = 0.0015; P < 0.0001; AST: P = 0.0106; P = 0.0395), while there was no statistical difference in the ALT and AST levels between the naïve group and the plateau group (ALT: P = 0.3170; AST: P > 0.9999).

[0040] To determine the activation levels of T cells in different treatment states, we analyzed the co-expression of HLA-DR and CD38, the key markers representing T cell activation, in activated HLA-DR + CD4 + T and CD8 + T cells and defined three subsets: HLA-DR + CD38 - , HLA-DR + CD38 dim and HLA-DR + CD38 hi . The percentage of HLA-DR + CD38 - CD4 + T cells in the plateau group was significantly lower than that in the naïve group and the NA-treated group (P = 0.0075; P < 0.0001). The percentage of HLA-DR + CD38 - CD4 + T cells in the NA-treated group was higher than that in the naïve group (P = 0.0289). The percentage of HLA-DR + CD38 dim CD4 +The percentage of T cells was significantly higher than that in the naïve group and the NA-treated group (P < 0.0001; P < 0.0001), while for HLA-DR + CD38 dim CD4 + there was no significant difference in the percentage of T cells between the naïve group and the NA-treated group (P > 0.9999). For HLA-DR + CD38 hi CD4 + the percentage of T cells, the levels in the plateau group were significantly higher than those in the naïve group and the NA-treated group (P < 0.0001; P < 0.0001), while there was no statistical difference in the percentage of HLA-DR + CD38 hi CD4 + T cells between the naïve group and the NA-treated group (P > 0.9999).

[0041] HLA-DR + CD38 - CD8 + T cells and HLA-DR + CD38 hi CD8 + T cells showed a similar trend of difference among different groups as CD4 + T cells. For HLA-DR + CD38 - CD8 + the percentage of T cells, the levels in the plateau group were significantly lower than those in the naïve group and the NA-treated group (P < 0.0001; P < 0.0001), and the percentage of HLA-DR + CD38 - CD8 + T cells in the NA-treated group was significantly increased compared with that in the naïve group (P = 0.0002). The percentage levels of HLA-DR + CD38 hi CD8 + T cells in the naïve group and the NA-treated group were significantly lower than those in the plateau group (P < 0.0001; P < 0.0001), and there was no significant difference in the percentage levels of HLA-DR + CD38 hi CD8 + T cells between the naïve group and the NA-treated group (P > 0.9999). The HLA-DR + CD38 dim CD8 +The percentage of CD4+ T cells was significantly lower than that in the naïve group and the NA-treated group (P = 0.0003; P < 0.0001), while there was no significant difference in the HLA-DR + CD38 dim CD8 + levels of CD4+ T cells between the naïve group and the NA-treated group (P > 0.9999) either. The data preliminarily showed that when patients reached the plateau stage after long-term IFN use, the phenomenon that the antiviral efficacy could not be sustained was related to the changes in the percentage of the co-expression subset of HLA-DR and CD38 on CD4 + + T cells and CD8 + + T cells.

[0042] This part of patients who started antiviral treatment with IFN was selected for follow-up in the naïve group and the NA-treated group. Compared with before IFN treatment, there were no obvious changes in the percentages of HLA-DR + CD38 - CD4 + + T cells in both the naïve group and the NA-treated group after IFN treatment. In the naïve group and the NA-treated group, the percentages of HLA-DR + CD38 dim CD4 + + T cells increased significantly after 4 weeks of IFN treatment (P = 0.002; P = 0.001). After 12 - 24 weeks of IFN treatment, there was no significant difference in the percentage of HLA-DR + CD38 dim CD4 + + T cells in the naïve group compared with that at 4 weeks of IFN treatment (P = 0.3660), but the percentage of HLA-DR + CD38 dim CD4 + + T cells in the NA-treated group decreased significantly compared with that at 4 weeks of IFN treatment (P = 0.0130).

[0043] During the use of IFN in patients in the naïve group and the NA-treated group, the changing trends of the percentages of HLA-DR + CD38 hi CD4 + + T cells were relatively consistent. At the initial stage of treatment, the percentages of HLA-DR + CD38 hi CD4 + + T cells increased compared with the baseline, but there were no statistical differences (P = 0.6060; P = 0.6580). At the late stage of IFN treatment, the HLA-DR + CD38 hi CD4 +The percentages of T cells were all significantly increased compared to the baseline (P < 0.0001; P = 0.0010).

[0044] In the naïve group and the NA-treated group, as the duration of IFN treatment prolonged, HLA-DR + CD38 - CD8 + The percentages of T cells gradually decreased (naïve group: 0 week vs. 4 week, P = 0.0070; 0 week vs. 12 - 24 week, P < 0.0001; 4 week vs. 12 - 24 week, P = 0.0390; NA-treated group: 0 week vs. 4 week, P < 0.0001; 0 week vs. 12 - 24 week, P < 0.0001; 4 week vs. 12 - 24 week, P = 0.0100). In the naïve group and the NA-treated group, HLA-DR + CD38 dim CD8 + The percentages of T cells both increased significantly after 4 weeks of IFN treatment (P = 0.0300; P = 0.0240). After 12 - 24 weeks of IFN treatment, HLA-DR + CD38 dim CD8 + The percentages of T cells were all significantly decreased compared to those at 4 weeks of IFN treatment (P = 0.0010; P = 0.0120). In addition, as the duration of IFN treatment prolonged, HLA-DR + CD38 hi CD8 + The changing trends of the percentages of T cells were also relatively consistent in the naïve group and the NA-treated group, and both increased significantly with the treatment duration (naïve group: 0 week vs. 4 week, P = 0.0050; 0 week vs. 12 - 24 week, P < 0.0001; 4 week vs. 12 - 24 week, P < 0.0001; NA-treated group: 0 week vs. 4 week, P = 0.0050; 0 week vs. 12 - 24 week, P < 0.0001; 4 week vs. 12 - 24 week, P = 0.0380).

[0045] The HLA-DR + CD38- CD4 + Percentage of T cells (all P > 0.9999), HLA-DR + CD38 dim CD4 + Percentage of T cells (P > 0.9999; P > 0.9999; P = 0.1700) and HLA-DR + CD38 hi CD4 + There were no significant differences in the percentage of T cells (all P > 0.9999). At the treatment baseline, HLA-DR in the NA-treated group + CD38 - CD8 + The percentage of T cells was significantly higher in the treatment-naive group than in the treatment-experienced group (P < 0.0001), while there were no significant differences between the two groups at 4 weeks of IFN treatment (P = 0.1010) and at 12 - 24 weeks of IFN treatment (P > 0.9999). HLA-DR in both groups of patients at the IFN treatment baseline, 4 weeks of IFN treatment, and 12 - 24 weeks of IFN treatment + CD38 dim CD8 + Percentage of T cells (P > 0.9999; P = 0.9150; P > 0.9999) and HLA-DR + CD38 hi CD8 + There were no significant differences in the percentage of T cells (P > 0.9999; P = 0.5600; P > 0.9999).

[0046] In the naïve group and the NA-experienced group, the HBsAg levels in each sample collected at 12 - 24 weeks of IFN treatment decreased by > 0.5 lg IU / mL compared with their respective treatment baselines. In the naïve group, the HBsAg and HBV DNA levels at 12 - 24 weeks of IFN treatment were significantly lower than those at baseline (P = 0.0010; P < 0.0001). At 12 - 24 weeks of IFN treatment, the HBsAg level in the NA-experienced group was significantly lower than that at baseline (P = 0.0410), and the HBV DNA level showed no significant change compared with the baseline (P = 0.191). The ALT and AST levels in both the naïve group and the NA-experienced group were significantly increased compared with their respective baselines in the early stage of IFN treatment (ALT: P = 0.0070; P = 0.0240; AST: P = 0.0020; P = 0.0440), but there were no significant differences compared with the baseline in the late stage of IFN treatment (ALT: P > 0.9999; P > 0.9999; AST: P = 0.8120; P = 0.7070). During the IFN treatment process, except that the baseline HBV DNA level in the NA-experienced group was significantly lower than that in the naïve group (P < 0.0001), there were no differences in the kinetics of HBsAg, ALT, and AST between the two groups.

[0047] Next, we analyzed the correlation between the dynamic changes of the co-expressing subsets of CD38 and HLA-DR on CD4 + T cells and CD8 + T cells and HBsAg using the longitudinal data of all patients in the two groups. HLA-DR + CD38 hi CD4 + T cell percentage and HLA-DR + CD38 hi CD8 + T cell percentage was negatively correlated with the HBsAg level (R = -0.277, P = 0.0117; R = -0.271, P = 0.0137). The percentages of HLA-DR + on CD4 + T cells and CD8 + T cells and HLA-DR - CD38 + CD38 dim on CD8 + T cells were not correlated with the HBsAg level. In summary, these results suggest that during IFN treatment, when patients do not enter the plateau phase, as the HBsAg level decreases, HLA-DR hi CD38 + on CD4+ CD38 hi CD8 + The percentage of T cells gradually increases.

[0048] To further explore whether the continuously increasing HLA-DR + CD38 hi CD4 + T cells and HLA-DR + CD38 hi CD8 + The percentage of T cells is related to entering the plateau phase after long-term use of IFN, we continued to analyze the data of the patients in the plateau phase group after IFN intermittent treatment and after IFN retreatment. The data showed that after 12 - 24 weeks of IFN intermittent treatment, HLA-DR + CD38 - CD4 + The percentage of T cells (P < 0.0001) and HLA-DR + CD38 dim CD4 + The percentage of T cells (P < 0.0001) both increased significantly compared with the plateau phase baseline, while at this time HLA-DR + CD38 hi CD4 + The percentage of T cells decreased significantly compared with the plateau phase baseline (P < 0.0001). After 12 - 24 weeks of IFN intermittent treatment, CD8 + Each corresponding subset on T cells also had a similar trend of change. Compared with the plateau phase baseline, after 12 - 24 weeks of IFN intermittent treatment, the patients' HLA-DR + CD38 - CD8 + The percentage of T cells (P < 0.0001) and HLA-DR + CD38 dim CD8 + The percentage of T cells (P < 0.0001) increased significantly, and HLA-DR + CD38 hi CD8 + The percentage of T cells decreased significantly (P < 0.0001).

[0049] Compared with after IFN intermittent treatment, after 12 - 24 weeks of IFN retreatment, HLA-DR + CD38 - CD4 + T cells and HLA-DR + CD38 dim CD4 +The percentages of T cells were all significantly decreased (P < 0.0001; P < 0.0001). However, HLA-DR after re-treatment with IFN + CD38 - CD4 + T cells and HLA-DR + CD38 dim CD4 + The percentages of T cells had no significant differences compared with the baseline at the plateau phase (P > 0.9999; P = 0.8890). After 12 - 24 weeks of re-treatment with IFN, HLA-DR + CD38 hi CD4 + The percentages of T cells were significantly increased compared with after intermittent IFN treatment (P < 0.0001), but were significantly decreased compared with the baseline at the plateau phase (P = 0.0040). Similarly, compared with after intermittent IFN treatment, HLA-DR after 12 - 24 weeks of re-treatment with IFN + CD38 - CD8 + T cells and HLA-DR + CD38 dim CD8 + The percentages of T cells were significantly decreased (P < 0.0001; P < 0.0001), but there were no significant differences compared with the baseline at the plateau phase (P > 0.9999; P > 0.9999). In addition, after re-treatment with IFN, HLA-DR + CD38 hi CD8 + The percentages of T cells were significantly increased compared with after intermittent IFN treatment (P < 0.0001), but were significantly lower than the baseline at the plateau phase (P = 0.0350).

[0050] Before and after the IFN intermittent treatment, there was no statistically significant difference in the HBsAg levels between the plateau phase groups (P > 0.9999). The HBsAg levels in each sample collected after the IFN retreatment in the plateau phase groups decreased by > 0.5 lg IU / mL compared with their respective levels after the IFN intermittent treatment, showing significant statistical differences (P = 0.0180), and were significantly lower than the plateau phase baseline levels (P = 0.0260). During the IFN intermittent treatment and IFN retreatment, there were no obvious changes in the HBV DNA levels in the plateau phase groups (P > 0.9999). Compared with the plateau phase baseline, the ALT (P = 0.0320) and AST (P = 0.0460) levels in the plateau phase groups were significantly decreased after the IFN intermittent treatment; compared with after the IFN intermittent treatment, the ALT (P = 0.0390) and AST (P = 0.0440) levels in the plateau phase groups were significantly increased after the IFN retreatment.

[0051] It should be noted that in this document, 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 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, such 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.

[0052] 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 do they 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 in the relevant technical field can well understand and utilize the present invention. The present invention is only limited by the claims and their full scope and equivalents.

Claims

1. Method for increasing the percentage of HLA-DR + CD38 dim subsets on T cells It is characterized in that The following steps are involved: Patients who had received PEG-IFNα 180 ug / week treatment but entered the plateau phase due to the inability to continuously decrease HBsAg were included in the plateau group; The patients with chronic hepatitis B who had not been treated with IFN were enrolled and then treated with PEG-IFNα. The patients with chronic hepatitis B who had not been treated with IFN were divided into the treatment-naive group and the NA-treated group. Blood samples were collected from patients in the plateau group, treatment-naive group, and NA-treated group at various follow-up time points; Determine the virological and serological indicators and biochemical indicators of HBV in blood samples, and detect the T cell-related phenotypes using a flow cytometer to analyze whether the percentage of HLA-DR + CD38 dim subsets increases.

2. Method for increasing the percentage of HLA-DR + CD38 dim subpopulation on T cells, Features: The treatment-naive group consisted of CHB patients who had never received antiviral treatment, and the NA-treated group consisted of CHB patients who had previously only received first-line oral antiviral drug treatment.

3. Method for increasing the percentage of HLA-DR on T cells according to claim 2 + CD38 dim subpopulation, Features: For patients in the plateau group, PEG-IFNα was discontinued, and IFN treatment was resumed after an interval of 12-24 weeks. During the interval, only first-line NAs were treated; the first-line oral antiviral drugs included one or more of ETV 0.5 mg / d, TDF 300 mg / d, and TAF 25 mg / d. For patients in the initial treatment group, subcutaneous injection of PEG-IFNα-2a 180 μg / week was started, and first-line oral antiviral drugs were selected for combination therapy based on their clinical data and personal wishes; for patients in the NA-treated group, subcutaneous injection of PEG-IFNα-2a 180 μg / week was started in combination with the original oral antiviral drugs.

4. A method for increasing the percentage of HLA-DR + CD38 dim subsets on T cells, It is characterized in that The inclusion criteria for the plateau group, the first-treatment group, and the NA-treated group were: Age 18-65 years old; Hepatitis B surface antigen positivity (HBsAg ≥ 0.05 IU / mL) for more than 6 months; For the treatment-naive group, they had never received antiviral treatment before; For the NA-treated group, they have only taken first-line oral antiviral drugs for more than 6 months; For the plateau group, the hepatitis B surface antigen level decreased to a plateau after IFN antiviral treatment for ≥6 months; The plateau phase means that the hepatitis B surface antigen level decreases by less than 0.5 lg IU / mL compared with the previous detection time point, and the interval compared with the previous detection time point is ≥3 months.

5. A method for increasing the percentage of HLA-DR on T cells according to claim 1 + CD38 dim subpopulation percentage It is characterized in that The exclusion criteria for the plateau group, the first-treatment group, and the NA-treated group were: Known viral hepatitis other than hepatitis B; Cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus, and non-hepatitis virus infections; Patients with concurrent liver diseases, including alcoholic hepatitis, autoimmune hepatitis, metabolic-related fatty liver disease, hepatolenticular degeneration, decompensated cirrhosis, and liver cancer; Chronic alcohol abuse and / or use of drugs that damage the liver; Mental illness; Severe heart, brain, lung, or kidney diseases; Use of hormones and / or immunosuppressants or hepatoprotective agents.

6. The method for increasing the percentage of HLA-DR on T cells according to claim 3 + CD38 dim subpopulation percentage It is characterized in that The steps of collecting blood samples from patients in the plateau group, the first-treatment group and the NA-treated group are as follows: Blood samples were collected from patients in the treatment-naive group and the NA-treated group at baseline before IFN treatment, at the first month of IFN treatment, and after the third to sixth months of IFN treatment; Blood samples were collected from the patients in the plateau phase at the plateau phase baseline, after 3 - 6 months of IFN intermittent treatment, and after the 3 - 6th month of resuming IFN treatment. The plateau phase baseline was when IFN was discontinued.

7. A method for increasing the percentage of HLA-DR + CD38 dim subpopulation on T cells, It is characterized in that Measuring HBV virological and serological indicators and biochemical indicators of the blood sample, detecting T cell-related phenotypes using a flow cytometer, and analyzing whether the percentage of HLA-DR + CD38 dim subsets is increased is as follows: PBMCs were obtained from the collected blood samples, and samples before detection on the machine were prepared using PBMCs. Data were collected using a FACSCanto flow cytometer, and data analysis was performed using FlowJo software; Clinical indicators were detected. The clinical indicators included HBsAg, HBeAg, HBV DNA, ALT, and AST, and all the obtained data were statistically analyzed.

8. A method for increasing the percentage of HLA-DR on T cells according to claim 7 + CD38 dim subpopulation percentage It is characterized in that The steps of obtaining PBMCs from the collected blood samples and preparing samples before detection on the machine using PBMCs are as follows: The collected peripheral venous blood of the patient was placed in an EDTA anticoagulation tube, and PBMCs were separated by density gradient centrifugation using lymphocyte separation medium; PBMCs were co - incubated with directly conjugated antibodies in the dark at 4°C for 20 minutes; 2 mL of 1×PBS was added to wash the cells. After spinning at 1200 rpm for 5 minutes, the supernatant was discarded; 200 mL of 1×PBS was added to prepare a sample before detection on the machine.

9. A method for increasing the percentage of HLA-DR on T cells according to claim 8 + CD38 dim subpopulation, It is characterized in that The directly conjugated antibodies include: anti - human CD4 - APC - Fire750 (Clone SK3), CD8 - BV510 (Clone SK1), HLA - DR - PE - Cy7 (Clone L243), CD38 - APC (Clone HB - 7).

10. A method for increasing the percentage of HLA-DR on T cells according to claim 9 + CD38 dim subpopulation percentage It is characterized in that The steps of detecting clinical indicators, where the clinical indicators include HBsAg, HBeAg, HBV DNA, ALT, and AST, and statistically analyzing all the obtained data are as follows: SPSS 26.0 software was used for statistical analysis. Categorical variables were expressed as frequency and percentage, and the chi - square test was used to compare differences between groups. The Shapiro - Wilk test was used to evaluate the normal distribution of continuous variables. Continuous variables that conformed to the normal distribution were expressed as mean ± standard deviation, and continuous variables that did not conform to the normal distribution were expressed as median and interquartile range; For baseline data, according to whether it conformed to normality, One - way ANOVA test and Kruskal - Wallis test were respectively used to compare between the plateau phase group, naïve group, and NA - treated group. Subsequently, Tukey's multiplicity test and Dunn's multiplicity test were respectively used for pairwise comparison between groups. A linear mixed - effects model was used to analyze the changes of each parameter at different time points within the plateau phase group, naïve group, and NA - treated group, and the Bonferroni method was used to adjust the P - value for multiplicity.

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