CD4 is adjusted based on the ICOS-FOXO1 control axis. + Methods and applications of T-cell immune function drugs

Abstract

The application provides a method for screening a CD4 + The application discloses a method for screening a CD4 + T cell immune function drug and application thereof, and belongs to the technical field of drug screening and immune regulation. + T cell immune function drug and application thereof, and belongs to the technical field of drug screening and immune regulation. + T cell immune function drug and application thereof, and belongs to the technical field of drug screening and immune regulation. + T cell immune function drug and application thereof, and belongs to the technical field of drug screening and immune regulation. The application can be used for screening an HBV-ACLF immune therapy candidate drug.

Description

Technical Field

[0001] This invention belongs to the field of drug screening and immune regulation technology, and particularly relates to screening for CD4 regulation based on the ICOS-FOXO1 regulatory axis. + Methods and applications of T-cell immune function drugs. Background Technology

[0002] Hepatitis B virus-associated acute-on-chronic liver failure (HBV-ACLF) is a critical liver disease syndrome characterized by a sudden and rapid deterioration of liver function on the basis of chronic hepatitis B. It is characterized by rapid disease progression, numerous complications, and extremely high short-term mortality. Its core pathogenesis is closely related to immune dysregulation. CD4 + T-cell immune dysfunction is a key factor mediating the immune imbalance in HBV-ACLF, and CD4+ in patients... + T cells commonly suffer from insufficient activation, limited proliferation, and defective effector cytokine secretion, leading to a significant decrease in viral clearance capacity and disordered inflammatory regulation. This directly exacerbates liver tissue damage and worsens patient prognosis. Therefore, restoring CD4+ is crucial. + Normal T-cell immune function is the core target of HBV-ACLF immunotherapy.

[0003] Currently, in clinical practice and research, the screening of immunomodulatory drugs mostly relies on the detection of single cell surface markers or single cytokines. This limited detection scope fails to comprehensively reflect the complex transcriptional regulatory network downstream of co-stimulatory signals, making it difficult to accurately assess the effects of candidate drugs on CD4. + The overall regulatory effect on T cell function leads to poor drug specificity and limited efficacy in drug screening, failing to meet clinical treatment needs. FOXO1 is a core transcription factor regulating T cell fate differentiation, proliferation, metabolism, migration, and effector function, and plays a crucial role in CD4+. + This ICOS-FOXO1 regulatory axis plays a crucial role in the regulation of T cell immune function. Currently, this axis is involved in HBV-ACLF / FCD4. + Downstream target genes, screening and evaluation systems, and drug translation and application systems for T cell function repair have not yet been established, resulting in significant technological gaps. Therefore, constructing a novel drug screening method targeting the ICOS-FOXO1 regulatory axis has important clinical value and research significance for accurately screening highly effective immunomodulatory drugs and improving HBV-ACLF immunotherapy regimens. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a CD4 filtering adjustment based on the ICOS-FOXO1 control axis. + In vitro screening methods for drugs that enhance T-cell immune function.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for adjusting CD4 based on the ICOS-FOXO1 control axis. + The in vitro screening method for T-cell immune function drugs includes the following steps: Intervention of CD4 with the drug to be tested + T cells, CD4 count after intervention + T cell activation level, proliferation level, cytokine secretion level, apoptosis level, FOXO1 signaling activity, and expression levels of target genes related to the ICOS-FOXO1 regulatory axis; select test drugs that meet at least 3 of the following criteria: compared with the negative control group, (1) cell viability is not less than 80% and / or cell death rate is not more than 20%; (2) CD4 + (2) The proportion of T cells positive for CD69 or CD25 increased by ≥20% and / or the fluorescence intensity increased by ≥1.5 times, and P<0.05; (3) Cell proliferation increased by ≥20%, and P<0.05; (4) Cytokine secretion level increased by ≥20% and / or fluorescence intensity increased by ≥1.5 times, and P<0.05; (5) The proportion of Annexin V positive apoptosis decreased by ≥20%, and P<0.05; (6) FOXO1 nuclear localization, FOXO1 phosphorylation or any one of FOXO1, the binding level of target gene promoters was significantly changed, the change range was ≥20%, and P<0.05; (7) Among the target genes related to the ICOS-FOXO1 regulatory axis, the expression changes of at least 2 target genes were consistent with the direction of ICOS activation positive control group, the difference in expression fold was ≥1.5 times or ≤0.67 times, and P<0.05.

[0006] Preferably, the screening method for target genes related to the ICOS-FOXO1 regulatory axis includes the following steps: CD4 + T cells were activated by ICOS signaling, and transcriptome sequencing was used to screen for differentially expressed genes related to ICOS signaling. Differentially expressed genes related to ICOS signaling met the following criteria: corrected P value < 0.05, and differential expression fold ≥ 1.5-fold or ≤ 0.67-fold. High-throughput sequencing of chromatin immunoprecipitation of FOXO1 transcription factor was performed to obtain the FOXO1 binding peak. The differentially expressed genes were analyzed in conjunction with the FOXO1 binding peak. The target genes related to the ICOS-FOXO1 regulatory axis were those that simultaneously met the following criteria: (1) The gene was a differentially expressed gene related to ICOS signaling; (2) The gene had a FOXO1 binding peak in any one or more regions of its promoter region, enhancer region, or other regulatory regions; (3) The FOXO1 binding peak met the criteria of q value < 0.05 and enrichment fold ≥ 2-fold; (4) The direction of the gene expression change was consistent with the direction of ICOS signaling affecting FOXO1 transcriptional regulation activity; (5) The gene participated in any one or more functions in T cell activation, proliferation, cytokine secretion, migration, survival, apoptosis, immune metabolism, or immune effect regulation.

[0007] Preferably, target genes associated with the ICOS-FOXO1 regulatory axis include Abca1 , St6gal1 , Runx2 , Sema4a and Brpf3 .

[0008] Preferred, CD4 + T cells can be derived from any of the following: peripheral blood mononuclear cells, lymph node cells, spleen cells, liver immune cells, in vitro induced differentiated T cells, T cell lines, or gene-edited T cells.

[0009] The present invention also provides an adjustment CD4 based on the ICOS-FOXO1 control axis. + An in vitro screening system for T-cell immune function drugs, the in vitro screening system comprising: a sample collection module, an indicator detection module, a screening module, and a result output module; The sample acquisition module collects CD4 after treatment with the drug to be tested. + T cells; The indicator detection module detects CD4. + The activation level, proliferation level, cytokine secretion level, apoptosis level, FOXO1 signaling activity, and expression level of target genes related to the ICOS-FOXO1 regulatory axis of T cells were detected. The screening module analyzes the detection indicators to determine whether the drug to be tested meets at least three of the screening criteria; the screening criteria are compared with the negative control group: (1) cell viability not less than 80% and / or cell death rate not more than 20%; (2) CD4 +(2) The proportion of T cells positive for CD69 or CD25 increased by ≥20% and / or the fluorescence intensity increased by ≥1.5 times, and P<0.05; (3) Cell proliferation increased by ≥20%, and P<0.05; (4) Cytokine secretion level increased by ≥20% and / or fluorescence intensity increased by ≥1.5 times, and P<0.05; (5) The proportion of Annexin V positive apoptosis decreased by ≥20%, and P<0.05; (6) FOXO1 nuclear localization, FOXO1 phosphorylation or any one of FOXO1, the binding level of target gene promoters was significantly changed, the change range was ≥20%, and P<0.05; (7) Among the target genes related to the ICOS-FOXO1 regulatory axis, the expression changes of at least 2 target genes were consistent with the direction of ICOS activation positive control group, the difference in expression was ≥1.5 times or ≤0.67 times, and P<0.05; The result output module outputs the results.

[0010] The present invention also provides the application of the in vitro screening method or the in vitro screening system in screening and preparing drugs for the prevention and / or treatment of hepatitis B virus-related acute-on-chronic liver failure.

[0011] Preferred drugs for the prevention and / or treatment of hepatitis B virus-associated acute-on-chronic liver failure include traditional Chinese medicine extracts, chemically synthesized compounds, natural product monomers, peptides, or nucleic acid drugs.

[0012] This invention also provides a method for evaluating drugs for hepatitis B virus-related acute-on-chronic liver failure based on the ICOS-FOXO1 regulatory axis, comprising the following steps: intervening in CD4 with the drug to be evaluated. + T cells, with CD4 cells treated with CD3 antibody + T cells served as a negative control. The efficacy of the drug was assessed based on one or more of the following indicators: CD4. + The activation level, proliferation level, cytokine secretion level, apoptosis level, FOXO1 signaling activity, and expression levels of target genes related to the ICOS-FOXO1 regulatory axis of T cells.

[0013] The present invention also provides an in vitro evaluation system for drugs for hepatitis B virus-related acute-on-chronic liver failure based on the ICOS-FOXO1 regulatory axis. The in vitro evaluation system includes: a sample collection module, an indicator detection module, an evaluation module, and a result output module. The sample collection module collects CD4 after the drug intervention to be evaluated. + T cells; The indicator detection module detects CD4. +The activation level, proliferation level, cytokine secretion level, apoptosis level, FOXO1 signaling activity, and expression level of target genes related to the ICOS-FOXO1 regulatory axis of T cells were detected. The evaluation module uses CD4 treated with CD3 antibody. + T cells served as a negative control. Various indicators were analyzed to evaluate the efficacy of the drug. The result output module outputs the results.

[0014] The present invention also provides the application of the evaluation method or the in vitro evaluation system in the preparation of drugs for hepatitis B virus-associated acute-on-chronic liver failure.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for adjusting CD4 based on the ICOS-FOXO1 control axis. + This invention relates to an in vitro screening method for T-cell immune function drugs, which integrates ICOS signaling, FOXO1 transcriptional regulation, and CD4. + This invention integrates T-cell function readings into the drug screening process. It uses CD4... + Using T cells as vectors, with CD4 + Using T cell activation, proliferation, cytokine secretion, apoptosis, FOXO1 signaling activity, and target gene expression levels related to the ICOS-FOXO1 regulatory axis as evaluation indicators, and by setting judgment thresholds, it is possible to rapidly and specifically identify those that enhance CD4+ expression by intervening in the ICOS-FOXO1 regulatory axis. + Candidate drugs for T-cell immune function possess high specificity, high reproducibility, and high-throughput screening potential. This invention can be used to screen candidate drugs for HBV-ACLF immunotherapy. This invention is applicable to both basic research and preclinical candidate drug screening and pharmacodynamic evaluation. Attached Figure Description

[0016] Figure 1 The influence of ICOS signal on CD4 + The immune-related functions of T cells; where A is the experimental design of Bulk RNA-seq; B is the PCA analysis of sequencing samples; C is a heatmap showing the expression of all differentially expressed genes among different samples; D is the correlation analysis between "group A and group C" and "group B and group D"; E is the correlation analysis between "group E and group C" and "group F and group D"; F is the GO enrichment analysis of ICOS-related differentially expressed genes. Figure 2 CD4 modulation for ICOS signal +The potential molecular mechanisms of T cell function; where A is a Venn plot showing the intersection between the FOXO1ChIP-seq dataset and the ICOS differentially expressed genes; B is a heatmap showing the distribution of 56 differentially expressed genes; Figure 3 To visualize FOXO1 using IGV Abca1 , St6gal1 , Runx2 , Sema4a and Brpf3 Distribution of binding peaks in the promoter regions of isogens; Figure 4 This is the validation result of immune-enhancing drugs in an HBV-ACLF-related in vitro cell model; where A represents the CD4 count of PBMCs after 5 hours of culture with anti-ICOS or PBS. + The expression level of IFN-γ in T cells; B represents the expression level of CD4 in PBMCs after 5 hours of culture with anti-ICOS or PBS. + The expression level of TNF-α in T cells; C represents the expression level of CD4 in PBMCs after 5 hours of culture with anti-ICOS or PBS. + The expression level of IL-2 in T cells; D represents the expression level of CD8 in PBMCs after 5 hours of culture with anti-ICOS or PBS. + The expression level of IFN-γ in T cells; E represents the expression level of CD8 in PBMCs after 5 hours of culture with anti-ICOS or PBS. + The expression level of TNF-α in T cells; F represents the expression level of CD8 in PBMCs after 5 hours of culture with anti-ICOS or PBS. + IL-2 expression levels in T cells; no statistically significant difference in ns, * P <0.05,** P <0.01, *** P <0.001; Figure 5 The results show the validation of immune-enhancing drugs in HBV-ACLF-related animal models; where A is a schematic diagram of the construction of the ACLF mouse model; B is the HE pathological staining of liver tissues from ACLF and control mice; C is... Icos Survival analysis of KO (n=10) and WT (n=10) mice in inducing ACLF model; D represents 24 and 48 hours after KP injection. Icos KP load in peripheral blood of KO and WT mice; E represents ACLF induced by the model. Icos Changes in body temperature and weight in KO and WT mice; no statistically significant difference was observed in ns, * P <0.05,** P <0.01, *** P <0.001. Detailed Implementation

[0017] This invention provides a method for adjusting CD4 based on the ICOS-FOXO1 control axis. + The in vitro screening method for drugs that enhance T-cell immune function includes the following steps: intervention with the drug to be tested on CD4. + T cells, CD4 count after intervention + T cell activation level, proliferation level, cytokine secretion level, apoptosis level, FOXO1 signaling activity, and expression levels of target genes related to the ICOS-FOXO1 regulatory axis; select test drugs that meet at least 3 of the following criteria: compared with the negative control group, (1) cell viability is not less than 80% and / or cell death rate is not more than 20%; (2) CD4 + (2) The proportion of T cells positive for CD69 or CD25 increased by ≥20% and / or the fluorescence intensity increased by ≥1.5 times, and P<0.05; (3) Cell proliferation increased by ≥20%, and P<0.05; (4) Cytokine secretion level increased by ≥20% and / or fluorescence intensity increased by ≥1.5 times, and P<0.05; (5) The proportion of Annexin V positive apoptosis decreased by ≥20%, and P<0.05; (6) FOXO1 nuclear localization, FOXO1 phosphorylation or any one of FOXO1, the binding level of target gene promoters was significantly changed, the change range was ≥20%, and P<0.05; (7) Among the target genes related to the ICOS-FOXO1 regulatory axis, the expression changes of at least 2 target genes were consistent with the direction of ICOS activation positive control group, the difference in expression fold was ≥1.5 times or ≤0.67 times, and P<0.05.

[0018] In this invention, FOXO1 signaling activity is evaluated by detecting one or more of the following: FOXO1 protein nuclear localization, FOXO1 phosphorylation level, FOXO1 binding level to target gene promoter region, FOXO1 downstream transcriptional regulatory activity, or FOXO1 reporter gene activity.

[0019] In this invention, the method for screening target genes related to the ICOS-FOXO1 regulatory axis includes the following steps: CD4 +T cells are activated by ICOS signaling, and transcriptome sequencing is used to screen for differentially expressed genes related to ICOS signaling. Differentially expressed genes related to ICOS signaling meet the following criteria: corrected P value < 0.05, and differential expression fold ≥ 1.5-fold or ≤ 0.67-fold. High-throughput sequencing of FOXO1 transcription factor with chromatin immunoprecipitation is performed to obtain FOXO1 binding peak. The differentially expressed genes and FOXO1 binding peak are analyzed together. The target genes related to the ICOS-FOXO1 regulatory axis are genes that meet the following criteria: (1) the gene is a differentially expressed gene related to ICOS signaling; (2) any one or more regions of the promoter region, enhancer region or other regulatory regions of the gene contain FOXO1 binding peak; (3) the FOXO1 binding peak meets the criteria of q value < 0.05 and enrichment fold ≥ 2-fold; (4) the direction of gene expression change is consistent with the direction of ICOS signal affecting FOXO1 transcriptional regulation activity; (5) the gene participates in any one or more functions in T cell activation, proliferation, cytokine secretion, migration, survival, apoptosis, immune metabolism or immune effect regulation.

[0020] In this invention, the target genes related to the ICOS-FOXO1 regulatory axis include Abca1 , St6gal1 , Runx2 , Sema4a and Brpf3 .

[0021] In this invention, CD4 + T cells can be derived from any of the following: peripheral blood mononuclear cells, lymph node cells, spleen cells, liver immune cells, in vitro induced differentiated T cells, T cell lines, or gene-edited T cells.

[0022] The present invention also provides an adjustment CD4 based on the ICOS-FOXO1 control axis. + An in vitro screening system for T-cell immune function drugs, comprising: a sample collection module, an indicator detection module, a screening module, and a result output module; the sample collection module collects CD4+ after treatment with the drug to be tested. + T cells; the indicator detection module detects CD4 + The activation level, proliferation level, cytokine secretion level, apoptosis level, FOXO1 signaling activity, and expression level of target genes related to the ICOS-FOXO1 regulatory axis of T cells were detected. The screening module analyzed the detection indicators to determine whether the drug to be tested met at least three of the screening criteria. The screening criteria were compared with the negative control group as follows: (1) cell viability not less than 80% and / or cell death rate not more than 20%; (2) CD4 +(2) The proportion of T cells positive for CD69 or CD25 increased by ≥20% and / or the fluorescence intensity increased by ≥1.5 times, and P<0.05; (3) Cell proliferation increased by ≥20%, and P<0.05; (4) Cytokine secretion level increased by ≥20% and / or fluorescence intensity increased by ≥1.5 times, and P<0.05; (5) Annexin V positive apoptosis rate decreased by ≥20%, and P<0.05; (6) FOXO1 nuclear localization, FOXO1 phosphorylation or any one of FOXO1, the binding level of FOXO1 to the promoter of the target gene was significantly changed, the change range was ≥20%, and P<0.05; (7) Among the target genes related to the ICOS-FOXO1 regulatory axis, the expression changes of at least 2 target genes were consistent with the direction of ICOS activation positive control group, the difference in expression fold was ≥1.5 times or ≤0.67 times, and P<0.05; The results output module outputs the results.

[0023] This invention also provides the application of the in vitro screening method or the in vitro screening system in screening and preparing drugs for the prevention and / or treatment of hepatitis B virus-related acute-on-chronic liver failure. These drugs include traditional Chinese medicine extracts, chemically synthesized compounds, natural product monomers, polypeptides, or nucleic acid drugs.

[0024] This invention also provides a method for evaluating drugs for hepatitis B virus-related acute-on-chronic liver failure based on the ICOS-FOXO1 regulatory axis, comprising the following steps: intervening in CD4 with the drug to be evaluated. + T cells, with CD4 cells treated with CD3 antibody + T cells served as a negative control. The efficacy of the drug was assessed based on one or more of the following indicators: CD4. + The activation level, proliferation level, cytokine secretion level, apoptosis level, FOXO1 signaling activity, and expression levels of target genes related to the ICOS-FOXO1 regulatory axis of T cells.

[0025] This invention also provides an in vitro evaluation system for drugs targeting hepatitis B virus-related acute-on-chronic liver failure based on the ICOS-FOXO1 regulatory axis. The in vitro evaluation system includes: a sample collection module, an indicator detection module, an evaluation module, and a result output module. The sample collection module collects CD4+ after treatment with the drug to be evaluated. + T cells; the indicator detection module detects CD4 + The evaluation module detected the activation level, proliferation level, cytokine secretion level, apoptosis level, FOXO1 signaling activity, and expression levels of target genes related to the ICOS-FOXO1 regulatory axis of T cells; the evaluation module used CD4 cells treated with CD3 antibody. +T cells served as a negative control. Various indicators were analyzed to assess the drug's efficacy; the results output module then output the results.

[0026] In this invention, the detection methods for each indicator can be selected as follows: Cell activation assay: After culturing for 24 h or 48 h, the expression of CD69, CD25 and CD38 was detected by flow cytometry; Cell proliferation assay: Cells were pre-stained with CFSE and the CFSE dilution ratio was measured after 72 h or 96 h of culture. Cytokine secretion detection: After culturing for 24-96 h, PMA, ionomycin and GolgiStop were added for stimulation for 4-6 h, and the expression of IFN-γ, TNF-α and IL-2 was detected. Apoptosis detection: After culturing for 24 h or 48 h, the apoptosis rate was detected by Annexin V / PI staining; FOXO1 signal detection: After 2 h, 6 h, 12 h or 24 h of culture, the phosphorylation level of FOXO1, the nuclear localization of FOXO1 or the binding level of FOXO1 to the promoter region of the target gene were detected. Target gene expression detection: After 12 h, 24 h or 48 h of culture, the expression levels of ABCA1, ST6GAL1, RUNX2, SEMA4A and BRPF3 were detected by qRT-PCR.

[0027] In this invention, to improve the comparability of screening and / or assessment results for hepatitis B virus-related acute-on-chronic liver failure, a comprehensive scoring system can be established. Each indicator is scored from 0 to 2 points. CD69 / CD25 elevation: No change is 0 points, 20%~50% elevation is 1 point, and ≥50% elevation is 2 points; CFSE dilution increase: no change is 0 points, an increase of 20%~50% is 1 point, and an increase of ≥50% is 2 points; Increased IFN-γ / TNF-α secretion: No change is 0 points, an increase of 20%~50% is 1 point, and an increase of ≥50% is 2 points; Annexin V apoptosis decrease: no change is 0 points, a decrease of 20%~50% is 1 point, and a decrease of ≥50% is 2 points; Changes in the FOXO1 axis: No significant change is 0 points, one significant change in one FOXO1 indicator is 1 point, and two or more significant changes in two or more FOXO1 indicators are 2 points. Changes in target gene expression: 0 points for changes in less than 2 target genes, 1 point for changes in 2–3 target genes, and 2 points for changes in 4–5 target genes.

[0028] A total score of ≥6 points is considered a positive candidate in the initial screening; a total score of ≥8 points is considered a preferred candidate; and a total score of ≥10 points is considered a highly preferred candidate.

[0029] The present invention also provides the application of the evaluation method or the in vitro evaluation system in the preparation of drugs for hepatitis B virus-associated acute-on-chronic liver failure.

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Unless otherwise specified, the following embodiments are all conventional methods.

[0032] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0033] Example 1 RNA-seq screening for differentially expressed genes associated with ICOS signaling: Based on CD4 + T-cell transcriptome sequencing was used to screen for differentially expressed genes associated with ICOS signaling.

[0034] 1. Experimental materials: The experimental animals used were 6-8 week old male C57BL / 6J wild-type mice and Icos Gene knockout mice. Cell culture medium was RPMI-1640 complete medium, consisting of RPMI-1640 basal medium, 10% fetal bovine serum, 1% penicillin-streptomycin antibiotics, and 1% HEPES buffer. The anti-mouse CD3 antibody was BioLegend's anti-mouse CD3 antibody, catalog number 100340; the anti-mouse CD28 antibody was BioLegend's anti-mouse CD28 antibody, catalog number 102116; and the ICOS agonist antibody was BioX Cell's InVivoMAb anti-mouse / human / rat / monkey ICOS antibody, catalog number BE0353. CD4 + The T-cell magnetic beads are Miltenyi Biotec's CD4 MicroBeads, catalog number 130-117-043.

[0035] 2. CD4 + T cell isolation and processing: Spleens and peripheral lymph nodes from wild-type mice and Icos knockout mice were collected, homogenized in pre-chilled PBS or flow cytometry buffer, filtered through a 70 μm cell sieve, and centrifuged at 300 g for 5–10 min. If a high concentration of red blood cells was found, red blood cell lysis buffer was added for 1–3 min, followed by termination of lysis with PBS and washing. CD44 was sorted using CD4 magnetic beads. + T cells were sorted and their purity was determined by flow cytometry, with CD4+ being a key requirement. + The purity of T cells is no less than 90%.

[0036] The sorted CD4 + T cells adjusted to 1×10 6 Cells were seeded at a concentration of 10 μg / mL in cell culture plates pre-coated with anti-CD3 antibody. The working concentration of anti-CD3 antibody was 0.5 μg / mL; anti-CD28 antibody was set at 0 μg / mL, 0.125 μg / mL, or 1 μg / mL to simulate no CD28 co-stimulation, low CD28 co-stimulation, and high CD28 co-stimulation states, respectively. The working concentration of ICOS agonist antibody was 10 μg / mL. PBS was used as a negative control.

[0037] Set up the following experimental groups: WT-CD3 / CD28low-PBS group (Group A): Wild-type CD4 + T cells, anti-CD3 0.5 μg / mL, anti-CD28 0.125 μg / mL, treated with PBS; WT-CD3 / CD28low-anti-ICOS group (Group E): Wild-type CD4 + T cells were treated with anti-CD3 0.5 μg / mL, anti-CD28 0.125 μg / mL, and anti-ICOS 10 μg / mL. WT-CD3 / CD28high-PBS group (Group C): wild-type mouse CD4 + T cells were treated with anti-CD3 0.5 μg / mL and anti-CD28 1.0 μg / mL in PBS. Icos KO-CD3 / CD28high-PBS group (Group D): Icos Gene knockout mice CD4 + T cells were treated with anti-CD3 0.5 μg / mL and anti-CD28 1.0 μg / mL in PBS.

[0038] IcosKO-CD3 / CD28low-PBS group (Group B): Icos Gene knockout mice CD4 + T cells, anti-CD3 0.5 μg / mL, anti-CD28 0.125 μg / mL, treated with PBS; Icos KO-CD3 / CD28low-anti-ICOS group (Group F): Icos Gene knockout mice CD4 + T cells were treated with anti-CD3 0.5 μg / mL, anti-CD28 0.125 μg / mL, and anti-ICOS 10 μg / mL.

[0039] Each group consisted of three biological replicates. Cells were cultured at 37°C and 5% CO2 for 24–48 h before collection. For detecting the effect of ICOS signaling on early transcriptional changes, cells were cultured for 24 h; for detecting more stable effector transcriptional changes, cells were cultured for 48 h.

[0040] 3. RNA extraction, quality control, and sequencing: After culturing for 48 hours, CD4 was collected from each group. + T cells were used to extract total RNA using the TRIzol method or a column-based RNA extraction kit. RNA samples should meet the following quality control standards: RNA integrity index (RIN) ≥ 7.0, preferably ≥ 8.0; OD260 / 280 1.8–2.1; OD260 / 230 ≥ 1.8; and total RNA volume sufficient for library construction. After mRNA enrichment or rRNA removal from qualified RNA samples, transcriptome libraries were constructed and sequenced using the Illumina platform with paired ends. The effective sequencing data volume for each sample should preferably be no less than 6 Gb, or at least 20 million effective reads per sample.

[0041] 4. Bioinformatics analysis: After quality control, adapter sequences, low-quality reads, and reads with excessively high N ratios were removed from the raw sequencing data. Clean reads were aligned to the mouse reference genome using Hisat2, STAR, or other commonly used alignment software. Gene expression quantification was performed using featureCounts and HTSeq. Differential expression analysis was conducted using DESeq2, edgeR, or limma-voom software.

[0042] The criteria for identifying differentially expressed genes are as follows: (1) Initial screening criteria: Corrected P value, i.e., FDR or adjusted P value < 0.05, and |log2 FoldChange| ≥ 0.585, i.e. expression change ≥ 1.5 times or ≤ 0.67 times; (2) Strict standard: FDR < 0.05, and |log2 Fold Change| ≥ 1, that is, the change factor is ≥ 2 times or ≤ 0.5 times; (3) For candidate genes related to immune function, if their FDR < 0.05 and show a stable and homologous change in the ICOS activation group, even if |log2 Fold Change| is between 0.585 and 1, they can be included in subsequent validation.

[0043] The selection of differentially expressed genes related to ICOS signaling was determined as follows: the WT-CD3 / CD28low-anti-ICOS group was compared with the WT-CD3 / CD28low-PBS group to screen for differentially expressed genes affected by ICOS signaling activation; simultaneously, Icos KO-related groups were used as specific controls and excluded. Icos Non-specific stimulatory genes that still undergo the same changes in a deletion context. If a gene in wild-type CD4... + T cells treated with anti-ICOS showed significant upregulation or downregulation, while Icos CD4 gene knockout + If the changes in T cells disappear or are significantly reduced, the gene is identified as an ICOS-dependent differentially expressed gene.

[0044] 5. Result Judgment Criteria: The RNA-seq screening results are considered reliable if all of the following conditions are met: (1) In the PCA analysis, the samples in the same group showed good clustering, and a separation trend was visible between different treatment groups; (2) Each group must have at least 3 biological replicates; (3) Differentially expressed genes satisfy FDR < 0.05 and |log2 Fold Change| ≥ 0.585; (4) GO enrichment results are related to immune functions such as T cell activation, immune response, leukocyte activation or cytokine regulation; (5) Select at least 5 to 10 representative differentially expressed genes for qRT-PCR verification, and the proportion of qRT-PCR results that are consistent with the direction of RNA-seq changes should not be less than 70%.

[0045] In this embodiment ( Figure 1PCA analysis showed good clustering of samples within the groups, indicating good reproducibility of the sequencing data. Differentially expressed gene analysis revealed 7084 differentially expressed genes across the analyzed groups. Further comparison of ICOS signaling-related groups showed 245 upregulated and 119 downregulated differentially expressed genes related to ICOS signal activation. GO enrichment analysis showed that the differentially expressed genes were mainly enriched in biological processes such as cell activation, leukocyte activation, regulation of immune effector processes, lymphocyte activation, T cell activation, and regulation of cytokine production in the immune response.

[0046] Example 2 FOXO1 ChIP-seq combined analysis to screen candidate target genes of the ICOS-FOXO1 axis: The RNA-seq screening results were combined with FOXO1 ChIP-seq data to obtain candidate target genes of the ICOS-FOXO1 regulatory axis.

[0047] 1. Experimental materials and sample processing: Using wild-type mouse CD4 obtained in Example 1 + T cells and Icos Gene knockout mice CD4 + T cells. The experimental grouping and treatment conditions for the cells were the same as in Example 1, and the cells were collected after culturing for 24-48 h.

[0048] If you conduct your own FOXO1 ChIP-seq experiments, collect at least 1×10⁻⁶ samples per group. 7 CD4 + T cells. If the cell count is insufficient, FOXO1 ChIP-qPCR verification can be performed first, or low-starting-volume chromatin binding analysis techniques such as CUT&RUN and CUT&Tag can be used.

[0049] 2. FOXO1 ChIP-seq experimental procedure: Collect and process CD4 + T cells were cross-linked with 1% formaldehyde at room temperature for 10 min, followed by the addition of glycine at a final concentration of 125 mmol / L to terminate the cross-linking for 5 min. Cells were washed with PBS and lysed to extract chromatin. Chromatin fragmentation to approximately 200–500 bp was performed using sonication. One percent of the samples were used as an input control, while the remaining samples underwent immunoprecipitation with FOXO1 antibody, and an IgG negative control was included. After immunoprecipitation, the cells were washed, reversed cross-linked, treated with proteinase K, and purified to obtain FOXO1-binding DNA fragments.

[0050] Purified DNA was used for library construction and sequencing. It is recommended to obtain at least 20 million valid reads per sample. Sequencing data were quality-controlled and aligned to a mouse reference genome, followed by peak calling using MACS2 or similar software.

[0051] 3. Criteria for judging FOXO1 binding peak: (1) The peak calling q value or FDR is <0.05, preferably q value <0.01; (2) The peak enrichment factor is ≥2 times that of the input control, preferably ≥4 times; (3) No significant enrichment peaks at the same position should appear in the IgG negative control; (4) The binding peak is preferentially located in the gene promoter region, that is, within the range of 2 kb upstream to 500 bp downstream of the transcription start site; it may also include enhancer regions, first intron regions or other regions with regulatory functions. (5) In IGV or similar genome browsers, the FOXO1 peak shape is clear, the background noise is low, and the peak position is consistent among duplicate samples.

[0052] 4. Combined analysis of RNA-seq and FOXO1 ChIP-seq: Intersection analysis was performed on the differentially expressed genes related to ICOS signaling obtained in Example 1 and the target genes obtained from FOXO1 ChIP-seq annotation. A gene was identified as a candidate target gene of the ICOS-FOXO1 regulatory axis if it simultaneously met the following conditions: (1) This gene belongs to the differentially expressed genes related to ICOS signaling; (2) The promoter region, enhancer region or other regulatory regions of the gene contain FOXO1 binding peaks; (3) The FOXO1 binding peak satisfies q value < 0.05 and enrichment factor ≥ 2; (4) The direction of this gene expression change is consistent with the direction of ICOS signal affecting FOXO1 transcriptional regulatory activity; (5) This gene is involved in T cell activation, proliferation, cytokine secretion, migration, survival, apoptosis, immune metabolism or immune effect regulation.

[0053] 5. Validation criteria for candidate target genes: Candidate target genes were validated using qRT-PCR, Western blot, flow cytometry, ChIP-qPCR, or reporter gene assays. The following criteria were used for evaluation: (1) In the qRT-PCR verification, the expression of candidate genes in the anti-ICOS treatment group was ≥1.5-fold or ≤0.67-fold compared with the PBS group, and P<0.05; (2) In the ChIP-qPCR verification, the enrichment level of FOXO1 in the promoter region of candidate genes was significantly higher than that of IgG control, with an enrichment fold of ≥2 times and P<0.05; (3) After ICOS activation, the binding level of FOXO1 in the promoter region of candidate genes changed significantly, with a change of ≥30% and P<0.05; (4) At least two candidate target genes have obtained consistent validation results in independent replicate experiments; (5) If the candidate drug treatment can replicate the changes in FOXO1 localization, FOXO1 binding level and candidate target gene expression caused by anti-ICOS activation, it suggests that the candidate drug may act on the ICOS-FOXO1 regulatory axis.

[0054] like Figure 2 and Figure 3 As shown, an intersection analysis was performed between the ICOS-related differentially expressed genes screened by RNA-seq and the target genes annotated by FOXO1 ChIP-seq, yielding a total of 56 candidate genes. Further IGV visualization analysis revealed altered expression in genes such as Abca1, St6gal1, Runx2, Sema4a, and Brpf3, with FOXO1 binding peaks detected in their promoter regions or adjacent regulatory regions. These results suggest that Abca1, St6gal1, Runx2, Sema4a, and Brpf3 could serve as candidate target genes for the ICOS-FOXO1 regulatory axis.

[0055] Example 3 Candidate drug screening process based on the ICOS-FOXO1 regulatory axis: A method for screening HBV-ACLF candidate immune-enhancing drugs based on the ICOS-FOXO1-target gene axis.

[0056] 1. Candidate Type: Candidates include, but are not limited to, one or more of the following: ICOS agonist antibodies, ICOS protein, ICOS-Fc fusion protein, polymerized ICOS, ICOS functional fragments, bispecific or multispecific ICOS agonists, ICOS signaling activating peptides, peptides, small molecule allosteric agonists, nucleic acid drugs encoding ICOS or functional fragments thereof, engineered cells expressing ICOS, extracellular vesicles carrying ICOS, and nanodelivery formulations loaded with the above active ingredients.

[0057] 2. Establishment of cell model: Isolate wild-type mouse CD4+ T cells were isolated from peripheral blood PBMCs of HBV-ACLF patients and analyzed for CD4+. + T cells.

[0058] For mouse CD4 + T cells were stimulated with anti-CD3 at 0.5 μg / mL, and anti-CD28 was set at 0 μg / mL or 0.125 μg / mL to simulate a state of insufficient CD28 co-stimulation.

[0059] For human PBMCs, stimulation was performed with an anti-human CD3 antibody (1 μg / mL), the anti-human CD3 antibody being Bio X Cell's InVivoMAb anti-human CD3 antibody, catalog number BE0001-2; candidate or control products were also added simultaneously.

[0060] The cell seeding density can be set to 1×10⁻⁶. 6 1 × 10⁻⁶ cells / mL. Add 1 × 10⁻⁶ cells / mL to each well of a 96-well plate. 5 ~2×10 5 5 × 10⁵ cells were added to each well of a 24-well plate. 5 ~1×10 6 Cells. Culture conditions: 37°C, 5% CO2.

[0061] 3. Group settings The screening experiment should include at least the following groups: (1) Negative control group: anti-CD3 stimulation with PBS or candidate solvent; (2) Positive control group: anti-CD3 stimulation plus ICOS agonist antibody, anti-ICOS 10 μg / mL; (3) Low-dose candidate group; (4) Candidate mid-dose group; (5) High-dose candidate group; (6) Cytotoxicity control group; (7) If necessary, set up an Icos gene knockout CD4+ T cell group or an ICOS blockade group to determine whether the effect of the candidate depends on ICOS signaling.

[0062] Small molecule candidates can be set with concentration gradients of 0.001 μmol / L, 0.01 μmol / L, 0.1 μmol / L, 1 μmol / L, 10 μmol / L, and 50 μmol / L. When candidates are dissolved in DMSO, the final concentration of DMSO should not exceed 0.1%. Protein or antibody candidates can be set with concentration gradients of 0.01 μg / mL, 0.1 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL. The dosage of nucleic acid drugs or nanoformulations should be optimized according to the delivery system, with at least three dosage gradients set.

[0063] 4. Initial screening test indicators and time points: After the candidate is added to the cell culture system, different culture times are set according to different detection indicators: (1) Cell activation detection: After culturing for 24 h or 48 h, the expression of CD69, CD25 and CD38 was detected by flow cytometry; (2) Cell proliferation detection: Cells were pre-stained with CFSE and the CFSE dilution ratio was detected after 72 h or 96 h of culture; (3) Detection of cytokine secretion: After culturing for 24-96 h, PMA (final concentration 50 ng / mL), ionomycin (final concentration 1 μg / mL) and GolgiStop (0.7 μL / mL) were added for stimulation for 4-6 h, and the expression of IFN-γ, TNF-α and IL-2 was detected. (4) Apoptosis detection: After culturing for 24 h or 48 h, the apoptosis rate was detected by Annexin V / PI staining; (5) FOXO1 signal detection: After 2 h, 6 h, 12 h or 24 h of culture, the phosphorylation level of FOXO1, the nuclear localization of FOXO1 or the binding level of FOXO1 to the promoter region of the target gene were detected. (6) Detection of target gene expression: After 12 h, 24 h or 48 h of culture, the expression levels of ABCA1, ST6GAL1, RUNX2, SEMA4A and BRPF3 were detected by qRT-PCR.

[0064] 5. Detection methods for FOXO1 and its target genes: Nuclear localization of FOXO1 can be detected using immunofluorescence, Western blot after separation of nuclear / cytoplasmic proteins, or imaging flow cytometry. FOXO1 phosphorylation can be detected using Western blot or flow cytometry to determine p-FOXO1 levels. The binding level of FOXO1 to the promoter region of target genes can be detected using ChIP-qPCR. Target gene expression levels can be detected using qRT-PCR, Western blot, or flow cytometry.

[0065] ChIP-qPCR is preferred for detecting the fragments containing FOXO1 binding peaks in the promoter regions of Abca1, St6gal1, Runx2, Sema4a, and Brpf3. Input was used as a normalization control, and IgG was used as a negative control.

[0066] 6. Criteria for determining a positive result in the initial screening: Candidates can be identified as positive candidates in the initial screening if they meet the following conditions: (1) Cell viability is not less than 80% of that of the negative control group, or cell death rate does not exceed 20%; (2) The proportion of CD69 or CD25 positive CD4+ T cells increased by ≥20% compared with the negative control group, or the average fluorescence intensity increased by ≥1.5 times, and P<0.05; (3) The CFSE dilution ratio was ≥20% higher than that of the negative control group, and P<0.05; (4) The positive rate of IFN-γ or TNF-α was ≥20% higher than that of the negative control group, or the average fluorescence intensity was ≥1.5 times higher, and P<0.05; (5) The proportion of Annexin V positive apoptosis was ≥20% lower than that of the negative control group, and P<0.05; (6) At least one of the following is significantly altered: FOXO1 nuclear localization, FOXO1 phosphorylation, or FOXO1 binding level to the target gene promoter, with a change of ≥20% and P<0.05; (7) The expression changes of at least two target genes among ABCA1, ST6GAL1, RUNX2, SEMA4A and BRPF3 were consistent with the direction of the ICOS activation positive control group, with a change fold of ≥1.5 times or ≤0.67 times, and P<0.05.

[0067] If a candidate drug meets the positive criteria in at least three of the above-mentioned functional indicators, and meets the positive criteria in at least one FOXO1 axis indicator and at least two target gene expression indicators, it is determined to be an ICOS-FOXO1 axis-related candidate immune-enhancing drug.

[0068] 7. Criteria for rescreening: For candidates that initially tested positive, repeat the screening test described above for secondary screening, requiring them to meet the following conditions: (1) Positive results are obtained repeatedly in at least 3 independent batch experiments; (2) It exhibits a dose-dependent or dose-response trend; (3) Compared with the positive control anti-ICOS antibody, its comprehensive effect reaches more than 30% of the positive control, preferably more than 50%; (4) Under the conditions of Icos gene knockout CD4+ T cells or ICOS blockade, the effect of the candidate on enhancing CD4+ T cell function was significantly weakened or disappeared, suggesting that its effect is ICOS-dependent. (5) The candidate does not cause significant cytotoxicity and cell viability is maintained at over 80%.

[0069] 8. Overall scoring criteria: To improve the comparability of screening results, a comprehensive scoring system can be established. Each indicator is scored from 0 to 2 points. (1) CD69 / CD25 increase: no change is 0 points, 20%~50% increase is 1 point, and ≥50% increase is 2 points; (2) Increase in CFSE dilution: No change is 0 points, an increase of 20%~50% is 1 point, and an increase of ≥50% is 2 points; (3) Increased IFN-γ / TNF-α secretion: No change is 0 points, an increase of 20%~50% is 1 point, and an increase of ≥50% is 2 points; (4) Decreased Annexin V apoptosis: No change is 0 points, a decrease of 20%~50% is 1 point, and a decrease of ≥50% is 2 points; (5) Changes in the FOXO1 axis: 0 points for no significant change, 1 point for a significant change in 1 FOXO1 indicator, and 2 points for a significant change in 2 or more FOXO1 indicators; (6) Changes in target gene expression: less than 2 changes in target genes result in 0 points, 2-3 changes in target genes result in 1 point, and 4-5 changes in target genes result in 2 points.

[0070] A total score of ≥6 points is considered a positive candidate in the initial screening; a total score of ≥8 points is considered a preferred candidate; and a total score of ≥10 points is considered a highly preferred candidate.

[0071] Example 4 Application of screening results in the development of HBV-ACLF candidate drugs: Validation method of candidate immune-enhancing drugs obtained by screening in Example 3 in HBV-ACLF-related in vitro cell models and ACLF animal models.

[0072] 1. In vitro validation of PBMCs from HBV-ACLF patients: Peripheral blood was collected from HBV-ACLF patients, and PBMCs were separated using Ficoll density gradient centrifugation. The PBMCs were then adjusted to a density of 1×10⁻⁶. 6 Cells were seeded at a density of 1 / mL in cell culture plates. Stimulation was performed with anti-human CD3 antibody (1 / 2 μg / mL), along with the candidate drug, a positive control (anti-ICOS antibody, 10 μg / mL), or a PBS negative control. CD4 counts were measured after 24 h of culture. +T cell expression of CD69, CD25, and CD38; CFSE dilution ratio detected after 96 h of culture; Annexin V / PI apoptosis detected after 24–48 h of culture; IFN-γ, TNF-α, and IL-2 expression detected after 5 h of culture or after 4–6 h of restimulation with PMA (final concentration 50 ng / mL), ionomycin (final concentration 1 μg / mL), and GolgiStop (0.7 μL / mL).

[0073] Cells were resuspended in RPMI-1640 complete medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% HEPES. Each patient's PBMCs were allocated equally to the PBS control group and the ICOS agonist antibody treatment group.

[0074] The ICOS agonist antibody used was an anti-mouse / human / rat / monkey ICOS antibody, clone number C398.4A, catalog number BE0353, Bio X Cell. The antibody was diluted to 10 μg / mL using sterile PBS, and 200 μL was added to each well of a 96-well flat-bottom culture plate, equivalent to 2 μg of antibody per well, and coated overnight. An equal volume of PBS was added to the PBS control wells, and the same conditions were applied. The coating solution was discarded the next day, and PBS washing was performed according to the original experimental records. The product information for this antibody states that it can be used for in vitro T cell stimulation and activation.

[0075] PBMCs were added to the coated culture wells, with 200 μL of cell suspension added to each well. All groups were stimulated with anti-human CD3 antibody to a final anti-CD3 concentration of 0.5 μg / mL. The anti-human CD3 antibody used was clone OKT3, catalog number BE0001-2, Bio X Cell. All groups were cultured at 37 ℃ under 5% CO2 conditions.

[0076] In the T-cell activation assay, seven independent PBMC samples from HBV-ACLF patients were used. Each sample was further divided into a PBS group and an anti-ICOS group. CD3 levels were measured after 24 h of culture. + CD4 + The positive rates and average fluorescence intensity of CD69, CD25 and CD38 in T cells.

[0077] In the T cell proliferation assay, 12 independent patient PBMC samples were used. PBMCs were labeled according to the CFSE reagent instructions before culture. Each sample was further divided into a PBS group and an anti-ICOS group. CD3 levels were measured after 96 h of culture. + CD4 +CFSE dilution ratio of T cells.

[0078] In the apoptosis assay, five independent patient PBMC samples were used, with each sample further divided into a PBS group and an anti-ICOS group. Cells were collected after 24 h of culture, stained with Annexin V and PI, and CD3+ was detected by flow cytometry. + CD4 + Annexin V in T cells + PI - Early apoptotic cells and Annexin V + The proportion of total apoptotic cells.

[0079] In the cytokine assay, five independent patient PBMC samples were used, with each sample further divided into a PBS group and an anti-ICOS group. After 5 h of culture, intracellular cytokine staining was performed, and CD3 was detected. + CD4 + The positive proportions and average fluorescence intensity of IFN-γ, TNF-α and IL-2 in T cells.

[0080] If a candidate drug meets the following criteria, it is considered to have an immune-enhancing effect in the HBV-ACLF patient PBMCs model: (1) CD4 + T cell CD69 or CD25 expression was ≥20% higher than in the PBS group, and P<0.05; (2) CD4 + The proportion of T cells diluted with CFSE increased by ≥20% compared to the PBS group, and P<0.05; (3) CD4 + The secretion of IFN-γ or TNF-α by T cells was ≥20% higher than that in the PBS group, and P<0.05; (4) CD4 + The proportion of T cells positive for Annexin V decreased by ≥20% compared with the PBS group, and P<0.05; (5) Cell viability is not less than 80%.

[0081] The positive control experiment of this invention showed that the ICOS agonist antibody can enhance peripheral blood CD4 in HBV-ACLF patients. + T cell activation, proliferation, and cytokine secretion, and reduction of CD4. + The proportion of T cell apoptosis. Specifically, compared with the PBS control group, the anti-ICOS treatment group had a higher CD4 count. + Increased expression of CD69 and CD25 on T cells, increased CFSE dilution ratio, increased expression of IFN-γ and TNF-α, and decreased Annexin V-positive apoptosis ratio. Figure 4 This result indicates that activation of ICOS signaling can enhance CD4 in HBV-ACLF patients. + T cell activation, proliferation, and expression of some effector cytokines, and a decrease in the proportion of apoptotic cells.

[0082] 2. ACLF animal model validation: An ACLF animal model was established using 6-8 week old male C57BL / 6J mice. Chronic liver injury was induced first by intraperitoneal injection of carbon tetrachloride (CCl4). CCl4 was diluted with olive oil to a 10% solution and administered at a dose of 0.2 mL / kg twice weekly for 8 weeks. After week 8, a double dose of CCl4 was administered as an acute shock at a dose of 0.4 mL / kg. 72 hours after the acute shock, Klebsiella pneumoniae was injected intraperitoneally at a dose of approximately 1500 CFU per mouse to simulate the bacterial infection state associated with ACLF.

[0083] Candidate drugs can be administered before, concurrently with, or after injection of Klebsiella pneumoniae. Routes of administration can include tail vein injection, intraperitoneal injection, subcutaneous injection, or other methods suitable for the drug's properties. Low, medium, and high doses can be set, such as 0.1 mg / kg, 1 mg / kg, and 10 mg / kg; for small molecule candidates, doses can be set based on pharmacokinetic results, such as 0.1 mg / kg, 1 mg / kg, 10 mg / kg, or higher. Dosing frequency can be set as a single dose, once daily, or once every 2–3 days, with observation for 7 consecutive days.

[0084] In this embodiment, the candidate drug was the ICOS antibody (BE0059, Bioxcell). Chronic liver injury was induced by continuous carbon tetrachloride (CCl4), and acute liver injury was induced by a single double dose of CCl4. An ACLF model was constructed by combining this with intraperitoneal injection of Klebsiella pneumoniae. The construction method is described in Xiang X, Feng D, Hwang S, Ren T, Wang X, TrojnarE, Matyas C, Mo R, Shang D, He Y, Seo W, Shah VH, Pacher P, Xie Q, Gao B. Interleukin-22 ameliorates acute-on-chronic liver failure by reprogramming impaired regeneration pathways in mice. J Hepatol. 2020 Apr;72(4):736-745. doi: 10.1016 / j.jhep.2019.11.013. Epub 2019 Nov 29. PMID: 31786256; PMCID: PMC7085428. and Yu X, Yang F, Shen Z, Zhang Y, Sun J, Qiu C, Zheng Y, Zhao W, Yuan S, Zeng D, Zhang S, Long J, Zhu M, Zhang + T-cell exhaustion. Nat Commun. 2024 Feb 28;15(1):1835. doi: 10.1038 / s41467-024-46047-8. PMID: 38418488; PMCID: PMC10901893.

[0085] Before Klebsiella pneumoniae inoculation, the body weight and temperature of mice in each group were recorded as baseline values ​​at 0 h post-infection. Animal survival and general condition were observed every 6 h from 0 to 48 h post-inoculation, every 12 h from 48 to 72 h, and daily thereafter until day 7 post-infection, with the time of death or reaching the predetermined humanitarian endpoint recorded. Body temperature was measured at 6, 12, 24, 36, 48, and 72 h post-infection, and body weight was measured at 24, 48, and 72 h, and on days 5 and 7 post-infection. Peripheral blood was collected 24 h post-infection, serially diluted, and plate-cultured. Peripheral blood bacterial load was expressed as colony-forming units (CFU / mL). All indicators were evaluated by researchers unaware of the animal's genotype. Icos Survival, body temperature, body weight, and peripheral blood bacterial load in gene knockout mice and wild-type mice.

[0086] 2. Observation indicators for animal models: (1) Survival rate: Record the survival status 7 days or 14 days after modeling and compare them using the Log-rank test; (2) Body temperature: The body temperature of mice was measured at a fixed time every day; (3) Weight: Weigh yourself at a fixed time every day; (4) Peripheral blood bacterial load: Blood samples were collected 24 h and 48 h after Klebsiella pneumoniae injection, serially diluted and cultured on agar plates, and CFU / mL was calculated; (5) Liver pathological damage: Liver tissue was taken for HE staining to evaluate the degree of inflammation, necrosis and structural damage; (6) Peripheral blood or liver CD4 + T cell function: Flow cytometry was used to detect indicators such as CD69, CD25, IFN-γ, TNF-α, and Annexin V. (7) If necessary, serum ALT, AST, TBil, inflammatory factors and other liver function indicators should be tested.

[0087] 4. Criteria for determining a positive result in animal experiments: If a candidate drug meets any three or more of the following criteria compared to the model control group, it is deemed to have therapeutic value for HBV-ACLF: (1) The 7-day survival rate increased by ≥20%, and the Log-rank test P<0.05; (2) Peripheral blood bacterial load decreased by ≥50%, and P<0.05; (3) The decrease in body temperature is reduced by ≥20%, or the recovery speed is faster; (4) The rate of weight loss is reduced by ≥20%, or the recovery rate is accelerated; (5) HE staining of liver tissue indicated a reduction in the degree of inflammation and necrosis; (6) CD4+ T cell expression of CD69, CD25, IFN-γ, or TNF-α increased by ≥20%, and P<0.05; (7) CD4 + The proportion of T cell apoptosis decreased by ≥20%, and P<0.05; (8) Serum ALT, AST or TBil decreased by ≥20% compared with the model control group, and P<0.05.

[0088] The results of this invention's animal model show that, compared with wild-type mice, Icos knockout mice have lower survival rates, higher peripheral blood bacterial loads, and more significant decreases in body temperature and weight in the ACLF model. Figure 5 This result suggests that ICOS signaling plays an important role in pathogen clearance and survival improvement in ACLF. Therefore, the candidate drugs obtained through screening in Example 3 that can activate the ICOS-FOXO1 target gene axis can be further developed as candidate drugs for the prevention and / or treatment of HBV-ACLF, reducing the risk of secondary infection, or improving prognosis.

[0089] 5. Actual drug screening and validation data recording methods: For the specific drugs actually obtained through screening, at least the following data should be recorded: (1) Candidate drug name, code, source, structural type and purity; (2) Dosage or concentration; (3) CD4+ T cell activity; (4) Expression levels of CD69, CD25 and CD38; (5) CFSE dilution ratio; (6) Expression levels of IFN-γ, TNF-α and IL-2; (7) Annexin V / PI apoptosis ratio; (8) FOXO1 nuclear localization, FOXO1 phosphorylation, or FOXO1 promoter binding level; (9) Expression levels of ABCA1, ST6GAL1, RUNX2, SEMA4A and BRPF3; (10) Validation results in PBMCs of HBV-ACLF patients; (11) Results of bacterial load, body temperature, body weight and survival rate in ACLF animal models.

[0090] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Based on ICOS-FOXO1 control axis, filter and adjust CD4 + The in vitro screening method for T-cell immune function drugs is characterized by, Includes the following steps: Intervention of CD4 with the drug to be tested + T cells, CD4 count after intervention + T cell activation level, proliferation level, cytokine secretion level, apoptosis level, FOXO1 signaling activity, and expression levels of target genes related to the ICOS-FOXO1 regulatory axis; select test drugs that meet at least 3 of the following criteria: compared with the negative control group, (1) cell viability is not less than 80% and / or cell death rate is not more than 20%; (2) CD4 + (2) The proportion of T cells positive for CD69 or CD25 increased by ≥20% and / or the fluorescence intensity increased by ≥1.5 times, and P<0.05; (3) Cell proliferation increased by ≥20%, and P<0.05; (4) Cytokine secretion level increased by ≥20% and / or fluorescence intensity increased by ≥1.5 times, and P<0.05; (5) The proportion of Annexin V positive apoptosis decreased by ≥20%, and P<0.05; (6) FOXO1 nuclear localization, FOXO1 phosphorylation or any one of FOXO1, the binding level of target gene promoters was significantly changed, the change range was ≥20%, and P<0.05; (7) Among the target genes related to the ICOS-FOXO1 regulatory axis, the expression changes of at least 2 target genes were consistent with the direction of ICOS activation positive control group, the difference in expression fold was ≥1.5 times or ≤0.67 times, and P<0.

05.

2. The in vitro screening method according to claim 1, characterized in that, The screening method for target genes related to the ICOS-FOXO1 regulatory axis includes the following steps: CD4 + T cells were activated by ICOS signaling, and transcriptome sequencing was used to screen for differentially expressed genes related to ICOS signaling. Differentially expressed genes related to ICOS signaling met the following criteria: corrected P value < 0.05, and differential expression fold ≥ 1.5-fold or ≤ 0.67-fold. High-throughput sequencing of chromatin immunoprecipitation of FOXO1 transcription factor was performed to obtain the FOXO1 binding peak. The differentially expressed genes were analyzed in conjunction with the FOXO1 binding peak. The target genes related to the ICOS-FOXO1 regulatory axis were those that simultaneously met the following criteria: (1) The gene was a differentially expressed gene related to ICOS signaling; (2) The gene had a FOXO1 binding peak in any one or more regions of its promoter region, enhancer region, or other regulatory regions; (3) The FOXO1 binding peak met the criteria of q value < 0.05 and enrichment fold ≥ 2; (4) The direction of the gene expression change was consistent with the direction of ICOS signaling affecting FOXO1 transcriptional regulation activity; (5) The gene participated in any one or more functions in T cell activation, proliferation, cytokine secretion, migration, survival, apoptosis, immune metabolism, or immune effect regulation.

3. The in vitro screening method according to claim 2, characterized in that, Target genes associated with the ICOS-FOXO1 regulatory axis include Abca1 , St6gal1 , Runx2 , Sema4a and Brpf3 .

4. The in vitro screening method according to claim 1, characterized in that, CD4 + T cells can be derived from any of the following: peripheral blood mononuclear cells, lymph node cells, spleen cells, liver immune cells, in vitro induced differentiated T cells, T cell lines, or gene-edited T cells.

5. An adjustment CD4 based on the ICOS-FOXO1 control axis + An in vitro screening system for T-cell immune function drugs, characterized in that... The in vitro screening system includes: a sample collection module, an indicator detection module, a screening module, and a result output module; The sample acquisition module collects CD4 after treatment with the drug to be tested. + T cells; The indicator detection module detects CD4. + The activation level, proliferation level, cytokine secretion level, apoptosis level, FOXO1 signaling activity, and expression level of target genes related to the ICOS-FOXO1 regulatory axis of T cells were detected. The screening module analyzes the detection indicators to determine whether the drug to be tested meets at least three of the screening criteria; the screening criteria are compared with the negative control group: (1) cell viability not less than 80% and / or cell death rate not more than 20%; (2) CD4 + (2) The proportion of T cells positive for CD69 or CD25 increased by ≥20% and / or the fluorescence intensity increased by ≥1.5 times, and P<0.05; (3) Cell proliferation increased by ≥20%, and P<0.05; (4) Cytokine secretion level increased by ≥20% and / or fluorescence intensity increased by ≥1.5 times, and P<0.05; (5) The proportion of Annexin V positive apoptosis decreased by ≥20%, and P<0.05; (6) FOXO1 nuclear localization, FOXO1 phosphorylation or any one of FOXO1, the binding level of target gene promoters was significantly changed, the change range was ≥20%, and P<0.05; (7) Among the target genes related to the ICOS-FOXO1 regulatory axis, the expression changes of at least 2 target genes were consistent with the direction of ICOS activation positive control group, the difference in expression was ≥1.5 times or ≤0.67 times, and P<0.05; The result output module outputs the results.

6. The use of the in vitro screening method according to any one of claims 1 to 4 or the in vitro screening system according to claim 5 in screening and preparing drugs for the prevention and / or treatment of hepatitis B virus-related acute-on-chronic liver failure.

7. The application according to claim 6, characterized in that, Drugs for the prevention and / or treatment of hepatitis B virus-associated acute-on-chronic liver failure include traditional Chinese medicine extracts, chemically synthesized compounds, natural product monomers, peptides, or nucleic acid drugs.

8. A method for evaluating drugs for hepatitis B virus-related acute-on-chronic liver failure based on the ICOS-FOXO1 regulatory axis, characterized in that, Includes the following steps: Using the drug intervention to be evaluated for CD4 + T cells, with CD4 cells treated with CD3 antibody + T cells served as a negative control. The efficacy of the drug was assessed based on one or more of the following indicators: CD4. + The activation level, proliferation level, cytokine secretion level, apoptosis level, FOXO1 signaling activity, and expression levels of target genes related to the ICOS-FOXO1 regulatory axis of T cells.

9. An in vitro evaluation system for drugs targeting hepatitis B virus-related acute-on-chronic liver failure based on the ICOS-FOXO1 regulatory axis, characterized in that, The in vitro evaluation system includes: a sample collection module, an indicator detection module, an evaluation module, and a result output module; The sample collection module collects CD4 after the drug intervention to be evaluated. + T cells; The indicator detection module detects CD4. + The activation level, proliferation level, cytokine secretion level, apoptosis level, FOXO1 signaling activity, and expression level of target genes related to the ICOS-FOXO1 regulatory axis of T cells were detected. The evaluation module uses CD4 treated with CD3 antibody. + T cells served as a negative control. Various indicators were analyzed to evaluate the efficacy of the drug. The result output module outputs the results.

10. The use of the evaluation method of claim 8 or the in vitro evaluation system of claim 9 in the preparation of drugs for hepatitis B virus-related acute-on-chronic liver failure.

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