Application of immune response biomarker in prognosis evaluation of mesenchymal stromal cell treatment of liver cirrhosis patient

Abstract

The invention provides application of an immune response biomarker in prognosis evaluation of mesenchymal stromal cells in treatment of liver cirrhosis patients, and relates to the technical field of biomedicine, the immune response biomarker comprises MX1 positive monocytes or / and LGALS2 positive monocytes, and the MX1 positive monocytes are MX1 positive monocytes or / and LGALS2 positive monocytes. Screening to obtain a potential biomarker LGALS2 positive mononuclear cell and an MX1 positive mononuclear cell for predicting the MSC treatment dose-effect relationship; compared with a healthy control, the proportion of baseline MX1 positive monocytes of a liver cirrhosis patient is remarkably increased (Plt; 0.01), while the proportion of LGALS2 positive monocytes is significantly reduced (Plt; 0.05) of the substrate (1); the change trend of the biomarker under the same MSC dose and the regulation effect on other immune cell subgroups are identified through sequencing analysis, the dose-effect relationship of MSC-mediated immune regulation can be accurately reflected, and patients with low MSC treatment prognosis recurrence risk can be identified.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of an immune response biomarker in the prognosis evaluation of patients with liver cirrhosis treated with mesenchymal stromal cells. Background Art

[0002] Decompensated cirrhosis (DLC) is the advanced stage of cirrhosis. Cirrhosis-associated immune dysfunction (CAID), manifesting as a paradoxical combination of systemic inflammation and immune deficiency, is a key factor in the progression of DLC. Immune dysfunction predisposes patients to frequent and severe infections and promotes bacterial translocation, exacerbating liver damage, leading to multi-organ failure and ultimately a poor prognosis.

[0003] MSCs are recognized as promising cell therapy candidates due to their easy access, low immunogenicity, differentiation potential, and strong immunomodulatory capacity. Since DLC is characterized by immune dysregulation and progressive tissue damage, it has become an ideal target for MSC therapy because MSCs have the potential to restore immune homeostasis. Multiple clinical studies have documented improvements in indicators such as model end-stage liver disease (MELD) score, prothrombin time, and liver function after MSC infusion. Although the immunomodulatory potential of MSCs has been recognized as a key therapeutic benefit, it is worth noting that the dose-dependent effects of MSCs in human DLCs have not been systematically studied, and the dose-effect relationship of MSC-mediated immunomodulation remains poorly understood. This knowledge gap is a major obstacle to the effective clinical application of MSCs in the treatment of DLC. It is important to predict patients' response to MSCs before treatment and select potential responders.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] One of the objectives of the present invention is to provide an immune response biomarker to at least solve the technical problem in the prior art of lacking an immune response biomarker that can reflect the dose-effect relationship of MSC-mediated immune regulation.

[0006] A second object of the present invention is to provide the use of the above-mentioned immune response biomarkers in the prognosis evaluation of patients with cirrhosis treated with mesenchymal stromal cells or in the preparation of a kit for monitoring the prognosis of patients with cirrhosis treated with mesenchymal stromal cells.

[0007] A third object of the present invention is to provide the use of the above-mentioned immune response biomarkers in the preparation of drugs for immunomodulation in the treatment of patients with liver cirrhosis using mesenchymal stromal cells.

[0008] A fourth object of the present invention is to provide the use of the above-mentioned immune response biomarkers in the preparation of drugs for treating immune dysfunction in patients with cirrhosis.

[0009] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0010] In a first aspect, the present invention provides an immune response biomarker, wherein the immune response biomarker comprises MX1-positive monocytes and / or LGALS2-positive monocytes.

[0011] Furthermore, the MX1-positive monocytes have IFN-stimulated gene expression characteristics;

[0012] Preferably, the LGALS2-positive monocytes are CD14-positive monocytes;

[0013] Preferably, the MX1-positive monocytes include CD14-positive / CD16-negative classical monocytes.

[0014] In a second aspect, the present invention provides the use of the above-mentioned immune response biomarkers in the prognosis assessment of patients with cirrhosis treated with mesenchymal stromal cells or in the preparation of a kit for monitoring the prognosis of patients with cirrhosis treated with mesenchymal stromal cells.

[0015] Furthermore, the prognostic assessment includes establishing a dose-effect relationship between mesenchymal stromal cells and immune response biomarkers, obtaining the ratio of immune response biomarkers in peripheral blood at different treatment times, and performing prognostic assessment based on the ratio of immune response biomarkers;

[0016] Preferably, the prognostic assessment based on the ratio of immune response biomarkers includes that compared with before treatment, if the ratio of MX1-positive monocytes is significantly decreased and the ratio of LGALS2-positive monocytes is significantly increased, then the treatment is effective and the prognosis recurrence risk is low; otherwise, the treatment is ineffective and the prognosis recurrence risk is high.

[0017] Furthermore, the prognostic assessment includes assessing the immunoregulatory strength of mesenchymal stromal cells, or / and assessing the depth of improvement in the prognostic liver disease score;

[0018] Preferably, the dosage of the mesenchymal stromal cells is ≥1.5×10 8 cells / times;

[0019] Preferably, the dosing interval is ≤ 7 days.

[0020] Furthermore, the effects include reducing the phagocytosis or antigen presentation function of MX1-positive monocytes;

[0021] Preferably, reducing the phagocytosis or antigen presentation function of MX1-positive monocytes comprises downregulating the expression of at least one of proinflammatory cytokines, interleukin-15, or macrophage stimulating factor 1 in MX1-positive monocytes; or / and,

[0022] Upregulate the expression of at least one of TNFSF10, TNFSF13 or TNFSF14.

[0023] Furthermore, the kit includes substances for detecting immune response biomarkers;

[0024] Preferably, the monitoring method comprises detecting the ratio of immune response biomarkers in peripheral blood at different times after treatment.

[0025] In a third aspect, the present invention provides the use of the above-mentioned immune response biomarkers in the preparation of a drug for immunomodulation in the treatment of patients with liver cirrhosis using mesenchymal stromal cells.

[0026] Furthermore, the immune regulation includes activating the CD86-CTLA4 signaling axis and downregulating the activity of regulatory T cells; or / and,

[0027] Promotes recruitment of cytotoxic T cells through ICAM1-mediated transendothelial migration and activates the immune activity of γδ T cells; or / and,

[0028] Enhancement of LGALS2 by secretion of ANXA1 protein + phagocytic function of monocytes;

[0029] Preferably, the immune response biomarker is MX1-positive monocytes.

[0030] In a fourth aspect, the present invention provides the use of the above-mentioned immune response biomarker in the preparation of a medicament for treating immune dysfunction in patients with liver cirrhosis;

[0031] Preferably, the immune response biomarker is MX1-positive monocytes.

[0032] The immune response biomarkers provided by the present invention are screened for potential biomarkers predicting the dose-effect relationship of MSC treatment by performing single-cell RNA sequencing or mass spectrometry before and after treatment in a clinical study of MSC treatment of DLC patients with single or multiple ascending doses. LGALS2-positive monocytes and MX1-positive monocytes are compared; compared with healthy controls, the baseline proportion of MX1-positive monocytes in patients with cirrhosis is significantly increased (P<0.01), while the proportion of LGALS2-positive monocytes is significantly decreased; sequencing analysis is used to identify the changing trends of biomarkers at the same MSC dose and the regulatory effects on other immune cell subsets, which can accurately reflect the dose-effect relationship of MSC-mediated immune regulation and identify patients with a low risk of recurrence after MSC treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0034] Figure 1 Flowchart of the study design and participant grouping provided for this invention;

[0035] Figure 2 A graph showing changes in Child-Pugh scores of participants before and after MSC treatment provided by the present invention;

[0036] Figure 3 The dynamic landscape of monocytic immune cells in DLC patients treated with MSCs provided by the present invention; wherein, a is a schematic diagram of multi-omics analysis of the clinical trial cohort and the validation cohort; b is a UMAP plot of cell lines and subpopulations; c is a dynamic box plot of major immune cell lines; c is UMAP and CD14 / FCGR3A expression of monocyte subpopulations; e is a KEGG pathway feature score heat map; f is the dynamics of monocyte subpopulation ratios; g is the ratio of LGALS2-positive and MX1-positive monocytes; h is the dynamics of CCR-positive monocytes in CyTOF; i is a violin plot of cytokine production scores of MX1-positive monocytes; j is a heat map of gene expression in co-culture of MX1-positive monocytes;

[0037] Figure 4 The present invention provides the dynamic single-cell characteristics of DLC patients during MSC treatment; wherein, a is the t-SNE map of monocyte subsets annotated in CyTOF; b is the pseudo-time trajectory of Monocle3 (left) and Slingshot (right) of monocyte differentiation; c is the heat map of gene expression changes along pseudo-time; d is the distribution density map of monocyte subsets along pseudo-time in the healthy cohort, baseline, and low-dose, high-dose, and ultra-high-dose groups after treatment; e is the heat map of cytokines expressed by monocyte subsets; # indicates a significant difference between baseline and healthy individuals. * indicates a significant difference between the time point after MSC treatment and the baseline; f is the MX1 grouped by Child-Pugh score changes. + Box plot of dynamic changes of monocytes;

[0038] Figure 5Schematic diagram of the analysis of other immune cells and their interactions with monocytes under MSC treatment provided by the present invention; wherein, a is the UMAP and dynamics of T cell subsets; b is the dynamics of T cell subsets in CyTOF; c is the ligand-receptor heat map; d is the immune cell correlation pie chart; e is a schematic diagram of the interaction between MSC immune regulation timing and MX1-positive monocytes;

[0039] Figure 6 The present invention provides the results of changes in other immune cells of DLC patients under MSC treatment; wherein a. The left side is the UMAP map of NK cell subsets, showing the expression of NCAM1 and FCGR3A. The right side is the dynamic change map of the main NK cell subsets. The cell ratio is calculated as a percentage of the total NK cell number. b. The left side is the tSNE map of NK cell subsets annotated by CyTOF, and the right side is the dynamic change map of three NK cell subsets detected by CyTOF. c. The left side is the UMAP map of B cell subsets, and the right side is the dynamic change map of the main B cell subsets. The cell ratio is calculated as a percentage of the total B cell number. d. The left side is the tSNE map of B cell subsets annotated by CyTOF, and the right side is the dynamic change map of three B cell subsets detected by CyTOF. **p<0.001, p<0.01, p<0.05, respectively indicate extremely significant difference, significant difference and significant difference. DETAILED DESCRIPTION

[0040] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "including" and other forms is non-limiting.

[0041] Generally, the nomenclature used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein and its technology are those well-known and commonly used in this area.Unless otherwise indicated, the methods and techniques of the present invention are generally according to those well-known in the art, and are carried out as described in various general and more specific references, which are cited and discussed throughout this specification.Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as commonly achieved in this area, or as described herein.The nomenclature used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry as described herein and its laboratory procedures and technology are those well-known and commonly used in this area.

[0042] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0043] In one aspect, the present invention provides an immune response biomarker, which comprises LGALS2-positive monocytes and / or MX1-positive monocytes.

[0044] By performing single-cell RNA sequencing or mass spectrometry before and after treatment in a clinical study of MSC treatment of DLC patients with single or multiple ascending doses, potential biomarkers for predicting the dose-effect relationship of MSC treatment were screened, namely LGALS2-positive monocytes and MX1-positive monocytes. Compared with healthy controls, the baseline proportion of MX1-positive monocytes in patients with cirrhosis was significantly increased (P<0.01), while the proportion of LGALS2-positive monocytes was significantly decreased. Sequencing analysis was used to identify the changing trends of biomarkers at the same MSC dose and their regulatory effects on other immune cell subsets, which can accurately reflect the dose-effect relationship of MSC-mediated immune regulation and identify patients with a low risk of recurrence after MSC treatment.

[0045] The present invention reveals for the first time that MX1-positive monocytes are the key regulatory targets and effector cells for MSC treatment of liver cirrhosis, and that the regulatory effect of MSC treatment on MX1-positive monocytes is significantly dose-related.

[0046] In some specific embodiments, the MX1-positive monocytes have IFN-stimulated gene expression characteristics; in some specific embodiments, the LGALS2-positive monocytes LGALS2-positive monocytes are CD14-positive monocytes; in some specific embodiments, the MX1-positive monocytes include CD14-positive / CD16-negative classical monocytes.

[0047] According to another aspect of the present invention, there is also provided a use of the above-mentioned immune response biomarker in evaluating the prognosis of patients with cirrhosis treated with mesenchymal stromal cells or in preparing a kit for monitoring the prognosis of patients with cirrhosis treated with mesenchymal stromal cells.

[0048] In some specific embodiments, the prognostic assessment includes establishing a dose-effect relationship between mesenchymal stromal cells and immune response biomarkers, obtaining the ratio of immune response biomarkers in peripheral blood at different treatment times, and performing prognostic assessment based on the ratio of immune response biomarkers;

[0049] In some specific embodiments, the prognostic assessment based on the ratio of immune response biomarkers includes that compared with before treatment, if the ratio of MX1-positive monocytes is significantly decreased and the ratio of LGALS2-positive monocytes is significantly increased, then the treatment is effective and the prognosis of recurrence risk is low; otherwise, the treatment is ineffective and the prognosis of recurrence risk is high.

[0050] In some specific embodiments, the prognostic assessment includes assessing the immunoregulatory strength of mesenchymal stromal cells, and / or assessing the depth of improvement in a prognostic liver disease score.

[0051] High-dose MSCs (≥1.5×10 cells / time) can significantly reduce the proportion of MX1-positive monocytes and improve their biological functions, and this effect lasts for 7 days. However, low-dose MSCs (≤1.0×10 cells / time) have limited effects. In some specific embodiments, the dose of mesenchymal stromal cells is ≥1.5×10 8 cells / time; in some specific embodiments, the dosing interval is ≤7 days.

[0052] In some specific embodiments, the effect includes reducing the phagocytosis or antigen presentation function of MX1-positive monocytes;

[0053] During MSC treatment of DLC, CCR4, CCR6, IL15, MST1, TNFSF10, TNFSF13, and TNFSF14 exhibit specific, dose-dependent changes in MX1-positive monocytes. In some specific embodiments, reducing the phagocytic activity or antigen presentation function of MX1-positive monocytes comprises downregulating the expression of at least one of the proinflammatory cytokines, interleukin-15, or macrophage stimulating factor 1 (MST1) in MX1-positive monocytes; and / or upregulating the expression of at least one of TNFSF10, TNFSF13, or TNFSF14.

[0054] In some specific embodiments, the kit includes a substance for detecting immune response biomarkers. In some specific embodiments, the substance for detecting immune response biomarkers includes a substance for detecting MX1-positive monocytes, or / and a substance for detecting LGALS2-positive monocytes.

[0055] In some specific embodiments, the monitoring method comprises detecting the ratio of immune response biomarkers in peripheral blood at different times after treatment.

[0056] By analyzing the cell communication between MX1+ monocytes and other immune cell subsets, it was revealed for the first time that MX1+ monocytes play the role of "immune regulatory center" in MSC treatment. According to another aspect of the present invention, the use of the above-mentioned immune response biomarkers in the preparation of drugs for immunomodulation in mesenchymal stromal cell treatment of patients with cirrhosis is also provided.

[0057] This invention reveals for the first time the core mechanism of stem cell therapy for liver cirrhosis through single-cell multi-omics technology: MX1+ monocytes act as immune regulatory hubs, significantly improving the immune imbalance of liver cirrhosis by dose-dependently regulating Treg activity, enhancing CD8+ T cell migration, and coordinating anti-inflammatory monocyte differentiation.

[0058] In some specific embodiments, the immune regulation includes activating the CD86-CTLA4 signaling axis, downregulating the activity of regulatory T cells (Treg), and relieving the excessive immune suppression state; or / and promoting the transendothelial migration of cytotoxic T cells (CD8 + Teff) recruitment, while activating the immune activity of γδT cells through the APP-CD74 pathway (the inherent immune activity of γδT cells); or / and, enhancing the phagocytic function of LGALS2+ monocytes by secreting ANXA1 protein to regulate anti-inflammatory monocytes.

[0059] In some specific embodiments, the immune response biomarker is MX1-positive monocytes.

[0060] According to another aspect of the present invention, there is also provided the use of the above-mentioned immune response biomarker in the preparation of a drug for treating immune dysfunction in patients with liver cirrhosis;

[0061] Preferably, the immune response biomarker is MX1-positive monocytes.

[0062] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0063] scRNA-seq: single-cell RNA sequencing.

[0064] CyTOF: time-of-flight mass cytometry.

[0065] Bulk RNAseq: Bulk RNA sequencing.

[0066] Example: Dose-effect relationship study based on immune response biomarkers in MSC treatment of DLC

[0067] 1. Patient recruitment

[0068] Phase Ia patients were recruited at the Fifth Medical Center, PLA General Hospital, from March 22, 2022, to July 5, 2023. After evaluation of phase Ia data, phase Ib recruitment continued from August 22, 2023, to March 22, 2024. Patients aged 18-75 years, with a Child-Pugh score of 7-12 and a diagnosis of DLC were included.

[0069] Diagnosis is based on clinical manifestations, laboratory tests, imaging findings, and / or representative pathological findings. In addition, patients must have at least one severe complication, such as hepatic encephalopathy, upper gastrointestinal bleeding caused by esophageal or gastric varices, spontaneous bacterial peritonitis, or ascites. To prevent potential recompensation interference, this study excluded patients who had received antiviral treatment for HBV infection for less than 12 months, had TIPS insertion in the past 6 months, or had received corticosteroid treatment for autoimmune cirrhosis for less than 6 months. To validate the immunological mechanism identified in the above cohort, this study also recruited 5 DLC patients who met the inclusion and exclusion criteria and did not receive MSC treatment as independent research subjects for verification.

[0070] 2. Research Design

[0071] like Figure 1 This Phase Ia / Ib clinical trial, based on an open-label, sequential, single-arm, dose-escalation approach, included single and multiple doses of MSCs according to a "3+3" strategy. The sample size was based on a Phase I dose-escalation design, prioritizing safety assessments while minimizing patient risk in accordance with regulatory guidelines. In Phase Ia, patients were assigned to one of four cohorts, each receiving a single dose of MSCs: Cohort I received 5.0×10 cells, Cohort II received 1.0×10 cells, Cohort III received 1.5×10 cells, and Cohort IV received 2.0×10 cells. Based on Phase Ia data, the safety, efficacy, immunomodulatory effects, and duration of effect of different doses of MSCs were evaluated. In Phase Ib, patients received three doses of MSCs, administered one week apart: Cohort A received 1.0×10 cells per dose, while Cohort B received 2.0×10 cells per dose. Follow-up assessments were performed at baseline, day 3 (D3), day 7 (D7), day 14 (D14), and day 28 (D28).

[0072] 3. Sample processing: Peripheral blood samples for scRNA-seq were collected at baseline, D3, D7, D14 and D28, and samples for CyTOF staining were collected at baseline, D3 and D7. The above samples were transported to the laboratory for processing within 12 hours at room temperature, and within 48 hours if stored at approximately 4°C. All samples were analyzed fresh and did not require freezing. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll density gradient centrifugation. The cell pellet was resuspended in 5 ml of pre-cooled fluorescence-activated cell sorting (FACS) buffer (1× phosphate-buffered PBS, supplemented with 0.5% bovine serum albumin) and then centrifuged at 400×g for 5 minutes at 4°C. After discarding the supernatant, the microspheres were resuspended in FACS buffer. Cell counts were performed, and samples for subsequent analysis needed to meet the following criteria: cell counts of at least 3×10 6 , the survival rate is higher than 85%.

[0073] 4. Single-cell transcriptome preprocessing:

[0074] Raw FASTQ files were mapped to the GRCh38 genome using Cell Ranger software (version 4.0.0) with default parameters. After alignment, a digital gene expression (DGE) matrix was generated. For each sample, a barcode file, a gene annotation file, and a raw count matrix file were generated. These data were then imported into R (version 4.2.1) using the Seurat package (version 4.2.0). Genes expressed in cells with <10 cells and <200 gene features or 1000 read counts were excluded from the count matrix. Cells with <10% mitochondrial gene expression were retained for further analysis. The count matrix was then normalized using a logarithmic transformation, and the top 2000 variable features were selected for dimensionality reduction. To eliminate batch effects arising from samples collected at different time points and from different patients, all Seurat objects were integrated using the FastMNN method. To remove doublets, the DoubletFinder package (version 2.0.3) was used. The proportion of double-positive cells increased by 0.8% for every 1000 additional cells in the sample. High-confidence doublets were then removed. Dimensionality reduction was performed using principal component analysis (PCA). The top 30 principal components were selected for clustering, and the cell clusters were visualized using uniform manifold approximation and projection (UMAP). Cell subtypes were obtained using the FindNeighbors and FindCluster functions in Seurat. Finally, subtypes were annotated based on marker genes to obtain immune cell subtypes. The signal pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) were downloaded using the 7.5.1 version of the msigdbr software package to facilitate the calculation of single-cell feature scores. The gene lists of these pathways were then provided through the "AddModuleScore" function in Seurat, and the signature scores were calculated with default parameters. The average signal pathway score for each cell subset was calculated using the "AverageExpression" function in Seurat.

[0075] Based on the expression levels of each receptor and ligand gene pair, the CellChat method was used to infer potential interactions between two cell types. Receptors and ligands expressed in more than 20% of cells in the corresponding subcluster were included in the analysis. Visualization was performed using various built-in functions of the CellChat package (with default parameters).

[0076] 5. Mass Spectrometry Staining and Data Acquisition

[0077] Antibody labeling was performed using the Maxpar Antibody Coupling Kit (Fluidigm), and the concentration of the mass-labeled antibody was determined using NanoDrop. The concentration of the labeled antibody was adjusted to 200 mg / mL using antibody stabilization buffer, and the optimal concentration was determined by titration. The obtained cells were washed with PBS and stained with 100 μL of 250 nM cisplatin (Fluidigm, South San Francisco, CA, USA) on ice for 5 minutes to exclude dead cells. The cells were then incubated in Fc receptor blocking solution and then stained with a mixture of surface antibodies on ice for 30 minutes. After PBS washing, individual samples were labeled for 30 minutes using a unique barcode isotope combination. The cells were washed twice with FACS buffer and fixed overnight with 200 μL of intercalation solution (Maxpar Fix and Perm buffer containing 250 nM 191 / 193 Ir, Fluidigm). The cells were then washed with FACS buffer and Perm buffer (eBioscience, San Diego, CA, USA) and stained with a mixture of intracellular antibodies on ice for 30 minutes. After staining, cells were washed and resuspended in deionized water, mixed with 20% EQ beads (Fluidigm), and analyzed using a Helios mass cytometer (Fluidigm). CyTOF experiments were performed by PLTTECH (Hangzhou, China). Before loading each batch, signal intensity was adjusted for each channel based on the same head signal (140Ce, 151Eu, 153Eu, 165Ho, and 175Lu). All samples were normalized before analysis to prevent batch effects.

[0078] Raw data for each sample were encoded using a double filtering scheme with a unique mass-labeled barcode. The fcs files were normalized using the header normalization method across batches. Manual gating was performed using FlowJo software (FlowJo, Ashland, OR, USA) to exclude debris, dead cells, and doublets, leaving only live single immune cells. The X-shift clustering algorithm was applied to divide cells into different phenotypes based on marker expression levels. Each cluster was then annotated based on its marker expression pattern and visualized on a heatmap. The high-dimensional data were analyzed using the t-distributed stochastic neighbor embedding (t-SNE) algorithm to reduce dimensionality and visualize the differences between cluster distributions, marker expression, and groups or sample types.

[0079] 6. Monocyte and MSC Co-culture and RNA Sequencing

[0080] Peripheral blood (10 mL) was collected from patients in an independent validation cohort, and plasma and PBMCs were separated. CD14-positive monocytes were then purified from PBMCs using magnetic bead sorting (MACS). The isolated monocytes were plated in 0.4 μm Transwell inserts and placed at a 1:1 cell ratio in 12-well plates pre-seeded with MSCs. Co-culture was maintained at 37°C for 24 hours. After incubation, monocytes were collected, and total RNA was extracted for RNA sequencing. Clean data were obtained by removing adapters and filtering out low-quality reads. Paired-end reads were aligned to the human genome (UCSC hg38) using HISAT2 (version 2.1.0). Gene annotation and read counts were performed using HTSeq (version 0.11.2). Protein-coding genes were selected, and normalized expression values ​​were calculated using the DESeq2 package (version 1.32.0) on the R platform (version 4.1.0). MX1-positive monocyte-specific genes were defined based on two criteria: (1) expression in more than 30% of MX1-positive monocytes and (2) an average log-fold change of expression greater than 0.2 between MX1-positive monocytes and other cells. Gene Ontology (GO) biological process terms were used to annotate the functions of MX1-positive monocyte-specific genes. Finally, differentially expressed genes after MSC co-culture were analyzed in three functional categories: (1) cytokine production, (2) phagocytosis, and (3) antigen processing and presentation, to explore the effects of MSC on MX1-positive monocytes and whether MX1-positive monocytes can transition to a healthy state.

[0081] 7. Statistical analysis:

[0082] Continuous variables are summarized as mean ± standard deviation (SD), and categorical variables are summarized as frequency and percentage. Categorical variables were compared between dose cohorts using the chi-square test or Fisher's exact test. Continuous variables were compared using the t-test or Wilcoxon signed-rank test. The corresponding 95% confidence intervals (95% CI) are provided.

[0083] To analyze changes in cell proportions across multiple cohorts and time points, three statistical strategies were used: (1) comparison of changes in cell proportions across time points across all patients using one-way analysis of variance followed by paired t-tests; (2) comparison of changes in cell proportions across time points within each patient group using one-way analysis of variance followed by paired t-tests; and (3) comparison of differences in cell proportions across time points across different patient groups using linear mixed models. A p-value < 0.05 was considered statistically significant. All clinical statistical analyses were performed using SAS 9.4 (SAS Institute, Cary, North Carolina, USA). Statistical analysis of the frequencies of annotated cell populations was performed using the Student's t-test using the R platform (version 4.2.2).

[0084] 8. Baseline clinical characteristics, as shown in Table 1:

[0085] Table 1 Baseline demographic and clinical characteristics of patients

[0086]

[0087]

[0088] BMI = weight (kg) / height (m) 2 .

[0089] 9. Result 1: Safety evaluation and Child-Pugh score results of the MSC-treated DLC clinical cohort.

[0090] like Figure 2 Results showed that MSC infusion was well tolerated, with no adverse hemodynamic or respiratory changes observed during or within 30 minutes after infusion. During the 28-day observation period, there were no dose-limiting toxic events, discontinuations due to adverse events, serious adverse events (SAEs), or suspected unexpected serious adverse reactions (SUSARs).

[0091] At day 28, 53.3% (n = 8) of participants in Phase Ia had a decrease in Child-Pugh score, while 13.3% (n = 2) had an increase in Child-Pugh score, limited to Cohorts I and II, with no increases recorded in Cohorts III and IV. In Phase Ib, 88.9% (n = 8) of participants had a decrease in Child-Pugh score, including 3 in Cohort A and 5 in Cohort B, with no increase in Child-Pugh score reported in either cohort. Overall, the high-dose group was more likely to have a decrease in Child-Pugh score in both phases.

[0092] 10. Result 2: Monocytes are the main effector cells in the peripheral blood immune cells of patients with cirrhosis treated with MSC.

[0093] The present invention performed single-cell RNA sequencing (scRNA-seq) on peripheral blood mononuclear cell (PBMC) samples from patients in different dosing cohorts at five time points (baseline, day 3 [D3], day 7 [D7], day 14 [D14], and day 28 [D28]). Figure 3 At the same time, PBMC data of 28 healthy donors from public databases were integrated to clarify the baseline characteristics of patients with cirrhosis-associated immune dysregulation (CAID). After strict quality control and batch effect correction, a total of 467,831 single cells were obtained (as shown in Figure 2). Figure 3 Through dimensionality reduction analysis and classic marker annotation, four major immune cell lineages were identified: T cells, B cells, NK cells and monocytes, as well as other myeloid immune cells (such as Figure 3 The above lineages can be further subdivided into subpopulations with higher resolution. By calculating the proportion of each cell lineage at baseline and its dynamic changes after MSC treatment, it was found that the proportion of monocytes and B cells increased at baseline, while the proportion of T cells and NK cells decreased (as shown in Figure 2b). Figure 3 (c) After MSC treatment, the proportions of these cells gradually approached healthy levels, with the most significant recovery at D7. This conclusion was confirmed by mass cytometry (CyTOF) results, which showed that among all immune cell subsets, monocytes showed the most significant differences between DLC patients and healthy individuals. This indicates that MSC treatment has the strongest effect on monocytes, while having limited effects on the proportions of other immune cells, suggesting that changes in monocytes are the primary mechanism by which MSCs exert their immunomodulatory functions.

[0094] 11. Result 3: Characteristic analysis of 5 monocyte subsets.

[0095] The present invention annotated and analyzed 5 monocyte subsets (such as Figure 3d) to elucidate the changes in monocyte subsets during MSC treatment. The results showed that versican-positive (VCAN+, c35) monocytes, galectin-2-positive (LGALS2+, c36) monocytes, and myxovirus resistance protein 1-positive (MX1+, c38) monocytes all expressed high levels of CD14 and low levels of Fcγ receptor IIIa (FCGR3A, CD16), consistent with classical monocyte characteristics. MX1+ monocytes also showed high expression of interferon-stimulated genes (ISGs). MHC class II molecule DPβ+ (HLA-DPB+, c37) monocytes expressed both FCGR3A and CD14 (transitional monocytes), while FCGR3A+ (c39) monocytes expressed high levels of FCGR3A (CD16) and low levels of CD14, corresponding to non-classical monocytes. KEGG pathway analysis showed that MX1+ monocytes had higher immune activity, and their highly expressed genes were enriched in chemokine receptor (CCR), mitogen-activated protein kinase (MAPK) and transforming growth factor-β (TGF-β) pathways (e.g. Figure 3 (as shown in e).

[0096] 12. Result 4: Characteristics of MX1+ monocytes and LGALS2+ monocytes in patients with cirrhosis and their response to MSC treatment.

[0097] Cell ratio analysis showed that MX1+ monocytes were significantly increased compared with healthy controls at baseline, while LGALS2+ monocytes were significantly decreased (e.g. Figure 3 After MSC treatment, both subpopulations showed a trend toward a healthy state, but there were differences between different cohorts: in the high-dose cohort, MSCs effectively reduced MX1+ monocytes and increased LGALS2+ monocytes, with the most significant effect at D7; the medium-dose cohort had a moderate regulatory effect; while no significant changes were observed in the low-dose cohort (as shown in Figure 5). Figure 3 The CyTOF data also identified a subpopulation with high expression of CCR4 and CCR6, which corresponds to MX1+ monocytes (as shown in g). Figure 4 The proportion of this subpopulation decreased on D3 and D7 (as shown in a). Figure 3 h in the figure).

[0098] Compared with healthy controls, the baseline proportion of MX1-positive monocytes in DLC patients was significantly increased (P<0.01), while the proportion of LGALS2-positive monocytes was significantly decreased (P<0.05).

[0099] 13. Result 5: High-dose MSC treatment effectively promoted the differentiation of monocytes from the VCAN+ monocyte population (differentiation starting point) to the FCGR3A+ monocyte population (terminal state).

[0100] Monocle3 pseudo-sequential analysis and Slingshot validation showed that VCAN+ monocytes were the starting point of differentiation and FCGR3A+ monocytes were the terminal state (e.g. Figure 4 As the differentiation process progresses, cells gradually express MHC class II molecules, such as Figure 4 As shown in Figure c, MHC class II molecules include HLA-DRA, HLA-DRB1, HLA-DQA1, HLA-DPA1, and HLA-DMA. At baseline, FCGR3A+ monocytes showed earlier phenotypic characteristics than those in the healthy state (e.g. Figure 4 d), and MSC treatment promoted their differentiation toward terminal phenotypes, especially in the high-dose cohort.

[0101] 14. Result 6: High-dose MSC treatment effectively exerts an immunomodulatory effect on MX1+ monocytes and is associated with clinical outcomes.

[0102] Cytokine secretion by MX1+ monocytes decreased after MSC treatment, with the most obvious downward trend in the high-dose cohort and the strongest effect at D7 (e.g. Figure 3 Further analysis showed that MSC treatment downregulated the expression of pro-inflammatory cytokines (IL15 and MST1) in MX1+ monocytes that were elevated at baseline in DLC patients, but increased the expression of tumor necrosis factor superfamily members (TNFSF10, TNFSF13, TNFSF14), which are known to activate T cells (e.g. Figure 4 (as shown in e).

[0103] By verifying the results of an independent cohort experiment (batch RNA-seq of peripheral blood CD14+ monocytes from 5 DLC patients after co-culture with MSCs), using scRNA-seq as a reference, it was found that after co-culture of MX1+ monocytes with MSCs, the expression of genes related to cytokine production, phagocytosis, and antigen presentation were all downregulated, confirming the immunomodulatory effect of MSCs on MX1+ monocytes (such as Figure 3 In addition, the patients were divided into four groups (+1 / +2, 0, -1, and -2) according to the changes in Child-Pugh scores. The analysis showed that the proportion of MX1+ monocytes in the "+1 / +2" group returned to above the baseline on D7; in the "0" group, it exceeded the baseline on D14; in the "-1" group, it was below the baseline on D3 and was comparable to the baseline on D7 / D14; and in the "-2" group, it remained below the baseline at all post-treatment time points (e.g., Figure 4 (f), indicating that the proportion of MX1+ monocytes is associated with clinical outcomes.

[0104] Therefore, the dose-effect of MX1+ monocytes was most obvious in the high-dose group (≥1.5×10 cells / time), and this effect lasted for 7 days, while there was no obvious effect in the medium and low-dose groups.

[0105] 15. Result 7: MX1+ monocytes mediate the immunoregulatory function of MSCs, acting as an "immune regulatory hub" in MSC treatment of liver cirrhosis

[0106] The present invention analyzed other immune cell subsets that were abnormally expressed in DLC patients and responded to MSC treatment. 223,953 T cells were divided into 17 subsets (e.g. Figure 5 (shown in a). Regulatory CD4+ T cells (c5_CD4Treg_FOXP3) and effector CD8+ T cells (c10_CD8Teff_NKG7) were both elevated at baseline. After MSC treatment, CD4Treg gradually decreased on D3 and D7, while the proportion of CD8Teff was not directly inhibited. CyTOF also identified the corresponding subpopulations ( Figure 5 (as shown in middle b), most of the patients with increased CD4 Treg proportion were from the high-dose group, and the change trend of CD8 Teff was consistent with that of scRNA-seq.

[0107] Among the eight NK cell subsets, c22_NK_GZMK was identified as CD56bright NK cells (e.g. Figure 6 It is worth noting that the proportion of this subgroup decreased in cohort I, while it increased in some patients in cohorts II / III / IV (as shown in a). Figure 6 Among the 9 B cell subsets, two naive B cell subsets (c26_NaiveB_IL4R and c27_NaiveB_TCL1A) and germinal center B cells (c33_gcB_CD38) were elevated at baseline, while three memory B cell subsets (c29_MemB_AIM2, c30_MemB_TNFRSF13B, and c31_MemB_FCRL5) were decreased (as shown in Figure 2b). Figure 6 After MSC treatment, the initial B cells remained stable and gradually decreased on D3 / D7 (as shown in Figure c). Figure 6 (as shown in c and d).

[0108] Intercellular communication analysis revealed that the signaling intensity of all immune cell subsets was increased at baseline compared with healthy controls, and was weakened after MSC treatment (e.g. Figure 3MX1+ monocytes mainly act as signal senders, while CD8 Teffs are the main receivers. Among the signals sent by MX1+ monocytes, the galectin pathway was enhanced in DLC patients but decreased on D7 in cohorts II / IV; the resistin pathway was decreased in patients but increased on D3 / D7 in cohort IV (as shown in Figure 2). Figure 3 As shown in b and c). Ligand-receptor pair analysis showed (as shown in Figure 5 Middle C and Figure 3 (as shown in d), at baseline, MX1+ monocytes can promote the transendothelial migration of CD4Treg, CD8Teff, γδT cells and LGALS2+ monocytes through intercellular adhesion molecule-1 (ICAM1), and interact with other subsets through the amyloid precursor protein (APP)-CD74 axis. MX1+ monocytes also enhance the suppressive function of CD4Treg through CD86-CTLA4 and strengthen the chemotaxis and phagocytic ability of LGALS2+ monocytes through annexin A1 (ANXA1). At D3 / D7 after MSC treatment, the chemotactic activity of MX1+ monocytes decreased while the antigen presentation ability increased, and they can regulate CD4Treg function through IL16-CD4. Correlation analysis showed (as shown in Figure 5 As shown in Figure d), MX1+ monocytes are positively correlated with CD4Treg, γδT cells and naive B cells, and negatively correlated with CD8Teff, LGALS2+ monocytes and memory B cells. Based on the above findings, a schematic diagram of the mechanism by which MSCs regulate immune cells was drawn (as shown in Figure d). Figure 5 (as shown in e).

[0109] 16. Result 8: Correlation between MX1-positive monocytes and clinical response.

[0110] Patients in groups I, II, and IV were divided into four groups based on changes in Child-Pugh scores (i.e., +1 / +2, 0, -1, -2), and changes in the proportion of MX1-positive monocytes were analyzed. The results showed that in the "+1 / +2" group, the proportion of MX1-positive monocytes increased by D7 compared with baseline, while in the "0" group, the proportion increased by D14 compared with baseline. In contrast, the proportion of patients in the "-1" group was lower than baseline on D3, while the proportion on D7 and D14 was comparable to baseline. In contrast, the difference between D7 and D14 in the "-1" group and baseline was not significant. More importantly, in the "-2" group, the proportion of MX1-positive monocytes remained consistently lower than baseline at all four time points after treatment. These findings suggest that the degree of MSC-mediated MX1+ monocyte suppression is positively correlated with the depth of improvement in liver function.

[0111] Application scenario description:

[0112] 1. Accurately regulate the immune balance of DLC patients and optimize clinical use

[0113] The MSC treatment in this invention mainly regulates MX1 in DLC patients. + The changes in the proportion of monocytes were consistent with the changes in Child-Pugh scores. Patients with a greater decrease in scores after treatment also had lower levels of MX1-positive monocytes, indicating that MX1 + Monocytes are targets and biomarkers of MSC therapy response.

[0114] We further demonstrated that higher MSC doses elicited stronger immunomodulatory effects, as reflected in the significant modulation of immune cell subsets critical for CAID pathogenesis. These effects persisted for 7 days after treatment but diminished by day 14, highlighting the importance of optimizing dose and treatment interval. + Monocytes are key immune mediators, and the dose of mesenchymal stromal cells is ≥1.5×10 8 Cells / time is a reasonable dosage for DLC patients. The dosage interval is once every 7 days, repeated 3 times. It can be used in the trial or actual treatment of MSC treatment of DLC to guide efficacy research.

[0115] 2. MX1 + Monocytes are used as biomarkers to predict or monitor treatment response. The proportion of monocytes in peripheral blood and the changes after MSC infusion can be detected by flow cytometry to predict the therapeutic effect on cirrhosis. For example, peripheral blood can be collected before infusion to quickly identify and quantify MX1. + Monocyte subpopulations; then according to the dynamic changes of the subpopulation ratio and functional state at different doses, the dose-effect relationship of the present invention was established, according to MX1 + The proportion of monocytes can predict the effect of MSC treatment, so as to adjust the treatment plan in time to achieve precise regulation of the immune balance of DLC patients and optimize the clinical efficacy.

[0116] 3. MX1 + Monocytes act as immune regulatory centers and regulate related application scenarios of other cells.

[0117] This study analyzed other immune cell subsets that are abnormally expressed in DLC patients and responsive to MSC therapy, demonstrating that MX1+ monocytes mediate the immunomodulatory function of MSCs. For example, during MSC therapy for DLC, MX1+ monocytes serve as key signaling centers, orchestrating systemic immune homeostasis through multiple ligand-receptor pathways. Furthermore, during MSC-induced immune remodeling, MX1+ monocytes play a central role in restoring systemic immune homeostasis by coordinating the function of multiple subsets, including Tregs, effector T cells, and B cells.

[0118] 4. In view of MX1 +Correlation between monocytes and liver disease scores, as assessed by MX1 + The proportion and function of monocytes can assess the severity of cirrhosis and cirrhosis-related immune dysfunction.

[0119] 5. MX1 + Monocytes can be used as targets and markers for treating cirrhosis-related immune dysfunction in a variety of treatment methods, including MSC therapy (chemotherapeutic drugs, cell therapy, etc.).

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An immune response biomarker, characterized in that The immune response biomarkers include MX1-positive monocytes and / or LGALS2-positive monocytes.

2. The immune response biomarker according to claim 1, characterized in that The MX1-positive monocytes have IFN-stimulated gene expression characteristics and active TGF-β and CCR pathway expression; Preferably, the LGALS2-positive monocytes are CD14-positive / CD16-negative monocytes; Preferably, the MX1-positive monocytes include CD14-positive / CD16-negative classical monocytes.

3. Use of the immune response biomarker according to claim 1 or 2 in the prognosis assessment of patients with cirrhosis treated with mesenchymal stromal cells or in the preparation of a kit for monitoring the prognosis of patients with cirrhosis treated with mesenchymal stromal cells.

4. The use according to claim 3, characterized in that The prognostic assessment includes establishing a dose-effect relationship between mesenchymal stromal cells and immune response biomarkers, obtaining the ratio of immune response biomarkers in peripheral blood at different treatment times, and performing prognostic assessment based on the ratio of immune response biomarkers; Preferably, the prognostic assessment based on the ratio of immune response biomarkers includes that compared with before treatment, if the ratio of MX1-positive monocytes is significantly decreased and the ratio of LGALS2-positive monocytes is significantly increased, then the treatment is effective and the prognosis recurrence risk is low; otherwise, the treatment is ineffective and the prognosis recurrence risk is high.

5. The use according to claim 4, characterized in that The prognostic evaluation includes evaluating the immune regulation strength of mesenchymal stromal cells, or / and evaluating the improvement depth of the prognostic liver disease score; Preferably, the dosage of the mesenchymal stromal cells is ≥1.5×10 8 cells / times; Preferably, the dosing interval is ≤ 7 days.

6. The use according to claim 4, characterized in that The effects include reducing the phagocytosis or antigen presentation function of MX1-positive monocytes; Preferably, reducing the phagocytosis or antigen presentation function of MX1-positive monocytes comprises downregulating the expression of at least one of proinflammatory cytokines, interleukin-15, or macrophage stimulating factor 1 in MX1-positive monocytes; or / and, Upregulate the expression of at least one of TNFSF10, TNFSF13 or TNFSF14.

7. The use according to claim 3, characterized in that The kit includes substances for detecting immune response biomarkers; Preferably, the monitoring method comprises detecting the ratio of immune response biomarkers in peripheral blood at different times after treatment.

8. Use of the immune response biomarker according to claim 1 or 2 in the preparation of a drug for immunomodulation in the treatment of patients with liver cirrhosis using mesenchymal stromal cells.

9. The use according to claim 8, characterized in that The immune regulation includes activating the CD86-CTLA4 signaling axis and downregulating the activity of regulatory T cells; or / and, Promotes recruitment of cytotoxic T cells through ICAM1-mediated transendothelial migration and activates the immune activity of γδ T cells; or / and, Enhancement of LGALS2 by secretion of ANXA1 protein + phagocytic function of monocytes; Preferably, the immune response biomarker is MX1-positive monocytes.

10. Use of the immune response biomarker according to claim 1 or 2 in the preparation of a medicament for treating immune dysfunction in patients with liver cirrhosis; Preferably, the immune response biomarker is MX1-positive monocytes.

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