Novel IgG4 related disease marker and application thereof

Abstract

The invention provides a novel marker for IgG4 related diseases and application of the novel marker, and particularly relates to application of OX40 serving as a biomarker to preparation of products for diagnosing IgG4-RD, evaluating disease activity of the IgG4-RD and evaluating recurrence risk prognosis of the IgG4-RD. On the basis of Olink proteomics analysis, expression of PD1, OX40, CCL19 and MMP12 in IgG4-RD patients is remarkably up-regulated, and the disease activity of the IgG4-RD patients is remarkably up-regulated. A verification queue verifies that the plurality of biomarkers have relatively high diagnosis efficiency, sensitivity and specificity, are in positive correlation with the IgG4 level, the inflammation index and the disease activity, and are in positive correlation with specific visceral organ enumeration. The invention finds that serum OX40 can be used as a baseline for predicting disease recurrence and evaluating a prediction index of recurrence risk.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a novel marker for IgG4-related diseases and application thereof. Background Art

[0002] IgG4-related disease (IgG4-RD) is a rare systemic disease characterized by chronic inflammation and fibrosis, which progresses insidiously and can be life-threatening in severe cases. IgG4-RD often affects multiple organs throughout the body, and the tissues present with inflammatory masses or fibrosis. Due to the diverse clinical features of IgG4-RD and the wide range of organs involved, as well as the lack of specific biomarkers, misdiagnosis and mistreatment of IgG4-RD often occur. Early diagnosis poses a huge challenge to clinicians.

[0003] IgG4-RD is a multi-organ fibro-inflammatory disease characterized by abnormally elevated serum IgG4 levels, the formation of inflammatory pseudotumors, and the production of a large number of IgG4-secreting plasmablasts in the affected organs. Currently, the understanding and research of IgG4-RD are still in the development stage, and there is also a lack of reliable indicators for diagnosing the disease and reflecting the disease activity. The existing diagnosis of IgG4-RD mainly relies on elevated serum IgG4, clinical manifestations, and pathological characteristics. However, on the one hand, the sensitivity and specificity of IgG4 are limited. Elevated serum IgG4 can also be seen in tumors, infections, and other autoimmune diseases, and some IgG4-RD patients have normal serum IgG4 levels (Carruthers MN, Khosroshahi A, Augustin T, et al. The diagnostic utility of serum IgG4 concentrations in IgG4-related disease. Ann Rheum Dis. 2015 Jan;74(1):14-8.). On the other hand, the characteristic pathological diagnostic criteria of the affected tissues depend on the quality of the samples and are limited to the affected organs, which increases the workload and technical difficulty of pathological diagnosis.

[0004] The existing diagnostic markers for IgG4-RD still have defects such as low sensitivity and specificity. Moreover, IgG4-RD affects multiple organs, such as the pancreas, biliary system, liver, lungs, kidneys, thyroid glands, salivary glands, and lymph nodes. Due to different organs, there will be differences in clinical, serological or pathological characteristics. For example, Kasashima S et al. (Kasashima S, Kawashima A, Kasashima F, Endo M, Matsumoto Y, Kawakami K. Inflammatory features, including symptoms, increased serum interleukin-6, and C-reactive protein, in IgG4-related vascular diseases. Heart Vessels. 2018 Dec;33(12):1471-1481.) pointed out in their study that clinical symptoms such as low fever, abdominal pain, back pain, and anemia were often detected in patients with IgG4-related aortic aneurysms, but these symptoms were rarely detected in patients with IgG4-related retroperitoneal fibrosis. A complete evaluation of the organs affected by IgG4-RD is crucial for determining the treatment plan. In addition, IgG4-RD has the characteristics of alternating clinical remission and relapse, and about 10.66% of patients experience clinical relapse during one year of treatment, which may lead to further deterioration of organ function (Peng Y, Li JQ, Zhang PP, et al. Clinical outcomes and predictive relapse factors of IgG4-related disease following treatment: a long-term cohort study. J Intern Med. 2019 Nov;286(5):542-552.). Due to these disease characteristics of IgG4-RD, there is an urgent need for biological markers for diagnosis, evaluation of disease activity and prognosis in clinical practice, and to reveal their relationship with different disease characteristics.

[0005] Morniga M lectin and / or Vicia villosa lectin were found to reflect the disease activity of IgG4-RD, and DSL lectin-binding glycan levels were used as biological markers for multi-organ involvement in IgG4-RD. However, these studies only performed differential analysis through omics without further validation and experimental verification in independent cohorts, so the conclusions are limited. At present, there is a lack of a validated marker that reflects the disease activity of IgG4-RD. Summary of the invention

[0006] In a first aspect of the present invention, a biomarker for IgG4-RD is provided, wherein the biomarker comprises OX40.

[0007] Preferably, the biomarkers also include PD1, CCL19, and MMP12.

[0008] In a specific embodiment of the present invention, the biomarker is OX40.

[0009] A second aspect of the present invention provides a use of OX40 as a biomarker in the preparation of a product for diagnosing IgG4-RD, assessing the disease activity of IgG4-RD and / or assessing the prognosis of IgG4-RD.

[0010] The biomarker is a protein or a gene.

[0011] In a specific embodiment of the present invention, the biomarker is a protein.

[0012] The OX40 is OX40 in serum or plasma.

[0013] The diagnosis of IgG4-RD, assessment of disease activity of IgG4-RD and / or prognosis assessment of IgG4-RD include detecting the presence or level of OX40.

[0014] The method for diagnosing IgG4-RD, evaluating the disease activity of IgG4-RD and / or evaluating the prognosis of IgG4-RD comprises: obtaining serum and detecting the level of OX40;

[0015] Preferably, OX40 levels significantly above the threshold value indicate the occurrence of IgG4-RD or more severe disease.

[0016] The product comprises a reagent for detecting OX40, preferably detecting the presence or level of OX40.

[0017] Further preferably, the reagent detects the presence or level of OX40 protein, or detects the presence or level of mRNA encoding OX40, namely TNFRSF4.

[0018] The product is a test kit, a test strip, a chip or a device.

[0019] The IgG4-RD involves inflammation of the affected organs.

[0020] The organs affected by IgG4-RD include sinuses, lungs and / or kidneys.

[0021] Preferably, the organs affected by IgG4-RD also include one or more of the following: eyes, sinuses, pituitary gland, dura mater, pia mater, pleura, liver, bile duct, gallbladder, aorta, pericardium, mediastinum, mesentery, breast, blood vessels, testis, lymph nodes, skin or nerves.

[0022] When the IgG4-RD involves the sinuses, it manifests as sinusitis or polyps; when it involves the lungs, it manifests as interstitial pneumonia or bronchial wall thickening; when it involves the kidneys, it manifests as ureteral wall thickening or renal pelvic tumors.

[0023] The OX40 level was positively correlated with the IgG4-RD RI, indicating that the expression level of OX40 can be used as an assessment of disease activity.

[0024] In a third aspect of the present invention, a kit for diagnosing IgG4-RD is provided, wherein the kit comprises a reagent, a test paper or a chip for detecting a biomarker, and the biomarker is OX40.

[0025] The test strip or chip contains molecules that bind to biomarkers.

[0026] In a fourth aspect, the present invention provides a method for diagnosing IgG4-RD, assessing the disease activity of IgG4-RD and / or evaluating the prognosis of IgG4-RD, wherein the method comprises detecting TNFRSF4 in a sample of a subject.

[0027] The sample is serum or plasma.

[0028] The method further comprises comparing the detected OX40 expression level with a threshold value. The threshold value is obtained in a previous experiment, that is, the threshold value is determined by the difference in biomarkers between IgG4-RD and healthy subjects and pancreatic cancer and ANCA-associated vasculitis through experiments and data analysis.

[0029] When the biomarkers described in this application are different or significantly different from the threshold (the difference is statistically significant, such as p<0.05, p<0.01, p<0.001, p<0.0001), it is determined that the disease or the disease is more severe or there is a risk of recurrence. For example:

[0030] OX40 above the threshold or significantly above the threshold indicates the occurrence of IgG4-RD or more severe disease or poor prognosis.

[0031] Preferably, the detection method can be selected from mass spectrometry, liquid chromatography or ELISA.

[0032] The organs affected by IgG4-RD include sinuses, lungs and / or kidneys;

[0033] Preferably, the organs affected by IgG4-RD also include one or more of the following: eyes, sinuses, pituitary gland, dura mater, pia mater, pleura, liver, bile duct, gallbladder, aorta, pericardium, mediastinum, mesentery, breast, blood vessels, testis, lymph nodes, skin or nerves.

[0034] The "disease activity" mentioned in the present invention refers to the activity or progression of the disease.

[0035] The term "diagnosis" as used herein refers to finding out whether a patient has had a disease or condition in the past, at the time of diagnosis, or in the future, or to finding out the progression of a disease or its possible future progression.

[0036] The "prognosis assessment" mentioned in the present invention refers to the assessment of the patient's response to treatment before treatment, and the risk of future recurrence.

[0037] The "subject" of the present invention is a human or non-human animal, and the non-human animal may be a wild animal, a zoo animal, an economic animal, a pet, an experimental animal, etc. Preferably, the non-human mammal includes, but is not limited to, pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels), monkeys, etc.

[0038] The abbreviation and full name of this application are shown in Table 1. Table 1 Abbreviation Full name AAV ANCA-associated small vessel vasculitis AUC Area under the ROC curve CCL19 CC motif chemokine ligand 19 CI Confidence interval CRP C-reactive protein C3 Complement C3 C4 Complement C4 DEP Differentially expressed proteins EO Eosinophils ESR Erythrocyte sedimentation rate GLB globulin GO Gene Ontology HC Healthy controls IgE Immunoglobulin E IgG IgG IgG1 IgG1 IgG4 IgG4 IgG4-RD IgG4-related disease IgG4-RD RI IgG4-RD Reaction Index IQR Interquartile range KEGG Kyoto Encyclopedia of Genes and Genomes MMP12 MMP-12 OR Odds ratio OX40 Tumor necrosis factor receptor superfamily member 4 PD1 Programmed death receptor 1 ROC Receiver operating characteristic curve SD Standard Deviation Tf Follicular helper T cells Th T helper cells Treg Regulatory T cells

[0039] Beneficial effects of the invention: This patent screened out four biomarkers based on omics difference analysis, and conducted experimental verification in a large sample independent cohort, further supporting the reliability of the conclusion. This patent found that these four markers are closely related to disease activity, clinical indicators, and the number and type of affected organs. At the same time, it revealed for the first time the clinical value of OX40 as a biomarker in predicting recurrence in IgG4-RD.

[0040] (1) Based on Olink proteomic analysis, the present invention found that in the discovery cohort, 25 proteins were differentially expressed between IgG4-RD patients and healthy controls, among which OX40, PD1, CCL19, and MMP12 were significantly upregulated in IgG4-RD patients. Subsequently, the four biomarkers were verified by the validation cohort and all had high diagnostic efficacy, sensitivity, and specificity.

[0041] (2) In a large sample cohort, through 5-fold cross-validation, the average AUC value of OX40 was 0.869, the average AUC value of CCL19 was 0.879, the average AUC value of PD1 was 0.902, and the AUC value of MMP12 was 0.699.

[0042] (3) The present invention screened candidate markers from the discovery cohort and validated them in an independent cohort. The validation cohort included 140 patients and 80 healthy subjects. Since the diagnosis of IgG4-RD is extremely challenging, it is very difficult to collect a large number of confirmed IgG4-RD patients. The validation cohort of the present invention has a large sample size, which further improves the credibility of OX40 as a diagnostic marker for IgG4-RD.

[0043] (4) Based on univariate / multivariate logistic regression analysis, the present invention found that baseline OX40 level was an independent risk factor for disease recurrence. IgG4-RD patients with high baseline OX40 serum levels were more likely to experience clinical recurrence, with an OR value of 12.615. The present invention provides an important target molecule for the elucidation and treatment of the disease mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 : Heat map of differential protein expression between healthy group and IgG4-RD disease group.

[0045] Figure 2 : Volcano plot of differential protein expression between the healthy group and the IgG4-RD disease group.

[0046] Figure 3 : The top 30 pathways of KEGG enrichment analysis of differentially expressed proteins between the healthy group and the IgG4-RD disease group.

[0047] Figure 4 : Enrichment of differentially expressed proteins between the healthy group and the IgG4-RD diseased group in biological processes, molecular functions, and cellular components in GO enrichment analysis.

[0048] Figure 5 : Volcano plot of differential protein expression before and after IgG4-RD treatment.

[0049] Figure 6 : GO enrichment analysis of differentially expressed proteins before and after IgG4-RD treatment.

[0050] Figure 7 : Trends of PD1, OX40, CCL19, MMP12 and serum IgG4 concentrations and disease activity RI scores in IgG4-RD patients with treatment.

[0051] Figure 8 : Heat map of the correlation between differential protein levels and immune indicators.

[0052] Fig. 9 : Expression levels of PD1, OX40, CCL19, and MMP12 in IgG4-RD patients, disease controls, and healthy subjects.

[0053] Fig.10: 5-fold cross-validation ROC curves of PD1, OX40, CCL19, and MMP12 in the IgG4-RD cohort.

[0054] Fig.11 : ROC curve of the 5-fold cross-validation of the combined 4-protein model in the IgG4-RD cohort.

[0055] Fig.12 : Correlation diagram between PD1, OX40, CCL19, MMP12 and IgG4 concentration and disease activity RI score.

[0056] Fig.13 : Expression of PD1, OX40, CCL19, and MMP12 in IgG4-RD patients with different organ involvement.

[0057] Fig.14 : Univariate logistic regression forest plot of clinical relapse in patients with IgG4-RD.

[0058] Fig.15 : Multivariate logistic regression forest plot of clinical relapse in patients with IgG4-RD.

[0059] Fig.16 : Expression of OX40 on CD4+T cells in IgG4-RD patients and healthy subjects.

[0060] Fig.17 : Expression of OX40 on Th cells in IgG4-RD patients and healthy subjects.

[0061] Fig.18 : Immunohistochemistry showed the expression of OX40 in the submandibular gland tissue affected by IgG4-RD.

[0062] Fig.19 :Single-cell sequencing shows dimensionality reduction clustering of IgG4-RD.

[0063] Fig. 20 :Single-cell sequencing shows the expression of OX40 in different populations. DETAILED DESCRIPTION

[0064] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0065] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0066] Unless otherwise specified, the reagents and materials used in the following examples can be obtained from commercial sources.

[0067] The experimental subject selection and data analysis methods involved in the embodiment are as follows:

[0068] 1. Research subjects: Patients with IgG4-RD who met the 2019 ACR / EULAR diagnostic criteria and were admitted to the Department of Rheumatology, Beijing Friendship Hospital, Capital Medical University were recruited. Disease controls, i.e., samples of patients with antineutrophilcytoplasmic antibody-associated vasculitis (AAV), were obtained from the Department of Rheumatology, and patients with pancreatic cancer were obtained from the Department of General Surgery. Healthy controls (HC) were also recruited from the Health Examination Center. This invention was approved by the Ethics Committee of Beijing Friendship Hospital, Capital Medical University, and all participants provided informed consent in accordance with the principles of the Declaration of Helsinki.

[0069] 2. Data analysis methods

[0070] Statistical analysis was performed using GraphPad Prism version 9, IBM SPSS Statistics version 25, and R version 4.3.3. The Shapiro-Wilk test confirmed normal distribution. Parametric data were analyzed using the Student's t-test, and nonparametric data were compared between the two groups using the Mann-Whitney U test. Categorical parameters were analyzed using the chi-square test or Fisher's exact test. Correlations between variables were analyzed using the Pearson rank test (normally distributed data) and the Spearman rank correlation test (non-normally distributed data). A two-tailed p value < 0.05 was considered statistically significant. Normally distributed data were expressed as mean ± standard deviation (SD), and continuous non-normally distributed data were expressed as median and interquartile range (IQR). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis used the KEGG library, and Gene Ontology (GO) pathway functional enrichment annotation was performed using Blast2Go. A logistic regression model was constructed using candidate proteins from the discovery cohort, and its validity was verified using the glm function of R-package stats and the forward stepwise method. The receiver operating characteristic (ROC) curve was used to analyze the score, and the area under the ROC curve (AUC), sensitivity, specificity, Youden index, cutoff value, and significance (p<0.05) were determined to evaluate the performance of the model. K-fold cross-validation was used for additional evaluation of the model. Univariate and multivariate logistic regression analysis was used to analyze the risk factors associated with recurrence, and forest plots were drawn based on the odds ratio (OR), confidence interval (CI), and P value of the logistic regression.

[0071] Example 1: Serum Olink proteomics study of IgG4-RD

[0072] 1. Research subjects

[0073] A total of 22 subjects were included in this study as a discovery cohort, including 11 IgG4-RD patients (untreated) and 11 healthy controls (age and gender matched with IgG4-RD patients).

[0074] 2. Research Methods

[0075] (1) Fasting fresh whole blood samples were collected from healthy controls and IgG4-RD patients at different treatment time points. After being placed at room temperature for 2 hours, they were centrifuged at 4°C and 2500g for 10 minutes. The supernatant was collected into 2 ml cryotubes and transferred to a -80°C refrigerator as soon as possible for storage.

[0076] (2) Olink uses proximity extension technology to connect protein-specific antibodies to DNA-encoded tags, converting protein information into amplifiable signals to obtain the abundance of key proteins. The present invention analyzes 92 immunological proteins.

[0077] (3) Use the normalized protein expression (NPX) software OlinkNPX Manager to perform quality control and data normalization with reference to internal and external controls. NPX value is used as a relative quantitative method to compare the expression levels of individual proteins under different conditions. Proteins with significant changes in NPX value are considered differentially expressed proteins (DEPs) (p < 0.05).

[0078] 3. Research Results

[0079] Olink proteomic analysis revealed differential protein expression between IgG4-RD patients and healthy controls in the cohort. Olink quality control results showed that serum proteomic results had good stability and accuracy. Ideal protein biomarkers are low or absent in the healthy control group and highly expressed in the IgG4-RD group. Figure 1 ) and volcano plots ( Figure 2 ), compared the relative levels of proteins in the normal group and IgG4-RD group, and excluded proteins with missing data > 75%. It was found that 11 of the 27 differentially expressed proteins were up-regulated in the IgG4-RD group, and 16 were down-regulated in the IgG4-RD group. The differentially expressed genes were mainly enriched in immune regulation-related pathways. In the enrichment analysis of differentially expressed proteins, KEGG (using KEGG library) and GO (using Blast2Go) pathway enrichment analysis showed that the differentially expressed proteins were mainly involved in the regulation of biological processes. Figure 3-4 Subsequently, we analyzed the differential proteins in IgG4-RD patients before and after treatment and found 10 differentially expressed genes ( Figure 5 ), and also enriched in immune regulation-related pathways ( Figure 6 ). The above indicated that immune regulation-related proteins were abnormally activated in IgG4-RD.

[0080] Example 2: Further screening of biomarkers and preliminary verification

[0081] The 11 IgG4-RD patients selected from the discovery cohort in Example 1 were treated, specifically receiving conventional treatment (glucocorticoids alone or in combination with immunosuppressants). Among them, 8 patients had clinical relapse within 1 year, and 3 patients had stable conditions. Clinical examinations, blood routine parameter assessments, and serum markers were performed at each visit (at onset, 4 / 12 / 24 / 36 / 52 weeks of treatment, and at clinical relapse). The 25 differentially expressed proteins in the 11 patients at different treatment time periods were detected along with the treatment trend ( Figure 7 ), found that the NPX expression levels of the four proteins PD1, OX40, CCL19 and MMP12 decreased after treatment and increased again when the disease relapsed clinically, and the levels of the two proteins showed synchronous changes with the serum IgG4 level and the disease activity IgG4-RD RI score. Correlation analysis of the differentially expressed proteins and IgG4-RD immunological indicators found that these candidate biomarkers were positively correlated with disease activity and inflammatory indicators (ESR, CRP) ( Figure 8 ), and negatively correlated with complement (C3 and C4). The above proves that these four proteins are closely related to disease progression.

[0082] Next, we recruited 30 IgG4-RD patients, 30 AAV patients, and 28 preoperative pancreatic cancer patients and verified the expression of PD1, OX40, CCL19, and MMP12 by ELISA ( Fig. 9 ). The results showed that the four proteins were significantly increased in IgG4-RD, and their expression was higher than that of other disease controls and healthy people. The results of high expression of the four proteins were consistent with the results of proteomics sequencing, suggesting their potential as biomarkers.

[0083] Example 3: Validation of IgG4-RD biomarkers

[0084] An additional 140 IgG4-RD patients and 80 healthy controls (matched in age and gender with IgG4-RD patients) were recruited as an independent validation cohort, and the diagnostic efficacy, sensitivity, specificity and other indicators of the biomarkers were evaluated by machine learning using the 5-fold cross-validation method. The 5-fold cross-validation method randomly divided the 220 samples into 5 subsets and repeated 5 times. The results are shown in Table 2. The average AUC values ​​of OX40, PD1, CCL19, and MMP12 were 0.869, 0.902, 0.879, and 0.699, respectively. Fig.10 In addition, OX40 has the highest sensitivity among the four markers (82.2%). When the four proteins were combined to build a model, the diagnostic performance of the model was better than that of independent protein detection ( Fig.11 ). Table 2 variable AUC Sensitivity Specificity p-value OX40 (pg / ml) 0.869 0.840 0.814 <0.0001 PD1 (pg / ml) 0.902 0.775 0.987 <0.0001 CCL19 (pg / ml) 0.879 0.822 0.848 <0.0001 MMP12 (ng / ml) 0.699 0.694 0.739 <0.0001 Protein Association Model 0.938 0.871 0.933 <0.0001

[0085] Example 4: Relationship of biomarkers to disease activity and clinical parameters

[0086] The Spearman correlation analysis was used to explore the correlation between biomarkers and disease activity IgG4-RD RI and clinical parameters in IgG4-RD patients (validation cohort in Example 3).

[0087] The protein levels of the four biomarkers were correlated with serum IgG4 levels and disease activity IgG4-RD RI ( Fig.12 ) was significantly positively correlated with inflammatory markers (ESR and CRP) and the number of affected organs, as shown in Table 3, and OX40 was positively correlated with other clinical parameters such as IgG, IgG1, GLB, IgE and eosinophil counts, and negatively correlated with complement C3 and C4 (p<0.05). These findings indicate that elevated OX40 levels are strongly correlated with severe inflammation and disease activity, suggesting that it may play a biological role in the disease. Table 3

[0088] Example 5: Relationship between OX40 and disease-affected organs

[0089] The differences in serum OX40 levels in patients with IgG4-RD in different affected organs were further explored. The cohort patients were grouped according to the different affected organs, including patients with IgG4-RD in the lungs (manifested as interstitial pneumonia or bronchial wall thickening), kidneys, glands, pancreas or retroperitoneum, sinuses, etc., and patients with IgG4-RD that did not involve the lungs, kidneys, glands, pancreas or retroperitoneum, sinuses. Serum was collected according to the steps in Example 3, and the serum levels of biomarkers were evaluated by ELISA.

[0090] Results showed that patients with involvement of the sinuses, lungs, and kidneys showed elevated serum OX40 concentrations ( Fig.13 ). However, there was no significant difference in OX40 expression in patients with glandular, pancreatic or retroperitoneal involvement. This finding about the correlation between organ and protein marker levels can help evaluate the organ involvement of patients in clinical work, especially for patients with the above-mentioned organ involvement. The detection of biomarkers can provide a new means of evaluating the organ involvement of patients and even reduce the possible invasive detection such as pathology.

[0091] Example 6: OX40 levels predict disease recurrence

[0092] In the above IgG4-RD cohort, there were 66 pre-treatment patients who were followed up for at least 1 year, of whom 20 relapsed within 1 year and 26 were stable within 1 year. There were no significant differences in the baseline demographic characteristics, laboratory test indicators, and disease activity between the relapse group and the non-relapse group. We performed a univariate logistic regression analysis of relapse ( Fig.14 ), indicating that high PD1, high OX40, high CCL19, high MMP12, and elevated eosinophil count at baseline are risk factors for recurrence, while treatment is a protective factor. Subsequently, we included these factors in multivariate logistic regression ( Fig.15 ), the results showed that among these biomarkers, only OX40 level was an independent risk factor, that is, IgG4-RD patients with high baseline serum OX40 levels were more likely to relapse. For the first time, we revealed the protein molecules associated with IgG4-RD relapse, providing a new possible molecular mechanism for the onset of the disease.

[0093] Example 7: Expression of OX40 in tissues and cells of IgG4-RD patients

[0094] Above, we revealed that serum OX40 levels are significantly upregulated in IgG4-RD and are closely related to disease activity as well as laboratory and clinical indicators. We continued to explore the expression of OX40 on peripheral blood lymphocytes in IgG4-RD patients and healthy subjects. Peripheral blood was collected from 30 IgG4-RD patients and 30 healthy subjects, peripheral blood mononuclear cells were extracted by Ficoll method, and flow cytometry was used to detect the expression of OX40 on CD4+T cells. The results showed that the expression of OX40 on peripheral blood CD4+T cells in IgG4-RD patients was upregulated, especially on regulatory T cells (Treg) ( Fig.16 ). The expression rate is increased on helper T cells (T helper cells, Th), especially Th2 cells ( Fig.17 ), suggesting that OX40 molecules may be involved in the inflammatory regulation and allergic response process of the disease.

[0095] According to the characteristics of IgG4-RD, 3 cases of affected submandibular gland tissues with typical pathological features of IgG4-RD and normal submandibular gland tissues were selected as controls, and the expression of OX40 in the tissues was compared by immunohistochemistry. The results showed that ( Fig.18), in the submandibular gland tissues affected by IgG4-RD patients, OX40 expression was significantly higher than that in normal submandibular gland tissues. In addition, the high expression of OX40 in IgG4-RD affected tissues was mainly distributed in the germinal center area. At the genetic level, we performed 10× single-cell sequencing on 3 submandibular gland tissues affected by IgG4-RD and 3 normal submandibular gland tissues, and identified 6 subgroups through dimensionality reduction clustering, of which Cluster2 and Cluster3 represent follicular helper T cells and Treg cells, respectively. Fig.19 We found that the gene TNFRSF4 encoding the OX40 protein is also highly expressed in disease-affected tissues, especially on the surface of Tfh and Treg cells ( Fig. 20 ).

[0096] In the above, we found 4 protein molecules that can identify and assist in the diagnosis of IgG4-RD, which are closely related to the inflammatory indicators of the disease, disease activity, and the number of affected organs, and revealed the clinical predictive value of the OX40 molecule for predicting the risk of recurrence. These findings can help clarify the pathogenesis of IgG4-RD and provide new targets for the future treatment of IgG4-RD.

[0097] The above description of the embodiments is to facilitate the understanding and use of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A marker for IgG4-related diseases, characterized in that The marker is OX40, and preferably, the OX40 is OX40 in serum or plasma.

2. The marker according to claim 1, characterized in that The markers also include one or more of PD1, CCL19, and MMP12.

3. The marker according to claim 1, characterized in that The marker is a protein.

4. The marker according to any one of claims 1 to 3, characterized in that The IgG4-related disease is selected from one or more of the diagnosis of IgG4-RD, assessment of disease activity of IgG4-RD and / or prognosis assessment. Preferably, the IgG4-related disease includes inflammation of the involved organs. Preferably, the involved organs include the lungs and / or kidneys. Preferably, the involved organs also include one or more of the eyes, sinuses, pituitary gland, dura mater, pia mater, pleura, liver, bile duct, gallbladder, aorta, pericardium, mediastinum, mesentery, breast, blood vessels, testicles, lymph nodes, skin or nerves.

5. Use of OX40 and / or PD1, CCL19, MMP12 in the preparation of products for diagnosing IgG4-RD, assessing the disease activity of IgG4-RD and / or prognostic assessment of IgG4-related diseases.

6. The use according to claim 5, wherein the product comprises a product for diagnosing IgG4-RD, assessing the disease activity of IgG4-RD and / or assessing the prognosis of IgG4-RD.

7. The use according to claim 6, characterized in that: The product is a test kit, a test strip, a chip or a device.

8. The use according to claim 1, characterized in that: The IgG4-RD involves inflammation of the affected organs.

9. The use according to claim 1, characterized in that: The organs affected by IgG4-RD include sinuses, lungs and / or kidneys.

10. The use according to claim 9, characterized in that: Organs affected by IgG4-RD include one or more of the eyes, sinuses, pituitary gland, dura mater, pia mater, pleura, liver, bile duct, gallbladder, aorta, pericardium, mediastinum, mesentery, breast, blood vessels, testis, lymph nodes, skin, or nerves.

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