A novel biomarker for IgG4-related disease and its application

Abstract

The present invention provides a novel biomarker for IgG4-related diseases and its applications, specifically the application of OX40 as a biomarker in the preparation of products for diagnosing IgG4-RD, evaluating the disease activity of IgG4-RD, and prognosticating the recurrence risk of IgG4-RD. Based on Olink proteomic analysis, the present invention found that the expressions of PD1, OX40, CCL19, and MMP12 were significantly upregulated in patients with IgG4-RD. After verification in a validation cohort, these biomarkers have high diagnostic efficacy, sensitivity, and specificity, are positively correlated with IgG4 levels, inflammatory indicators, and disease activity, and are related to specific organ involvement. The present invention found that serum OX40 can be used as a predictive indicator for baseline prediction of disease recurrence and evaluation of recurrence risk.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and particularly relates to a novel biomarker for IgG4-related disease and its application. Background Art

[0002] IgG4-related disease (IgG4-RD) is a rare systemic disease characterized by chronic inflammation and fibrosis, with insidious progression and severe cases that can be life-threatening. IgG4-RD often affects multiple organs throughout the body, and the tissues present inflammatory masses or fibrosis. Due to the diverse clinical characteristics of IgG4-RD and the extensive involvement of organs, the lack of specific biomarkers has led to frequent misdiagnosis and mistreatment of IgG4-RD. Early diagnosis poses a great challenge to clinicians.

[0003] IgG4-RD is a multi-organ fibroinflammatory disease characterized by an abnormal increase in the level of serum IgG4 in patients, the formation of inflammatory pseudotumors, and the production of a large number of IgG4-secreting plasmablasts in the affected organs. At present, the understanding and research of IgG4-RD are both in the development stage, and there is also a lack of reliable indicators for diagnosing the disease and reflecting its activity. The current diagnosis of IgG4-RD mainly relies on elevated serum IgG4, clinical manifestations, and pathological features. However, on the one hand, the sensitivity and specificity of IgG4 are limited, and an increase in 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 for affected tissues depend on the quality of the samples and are limited to some of the affected organs, increasing the workload and technical difficulty of pathological diagnosis.

[0004] In the prior art, the markers for diagnosing IgG4-RD still have defects such as low sensitivity and specificity. Moreover, IgG4-RD affects multiple organs, such as the pancreas, biliary system, liver, lung, kidney, thyroid, salivary gland, and lymph nodes. Due to the differences in organs, there will be differences in clinical, serological, or pathological features. For example, in the study by 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.), it was pointed out that clinical symptoms such as low fever, abdominal pain, low back pain, and anemia were often detected in patients with IgG4-related aortic aneurysm, but these symptoms were rarely detected in patients with IgG4-related retroperitoneal fibrosis. A complete assessment of the organs involved in IgG4-RD is crucial for determining the disease treatment plan. In addition, IgG4-RD is characterized by alternating clinical remission and relapse. Approximately 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 in clinical practice for biological markers for diagnosing, evaluating disease activity, and prognosis, and to reveal their relationships with different disease characteristics.

[0005] Morniga M lectin and / or Vicia villosa lectin were found to be able to reflect the disease activity of IgG4-RD. The DSL lectin-binding glycan level is used as a biological marker for multi-organ involvement in IgG4-RD. However, these studies only performed differential analysis through omics, without further verification in an independent cohort and experimental verification, 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, and the biomarker comprises OX40.

[0007] Preferably, the biomarker further comprises PD1, CCL19, and MMP12.

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

[0009] In a second aspect of the present invention, an application of OX40 as a biomarker in the preparation of a product for diagnosing IgG4-RD, evaluating the disease activity of IgG4-RD, and / or prognosticating IgG4-RD is provided.

[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 diagnosing IgG4-RD, evaluating the disease activity of IgG4-RD, and / or prognosticating IgG4-RD includes detecting the presence or absence or level of OX40.

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

[0015] Preferably, a significantly higher OX40 level than the threshold indicates the occurrence of IgG4-RD or a more severe disease.

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

[0017] More preferably, the reagent detects the presence or absence or level of the OX40 protein, or detects the presence or absence or level of the mRNA encoding OX40, i.e., TNFRSF4.

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

[0019] The IgG4-RD includes inflammation of the involved organs.

[0020] The organs involved in the IgG4-RD include the sinuses, the lungs, and / or the kidneys.

[0021] Preferably, the organs involved in IgG4-RD also include one or more of the eye, nasal sinus, pituitary gland, dura mater, pia mater, pleura, liver, bile duct, gallbladder, aorta, pericardium, mediastinum, mesentery, breast, blood vessel, testis, lymph node, skin or nerve.

[0022] When IgG4-RD involves the nasal sinus, it presents as sinusitis or polyps; when it involves the lung, it presents as interstitial pneumonia or thickening of the bronchial wall; when it involves the kidney, it presents as thickening of the ureteral wall or a mass in the renal pelvis.

[0023] The level of OX40 is 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, there is provided a kit for diagnosing IgG4-RD, the kit comprising a reagent, test strip or chip for detecting a biomarker, and the biomarker is OX40.

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

[0026] In a fourth aspect of the present invention, there is provided a method for diagnosing IgG4-RD, evaluating the disease activity of IgG4-RD and / or prognosticating IgG4-RD, the method comprising detecting TNFRSF4 in a sample from a subject.

[0027] The sample is serum or plasma.

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

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

[0030] OX40 being higher than the threshold value or significantly higher than the threshold value indicates the occurrence of IgG4-RD, or the disease is more severe or the prognosis is poor.

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

[0032] The organs involved in IgG4-RD include the nasal sinus, lung and / or kidney;

[0033] Preferably, the organs involved in IgG4-RD further include one or more of the eye, nasal sinus, pituitary gland, dura mater, pia mater, pleura, liver, bile duct, gallbladder, aorta, pericardium, mediastinum, mesentery, breast, blood vessel, testis, lymph node, skin or nerve.

[0034] As used herein, "disease activity" refers to the degree of activity or progression state of a disease.

[0035] As used herein, "diagnosis" means to determine whether a patient has had, has, or will have a disease or disorder, or to determine the progression or potential future progression of a disease.

[0036] As used herein, "prognosis assessment" refers to assessing the response of a patient to treatment before treatment and the future risk of recurrence.

[0037] As used herein, "subject" is a human or non-human animal, and the non-human animal can be a wild animal, zoo animal, economic animal, pet, experimental animal, etc. Preferably, the non-human mammals include, but are not limited to, pigs, cows, sheep, horses, donkeys, foxes, raccoons, minks, camels, dogs, cats, rabbits, rats (such as rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels) or monkeys, etc.

[0038] The abbreviations and full names in this application are shown in Table 1.

[0039] Table 1

[0040] Abbreviation Full name AAV ANCA-associated 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 protein EO Eosinophil ESR Erythrocyte sedimentation rate GLB Globulin GO Gene Ontology HC Healthy control IgE Immunoglobulin E IgG Immunoglobulin G IgG1 Immunoglobulin G1 IgG4 Immunoglobulin G4 IgG4-RD IgG4-related disease IgG4-RD RI IgG4-RD response index IQR Interquartile range KEGG Kyoto Encyclopedia of Genes and Genomes MMP12 Matrix metalloproteinase 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 Tfh Follicular helper T cell Th Helper T cell Treg Regulatory T cell

[0041] Advantages of the present invention: Based on omics difference analysis, four biomarkers were screened out in this patent and experimentally verified in a large independent cohort, further supporting the reliability of the conclusion. This patent discovered the close relationship between these four biomarkers and disease activity, clinical indicators, as well as the number and types of involved organs. At the same time, for the first time, it revealed the clinical value of OX40 as a biomarker in predicting recurrence in IgG4-RD.

[0042] (1) Based on Olink proteomics analysis, it was found that in the discovery cohort, there were 25 differentially expressed proteins between IgG4-RD patients and healthy individuals. Among them, the expressions of OX40, PD1, CCL19, and MMP12 were significantly upregulated in IgG4-RD patients. After verification in the validation cohort, all four biomarkers had high diagnostic efficacy, sensitivity, and specificity.

[0043] (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.

[0044] (3) The present invention screens candidate markers from the discovery cohort and validates them in an independent cohort. The validation cohort includes 140 patients and 80 healthy individuals. Since there are great challenges in diagnosing IgG4-RD, it is very difficult to collect a large number of confirmed IgG4-RD patients. The large sample size of the validation cohort of the present invention further improves the credibility of OX40 as a diagnostic marker for IgG4-RD.

[0045] (4) Based on single / multivariate logistic regression analysis, the present invention finds that the baseline OX40 level is an independent risk factor for disease recurrence. IgG4-RD patients with high baseline OX40 serum levels are more likely to have clinical recurrence, and the OR value is 12.615. The present invention provides an important target molecule for elucidating the disease mechanism and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 : Heat map of differential protein expression between the healthy group and the IgG4-RD patient group.

[0047] Figure 2 : Volcano plot of differential protein expression between the healthy group and the IgG4-RD patient group.

[0048] Figure 3 : Top 30 pathways of KEGG enrichment analysis of differential proteins between the healthy group and the IgG4-RD patient group.

[0049] Figure 4 : Enrichment of differential proteins between the healthy group and the IgG4-RD patient group in biological processes, molecular functions, and cellular components of GO enrichment analysis.

[0050] Figure 5 : Volcano plot of differential protein expression before and after treatment of IgG4-RD.

[0051] Figure 6 : GO enrichment analysis of differential proteins before and after treatment of IgG4-RD.

[0052] Figure 7 : Trend chart of the changes of PD1, OX40, CCL19, MMP12 and serum IgG4 concentration and disease activity RI score during treatment in IgG4-RD patients.

[0053] Figure 8 : Heat map of the correlation between differential protein levels and immune indexes.

[0054] Figure 9 : Expression levels of PD1, OX40, CCL19, MMP12 in IgG4-RD patients, disease controls and healthy individuals.

[0055] Figure 10 : ROC curves of 5-fold cross-validation for PD1, OX40, CCL19, and MMP12 in the IgG4-RD cohort.

[0056] Figure 11 : ROC curves of 5-fold cross-validation for the combined 4-protein model in the IgG4-RD cohort.

[0057] Figure 12 : Correlation diagrams of PD1, OX40, CCL19, MMP12 with IgG4 concentration and disease activity RI score.

[0058] Figure 13 : Expression of PD1, OX40, CCL19, and MMP12 in IgG4-RD patients with different organ involvements.

[0059] Figure 14 : Univariate logistic regression forest plot of clinical recurrence in IgG4-RD patients.

[0060] Figure 15 : Multivariate logistic regression forest plot of clinical recurrence in IgG4-RD patients.

[0061] Figure 16 : Expression of OX40 on CD4+ T cells in IgG4-RD patients and healthy individuals.

[0062] Figure 17 : Expression of OX40 on Th cells in IgG4-RD patients and healthy individuals.

[0063] Figure 18 : Immunohistochemistry showing the expression of OX40 in IgG4-RD involved submandibular gland tissue.

[0064] Figure 19 : Single-cell sequencing showing the dimensionality reduction clustering of IgG4-RD.

[0065] Figure 20 : Single-cell sequencing showing the expression of OX40 in different clusters. Detailed implementation manners

[0066] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only partial embodiments of the present invention, rather than all. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0067] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified.

[0068] The reagents, materials, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0069] The experimental subject selection and data analysis methods involved in the examples are as follows:

[0070] 1. Research subjects: IgG4-RD patients who met the 2019 ACR / EULAR diagnostic criteria and were treated in the Rheumatology Department of Beijing Friendship Hospital, Capital Medical University were recruited. Disease controls, namely antineutrophil cytoplasmic antibody-associated vasculitis (AAV) patient samples, were from the rheumatology outpatient clinic, pancreatic cancer patients were from the general surgery department, and healthy controls (HC) were 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.

[0071] 2. Data analysis methods

[0072] 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 a normal distribution. Parametric data were analyzed using the Student's t-test, and non-parametric data were compared between two groups using the Mann-Whitney U test. Categorical parameters were analyzed using the chi-square test or Fisher's exact test. Correlation analysis between variables was performed using Pearson's rank test (for normally distributed data) and Spearman's rank correlation test (for 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 was performed using 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 the R-package stats and the forward stepwise method. Receiver Operating Characteristic curve (ROC) analysis was used to score, and the area under the ROC curve (AUC), sensitivity, specificity, Youden index, cut-off 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. Recurrence-related risk factors were analyzed using univariate and multivariate logistic regression, and a forest plot was drawn based on the odds ratio (OR), confidence interval (CI), and P value of the logistic regression.

[0073] Example 1: Serum Olink Proteomics Study of IgG4-RD

[0074] 1. Research Subjects

[0075] A total of 22 subjects were included in this study as the discovery cohort, including 11 IgG4-RD patients (untreated) and 11 healthy controls (matched for age and sex to the IgG4-RD patients).

[0076] 2. Research Methods

[0077] (1)Collect fasting fresh whole blood samples from healthy controls and IgG4-RD patients at different treatment time points. After standing at room temperature for 2 hours, centrifuge at 4°C and 2500g for 10 minutes, collect the supernatant into 2 ml cryotubes, and transfer them to an -80°C refrigerator for storage as soon as possible.

[0078] (2)Olink uses the proximity extension technique to link protein-specific antibodies with DNA-encoded tags, converting protein information into amplifiable signals to obtain the abundances of key proteins. In this invention, 92 immunological proteins were analyzed.

[0079] (3)Use the Normalized Protein Expression (NPX) software OlinkNPX Manager to perform quality control and data normalization with reference to internal and external controls. The NPX value, as a relative quantification method, is used to compare the expression levels of individual proteins under different conditions. Proteins with significantly changed NPX values are considered differentially expressed proteins (DEPs) (p < 0.05).

[0080] 3. Research Results

[0081] Through Olink proteomics analysis, the expression of differential proteins between IgG4-RD patients and healthy controls in the cohort was characterized. The Olink quality control results showed that the serum proteomics results had good stability and accuracy. Ideal protein biomarkers are characterized by low or no expression in the healthy control group and high expression in the IgG4-RD group. By plotting a heat map ( Figure 1 )and a volcano plot ( Figure 2 ), comparing the relative levels of proteins in the normal group and the IgG4-RD group, after excluding proteins with missing data > 75%, it was found that among the 27 differential proteins, 11 were upregulated in the IgG4-RD group and 16 were downregulated in the IgG4-RD group. The differential genes were mainly enriched in immune regulation-related pathways. In the enrichment analysis of differential proteins, KEGG (using the KEGG library) and GO (using Blast2Go) pathway enrichment analyses showed that the biological process regulations mainly participated in by differential proteins are shown in Figure 3-4 . Subsequently, we analyzed the differential proteins before and after treatment in IgG4-RD patients and found 10 differential genes ( Figure 5 ), which were also enriched in immune regulation-related pathways ( Figure 6 ). The above suggests that there is abnormal activation of immune regulation-related proteins in IgG4-RD.

[0082] Example 2: Further Screening of Biomarkers and Preliminary Verification

[0083] Eleven IgG4-RD patients selected from the discovery cohort in Example 1 were treated, specifically with conventional treatment (using glucocorticoids alone or in combination with immunosuppressants). Among them, 8 patients had clinical recurrence within 1 year, and 3 patients had stable conditions. Clinical examinations, blood routine parameter evaluations, and serum biomarker detections were performed at each visit (at onset, at 4 / 12 / 24 / 36 / 52 weeks of treatment, and at clinical recurrence). By detecting the changing trends of 25 differential proteins in 11 patients at different treatment time periods ( Figure 7 ), it was found that the NPX expression levels of four proteins, PD1, OX40, CCL19, and MMP12, decreased after treatment and then increased again at the time of clinical recurrence of the disease. Moreover, the levels of these two proteins showed synchronous changes with the serum IgG4 level and the disease activity IgG4-RD RI score. Correlation analysis was performed between the differential proteins and the immunological indexes of IgG4-RD, and it was found that these candidate biomarkers were positively correlated with the disease activity and inflammatory indexes (ESR, CRP) ( Figure 8 ), and negatively correlated with the complement (C3 and C4). Above, it was proved that these four proteins were closely related to the disease progression.

[0084] Next, we recruited 30 IgG4-RD patients, 30 AAV patients, and 28 preoperative pancreatic cancer patients, and verified the expressions of PD1, OX40, CCL19, and MMP12 by ELISA ( Figure 9 ). The results showed that the expressions of all four proteins were significantly increased in IgG4-RD, higher than those in other disease controls and healthy people. The results of the high expression of the four proteins were consistent with the results of proteomic sequencing, suggesting their potential as biomarkers.

[0085] Example 3: Verification of Biomarkers for IgG4-RD

[0086] An additional 140 IgG4-RD patients and 80 healthy controls (matched with IgG4-RD patients in age and gender) were recruited as an independent verification cohort, and the diagnostic efficacy, sensitivity, specificity, and other indexes of the biomarkers were evaluated by machine learning using the 5-fold cross-validation method. For 5-fold cross-validation, 220 samples were randomly divided into 5 subsets and repeated 5 times. The results are shown in Table 2. The average AUC of OX40 was 0.869, the average AUC of PD1 was 0.902, the average AUC of CCL19 was 0.879, and the average AUC value of MMP12 was 0.699 ( Figure 10 ). In addition, OX40 had the highest sensitivity (82.2%) among the four markers. When a model was established by combining the four proteins, the diagnostic performance of the model was better than that of independent protein detection ( Figure 11 ).

[0087] Table 2

[0088] 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 combined model 0.938 0.871 0.933 <0.0001

[0089] Example 4: Relationship between Biomarkers and Disease Activity and Clinical Parameters

[0090] The Spearman correlation analysis method was used to explore the correlation between biomarkers and the disease activity IgG4-RD RI and clinical parameters in IgG4-RD patients (validation cohort in Example 3).

[0091] The protein levels of the 4 biomarkers were all significantly positively correlated with the serum IgG4 level and the disease activity IgG4-RD RI ( Figure 12 ), with p < 0.001. Table 3 shows that OX40 was positively correlated with the inflammatory indicators (ESR and CRP) and the number of involved organs, and was positively correlated with other clinical parameters such as IgG, IgG1, GLB, IgE, and eosinophil count, and negatively correlated with complement C3 and C4 (p < 0.05). These findings indicate that an increase in OX40 level is strongly correlated with severe inflammation and disease activity, suggesting its possible biological role in the disease.

[0092] Table 3

[0093]

[0094] Example 5: Relationship between OX40 and Involved Organs of the Disease

[0095] The difference in serum OX40 level in IgG4-RD patients with different involved organs was further explored. The cohort patients were grouped according to different involved organs, including IgG4-RD patients with lungs (manifested as interstitial pneumonia or bronchial wall thickening), kidneys, glands, pancreas or retroperitoneum, sinuses, etc., and IgG4-RD patients without involvement of 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.

[0096] The results showed that patients with involvement of the sinuses, lungs, and kidneys showed an increase in serum OX40 concentration ( Figure 13 ). However, there was no significant difference in OX40 expression in patients with involvement of the glands, pancreas, or retroperitoneum. This finding regarding the correlation between organs and protein biomarker levels can help in evaluating organ involvement in clinical work. Especially for patients with involvement of the above-mentioned several organs, the detection of biomarkers can provide a new means to evaluate organ involvement in patients, and even reduce invasive detections such as possible pathology.

[0097] Example 6: Prediction of Disease Recurrence by OX40 Level

[0098] There were 66 pre-treatment patients in the above IgG4-RD cohort who were followed up for at least 1 year. Among them, 20 relapsed within 1 year and 26 patients were stable within 1 year. There were no significant differences in demographic characteristics, laboratory test indicators, and disease activity at baseline between the relapse group and the non-relapse group. We performed univariate logistic regression for relapse ( Figure 14 ), suggesting that high PD1, high OX40, high CCL19, high MMP12, and elevated eosinophil count at baseline were all risk factors for relapse, while the treatment method was a protective factor. Subsequently, we included these factors in multivariate logistic regression ( Figure 15 ), and the results showed that among these biomarkers, only the OX40 level was an independent risk factor, that is, IgG4-RD patients with high OX40 levels in the baseline serum were more likely to relapse. We first revealed the protein molecules related to IgG4-RD relapse, providing new possible molecular mechanisms for disease pathogenesis.

[0099] Example 7: Expression of OX40 in tissues and cells of IgG4-RD patients

[0100] As mentioned above, we revealed that the serum OX40 level was significantly up-regulated in IgG4-RD and was closely related to disease activity, laboratory, and clinical indicators. We continued to explore the expression of OX40 on peripheral blood lymphocytes of IgG4-RD patients and healthy people. Peripheral blood of 30 IgG4-RD patients and 30 healthy people was collected. Peripheral blood mononuclear cells were extracted by the Ficoll method, and the expression of OX40 on CD4+ T cells was detected by flow cytometry. The results showed that the expression of OX40 was up-regulated on CD4+ T cells in the peripheral blood of IgG4-RD patients, especially on regulatory T cells (Treg) ( Figure 16 ). The expression ratio increased on T helper cells (Th), especially Th2 cells ( Figure 17 ), suggesting that the OX40 molecule may be involved in the inflammatory regulation and allergic reaction process of the disease.

[0101] According to the disease characteristics of IgG4-RD, 3 cases of involved 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 ( Figure 18), in the submandibular gland tissues involved in IgG4-RD patients, the expression of OX40 was significantly higher than that in normal submandibular gland tissues. In addition, the high expression of OX40 in IgG4-RD involved tissues was mainly distributed in the germinal center region. At the gene level, we performed 10× single-cell sequencing on 3 submandibular gland tissues involved in IgG4-RD and 3 normal submandibular gland tissues. A total of 6 subpopulations were identified through dimensionality reduction and clustering. Among them, Cluster2 and Cluster3 represent follicular helper T cells and Treg cells respectively ( Figure 19 ). We found that the gene TNFRSF4 encoding the OX40 protein was also highly expressed in the diseased tissues, especially on the surface of Tfh and Treg cells ( Figure 20 ).

[0102] Above, we discovered 4 protein molecules that can be used to assist in the diagnosis of IgG4-RD. They are closely related to the inflammatory indicators, disease activity, and the number of affected organs of the disease, and revealed the clinical predictive value of the OX40 molecule with the risk of predicting recurrence. These findings can help clarify the pathogenesis of IgG4-RD and provide new targets for the future treatment of IgG4-RD.

[0103] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the present invention. 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 efforts. Therefore, the present invention is not limited to the above embodiments. Modifications and improvements 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. Use of a marker of IgG4-RD in the preparation of a reagent for evaluating the disease activity and / or recurrence risk prognosis of IgG4-RD, characterized in that: The markers are OX40, PD1, CCL19 and MMP12 in serum.

2. The use according to claim 1, wherein: The IgG4-RD includes inflammation of the affected organs, wherein the affected organs include one or more of the sinuses, lungs, and kidneys.

3. The use according to claim 1 or 2, characterized in that: The reagent is a test kit, a test paper, a chip or a device.

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