Use of reagents for detecting b cells expressing cd226 protein in the preparation of a medicament for aiding in the diagnosis of autoimmune diabetes

Abstract

The application discloses application of a reagent for detecting B cell expression of CD226 protein in preparation of an autoimmune diabetes auxiliary diagnosis preparation. Research finds that the B cell expression of CD226 is significantly related to the islet function and blood sugar level of an autoimmune diabetes patient, and can be used as a marker reflecting the disease progress of the autoimmune diabetes patient. The CD226 can be used for auxiliary diagnosis of the autoimmune diabetes, has good sensitivity and specificity, provides a new clue and basis for precise typing of the diabetes, and has important clinical application value for diagnosis and treatment of the autoimmune diabetes. The marker is from peripheral blood, the method is rapid, efficient, simple, feasible and high in sensitivity, and can improve the diagnosis and treatment efficiency of the autoimmune diabetes.

Description

Technical fields:

[0001] This invention belongs to the field of biomedical technology, specifically, it relates to the application of a reagent for detecting CD226 protein expression in B cells in the preparation of an auxiliary diagnostic agent for autoimmune diabetes. Background technology:

[0002] Autoimmune diabetes includes type 1 diabetes (T1D) and latent autoimmune diabetes in adults (LADA). T1D occurs due to damage to pancreatic beta cells responsible for insulin production caused by an autoimmune response. This disease is characterized by insufficient natural insulin secretion, requiring lifelong exogenous insulin supplementation. LADA is a slower-progressing form of autoimmune diabetes compared to T1D, characterized by gradual destruction of pancreatic beta cells and the presence of diabetes-related autoantibodies. LADA typically occurs in adulthood, lasting at least 6 months in its initial stages, and does not require insulin treatment. LADA shares characteristics with both T1D and type 2 diabetes (T2D) in its pathogenesis and clinical manifestations, and is therefore sometimes referred to as type 1.5 diabetes. If autoimmune diabetes is not well controlled, patients will be in a hyperglycemic environment for a long time, leading to chronic complications such as macrovascular and microvascular damage, retinal, kidney, and nerve damage. In severe cases, acute complications such as ketoacidosis may occur, placing a significant medical burden on patients. In recent years, the incidence of autoimmune diabetes has been increasing annually.

[0003] CD226 is an activating receptor that can be found on the surface of B cells. An increased percentage of CD226+ B cells is positively correlated with disease activity in systemic lupus erythematosus (SLE). However, the role of CD226+ B cells in the progression of autoimmune diabetes has not been reported.

[0004] Currently, autoimmune diabetes is often diagnosed clinically based on patients' clinical characteristics and laboratory tests such as C-peptide and pancreatic autoantibodies (glutamate decarboxylase antibody (GADA), protein tyrosine phosphatase antibody (IA-2A), zinc transporter 8 antibody (ZnT8A)). However, this diagnostic model faces several challenges: C-peptide only reflects disease characteristics in cases of severe pancreatic islet damage; some patients are negative for autoantibodies, while in others, antibody titers gradually decrease or even turn negative as the disease progresses; there are also cases where susceptible individuals with positive autoantibodies do not develop autoimmune diabetes; and autoantibody testing technology is not yet widely available, with many hospitals unable to perform such tests or possessing inadequate testing techniques, leading to the possibility of false positives and false negatives. Considering the high cost, time-consuming nature, and inconvenience of autoantibody testing, there is an urgent need in this field to find a new biomarker for clinical guidance in the accurate subtyping of diabetes, which has significant scientific and social value in reducing the medical burden and serving public health. Summary of the Invention:

[0005] This invention found that the level of CD226 expression in B cells is significantly correlated with pancreatic function and blood glucose levels in patients with autoimmune diabetes, and can serve as a biomarker reflecting the progression of the disease in these patients.

[0006] The primary objective of this invention is to overcome the shortcomings of existing technologies and provide a reagent for detecting the level of CD226 protein expression in B cells, which is used in the preparation of agents to assist in the diagnosis of autoimmune diabetes.

[0007] To achieve the purpose of the invention, the technical solution adopted by the present invention is as follows:

[0008] The reagents used to detect the expression level of CD226 protein on B cells include reagents for detecting the ratio of the number of CD226-positive B cells (CD226+ B cells) to the total number of B cells.

[0009] Furthermore, the autoimmune diabetes mellitus mentioned includes T1D and LADA.

[0010] Furthermore, the reagent used to detect the level of CD226 protein expression in B cells is an antibody that can specifically bind to CD226.

[0011] A second objective of this invention is to provide the application of a reagent for detecting the expression level of the CD226 gene in B cells in the preparation of a diagnostic agent for autoimmune diabetes.

[0012] For example, PCR can be used to detect the gene expression level of CD226 in B cells.

[0013] A third objective of this invention is to provide a reagent for detecting the level of CD226 protein expression in B cells for use in the preparation of a formulation for detecting fasting C-peptide levels in patients with autoimmune diabetes.

[0014] A fourth objective of this invention is to provide the application of a reagent for detecting the level of CD226 protein expression in B cells in the preparation of a formulation for detecting fasting blood glucose levels in patients with autoimmune diabetes.

[0015] The fifth objective of this invention is to provide a reagent for detecting the level of CD226 protein expression in B cells and its application in the preparation of a formulation for detecting glycated albumin levels in patients with autoimmune diabetes.

[0016] The sixth objective of this invention is to provide the application of a reagent for detecting the level of CD226 protein expression in B cells in the preparation of a formulation for detecting glycated hemoglobin levels in patients with autoimmune diabetes.

[0017] This study found that, using flow cytometry analysis, CD226 expression in peripheral blood B lymphocytes of patients with autoimmune diabetes was higher than that in the HC and T2D groups. The level of CD226 protein expression in B cells was negatively correlated with the patient's fasting C-peptide level, suggesting that patients with a higher proportion of CD226+ B cells had poorer pancreatic function. Furthermore, the level of CD226 protein expression in B cells was positively correlated with fasting blood glucose, glycated albumin, and glycated hemoglobin levels, indicating that patients with a higher proportion of CD226+ B cells had poorer glycemic control. This suggests that CD226+ B cells can reflect disease progression and can serve as a biomarker for the diagnosis and prognosis of autoimmune diabetes.

[0018] The seventh objective of this invention is to provide a reagent for detecting the level of CD226 protein expression in B cells for use in the preparation of a formulation for detecting the levels of TNF-α, IFN-γ, IL-6, and IL-12 secreted by B cells in patients with autoimmune diabetes.

[0019] This study found that compared to CD226-negative (CD226-) groups, CD226-positive (CD226+) B cells produce increased levels of TNF-α, IFN-γ, IL-6, and IL-12, indicating that CD226+ B cells secrete more pro-inflammatory cytokines and participate in the development and progression of autoimmune diabetes.

[0020] Compared with the prior art, the present invention has the following advantages: 1) It is applicable to all patients with autoimmune diabetes; 2) The detection indicators are significantly correlated with disease progression, with high sensitivity and specificity, single detection indicators, convenient and quick, easy to repeat, and broad application prospects; 3) The reagents and experimental methods used are relatively common, and the cost is controlled within a feasible range. Attached image description:

[0021] Figure 1 This is a flow cytometry representation of the higher proportion of CD226+ B cells in peripheral blood of autoimmune diabetic patients (T1D and LADA) compared to HC and T2D. Figure 1 A) and statistical charts ( Figure 1 B).

[0022] Figure 2 This is a graph showing the correlation between the proportion of CD226+ B cells and clinical indicators in patients with autoimmune diabetes. It also shows a negative correlation between the proportion of CD226+ B cells and fasting C-peptide (FCP) levels in these patients. Figure 2 A); A graph showing a positive correlation between the proportion of CD226+ B cells and fasting blood glucose (FBG) levels in autoimmune diabetic patients. Figure 2 B); A graph showing a positive correlation between the proportion of CD226+ B cells and glycated albumin (GA) levels in autoimmune diabetic patients. Figure 2C); A graph showing a positive correlation between the proportion of CD226+ B cells and glycated hemoglobin (HbA1c) levels in autoimmune diabetic patients. Figure 2 D).

[0023] Figure 3 This is the ROC curve for predicting autoimmune diabetes by the proportion of CD226+ B cells in patients with autoimmune diabetes.

[0024] Figure 4 This is a flow cytometry representation of the higher levels of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-6, IL-12) secreted by CD226+ B cells compared to CD226-negative B cells (CD226-B cells) in autoimmune diabetic patients. Figure 4 A) and statistical charts ( Figure 4 B).

[0025] Figure 5 This is a flow cytometry representation of the amount of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-6, IL-12) secreted by CD226+ B cells in the spleen (SP) and pancreatic draining lymph nodes (PLN) of NOD mice compared to CD226-B cells. Figure 5 A) and statistical charts ( Figure 5 B). Detailed implementation method:

[0026] All trials in the following examples were conducted with the informed consent of each subject. Demographic data and clinical parameters of all participants were obtained through physical examination and laboratory tests. There were no statistically significant differences in general patient information.

[0027] Example 1: The expression level of CD226 on the surface of B cells is elevated in autoimmune diabetic patients and is associated with disease progression.

[0028] Diabetes mellitus is diagnosed based on World Health Organization (WHO) criteria, including a fasting blood glucose (FBG) ≥7.0 mmol / L or a 2-hour postprandial blood glucose (2hBG) ≥11.1 mmol / L after a 75g oral glucose tolerance test (OGTT), accompanied by at least one significant symptom of diabetes (polydipsia, polyuria, or unexplained weight loss). The inclusion criteria for type 1 diabetes (T1D) are: a diagnosis of diabetes; acute ketosis or ketoacidosis requiring immediate insulin replacement therapy; a positive result for at least one typical islet autoantibody (glutamate decarboxylase antibody (GADA), protein tyrosine phosphatase antibody (IA-2A), zinc transporter 8 antibody (ZnT8A)); or impaired islet function. The inclusion criteria for type 1 diabetes (T2D) are: a diagnosis of diabetes; no ketosis or ketoacidosis, and no need for insulin for at least 6 months after diagnosis; a positive result for at least one islet autoantibody (GADA, IA-2A, ZnT8A); and an age of onset of diabetes after 30 years of age. The inclusion criteria for type 2 diabetes (T2D) are: a diagnosis of diabetes; no dependence on insulin therapy; and a negative islet autoantibody. Inclusion criteria for HC were normal glucose tolerance and no history of diabetes. HC and T2D were matched for T1D and LADA by sex and age, respectively. Autoantibody detection was performed using the radioligand method, with the positive cutoff criteria being the 99th percentile of the antibody index in 405 healthy individuals: GADA ≥ 0.05, IA-2A ≥ 0.0078, ZnT8A ≥ 0.011. Exclusion criteria included: gestational diabetes, other specific types of diabetes; severe infection, trauma, surgery, or other stressful conditions; other autoimmune diseases; malignant tumors; severe cardiovascular or cerebrovascular diseases; pregnant or lactating women; and liver or kidney dysfunction.

[0029] Peripheral blood (5 mL) was collected from each subject, including 40 patients with HC, 20 with T2D, and 60 patients with autoimmune diabetes (40 with T1D and 20 with LADA). PBMCs were isolated using density gradient centrifugation and the cell number was adjusted to 1 × 10⁻⁶ cells / mL. 6 Each flow cytometer was used. 1 μL of CD226 antibody (Cat#338306, BioLegend) and 1 μL of CD19 antibody (Cat#566396, BD) were added to a 100 μL system. After mixing, the mixture was incubated on ice in the dark for 30 minutes. The reaction was stopped with 1 mL of FACS buffer. The cells were centrifuged at 500 g for 5 minutes. The remaining 200 μL was analyzed by flow cytometry, and the proportion of CD226+ cells to CD19+ B cells was determined by gating.

[0030] The results are as follows Figure 1 As shown, the proportion of peripheral blood CD226+ B cells in autoimmune diabetic patients (including T1D and LADA) is higher than that in HC and T2D. Figure 1A) indicates that peripheral blood B cell CD226 expression is elevated in autoimmune diabetic patients, which can be used as an indicator for diagnosing autoimmune diabetes. Correlation analysis between the proportion of CD226+ B cells and clinical indicators in autoimmune diabetic patients revealed a negative correlation between the proportion of CD226+ B cells and fasting C-peptide levels. Figure 2 A) suggests that patients with a higher proportion of CD226+ B cells have poorer pancreatic function; the proportion of CD226+ B cells and fasting blood glucose ( Figure 2 B) Glycated albumin ( Figure 2 C) and glycated hemoglobin ( Figure 2 D) The levels are positively correlated. Glycated albumin reflects the patient's blood glucose control over 2-3 weeks, while glycated hemoglobin reflects the patient's blood glucose control over 2-3 months. This suggests that patients with a higher proportion of CD226+B cells have poorer blood glucose control, indicating that CD226+B cells can reflect disease progression. CD226+B cells can serve as a marker for the diagnosis and prognosis of autoimmune diabetes.

[0031] Example 2: Using the proportion of CD226+ B cells to assist in the diagnosis of autoimmune diabetes.

[0032] This invention can be used to differentiate between type 1 diabetes (T1D) and common hyperthyroidism (HC), as well as between latent diabetes mellitus (LADA) and type 2 diabetes (T2D). T1D typically presents in childhood, so accurately distinguishing it from healthy children (HC) is crucial for early diagnosis and treatment. Meanwhile, LADA patients often develop the disease in adulthood and are frequently misdiagnosed as having T2D due to their similar clinical symptoms. Therefore, differentiating between LADA and T2D is of great clinical significance for achieving accurate diabetes subtyping and providing precision medicine.

[0033] Using the expression level of peripheral blood CD226+ B cells in the HC group as a control, ROC curve analysis was performed using GraphPad Prism software based on the expression level of peripheral blood CD226+ B cells in the T1D group. Figure 3A). The results showed that the area under the ROC curve (AUC) was 0.9591 (95% confidence interval 0.9235–0.9946, P < 0.0001), indicating that the expression level of peripheral blood CD226+ B cells in the T1D group could accurately predict T1D. The optimal value on the ROC curve is the threshold. Considering both sensitivity and specificity, the sensitivity should be as high as possible while maintaining the highest specificity. Based on this method, the optimal value of the ROC curve was 27.85, i.e., the threshold was 27.85. At this threshold, the expression level of CD226+ B cells showed 85% sensitivity and 92.5% specificity, indicating that the model has a good ability to distinguish between T1D and HC, and thus the expression level of CD226+ B cells can assist in the diagnosis of T1D. If the expression level of CD226+ B cells in the peripheral blood of the test subject is greater than 27.85%, the test subject is or is suspected of being a T1D patient; if the expression level of CD226+ B cells in the peripheral blood of the test subject is less than 27.85%, the test subject is not or is not suspected of being a T1D patient.

[0034] Using the expression level of peripheral blood CD226+ B cells in the T2D group as a control, ROC curve analysis was performed using GraphPad Prism software based on the expression level of peripheral blood CD226+ B cells in the LADA group. Figure 3 B). The results showed that the area under the ROC curve (AUC) was 0.9475 (95% confidence interval 0.8805–1.0000, P < 0.0001), indicating that the expression level of peripheral blood CD226+ B cells in the LADA group could accurately predict LADA. The optimal value on the ROC curve is the threshold. Considering both sensitivity and specificity, the sensitivity should be as high as possible while maintaining the highest specificity. Based on this method, the optimal value of the ROC curve was 26.80, i.e., the threshold was 26.80. At this point, the expression level of CD226+ B cells showed 90% sensitivity and 90% specificity, indicating that the model has a good ability to distinguish between LADA and T2D. Therefore, the expression level of CD226+ B cells can assist in the diagnosis of LADA. If the expression level of CD226+ B cells in the peripheral blood of the test subject is greater than 26.80%, the test subject is or is suspected of being a LADA patient; if the expression level of CD226+ B cells in the peripheral blood of the test subject is less than 26.80%, the test subject is not or is not suspected of being a LADA patient.

[0035] To further validate the diagnostic predictive efficacy and reliability of CD226+ B cell expression levels for autoimmune diabetes, the inventors randomly collected peripheral blood samples from another independent validation cohort (20 cases of HC, 20 cases of T2D, 20 cases of T1D, and 20 cases of LADA) and detected the expression level of peripheral blood CD226+ B cells in each individual of the validation cohort. Using the expression level of peripheral blood CD226+ B cells in the HC group as a control, ROC curve analysis was performed using GraphPad Prism software based on the expression level of peripheral blood CD226+ B cells in the T1D group. Figure 3 C), the diagnostic efficacy AUC for T1D in the validation cohort was calculated to be 0.8250 (95% confidence interval 0.6993–0.9507, P = 0.0004), with a sensitivity of 60% and a specificity of 90% at the maximum Youden index. The expression level of peripheral blood CD226+ B cells in the T2D group was used as a control. ROC curve analysis was performed using GraphPad Prism software based on the expression level of peripheral blood CD226+ B cells in the LADA group. Figure 3 D) The AUC of its diagnostic efficacy for LADA in the validation cohort was calculated to be 0.8903 (95% confidence interval 0.7901–0.9905, P<0.0001), with a sensitivity of 96.8% and a specificity of 84% at the maximum Youden index. Therefore, this indicates that CD226+ B cells also have good discriminatory ability for autoimmune diabetes in the validation cohort.

[0036] Example 3: Peripheral blood CD226+ B cells in autoimmune diabetic patients secrete more pro-inflammatory cytokines

[0037] Peripheral blood (5 ml) was collected from 5 patients with T1D and 3 patients with LADA, and PBMCs were separated using density gradient centrifugation. The cell number was adjusted to 1×10⁻⁶. 61 per flow cytometer. Add Leukocyte Activation Cocktail (Cat#550583, BD) and incubate at 37°C for 5 hours. Perform cell surface marker staining first. Add 1 μl each of CD226 antibody (Cat#338306, BioLegend; Cat#338334, BioLegend) and CD19 antibody (Cat#302230, Biolegend) to a 100 μl system, mix well, and incubate on ice in the dark for 30 minutes. Add 1 ml of FACS buffer and centrifuge at 500g for 5 minutes. Add 300 μl of IC Fixation Buffer (Cat#88-8824, eBioscience) to each tube and incubate at room temperature in the dark for 30 minutes. Then, intracellular staining was performed. In a 100 μl system of 1×Permeabilization Buffer (Cat#88-8824, eBioscience), 1 μl each of TNF-α (Cat#502946, BioLegend), IFN-γ (Cat#502540, BioLegend), IL-6 (Cat#501122, BioLegend), and IL-12 (Cat#46-7235-42, eBioscience) antibodies were added. The reaction tubes were incubated at room temperature in the dark for 30 minutes. Finally, 2 ml of 1×Permeabilization Buffer was added, and the mixture was centrifuged at 500g for 5 minutes. The remaining 200 μl was then analyzed by flow cytometry. In PBMCs of autoimmune diabetic patients, B cells were divided into CD226-positive and CD226-negative populations based on CD226 expression. It was found that CD226+ B cells secreted more pro-inflammatory cytokines such as TNF-α, IFN-γ, IL-6, and IL-12. Figure 4 This suggests that CD226+ B cells have pro-inflammatory functions and participate in the disease's development and progression.

[0038] Example 4: CD226+ B cells in the spleen and pancreatic draining lymph nodes of NOD mice secrete more pro-inflammatory cytokines.

[0039] NOD mice are an ideal animal model widely used in the study of autoimmune diabetes. Spleens and pancreatic drainage lymph nodes were collected from five NOD mice, ground to obtain a single-cell suspension, and the cell number was adjusted to 1×102. 61 sample per flow cytometer. Add Leukocyte Activation Cocktail (Cat#550583, BD) and incubate at 37°C for 5 hours. Perform cell surface marker staining first. Add 1 μl each of CD226 antibody (Cat#133613, BioLegend) and B220 antibody (Cat#103235, BioLegend) to a 100 μl system, mix well, and incubate on ice in the dark for 30 minutes. Add 1 ml of FACS buffer and centrifuge at 500g for 5 minutes. Add 300 μl of ICFixation Buffer (Cat#88-8824, eBioscience) to each tube and incubate at room temperature in the dark for 30 minutes. Then, intracellular staining was performed. In a 100 μl system of 1×Permeabilization Buffer (Cat#88-8824, eBioscience), 1 μl each of TNF-α (Cat#506306, BioLegend), IFN-γ (Cat#505836, BioLegend), IL-6 (Cat#504508, BioLegend), and IL-12 (Cat#505209, BioLegend) antibodies were added. The reaction tubes were incubated at room temperature in the dark for 30 minutes. Finally, 2 ml of 1×Permeabilization Buffer was added, and the mixture was centrifuged at 500g for 5 minutes. The remaining 200 μl was then analyzed by flow cytometry. In the spleen and pancreatic draining lymph nodes of NOD mice, B cells were divided into CD226-positive and CD226-negative groups based on CD226 expression. It was found that CD226+ B cells secreted more pro-inflammatory cytokines such as TNF-α, IFN-γ, IL-6, and IL-12. Figure 5 This suggests that CD226+ B cells have pro-inflammatory functions and participate in the disease's development and progression.

Claims

1. The application of a reagent for detecting the level of CD226 protein expression in B cells in the preparation of an auxiliary diagnostic agent for autoimmune diabetes; wherein the reagent for detecting the level of CD226 protein expression in B cells is a reagent for detecting the ratio of the number of CD226-positive B cells to the total number of B cells; wherein the autoimmune diabetes is T1D and LADA.

2. The application according to claim 1, characterized in that, The reagent is an antibody that can specifically bind to CD226.

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