Application of NKG2A positive DNT cell in preparation of product for diagnosing and / or treating type 1 diabetes

Abstract

The invention discloses application of NKG2A positive DNT cells in preparation of a product for diagnosing and / or treating type 1 diabetes mellitus. According to the invention, the proportion of NKG2A positive DNT cells is proposed as an index for diagnosing the type 1 diabetes for the first time, the detection sensitivity is high, the specificity is good, and the index can better guide the treatment of the type 1 diabetes. Meanwhile, it is found through experiments that the NKG2A positive DNT cell can be effectively applied to immunotherapy of the type 1 diabetes for the first time, the NKG2A positive DNT cell and an immunotherapy method thereof are used for immunotherapy of the type 1 diabetes, and the NKG2A positive DNT cell and the immunotherapy method have the advantages of reducing blood sugar, delaying morbidity, inhibiting immune cell activity of spleen and pancreas drainage lymph nodes, relieving pancreas islet inflammation and the like. The traditional Chinese medicine is suitable for comprehensive treatment of type 1 diabetes mellitus.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the use of NKG2A-positive DNT cells in the preparation of products for the diagnosis and / or treatment of type 1 diabetes. Background Technology

[0002] Type 1 diabetes (T1D) is a chronic metabolic disease mediated by an autoimmune response, characterized by absolute insulin deficiency due to the destruction of pancreatic β-cells. The pathogenesis of T1D involves the interaction of genetic susceptibility, environmental triggers, and immune dysregulation, ultimately leading to loss of immune tolerance to autoantigens. Despite significant advances in immunological research in recent years, the immunopathological mechanisms of T1D remain unclear, and patients still require lifelong insulin therapy. Currently, the anti-CD3 monoclonal antibody teplizumab is the first and only immunomodulatory drug approved by the US Food and Drug Administration (FDA) for delaying the onset of T1D; however, its clinical application is limited due to its non-specific immunosuppressive effects and potential adverse reactions. Therefore, there is an urgent need to find new approaches that can provide better solutions for the diagnosis, prediction, and treatment of T1D.

[0003] Although most research focuses on conventional T cells, mounting evidence suggests that double-negative T (DNT) cells also play a crucial role in cancer and autoimmune diseases. DNT cells are a subset of T cells lacking the expression of CD4, CD8, or natural killer (NK) cell markers on their surface, and primarily rely on perforin and granzyme B (GZMB) to regulate immune responses. The function of DNT cells is highly complex, possessing potent immunomodulatory and effector functions, playing contradictory roles in diseases: in some autoimmune diseases and transplantation, DNT cells have been shown to prevent tissue damage or graft rejection by suppressing the overactivation of autoreactive T cells and B cells through the secretion of inhibitory cytokines; however, in certain inflammatory environments, DNT cells can produce pro-inflammatory cytokines and cytotoxic effector molecules to kill target cells, exerting pathogenic effects.

[0004] NKG2A is an inhibitory receptor expressed on the surface of NK cells and T cells. It forms a complex with CD94 and transmits self-protective signals by recognizing HLA-E molecules on the surface of healthy cells, thereby preventing the immune system from attacking normal tissues. In tumor and viral infection environments, abnormal cells utilize this mechanism to upregulate HLA-E expression to overactivate the NKG2A inhibitory pathway, achieving immune evasion. Therefore, NKG2A has become an important immune checkpoint. Monoclonal antibody drugs developed targeting it can block this signaling pathway, relieve the inhibitory state of immune cells, and reactivate their ability to kill diseased cells, providing a new direction for cancer immunotherapy. However, NKG2A-positive DNT cells have not yet been reported in autoimmune diseases, especially T1D. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes the application of a reagent for detecting the proportion of NKG2A-positive DNT cells in the preparation of products for diagnosing or assisting in the diagnosis of type 1 diabetes.

[0006] The present invention also proposes a product.

[0007] The present invention also proposes a detection system.

[0008] This invention also proposes a method for preparing NKG2A-positive DNT cells.

[0009] The present invention also proposes NKG2A-positive DNT cells prepared by the above preparation method.

[0010] This invention also proposes the application of NKG2A-positive DNT cells in the preparation of products for treating type 1 diabetes.

[0011] The present invention also proposes a drug comprising the above-mentioned NKG2A positive DNT cells.

[0012] According to a first aspect of the present invention, the use of a reagent for detecting the proportion of NKG2A-positive DNT cells in the preparation of products for diagnosing or assisting in the diagnosis of type 1 diabetes is proposed.

[0013] In some embodiments of the present invention, the proportion of NKG2A-positive DNT cells refers to the proportion of NKG2A-positive DNT cells in the peripheral blood mononuclear cells of the subject to the total number of DNT cells.

[0014] In some embodiments of the present invention, the NKG2A-positive DNT cells are DNT cells expressing NKG2A.

[0015] In some embodiments of the present invention, the detection reagent is capable of detecting the expression level of NKG2A protein in a sample.

[0016] In some embodiments of the present invention, the reagent is selected from: Probes that specifically recognize the KLRC1 gene; or Primers for specific amplification of the KLRC1 gene; or A binding agent that specifically binds to the NKG2A protein.

[0017] In some embodiments of the present invention, the KLRC1 gene encodes the NKG2A protein.

[0018] In some embodiments of the present invention, the KLRC1 gene has a Gene ID of 3821 in NCBI.

[0019] In some embodiments of the present invention, the NCBI number of the NKG2A protein is NP_001291377.1.

[0020] In some embodiments of the present invention, the NKG2A protein sequence is shown as SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0021] In some embodiments of the present invention, the binding agent includes an antibody that specifically binds to the NKG2A protein, an antibody functional fragment, or a conjugated antibody.

[0022] In some embodiments of the present invention, the product includes reagents for detecting the expression level of the NKG2A protein using nucleic acid hybridization technology, nucleic acid amplification technology, protein immunoassay technology, sequencing technology, chromatography technology, and mass spectrometry technology.

[0023] In some embodiments of the present invention, the product includes a chip, kit, or nucleic acid membrane strip capable of detecting the expression level of the NKG2A protein.

[0024] In some embodiments of the present invention, the product includes a protein chip, a protein detection kit, or a protein immunoblot strip capable of detecting the expression level of the NKG2A protein.

[0025] In some embodiments of the present invention, the test sample targeted by the test reagent is selected from tissues, blood, cell lines, and cell cultures.

[0026] In some embodiments of the present invention, the test reagent is used to test blood.

[0027] In some embodiments of the present invention, the product also has any one of the following functions: A1) Detect fasting C-peptide levels; A2) Detect blood glucose levels; A3) Detect the levels of anti-inflammatory cytokines and cytotoxic molecules.

[0028] This invention reveals that, using flow cytometry analysis, NKG2A expression in peripheral blood DNT cells of type 1 diabetes patients was significantly lower than in the healthy control (HC) group. The expression level of NKG2A protein in DNT cells was positively correlated with the fasting C-peptide level in patients, suggesting that patients with a lower proportion of NKG2A-positive DNT cells (NKG2A+DNT cells) have poorer pancreatic function. Furthermore, the expression level of NKG2A protein in DNT cells was negatively correlated with 2-hour postprandial blood glucose levels, indicating that patients with a lower proportion of NKG2A+DNT cells have poorer glycemic control. This demonstrates that NKG2A+DNT cells can reflect disease progression and can serve as a biomarker for the diagnosis and prognosis of type 1 diabetes. This biomarker can be derived from the patient's peripheral blood; direct blood sample testing reduces patient discomfort compared to complex procedures involving multiple indicators such as biochemistry, coagulation, and blood oxygen saturation, and is less invasive and highly sensitive.

[0029] In some embodiments of the present invention, the anti-inflammatory cytokines include TGF-β and IL-1. 10.

[0030] In some embodiments of the present invention, the lethal effector molecules include GZMB and IFN. γ.

[0031] According to a second aspect of the present invention, a product for detecting the proportion of NKG2A-positive DNT cells is provided, the product comprising a reagent for detecting the proportion of NKG2A-positive DNT cells, wherein the proportion of NKG2A-positive DNT cells refers to the proportion of the number of NKG2A-positive DNT cells in the peripheral blood mononuclear cells of a subject to the total number of DNT cells.

[0032] In some embodiments of the present invention, the detection reagent is capable of detecting the expression level of NKG2A protein in a sample.

[0033] In some embodiments of the present invention, the reagent is selected from: Probes that specifically recognize the KLRC1 gene; or Primers for specific amplification of the KLRC1 gene; or A binding agent that specifically binds to the NKG2A protein.

[0034] In some embodiments of the present invention, the KLRC1 gene encodes the NKG2A protein.

[0035] In some embodiments of the present invention, the binding agent includes an antibody that specifically binds to the NKG2A protein, an antibody functional fragment, or a conjugated antibody.

[0036] In some embodiments of the present invention, the product includes reagents for detecting the expression level of the NKG2A protein using nucleic acid hybridization technology, nucleic acid amplification technology, protein immunoassay technology, sequencing technology, chromatography technology, and mass spectrometry technology.

[0037] In some embodiments of the present invention, the product includes a chip, kit, or nucleic acid membrane strip capable of detecting the expression level of the NKG2A protein.

[0038] In some embodiments of the present invention, the product includes a protein chip, a protein detection kit, or a protein immunoblot strip capable of detecting the expression level of the NKG2A protein.

[0039] In some embodiments of the present invention, the test sample targeted by the test reagent is selected from tissues, blood, cell lines, and cell cultures.

[0040] In some embodiments of the present invention, the test sample is blood.

[0041] In some embodiments of the present invention, the product further includes reagents for processing test samples.

[0042] In some embodiments of the present invention, the product also has any one of the following functions: A1) Detect fasting C-peptide levels; A2) Detect blood glucose levels; A3) Detect the levels of anti-inflammatory cytokines and cytotoxic molecules.

[0043] In some embodiments of the present invention, the anti-inflammatory cytokines include TGF-β and IL-1. 10.

[0044] In some embodiments of the present invention, the lethal effector molecules include GZMB and IFN. γ.

[0045] According to a third aspect of the present invention, a detection system is provided, comprising: B1) Detection module: Collects samples from patients to be tested, measures the proportion of NKG2A-positive DNT cells, and outputs the measurement results to the analysis module; B2) Analysis module: Obtain the above test results and determine the diagnostic results based on the proportion of NKG2A positive DNT cells.

[0046] In some embodiments of the present invention, the proportion of NKG2A-positive DNT cells refers to the proportion of NKG2A-positive DNT cells in the peripheral blood mononuclear cells of the subject to the total number of DNT cells.

[0047] According to a fourth aspect of the present invention, a method for preparing NKG2A-positive DNT cells is provided, the method comprising the following steps: isolating and purifying NKG2A-positive DNT cells from peripheral blood mononuclear cells of patients with type 1 diabetes by flow cytometry sorting.

[0048] In some embodiments of the present invention, the method of sorting and purifying by flow cytometry specifically includes: removing lineage-positive cells (CD4+, CD8+, TCRγδ+, CD19+, CD56+) using antibodies conjugated with magnetic beads, and then obtaining NKG2A-positive DNT cells by flow cytometry sorting.

[0049] In some embodiments of the present invention, a step of in vitro culture of the obtained NKG2A-positive DNT cells is further included, the step comprising: adding NKG2A-positive DNT cells to a solution containing anti-CD3 antibody, anti-CD28 antibody and IL. Stimulated amplification culture was carried out in the culture medium of 2.

[0050] In some embodiments of the present invention, the concentration of the anti-CD3 antibody is 2-3 μg / mL.

[0051] In some embodiments of the present invention, the concentration of the anti-CD28 antibody is 2-3 μg / mL.

[0052] In some embodiments of the present invention, the final concentration of IL-2 in the system is 8-12 ng / mL.

[0053] In some embodiments of the present invention, the culture apparatus further includes a culture medium, wherein the basal culture medium of the culture medium includes RPMI-1640 basal culture medium.

[0054] In some embodiments of the present invention, the culture medium further includes 8-12% FBS, 0.5-1.5% penicillin / streptomycin, 8-12mM HEPES, 0.5-1.5mM sodium pyruvate, 1-3mM GlutaMAX, and 0.3-0.7mM β-mercaptoethanol.

[0055] In some embodiments of the present invention, the stimulation amplification culture time is 5-7 days.

[0056] In some embodiments of the present invention, the temperature of the stimulation amplification culture is 35-39°C.

[0057] According to a fifth aspect of the present invention, the NKG2A-positive DNT cells prepared by the above method are...

[0058] According to a sixth aspect of the present invention, the use of NKG2A-positive DNT cells in the preparation of products for treating type 1 diabetes is proposed.

[0059] In some embodiments of the present invention, the product has any one of the following functions: (1) Secretion of cytotoxic molecules; (2) Anti-inflammatory; (3) Regulates blood sugar levels; (4) Regulates fasting C-peptide levels; (5) Reduces pancreatic inflammation; (6) Inhibits the immune cell activity of the spleen and pancreatic draining lymph nodes.

[0060] In some embodiments of the present invention, the lethal effector molecules include GZMB and IFN. γ.

[0061] In some embodiments of the present invention, the anti-inflammatory effect includes the secretion of anti-inflammatory cytokines.

[0062] In some embodiments of the present invention, the anti-inflammatory cytokines include TGF-β and IL-1. 10.

[0063] In some embodiments of the present invention, the regulation of blood glucose levels includes lowering blood glucose levels.

[0064] In some embodiments of the present invention, regulating fasting C-peptide levels includes increasing fasting C-peptide levels.

[0065] In some embodiments of the present invention, the product is used via intravenous infusion or intraperitoneal injection of NKG2A-positive DNT cells.

[0066] In some embodiments of the present invention, the intravenous infusion or intraperitoneal injection of NKG2A-positive DNT cells may involve extracting autologous peripheral blood, isolating NKG2A-positive DNT cells, expanding them in vitro, and then intravenously infusing or injecting them into the patient's own body to achieve the purpose of eliminating autoreactive T cells and B cells.

[0067] In some embodiments of the present invention, the peripheral blood is derived from the patient's own blood or from the peripheral blood of a subject whose blood type matches that of the patient.

[0068] In some embodiments of the present invention, the NKG2A-positive DNT cells are isolated by flow cytometry.

[0069] In some embodiments of the present invention, the product is a pharmaceutical product.

[0070] According to some embodiments of the present invention, the dosage form of the medicine is a pharmaceutically acceptable injectable dosage form.

[0071] According to some embodiments of the present invention, the pharmaceutical product further includes a pharmaceutically acceptable carrier or excipient.

[0072] According to a seventh aspect of the present invention, a medicine comprising the above-described NKG2A-positive DNT cells is provided.

[0073] According to some embodiments of the present invention, the dosage form of the medicine is a pharmaceutically acceptable injectable dosage form.

[0074] According to some embodiments of the present invention, the pharmaceutical product further includes a pharmaceutically acceptable carrier or excipient.

[0075] According to embodiments of the present invention, at least the following beneficial effects are achieved: The present invention proposes for the first time that the proportion of NKG2A-positive DNT cells be used as a diagnostic indicator for detecting type 1 diabetes. The detection has high sensitivity and good specificity, and this indicator can better guide the treatment of type 1 diabetes.

[0076] Simultaneously, this invention, through experiments, first discovered that NKG2A-positive DNT cells can be effectively applied to immunotherapy for type 1 diabetes. Immunotherapy for type 1 diabetes using NKG2A-positive DNT cells and its associated immunotherapy methods has advantages such as lowering blood glucose, delaying onset, inhibiting the activity of immune cells in the spleen and pancreatic draining lymph nodes, and reducing pancreatic islet inflammation, making it suitable for comprehensive treatment of type 1 diabetes.

[0077] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0078] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 These are flow cytometry representation plots and statistical graphs showing that the proportion of NKG2A+DNT cells in the peripheral blood of patients with type 1 diabetes (T1D) is lower than that of healthy controls (HC). In this graph, A is the flow cytometry representation plot and B is the statistical graph. Figure 2This is a graph showing the correlation between the proportion of NKG2A+DNT cells and clinical indicators in T1D patients. In the graph, A shows a positive correlation between the proportion of NKG2A+DNT cells and the fasting C-peptide (FCP) level in T1D patients, and B shows a negative correlation between the proportion of NKG2A+DNT cells and the postprandial blood glucose (PBG) level in T1D patients. Figure 3 The ROC curves for predicting T1D based on the proportion of NKG2A+DNT cells in T1D patients and the ROC curves for the validation cohort are shown. In the figure, A is the predicted ROC curve and B is the validation ROC curve. Figure 4 In patients with type 1 diabetes (T1D), NKG2A+ DNT cells secrete more cytotoxic effector molecules (IFN) than NKG2A-negative DNT cells (NKG2A-DNT cells). Representative flow cytometry TSNE plots and statistical plots of γ, GZMB and more anti-inflammatory cytokines (TGF-β, IL-10), where A is a representative flow cytometry plot and B is a statistical plot; Figure 5 This is a graph showing the effect of NKG2A+DNT cell reinfusion on type 1 diabetes in NOD mice. A is a schematic diagram of NOD mouse models treated with intraperitoneal injection of NKG2A+DNT or DNT cells and phenotypic analysis; B is the survival curves for diabetes development in the three groups of mice; C is the blood glucose level monitoring in the three groups of mice; D is the body weight monitoring in the three groups of mice; and E is the histopathological staining results of islet tissue in the three groups of mice (scale bar: 50 μm) and a statistical graph of islet inflammation scores based on islet inflammatory cell infiltration. Figure 6 The graphs show the results of the detection of the effects of NKG2A+DNT cells on the proportion and function of B cells and T cells. Among them, A and B are flow cytometry representative graphs and statistical graphs of the proportion of B cells and activation and proliferation functions (CD86, CD69 and Ki-67) of NOD mice treated in the Mock group, DNT cell group and NKG2A+DNT cell group; C and D are flow cytometry representative graphs and statistical graphs of the proportion of T cells of NOD mice treated in the Mock group, DNT cell group and NKG2A+DNT cell group; E and F are flow cytometry representative graphs and statistical graphs of the activation and proliferation functions (CD69 and Ki-67) of CD8+ T cells of NOD mice treated in the Mock group, DNT cell group and NKG2A+DNT cell group. Figure 7This is a graph showing the results of the in vitro immunomodulatory effects of NKG2A+DNT cells on B cells and T cells. A shows the flow cytometry representation and statistical graph of the expression of activation markers (CD80, CD86, MHC-II) of B cells when co-cultured with or alone with NKG2A+DNT cells; B shows the flow cytometry representation and statistical graph of B cell proliferation detected by CFSE when co-cultured with or alone with NKG2A+DNT cells; C shows the flow cytometry representation and statistical graph of the expression of activation marker (CD69) of T cells when co-cultured with or alone with NKG2A+DNT cells; and D shows the flow cytometry representation and statistical graph of T cell proliferation detected by CFSE when co-cultured with or alone with NKG2A+DNT cells. In the results graph of the embodiments of the present invention, * indicates the significance level. p <0.05, ** indicates significance level p <0.01, *** indicates significance level p <0.001, **** indicates significance level p <0.0001. Detailed Implementation

[0079] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0080] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0081] All trials in the following examples were conducted with the informed consent of all participants. 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. Statistical analysis of the examples was performed using GraphPad Prism 9.0.

[0082] The animals used in this invention were housed in the Specific Pathogen-Free Animal Facility of the Experimental Animal Center, Central South University (Changsha, China). All animals had free access to water and food and were kept under constant temperature and humidity conditions with 12-hour light-dark cycles, and in a well-ventilated and clean environment. All animal experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of the Experimental Animal Institution of Central South University.

[0083] The DNT cells used in the examples are double negative T (DNT) cells with the phenotype CD3+TCRβ+CD4-CD8-CD56- (human) or CD3+TCRβ+CD4-CD8-NK1.1- (mouse).

[0084] The NKG2A protein sequence is as follows: NKG2A protein sequence (human): MDNQGVIYSDLNLPPNPKRQQRKPKGNKNSILATEQEITYAELNLQKASQDFQGNDKTYHCKDLPSAPEKLIVGILGIICLILMASVVTIVVIPSTLIQRHNNSSLNTRTQKARHCGH CPEEWITYSNSCYYIGKERRTWEESLLACTSKNSSLLSIDNEEEMKFLSIISPSSWIGVFRNSSHHPWVTMNGLAFKHEIKDSDNAELNCAVLQVNRLKSAQCGSSIIYHCKHKL (SEQ ID NO:1); NKG2A protein sequence (mouse) 1: MSNERVTYAELKVAKNSRNQHRKPRGPRSSISVIEQEIIYSDFSFQNPSQEHPWICRNCPCKGFPSPPEKLIAGTLGLICFVLIVAVVVITTVATPYNEANAQINSSMTRTHRDINYTLSSAQP CPHCPKEWISYSHNCYFIGMERKSWNDSLVSCISKNCSLLHIDSEEEQDFLQSLSLVSWTGILRKGRGQPWVWKKDSIFKPKIAEILHDECNCAMMSASGLTADSCTTLHPYLCKCKFPI (SEQ ID NO:2); NKG2A protein sequence (mouse) 2: MSNERVTYAELKVAKNSRNQHRKPRGPRSSISVIEQEIIYSDFSFQNPSQEHPWICRNCPCKGFPSPPEKLIAGTLGLICFVLIVAVVVITTVATPYTEAKAQINSSMTRTHRDINYTLSSAQP CPHCPKEWISYSHNCYFIGMERKSWNDSLVSCISKNCSLLYIDSEEEQDFLQSLSLISWTGILRKGRGQPWVWKEDSIFKPKIAEILHDECNCAMMSASGLTADNCTTLHPYLCKCKFPI (SEQ ID NO:3).

[0085] Example 1: Application of NKG2A+DNT cells in the preparation of products for T1D diagnosis and prognosis This embodiment demonstrates the application of NKG2A+DNT cells in the preparation of products for the diagnosis and prognosis of T1D. The specific research steps are as follows: 1. The expression level of NKG2A on the surface of DNT cells is decreased in patients with type 1 diabetes and is associated with disease progression. (1) Sample information This study was approved by the hospital's ethics committee, and informed consent was obtained from the patients.

[0086] Inclusion and exclusion criteria for patients: Diabetes is diagnosed based on World Health Organization (WHO) criteria, including fasting blood glucose (FBG) ≥7.0 mmol / L, or 2-hour postprandial blood glucose (2hBG) ≥11.1 mmol / L in an oral glucose tolerance test (OGTT) with at least one marked symptom of diabetes (polydipsia, polyuria, or unexplained weight loss).

[0087] The inclusion criteria for T1D are: a diagnosis of diabetes; acute ketosis or ketoacidosis requiring immediate insulin replacement therapy; and at least one typical pancreatic autoantibody such as glutamate decarboxylase antibody (GADA) or protein tyrosine phosphatase antibody (IA). 2A) Positive zinc transporter 8 antibody (ZnT8A); or impaired pancreatic islet function. Healthy controls (HC) were matched with T1D patients by sex and age.

[0088] Autoantibody detection was performed using the radioligand method. The positive criterion was: the 99th percentile of the antibody index in 405 healthy individuals was used as the positive threshold: GADA ≥ 0.05, IA 2A≥0.02, ZnT8A≥0.011.

[0089] Exclusion criteria: gestational diabetes, other special types of diabetes; severe infection, trauma, surgery or other stress conditions; other autoimmune diseases; malignant tumors; severe cardiovascular and cerebrovascular diseases; pregnant or lactating women; liver and kidney dysfunction, etc.

[0090] (2) Detection of NKG2A expression level on the surface of DNT cells in patients with type 1 diabetes. Peripheral blood of 5 mL was collected from each subject, including 30 cases of HC and 30 cases of T1D.

[0091] Peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation, and the cell number was adjusted to 1×10⁻⁶. 6 Add 50 μL of FACS buffer (D-Hanks + 0.4% BSA) containing 0.2 μL of Fc block to each flow cytometer. Incubate at 4°C in the dark for 10 min to block nonspecific binding of the Fc receptor. Then, add 100 μL of a pre-prepared fluorescently labeled antibody mixture containing ZombieAqua (Cat#423102, Biolegend), CD3 (Cat#344818, Biolegend), CD19 (Cat#566396, BD), TCRαβ (Cat#306706, Biolegend), TCRγδ (Cat#46-9959-42, Invitrogen), CD4 (Cat#344661, Biolegend), CD8 (Cat#344730, Biolegend), CD56 (Cat#564058, BD), and NKG2A (Cat#375122, Biolegend). Incubate on ice in the dark for 30 min, then stop the reaction with 1 mL of FACS buffer. Centrifuge at 500 g for 5 min, discard the supernatant, and add 200 μL of the mixture. After mixing with FACS buffer, the cells were analyzed by flow cytometry, and the proportion of NKG2A+ cells to DNT cells was determined by gating.

[0092] The results are as follows Figure 1-2 As shown in the figure, the proportion of NKG2A+DNT cells in the peripheral blood of T1D patients is significantly lower than that of HC ( Figure 1 (AB) indicates that NKG2A expression in peripheral blood DNT cells is decreased in T1D patients, which can be used as an indicator for diagnosing T1D. Correlation analysis between the proportion of NKG2A+DNT cells in T1D patients and clinical indicators revealed a positive correlation between the proportion of NKG2A+DNT cells and fasting C-peptide levels in T1D patients. Figure 2 A) suggests that patients with a lower proportion of NKG2A+DNT cells have poorer pancreatic function; the proportion of NKG2A+DNT cells is negatively correlated with 2-hour postprandial blood glucose levels. Figure 2(B) This suggests that patients with a lower proportion of NKG2A+DNT cells have poorer glycemic control, indicating that NKG2A+DNT cells can reflect disease progression and can serve as a biomarker for the diagnosis and prognosis of T1D.

[0093] 2. Application of the proportion of NKG2A+DNT cells in peripheral blood in the preparation of products for the auxiliary diagnosis of T1D.

[0094] This embodiment provides the ratio of NKG2A+DNT cells, which can be used to differentiate between T1D and HC. T1D typically develops in childhood, so accurately distinguishing it from healthy children (HC) is crucial for early diagnosis and treatment.

[0095] (1) Discovery phase Peripheral blood of 5 mL was collected from each subject, including 30 cases of HC and 30 cases of T1D.

[0096] The expression level of NKG2A+DNT cells in peripheral blood of the HC group was used as a control, and ROC curve analysis was performed using GraphPad Prism 9.0 software based on the expression level of NKG2A+DNT cells in peripheral blood of the T1D group.

[0097] T1D diagnosis criteria: If the expression level of NKG2A+DNT cells in the peripheral blood of the test subject is less than 26.55%, the test subject is a T1D patient or is suspected of having T1D; if the expression level of NKG2A+DNT cells in the peripheral blood of the test subject is greater than 26.55%, the test subject is not a T1D patient or is suspected of not having T1D.

[0098] The results are as follows Figure 3 As shown in Figure A, the area under the ROC curve (AUC) is 0.8489 (95% confidence interval 0.7548–0.9430). P The value <0.0001 indicates that the expression level of peripheral blood NKG2A+DNT cells in the T1D group can accurately predict T1D. The optimal value on the ROC curve is the threshold. Considering both sensitivity and specificity, the sensitivity is maximized while maintaining the highest specificity. Based on this method, the optimal value of the ROC curve is 0.2655, i.e., the threshold is 0.2655. At this point, the expression level of NKG2A+DNT cells shows a sensitivity of 63% and a specificity of 97%, indicating that the model has a good ability to distinguish between T1D and HC. Therefore, the expression level of NKG2A+DNT cells can assist in the diagnosis of T1D.

[0099] (2) Verification phase To further validate the diagnostic predictive efficacy and reliability of NKG2A+DNT cell expression levels for T1D, the inventors randomly collected peripheral blood samples from another independent validation cohort (20 cases of HC and 20 cases of T1D) and detected the expression level of NKG2A+DNT cells in the peripheral blood of each individual in the validation cohort. Using the expression level of NKG2A+DNT cells in the HC group as a control, ROC curve analysis was performed using GraphPad Prism 9.0 software based on the expression level of NKG2A+DNT cells in the T1D group to calculate its diagnostic efficacy (AUC) for T1D in the validation cohort.

[0100] The ROC curve analysis results are as follows: Figure 3 As shown in Figure B, the AUC is 0.8288 (95% confidence interval 0.6961–0.9614). P =0.0004), with a maximum Youden index of 75% sensitivity and 90% specificity. Therefore, this indicates that NKG2A+DNT cells also have good discriminative ability against T1D in the validation cohort.

[0101] Example 2: A study on the secretion of cytotoxic effector molecules and anti-inflammatory cytokines by peripheral blood NKG2A+ DNT cells in patients with T1D. Peripheral blood (5 mL) was collected from 8 patients with type 1 diabetes mellitus (T1D) and 8 patients with hepatitis C (HC). Peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation. The cell number was adjusted to 1 × 10⁻⁶ cells / mL. 6 Flow cytometry tubes. Add Leukocyte Activation Cocktail (Cat#550583, BD) and incubate at 37°C for 5 hours. First, stain for cell surface markers by adding 50 μL of FACS buffer containing 0.2 μL of Fc block and incubating at 4°C in the dark for 10 min to block non-specific binding of the Fc receptor. Then, add a pre-prepared fluorescently labeled antibody mixture (Zombie Aqua, CD3, CD19, TCRαβ, TCRγδ, CD4, CD8, CD56, NKG2A) to 100 μL of the mixture. After incubating on ice in the dark for 30 minutes, stop the reaction with 1 mL of FACS buffer, centrifuge at 500g for 5 minutes, and discard the supernatant. Add 300 μL of IC Fixation Buffer (Cat#88) to each tube. Incubate at room temperature in the dark for 30 minutes (Cat#8824, eBioscience). Then perform intracellular staining in 100 μL of 1×Permeabilization Buffer (Cat#88). In the 8824, eBioscience system, GZMB, IFN γ, TGF-β, IL Add 1 μL of antibody to each of the 10 reaction tubes and incubate the reaction tubes at room temperature in the dark for 30 minutes. Finally, add 2 mL of 1×Permeabilization Buffer, centrifuge at 500g for 5 minutes, and analyze the remaining 200 μL of the mixture using flow cytometry.

[0102] The results are as follows Figure 4 As shown in the figure, in the PBMCs of T1D patients, DNT cells were divided into NKG2A-positive and NKG2A-negative groups based on NKG2A expression. It was found that NKG2A+ DNT cells secreted more GZMB and IFN. γ-killing effector molecules and TGF-β, IL The presence of 10 anti-inflammatory cytokines suggests that NKG2A+DNT cells possess cytotoxic and anti-inflammatory functions and participate in the disease development process.

[0103] Example 3: Application of NKG2A+DNT cells in the preparation of drugs for treating type 1 diabetes This embodiment provides the application of NKG2A+DNT cells in the preparation of drugs for treating type 1 diabetes. NKG2A+DNT cells are reinfused for immunotherapy of type 1 diabetes. The experimental flowchart is shown below. Figure 5 As shown in Figure A, the specific steps are as follows: 1. Obtaining and culturing NKG2A+DNT cells (1) Obtaining NKG2A+DNT cells: NKG2A+DNT cells were isolated from the spleen and pancreatic draining lymph nodes of 10-week-old female NOD / LtJ mice (purchased from Beijing Huafukang Biotechnology Co., Ltd.) weighing approximately 20g, and sorted and purified by flow cytometry. The specific steps were as follows: First, lineage-positive cells (CD4+, CD8+, TCRγδ+, NK1.1+, CD19+) were removed using antibodies conjugated with magnetic beads, and then high-purity NKG2A+DNT cells were obtained by flow cytometry sorting.

[0104] (2) In vitro culture of NKG2A+DNT cells: 5×10 5 Or 2×10 5NKG2A+ DNT cells were expanded and stimulated in 48-well or 96-well plates containing anti-CD3 antibody (2.5 μg / mL), anti-CD28 antibody (2.5 μg / mL), and IL-2 (10 ng / mL). The culture medium consisted of RPMI-1640 (Cat#C11875500BT, Gibco) as the basal medium, supplemented with 10% FBS, 1% penicillin / streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM GlutaMAX, and 0.5 mM β-mercaptoethanol. After incubation at 37°C for 5-7 days (6 days in this example), NKG2A+ DNT cells were purified again by flow cytometry before intraperitoneal injection experiments.

[0105] 2. NKG2A+DNT cell immunotherapy for type 1 diabetes Grouping: Ten-week-old female NOD / LtJ mice were randomly divided into three groups (n=10 per group): solvent control group (Mock group), whole DNT cell therapy group (DNT group), and NKG2A+DNT cell therapy group (NKG2A+DNT group; N+DNT group). A cyclophosphamide-accelerated NOD mouse diabetes model was established.

[0106] Experimental procedure: On day 0, all three groups of mice were intraperitoneally injected (ip) with cyclophosphamide 200 mg / kg to establish a cyclophosphamide-accelerated NOD mouse diabetes model; on day 2, the DNT group and the N+DNT group were intraperitoneally injected again with 2 × 10⁻⁶ DNT or NKG2A+DNT cells expanded in vitro. 6 The Mock group received an injection of an equal volume of PBS. From the start of injection until day 30, blood glucose and weight were monitored every 3 days. A blood glucose level ≥13.9 mmol / L for two consecutive days was considered a diagnosis of diabetes.

[0107] On day 15 after cyclophosphamide injection, four mice from each group were randomly anesthetized and sacrificed. Pancreatic tissue was collected, washed with PBS, fixed with 4% paraformaldehyde, embedded in paraffin, cleared, sectioned, and stained with H&E to analyze the infiltration of inflammatory cells in the islets, assess the islet inflammation score, and quantify the lymphocyte infiltration.

[0108] The inflammation severity stages of islet inflammation in this embodiment are as follows: Stage 0: Normal islet morphology, no immune cell infiltration; Stage 1: Immune cell infiltration around the islets, but less than 25% of the islets have immune cell infiltration; Stage 2: Approximately 25%. Phase 3: Immune cell infiltration occurs in 50% of the islets; Phase 4: Immune cell infiltration occurs in more than 50% of the islets.

[0109] The monitoring results of the incidence of diabetes in mice are as follows: Figure 5 As shown in Figure B, the blood glucose monitoring results are as follows: Figure 5 As shown in Figure C, the weight monitoring results are as follows: Figure 5 As shown in Figure D, the experimental results indicate that, compared to the Mock group mice, both cell infusion methods significantly inhibited the occurrence of type 1 diabetes and alleviated hyperglycemia, without affecting body weight. Furthermore, the NKG2A+DNT group showed more significant disease relief, with a significantly delayed onset of disease, reduced incidence, and lower blood glucose levels compared to the DNT group.

[0110] In addition, pancreatic islet tissue was collected from three groups of mice and subjected to histopathological staining. The staining results are as follows: Figure 5 As shown in Figure E, the islet inflammation status in mice was scored based on the infiltration of immune cells. The results showed that compared to the Mock group, the DNT and NKG2A+DNT groups exhibited significantly reduced islet inflammatory cell infiltration and lower islet inflammation scores. Furthermore, the NKG2A+DNT group significantly alleviated islet inflammatory cell infiltration and reduced the incidence of severe islet inflammation in type 1 diabetic mice compared to the DNT group. In conclusion, the results suggest that reinfusion of NKG2A+DNT cells is superior to reinfusion of total DNT cells in the prevention and treatment of T1D.

[0111] 3. Effects of NKG2A+DNT cells on the ratio and function of pathogenic B cells and T cells NOD mice were divided into a solvent control group (Mock group), a whole DNT cell therapy group (DNT group), and an NKG2A+DNT cell therapy group (NKG2A+DNT group; N+DNT group). NOD mice were treated with NKG2A+DNT cell immunotherapy for type 1 diabetes according to the method of 2. After reinfusion, pancreatic drainage lymph nodes were collected 12 days later for flow cytometry to evaluate the pathogenic B cell and T cell response.

[0112] The results are as follows Figure 6 As shown in the figure, compared with the Mock group, the DNT group reduced the proportion of B cells and T cells and the related pathogenic response, while the inhibitory effect of the N+DNT group was more significant: the proportion of B cells decreased further, and the activation and proliferation functions of B cells were significantly reduced. Figure 6 A–B), while the ratio of CD4+ to CD8+ T cells decreased ( Figure 6 C–D), and the activation and proliferation of CD8+ T cells were significantly reduced ( Figure 6 E–F).

[0113] In summary, NKG2A+DNT cell reinfusion can simultaneously inhibit the proliferation and activation of pathogenic B cells and T cells in vivo, weaken immune-mediated attack, and thus produce a stronger protective effect against the occurrence and development of T1D. Moreover, its overall efficacy is superior to that of reinfusing unsorted total DNT cell population.

[0114] 4. Verification of the in vitro immunomodulatory effects of NKG2A+DNT cells on B cells and T cells. To functionally validate the immunomodulatory capacity of NKG2A+DNT cells against key pathogenic effector cells (B cells and T cells) in type 1 diabetes, B cells and T cells were selected as target cells. An in vitro co-culture system was established, and activation and proliferation functions were evaluated. The specific experimental steps are as follows: Freshly sorted NKG2A+ DNT cells from NOD mice were co-cultured with B cells or T cells at a ratio of 1:4. Before co-culture, B cells were activated by stimulation with lipopolysaccharide (LPS, 10 μg / mL); T cells were activated by stimulation with anti-CD3 antibody (2.5 μg / mL), anti-CD28 antibody (2.5 μg / mL), and IL-2 (10 ng / mL). All cells were cultured under the same medium conditions. After 18 hours of co-culture, the expression of target cell activation markers was detected: CD80, CD86, and MHC II were detected in B cells; CD69 was detected in T cells.

[0115] In the CFSE proliferation assay, CFSE-labeled target cells were co-cultured under the same conditions for 4 days to assess the proliferation levels of B cells and T cells.

[0116] The results are as follows Figure 7 As shown in the figure, compared with the control group, the addition of NKG2A+DNT cells significantly reduced B cell activation and antigen presentation, as evidenced by decreased expression of CD80, CD86, and MHC II. Figure 7 A); at the same time, the proliferation function of B cells decreases (A); Figure 7 B). NKG2A+DNT cells also significantly inhibited the activation of CD4+ and CD8+ T cells, as evidenced by a decrease in the proportion of CD69+ cells. Figure 7 C), and inhibit its proliferation level (C), Figure 7 D). The above results indicate that NKG2A+DNT cells can simultaneously inhibit the activation and expansion of T1D-related B cells and T cells, suggesting that they have clear immunomodulatory potential and application value as an immune intervention strategy or cell preparation.

[0117] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. The application of a reagent for detecting the proportion of NKG2A-positive DNT cells in the preparation of products for diagnosing or assisting in the diagnosis of type 1 diabetes, characterized in that, The proportion of NKG2A-positive DNT cells refers to the proportion of NKG2A-positive DNT cells in the peripheral blood mononuclear cells of the subject to the total number of DNT cells.

2. The application according to claim 1, characterized in that, The detection reagent can detect the expression level of NKG2A protein in the sample.

3. The application according to any one of claims 1-2, characterized in that, The reagents are selected from: Probes that specifically recognize the KLRC1 gene; or Primers for specific amplification of the KLRC1 gene; or A binding agent that specifically binds to the NKG2A protein; Preferably, the binding agent comprises an antibody that specifically binds to the NKG2A protein, an antibody functional fragment, or a conjugated antibody.

4. The application according to claim 1, characterized in that, The product includes reagents for detecting the expression level of the NKG2A protein using nucleic acid hybridization technology, nucleic acid amplification technology, protein immunoassay technology, sequencing technology, chromatography technology, and mass spectrometry technology. And / or, the product includes a chip, kit, or nucleic acid membrane strip capable of detecting the expression level of the NKG2A protein; And / or, the test samples targeted by the test reagent are selected from tissues, blood, cell lines, and cell cultures; And / or, the product also has any of the following functions: A1) Detect fasting C-peptide levels; A2) Detect blood glucose levels; A3) Detect the levels of anti-inflammatory cytokines and cytotoxic effector molecules; Preferably, the anti-inflammatory cytokines include TGF-β and IL-12. 10; Preferably, the lethal effector molecules include GZMB and IFN. γ.

5. A product for detecting the proportion of NKG2A-positive DNT cells, characterized in that, The product includes a reagent for detecting the proportion of NKG2A-positive DNT cells; the proportion of NKG2A-positive DNT cells refers to the proportion of NKG2A-positive DNT cells in the peripheral blood mononuclear cells of the subject to the total number of DNT cells; Preferably, the detection reagent is capable of detecting the expression level of NKG2A protein in the sample; Preferably, the reagent is selected from: Probes that specifically recognize the KLRC1 gene; or Primers for specific amplification of the KLRC1 gene; or A binding agent that specifically binds to the NKG2A protein; More preferably, the binding agent includes an antibody that specifically binds to the NKG2A protein, an antibody functional fragment, or a conjugated antibody; Preferably, the product includes reagents for detecting the expression level of the NKG2A protein using nucleic acid hybridization technology, nucleic acid amplification technology, protein immunoassay technology, sequencing technology, chromatography technology, and mass spectrometry technology; Preferably, the product includes a chip, kit, or nucleic acid membrane strip capable of detecting the expression level of the NKG2A protein.

6. A detection system, characterized in that, include: B1) Detection module: Collects samples from patients to be tested, measures the proportion of NKG2A-positive DNT cells, and outputs the measurement results to the analysis module; B2) Analysis module: Obtain the measurement results data described in B1) and determine the diagnostic results based on the proportion of NKG2A positive DNT cells.

7. A method for preparing NKG2A-positive DNT cells, characterized in that, The method includes the following steps: isolating and purifying NKG2A-positive DNT cells from peripheral blood mononuclear cells of patients with type 1 diabetes by flow cytometry.

8. An NKG2A-positive DNT cell, characterized in that, Prepared according to the method described in claim 7.

9. Application of NKG2A-positive DNT cells in the preparation of products for treating type 1 diabetes; Preferably, the product has any one of the following functions: (1) Secretion of cytotoxic molecules; (2) Anti-inflammatory; (3) Regulates blood sugar levels; (4) Regulates fasting C-peptide levels; (5) Reduces pancreatic inflammation; (6) Inhibits the immune cell activity of the spleen and pancreatic draining lymph nodes; Preferably, the lethal effector molecules include GZMB and IFN. γ; Preferably, the anti-inflammatory effect includes secreting anti-inflammatory cytokines; More preferably, the anti-inflammatory cytokines include TGF-β and IL-12.

10. Preferably, the product is used via intravenous infusion or intraperitoneal injection of NKG2A-positive DNT cells.

10. A medicine, characterized in that, Including NKG2A-positive DNT cells; Preferably, the drug further includes a pharmaceutically acceptable carrier or excipient.

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