Immune tolerance implementation method based on immune cell negative selection

Abstract

The invention discloses an immune tolerance realization method based on immune cell negative selection, which comprises the following steps: step 1, separating mononuclear cells from peripheral blood of a patient, and inducing and differentiating the mononuclear cells into DCs in vitro; 2, introducing the Aire gene into DCs by using a virus vector adeno-associated virus (AAV) or a lentivirus; 3, over-expressing Aire protein by the modified DCs, so that the modified DCs can express a plurality of autoantigens; and step 4, injecting the DCs subjected to Aire overexpression back into the body of the patient, and inducing immune tolerance. The preparation method has the beneficial effects that the immune regulation function of DCs is enhanced through the Aire gene, the activity of autoreactive T cells is inhibited, and the damage to pancreatic beta cells is reduced; dependence on a traditional immunosuppressor is reduced, and side effects are reduced; a novel treatment method is provided for autoimmune diseases such as type 1 diabetes, and high safety and specificity are achieved.

Description

Technical Field

[0001] The present invention relates to the field of biological immunity technology, and in particular to a method for realizing immune tolerance based on negative selection of immune cells. Background Art

[0002] Autoimmune tolerance is one of the important physiological functions of the immune system. The body's normal immune system has the ability to distinguish "self" from "non-self" through the mechanism of autoimmune tolerance, that is, it can only recognize and respond to exogenous antigens or changed self-antigens, but not to its own normal tissues, thus maintaining its own immune tolerance state and avoiding the immune system's attack on normal self-tissues.

[0003] However, once this state of autoimmune tolerance is broken, the immune system will produce an excessive immune response to self-antigens, causing self-cell destruction, tissue damage or dysfunction, which will lead to the occurrence of autoimmune diseases. As people continue to deepen their research in the field of autoimmune tolerance mechanisms, more and more researchers are beginning to try to use this mechanism to artificially manipulate the formation of immune tolerance to specific antigens, hoping to achieve the purpose of effectively inducing immune tolerance.

[0004] Establishing an effective method of inducing immune tolerance will have important biological significance and clinical application value for the prevention and treatment of pathological immune responses such as autoimmune diseases, transplant rejection, and allergic reactions.

[0005] The thymus can eliminate autoreactive T cells through negative selection, and also maintain immune tolerance by producing regulatory T cells (Treg) with immunosuppressive properties. Among them, the negative selection process of the thymus is an important mechanism for eliminating autoreactive T cells.

[0006] It has been found that autoreactive T cells destroy normal tissue cells, such as pancreatic β cells, leading to insufficient insulin secretion, which may be an important mechanism for the occurrence of autoimmune diseases such as type 1 diabetes (T1D). The treatment of type 1 diabetes is a major clinical challenge. The use of traditional glucocorticoids and immunosuppressants can control the disease and improve the survival rate of patients, but long-term use will cause adverse reactions, seriously affecting the quality of life of patients and even threatening their lives. Summary of the invention

[0007] The purpose of the present invention is to provide a method for achieving immune tolerance based on negative selection of immune cells to solve the technical problem that the existing treatment methods for type 1 diabetes mentioned above seriously affect the quality of life of patients and even threaten their lives.

[0008] In order to solve the above technical problems, the present invention provides a method for achieving immune tolerance based on negative selection of immune cells, which comprises the following steps:

[0009] Step 1: Isolate monocytes from the patient's peripheral blood and induce them to differentiate into DCs in vitro;

[0010] Step 2: Use viral vectors such as adeno-associated virus (AAV) or lentivirus to introduce the Aire gene into DCs;

[0011] Step 3: The modified DCs overexpress Aire protein, enabling them to express multiple self-antigens;

[0012] Step 4: Inject Aire-overexpressing DCs back into the patient to induce immune tolerance.

[0013] Furthermore, the step 1 specifically includes the following steps:

[0014] Step a, collecting peripheral blood mononuclear cells (PBMCs) from patients or healthy donors;

[0015] Step b, separating PBMCs using Ficoll separation method;

[0016] Step c, inducing monocytes to differentiate into immature DCs using cytokine IL-4 in vitro;

[0017] Step d, culturing DCs in culture medium.

[0018] Furthermore, in step 2, the Aire gene is inserted into the viral vector adeno-associated virus AAV; a CMV promoter is added to the vector to drive the efficient expression of the Aire gene; and a selection marker fluorescent protein or an antibiotic resistance gene is added to the vector to screen successfully transfected cells.

[0019] Furthermore, the step 2 specifically includes the following steps:

[0020] Step a, inoculating DCs, using RPMI 1640 medium containing FBS;

[0021] Step b, use high titer virus (≥1×10^ 8 TU / mL), diluted to MOI 10-100;

[0022] Step c, add the virus suspension, centrifuge at 1200-1500g, 30-60 minutes, room temperature;

[0023] Step d: After 4-6 hours of culture, the medium was changed, and the culture was continued for 24-48 hours to detect the transfection efficiency.

[0024] Furthermore, in step a, DCs are inoculated and the cells are cultured in RPMI 1640 medium containing 10% FBS at 37° C. and 5% CO 2 .

[0025] Furthermore, in step d, the cells are returned to a 37° C., 5% CO 2 incubator for further culturing for 4-6 hours, fresh culture medium is replaced, and the culture is continued for 24-48 hours. The transfection efficiency is detected using a fluorescence microscope or flow cytometry.

[0026] The beneficial effects of the present invention are as follows: Compared with the prior art, a method for achieving immune tolerance based on negative selection of immune cells separates monocytes from peripheral blood and induces differentiation into dendritic cells (DCs), and introduces the Aire gene into DCs using a lentiviral vector, so that the DCs overexpress the Aire protein and promote DCs to express a variety of autoantigens. After in vitro amplification and functional verification, DCs overexpressing Aire are re-infused into the patient to induce immune tolerance. The specific steps include: 1) isolation of peripheral blood monocytes and induction of differentiation of DCs; 2) Aire gene cloning and lentiviral vector construction; 3) virus packaging and purification; 4) virus infection and screening of DCs; 5) DCs re-infusion and monitoring of treatment effects. The Aire gene enhances the immune regulation function of DCs, inhibits the activity of autoreactive T cells, and reduces the damage to pancreatic beta cells; reduces dependence on traditional immunosuppressants and reduces side effects; and provides a new treatment method for autoimmune diseases such as type 1 diabetes, which has high safety and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the location of human AIRE in chromosomes and its domain structure.

[0028] Figure 2 Schematic diagram of the structure of AIRE eliminating autoreactive T cells by regulating the expression of TSA in DCs.

[0029] Figure 3 Schematic diagram of the method for achieving immune tolerance.

[0030] Figure 4 Schematic diagram of the DCs virus transfection process. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiment is only one embodiment of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the protection scope of the present invention.

[0032] In order to make the objectives, technical solutions and advantages of the present application more clear, the present application is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0033] In the following description, references to "one embodiment", "an embodiment", "an example", "an example", etc. indicate that the embodiment or example described in this way may include specific features, structures, characteristics, properties, elements or limitations, but not every embodiment or example necessarily includes the specific features, structures, characteristics, properties, elements or limitations. In addition, repeated use of the phrase "according to one embodiment of the present application" may refer to the same embodiment, but does not necessarily refer to the same embodiment.

[0034] In this embodiment: Figure 3 As shown, the following steps are included:

[0035] Specific experimental methods: Step 1: Isolation and culture of DCs; First, peripheral blood (20-50 mL) is collected from patients or healthy donors using sterile sodium heparin anticoagulant tubes to avoid blood coagulation; then peripheral blood mononuclear cells (PBMCs) are separated using Ficoll density gradient centrifugation;

[0036] Then dilute the peripheral blood with PBS at a ratio of 1:1, mix gently, and slowly add the diluted blood to the Ficoll liquid surface to avoid mixing. Centrifuge at 400g, room temperature, 30 minutes.

[0037] PBMCs were located in the white layer at the Ficoll interface and were washed twice with PBS (300 g, 10 min) to remove platelets and residual Ficoll.

[0038] PBMCs were seeded in 6-well plates at a density of 1×10^ 6 RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin was used.

[0039] Finally, 50ng / mL GM-CSF and 20ng / mL IL-4 cytokines were added. The culture conditions were 37°C and 5% CO2 for 5-7 days, and the culture medium was replaced and cytokines were supplemented every 2 days. After 5-7 days of culture, the number of DCs could be expanded to 5-10 times that of the initial PBMCs.

[0040] Step 2: Cloning of Aire gene and construction of viral vector; first, clone the Aire gene (full length about 1.5 kb) from the human cDNA library; use restriction endonucleases (EcoRI and BamHI) to insert the Aire gene into the viral vector (lentiviral vector pLVX).

[0041] Then, a strong promoter (i.e., CMV promoter) is added to the vector to drive the efficient expression of the Aire gene; a selection marker (GFP or puromycin resistance gene) is added to the vector. After the vector construction is completed, the correct insertion of the Aire gene is verified by sequencing, and a high-purity plasmid is prepared using an endotoxin-free plasmid extraction kit to avoid cytotoxicity during transfection.

[0042] Step 3: First, co-transfect the constructed viral vector and packaging plasmid (psPAX2 and pMD2.G) into HEK293T cells.

[0043] Polyethyleneimine (PEI) was used as the transfection reagent, wherein the ratio of DNA to PEI was 1:3; the transfection conditions were 37° C., 5% CO 2 , and cultured for 48 hours; then the cell supernatant was collected 48 hours after transfection and filtered through a 0.45 μm filter.

[0044] The virus particles were purified by ultracentrifugation at 70,000 g, 4°C, for 2 hours. The virus titer was determined by qPCR, which could reach 1×10^ 8 -1×10^ 9 TU / mL (transduction units / mL).

[0045] Step 4: Aire gene was introduced into DCs and DCs were seeded in 6-well plates at a density of 1×10^ 6 cells / mL; add purified virus particles, MOI (multiplicity of infection) is 10-50; add 8μg / mL Polybrene to enhance the virus infection efficiency; infection conditions are 37°C, 5% CO2, and culture for 24 hours.

[0046] Successfully transfected DCs were then screened using puromycin (2 μg / mL) for 48 hours, or GFP-positive DCs were sorted by flow cytometry.

[0047] The screened DCs were cultured for 3-5 days and expanded to the required number; the expression level of Aire was verified by qPCR, Western Blot, flow cytometry and other methods; the expression level of Aire mRNA can usually be increased by 10-50 times; Western Blot was used to detect the expression of Aire protein; flow cytometry was used to detect the expression of DCs surface markers (CD11c or HLA-DR) and Aire.

[0048] Step 5: Perform strict quality testing on Aire-overexpressing DCs to ensure their safety, purity and functionality. Wash DCs with saline before reinfusion to remove residual components in the culture medium; detect the survival rate of DCs (usually >90%) and the expression level of Aire.

[0049] Aire-overexpressing DCs were intravenously injected back into the patient at a dose of 1×10^ 6 -1×10^ 7 cells / kg body weight; the immune indicators (Tregs ratio, autoreactive T cell activity) and metabolic indicators (blood sugar level, insulin secretion) of the patients were monitored at the same time; peripheral blood of the patients was collected regularly to detect the survival and function of DCs.

[0050] Further, the efficiency of successful viral transfection of DCs was improved.

[0051] First, DCs were inoculated into a 24-well plate at a cell density of 1×10^5 cells / mL; cells were cultured in RPMI1640 medium containing 10% FBS at 37°C and 5% CO2; high-titer viral vectors (lentivirus or adenovirus) were used, and the viral titer should be ≥1×10^8TU / mL.

[0052] According to the experimental requirements, the virus is diluted to an appropriate MOI (Multiplicity of Infection), usually in the range of 10-100. Centrifugal enhanced transfection can achieve higher transfection efficiency at a lower MOI, thereby reducing the amount of virus used and reducing costs.

[0053] Add the virus suspension to the DCs culture wells, adding 100-500 μL of virus suspension to each well. The centrifugal force can quickly sediment the virus particles to the cell surface, increasing the probability of contact between the virus and the cell membrane, thereby improving the infection efficiency. For cells such as DCs that are difficult to transfect, centrifugation-enhanced transfection can significantly improve the infection efficiency of viral vectors.

[0054] Place the culture plate in a centrifuge, use a horizontal rotor, set the centrifugation conditions to 1200-1500g, 30-60 minutes, room temperature; after the centrifugation is completed, gently take out the culture plate to avoid violent shaking. After the centrifugation conditions are optimized, the activity of DCs is less affected, and the cell survival rate is usually maintained above 90%.

[0055] The cells were returned to a 37°C, 5% CO2 incubator and cultured for 4-6 hours; fresh culture medium was replaced and cultured for 24-48 hours. The transfection efficiency (GFP expression) was detected using a fluorescence microscope or flow cytometry. In addition, the present invention has the following explanations:

[0056] Immune tolerance: Immune tolerance refers to the state in which the immune system does not produce an immune response to specific antigens (self-antigens or foreign antigens); immune tolerance is an important mechanism for maintaining the balance of the immune system, preventing the immune system from attacking its own tissues (autoimmunity) or overreacting to harmless foreign substances (such as food or symbiotic microorganisms) (allergy).

[0057] Positive selection: Positive selection is a key step in the development of T cells in the thymus, which occurs at the CD4+CD8+ double-positive stage. By interacting with MHC molecules (MHC-I or MHC-II) on the surface of thymic epithelial cells, T cells that bind with appropriate affinity to the T cell receptor (TCR) receive survival signals and continue to differentiate into CD4+ or CD8+ single-positive T cells; while T cells that fail to bind or bind too strongly / weakly are eliminated through apoptosis. Positive selection ensures that T cells are MHC-restricted, that is, they can only recognize antigenic peptides bound to their own MHC molecules, while maintaining immune diversity.

[0058] Negative selection: Negative selection is a key step in the development of T cells in the thymus, which occurs at the CD4+CD8+ double positive or single positive stage. Through the interaction with the self-antigen-MHC complex on the surface of antigen presenting cells (such as dendritic cells and macrophages) in the medullary region of the thymus, T cells that bind with high affinity to the T cell receptor (TCR) (i.e., autoreactive T cells) are eliminated through apoptosis, while T cells that bind with low affinity survive and enter the periphery. Negative selection maintains immune tolerance and avoids the occurrence of autoimmune diseases by eliminating autoreactive T cells and preventing the immune system from attacking its own tissues.

[0059] Dendritic cells (DCs) are the most powerful professional antigen-presenting cells in the immune system and are widely distributed in peripheral tissues and lymphoid organs. DCs activate naive T cells and initiate adaptive immune responses by capturing, processing and presenting antigens. They highly express MHC molecules and co-stimulatory molecules (such as CD80 and CD86), and can secrete cytokines (such as IL-12) to regulate the direction of immune responses.

[0060] DCs also play a key role in immune tolerance by presenting self-antigens to induce T cell anergy or apoptosis, thereby preventing autoimmune reactions.

[0061] Sterile sodium heparin anticoagulant tube: Sterile sodium heparin anticoagulant tube is a laboratory consumable used to collect and preserve blood samples. The tube is pre-coated with sodium heparin (an anticoagulant), which can prevent blood coagulation and keep the blood in a liquid state.

[0062] Ficoll density gradient centrifugation: Ficoll density gradient centrifugation is a commonly used cell separation technique, mainly used to separate cells of a specific density from whole blood or tissue samples. This method uses the density gradient of the Ficoll solution to separate cells of different densities.

[0063] PBS: PBS (Phosphate-Buffered Saline) is a commonly used biological buffer because of its simple composition, stable pH, and similar osmotic pressure and ion concentration to biological fluids.

[0064] Adeno-associated virus AAV: Adeno-associated virus (AAV) is a small, non-enveloped single-stranded DNA virus belonging to the genus Dependoparvovirus.

[0065] TSA: TSA refers to tissue-specific antigen. AIRE promotes the expression of TSA, helping the immune system to identify and eliminate autoreactive T cells, thereby maintaining immune tolerance and preventing autoimmune diseases.

[0066] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for achieving immune tolerance based on negative selection of immune cells, characterized in that: The following steps are involved: Step 1: Isolate monocytes from the patient's peripheral blood and induce them to differentiate into DCs in vitro; Step 2: Use viral vectors such as adeno-associated virus (AAV) or lentivirus to introduce the Aire gene into DCs; Step 3: The modified DCs overexpress Aire protein, enabling them to express multiple self-antigens; Step 4: Inject Aire-overexpressing DCs back into the patient to induce immune tolerance.

2. The method for achieving immune tolerance based on negative selection of immune cells according to claim 1, characterized in that: The step 1 specifically includes the following steps: Step a, collecting peripheral blood mononuclear cells (PBMCs) from patients or healthy donors; Step b, separating PBMCs using Ficoll separation method; Step c, inducing monocytes to differentiate into immature DCs using cytokine IL-4 in vitro; Step d, culturing DCs in culture medium.

3. The method for achieving immune tolerance based on negative selection of immune cells according to claim 1, characterized in that: The second step is to insert the Aire gene into the viral vector adeno-associated virus AAV; add a CMV promoter to the vector to drive the efficient expression of the Aire gene; and add a selection marker fluorescent protein or an antibiotic resistance gene to the vector to screen successfully transfected cells.

4. The method for achieving immune tolerance based on negative selection of immune cells according to claim 3, characterized in that: The step 2 specifically includes the following steps: Step a, inoculating DCs, using RPMI 1640 medium containing FBS; Step b, use high titer virus (≥1×10^ 8 TU / mL), diluted to MOI 10-100; Step c, add the virus suspension, centrifuge at 1200-1500g, 30-60 minutes, room temperature; Step d: After 4-6 hours of culture, the medium was changed, and the culture was continued for 24-48 hours to detect the transfection efficiency.

5. The method for achieving immune tolerance based on negative selection of immune cells according to claim 4, characterized in that: In the step a, DCs are inoculated and the cells are cultured in RPMI 1640 medium containing 10% FBS at 37° C. and 5% CO 2 .

6. The method for achieving immune tolerance based on negative selection of immune cells according to claim 4, characterized in that: In step d, the cells are placed back in a 37° C., 5% CO 2 incubator for further culturing for 4-6 hours, fresh culture medium is replaced, and the culture is continued for 24-48 hours. The transfection efficiency is detected using a fluorescence microscope or flow cytometry.

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