Tolerant dendritic cell induced in vitro as well as preparation method and application thereof

Abstract

The invention discloses an in-vitro induced tolerant dendritic cell as well as a preparation method and application thereof. The STAT1 or p-STAT1 protein level of the tolerant dendritic cell is deleted. In the in-vitro dendritic cell differentiation process, the STAT1 gene / protein expression level of immature dendritic cells or STAT1 protein phosphorylation is inhibited, and the tolerant dendritic cells are prepared through induction. The tolerant dendritic cell low-expression surface costimulatory molecules CD80, CD86 and MHC-II secrete high-level IL-10 and TGF-beta and low-level INF-gamma and TNF-a, has an obvious negative immunoregulation effect, and can effectively treat or delay the progression of autoimmune diseases such as T1D and the like by inducing a receptor to generate a CD4 + Treg cell subset in vivo and inhibiting a Th1 cell subset.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of immune cells for autoimmune diseases, and particularly relates to an in vitro induced tolerogenic dendritic cell, a preparation method thereof and an application thereof. Background Art

[0002] Type 1 diabetes (T1D) is an autoimmune disease caused by immune cell infiltration and destruction of β cells. The absolute lack and insufficient replacement of insulin lead to complex metabolic disorders and metabolomic abnormalities. T1D mostly occurs in children and adolescents, seriously affecting the quality of life and life expectancy of patients, and bringing a heavy economic burden to families and society. Therefore, establishing new and effective intervention and treatment measures for T1D has important scientific significance and social value.

[0003] Signal transducer and activator of transcription 1 (STAT1) is a key effector molecule in the IFN-γ signaling pathway, which can promote Th1 immune response. The high expression of STAT1 in CD4⁺ T cells will enhance their diabetogenic ability. In addition to its function in adaptive immunity, STAT1 is also widely expressed in innate immune cell populations, playing a core signaling node role. A single-cell transcriptome sequencing analysis of peripheral blood mononuclear cells (PBMCs) from T1D patients showed that STAT1 was highly expressed not only in T cells, but also in CD16⁻ monocytes and dendritic cell subsets, suggesting that STAT1 may regulate innate immune plasticity in the context of T1D.

[0004] As a bridge between innate immunity and adaptive immunity, the origin, phenotype and function of DCs all have obvious diversity and heterogeneity. In the process of autoimmune T cells killing pancreatic islet β cells, dendritic cells (DCs) play an important role as antigen-presenting cells. DCs can not only stimulate T cell activation by presenting antigens and promote immune responses, but also induce T cell anergy and the generation of regulatory T cells (Tregs) by contacting or secreting pleiotropic cytokines (IL-10, IL-27 and TGF-β) and metabolites, inhibiting the function of effector T cells, and then inducing immune tolerance, which is also called tolerogenic dendritic cells (tolDCs). Considering that tolDCs can specifically inhibit autoimmune responses without causing general immunosuppression, developing a T1D treatment method centered on tolDCs has good development and clinical application prospects.

[0005] At present, the method for preparing tolerogenic dendritic cells (tolDC) mainly involves inducing DCs to transform into tolDCs in vitro. However, there are still relatively few reports on tolDCs that can be used in clinical treatment, have a strong immunosuppressive effect, and have stable functions. In addition, the immunosuppressive function and activity of in vitro-induced tolDCs are affected by multiple factors such as induction targets and sample sources, and there is still little research on such technologies. Therefore, it is of great significance to develop tolDC induction production processes targeting different targets. Summary of the Invention

[0006] The purpose of the present invention is to provide an in vitro-induced tolerogenic dendritic cell, its preparation method and application. Compared with mature dendritic cells (DC), the tolerogenic dendritic cell (tolDC) of the present invention has low expression of surface co-stimulatory molecules CD80, CD86 and MHC-II, secretes higher levels of IL-10, TGF-β and low levels of INF-γ, TNF-a; can induce the generation of regulatory immune cells (Treg), and has an obvious negative immunomodulatory effect; the induction method is efficient and stable, the cell purity is high, and the cost is relatively low, enabling the supply of a large number of tolerogenic dendritic cells. At the same time, the present invention uses methods such as RNA-seq of second-generation sequencing technology, DC-T cell co-culture and animal models to clarify the transcriptional profile, molecular characteristics and negative immunoregulatory effects of this tolDC, laying a foundation for the development of a T1D treatment method centered on tolDC.

[0007] To achieve the above purpose, the present application adopts the following technical solutions: In the first aspect, the present invention provides an in vitro-induced tolerogenic dendritic cell, in which the protein level of STAT1 or p-STAT1 in the tolerogenic dendritic cell is absent.

[0008] The tolerogenic dendritic cell of the present invention has a transcriptional profile with down-regulated genes such as H2-Aa, H2-Ab1, H2-M2, CD86, CD80, IL-1β, IL-6, TNF-a, and the protein levels of STAT1 and p-STAT1 are absent, having a selective negative immunomodulatory effect. The tolerogenic dendritic cell of the present invention highly expresses the surface marker molecule CD11c of DC. Compared with mature DC, it has low expression of surface co-stimulatory molecules CD80, CD86 and MHC-II, secretes higher levels of IL-10, TGF-β and low levels of INF-γ, TNF-a; can induce the generation of CD4+ regulatory T cells (Treg) with immunosuppressive effects.

[0009] Second aspect, the present invention provides a method for preparing in vitro-induced tolerogenic dendritic cells. During the in vitro dendritic cell differentiation process, the expression level of STAT1 gene / protein in immature dendritic cells is inhibited or the phosphorylation of STAT1 protein is inhibited to induce and prepare the tolerogenic dendritic cells.

[0010] Among them, during the in vitro induction and differentiation process, DCs that are not stimulated and activated by LPS are called immature dendritic cells.

[0011] In the above technical solution, the method for inhibiting the expression level of STAT1 gene / protein is to transfect immature dendritic cells with STAT1-shRNA lentivirus.

[0012] In the above technical solution, the target sequence of the STAT1-shRNA is shown as SEQ ID No. 1, which is 5'-GCTGTTACTTTCCCAGATATT-3'.

[0013] In the above technical solution, the specific method for transfecting immature dendritic cells with STAT1-shRNA lentivirus is: transfect immature dendritic cells (im-DC) with STAT1-shRNA lentivirus STAT1-shRNA-LV, the MOI of STAT1-shRNA-LV transfection is 40, the concentration of transfection enhancer Polybrene is 5 μg / mL. After transfection for a period of time, change the medium, wash away the transfection enhancer, maintain cell viability, and continue culturing to obtain the tolerogenic dendritic cells.

[0014] As an alternative specific implementation: Transfect the prepared STAT1-shRNA lentivirus (STAT1-shRNA-LV) into im-DC, the MOI of STAT1-shRNA-LV transfection is 40, and the concentration of transfection enhancer Polybrene is 5 μg / mL. Change the medium 6 hours after transfection, wash away the transfection reagent, maintain cell viability, and continue culturing; 48 - 72 hours (preferably 48 hours) after transfection, obtain tolerogenic dendritic cells (tolDC).

[0015] In the above technical solution, the expression level of STAT1 gene / protein is inhibited or the phosphorylation of STAT1 protein is inhibited by a chemical inhibitor.

[0016] In the above technical solution, the chemical inhibitor is Fludarabine.

[0017] In the above technical solution, the use concentration of Fludarabine is not less than 5 μM.

[0018] Preferably, the use concentration is 5 - 10 μM. Considering factors such as cost, it is recommended to preferably use a concentration of 5 μM.

[0019] In the above technical solution, the specific method for inducing and preparing the tolerogenic dendritic cells using Fludarabine is as follows: Add the inhibitor Fludarabine on the 5th day during the induction process of immature dendritic cells, and continue culturing to obtain the tolerogenic dendritic cells.

[0020] As an alternative specific implementation: Add the inhibitor Fludarabine (with a use concentration of 5 - 10 μM) on the 5th day during the im-DC induction process, and continue culturing until the 7th day to obtain tolerogenic dendritic cells (the above im-DC can be derived from bone marrow or peripheral blood PBMC).

[0021] In a third aspect, the present invention provides the use of the above tolerogenic dendritic cells in the preparation of a medicament for treating type 1 diabetes (T1D).

[0022] By adoptive infusion of tolerogenic dendritic cells induced by Fludarabine in vitro, it promotes the differentiation of regulatory T cells and inhibits the function of Th1 cell subsets, specifically inhibits the autoimmune response, protects pancreatic islet β cells, and thus achieves the ultimate goal of treating or delaying the progression of T1D disease.

[0023] The beneficial effects of the present invention are as follows: The present invention creatively prepares tolerogenic dendritic cells by regulating the STAT1 target in im-DC. The preparation method is efficient and stable, the cell purity can reach more than 95%, and the cost is relatively low, enabling the supply of a large number of tolerogenic dendritic cells. The tolerogenic dendritic cells obtained by the preparation method of the present invention have low expression of surface co-stimulatory molecules CD80, CD86 and MHC-II, secrete relatively high levels of IL-10, TGF-β and low levels of INF-γ, TNF-a, and have an obvious negative immunomodulatory effect. By inducing the generation of CD4+Treg cell subsets in the receptor body and inhibiting Th1 cell subsets, it can effectively treat or delay the progression of autoimmune diseases such as T1D. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram of the preparation process for in vitro induction of tolerogenic DC.

[0025] Figure 2 It is an experimental diagram (one) for determining STAT1 as an intervention target by single-cell sequencing of clinical samples.

[0026] Figure 3 It is an experimental diagram (two) for determining STAT1 as an intervention target by single-cell sequencing of clinical samples.

[0027] Figure 4It is an experimental graph showing the inhibitory effects of different concentrations of Fludarabine on the protein levels of STAT1 and p-STAT1.

[0028] Figure 5 It is a graph (I) showing the tolerance phenotype of tolDC induced in vitro detected by flow cytometry.

[0029] Figure 6 It is a graph (II) showing the tolerance phenotype of tolDC induced in vitro detected by flow cytometry.

[0030] Figure 7 It is a graph showing the tolerance phenotype of tolDC induced in vitro detected by RT-PCR.

[0031] Figure 8 It is an experimental graph (I) showing that the tolDC induced in vitro has a stable negative regulatory effect verified by DC-T co-culture.

[0032] Figure 9 It is an experimental graph (II) showing that the tolDC induced in vitro has a stable negative regulatory effect verified by DC-T co-culture.

[0033] Figure 10 It is a transcriptome sequencing revealing the molecular characteristics of DC Flu and the experimental graph (I) of the tolerance-related transcription profile.

[0034] Figure 11 It is a transcriptome sequencing revealing the molecular characteristics of DC Flu and the experimental graph (II) of the tolerance-related transcription profile.

[0035] Figure 12 It is an experimental graph showing that adoptive transfer of DC Flu can inhibit the progression of T1D disease in an adoptive transfer animal experiment (I).

[0036] Figure 13 It is an experimental graph showing that adoptive transfer of DC Flu can inhibit the progression of T1D disease in an adoptive transfer animal experiment (II).

[0037] Figure 14 It is an experimental graph showing that adoptive transfer of DC Flu can inhibit the progression of T1D disease in an adoptive transfer animal experiment (III).

[0038] The figure legends used in the statistical analysis in the attached figures, represent p < 0.05, represent p < 0.01, represent p < 0.001, represent p < 0.0001, and "ns" represents that the result has no statistical difference. Detailed implementation manners

[0039] To better illustrate the objectives, technical solutions, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the present invention to those skilled in the art. The present invention will be defined only by the claims.

[0040] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0041] C57BL / 6 mice were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.

[0042] Streptozotocin (STZ) was purchased from Sigma (S0130-1G).

[0043] STAT1-shRNA lentivirus (STAT1-shRNA-LV) was produced by Shanghai Dianjun Biotechnology.

[0044] The chemical inhibitor Fludarabine was purchased from MedChemExpress (HY-B0069).

[0045] The experimental animals were treated according to the "Implementing Rules for the Administration of Medical Laboratory Animals", and C57BL / 6 mice were maintained in a SPF-class animal facility.

[0046] Example 1: Preparation and phenotypic identification of Fludarabine-induced tolerogenic dendritic cells in vitro 1.1. Single-cell sequencing analysis of clinical samples to identify STAT1 as an intervention target First, we performed single-cell RNA sequencing analysis on peripheral blood mononuclear cells (PBMCs) from 5 newly diagnosed type 1 diabetes (T1D) patients and 3 healthy controls (dataset GSE221297). Based on the differential expression patterns of specific marker genes, the cells were divided into 11 subpopulations ( Figure 2 A, B). An overview of the cell composition showed significant differences between the healthy control group and the newly diagnosed group, especially in the proportions of CD16⁻ monocytes and conventional dendritic cells (cDCs) ( Figure 2 C). Further, we systematically evaluated the expression abundance of STAT1 in each subpopulation of PBMCs. Notably, STAT1 was highly enriched in CD16⁻ monocytes. CD16⁻ monocytes have the ability to migrate to peripheral tissues and differentiate into inflammatory dendritic cells (infDCs) or monocyte-derived macrophages (moMACs) ( Figure 3A, B). Meanwhile, upregulation of STAT1 expression was also observed in dendritic cells (DCs) ( Figure 3 A, B).

[0047] At present, the method for preparing tolerogenic dendritic cells (tolDC) is mainly to induce DCs to transform into tolDCs in vitro. However, there are still few reports on tolDCs that can be used in clinical treatment, have a strong immunosuppressive effect and stable function. Based on the above single-cell data analysis of clinical samples, we selected STAT1 in DCs as the intervention target and developed a new method for in vitro induction of tolDCs based on this. In the present invention, the STAT1-specific inhibitor fludarabine was used for intervention. Through in vitro experiments, 5 μM was determined as the appropriate concentration of action ( Figure 4 ).

[0048] Figure 2 A: UMAP dimensionality reduction visualization analysis of PBMCs from newly diagnosed T1D patients and healthy controls, and each cell subset is labeled with the corresponding annotation.

[0049] Figure 2 B: Dot plot showing the expression levels of classical marker genes in each cell type.

[0050] Figure 2 C: Composition of the main cell types in newly diagnosed T1D patients and healthy controls. The cell types marked with colors correspond to Figure 2 A. pDC: plasmacytoid dendritic cell; cDC: conventional dendritic cell; NK: natural killer cell.

[0051] Figure 3 A: UMAP plots respectively show the expression levels of STAT1 in 11 cell subsets.

[0052] Figure 3 B: Violin plots show the transcript expression levels of STAT1 in 11 cell subsets.

[0053] Figure 4 : Inhibitory effects of different concentrations of Fludarabine on the protein levels of STAT1 and p-STAT1.

[0054] 1.2. Preparation of mouse bone marrow-derived dendritic cells (BMDC) The specific method for preparing mouse BMDCs is as follows: (1)The mice were sacrificed, soaked in 70% alcohol for 5 min, and then taken out and placed on the cell table. Take the hind limbs of both sides, skin them, and keep the thighs. If more cells are needed, the front legs can also be taken off together. (Cut a little bit backward along the spinal cord connection, and be careful not to cut the two ends of the bone to avoid contaminating the bone marrow. The cells will also be killed during the alcohol soaking process.) (2)After cutting, place them in 70% alcohol (in Dish No. 1) for temporary storage and disinfection.

[0055] (3)Wrap the legs soaked in alcohol with gauze, tear the muscles clean, and place the clean bones in 1640 (in Dish No. 2) for temporary storage.

[0056] (4)Take clean scissors and cut both ends of the bone. (If there are many samples, after cutting, they can be placed in 1640 (in Dish No. 3) for temporary storage first, and then rinsed together.) Then use a 1 ml syringe to draw 1640 (from Dish No. 3) and insert it into the bone marrow cavity, and rinse repeatedly until the bone turns grayish-white.

[0057] (5)Filter the 1640 obtained by rinsing through a 70 μm filter, grind it appropriately, and rinse the culture dish with a small amount of 1640 to save cells.

[0058] (6)Centrifuge at 300G for 5 min, discard the supernatant, add 2 ml of red blood cell lysate to lyse red blood cells for 5 minutes, terminate with 5 - 10 times the volume of 1640, and centrifuge at 300G for 5 min.

[0059] (7)Prepare DC medium, 1640 + 10% serum, take 12 ml and add GM - CSF (10 ng / ml) + IL - 4 (10 ng / ml) for inducing DC.

[0060] (8)Resuspend with 1 ml, pipette and mix well, then make up to 6 ml, spread it on a DC - specific low - attachment 6 - well plate (Corning, 3471), add 1 ml to each well, and place it in the incubator.

[0061] (9)Replace all the medium on the third day and replace half of the medium on the fifth day (do not collect the upper - layer cells during both medium replacements, just operate gently). Harvest the cells on the seventh day to obtain DC (cDC). Note: At this time, the cells in the supernatant need to be collected together.

[0062] 1.3. Inducing tolerogenic dendritic cells using STAT1 - shRNA lentivirus As Figure 1As shown in the flow chart, the mice were sacrificed, soaked in 70% alcohol for 5 min, and then taken out and placed on the cell stage. The two hind limbs were taken, the muscle tissues were removed, and the bones of the two hind limbs were left. Clean scissors were taken to cut both ends of the bone and place it in clean 1640. Then, 1640 was aspirated with a 1 ml syringe and inserted into the bone marrow cavity, and rinsed repeatedly until the bone marrow was flushed out and the bone became grayish white. The bone marrow suspension was filtered through a 70 μm filter, ground appropriately, centrifuged at 300G for 5 min, then the red blood cells were lysed for 5 minutes, terminated with 5 - 10 volumes of 1640, and centrifuged at 300G for 5 min. DC medium was prepared, 1640 + 10% serum + 1% double antibody, and 12 ml was taken and added with GM - CSF (10 ng / ml) + IL - 4 (10 ng / ml) for inducing DC. The cells were resuspended with DC medium and plated on a DC - specific low - attachment 6 - well plate, divided into two groups, A and B.

[0063] During the differentiation process, the medium was completely changed on the third day, and half of the medium was changed on the fifth day. Immature dendritic cells (im - DC) were obtained after half of the medium was changed on the fifth day. In group A, the prepared STAT1 - shRNA lentivirus (STAT1 - shRNA - LV) was transfected into im - DC. The MOI of STAT1 - shRNA - LV transfection was 40, and the concentration of the transfection enhancer Polybrene was 5 μg / mL. The medium was changed 6 hours after transfection to wash away the transfection reagent and maintain cell viability for continued culture; 48 - 72 hours (preferably 48 hours) after transfection, tolerogenic dendritic cells (tolDC) were obtained, and LPS was added at a final concentration of 100 ng / ml and stimulated for 12 h for subsequent detection and experiments.

[0064] 1.4. Inducing tolerogenic dendritic cells using the inhibitor Fludarabine As Figure 1 As shown in the flow chart of B, the mice were sacrificed, soaked in 70% alcohol for 5 min, and then taken out and placed on the cell stage. The two hind limbs were taken, the muscle tissues were removed, and the bones of the two hind limbs were left. Clean scissors were taken to cut both ends of the bone and place it in clean 1640. Then, 1640 was aspirated with a 1 ml syringe and inserted into the bone marrow cavity, and rinsed repeatedly until the bone marrow was flushed out and the bone became grayish white. The bone marrow suspension was filtered through a 70 μm filter, ground appropriately, centrifuged at 300G for 5 min, then the red blood cells were lysed for 5 minutes, terminated with 5 - 10 volumes of 1640, and centrifuged at 300G for 5 min. DC medium was prepared, 1640 + 10% serum + 1% double antibody, and 12 ml was taken and added with GM - CSF (10 ng / ml) + IL - 4 (10 ng / ml) for inducing DC. The cells were resuspended with DC medium and plated on a DC - specific low - attachment 6 - well plate, divided into two groups, C and D.

[0065] During the differentiation process, the medium was completely changed on the third day, and half of the medium was changed on the fifth day. Immature dendritic cells (im - DC) were obtained after half of the medium was changed on the fifth day. In group C, the inhibitor Fludarabine was added (the usage concentration was 5 - 10 μM, and the inhibitory effect was as Figure 4As shown, continue culturing until the 7th day to obtain tolerogenic dendritic cells, and add LPS at a final concentration of 100 ng / ml to stimulate for 12 h for subsequent detection and experiments.

[0066] 1.5. Identification of the tolerogenic phenotype of tolerogenic dendritic cells induced by fludarabine 1.5.1. Detection of the tolerogenic phenotype of tolDC by flow cytometry (I) Experimental method 1. As shown in the operations of 1.2 and 1.4 in Example 1, obtain DC cells with reduced STAT1 expression (DC Flu ), and DC cells in the negative control group (DC WT ).

[0067] 2. After centrifuging the obtained DC cells, resuspend them in 1 ml of flow cytometry buffer, and dispense 100 μl per tube into flow cytometry tubes for preparation of staining.

[0068] 3. Surface staining: Dilute the antibodies to form a system (diluted with flow cytometry buffer), and add CD11C, MHC II, CD86, and CD80 at 1 μl (original concentration) of antibody per tube. Add 100 μl of the diluted antibody to each tube, and stain in the dark on ice for 30 min (when adding the staining antibody, if the number of staining antibodies per tube is different or there is a tube without staining, add an equal volume of antibody diluent to control variables).

[0069] 4. Add 3 volumes of 1640 to terminate the staining, centrifuge at 300 G for 5 min, discard the supernatant, resuspend in 200 μl of flow cytometry buffer, and perform detection on the machine.

[0070] 5. Intracellular staining: First, add Cell Activation Cocktail (with Brefeldin A) to stimulate DC cells, and collect the cells after culturing for 6 h.

[0071] 6. First, dilute the antibodies for surface staining to form a system, add CD11C and MHCII at 1 μl (original concentration) of antibody per tube, add 100 μl of the diluted antibody to each tube, stain in the dark on ice for 30 min, add 3 volumes of 1640 to terminate the staining, centrifuge at 300 G for 5 min, and discard the supernatant.

[0072] 7. After the surface staining is completed, add 200 μl of the prepared cytofix to each tube and fix for 30 min (200 μl of fix for the centrifuged cells, gently shake to resuspend the cells), add 3 volumes of perm to terminate, centrifuge at 300 G for 5 min, and discard the supernatant.

[0073] 8. Add the intracellular antibody dyes IL-6, TNF-α, IL-10, and TGF-β diluted with cytoperm for membrane permeabilization, and perform membrane permeabilization staining for 1 h.

[0074] 9. Terminate with 3 volumes of 1640, centrifuge at 300 G for 5 min, and discard the supernatant.

[0075] 10. Resuspend with 200 μl of flow cytometry buffer and prepare for flow cytometry analysis.

[0076] (II) Experimental Results The results of flow cytometry analysis are as Figures 5 - 6 shown.

[0077] Figure 5 A: Detection of DC differentiation ratio showed that there was no significant difference in the DC differentiation ratio between DC Flu and DC WT groups, and both were above 93%.

[0078] Figure 5 B: After LPS stimulation, DC Flu still showed low expression of CD86 and MHC-II. Compared with DC WT , DC Flu had the ability to resist LPS-stimulated maturation.

[0079] Figure 5 C: The results showed that the level of IL-6 secreted by DC Flu was significantly reduced.

[0080] Figure 5 D: The results showed that the level of TNF-α secreted by DC Flu was significantly reduced.

[0081] Figure 6 A: The results showed that the level of IL-10 secreted by DC Flu was significantly increased.

[0082] Figure 6 B: The results showed that the level of TGF-β secreted by DC Flu was significantly increased.

[0083] The ability to resist the activation of LPS stimulation and the secretion levels of inflammatory cytokines and anti-inflammatory cytokines are important evaluation indicators for tolerogenic dendritic cells. Therefore, the experiments in this example demonstrated that STAT1 is an important target for regulating DC tolerance, and knocking down the STAT1 expression level or inhibiting its protein function can induce the generation of tolerogenic dendritic cells.

[0084] 1.5.2. Detection of tolDC tolerance phenotype by RT-PCR (I) Experimental Method 1. As shown in the operations of 1.2 and 1.4 in Example 1, obtain DC cells with reduced STAT1 expression (DC Flu), the DC cells of the negative control group (DC WT ), collect the cell samples, add 1 ml of Trizol, and lyse on ice for more than 30 minutes.

[0085] (1) The lysis time with Trizol can be very long because it contains an RNase inhibitor and will not damage RNA. Note that it should be stored in the dark. Trizol is toxic, carcinogenic upon contact with the skin. Wear gloves and a mask and operate in a fume hood.

[0086] (2) If there are clumps, it will affect the lysis effect (lysis on ice, slow rate). If necessary, sonication can be performed, two cycles are sufficient.

[0087] 2. After lysis is complete, add 200 μl of chloroform to each tube (Trizol:chloroform = 5:1), gently invert the tube up and down about 5 times (observe whether it is completely mixed), let it stand for 5 - 10 minutes, centrifuge at 4°C, 12000 G for 15 minutes (chloroform plays an extraction role to make RNA enter the aqueous phase. Chloroform is toxic and volatile, the operation should be rapid).

[0088] 3. Take another EP tube and number it, transfer the upper aqueous phase to the corresponding EP tube (avoid sucking the lower chloroform, a part of the aqueous phase can be left and discarded).

[0089] 4. Add ammonium acetate (5 M) at 0.1 times the volume of the aqueous phase, and then add 2 μl of glycogen to assist in nucleic acid precipitation.

[0090] 5. Add isopropanol (pre-cooled at -80°C) equivalent to the above total volume to precipitate RNA.

[0091] 6. Gently invert the tube up and down about 5 times, and let it stand for 10 minutes.

[0092] 7. Centrifuge at 4°C, 12000 G for 10 minutes.

[0093] 8. Discard the supernatant (either aspirate or pour it out, prepare a tissue to dry the liquid at the centrifuge tube mouth), add 1 ml of 75% ethanol (-80°C) for washing, gently shake to suspend the precipitate (until the precipitate is suspended, do not pipette the precipitate clumps).

[0094] 9. Centrifuge at 4°C, 12000 G for 5 minutes.

[0095] 10. Repeat steps 8 - 9 for sufficient washing, dry at room temperature for 5 - 10 minutes, and the remaining moisture can be wiped off with a sterile cotton swab. Remember, do not touch the precipitate clumps.

[0096] 11. Add 20 μl of DEPC (RNase inhibitor-treated water) to complete the extraction, and measure the RNA concentration.

[0097] 12. Reverse transcribe into cDNA. According to the Kit operation, carefully dilute the original RNA solution to an appropriate concentration in advance (dilute with autoclaved dd water).

[0098] 13. Take the reverse-transcribed cDNA for RT-PCR to detect the expression levels of IL-6, IL-1β, IL-23, TNF-α, IL-10, TGF-β, PD-L1, HO-1, etc. in the DC-STAT1 and DC-shame groups.

[0099] The primer sequences corresponding to RT-PCR are as follows: (2) Experimental results Figure 7 A: The RT-PCR results showed that DC Flu significantly decreased the secretion levels of inflammatory cytokines such as IL-6, IL-1β, IL-23, and TNF-α.

[0100] Figure 7 B: The RT-PCR results showed that DC Flu significantly increased the secretion levels of anti-inflammatory cytokines such as IL-10, TGF-β, PD-L1, and HO-1.

[0101] The secretion levels of inflammatory cytokines and anti-inflammatory cytokines are important evaluation indicators for tolerogenic dendritic cells. The RT-PCR experiment once again confirmed that compared with DC WT , DC Flu significantly decreased the secretion levels of inflammatory cytokines such as IL-6, IL-1β, IL-23, and TNF-α; significantly increased the secretion levels of anti-inflammatory cytokines such as IL-10, TGF-β, PD-L1, and HO-1. Therefore, the experiment in this example proved that STAT1 is an important target for regulating DC tolerance, and knocking down the STAT1 expression level or inhibiting its protein function can induce the generation of tolerogenic dendritic cells.

[0102] Example 2: Tolerogenic dendritic cells induced by fludarabine treatment have a stable negative regulatory effect (1) Experimental methods 1. Take one OT II (B6.Cg-Tg(TcraTcrb)425Cbn / J) mouse, take the spleen, grind it through a filter mesh, centrifuge at 300G for 5 min, lyse red blood cells, terminate with 5 volumes of medium, then centrifuge at 300G for 5 min, and resuspend with 200 μl of serum-free medium.

[0103] 2. Use the Miltenyi KIT to sort naive T cells Add 20 μl of CD4+ biotin cocktail per mouse, and incubate in the dark on ice for 5 min; Add 40 μl of Reagent 2 (microbeats) per mouse, and incubate in the dark on ice for 10 min; After diluting to a volume of 2.5 ml, perform magnetic sorting using an LS column, negative selection, collect the cells not adsorbed by the column, wash the column with 2 ml of RPMI 1640 (then wash the column twice), centrifuge at 300 G for 5 min, and resuspend with 200 μl of serum-free medium; Add 20 μl of Reagent 3 (CD44+ microbeats) per mouse, incubate in the dark on ice for 10 min, pass through the column, perform negative selection, collect the cells not adsorbed by the column, and wash the column with 2 ml of RPMI 1640 (then wash the column twice) to obtain naive T cells.

[0104] 3. Stain the sorted naive T cells with CTV (CellTrace™ Violet), add CTV at a concentration of 10 6 cells / μl, and incubate in a cell culture incubator for 15 min.

[0105] 4. During the incubation, take the DC cells with reduced STAT1 expression obtained from steps 1.2 and 1.4 in Example 1 (DC Flu ), and the DC cells in the negative control group (DC WT ), add OVA Peptide (323-339) and LPS in advance and stimulate for 24 h; Digest with trypsin, add 300 μl per well in a 6-well plate, and incubate in the incubator for 30 s (start timing when putting in). After digestion, add 1.5 ml (5-fold volume) of RPMI 1640 to terminate digestion, and immediately collect the cells to prevent re-adhesion.

[0106] 5. After 15 min, take out the naive T cells stained with CTV, wash twice with RPMI 1640 to avoid excessive fluorescence. Take 20 μl for flow cytometry to detect whether the cells are stained with CTV and perform cell counting.

[0107] 6. Mix DC and naive T cells at a ratio of 1:5, and supplement the system with 1 μg / ml of OVA Peptide (323-339); also add 10 ng / ml of IL-2 to assist T cell growth; at the same time, add the corresponding cytokines according to the direction of induced differentiation to induce Th1 and Treg differentiation.

[0108] 7. Incubate together in the incubator for 5 days, perform flow cytometry staining, and detect cell proliferation and the induced differentiation of T cell subsets.

[0109] (2) Experimental Results Figure 8 A: Schematic diagram of the DC-T co-culture process.

[0110] Figure 8B: The results of DC-T co-culture proliferation showed that DC Flu could inhibit T cell proliferation.

[0111] Figure 8 C: The results of apoptosis staining showed that DC Flu and DC WT There was no significant difference in T cell apoptosis between the two groups, and the T cell activity was good.

[0112] Figure 9 A: The results showed that compared with the DC WT group, DC Flu could inhibit the activation of naïve T cells.

[0113] Figure 9 B: The results showed that compared with the DC WT group, DC Flu could inhibit the activation of naïve T cells.

[0114] Figure 9 C: The results of DC-T co-culture showed that DC Flu could inhibit Th1 cell differentiation.

[0115] Figure 9 D: The results of DC-T co-culture showed that DC Flu could promote Treg cell differentiation.

[0116] The results of DC-T co-culture showed that DC treated with fludarabine did not affect T cell activity, and the apoptosis levels of both groups were within the normal range; compared with the control group (DC WT ), DC Flu significantly inhibited T cell activation and proliferation, inhibited the proportion of CD4+IFN-γ+ Th1 subset, and at the same time could up-regulate the proportion of CD4+CD25+ Treg subset; Fludarabine-induced tolerogenic dendritic cells had a stable negative regulatory effect.

[0117] Example 3: Second-generation transcriptome sequencing technology to clarify the molecular characteristics and transcriptional profiles of fludarabine-treated tolerogenic dendritic cells DC cells with reduced STAT1 expression (DC Flu ) were prepared using the 1.4 protocol in Example 1, and DC cells of the negative control group (DC WT ). After centrifugation and discarding the supernatant, 1×10 6For the above target cells, total RNA was extracted using the Trizol method shown in 1.4 of Example 1, and then RNA sequencing was performed using the Illumina NovaSeq Reagent Kit on the NovaSeq X Plus sequencing platform (commissioned Wuhan Aibai Biotechnology Co., Ltd. for second-generation transcriptome sequencing).

[0118] The raw sequencing data was first subjected to quality control analysis using FastQC to ensure data integrity. The quality-controlled clean reads were then aligned with the reference genome using the STAR alignment software (Spliced Transcripts Alignment to a Reference). Gene expression quantification was completed using the featureCounts tool to obtain the raw read counts for each gene. To eliminate technical biases such as library size, the read counts were normalized using the DESeq2 software package. The screening criteria for differential expression analysis were set as follows: the difference in log2-transformed expression levels among the six samples was ≥1-fold, and the FDR-corrected P value was <0.05.

[0119] Figure 10 A: Transcriptome sequencing transcript profile and molecular feature result diagram.

[0120] Figure 10 B: KEGG enrichment analysis.

[0121] Figure 11 A: GSEA enrichment analysis of the Antigen processing and presentation pathway.

[0122] Figure 11 B: GSEA enrichment analysis of the Cytokine-cytokine receptor interaction pathway.

[0123] Figure 11 C: Heatmap of Antigen presentation and activation marker expression.

[0124] Figure 11 D: Heatmap of Inflammatory cytokines expression.

[0125] The sequencing results indicated that compared with DC WT compared with DC Flu in the Antigen processing and presentation and Cytokine-cytokine receptor interaction pathways, the expression of major genes was downregulated ( Figures 10 - 11), genes such as H2-Aa, H2-Ab1, H2-M2, CD86, CD80, IL-1β, IL-6, TNF-a were significantly down-regulated; this suggests that tolerogenic DCs targeting STAT1 inhibition have special differentiation pathways and immune tolerance characteristics.

[0126] Example 4: The adoptive transfer model of DCs shows that tolerogenic dendritic cells induced by fludarabine treatment can inhibit the disease progression of T1D Use the inhibitor fludarabine to induce tolerogenic dendritic cells to prepare the donor cells for the adoptive transfer model. Using wild-type C57 mice as recipients, inject 2×10 6 DC cells with the number of cells into the recipient mice through the tail vein Flu ; at the same time, set up a control group and inject DCs with the same number of cells WT . After the operation, the mice were normally raised for 3 days.

[0127] During this period, prepare sodium citrate buffer: Weigh 2.1 g of citric acid (Mw: 210.14) and add it to 100 mL of double-distilled water and dissolve it fully to prepare solution A; Weigh 2.94 g of sodium citrate (Mw: 294.10) and add it to 100 mL of double-distilled water and dissolve it fully to prepare solution B; Mix solutions A and B in a ratio of 1:1.32, measure and adjust the pH = 4.2 - 4.5 with a pH meter, and filter out impurities with a 0.22 µm or 0.45 µm filter membrane, which is the citric acid buffer for preparing STZ.

[0128] After 3 days, fast the mice for 12 h, inject STZ intraperitoneally at 40 mg / kg body weight once a day for 5 consecutive days to induce the T1D model (where STZ needs to be freshly prepared with sodium citrate buffer and injected within 30 min).

[0129] Start detecting the blood glucose of the mice from the first day of STZ injection once a day. If the blood glucose is 250 mg / dl for two consecutive times, it is considered that the mice have developed T1D. Continuously monitor the incidence of T1D in the two groups of mice. When the incidence of any group reaches 80%, the experiment is terminated. Subsequently, use HE and immunofluorescence pancreatic pathology detection, ELISA, flow cytometry, etc. to compare the T1D-related indicators of the two groups of mice.

[0130] Figure 12 A: Schematic diagram of the DC adoptive transfer model process.

[0131] Figure 12 B: Statistical chart of the incidence of T1D in the adoptive transfer model.

[0132] Figure 12 C, D: ELISA detection results of insulin and C-peptide levels in mouse serum.

[0133] Figure 12E, F: ELISA test results of cytokine levels in mouse serum.

[0134] Figure 13 A, B: HE and immunofluorescence pathological examination results of the pancreas. The scale bar in the figure is 100 μm for all.

[0135] Figure 13 C, 14A, 14B, 14C: Flow cytometry test results of immune cells.

[0136] As Figure 12 shown in Flu Figure B, in the adoptive transfer group of DC, the incidence of T1D decreased and the onset time was delayed, indicating that the tolerogenic phenotype of DC Flu could delay the progression of T1D; Figure 12 As shown in Flu Figures C and D, the levels of serum insulin and C-peptide in the adoptive transfer group of DC were significantly higher than those in the control group, suggesting that Flu in the adoptive transfer group of DC, the islet destruction was milder and the islet β cells were better preserved. Figure 13 The HE and immunofluorescence pathological examination results of the pancreas in Figures A and 13B also illustrated this point; Figure 12 As shown in Flu Figures E and F, the levels of inflammatory cytokines IFN-γ and TNF-α in the serum of the adoptive transfer group of DC were lower; Figure 13 The flow cytometry test results of immune cells in Figures C, 14A, 14B, and 14C showed that, compared with the control group, Flu in the adoptive transfer group of DC, the overall activation level of CD4+ T cells was lower, the proportion of CD4+IFN-γ+ Th1 subset decreased, and at the same time, the proportion of CD4+CD25+ Treg subset increased significantly.

[0137] The above results indicate that the tolerogenic dendritic cells induced by fludarabine treatment have stable immunosuppressive functions and can inhibit the progression of T1D.

[0138] In addition, it should be noted that in the present invention, the tolerogenic dendritic cells prepared by STAT1-shRNA lentiviral transfection have the same experimental effects as the tolerogenic dendritic cells induced by fludarabine treatment and can also inhibit the progression of T1D.

[0139] Obviously, the above embodiments are only examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. An in vitro-induced tolerogenic dendritic cell, characterized in that: The STAT1 or p-STAT1 protein level of the tolerogenic dendritic cells is absent.

2. A method for preparing in vitro-induced tolerogenic dendritic cells, characterized in that: During the in vitro dendritic cell differentiation process, the STAT1 gene / protein expression level of immature dendritic cells is inhibited or the phosphorylation of STAT1 protein is inhibited to induce and prepare the tolerogenic dendritic cells.

3. The preparation method according to claim 2, wherein: The method for inhibiting the STAT1 gene / protein expression level is to transfect immature dendritic cells with STAT1-shRNA lentivirus.

4. The preparation method according to claim 3, wherein: The target sequence of the STAT1-shRNA is shown as SEQ ID No. 1, which is 5'-GCTGTTACTTTCCCAGATATT-3'.

5. The preparation method according to claim 3, characterized in that: The specific method for transfecting immature dendritic cells with STAT1-shRNA lentivirus is as follows: Transfect immature dendritic cells with STAT1-shRNA lentivirus STAT1-shRNA-LV. The MOI of STAT1-shRNA-LV transfection is 40, the concentration of the transfection enhancer Polybrene is 5 μg / mL. After transfection for a period of time, change the medium, wash away the transfection enhancer, maintain the cell viability, and continue culturing to obtain the tolerogenic dendritic cells.

6. According to the preparation method described in claim 2, characterized in that: The STAT1 gene / protein expression level is inhibited or the phosphorylation of STAT1 protein is inhibited by a chemical inhibitor.

7. The preparation method according to claim 6, wherein: The chemical inhibitor is Fludarabine.

8. The preparation method according to claim 7, characterized in that: The use concentration of Fludarabine is not less than 5 μM.

9. According to the preparation method described in claim 7, characterized in that: The specific method for inducing and preparing the tolerogenic dendritic cells with Fludarabine is as follows: Add the inhibitor Fludarabine on the 5th day during the induction process of immature dendritic cells, and continue culturing to obtain the tolerogenic dendritic cells.

10. Use of the tolerogenic dendritic cells according to claim 1 in the preparation of a medicament for treating type 1 diabetes.

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