A method for inducing CD4+Foxp3+CD69+Treg expansion in vitro and its use

Abstract

The present invention provides an in vitro induced CD4+Foxp3+CD69+Treg cell and a preparation method thereof, as well as related pharmaceutical compositions and uses. The 10 μM proteasome inhibitor MG132 (Z-Leu-Leu-Leu-CHO) used in the present invention pre-treats the initial CD4+T cells, and can obtain at least 65% of the CD4 + Foxp3 + CD69 + Treg; compared with the control group, it increased by 91.4 percentage points; the induced MG132Treg cells after pretreatment have stronger immunosuppressive activity, inducing the reconstruction of the body's immune tolerance, thereby preventing and / or treating autoimmune diseases, especially inhibiting the occurrence and development of inflammatory bowel disease. In addition, the method of the present invention is easy to culture and obtain in vitro, with low cost and high efficiency, which is conducive to the clinical application and development of induced Treg cells in the treatment of autoimmune inflammatory diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to CD69-positive regulatory T cells with enhanced immunosuppressive activity, a preparation method thereof, and related pharmaceutical compositions and applications. Background Art

[0002] Regulatory T cells (Treg) are a group of T cells that have immunosuppressive function and play an important role in maintaining immune balance in the body. Their surface markers are CD4, CD25 and Foxp3, and they usually express CD62L, CD44, CD28, OX40, FR4, CTLA-4 and GITR. Although Treg has strong immunoregulatory ability, it is easy for Treg to transform into Th17 cells in the inflammatory microenvironment, which greatly impairs its immunosuppressive function; and inflammatory cytokines will cause the downregulation of Foxp3 expression in Treg, which in turn causes Treg to transform into inflammatory cells. CD4 + CD69 + Foxp3 + Treg cells are regulatory T cells that can express CD69. + CD69 + Foxp3 + Treg can secrete high levels of IL-10 and TGF-β1, and has greater stability under inflammatory conditions and is not easy to differentiate into inflammatory cells, which has attracted widespread attention. By reinfusing suppressive immune cells, it can inhibit abnormally activated immune cells and become a new treatment for autoimmune diseases.

[0003] CD69 is an early leukocyte activation molecule and an immune regulatory molecule. CD69 downregulates immune responses by promoting the production of TGF-β1. - / - Mouse-derived Tregs have a reduced ability to inhibit T cell proliferation in vitro, and in vivo experiments have found that OVA-induced CD69 - / -Asthma inflammatory symptoms in mice are more severe (Cortés, José R., et al. J Autoimmun. 55, 51-62, (2014).); Other studies have shown that CD69 deficiency can lead to aggravation of collagen-induced arthritis animal models (Sancho, D. et al. J. Clin. Invest. 112, 872-882 (2003).). Inflammatory bowel disease (IBD) is one of the most common diseases of intestinal immune imbalance. The onset of IBD involves multiple processes at the cellular level. Due to the complex pathogenesis, current clinical treatment drugs are often ineffective, the disease is prone to recurrence, and there are adverse drug side effects. The literature refers to CD4 + Foxp3 + CD69 + Treg and CD4 + Foxp3 + CD69 - Tregs were adopted into IBD mice and a small number of CD4 + Foxp3 + CD69 + Treg can significantly inhibit the development of IBD, while CD4 + Foxp3 + CD69 - Tregs have poor disease alleviation ability. The results showed that CD69 plays a regulatory role in IBD and can be used as a target for the treatment of IBD (Yu, L, et al. Cell Death Dis 9.9, 1-14 (2018).).

[0004] Tregs come from the body, have not been chemically modified, are easily accepted by the body, are not prone to toxic effects caused by rejection reactions, and have high safety. Due to the small proportion of Treg cells in peripheral blood (<10%), low separation efficiency, and difficulty in amplification, the low cell yield is also the main bottleneck of Treg cell immunotherapy methods. In clinical practice, existing cell immunotherapy technologies mostly use direct blood drawing methods. If the therapeutic dose is to be achieved, the blood collection volume is required to be more than 200mL each time. Even so, up to 1 / 3 of patients cannot obtain enough therapeutic doses of cells after blood collection and cannot undergo subsequent treatment; and multiple large-scale blood collections will have adverse effects on the patient's body. Therefore, inducing culture to produce more stable and efficient Treg subpopulations is crucial for the application of cell vaccines and the treatment of autoimmune diseases. At present, the expression ratio of CD4+Foxp3+CD69+Treg in Treg induced by traditional methods is only about 35%, which limits its wide application in the field of disease treatment. Summary of the invention

[0005] In view of the shortcomings of the prior art, the technical problem to be solved by the present invention is to provide a method for inducing CD4 + Foxp3 + CD69 + Treg amplification method, related pharmaceutical composition and application; also provides a CD4 + Foxp3 + CD69 + The present invention overcomes the problem that the traditional Treg culture method has CD4 + Foxp3 + CD69 + The problem of low Treg expression ratio. The present invention can obtain at least 42% of CD4 + Foxp3 + CD69 + Treg cells increased by at least 20 percentage points compared with the control group; further, after treatment with 10μM MG132, up to 67% of CD4+Foxp3+CD69+Treg cells could be obtained in vitro, an increase of 91.4 percentage points compared with the control group. The excellent characteristics of low cost and high efficiency have accelerated the clinical application and development of CD4+Foxp3+CD69+Treg cells in autoimmune inflammatory diseases.

[0006] Preferably, the MG132 Treg cells induced in vitro with immunosuppressive function and high stability of the present invention contain high levels of CD4 + Foxp3 + CD69 + Treg is easy to use and / or more efficient, laying an experimental foundation for the clinical application of induced Treg (iTreg) cells to treat autoimmune inflammatory diseases. Further, the present invention provides a new use of MG132-induced Treg for the prevention or treatment of inflammatory bowel disease and related diseases, and provides a novel clinically administered Treg containing more

[0007] CD4 + Foxp3 + CD69 + Treg induction methods and related preparations.

[0008] The first object of the present invention is to provide a method for inducing CD4 + Foxp3 + CD69 + Treg expansion method, the expansion method uses proteasome inhibitor to pre-treat the initial CD4 +T cells were then cultured under conventional Treg induction conditions to obtain cells containing at least 42% CD4 + Foxp3 + CD69 + Treg cells; preferably, obtained containing at least 50% CD4 + Foxp3 + CD69 + Treg cells; more preferably, a cell population containing at least 55% CD4 + Foxp3 + CD69 + Treg cells (see Table 1)

[0009] The percentages are shown as follows: CD4 + Foxp3 + CD69 + The number ratio of Treg cells was used as the CD4 + Foxp3 + CD69 + Treg percentage.

[0010] Preferably, the initial CD4 + The T cells are derived from at least one of the spleen, mesenteric lymph nodes, inguinal lymph nodes and other lymphoid tissues.

[0011] Preferably, the initial CD4 + T cells need to be obtained through magnetic bead or flow sorting;

[0012] Furthermore, the initial CD4 + T cells were pretreated with MG132 to induce Treg cells (MG132 Treg), which inhibited CD4 + Enhanced T cell proliferation

[0013] Preferably, the proteasome inhibitor pretreatment time is 1h-2.5h,

[0014] Preferably, the proteasome inhibitor and the initial CD4 + The temperature for T cell co-incubation was 37°C;

[0015] Preferably, the proteasome inhibitor can be selected from MG132 (Z-Leu-Leu-Leu-CHO), Bortezomib, Carfilzomib; further, preferably

[0016] MG132(Z-Leu-Leu-Leu-CHO).

[0017] Preferably, the concentration of the proteasome inhibitor MG132 is 0.1-20 μM, the concentration of Bortezomib is 0.01-0.5 μM, and the concentration of Carfizomib is 0.001-0.01 μM.

[0018] Preferably, the Treg obtained after MG132 pretreatment contains at least 55% CD4 + Foxp3 + CD69 + Treg; further, preferably, at least 65% of CD4 + Foxp3 + CD69 + Tregs;

[0019] Preferably, the proteasome inhibitor promotes CD69 expression not limited to CD4 + T cells;

[0020] Preferably, the traditional method is: taking lymphoid tissue, grinding and filtering to form a single cell suspension, discarding the supernatant after centrifugation, adding the precipitate to the cell sorting solution and counting, resuspending it in T lymphocyte culture medium after sorting, adding cytokines and activation antibodies at the same time, and detecting Treg expression after induction culture for a certain period of time.

[0021] Preferably, the lymphoid tissue originates from at least one of spleen, mesenteric lymph nodes, inguinal lymph nodes and other lymphoid tissues.

[0022] Preferably, the culture medium used under the conventional Treg induction conditions contains at least one of a cytokine or an activating antibody;

[0023] Further, preferably, the cytokine is TGF-β1, with a concentration of 5-20 ng / mL; more preferably, the cytokine TGF-β1 concentration is 10 ng / mL

[0024] Further, preferably, the cytokine is IL-2 with a concentration of 50-100 IU; more preferably, the concentration of cytokine IL-2 is 50 IU.

[0025] Further, preferably, the concentration of the activating antibody anti-CD3 / CD28 is 0.5-2 μg / mL; more preferably, the concentration of the activating antibody anti-CD3 / CD28 is 2 μg / mL.

[0026] Further, preferably, the induction time is 72-96h;

[0027] The second object of the present invention is to provide a Treg cell with enhanced in vitro immunosuppressive activity. The enhanced immune activity refers to the CD4 + Foxp3 + CD69 + The proportion of Treg cells is increased; preferably, the Treg cells with enhanced in vitro immunosuppressive activity contain at least 42% CD4 + Foxp3 + CD69 + Treg cells; preferably, the CD4 + Foxp3 + CD69 + Treg cells are obtained by any of the above-described expansion methods; preferably, the Treg cells obtained after MG132 pretreatment contain at least 55% CD4 + Foxp3 + CD69 + Treg; further, preferably, at least 67% of CD4 + Foxp3 + CD69 + Tregs;

[0028] Preferably, the Treg secretes IL-10 and TGF-β1 at levels higher than those induced by traditional methods, and further, the immunosuppression-related molecules GITR and ICOS are higher than those of Treg induced by traditional methods.

[0029] The third object of the present invention is to provide a Treg with high expression of CD69 molecules. The high expression of CD69 molecules means that Treg contains at least 42% CD4 + Foxp3 + CD69 + Treg cells; preferably, carfilzomib treatment can obtain at least 42% of CD4 + Foxp3 + CD69 + Treg cells; more preferably, 55% of CD4 + Foxp3 + CD69 + Treg cells.

[0030] Preferably, Bortezomib treatment can obtain at least 50% of CD4 + Foxp3 + CD69 +Treg cells; more preferably, 55% of CD4 + Foxp3 + CD69 + Treg cells.

[0031] Preferably, MG132 (Z-Leu-Leu-Leu-CHO) treatment can obtain at least 55% CD4 + Foxp3 + CD69 + Treg cells; more preferably, 67% of CD4 + Foxp3 + CD69 + Treg cells.

[0032] The fourth object of the present invention is to provide an in vitro MG132 Treg cell, wherein the MG132 Treg cell is a cell that activates HSF1 and promotes its binding to the CD69 promoter region, specifically promoting the binding of HSF1 to the HSE sequence to interact with each other, thereby promoting the induction of CD4+ T cells by the initial CD4+ T cells. + Foxp3 + CD69 + obtained by upregulation of Treg expression;

[0033] Preferably, the HSE sequence is a GAAnnTTC structure.

[0034] Preferably, the more is obtained by comparing the experimental group with the control group.

[0035] Preferably, the up-regulated expression is obtained by comparing the experimental group with the control group.

[0036] Preferably, the in vitro MG132 Treg cells are in vitro MG132 CD69 + Treg cells.

[0037] A fifth object of the present invention is to provide an in vitro MG132 Treg cell, wherein the in vitro MG132 Treg cell has the following characteristics:

[0038] (a) The immunosuppressive Tregs are initial CD4 + T cells were first pretreated with proteasome inhibitors and then cultured under traditional Treg induction. The obtained Treg cells expressed a higher proportion of CD4 than Treg induced by traditional methods alone (Control Treg). + Foxp3 +CD69 + Tregs;

[0039] (b) MG132 Treg has stronger immunosuppressive activity, as shown by the higher levels of IL-10 and TGF-β1 secreted by MG132 Treg, the higher levels of GITR and ICOS in MG132 Treg immunosuppressive molecules than those in Control Treg, and the inhibition of CD4 + The ability of T cells to proliferate is better than that of Control Treg;

[0040] (c) MG132 Treg can inhibit the occurrence and development of inflammatory bowel disease, and its ability to alleviate enteritis is better than that of Control Treg. The MG132 Treg of the present invention can inhibit immune response and induce the reconstruction of immune tolerance in the body, thereby preventing and / or treating autoimmune diseases.

[0041] Preferably, the autoimmune disease is selected from at least one of inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, diabetes, and systemic lupus erythematosus.

[0042] Preferably, the MG132 can activate and promote the initial CD4 + T cell-induced CD4 + Foxp3 + CD69 + Treg expression is upregulated.

[0043] Preferably, the in vitro MG132 Treg cells are in vitro MG132 CD69 + Treg cells.

[0044] The sixth object of the present invention is to provide a Treg cell in vitro expansion medium that promotes the expression of Treg cell membrane protein CD69 in vitro, wherein the Treg cell in vitro expansion medium formula includes at least one or more of Hepes, sodium pyruvate, β-mercaptoethanol and antibiotics.

[0045] Preferably, the main component of the Treg cell in vitro expansion culture medium is a T cell-specific culture medium of 1640RPMI containing 10% serum.

[0046] Preferably, the concentration of Hepes in the Treg cell in vitro expansion medium is 10 mM, the concentration of sodium pyruvate is 1 mM, and the concentration of β-mercaptoethanol is 5 μM.

[0047] Preferably, the antibody is selected from at least one of CD3 and CD28; further, preferably, the amount of the antibody added is 0.5-10 μg / mL.

[0048] Preferably, the cytokine is selected from at least one of TGF-β1 and IL-2; further, preferably, the added amount of TGF-β1 is 5-20 ng / mL, and the added amount of IL-2 is 50-100 IU.

[0049] The seventh object of the present invention is to provide a pharmaceutical composition, which contains a safe and effective amount of the above-mentioned MG132 Treg or an effective amount of MG132 CD69 + Treg cells are at least one of the active ingredient and the pharmaceutically acceptable carrier;

[0050] Preferably, in a specific example of the present invention, the pharmaceutically effective amount of MG132 Treg or a salt thereof according to the present invention may be 2.5×10 7 -5×10 8 / d / kg; further, preferably 2.5×10 8 / d / kg.

[0051] Preferably, the "effective amount" refers to the amount of therapeutic agent to treat, alleviate or prevent the target disease or condition, or the amount that exhibits a detectable therapeutic or preventive effect; preferably, the precise effective amount for a subject depends on the subject's size and health condition, the nature and extent of the symptoms, and the therapeutic agent and / or combination of therapeutic agents selected for administration.

[0052] Preferably, the pharmaceutical composition further contains an immunosuppressive adjuvant, and more preferably, the immunosuppressive adjuvant is PBS.

[0053] Preferably, the Treg cells or MG132 Treg cells in the pharmaceutical composition are autologous or allogeneic.

[0054] Preferably, the pharmaceutical composition can be prepared as an injectable, such as a liquid solution or suspension; it can also be prepared as a solid form suitable for being mixed with a solution or suspension or a liquid carrier before injection.

[0055] Preferably, the pharmaceutical composition is administered by at least one of intravenous and intraperitoneal administration.

[0056] Preferably, the pharmaceutical composition is administered by intravenous injection, and more preferably, the therapeutic dosage regimen is selected from at least one of a single-dose regimen and a multiple-dose regimen.

[0057] Preferably, the pharmaceutical composition can be directly administered to a subject, and the subject to be prevented or treated is a non-human animal; more preferably, the non-human animal includes at least one of a rat or a mouse.

[0058] Preferably, the acceptable carrier refers to a carrier used for administering a therapeutic agent or a pharmaceutical carrier in a therapeutic pharmaceutical composition; preferably, it refers to some pharmaceutical carriers that do not induce the production of antibodies harmful to the individual receiving the composition and do not have excessive toxicity after administration.

[0059] Preferably, the acceptable carrier is a liquid; more preferably, the carrier is selected from at least one of PBS or physiological saline.

[0060] Preferably, the acceptable carrier may also contain auxiliary substances such as pH buffer substances.

[0061] The pharmaceutical composition of the present invention can be prepared by using, in addition to MG132 as an active ingredient, a pharmaceutically suitable and physiologically acceptable auxiliary agent, which can be dissolved in dimethyl sulfoxide (DMSO), whose chemical formula is C2H6OS.

[0062] The eighth object of the present invention is to provide a CD4 + Foxp3 + CD69 + Use of at least one of Treg cells, MG132 Treg cells in any form as described above, and pharmaceutical compositions in any form as described above in the field of treating autoimmune diseases.

[0063] Preferably, the autoimmune disease refers to a disease caused by an immune response of the body to its own antigens; further, the disease is related to the following factors:

[0064] (a) Loss of immune tolerance and exposure of hidden antigens;

[0065] (b) Trauma and infection trigger autoimmune responses;

[0066] (c) Genetic factors.

[0067] Furthermore, preferably, the autoimmune disease is selected from inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, diabetes, and systemic lupus erythematosus.

[0068] Preferably, in one embodiment of the present invention, the concentration of MG132 Treg cells used for adoptive transfusion treatment of IBD in mice can be 2.5×10 5 -1×10 7 / mL.

[0069] Preferably, the treatment, unless otherwise specified, refers to reversing or alleviating a disorder or disease, or one or more symptoms of the disorder or disease, or inhibiting or preventing its development.

[0070] In particular, the treatment in one embodiment of the present invention refers to the treatment behavior based on the above-mentioned definition. In mammals, the "treatment" or "treatment method" of inflammatory bowel disease and diseases related thereto may include one or more of the following situations.

[0071] (1) Prevent the development of inflammatory bowel disease and related diseases;

[0072] (2) Prevent the spread of inflammatory bowel disease and related diseases;

[0073] (3) Alleviate inflammatory bowel disease and related diseases;

[0074] (4) Prevent the recurrence of inflammatory bowel disease and related diseases;

[0075] (5) Relieve the symptoms of inflammatory bowel disease and related diseases.

[0076] Beneficial Effects

[0077] The present invention optimizes and improves the existing Treg induction expression system, establishes a more efficient induction expression technology, saves experimental cost and time, and shortens the experimental cycle. Preferably, compared with the traditional Treg induction method, MG132 pretreatment of the initial CD4 + T cells, then cultured under Treg induction conditions, promote more CD4 + Foxp3 + CD69 + Treg differentiation, secreting higher levels of IL-10 and TGF-β1; the MG132 Treg of the present invention can effectively inhibit the proliferation and differentiation of effector T cells; further, the MG132 Treg of the present invention is a biological preparation, with a small rejection reaction and a good immunosuppressive effect; the treatment cost is low, the number of injections is small, and usually only one injection is required every few weeks (such as 1-2 weeks), thereby reducing the pain of injection treatment and facilitating patient recovery. MG132 promotes more CD4 + Foxp3 + CD69 + MG132 Treg can prevent or treat inflammatory bowel disease and related autoimmune diseases; it has no drug toxicity and side effects, has a stable effect, and can be taken for a long time. In the present invention, the term "Treg" refers to CD4 + Foxp3 + Regulatory T cells;

[0078] In the present invention, the term "CD4 + Foxp3 + CD69 +“Treg” refers to the Treg cell subset expressing CD69;

[0079] In the present invention, the term "CD4 + Foxp3 + CD69 - “Treg” refers to the Treg cell subset that does not express CD69;

[0080] In the present invention, the term "CD69 - / - " refers to mice with CD69 knockout gene; in the present invention, the term "MG132Treg" refers to the initial CD4 + Tregs cultured under traditional Treg induction conditions after T cells;

[0081] In the present invention, the term "MG132 CD69 + "Treg" refers to Treg expressing CD69 sorted by flow cytometry from MG132 Treg, and MG132 CD69 + Tregs are all CD4 + Foxp3 + CD69 + Treg cells;

[0082] In the present invention, the term “Control Treg” refers to Treg induced by traditional methods;

[0083] In the present invention, the term "iTreg" refers to in vitro induced Treg (induced regulatory Tcell);

[0084] In the present invention, the term "Control CD69 + "Treg" refers to CD69 Tregs sorted by flow cytometry from control Tregs + Tregs;

[0085] In the present invention, the term " CD4 + "T cell" refers to CD4 + CD62L + Naive T cells;

[0086] In the present invention, the term "IBD" refers to inflammatory bowel disease.

[0087] In the present invention, the term "TGF-β1" refers to

[0088] transforming growth factor-β1, transforming growth factor 1;

[0089] In the present invention, the term "HSF1" refers to heat shock factor 1, heat shock transcription factor 1;

[0090] In the present invention, the term "HSE" refers to heat shock response elements;

[0091] In the present invention, the term "CFSE" refers to a cell marker, which refers to 5,6-carboxyfluorescein diacetate, succinimidyl ester, a fluorescent dye that can penetrate cell membranes, has a succinimidyl ester group that specifically binds to cells and a hydroxyfluorescein diacetate group that has non-enzymatic hydrolysis.

[0092] In the present invention, the term "flow sorting" refers to the use of a flow cytometer to perform multi-parameter, quantitative analysis on single cells or biological particles in flow and sort specific populations at the same time, with a sorting purity of up to 99%.

[0093] In the present invention, the term "ELISA" refers to an enzyme-linked immunosorbent assay, which is a technique in which known antigens or antibodies are adsorbed on the surface of a solid phase carrier and enzyme-labeled antigens or antibodies are reacted on the solid phase surface for quantitative analysis.

[0094] In the present invention, the cytokines used were purchased from R&D Company;

[0095] In the present invention, the flow cytometry antibodies used were purchased from ebioscience;

[0096] In the present invention, the ELISA kit used was purchased from Thermo Fisher;

[0097] In the present invention, the initial CD4 + T cell sorting kit was purchased from STEM Cell;

[0098] In the present invention, proteasome inhibitors were purchased from Selleck and Sigma;

[0099] In the present invention, the primers were synthesized by Shanghai Jierui Biological Company;

[0100] In the present invention, the CHIP kit is purchased from CST. In the present invention, the MG132 may be a compound represented by the following chemical formula 1. In particular, the MG132 compound may be a commercially available MG132 compound, and may not be a naturally isolated MG132 compound or a MG132 compound produced by a synthetic method known in the art.

[0101] BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1 .ELISA detection of IL-10 and TGF-β1 expression analysis.

[0103] Figure 2 . Heat map of the expression levels of immunosuppression-related genes in Control Treg and MG132 Treg after gene sequencing;

[0104] Figure 3 . Figure 3 A Flow cytometry analysis of MG132 Treg, Control Treg, and MG132 CD69 + Treg and Control CD69 + Schematic diagram of the expression of Treg immunosuppression-related molecules; Figure 3 B Flow cytometry analysis of MG132 Treg, Control Treg, and Control CD69 + Treg, MG132, and CD69 + Tregs inhibit CFSE-labeled CD4 + Schematic diagram of T proliferation.

[0105] Figure 4 . Figure 4 A Schematic diagram of the expression of HSF1 and HSF1 activation-related genes after MG132 pretreatment of CD4+T cells; Figure 4 B Schematic diagram of the transfer of HSF1 from the cell membrane to the nucleus after CD4+T cells were pretreated with MG132 using fluorescence confocal microscopy.

[0106] Figure 5 .Flow cytometry analysis of HSF1 + / + and HSF1 + / - After mice were injected with MG132, CD4 + Foxp3 + CD69 + Schematic diagram of Treg expression in mouse spleen, mesenteric lymph nodes and intestinal lamina propria lymph nodes.

[0107] Figure 6 Flow cytometry analysis of CD4+ naive T cells from HSF1+ / + or HSF1+ / - mice pretreated with MG132 and induced CD4+ naive T cells from HSF1+ / + mice after blocking HSF1 expression with KRIBB11 + Foxp3 + CD69 + Schematic diagram of Treg expression.

[0108] Figure 7 .Schematic diagram of the interaction between HSF1 protein and CD69 gene promoter.

[0109] Figure 8 .Adoptive transfer of MG132 Treg, Control Treg, MG132 CD69+Treg, and Control CD69+Treg into IBD mice, detection evaluation, and comparison of treatment effects.

[0110] Fig. 9 .Comparison of Th1 and Th17 downregulation in mice in the MG132 Treg, Control Treg, MG132 CD69+Treg, and Control CD69+Treg adoptive transfer treatment groups.

[0111] Special note: In order to make the figure clear and proportional, the annotation in the figure is CD69 + Treg refers to CD4 + Foxp3 + CD69 + Treg. DETAILED DESCRIPTION

[0112] The present invention is further described below in conjunction with the accompanying drawings and examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0113] Example 1 Proteasome inhibitors promote CD4 + Foxp3 + CD69 + Differentiation of Tregs

[0114] The CD4 + Foxp3 + CD69 + The expression ratio of Treg is only about 35%. In Example 1, the effect of proteasome inhibitors on CD4 + Foxp3 + CD69 + The effect of Treg production was induced by pre-treatment of mouse naive T cells with MG132, bortezomib, and carfilzomib, and then induced under the conditions of traditional Treg culture, which effectively promoted the production of anti-inflammatory CD4 + Foxp3 + CD69 + To determine the optimal concentration of each proteasome inhibitor pretreatment, CD4 Treg cells from mouse spleen were sorted by magnetic beads. + CD62L After counting the cells, 1 μM and 10 μM MG132, 0.01 μM and 0.1 μM Bortezomib, 0.001 μM and 0.01 μM Carfizomib were added respectively and placed in a 37°C cell culture incubator for pretreatment for 90 min. The DMSO group was used as the negative control group and pretreated in the same way as above. The cells were then washed three times with serum-free medium at 1640 °C and 2 × 10 6 / mL was resuspended in T lymphocyte culture medium, and cytokine TGF-β1, cytokine IL-2, and anti-CD3 / CD28 antibody were added. Three days later, the cultured cells were collected and flow cytometry was used to detect Treg and CD4 + Foxp3 + CD69 + Treg expression. Table 1 shows that each group of proteasome inhibitors can successfully induce Treg differentiation, with a percentage ranging from 42% to 67%. Further comparison of CD4 + Foxp3 + CD69 + The proportion of Tregs was found to be CD4 after pretreatment with the proteasome inhibitor MG132 + Foxp3 + CD69 + The proportion of Treg was 20%-32% higher than that of the control group. The best result was achieved in the 10μM MG132-treated group, which was 91.4 percentage points higher than that of the control group.

[0115] Both 0.01μM and 0.1μM Bortezomib can induce CD4 + Foxp3 + CD69 + Treg expression was high, 51-57 percentage points higher than that of the control group. 0.001μM Carfizomib could induce CD4 + Foxp3 + CD69 + Treg was upregulated by about 20% compared with the control group. The induction effect of 0.01μM Carfizomib was not obvious, only increasing by 7% compared with the control group.

[0116] ELISA kits were used to detect the levels of TGF-β1 and IL-10 secreted by the above cultured cells, showing that 10μM MG132 had the strongest ability to promote the secretion of IL-10 and TGF-β1 by Treg. In addition, its IL-10 and TGF-β1 were 1.5-2 times that of the control group. The IL-10 expression level in the 0.01μM, 0.1μM Bortezomib and 0.001μM Carfizomib groups was also upregulated ( Figure 1 ).

[0117] During the culture process, a T lymphocyte culture medium was used, and the preparation method thereof was as follows: β-mercaptoethanol with a final concentration of 5 μM, 1 mM sodium pyruvate, 10 mM Hepes were added to a 1640 culture medium containing 10% FBS, and the tertiary antibodies were added.

[0118] In this example, the induced initial CD4 + T cells differentiate into CD4 + Foxp3 + The number ratio of cells was taken as the percentage of Treg; the induced CD4 + Foxp3 + CD69 + The number ratio of Treg cells was used as the CD4 + Foxp3 + CD69 + Treg percentage.

[0119] Table 1 Effects of different concentrations of proteasome inhibitors on Treg and CD4 + Foxp3 + CD69 + Effect of Treg ratio

[0120]

[0121] Example 2. Differences in gene expression between MG132 Treg and Control Treg

[0122] In order to clarify the difference in gene expression between MG132 Treg and Control Treg, a flow sorter (instrument model: BD FACS S ORP ARIA II) was used to sort the MG132 Treg induced by 10 μM MG132 pretreatment in Example 1 and the control group Control Treg for gene sequencing. Figure 2 The results showed that the expression levels of MG132 Treg immunosuppression-related genes such as IL-10, TGF-β1, SOCS2, HSP90AA1, GITR, Gzmb, and CTLA-4 were significantly higher than those of Control Treg. At the same time, the expression of pro-inflammatory factors IL-17A and IL-17F in the MG132 Treg group was downregulated compared with that in the Control Treg group.

[0123] The flow cytometry sorting buffer is PBS, pH 7.2, with a final concentration of 0.5% BSA and 2 mM EDTA added, and the cells are filtered and packaged.

[0124] The loading buffer for flow cytometry sorting was: HBSS, pH 7.2, with a final concentration of 1% BSA and 4× double antibody.

[0125] The flow cytometry sorting collection buffer is: RPIM 1640 medium, with a final concentration of 30% serum and 4× double antibody.

[0126] Example 3. Analysis of MG132 Treg characteristics

[0127] Cells from each group were collected, 1×10 6 The MG132 Treg cells induced by 10 μM MG132 pretreatment and the control group ControlTreg cells were resuspended in 100 μl PBS, and the corresponding doses of fluorescent labeled antibodies were added according to the instructions: CD4, Foxp3, CD69, CTLA-4, ICOS, GITR, CD44, CXCR4, CCR7 and corresponding isotype control antibodies. They were incubated on ice in the dark for 30 min, and then washed three times with PBS. The Treg / CD4 expression after MG132 induction was detected by flow cytometry. + Foxp3 + CD69 + Treg and Treg / CD4 induced by traditional methods + Foxp3 + CD69 + Expression of Treg immunosuppression-related molecules, Figure 3 A shows that both groups of Treg highly expressed immunosuppressive molecules. However, MG132Treg immunosuppressive molecules CTLA-4 and ICOS were higher than those of ControlTreg, while Control CD69 + Treg and MG132CD69 + There were no significant differences in the immunosuppression-related molecules and chemokine receptors CCR7 and CXCR4 of Treg. + T cells were labeled with CFSE at a final concentration of 10 mM, washed twice with serum-free medium 1640, and anti-CD3 / CD28 antibody was added after adjusting the concentration to plate on a 96-well plate. Then, induced MG132 Treg, Control Treg, MG132 CD69 + Treg and Control CD69 + Treg, effector-target ratio was 1:1, 2:1, 4:1, each group had three replicate wells, and the fluorescence intensity was detected by FACS after incubation at 37℃ for 3 days. Cell proliferation experiment showed that CD4 +After T cells were activated by anti-CD3 / CD28 antibodies, their proliferation rate was about 90%. + When T cells were co-cultured at a 1:1 ratio, CD4 + The proliferation rate of T cells was 37%, and the control Treg and CD4 + When T cells were co-cultured at a 1:1 ratio, CD4 + The proliferation rate of T cells was 44%. Under different effector-target ratios, Control CD69 + Treg and MG132CD69 + Tregs suppress CD4 + T cell capacity is superior to that of Control Treg and MG132Treg. Importantly, MG132 Treg inhibits CD4 + T cell proliferation ability is better than Control Treg (see Figure 3 B) Preliminary results suggest that MG132 Treg has stronger immunosuppressive function than Control Treg. + Treg and MG132CD69 + There was no significant difference in the immunosuppressive function of Treg.

[0128] Example 4 MG132 promotes the activation of HSF1 and related genes

[0129] CD4 cells were pretreated with 10 μM MG132 + T cells were washed three times with serum-free 1640 medium for 90 minutes and then cultured in an incubator. Cells were collected at 12h and 24h, and a group without any treatment was set as a control.

[0130] Real-time PCR detection of CD4 + The expression of HSF1 in T cells and the expression of genes related to HSF1 activation (the primers used are shown in Table 2). Figure A shows that MG132 can promote the expression of HSF1 and its related genes HSP90A, DNAJB1, and HSPA1A, and the increase is more than 10 times that of the control group. At the same time, MG132 can also promote the expression of CD69 (see Figure 4 A). Compared with the control, MG132 pretreatment can promote the expression level of CD69 gene to increase by 4 times. HSF1 is mainly distributed in the cell membrane in the resting state. After MG132 treatment, immunofluorescence confocal microscopy showed that HSF1 entered the cell nucleus from the cell membrane and became activated (see Figure 4 B).

[0131] Table 2 Primers used in Real-time PCR

[0132]

[0133] Example 5. MG132 promotes CD69 expression of Treg in vivo and is dependent on HSF1

[0134] To further clarify the role of MG132 in promoting CD69 + Treg differentiation mechanism, using HSF1 + / + and HSF1 + / - Mice were injected with 15 μM / kg MG132 via the tail vein. 7 days later, the spleen, inguinal lymph nodes, mesenteric lymph nodes, and intestinal lamina propria lymph nodes were isolated and CD4 + Foxp3 + CD69 + The expression of Treg was used to determine the effect of MG132 on Treg differentiation and CD4 + Foxp3 + CD69 + To investigate the effect of HSF1 on Treg expression and whether the loss of HSF1 affects the increase of CD69. Figure 5 It shows that there is a certain proportion of CD4 in normal mice + Foxp3 + CD69 + Tregs are present in small amounts in the spleen and lymph nodes, but account for up to 68% of the total in the intestinal lamina propria. MG132 can upregulate HSF1 after in vivo injection. + / + CD4 + Foxp3 + CD69 + Treg expression was upregulated by about 15%, but HSF1 + / - Mouse CD4 + Foxp3 + CD69 + Treg had no significant effect, but MG132 could promote HSF1 + / + MG132 was unable to promote the expression of CD69 on Treg cells, despite this + / - Mouse CD4 + Foxp3 + CD69 + Treg expression, further indicating that MG132-promoting CD69 expression requires the participation of HSF1.

[0135] In this embodiment, the "HSF1 + / + " refers to wild-type mice in which the heat shock factor 1 (HSF1) gene is normally expressed; the same applies to the following embodiments.

[0136] In this embodiment, the "HSF1+ / + ” refers to mice lacking half of the heat shock factor 1 (HSF1) gene; the same applies to the following embodiments.

[0137] Example 6. MG132 induces CD4 in vitro via HSF1 + Foxp3 + CD69 + Expression of Treg

[0138] Sorting of HSF1 + / + or HSF1 + / - Mouse CD4 + Initial T cells were pretreated with 10 μM MG132 to induce Treg. The group without MG132 pretreatment was used as a control. Treg was induced under Treg polarization conditions. At the same time, the HSF1 inhibitor KRIBB11 (KRIBB11 is an inhibitor of HSF1 and can block the induction of HSF1 downstream target proteins such as HSP27 and HSP70) was added to block the expression of HSF1. Three days later, CD4 + Foxp3 + CD69 + Treg expression; inhibiting initial CD4 + After HSF1 expression in T cells, MG132 could not promote CD4 + Foxp3 + CD69 + Treg expression (see Figure 6 ), indicating that HSF deficiency affects the induction of CD4 + Foxp3 + CD69 + Regarding the expression level of Treg cells, after the action of HSF1 inhibitor, MG132 could not induce the upregulation of CD69 expression in Treg cells, indicating that the upregulation of CD69 caused by MG132 was HSF1-dependent.

[0139] Example 7. MG132 promotes HSF1 binding to the CD69 promoter region

[0140] By analyzing the CD69 promoter region sequence, it was found that there are two classic HSE (Heat Shock response elements) sequences that can bind to HSF1 in front of its promoter, namely the GAAnnTTC structure, see Figure 7 A, further through chromatin immunoprecipitation technique

[0141] The CHIP (Chromatin immunoprecipitation) experiment verified the interaction between HSF1 and CD69, and sorted CD4 +T cells, with or without MG132 pretreatment, were cultured under Treg induction conditions for 24 hours. The cells were fixed with 1% formaldehyde at 37°C for 10 minutes and then sonicated. The sonicated DNA fragments were incubated with HSF1 antibodies at 4°C overnight, and the IgG antibody group was set as a negative control group. The antibody and chromatin complexes were then precipitated with A / G protein agarose microspheres and incubated at 65°C for 4 hours. The DNA was further purified and PCR was used to detect the expression of the CD69 gene in each group. A total of 5 pairs of primers were designed around the CD69 promoter region. Figure 7 B shows that the fourth pair of primers showed that after MG132, more HSF1 bound to CD69, which was twice that of the group without MG132 pretreatment. The primer amplification sequence contained the HSE sequence close to the promoter region, and its primer sequence was 5'-AAATGAGCAAGGGATGATGA-3, 3'-TCTTTCAAGTGTCAGGAGTT-5'. In addition, the first pair of primers sequence was 5'-AGCCAACTTATAGCAGAGG-3'; 3'-AAGCAGGAAGGTCCAGAT-5'; the second pair of primers sequence was 5'-AGAACCGCTAAGACACAAC-3'; 3'-GCCAAGAAGAACCTCTACAT-5'; the third pair of primers sequence was 5'-GCACATTAGGACCAGCAT-3'; 3'-AATAGAGCAGAGAATGGAGAG-5'; the fifth pair of primers sequence was 5'-GTGGTGCTCAATAACTTATCTG-3';

[0142] 3′-TGTCATCCTCCACTGTCAA-5′.

[0143] Example 8. MG132 Treg / MG132 CD69 + Treg inhibits the occurrence and development of IBD in mice

[0144] Since MG132 can promote CD4 + Foxp3 + CD69 + We further compared MG132Treg / CD4 + Foxp3 + CD69 + Treg and Control Treg / CD4 + Foxp3 + CD69 + The therapeutic effect of Treg on IBD, magnetic bead separation kit to separate CD4 + CD62L + T cells were pretreated with 10 μM MG132 to induce Treg. The group without MG132 pretreatment was set as the treatment control group, and the PBS group was set as the blank control group. C57BL / 6 mice were fed 2% DSS to establish an IBD model. Two days after modeling, MG132-induced Treg (5×10 5 / mouse) and Treg induced by traditional methods (5×10 5 / mouse), see Figure 8 A. Observe and record the changes in the weight of mice, detect changes in pathological indicators, and detect changes in intestinal length in each group. The weight of mice began to decrease on the 5th day after drinking 2% DSS, and dropped to about 65% of the original weight on the 9th day. After adoptive transfusion of MG132 Treg, the weight loss of IBD mice was significantly alleviated. On the 9th day after treatment, their weight was maintained at 95% of the original weight. In contrast, the weight loss of the Control Treg group was about 20%. ( Figure 8 BC).MG132CD69 + Treg and Control CD69 + The body weight of the Treg group was basically maintained at a normal level. Consistent with this, after MG132 Treg adoptive infusion therapy, the shortening of intestinal length was less than that of the Control Treg group ( Figure 8 D), and the pathological section results showed that the degree of intestinal inflammation in the MG132 Treg group was alleviated compared with the PBS group and the Control Treg group.

[0145] MG132 CD69 + Treg and Control CD69 + There was no significant difference between the Treg group and the normal control group ( Figure 8 E). The results showed that the intestinal length did not shorten significantly. In the experimental group of inflammatory bowel disease animal model mice given MG132 Treg by intravenous administration, the number of lymphocytes in the intestinal lamina propria decreased and the arrangement of intestinal mucosal glands was intact.

[0146] The histological scoring criteria for mouse colitis are mainly based on the degree of inflammation, lesion depth, crypt destruction, regeneration index and lesion range (%), specifically: 0 points, no inflammation; 1 point, mild inflammation, 2 points, moderate inflammation, 3 points, severe inflammation. 0 points, no lesion depth; 1 point, involving the mucosal layer; 2 points, involving the mucosal layer and submucosal layer; 3 points, full-thickness inflammation. 0 points, no crypt destruction; 1 point, basal 1 / 3 crypts are destroyed; 2 points, basal 2 / 3 crypts are destroyed; 3 points, only the surface epithelium is intact; 4 points, all crypts and epithelium are destroyed. 0 points, complete regeneration or normal tissue; 1 point, almost complete regeneration; 2 points, regeneration with crypt defects; 3 points, incomplete surface epithelium; 4 points, no repair. 1 point, lesion range 1-25%; 2 points, lesion range 26-50%; 3 points, lesion range 51-75%; 4 points, lesion range 76-100%.

[0147] Example 9. MG132 Treg / MG132 CD69 + Treg inhibits the expression of Th1 and Th17 in mice

[0148] Inflammatory Th1 (CD4 + IFN-γ + ) and Th17 (CD4 + IL-17 + ) plays a vital role in the development of IBD in mice. In order to clarify the + The therapeutic mechanism and effect of Treg on IBD were analyzed. The expression of Th1 and Th17 in the spleen, mesenteric lymph nodes (MLN) and intestinal lamina propria lymph nodes (LPL) of each group of mice was analyzed. The spleen and lymph nodes of normal mice only expressed a small amount of Th1 and Th17, of which Th1 accounted for about 1% of the total CD4 + Th17 cells accounted for approximately 1.4% and 2.8% of the total CD4 T cells in the spleen and mesenteric lymph nodes, respectively. + The percentages of Th1 and Th17 in the intestinal lamina propria increased to about 20% and 15%, respectively. After inducing IBD, the expression of Th1 in the spleen, mesenteric lymph nodes and intestinal lamina propria lymph nodes increased by 3-4 times, and Th17 increased by 2-3 times. After treatment with MG132 Treg and Control Treg, Th1 and Th17 decreased to varying degrees. The MG132 Treg treatment group had the best therapeutic effect. + Treg and Control CD69 + The downregulation of Th1 and Th17 was most obvious in the Treg treatment group. Fig. 9 .

[0149] In summary, it was found that CD4 + Foxp3 + CD69 + Treg has stronger immunosuppressive function. The proteasome inhibitor MG132 can promote the expression of Treg cell membrane protein CD69. Compared with Control Treg, MG132 Treg can secrete higher levels of IL-10 and TGF-β1, and has stronger immunosuppressive function. Infusing MG132 Treg into IBD mice can effectively inhibit local inflammation. In addition, given that MG132 can activate HSF1, HSF1 deficiency leads to induced CD4 + Foxp3 + CD69 + Treg expression is reduced, and MG132 may regulate CD69 expression through HSF1, thereby giving Treg a stronger immunosuppressive function, making it likely to become an effective cell vaccine for the clinical treatment of IBD, and thus it is expected to become a universal preparation for the treatment of autoimmune diseases.

[0150] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An in vitro induction of CD4 + Foxp3 + CD69 + The method for expanding Treg cells is characterized by: The expansion method described herein uses proteasome inhibitor pretreatment of naive CD4 + T cells were then cultured under conventional Treg induction conditions to obtain cells containing at least 42% CD4 + Foxp3 + CD69 + Treg cells, the proteasome inhibitor is selected from at least one of MG132 (Z-Leu-Leu-Leu-CHO), bortezomib, and carfilzomib, the concentration of MG132 (Z-Leu-Leu-Leu-CHO) of the proteasome inhibitor is 0.1-20 μM, the concentration of bortezomib is 0.01-0.5 μM, the concentration of carfilzomib is 0.001-0.01 μM, the pretreatment time of the proteasome inhibitor is 1 h-2.5 h, and the pretreatment temperature of the proteasome inhibitor is 37° C.

2. The amplification method according to claim 1, characterized in that The amplification method described herein obtains cells containing at least 50% CD4 + Foxp3 + CD69 + Treg cells.

3. The amplification method according to claim 1, characterized in that The amplification method described herein obtains cells containing at least 55% CD4 + Foxp3 + CD69 + Treg cells.

4. The amplification method according to claim 1, characterized in that: The initial CD4 + The T cells are derived from at least one of the spleen, mesenteric lymph nodes, inguinal lymph nodes and other lymphoid tissues.

5. The amplification method according to claim 1, characterized in that: The initial CD4 + The T cell sorting method includes at least one of magnetic bead sorting or flow cytometry sorting.

6. The amplification method according to claim 1, characterized in that: The proteasome inhibitor is MG132 (Z-Leu-Leu-Leu-CHO).

7. The amplification method according to claim 1, characterized in that: Tregs obtained after MG132 pretreatment contained at least 55% CD4 + Foxp3 + CD69 + Treg.

8. The amplification method according to claim 1, characterized in that: At least 65% of CD4 + Foxp3 + CD69 + Treg.

9. The amplification method according to claim 1, characterized in that: The proteasome inhibitor promotes CD69 expression not limited to CD4 + T cells.

10. The amplification method according to any one of claims 1 to 9, characterized in that: The culture medium used in the conventional Treg induction condition contains at least one of cytokines or activating antibodies.

11. The amplification method according to claim 10, characterized in that: The Treg induction time is 72-96h.

12. The amplification method according to claim 10, characterized in that: The cytokine is TGF-β1, with a concentration of 5-20 ng / mL.

13. The amplification method according to claim 10, characterized in that: The concentration of cytokine TGF-β1 was 10 ng / mL.

14. The amplification method according to claim 10, characterized in that: The cytokine is IL-2, with a concentration of 50-100 IU.

15. The amplification method according to claim 10, characterized in that: The concentration of cytokine IL-2 was 50 IU.

16. The amplification method according to claim 10, characterized in that: The concentration of the activation antibody anti-CD3 / CD28 is 0.5-2 μg / mL.

17. The amplification method according to claim 10, characterized in that: The concentration of the activating antibody anti-CD3 / CD28 was 2 μg / mL.

18. A Treg cell with enhanced in vitro immunosuppressive activity, characterized in that: It contains at least 42% of the in vitro induced CD4 + Foxp3 + CD69 + CD4 Treg cell expansion method + Foxp3 + CD69 + Treg cells.

19. The Treg cell with enhanced in vitro immunosuppressive activity according to claim 18, characterized in that: The CD4 + Foxp3 + CD69 + The Treg cells are obtained by the expansion method according to any one of claims 1 to 13 or claims 15 to 17.

20. The Treg cell with enhanced in vitro immunosuppressive activity according to claim 18, characterized in that: The CD4 + Foxp3 + CD69 + Treg cells are obtained by the expansion method according to claim 14.

21. The Treg cell with enhanced in vitro immunosuppressive activity according to claim 18, characterized in that: Tregs obtained after MG132 pretreatment contained at least 65% CD4 + Foxp3 + CD69 + Treg.

22. The Treg cell with enhanced in vitro immunosuppressive activity according to claim 18, characterized in that: At least 67% of CD4 + Foxp3 + CD69 + Treg.

23. The Treg cell with enhanced in vitro immunosuppressive activity according to claim 18, characterized in that: The Tregs secrete IL-10 and TGF-β1 at levels higher than those induced by traditional methods.

24. The Treg cell with enhanced in vitro immunosuppressive activity according to claim 18, characterized in that: The immunosuppression-related molecules GITR and ICOS were higher than Treg induced by traditional methods.

25. A method of using the in vitro induced CD4 + Foxp3 + CD69 + The in vitro MG132-Treg cells obtained by the Treg cell expansion method are characterized in that: The MG132-Treg cells are MG132 activated HSF1 and promoted its binding to the CD69 promoter region, specifically promoting the binding of HSF1 and HSE sequence to interact, thereby promoting the initial CD4 + T cell-induced CD4 + Foxp3 + CD69 + The results were obtained by upregulating Treg expression.

26. The in vitro MG132-Treg cell according to claim 25, characterized in that The HSE sequence is a GAAnnTTC structure.

27. The in vitro MG132-Treg cell according to claim 25, characterized in that The in vitro MG132-Treg cells are MG132 pretreated primary CD4 + T cells induced by conventional methods contain high levels of CD69 + Treg cells.

28. An in vitro MG132-Treg cell, characterized in that The MG132-Treg cells are derived from the initial CD4 + T cells are obtained by first pre-treating the initial CD4+T cells with MG132 and then culturing them under traditional Treg induction method.

29. The in vitro MG132-Treg cell according to claim 28, characterized in that MG132 promotes CD69 expression through HSF1 activation, promoting the initial CD4 + T cell-induced CD4 + Foxp3 + CD69 + Treg expression is upregulated.

30. The in vitro MG132-Treg cell according to claim 28, characterized in that The concentration of MG132-Treg cells was 2.5×10 5 -1×10 7 / mL.

31. The in vitro MG132-Treg cell according to claim 28, characterized in that The in vitro MG132-Treg cells contain at least 60% CD69 + Treg cells.

32. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a safe and effective amount of the Treg described in any one of claims 18 to 24, the MG132-Treg described in any one of claims 25 to 31, and the CD4 obtained by the amplification method described in any one of claims 1 to 9. + Foxp3 + CD69 + At least one of the Tregs is an active ingredient.

33. The pharmaceutical composition according to claim 32, characterized in that The pharmaceutical composition further comprises an acceptable carrier.

34. The pharmaceutical composition according to claim 32, characterized in that The pharmaceutical composition also contains an immunosuppressive adjuvant.

35. The pharmaceutical composition according to claim 34, characterized in that The immunosuppressive adjuvant is selected from at least one of PBS and physiological saline.

36. The pharmaceutical composition according to claim 32, characterized in that The Treg cells or MG132-Treg cells are autologous or allogeneic.

37. The pharmaceutical composition according to claim 32, characterized in that The pharmaceutically effective amount of MG132-Treg or its salt is 2.5×10 7 -5×10 8 / d / kg.

38. The pharmaceutical composition according to claim 32, characterized in that The pharmaceutically effective amount of MG132-Treg or its salt is 2.5×10 8 / d / kg.

39. The pharmaceutical composition according to claim 32, characterized in that The pharmaceutical composition is administered by at least one of intravenous and intraperitoneal administration.

40. The pharmaceutical composition according to claim 32, characterized in that The pharmaceutical composition is applied by intravenous injection.

41. The pharmaceutical composition according to claim 32, characterized in that The therapeutic dosage regimen is selected from at least one of a single-dose regimen or a multiple-dose regimen.

42. The pharmaceutical composition according to claim 32, characterized in that The pharmaceutical composition may be prepared in the form of at least one of an injectable or solid form suitable for solution or suspension in liquid prior to injection, and a liquid carrier.

43. The pharmaceutical composition according to claim 42, characterized in that The carrier refers to a carrier used for administration of a therapeutic agent or a pharmaceutical carrier in a therapeutic pharmaceutical composition.

44. The pharmaceutical composition according to claim 33, characterized in that The carrier refers to some pharmaceutical carrier that does not induce the production of antibodies harmful to the individual receiving the composition and does not cause excessive toxicity after administration.

45. The pharmaceutical composition according to claim 33, characterized in that The acceptable carrier is a liquid.

46. ​​The pharmaceutical composition according to claim 45, characterized in that The carrier is selected from at least one of PBS or physiological saline.

47. The pharmaceutical composition according to claim 32, characterized in that The pharmaceutical composition can be directly administered to a subject, and the subject to be prevented or treated is a non-human animal.

48. The pharmaceutical composition according to claim 47, characterized in that The non-human animal comprises at least one of a rat or a mouse.

49. The CD4 obtained by the amplification method according to any one of claims 1 to 9. + Foxp3 + CD69 + Use of at least one of Treg cells, MG132-Treg cells according to any one of claims 18 to 31, and the pharmaceutical composition according to any one of claims 32 to 48 in the preparation of an agent for treating an autoimmune disease.

50. Use of at least one of the pharmaceutical compositions of claim 49 in the preparation of an agent for treating an autoimmune disease, characterized in that: The autoimmune disease refers to a disease caused by the body's immune response to its own antigens.

51. Use of at least one of the pharmaceutical compositions of claim 50 in the preparation of an agent for treating an autoimmune disease, characterized in that: The autoimmune disease is selected from inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, diabetes, and systemic lupus erythematosus.

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