WO2022022599A1

WO2022022599A1 - Method for amplifying in-vitro induced cd4 +foxp3 +cd69 +treg and use thereof

Info

Publication number: WO2022022599A1
Application number: PCT/CN2021/109085
Authority: WO
Inventors: 金洪传; 余磊; 王娴; 冯利锋
Original assignee: 浙江大学
Priority date: 2020-07-28
Filing date: 2021-07-28
Publication date: 2022-02-03

Abstract

An in-vitro induced CD4+Foxp3+CD69+Treg cell and a preparation method therefor, and a related pharmaceutical composition and use. Naive CD4+T cells are pretreated by using 10 μM of proteasome inhibitor MG132 (Z-Leu-Leu-Leu-CHO), so as to obtain at least 65% of CD4 ⁺Foxp3 ⁺CD69 ⁺Treg. Compared with a control group, same is higher by 91.4 percentage points. Induced MG132 Treg cells after pretreatment have a stronger immunosuppressive activity, and induce the reestablishment of immune tolerance in the body, thereby preventing and/or treating autoimmune diseases, in particular inhibiting the development and progression of inflammatory bowel disease.

Description

an in vitro induced CD4 +Foxp3 +CD69 Amplification method of +Treg and its use

technical field

The invention relates to the technical field of biomedicine, in particular to a CD69 positive regulatory T cell with enhanced immunosuppressive activity, a preparation method, and a related pharmaceutical composition and application thereof.

Background technique

Regulatory T cells (Treg) are a group of T cells with immunosuppressive function and play an important role in maintaining immune balance in the body. OX40, FR4, CTLA-4 and GITR. Although Treg has strong immune regulation ability, in the inflammatory microenvironment, Treg is easily transformed into Th17 and other cells, resulting in greatly impaired immunosuppressive function; and inflammatory cytokines can promote the down-regulation of Foxp3 expression in Treg, which in turn leads to Treg Transformation to inflammatory cells. CD4 ⁺ CD69 ⁺ Foxp3 ⁺ Treg cells are regulatory T cells capable of expressing CD69-positive, however, CD4 ⁺ CD69 ⁺ Foxp3 ⁺ Treg are capable of secreting high levels of IL-10 and TGF-β1 and are more potent under inflammatory conditions It has attracted widespread attention because of its stability and inability to differentiate into inflammatory cells. It has become a new therapy for autoimmune diseases by suppressing abnormally activated immune cells by infusing inhibitory immune cells.

CD69 is an early leukocyte activation molecule and an immunomodulatory molecule. CD69 downregulates the immune response by promoting the production of TGF-β1. It has been reported that Treg derived from CD69 ^-/- mice has a reduced ability to inhibit T cell proliferation in vitro. In vivo experiments found that OVA-induced CD69 ^-/- mice had more asthma symptoms Severe (Cortés, José R., et al. J Autoimmun. 55, 51-62, (2014).); CD69 deficiency has also been shown to lead to exacerbation in animal models of collagen-induced arthritis (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 regulation imbalance. The pathogenesis of IBD involves multiple processes at the cellular level. Due to the complex pathogenesis, the curative effect of current clinical treatment drugs is often poor, the disease is easy to repeat, and there are adverse drug side effects. In the literature, CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg and CD4 ⁺ Foxp3 ⁺ CD69 ^- Treg were adoptively infused into IBD mice, and it was found that a small amount of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg could significantly inhibit the development of IBD, while CD4 ⁺ Foxp3 ⁺ CD69 ^- Treg has poor disease remission ability. The results show 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).).

Treg is derived from the body, has not been chemically modified, is easily accepted by the body, is not easy to cause toxicological effects caused by rejection reactions, and has high safety. Difficulty in expansion, resulting in low cell yield is also the main bottleneck of Treg cell immunotherapy. In clinical practice, most of the existing cellular immunotherapy technologies use the method of direct blood drawing. If the final therapeutic dose is required, the amount of blood drawn each time is required to be more than 200 mL. Therapeutic doses of cells cannot be used for subsequent treatment; and multiple blood draws can have adverse effects on the patient's body. Therefore, the production of Treg subsets with better stability and efficiency in induced culture 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

Aiming at the deficiencies 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 expansion in vitro, related pharmaceutical compositions and applications; and also to provide a CD4 + immunosuppressive activity enhanced CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg. The invention overcomes the problem of low expression ratio of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg in the traditional Treg culture method. After pretreatment with proteasome inhibitor, at least 42% of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg cells can be obtained in the present invention, which is at least 20 percentage points higher than that of the control group; further, after 10 μM MG132 treatment, up to 67% of CD4 + Foxp3 + CD69 + Treg cells can be obtained in vitro Compared with the control group, the percentage of CD4+Foxp3+CD69+Treg cells increased by 91.4 percentage points. The excellent features of low cost and high efficiency accelerated the clinical application and development of CD4+Foxp3+CD69+Treg cells in autoimmune inflammatory diseases.

Preferably, the MG132 Treg cells with immunosuppressive function and high stability induced in vitro by the present invention contain high levels of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg, are easy to use and/or have higher efficiency, and are suitable for clinical application of inducible Treg (iTreg) cells. ) cell therapy for autoimmune inflammatory diseases to lay the experimental foundation. 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 a novel clinically administered containing more CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg induction method and related preparations.

The first object of the present invention is to provide a method for inducing CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg expansion in vitro, the expansion method uses proteasome inhibitors to pretreat naive CD4 ⁺ T cells, and then induce Treg in a traditional way Culturing under conditions to obtain at least 42% CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg cells; preferably, to obtain at least 50% CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg cells; more preferably, to obtain at least 55% CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg cells (see Table 1)

The percentage is the percentage of CD4 ^{+ Foxp3 + CD69 + Treg cells induced by flow cytometry as the percentage of CD4 +} ^{Foxp3 +} ^{CD69 +} ^Treg ^cells ^.

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

Preferably, the initial CD4 ⁺ T cells need to be obtained by magnetic beads or flow sorting;

Further, the Treg cells (MG132 Treg) induced by the initial CD4 ⁺ T cells pretreated with MG132, the ability of the MG132 Treg cells to inhibit the proliferation of CD4 ⁺ T cells is enhanced

Preferably, the pretreatment time of the proteasome inhibitor is 1h-2.5h,

Preferably, the temperature at which the proteasome inhibitor and the initial CD4 ⁺ T cells are co-incubated is 37°C;

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

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.

Preferably, Tregs obtained by pretreatment with MG132 comprise at least 55% CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Tregs; further, preferably, at least 65% CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Tregs are obtained by pretreatment with MG132 at a concentration of 10 μM;

Preferably, the proteasome inhibitor promoting CD69 expression is not limited to CD4 ⁺ T cells;

Preferably, the traditional method is as follows: take lymphoid tissue, grind and filter it to form a single-cell suspension, centrifuge and discard the supernatant, add the precipitation to a cell sorting solution for counting, and resuspend it in T lymphocytes after sorting. In the medium, cytokines and activating antibodies were added at the same time, and the expression of Treg was detected after culturing and inducing for a certain period of time.

Preferably, the lymphoid tissue is derived from at least one of spleen, mesenteric lymph nodes, inguinal lymph nodes and other lymphoid tissues.

Preferably, the medium used under the Treg induction condition in the traditional manner comprises at least one of cytokines or activating antibodies;

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

Further, preferably, the cytokine is IL-2 at a concentration of 50-100 IU; more preferably, the cytokine IL-2 is at a concentration of 50 IU.

Further, preferably, the activated antibody anti-CD3/CD28 concentration is 0.5-2 μg/mL; more preferably, the activated antibody anti-CD3/CD28 concentration is 2 μg/mL.

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

The second object of the present invention is to provide a Treg cell with enhanced immunosuppressive activity in vitro. The enhanced immune activity refers to an increase in the proportion of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg treated with a proteasome inhibitor; preferably, the Treg cells with enhanced immunosuppressive activity in vitro comprise at least 42% of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg cells; preferably, the CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg cells are obtained by any one of the aforementioned expansion methods; preferably, the Treg obtained by pretreatment with MG132 contains at least 55% CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg; further, preferably, at least 67% of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg are pretreated with MG132 at a concentration of 10 μM;

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

The third object of the present invention is to provide a Treg with high expression of CD69 molecule. The high expression of CD69 molecule means that Treg contains at least 42% CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg cells; preferably, carfilzomib treatment can obtain at least 42% or more CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg cells; More preferably, 55% of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg cells can be obtained when treated at a concentration of 0.001 μM.

Preferably, Bortezomib treatment can obtain at least 50% CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg cells; more preferably, 55% CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg cells can be obtained when the concentration is 0.1 μM.

Preferably, MG132 (Z-Leu-Leu-Leu-CHO) treatment can obtain at least 55% CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg cells; more preferably, 67% CD4 ⁺ Foxp3 ⁺ CD69 can be obtained when the concentration is 10 μM ⁺ Treg cells.

The fourth object of the present invention is to provide an in vitro MG132 Treg cell, the MG132 Treg cell is the interaction of MG132 by activating HSF1 and promoting its more binding to the CD69 promoter region, specifically promoting the binding of HSF1 to the HSE sequence , and then promote the up-regulation of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg expression induced by naive CD4 + T cells;

Preferably, the HSE sequence is a GAAnnTTC structure.

Preferably, the above is obtained by comparing the experimental group with the control group (control group).

Preferably, the expression up-regulation is obtained by comparing the experimental group with the control group (control group).

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

The fifth object of the present invention is to provide an in vitro MG132 Treg cell, the in vitro MG132 Treg cell has the following characteristics:

(a) The Treg with immunosuppressive function is that the initial CD4 ⁺ T cells are first pretreated with proteasome inhibitors, and then cultured under the traditional Treg induction method. Express a higher proportion of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg;

(b) MG132 Treg has stronger immunosuppressive activity, which is manifested in that MG132 Treg secretes higher levels of IL-10 and TGF-β1, MG132 Treg immunosuppression-related molecules GITR and ICOS are higher than Control Treg, and MG132 Treg inhibits CD4 ⁺ T The ability of cell proliferation is better than Control Treg;

(c) MG132 Treg can inhibit the occurrence and development of inflammatory bowel disease, and its relieving ability on 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 of the body, thereby preventing and/or treating autoimmune diseases.

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

Preferably, the MG132 can be activated to promote the up-regulation of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg expression induced by naive CD4 ⁺ T cells.

The sixth object of the present invention is to provide an in vitro expansion medium for Treg cells that promotes the expression of Treg cell membrane protein CD69 in vitro, the Treg cell in vitro expansion medium formulation includes Hepes, sodium pyruvate, β-mercaptoethanol and antibiotics at least one or more of them.

Preferably, the main body of the Treg cell in vitro expansion medium is a special medium for T cells containing 10% serum at 1640 RPMI.

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.

Preferably, the antibody is selected from at least one of CD3 or CD28; further, preferably, the antibody is added in an amount of 0.5-10 μg/mL,

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

The seventh object of the present invention is to provide a pharmaceutical composition comprising at least a safe and effective amount of the above-mentioned MG132 Treg or an effective amount of MG132 CD69 ⁺ Treg cells as an active ingredient and a pharmaceutically acceptable carrier A sort of;

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

Preferably, the "effective amount" refers to the amount of the therapeutic agent to treat, alleviate or prevent the target disease or condition, or to exhibit a detectable therapeutic or prophylactic effect; preferably, the precise effective amount for a subject depends on The size and health of the subject, the nature and extent of the symptoms, and the choice of therapeutic agent and/or combination of therapeutic agents to administer.

Preferably, the pharmaceutical composition further contains an immunosuppressive adjuvant, more preferably, the immunosuppressive adjuvant is PBS.

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

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 preparation into a solution or suspension, a liquid carrier before injection.

Preferably, the pharmaceutical composition is administered by at least one of intravenous and intraperitoneal methods.

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 or a multi-dose regimen.

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 rats or mice.

Preferably, the acceptable carrier refers to a carrier used for the administration of a therapeutic agent or a pharmaceutical carrier in a therapeutic pharmaceutical composition; preferably, it refers to a carrier that does not itself induce the production of antibodies that are detrimental to the individual receiving the composition, And some pharmaceutical carriers are not excessively toxic after administration.

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

Preferably, the acceptable carrier may also have auxiliary substances such as pH buffer substances and the like.

For the pharmaceutical composition of the present invention, in addition to using MG132 as an active ingredient, it can also be prepared by using a pharmaceutically suitable and physiologically acceptable adjuvant, and the adjuvant can be dissolved in dimethyl sulfoxide DMSO, Its chemical formula is C ₂ H ₆ OS.

The eighth object of the present invention is to provide CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg cells obtained by any one of the aforementioned expansion methods, MG132 Treg cells of any one of the aforementioned forms, and the aforementioned At least one use of a pharmaceutical composition in any one form in the field of autoimmune disease treatment.

Preferably, the autoimmune disease refers to a disease caused by the body's immune response to self-antigens; further, the disease is related to the following factors:

(a) loss of immune tolerance and cryptic antigen exposure;

(b) Trauma and infection trigger an autoimmune response;

(c) Genetic factors.

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

Preferably, in an embodiment of the present invention, the concentration of MG132 Treg cells used for adoptive infusion to treat IBD in mice may be 2.5×10 ⁵ -1×10 ⁷ /mL.

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.

In particular, the treatment in one embodiment of the present invention refers to the action of treatment based on the above-mentioned definition. In mammals, "treatment" or "method of treatment" for inflammatory bowel disease and diseases associated therewith may include more than one of the following.

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

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

(3) Alleviate inflammatory bowel disease and related diseases;

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

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

beneficial effect

By optimizing and improving the existing Treg inducible expression system, the present invention establishes a more efficient inducible expression technology, saves the experimental cost and time, and shortens the experimental period. Preferably, compared with traditional Treg induction methods, MG132 pretreatment of naive CD4 ⁺ T cells and then cultured under Treg induction conditions will promote more CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg differentiation and secrete 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 small rejection reaction and good immunosuppressive effect; Only one injection is required for several weeks (eg 1-2 weeks), which reduces the pain of injection treatment and facilitates the recovery of the patient. By promoting the expression of more CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg, MG132 Treg can prevent or treat inflammatory bowel disease and related autoimmune diseases; it has no toxicity and side effects of drugs, and the effect is stable and can be taken for a long time. . In the present invention, the term "Treg" refers to CD4 ⁺ Foxp3 ⁺ regulatory T cells;

In the present invention, the term "CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg" refers to a subset of Treg cells expressing CD69;

In the present invention, the term "CD4 ⁺ Foxp3 ⁺ CD69 ^- Treg" refers to a subset of Treg cells that do not express CD69;

In the present invention, the term "CD69 ^-/- " refers to a mouse knockout of CD69; in the present invention, the term "MG132 Treg" refers to naive CD4 ⁺ T cells pretreated with MG132 and cultured under traditional Treg induction conditions Tregs;

In the present invention, the term "MG132 CD69 ⁺ Treg" refers to Treg expressing CD69 from MG132 Treg by flow sorting, and all MG132 CD69 ⁺ Treg are CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg cells;

In the present invention, the term "Control Treg" refers to traditionally induced Treg;

In the present invention, the term "iTreg" refers to Treg (induced regulatory T cell) induced in vitro;

In the present invention, the term "Control CD69 ⁺ Treg" refers to the CD69 ⁺ Treg sorted out from Control Treg by flow;

In the present invention, the term "

CD4 ⁺ T cell" refers to CD4 ⁺ CD62L ⁺ naive T cells;

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

In the present invention, the term "TGF-β1" refers to transforming growth factor-β1, transforming growth factor 1;

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

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

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

In the present invention, the term "flow sorting" refers to the use of a flow cytometry sorter to perform multi-parameter and quantitative analysis on single cells or biological particles in flow, while sorting specific populations and sorting purity. up to 99%.

In the present invention, the term "ELISA" refers to an enzyme-linked immunosorbent assay, a technique in which a known antigen or antibody is adsorbed on the surface of a solid phase carrier, and the enzyme-labeled antigen or antibody is reacted on the solid phase surface for quantitative technology.

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

In the present invention, the flow antibody used was purchased from ebioscience;

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

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

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

In the present invention, the primers are synthesized by Shanghai Jierui Biological Company;

In the present invention, the CHIP kit was purchased from CST. In the present invention, the MG132 may be a compound represented by the following Chemical Formula 1. In particular, the commercially available MG132 compound can be used as the MG132 compound, and the MG132 compound that is naturally isolated or produced by synthetic methods known in the art cannot be used.

Description of drawings

Figure 1. Analysis of the expression of IL-10 and TGF-β1 detected by ELISA.

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

Figure 3. Figure 3A Schematic diagram of the expression of immunosuppression-related molecules in MG132 Treg, Control Treg, MG132 CD69 ⁺ Treg and Control CD69 ⁺ Treg by flow cytometry in Figure 3A; Figure 3B Flow cytometry analysis of MG132 Treg, Control Treg, Control CD69 ⁺ Treg, and MG132 Schematic diagram of CD69 ⁺ Treg inhibiting the proliferation of CFSE-labeled CD4 ⁺ T.

Figure 4. Schematic diagram of the expression of HSF1 and HSF1 activation-related genes after MG132 pretreatment of CD4+ T cells; Figure 4B Schematic diagram of the transfer of HSF1 from cell membrane to nucleus after MG132 pretreatment of CD4+ T cells by fluorescence confocal detection.

Figure 5. Flow cytometry analysis of the expression of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg in spleen, mesenteric lymph nodes and intestinal lamina propria lymph nodes after in vivo injection of MG132 in HSF1 ^+/+ and HSF1 ^+/- mice.

Figure 6. Flow analysis of induction of CD4 ⁺ Foxp3 ⁺ CD69 after pretreatment of CD4+ naive T cells from HSF1+/+ or HSF1+/- mice with MG132 and blockade of HSF1 expression in CD4+ naive T cells from HSF1+/+ mice with KRIBB11 ⁺ Schematic diagram of Treg expression.

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

Figure 8. Adoptive transfusion of MG132 Treg, Control Treg, MG132 CD69+Treg, and Control CD69+Treg to IBD mice, the comparison of detection, evaluation and treatment effects.

Figure 9. Comparison of Th1 and Th17 downregulation in MG132 Treg, Control Treg, MG132 CD69+Treg, Control CD69+Treg adoptive infusion therapy group.

In particular, for the sake of clarity and appropriate proportions in the drawings, the annotated CD69 ⁺ Treg in the drawings refers to CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg.

detailed description

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

Example 1 Proteasome inhibitors promote the differentiation of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg

At present, the expression ratio of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg in traditionally induced Treg is only about 35%. In Example 1, the effect of proteasome inhibitors on CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg production was examined, Treg cultured in a conventional manner after pretreatment of mouse naive T cells with MG132, bortezomib, carfilzomib Induction under the same conditions effectively promoted the generation of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg as an anti-inflammatory (Table 1). CD4 ⁺ CD62L

For T cells, 1μM, 10μM of MG132, 0.01μM, 0.1μM Bortezomib, 0.001μM, 0.01μM Carfizomib were added after cell counting, and then placed in a 37°C cell incubator for 90 minutes of pretreatment. The DMSO group was used as a negative control group. The method is the same as above, then washed three times in serum-free medium 1640, resuspended in T lymphocyte medium at 2×10 ⁶ /mL, and added cytokine TGF-β1, cytokine IL-2, anti-CD3/CD28 antibody at the same time . Three days later, the cultured cells were collected, and the expression of Treg and CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg was detected by flow cytometry. Table 1 shows that each group of proteasome inhibitor pairs can successfully induce the differentiation of Treg, and the percentage is between 42% and 67%. Further comparison of the proportion of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg induced by different ways shows that proteasome inhibitor The proportion of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg after pretreatment with MG132 was 20%-32% higher than that in the control group, and the 10 μM MG132 treatment group was the best, which was 91.4% higher than that in the control group. Both 0.01μM ^and 0.1μM Bortezomib can induce high expression of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg ^, which is 51-57% higher than that of Control Treg in the control group ^. , the induction effect of 0.01μM Carfizomib was not obvious, only 7% higher than that of the control group.

The levels of TGF-β1 and IL-10 secreted by the above cultured cells were detected by ELISA kit, and it showed that 10 μM MG132 had the strongest ability to promote the secretion of IL-10 and TGF-β1 in Treg, and its IL-10 and TGF-β1 were the control group. 1.5-2 times, 0.01 μM, 0.1 μM Bortezomib and 0.001 μM Carfizomib groups also increased the expression level of IL-10 (Figure 1).

The T lymphocyte medium was used in the culture process, and the preparation method was as follows: adding β-mercaptoethanol with a final concentration of 5 μM, 1 mM sodium pyruvate, 10 mM Hepes to the 1640 medium containing 10% FBS, and adding three anti.

In this example, the ratio of the number of initial CD4 ⁺ T cells differentiated into CD4 ⁺ Foxp3 ⁺ cells induced by flow cytometry was taken as the percentage of Treg; the ratio of the number of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg cells induced by flow cytometry As CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg percentage.

Table 1 Effects of different concentrations of proteasome inhibitor treatment on the ratio of Treg and CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg

The gene expression difference of embodiment 2.MG132 Treg and Control Treg

In order to clarify the difference in gene expression between MG132 Treg and Control Treg, the MG132 Treg induced by 10 μM MG132 pretreatment in Example 1 and the control group were sorted by a flow sorter (instrument model: BD FACS S ORP ARIA II). Control Treg, gene sequencing was performed, and the results in Figure 2 showed that the expression levels of MG132 Treg immunosuppression-related genes IL-10, TGF-β1, SOCS2, HSP90AA1, GITR, Gzmb, CTLA-4 were significantly higher than those in Control Treg. The expressions of inflammation-related factors IL-17A and IL-17F were down-regulated compared with Control Treg.

Among them, the flow cytometry sorting buffer was: PBS, pH 7.2, BSA and 2mM EDTA with a final concentration of 0.5% were added, and the samples were filtered and packaged.

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

Flow cytometric sorting and collection buffer: RPIM 1640 medium, serum with a final concentration of 30%, and 4× double antibody.

Embodiment 3. MG132 Treg characteristic analysis

Collect cells from each group, MG132 Treg cells induced by 1×10 ⁶ pretreatment with 10 μM MG132 and Control Treg cells from the control group were resuspended in 100 μl PBS, and the corresponding doses of fluorescently labeled antibodies were added according to the instructions: CD4, Foxp3, CD69, CTLA- 4. ICOS, GITR, CD44, CXCR4, CCR7 and corresponding isotype control antibodies were incubated on ice for 30 min in the dark, then washed three times with PBS, and then Treg/CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg induced by MG132 were detected by flow cytometry and traditional methods The expression of immunosuppression-related molecules in induced Treg/CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg, Figure 3A shows that both groups of Treg highly expressed immunosuppression-related molecules, although MG132 Treg immunosuppression-related molecules CTLA-4, ICOS higher than Control Treg, Control CD69 ⁺ Treg and MG132 CD69 ⁺ Treg immunosuppression-related molecules and chemokine receptors CCR7 and CXCR4 were not significantly different. C57BL/6 mouse CD4 ⁺ T cells were further flow-sorted, labeled with CFSE at a final concentration of 10 mM, washed twice with serum-free medium 1640, and then added anti-CD3/CD28 antibody to 96 wells after adjusting the concentration. Then, the induced MG132 Treg, Control Treg, MG132 CD69 ⁺ Treg and Control CD69 ⁺ Treg were added to the plate, and the ratio of effector to target was 1:1, 2:1, 4; 1. Three duplicate wells were set in each group, and incubated at 37°C for 3 days. Fluorescence intensity was detected by FACS. Cell proliferation experiments showed that the proliferation rate of CD4 ⁺ T cells alone was about 90% after activation by anti-CD3/CD28 antibodies. When MG132 Treg and CD4 ⁺ T cells were co-cultured under 1:1 conditions, CD4 ⁺ T cells The proliferation rate was 37%, and the proliferation rate of CD4 ⁺ T cells was 44% when Control Treg and CD4 ⁺ T cells were co-cultured under 1:1 conditions. Under different effector-target ratio conditions, Control CD69 ⁺ Treg and MG132 CD69 ⁺ Treg inhibited The ability of CD4 ⁺ T cells was better than that of Control Treg and MG132 Treg, and importantly, the ability of MG132 Treg to inhibit the proliferation of CD4 ⁺ T cells was better than that of Control Treg (see Figure 3B). It is preliminarily suggested that MG132 Treg has stronger immunosuppressive function than Control Treg, and there is no significant difference in the immunosuppressive function of Control CD69 ⁺ Treg and MG132 CD69 ⁺ Treg.

Example 4 MG132 promotes activation of HSF1 and related genes

CD4 ⁺ T cells were pretreated with 10 μM MG132 for 90 min, washed three times with serum-free 1640 medium, and then cultured in an incubator. The cells were collected at 12 h and 24 h, respectively. No treatment group was set as a control. Real-time PCR detected the expression of HSF1 in CD4 ⁺ T cells and the expression of HSF1 activation-related genes (see Table 2 for the primers used). Figure A shows that MG132 can promote the expression of HSF1 and its related genes HSP90A, DNAJB1, and HSPA1A. The multiples were more than 10 times that of the control group, and MG132 could also promote the expression of CD69 (see Figure 4A). Compared with the control, pretreatment with MG132 can increase the expression level of CD69 gene by 4 times, and HSF1 is mainly distributed in the cell membrane in the resting state. state (see Figure 4B).

Table 2 Primers used in Real-time PCR

Example 5. MG132 promotes Treg CD69 expression in vivo dependent on HSF1

To further clarify the mechanism by which MG132 promotes the differentiation of CD69 ⁺ Treg, HSF1 ^+/+ and HSF1 ^+/- mice were injected with 15 μM/kg MG132 via the tail vein, and the spleen, inguinal lymph nodes, mesenteric lymph nodes, and intestinal lamina propria were isolated 7 days later. , detect the expression of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg, determine the effect of MG132 on Treg induction and differentiation in vivo and the expression of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg, and determine whether HSF1 deletion affects the increase of CD69. Figure 5 shows that in normal small There is a certain proportion of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg in mice, and there are a small amount in the spleen and lymph nodes, but the proportion is as high as 68% in the intestinal lamina propria. After in vivo injection of MG132, it can up-regulate HSF1 ^+/+ intestinal lamina propria lymph nodes The expression of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg was up-regulated by about 15%, but had no significant effect on CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg in HSF1 ^+/- mice, MG132 could promote the expression of CD69 in HSF1 ^{+ / +} Treg cells in vivo. Expression, however, MG132 was unable to promote CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg expression in HSF1 ^+/- mice, further suggesting that HSF1 is required for MG132 to promote CD69 expression.

In this example, the "HSF1 ^+/+ " refers to wild-type mice with normal expression of heat shock factor 1 (HSF1) gene; the same is true in the following examples.

In this example, the “HSF1 ^+/+ ” refers to mice with half of the heat shock factor 1 (HSF1) gene deleted; the same is true in the following examples.

Example 6. MG132 induces the expression of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg in vitro through HSF1

The CD4 ⁺ naive T cells of HSF1 ^+/+ or HSF1 ^+/- mice were sorted, and Treg was induced after pretreatment with 10 μM MG132. The pretreatment group without MG132 was used as a control, and Treg was induced under the condition of Treg polarization, while adding HSF1 to inhibit The agent KRIBB11 (KRIBB11 is an inhibitor of HSF1, can block the induction of HSF1 downstream target proteins such as HSP27 and HSP70.) Block the expression of HSF1, three days later, the expression of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg was analyzed by flow cytometry; the initial CD4 was inhibited After the expression of HSF1 in ⁺ T cells, MG132 did not promote the expression of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg (see Figure 6), indicating that HSF deletion will affect the induced expression level of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg. After HSF1 inhibitor, MG132 It also failed to induce the up-regulation of CD69 expression in Treg cells, indicating that the up-regulation of CD69 by MG132 was HSF1-dependent.

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

By analyzing the sequence of the CD69 promoter region, it was found that there are two classical HSE (Heat Shock response elements) sequences that can bind to HSF1 in front of its promoter, namely the GAAnnTTC structure, as shown in Figure 7A, and further through the chromatin immunoprecipitation technology. (Chromatin immunoprecipitation) experiments to verify the interaction between HSF1 and CD69, sorting CD4 ⁺ T cells, with or without MG132 pretreatment, cultured under Treg-inducing conditions for 24 hours, cells were fixed with 1% formaldehyde at 37 °C for 10 min After sonication, the sonicated DNA fragments were incubated with HSF1 antibody overnight at 4°C, and the IgG antibody group was set as the 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 the expression of CD69 gene in each group was detected by PCR. A total of 5 pairs of primers were designed around the CD69 promoter region. Figure 7B shows that after the fourth pair of primers showed the effect of MG132, more HSF1 bound to CD69, which was 2 times that of the group without MG132 pretreatment. The amplified sequence of this primer contained The HSE sequence near the promoter region, its primer sequences are 5'-AAATGAGCAAGGGATGATGA-3, 3'-TCTTTCAAGTGTCAGGAGTT-5'. In addition, the first pair of primer sequences are 5'-AGCCAACTTATAGCAGAGG-3';3'-AAGCAGGAAGGTCCAGAT-5'; the second pair of primer sequences are 5'-AGAACCGCTAAGACACAAC-3';3'-GCCAAGAAGAACCTCTACAT-5'; the third pair of primers The sequences were 5'-GCACATTAGGACCAGCAT-3';3'-AATAGAGCAGAGAATGGAGAG-5'; the sequences of the fifth pair of primers were 5'-GTGGTGCTCAATAACTTATCTG-3';3'-TGTCATCCTCCACTGTCAA-5'.

Example 8. MG132 Treg/MG132 CD69 ⁺ Treg inhibit the occurrence and development of IBD in mice

Since MG132 can promote the differentiation of CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg, we further compared the therapeutic effect of MG132 Treg/CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg and Control Treg/CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg on IBD, magnetic bead sorting kit sorting

T cells were pretreated with 10μM MG132 to induce Treg. At the same time, the pretreatment group without MG132 was set as the treatment control group, and the PBS group was the blank control group. C57BL/6 mice drank 2% DSS to establish an IBD model. Two days after modeling, on the second day, MG132-induced Treg (5×10 ⁵ /mouse) and traditional method-induced Treg ( 5×10 ⁵ /mice), see Figure 8A for details. Changes in body weight of mice were observed and recorded, and changes in pathological indexes were detected, and changes in intestinal length in each group were detected. The body weight of mice started to decrease on the 5th day after drinking 2% DSS, until it dropped to about 65% of the original body weight on the 9th day. After adoptive infusion of MG132 Treg, the weight loss of IBD mice was significantly relieved. On the 9th day after treatment, their body weight Maintained at 95% of the original body weight, in contrast, the Control Treg group showed a weight loss of approximately 20%, (Figure 8B-C). The body weights of MG132 CD69 ⁺ Treg and Control CD69 ⁺ Treg groups were basically maintained at normal levels. Consistent with this, after adoptive infusion of MG132 Treg, the shortening of intestinal length was less than that in the Control Treg group (Fig. 8D). At the same time, the pathological section results showed that the intestinal inflammation in the MG132 Treg group was higher than that in the PBS group and the Control Treg group. eased. The MG132 CD69 ⁺ Treg and Control CD69 ⁺ Treg groups were not significantly different from the normal control group (Fig. 8E). The results showed that the length of the intestinal tract was not significantly shortened. In the experimental group of inflammatory bowel disease animal model mice administered MG132 Treg intravenously, lymphocytes in the intestinal lamina propria were decreased, and the intestinal mucosal glands were completely arranged.

The histological scoring standard of colitis in mice is mainly defined according to 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 Severe inflammation, 3 points, severe inflammation. 0 points, no depth of lesions; 1 point, involving the mucosal layer; 2 points, involving the mucosal layer and submucosa; 3 points, pan-thickness inflammation. 0, no crypt destruction; 1, basal 1/3 crypts destroyed; 2, basal 2/3 crypts destroyed; 3, only the superficial epithelium intact; 4, all crypts and epithelium destroyed. 0 points, complete regeneration or stasis; 1 point, almost complete regeneration; 2 points, regeneration with crypt defect; 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%.

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

Inflammatory Th1 (CD4 ⁺ IFN-γ ⁺ ) and Th17 (CD4 ⁺ IL-17 ⁺ ) play a crucial role in the occurrence and development of IBD in mice ^. For the treatment mechanism and effect of IBD, the expression of Th1 and Th17 in the spleen (Spleen), mesenteric lymph node (MLN) and intestinal lamina propria lymph node (LPL) of each group of mice were analyzed. The spleen and lymph nodes of normal mice only expressed A small amount of Th1 and Th17, of which Th1 accounted for about 1.4% and 2.8 of the total CD4 ⁺ T cells in the spleen and mesenteric lymph nodes, respectively, and Th17 accounted for about 0.63% and 0.63% of the total CD4 ⁺ T cells in the spleen and mesenteric lymph nodes, respectively. About 1.17%, the proportion of Th1 and Th17 in the intestinal lamina propria increased, about 20% and 15%, respectively. After induction of IBD, the expression of Th1 in the spleen, mesenteric lymph nodes and intestinal lamina propria lymph nodes can be increased by 3-4 times. Th17 can be increased by 2-3 times. After MG132 Treg and Control Treg treatment, both Th1 and Th17 decreased to varying degrees. The MG132 Treg treatment group had the best therapeutic effect, while the MG132 CD69 ⁺ Treg and Control CD69 ⁺ Treg treatment groups The most obvious down-regulation of Th1 and Th17 is shown in Figure 9.

To sum up, it was found that CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg has stronger immunosuppressive function, and 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 have stronger immunosuppressive function; re-infusion of MG132 Treg to IBD mice can effectively inhibit local inflammation. In addition, given that MG132 can activate HSF1, and the loss of HSF1 leads to the reduction of induced CD4 ⁺ Foxp3 ⁺ CD69 ⁺ Treg expression, MG132 may regulate the expression of CD69 through HSF1, thereby endowing Treg with stronger immunosuppressive function, making it a promising candidate for the clinical treatment of IBD. An effective cellular vaccine, which is expected to become a universal preparation for the treatment of autoimmune diseases.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content 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 appended claims of the present application.

Claims

An expansion method for inducing CD4 + Foxp3 + CD69 + Treg cells in vitro, characterized in that the expansion method uses proteasome inhibitors to pretreat initial CD4 + T cells, and then culture them under traditional Treg induction conditions, Obtain CD4 + Foxp3 + CD69 + Treg cells comprising at least 42%; preferably, obtain CD4 + Foxp3 + CD69 + Treg cells comprising at least 50%; more preferably, obtain CD4 + Foxp3 + CD69 + Treg cells comprising at least 55% cell;

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

Preferably, the method for sorting naive CD4 + T cells includes at least one of magnetic beads or flow sorting.
The amplification method of claim 1, wherein the proteasome inhibitor is selected from the group consisting of MG132 (Z-Leu-Leu-Leu-CHO), Bortezomib, Carfilzomib At least one of ; further, preferably MG132 (Z-Leu-Leu-Leu-CHO);

Preferably, the proteasome inhibitor pretreatment time is 1h-2.5h;

Preferably, the temperature of the proteasome inhibitor pretreatment is 37°C;

Preferably, 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, and the concentration of Carfilzomib is 0.001-0.01μM;

Preferably, Tregs obtained by pretreatment with MG132 comprise at least 55% CD4 + Foxp3 + CD69 + Tregs; further, preferably, at least 65% CD4 + Foxp3 + CD69 + Tregs are obtained by pretreatment with MG132 at a concentration of 10 μM;

Preferably, the proteasome inhibitor promoting CD69 expression is not limited to CD4 + T cells.
The amplification method according to any one of claims 1 or 2, wherein the medium used under the Treg induction condition in the traditional manner comprises at least one of cytokines or activating antibodies;

Preferably, the Treg induction time is 72-96h;

Preferably, the cytokine is TGF-β1 at a concentration of 5-20ng/mL; more preferably, the cytokine TGF-β1 is at a concentration of 10ng/mL; further, preferably, the cytokine is IL-2 at a concentration of 50- 100IU; more preferably, the concentration of cytokine IL-2 is 50IU; further, preferably, the concentration of the activated antibody anti-CD3/CD28 is 0.5-2 μg/mL; more preferably, the concentration of the activated antibody anti-CD3/CD28 is 2 μg/mL.
A Treg cell with enhanced immunosuppressive activity in vitro , characterized in that it comprises at least 42% CD4 + Foxp3 + CD69 + Treg cells; The amplification method described in the above item is obtained; preferably, the Treg obtained by MG132 pretreatment contains at least 55% CD4 + Foxp3 + CD69 + Treg; further, preferably, at least 67% CD4 when pre-treated with MG132 at a concentration of 10 μM + Foxp3 + CD69 + Treg;

Preferably, the Treg secretes higher levels of IL-10 and TGF-β1 than those induced by traditional methods. Further, preferably, the immunosuppression-related molecules GITR and ICOS are higher than Tregs induced by traditional methods.
An in vitro MG132 Treg cell, characterized in that, the MG132 Treg cell is that MG132 activates HSF1 and promotes its more binding to the CD69 promoter region, specifically, promotes the interaction between HSF1 and HSE sequence, thereby promoting the initial CD4 + T cells-induced up-regulation of CD4 + Foxp3 + CD69 + Treg expression; preferably, the HSE sequence is GAAnnTTC structure; Cells containing high levels of CD69 + Treg were induced in a conventional manner.
An in vitro MG132 Treg cell, characterized in that the MG132 Treg cell is derived from an initial CD4 + T cell, and the initial CD4 + T cell is first pretreated with MG132, and then cultured under a traditional Treg induction method;

Preferably, the MG132 promotes the expression of CD69 through HSF1 activation, and promotes the up-regulation of CD4 + Foxp3 + CD69 + Treg expression induced by naive CD4 + T cells;

Preferably, the MG132 Treg can inhibit the occurrence and development of autoimmune diseases; preferably, the MG132 Treg cell concentration is 2.5×10 5 -1×10 7 /mL;

Preferably, the in vitro MG132 Treg cells are in vitro MG132 CD69 + Treg cells;

Further, preferably, the autoimmune disease is selected from at least one of inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, diabetes, and systemic lupus erythematosus.
A Treg cell in vitro expansion medium for promoting the expression of Treg cell membrane protein CD69, the Treg cell in vitro expansion medium formulation comprises at least one or more of Hepes, sodium pyruvate, β-mercaptoethanol, antibodies, and cytokines ;

Preferably, the main body of the Treg cell in vitro expansion medium is a 1640 RPMI special medium for T cells containing 10% serum;

The concentration of β-mercaptoethanol is 5 μM;

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

Preferably, the cytokine is selected from at least one of TGF-β1 or IL-2; further, preferably, the addition amount of the cytokine TGF-β1 is 5-20 ng/mL, and the addition amount of the cytokine IL-2 50-100IU.
A pharmaceutical composition, characterized in that the pharmaceutical composition comprises a safe and effective amount of the Treg described in claim 4, the MG132 Treg described in any one of claims 5 or 6, and any one described in claim 1-3. At least one of CD4 + Foxp3 + CD69 + Treg obtained by the amplification method is an active ingredient; preferably, the pharmaceutical composition further comprises an acceptable carrier;

Preferably, the pharmaceutical composition further contains an immunosuppressive adjuvant, more preferably, the immunosuppressive adjuvant is selected from at least one of PBS and physiological saline;

Preferably, the Treg cells or MG132 Treg cells are autologous or allogeneic; preferably, the pharmaceutically effective amount of MG132 Treg or its salt is 2.5×10 7 -5×10 8 /d/kg; further, preferably 2.5×10 8 /d/kg;

Preferably, the pharmaceutical composition is administered by at least one of intravenous and intraperitoneal methods; preferably, the pharmaceutical composition is administered by intravenous injection, more preferably, the therapeutic dosage regimen is selected from a single-dose regimen or multiple at least one of the dosage regimens; preferably, the pharmaceutical composition can be made into at least one of solid forms including injectables or solid forms suitable for preparation into solutions or suspensions and liquid carriers before injection; further, preferably , the carrier refers to a carrier used for the administration of a therapeutic agent or a pharmaceutical carrier in a therapeutic pharmaceutical composition; preferably, the desired carrier refers to an antibody that does not itself induce the production of an antibody detrimental to an individual receiving the composition, and Some pharmaceutical carriers are not excessively toxic after administration; further, preferably, the acceptable carrier is a liquid; more preferably, the carrier is selected from at least one of PBS or physiological saline;

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.
At least one of the CD4 + Foxp3 + CD69 + Treg cells obtained by the expansion method according to any one of claims 1-3, the MG132 Treg cells according to claim 5 or 6, and the pharmaceutical composition according to claim 8 is in itself. Use in the field of immune disease therapy.
The autoimmune disease according to claim 9, wherein the autoimmune disease refers to a disease caused by the body's immune response to self-antigens; preferably, the autoimmune disease is selected from inflammatory bowel disease , rheumatoid arthritis, multiple sclerosis, diabetes, systemic lupus erythematosus.