CN113825832A

CN113825832A - Amplification method for in vitro induction of CD4+ Foxp3+ CD69+ Treg and application thereof

Info

Publication number: CN113825832A
Application number: CN202180003004.3A
Authority: CN
Inventors: 金洪传; 余磊; 王娴; 冯利锋
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2020-07-28
Filing date: 2021-07-28
Publication date: 2021-12-21

Abstract

The invention provides an in vitro induced CD4+ Foxp3+ CD69+ Treg cell, a preparation method thereof, a related pharmaceutical composition and application thereof. The 10 mu M proteasome inhibitor MG132(Z-Leu-Leu-Leu-CHO) adopted by the invention and the initial CD4+ T are pretreatedCells capable of acquiring at least 65% of CD4⁺Foxp3⁺CD69⁺Treg; compared with a control group, the percentage is increased by 91.4 percent; the MG132Treg cells induced by the pretreatment have stronger immunosuppressive activity, and induce the reconstruction of the immune tolerance of the organism, thereby preventing and/or treating autoimmune diseases, in particular inhibiting the occurrence and development of inflammatory bowel diseases. The method of the invention is easy to realize in vitro culture and acquisition, has low cost and high efficiency, and is beneficial to the clinical application and development of the induced Treg cells for treating autoimmune inflammatory diseases.

Description

In vitro induced CD4+Foxp3+CD69+Treg expansion method and application thereof

Technical Field

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

Background

Regulatory T cells (tregs) are a group of T cells that have immunosuppressive functions and play an important role in maintaining immune balance in vivo, and their surface markers are CD4, CD25 and Foxp3, which typically express CD62L, CD44, CD28, OX40, FR4, CTLA-4 and GITR. Although Tregs have strong immunoregulation capability, Tregs are easily transformed into cells such as Th17 and the like in an inflammatory microenvironment, so that the immunosuppressive function of the Tregs is greatly damaged; moreover, inflammatory cytokines can cause the Foxp3 expression of Tregs to be down-regulated, thereby causing the Tregs to be transformed into inflammatory cells. CD4⁺CD69⁺Foxp3⁺Treg cells are regulatory T cells that are capable of expressing CD69 positive, however,CD4⁺CD69⁺Foxp3⁺tregs can secrete high-level IL-10 and TGF-beta 1, have the characteristics of stronger stability and difficulty in differentiating to inflammatory cells under inflammatory conditions, are attracted extensive attention, and inhibit abnormally activated immune cells through reinfusion of the inhibitory immune cells to become a new therapy for autoimmune diseases.

CD69 is an early leukocyte activation molecule and is also an immune modulatory molecule. CD69 downregulates immune responses by promoting the production of TGF-. beta.1, and has been reported to be CD69^-/-The ability of Tregs derived from mice to inhibit T cell proliferation in vitro is reduced, and in vivo experiments show that OVA-induced CD69^-/-The inflammatory symptoms of asthma in mice are more severe (Cort es, Jos er., et al.j autoimmun.55,51-62, (2014)); there are also studies that have shown that a deficiency in CD69 leads to an increased disease in 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 with an unbalanced intestinal immunoregulation. The pathogenesis of IBD involves multiple processes at the cellular level, and due to the complex pathogenesis, the curative effect of the current clinical treatment drugs is not good enough, the disease condition is easy to repeat, and the current clinical treatment drugs have adverse drug side effects. Separately from the literature, CD4⁺Foxp3⁺CD69⁺Treg and CD4⁺Foxp3⁺CD69^-Treg were adoptively reinfused into IBD mice and small amounts of CD4 were found⁺Foxp3⁺CD69⁺Treg can obviously inhibit the development of IBD, and CD4⁺Foxp3⁺CD69^-Tregs have a poor capacity to alleviate disease. The results indicate that CD69 has a regulatory effect on IBD and can be used as a target for treating IBD (Yu, L, et al. cell Death Dis 9.9,1-14 (2018)).

Tregs are derived from the human body, are not chemically modified, are easily accepted by the organism, are not easy to cause the toxicological effect generated by rejection, and have higher safety; the low cell yield caused by the small proportion (< 10%) of the Treg cells in peripheral blood, low separation efficiency and difficult expansion is also a main bottleneck of the Treg cell immunotherapy method. In clinic, the existing cellular immunotherapy technology mostly adopts a direct blood drawing method, if the blood drawing amount is required to reach the therapeutic dose finally, the blood drawing amount is required to be more than 200mL each time, and even if the blood drawing amount of a patient as high as 1/3 cannot obtain enough cells of the therapeutic dose finally, the subsequent treatment cannot be carried out; and a large amount of blood sampling for many times can have adverse effects on the body of a patient. Therefore, the induction culture to produce a more stable and efficient Treg subgroup is crucial for the application of cell vaccines and the treatment of autoimmune diseases. The expression ratio of CD4+ Foxp3+ CD69+ Treg in the Treg induced by the traditional method is only about 35%, so that the wide application of the Treg in the field of disease treatment is limited.

Disclosure of Invention

Aiming at the defects of the prior art, the technical problem to be solved by the invention is to provide an in vitro induced CD4⁺Foxp3⁺CD69⁺Treg expansion methods, related pharmaceutical compositions and uses; also provided is a CD4 with enhanced immunosuppressive activity⁺Foxp3⁺CD69⁺And (4) Tregs. The invention overcomes the defect that the traditional Treg culture method has CD4⁺Foxp3⁺CD69⁺Low Treg expression ratio. The invention can obtain at least more than 42 percent of CD4 by the pretreatment of proteasome inhibitor⁺Foxp3⁺CD69⁺Treg cells, increased by at least 20 percentage points compared to control; further, by the treatment of 10 mu M MG132, CD4+ Foxp3+ CD69+ Treg cells with the concentration of 67% can be obtained in vitro, which is increased by 91.4% compared with a control group, and the excellent characteristics of low cost and high efficiency accelerate the clinical application and development of the CD4+ Foxp3+ CD69+ Treg cells in autoimmune inflammatory diseases.

Preferably, the in vitro induced MG132Treg cells with immunosuppressive function and high stability comprise high level of CD4⁺Foxp3⁺CD69⁺Tregs are convenient to use and/or have higher efficiency, and an experimental foundation is laid for clinically applying induced Treg (iTreg) cells to treat autoimmune inflammatory diseases. Further, the present invention provides a novel use of MG 132-induced tregs for preventing or treating diseases associated with inflammatory bowel disease, and a novel clinically administered formulation comprising more CD4⁺Foxp3⁺CD69⁺Methods of induction of tregs and related formulations.

The first purpose of the invention is to provide an in vitro induced CD4⁺Foxp3⁺CD69⁺Treg expansion method using proteasome inhibitor to pre-treat naive CD4⁺T cells, then cultured under conventional manner Treg inducing conditions, obtained contain at least 42% CD4⁺Foxp3⁺CD69⁺Treg cells; preferably, the obtained product comprises at least 50% of CD4⁺Foxp3⁺CD69⁺Treg cells; more preferably, the obtained material comprises at least 55% of CD4⁺Foxp3⁺CD69⁺Treg cells (see Table 1)

The percentage is CD4 induced by flow detection⁺Foxp3⁺CD69⁺The ratio of the number of Treg cells was taken as CD4⁺Foxp3⁺CD69⁺Percentage of tregs.

Preferably, the initial CD4⁺The 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 through magnetic bead or flow sorting;

further, the initial CD4⁺Treg cells (MG132 Tregs) induced by T cells after MG132 pretreatment, and MG132Treg cells inhibit CD4⁺Enhanced T cell proliferation capacity

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

preferably, the proteasome inhibitor and the primary CD4⁺The temperature of T cell co-incubation was 37 ℃;

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

Preferably, the concentration of the proteasome inhibitor MG132 is 0.1-20. mu.M, the concentration of Bortezomib is 0.01-0.5. mu.M, and the concentration of Carfizomib is 0.001-0.01. mu.M.

Preferably, the tregs obtained after MG132 pretreatment comprise at least 55% CD4⁺Foxp3⁺CD69⁺Treg; further, it is preferred that at least 65% of CD4 is obtained when pre-treated with MG132 at a concentration of 10. mu.M⁺Foxp3⁺CD69⁺Treg；

Preferably, the proteasome inhibitor promotes CD69 expression not only in CD4⁺A T cell;

preferably, the conventional method is as follows: taking lymph tissue, grinding, filtering to form single cell suspension, centrifuging, removing supernatant, adding cell sorting solution into the precipitate, counting, sorting, suspending in T lymphocyte culture medium, adding cytokine and activating antibody, culturing, inducing for a certain time, and detecting Treg expression.

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

Preferably, the medium used under Treg-inducing conditions in the traditional manner comprises at least one of a cytokine or an activated antibody;

further, preferably, the cytokine is TGF-beta 1, and the concentration is 5-20 ng/mL; more preferably, the concentration of the cytokine TGF-beta 1 is 10ng/mL

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

Further, preferably, the concentration of the activated antibody anti-CD3/CD28 is 0.5-2 μ g/mL; more preferably, the concentration of the activating antibody anti-CD3/CD28 is 2. mu.g/mL.

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

a second object of the invention is to provide a Treg cell with enhanced immunosuppressive activity in vitro. The immunological activity enhancement refers to CD4 treated by proteasome inhibitor⁺Foxp3⁺CD69⁺The proportion of Tregs is increased; preferably, the Treg cells with enhanced immunosuppressive activity in vitro comprise at least 42% of CD4⁺Foxp3⁺CD69⁺Treg cells; preferably, said CD4⁺Foxp3⁺CD69⁺Treg cell passageAn amplification method according to any of the forms described above; preferably, the tregs obtained after MG132 pretreatment comprise at least 55% CD4⁺Foxp3⁺CD69⁺Treg; further, it is preferred that at least 67% of the CD4 be pretreated with MG132 at a concentration of 10. mu.M⁺Foxp3⁺CD69⁺Treg；

Preferably, the tregs secrete higher levels of IL-10 and TGF- β 1 than those induced by the traditional means, and further, the immunosuppression-associated molecules GITR, ICOS are higher than those induced by the traditional means.

The third purpose of the invention is to provide a Treg with high expression of CD69 molecules. The high expression of the CD69 molecule means that Tregs contain at least 42% of CD4⁺Foxp3⁺CD69⁺Treg cells; preferably, Carfilzomib (Carfilzomib) treatment results in at least 42% or more CD4⁺Foxp3⁺CD69⁺Treg cells; more preferably, 55% CD4 was obtained when treated at a concentration of 0.001. mu.M⁺Foxp3⁺CD69⁺Treg cells.

Preferably, Bortezomib (Bortezomib) treatment results in at least 50% or more CD4⁺Foxp3⁺CD69⁺Treg cells; more preferably, 55% CD4 was obtained when treated at a concentration of 0.1. mu.M⁺Foxp3⁺CD69⁺Treg cells.

Preferably, MG132(Z-Leu-Leu-Leu-CHO) treatment yields at least 55% or more of CD4⁺Foxp3⁺CD69⁺Treg cells; more preferably, 67% CD4 is obtained when treated at a concentration of 10. mu.M⁺Foxp3⁺CD69⁺Treg cells.

A fourth object of the present invention is to provide an in vitro MG132Treg cell, wherein the MG132Treg cell is a cell that promotes initial CD4+ T cell-induced CD4 by activating HSF1 and promoting more binding thereof to the CD69 promoter region, specifically promoting binding of HSF1 to HSE sequence to interact therewith⁺Foxp3⁺CD69⁺Treg expression is up-regulated;

preferably, the HSE sequence is a GAAnnTTC structure.

Preferably, the more of the experimental group is compared with the control group (control group).

Preferably, the expression is up-regulated in comparison between the experimental group and the control group (control group).

Preferably, the in vitro MG132Treg cell is in vitro MG132CD69⁺Treg cells.

A fifth object of the present invention is to provide an in vitro MG132Treg cell, wherein the in vitro MG132Treg cell has the following characteristics:

(a) the immunosuppressive function of the Treg is naive CD4⁺The T cells are pretreated by a proteasome inhibitor and then cultured in a traditional Treg induction mode, and the obtained Treg cells have higher proportion of CD4 than Tregs (control Tregs) induced by a simple traditional method⁺Foxp3⁺CD69⁺Treg；

(b) The MG132Treg has stronger immunosuppressive activity, which is shown in that the MG132Treg secretes higher level of IL-10 and TGF-beta 1, the MG132Treg immunosuppressive related molecules GITR and ICOS are higher than those of Control Treg, and the MG132Treg inhibits CD4⁺The proliferation capacity of T cells is better than that of Control Tregs;

(c) the MG132Treg can inhibit the occurrence and the development of inflammatory bowel diseases, and the remission capacity of the MG132Treg on enteritis is better than that of Control Treg. The MG132Treg can inhibit immune response and induce the reconstruction of immune tolerance of an organism, 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, systemic lupus erythematosus.

Preferably, the MG132 is capable of activating and promoting incipient CD4⁺T cell induced CD4⁺Foxp3⁺CD69⁺Treg expression is upregulated.

Preferably, the in vitro MG132Treg cell is in vitro MG132CD69⁺Treg cells.

The sixth purpose of the invention is to provide an in vitro amplification culture medium for the Treg cells for promoting the expression of the Treg cell membrane protein CD69, wherein the formulation of the in vitro amplification culture medium for the Treg cells comprises at least one or more of Hepes, sodium pyruvate, beta-mercaptoethanol and antibiotics.

Preferably, the main body of the Treg cell in-vitro expansion culture medium is a T cell special culture medium containing 1640RPMI with 10% serum.

Preferably, the concentration of Hepes in the Treg cell in-vitro amplification culture medium is 10mM, the concentration of sodium pyruvate is 1mM, and the concentration of beta-mercaptoethanol is 5 mu M.

Preferably, the antibody is selected from at least one of CD3 or CD 28; further, preferably, the amount of the antibody added is 0.5 to 10. mu.g/mL,

preferably, the cytokine is selected from at least one of TGF-beta 1 or IL-2; furthermore, the addition amount of the TGF-beta 1 is preferably 5-20ng/mL, and the IL-2 is preferably 50-100 IU.

The seventh purpose of the invention is to provide a pharmaceutical composition, which contains the safe and effective amount of the MG132Treg or the effective amount of MG132CD69⁺The Treg cells are at least one of active ingredients and pharmaceutically acceptable carriers;

preferably, in a specific example of the invention, the pharmaceutically effective amount of MG132Treg or a salt thereof according to the invention may be 2.5 x 10⁷-5×10⁸D/kg; further, it is preferably 2.5X 10⁸/d/kg。

Preferably, the "effective amount" refers to an amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect; preferably, the precise effective amount for a subject will depend upon the size and health of the subject, the nature and extent of the condition, and the therapeutic agent and/or combination of therapeutic agents selected for administration.

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

Preferably, the Treg cells or MG132Treg cells in the pharmaceutical composition are autologous or allogeneic.

Preferably, the pharmaceutical composition will be formulated as an injectable preparation, such as a liquid solution or suspension; or in solid form suitable for constitution with a solution or suspension, or liquid carrier, before injection.

Preferably, the pharmaceutical composition is administered at least one of intravenously and intraperitoneally.

Preferably, the pharmaceutical composition is administered intravenously, and more preferably, the therapeutic dosage regimen is selected from at least one of a single dose regimen or a multiple dose regimen.

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

Preferably, the acceptable carrier refers to a carrier for administration of a therapeutic agent or a pharmaceutical carrier in a therapeutic pharmaceutical composition; preferably, it is meant that the pharmaceutical carrier does not itself induce the production of antibodies harmful to the individual receiving the composition and is not unduly 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 contain auxiliary substances such as pH buffering substances and the like.

The pharmaceutical composition of the present invention can be prepared by using pharmaceutically suitable and physiologically acceptable auxiliary agents, which can be dissolved in Dimethylsulfoxide (DMSO) and has a chemical formula C, in addition to MG132 as an active ingredient₂H₆OS。

It is an eighth object of the present invention to provide CD4 obtained by any one of the amplification methods described above⁺Foxp3⁺CD69⁺Use of at least one of Treg cells, MG132Treg cells in any of the forms described above, a pharmaceutical composition in any of the forms described above in the treatment of an autoimmune disease.

Preferably, the autoimmune disease refers to a disease caused by an immune response of the body to an autoantigen; further, the disease is associated with the following factors:

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

(b) trauma and infection provoke autoimmune responses;

(c) genetic factors.

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

Preferably, in one embodiment of the invention, the concentration of MG132Treg cells used to treat IBD in mice by adoptive back infusion may be 2.5 × 10⁵-1×10⁷/mL。

Preferably, the treatment, unless otherwise specified, means reversing or alleviating the condition or disease, or one or more symptoms of the condition or disease, or inhibiting or preventing its development.

In particular, the treatment refers in one embodiment of the invention to a treatment behaviour based on the definition as described above. In mammals, "treatment" or "methods of treatment" of inflammatory bowel disease and diseases associated therewith may include one or more of the following.

(1) Preventing the development of inflammatory bowel disease and diseases associated therewith;

(2) preventing the spread of inflammatory bowel disease and diseases associated therewith;

(3) reducing inflammatory bowel disease and diseases associated therewith;

(4) preventing the reoccurrence of inflammatory bowel disease and diseases associated therewith;

(5) relieving symptoms of inflammatory bowel disease and diseases associated therewith.

Advantageous effects

According to the invention, the existing Treg inducible expression system is optimized and improved, a more efficient inducible expression technology is established, the experiment cost and time are saved, and the experiment period is shortened. Preferably, MG132 pre-treatment of the initial CD4 compared to traditional Treg induction methods⁺T cells, later cultured under Treg-inducing conditions, promote more CD4⁺Foxp3⁺CD69⁺Tregs differentiate, secreting higher levels of IL-10 and TGF- β 1; the MG132Treg can effectively inhibit the proliferation and differentiation of effector T cells; furthermore, the MG132Treg is a biological agent, has small rejection reaction and good immunosuppressive effect; the treatment cost is low, the injection frequency is less, and only a plurality of weeks (such as 1-2 weeks) of injection are needed, so that the pain of injection treatment is reduced, and the recovery of patients is facilitated. MG132 promotes more CD4⁺Foxp3⁺CD69⁺Expression of tregs, MG132 tregs are capable of preventing or treating inflammatory bowel disease and autoimmune diseases associated therewith; and has no toxicity and side effect of the medicine, stable effect and long-term administration. In the present invention, the term "Treg" refers to CD4⁺Foxp3⁺Regulatory T cells;

in the present invention, the term "CD 4⁺Foxp3⁺CD69⁺Treg "refers to a subpopulation of Treg cells that express CD 69;

in the present invention, the term "CD 4⁺Foxp3⁺CD69^-Treg "refers to a subpopulation of Treg cells that do not express CD 69;

in the present invention, the term "CD 69^-/-"refers to a mouse with CD69 knocked out; in the present invention, the term "MG 132 Treg" refers to initial CD4 pretreated with MG132⁺Tregs cultured under traditional Treg-inducing conditions after T cells;

in the present invention, the term "MG 132CD69⁺Treg "refers to CD69 expressing tregs from MG132 tregs by flow sorting, and MG132CD69⁺Tregs are all CD4⁺Foxp3⁺CD69⁺Treg cells;

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

in the present invention, the term "iTreg" refers to an induced regulatory T cell induced in vitro;

in the present invention, the term "Control CD69⁺Treg "refers to CD69 that is separated from Control Treg by stream⁺Treg；

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-. beta.1" refers to transforming growth factor-. beta.1, transforming growth factor 1;

in the present invention, the term "HSF 1" refers to heat shock factor 1, heat shock transcription factor 1;

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

in the present invention, the term "CFSE" refers to a cell marker, which means hydroxyfluorescein diacetate succinimidyl ester (5, 6-carboxyfluorescein diacetate, succinimidyl ester), a fluorescent dye that penetrates cell membranes, has a succinimidyl group that specifically binds to cells and a hydroxyfluorescein diacetate group that has non-enzymatic hydrolysis.

In the present invention, the term "flow sorting" refers to sorting a specific population while performing multi-parameter, quantitative analysis on single cells or biological particles in flow by using a flow cytometer, and the sorting purity is as high as 99%.

In the present invention, the term "ELISA" refers to an enzyme-linked immunosorbent assay, which is a technique of performing quantification by adsorbing a known antigen or antibody on a solid phase carrier surface and reacting the enzyme-labeled antigen or antibody on the solid phase surface.

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

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

in the invention, the ELISA kit is purchased from Saimerfei;

in the present invention, the original CD4⁺T Cell sorting kit purchased from STEM Cell;

in the present invention, proteasome inhibitors are purchased from seleck and Sigma;

in the present invention, the primers were synthesized by Shanghai Czeri Bio Inc.;

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, as the MG132 compound, a commercially available MG132 compound may be used, and a MG132 compound that is naturally isolated or produced by a synthetic method known in the art may not be used.

Drawings

FIG. 1 is an analysis chart of ELISA detection of IL-10 and TGF-beta 1 expression.

FIG. 2 heatmap of the expression levels of immunosuppressive-associated genes for Control Treg and MG132Treg following gene sequencing;

FIG. 3A flow analysis of MG132Treg, Control Treg, MG132CD69 in FIG. 3A⁺Treg and Control CD69⁺A schematic representation of the expression of Treg immunosuppressive-associated molecules; FIG. 3B flow analysis of MG132Treg, Control CD69⁺Treg, and MG132CD69⁺Treg inhibits CFSE-labeled CD4⁺Schematic representation of T proliferation.

FIG. 4 is a schematic representation of HSF1 and HSF1 activation-related gene expression after pretreatment of CD4+ T cells with MG 132; FIG. 4B is a diagram of HSF1 cell membrane-to-cell nucleus transfer after MG132 pre-treated CD4+ T cells using confocal fluorescence detection.

FIG. 5 flow analysis HSF1^+/+And HSF1^+/-Mice were injected in vivo with MG132, CD4⁺Foxp3⁺CD69⁺Treg expression in mouse spleen, mesenteric lymph nodes and mesenteric lymph nodes is shown schematically.

FIG. 6 flow analysis of induced CD4 following MG132 pretreatment of CD4+ naive T cells from HSF1+/+ or HSF1 +/-mice and blocking of CD4+ naive T cell HSF1 expression from HSF1+/+ mice with KRIBB11⁺Foxp3⁺CD69⁺Schematic representation of Treg expression.

FIG. 7 is a schematic diagram showing the interaction between the HSF1 protein and the CD69 gene promoter.

FIG. 8 shows the detection, evaluation and comparison of therapeutic effects of adoptively transfused MG132Treg, Control Treg, MG132CD69 + Treg, Control CD69+ Treg to IBD mice.

FIG. 9 is a comparison graph of Th1 and Th17 downregulation in MG132Treg, Control Treg, MG132CD69 + Treg, Control CD69+ Treg adoptively-returned treatment group mice.

In particular, for clarity of illustration and scale in the drawings, the notation is CD69⁺Treg refers to CD4⁺Foxp3⁺CD69⁺Treg。

Detailed Description

The invention is further illustrated below with reference to the figures and examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1 proteasome inhibitor promotes CD4⁺Foxp3⁺CD69⁺Differentiation of Tregs

CD4 in Treg induced by traditional mode at present⁺Foxp3⁺CD69⁺The Treg expression ratio is only around 35%. In example 1, the proteasome inhibitor pair CD4 was examined⁺Foxp3⁺CD69⁺The influence of Treg generation is induced under the condition of culturing Treg in the traditional mode after the initial T cells of the mice are pretreated by MG132, bortezomib and carfilzomib, thereby effectively promoting the generation of the anti-inflammatory CD4⁺Foxp3⁺CD69⁺Treg generation (Table 1), to determine the optimal concentration for each proteasome inhibitor pretreatment, mice were sorted from spleen-derived CD4 using magnetic beads⁺CD62L

T cells are counted, 1 mu M MG132, 0.01 mu M Bortezomib, 0.1 mu M Bortezomib,0.001 mu M Carfizomib and 0.01 mu M Carfizomib are added into the cells respectively, the cells are placed in a 37 ℃ cell culture box for pretreatment for 90min, a DMSO group is used as a negative control group, the pretreatment mode is the same as the above, then the cells are washed for three times by a serum-free culture medium 1640, and the washing time is 2 multiplied by 10⁶The cell factor TGF-beta 1 is added into the suspension while the suspension is resuspended in the T lymphocyte culture mediumThe daughter IL-2, anti-CD3/CD28 antibody. Three days later, the cultured cells are collected, and Tregs and CD4 are detected in a flow mode⁺Foxp3⁺CD69⁺Treg expression profile. Table 1 shows that each group of proteasome inhibitor pairs can successfully induce the differentiation of Tregs, the percentage of the Tregs is 42-67%, and CD4 induced by different modes is further compared⁺Foxp3⁺CD69⁺Treg ratio, CD4 after proteasome inhibitor MG132 pretreatment⁺Foxp3⁺CD69⁺The proportion of Treg is 20% -32% higher than that of Control Treg, the 10 mu M MG132 treatment group is the best, and the proportion is 91.4% higher than that of Control Treg of the Control group. Both 0.01. mu.M and 0.1. mu.M Bortezomib induced CD4⁺Foxp3⁺CD69⁺Treg is highly expressed, 51-57 percentage points higher than Control Treg, and 0.001 mu M Carfizomib can induce CD4⁺Foxp3⁺CD69⁺The Treg is up-regulated by about 20 percent compared with the control group, the induction effect of 0.01 mu M Carfizomib is not obvious, and the induction effect is only improved by 7 percent compared with the control group.

The ELISA kit detects the level of TGF-beta 1 and IL-10 secreted by the cultured cells, and shows that 10 mu M MG132 has the strongest secretion capacity of IL-10 and TGF-beta 1 of Treg, and the IL-10 and TGF-beta 1 are 1.5-2 times of those of a control group, and the IL-10 expression level of 0.01 mu M, 0.1 mu M Bortezomib and 0.001 mu M Carfizomib groups is also up-regulated (figure 1).

The culture process uses T lymphocyte culture medium, and its preparation method is: to a 1640 medium containing 10% FBS, beta-mercaptoethanol at a final concentration of 5. mu.M, 1mM sodium pyruvate, 10mM Hepes, and Triantibody were added.

In this example, the initial CD4 induced by flow assay⁺Differentiation of T cells into CD4⁺Foxp3⁺The proportion of the number of the cells is used as the percentage of Tregs; induced CD4 by flow assay⁺Foxp3⁺CD69⁺The ratio of the number of Treg cells was taken as CD4⁺Foxp3⁺CD69⁺Percentage of tregs.

Table 1 different concentrations of proteasome inhibitor treatment on Treg and CD4⁺Foxp3⁺CD69⁺Influence of Treg proportion

Example 2 Gene expression differential between MG132Treg and Control Treg

In order to determine the difference between the MG132Treg and the Control Treg in the aspect of gene expression, a flow sorter (instrument model: BD FACS ORP ARIA II) is adopted to sort the MG132Treg and the Control Treg which are induced by 10 mu M MG132 pretreatment in example 1, and gene sequencing is carried out, and the result in FIG. 2 shows that the expression levels of MG132Treg immunosuppression related genes IL-10, TGF-beta 1, SOCS2, HSP90AA1, GITR, Gzmb, CTLA-4 and the like are obviously higher than those of the Control Treg, and meanwhile, the expression of proinflammatory related factors IL-17A, IL-17F in the MG132Treg group is reduced compared with that of the Control Treg.

Wherein, the flow cell sorting buffer is as follows: PBS, pH 7.2, BSA at a final concentration of 0.5% and 2mM EDTA were added, filtered and aliquoted.

The flow cytometric buffer was HBSS, pH 7.2, and BSA, 4 × double antibody, at a final concentration of 1%, was added.

The buffer collected by flow cell sorting is RPIM 1640 culture medium, and serum with final concentration of 30% and 4 Xdouble antibody are added.

Example 3 analysis of MG132Treg characteristics

Collection of each cell group, 1X 10⁶The 10 μ M MG132 pre-treatment induced MG132Treg and Control Treg cells were resuspended in 100 μ l PBS and the corresponding dose of fluorescently labeled antibody was added as per the instructions: CD4, Foxp3, CD69, CTLA-4, ICOS, GITR, CD44, CXCR4, CCR7 and corresponding isotype control antibody, incubating for 30min in ice in dark place, washing three times with PBS, and performing flow detection on Treg/CD4 induced by MG132⁺Foxp3⁺CD69⁺Treg and traditional method induced Treg/CD4⁺Foxp3⁺CD69⁺The expression of the immunosuppressive-associated molecules of Tregs, FIG. 3A shows that both groups of Tregs highly express the immunosuppressive-associated molecules, however, the MG132Treg immunosuppressive-associated molecules CTLA-4, ICOS are higher than those of Control Tregs, and Control CD69⁺Treg and MG132CD69⁺Immunosuppressive associated molecules of TregsAnd the chemokine receptors CCR7 and CXCR4 were not significantly different. Further flow sorting C57BL/6 mouse CD4⁺Labeling T cells with 10mM CFSE, washing twice with serum-free medium 1640, adjusting the concentration, adding anti-CD3/CD28 anti body, spreading on 96-well plate, adding induced MG132Treg, Control Treg, and MG132CD69⁺Treg and Control CD69⁺Treg, the effective target ratio is 1:1, 2:1 and 4; 1, each group was set with three wells, and fluorescence intensity was measured by FACS after 3 days of incubation at 37 ℃. Cell proliferation experiments showed CD4 alone⁺After T cells are activated by anti-CD3/CD28 antibody, the proliferation rate of the T cells is about 90 percent, and MG132Treg and CD4⁺CD4 when T cells were co-cultured at 1:1 conditions⁺The T cell proliferation rate is 37%, and Control Treg and CD4⁺CD4 when T cells were co-cultured at 1:1 conditions⁺The T cell proliferation rate is 44%, and under the condition of different effective target ratios, the Control CD69⁺Treg and MG132CD69⁺Treg inhibits CD4⁺The T cell capacity is superior to that of Control Treg and MG132Treg, and importantly, the MG132Treg inhibits CD4⁺T cell proliferation capacity was superior to Control tregs (see figure 3B). Preliminarily suggests that MG132Treg has stronger immunosuppressive function than Control Treg, Control CD69⁺Treg and MG132CD69⁺There was no significant difference in the immunosuppressive function of tregs.

Example 4 MG132 promotes HSF1 and related Gene activation

10 μ M MG132 Pre-treatment of CD4⁺T cells are cultured in an incubator after being washed for three times by serum-free 1640 medium for 90min, and the cells are collected for 12h and 24h respectively, and a treatment group without any treatment is used as a control. Real-time PCR detection of CD4⁺The expression of HSF1 in T cells and the expression of HSF1 activation-related genes (see Table 2 for primers used), and the graph A shows that MG132 can promote the expression of HSF1 and its related genes HSP90A, DNAJB1 and HSPA1A by more than 10 times of the control group, and MG132 can also promote the expression of CD69 (see FIG. 4A). Wherein compared with control, MG132 pretreatment can promote CD69 gene expression level to increase 4 times, HSF1 is mainly distributed on cell membrane in resting state, and after MG132 and treatment, immunofluorescence confocal HSF1 enters cell nucleus from cell membraneAnd becomes an active state (see fig. 4B).

TABLE 2 primers used for Real-time PCR

Example 5 MG132 promotes in vivo expression of CD69 of Tregs dependent on HSF1

To further clarify that MG132 promotes CD69⁺The differentiation mechanism of Treg adopts HSF1^+/+And HSF1^+/-Mice were injected via tail vein with 15. mu.M/kg MG132, and 7 days later, mice were isolated with spleen, inguinal lymph node, mesenteric lymph node, and intestinal lamina propria lymph node, and tested for CD4⁺Foxp3⁺CD69⁺Treg expression, and the judgment that MG132 induces Treg differentiation and CD4 in vivo⁺Foxp3⁺CD69⁺Influence of Treg expression, to determine whether deletion of HSF1 affected an increase in CD69, fig. 5 shows that a proportion of CD4 was present in normal mice⁺Foxp3⁺CD69⁺Tregs, present in small amounts in both the spleen and lymph nodes, but in the lamina propria in the intestine in a proportion of up to 68%, up-regulated HSF1 following in vivo injection of MG132^+/+CD4 of intestinal lamina propria lymph node⁺Foxp3⁺CD69⁺Treg expression, presumably up-regulated by 15%, but on HSF1^+/-Mouse CD4⁺Foxp3⁺CD69⁺Treg has no obvious influence, and MG132 can promote HSF1 in vivo^+/+Treg cells expressed CD69, however MG132 did not promote HSF1^+/-Mouse CD4⁺Foxp3⁺CD69⁺Treg expression, further suggesting that HSF1 is required for MG132 to promote CD69 expression.

In this embodiment, the term "HSF 1^+/+"refers to a wild-type mouse in which the heat shock factor 1(HSF1) gene is normally expressed; the following examples are the same.

In this embodiment, the term "HSF 1^+/+"refers to a mouse with half of the heat shock factor 1(HSF1) gene deleted; the following examples are the same.

Example 6 in vitro Induction of CD4 by MG132 through HSF1⁺Foxp3⁺CD69⁺Expression of Tregs

Sorting HSF1^+/+Or HSF1^+/-Mouse CD4⁺Initial T cells, induced Treg after 10 μ M MG132 pretreatment, and with the group without MG132 pretreatment as a control, induced Treg under Treg polarization conditions, with the addition of HSF1 inhibitor KRIBB11(KRIBB11 is an inhibitor of HSF1 and is capable of blocking the induction of HSF1 downstream target proteins such as HSP27 and HSP 70) to block HSF1 expression, three days later, CD4 was flow analyzed⁺Foxp3⁺CD69⁺The expression profile of tregs; inhibition of primary CD4⁺After expression of HSF1 in T cells, MG132 did not promote CD4⁺Foxp3⁺CD69⁺Treg expression (see fig. 6), suggesting that HSF deletion affects induced CD4⁺Foxp3⁺CD69⁺The expression level of tregs and MG132 could not induce the upregulation of CD69 expression of Treg cells after the action of HSF1 inhibitor, indicating that CD69 upregulation by MG132 is HSF1 dependent.

Example 7 MG132 promotes HSF1 binding to the CD69 promoter region

Through analyzing the sequence of the CD69 promoter region, two classic HSE (Heat Shock stress elements) sequences capable of binding with HSF1 are found in front of the promoter, namely the GAAnnTTC structure, as shown in FIG. 7A, and the interaction condition of HSF1 and CD69 is further verified through the chromatin immunoprecipitation technology, namely CHIP (chromosome immunoprecipitation) experiment, and CD4 is sorted⁺T cells are cultured for 24 hours under the condition of Treg induction after being pretreated by MG132 or not, the cells are subjected to ultrasonic treatment after being fixed for 10min at 37 ℃ by 1% formaldehyde, DNA fragments after ultrasonic treatment and HSF1 antibody are incubated overnight at 4 ℃, and meanwhile, an IgG antibody group is set as a negative control group. Then the antibody and chromatin complex were precipitated by A/G protein agarose beads, incubated at 65 ℃ for 4 hours, and the DNA was further purified and then PCR-detected for each set of CD69 gene expression. A total of 5 primers were designed around the region near the CD69 promoter, FIG. 7B shows that after the 4 th primer showed MG132 action, more HSF1 bound CD69 than 2-fold in the non-MG 132-pretreated group, and the primer amplification sequence contained HSE sequence near the promoter region with primer sequence 5 '-AAATGAGCAAGGGATGATGA-3, 3' -TCTTTCAAGTGTCAGGAGTT-5'. Furthermore, primer sequence No. 1 was 5'-AGCCAACTTATAGCAGAGG-3'; 3 '-AAGCAGGAAGGTCCAGAT-5'; the 2 nd pair of primer sequences is 5'-AGAACCGCTAAGACACAAC-3'; 3 '-GCCAAGAAGAACCTCTACAT-5'; the 3 rd pair of primer sequences is 5'-GCACATTAGGACCAGCAT-3'; 3 '-AATAGAGCAGAGAATGGAGAG-5'; the 5 th pair of primers has the sequence of 5'-GTGGTGCTCAATAACTTATCTG-3'; 3 '-TGTCATCCTCCACTGTCAA-5'.

Example 8 MG132 Treg/MG132 CD69⁺Treg inhibits the generation and development of mouse IBD

Since MG132 can promote CD4⁺Foxp3⁺CD69⁺Treg differentiation, we further compared MG132Treg/CD4⁺Foxp3⁺CD69⁺Treg and Control Treg/CD4⁺Foxp3⁺CD69⁺Therapeutic effect of Tregs on IBD, magnetic bead sorting kit sorting CD4⁺CD62L⁺

T cells induce Treg after 10 mu M MG132 pretreatment, and meanwhile, a group without MG132 pretreatment is used as a treatment control group, and a PBS group is used as a blank control group. IBD model was constructed by drinking 2% DSS from C57BL/6 mice, and MG 132-induced Treg (5X 10) was returned via tail vein after two days of modeling, i.e., day 2⁵Mice) and traditional induced tregs (5 × 10)⁵Mice), see in particular fig. 8A. The change of the body weight of the mice is observed and recorded, the change of pathological indexes is detected, and the change of the intestinal length of each group is detected. Mice began to lose weight on day 5 after drinking 2% DSS until day 9, after adoptive return of MG132Treg, IBD mice had significantly reduced weight loss, which remained at 95% of their original weight on day 9 after treatment, compared to the Control Treg group, which showed about 20% weight loss (fig. 8B-C). MG132CD69⁺Treg and Control CD69⁺Treg body weight was maintained at essentially normal levels. In line with this, the decrease in intestinal length was reduced compared to the Control Treg group after adoptive reinfusion treatment with MG132Treg (fig. 8D), and the pathological section results showed that the MG132Treg group had a lower degree of intestinal inflammation than the PBS group and the Control Treg. MG132CD69⁺Treg and Control CD69⁺The Treg group was not significantly different from the normal control group (fig. 8E). The results show that the intestinal tract length is not obviously shortened, and in an experimental group of animal model mice of inflammatory bowel diseases for intravenously supplying MG132Treg, the intestinal tract lamina propria lymphocyte is reduced, and the arrangement of intestinal mucosa glands is complete.

The histological scoring standard of the colitis of the mice is mainly defined according to the inflammation degree, the lesion depth, the crypt destruction, the regeneration index and the lesion range (%), and specifically comprises the following steps: 0 point, no inflammation; 1 point, mild inflammation, 2 points, moderate inflammation, 3 points, severe inflammation. Score 0, no depth of lesion; 1 minute, affecting the mucous membrane layer; 2 min, affecting the mucosa layer and the submucosa; score 3, panniculitis. 0min, no crypt destruction; score 1, the crypt of basement 1/3 was destroyed; 2 part basis 2/3 crypts were destroyed; score 3, only the surface epithelium is intact; in 4 points, all crypts and epithelium were destroyed. Score 0, fully regenerated or arrhythmic tissue; 1 minute, almost complete regeneration; 2, regeneration with crypt defect; score 3, incomplete surface epithelium; 4 min, no repair. 1 point, with a lesion range of 1-25%; 2 points, the range of pathological changes is 26-50%; 3 points, the range of pathological changes is 51-75%; 4 points, the range of pathological changes is 76-100%.

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

Inflammatory Th1(CD 4)⁺IFN-γ⁺) And Th17(CD 4)⁺IL-17⁺) The invention plays a crucial role in the development of IBD (inflammatory bowel disease) in mice, and aims to clarify MG132 Treg/MG132 CD69⁺The therapeutic mechanism and effect of Treg on IBD are analyzed by analyzing the expression of Th1 and Th17 in Spleen (Spleen), Mesenteric Lymph Node (MLN) and intestinal lamina propria lymph node (LPL) of each group of mice, and the Spleen and lymph node of normal mice only express a small amount of Th1 and Th17, wherein Th1 accounts for total CD4 in Spleen and mesenteric lymph node respectively⁺About 1.4% and 2.8% of T cells, Th17 accounted for approximately total CD4 in spleen and mesenteric lymph nodes, respectively⁺About 0.63% and 1.17% of T cells, about 20% and 15% of Th1 and Th17 in the lamina propria of the intestine, respectively, and Th1 in the spleen, mesenteric lymph nodes and the lamina propria of the intestine after induction of IBDThe expression of baryon can be increased by 3-4 times, Th17 can be increased by 2-3 times, Th1 and Th17 are decreased in different degrees after MG132Treg and Control Treg treatment, the treatment effect of the MG132Treg treatment group is good, and MG132CD69⁺Treg and Control CD69⁺The down-regulation of Th1 and Th17 was most pronounced in the Treg treatment group, see in particular fig. 9.

In summary of the examples, CD4 was found⁺Foxp3⁺CD69⁺The Treg has a stronger immunosuppressive function, the proteasome inhibitor MG132 can promote the expression of a Treg cell membrane protein CD69, and compared with a Control Treg, the MG132Treg can secrete a higher level of IL-10 and TGF-beta 1, so that the immunosuppressive function is stronger; the local inflammation can be effectively inhibited by feeding back MG132Treg to IBD mice. Furthermore, whereas MG132 is able to activate HSF1, deletion of HSF1 results in induced CD4⁺Foxp3⁺CD69⁺The expression of Tregs is reduced, and MG132 can regulate the expression of CD69 through HSF1, so that Tregs are endowed with a stronger immunosuppressive function, and are expected to become effective cell vaccines for the clinical treatment of IBD, and the Tregs are expected to become a general preparation for treating autoimmune diseases.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. In vitro induced CD4⁺Foxp3⁺CD69⁺Method for the expansion of Treg cells, characterized in that said expansion method uses a proteasome inhibitor to pre-treat the starting CD4⁺T cells, then cultured under conventional manner Treg inducing conditions, obtained contain at least 42% CD4⁺Foxp3⁺CD69⁺Treg cells; preferably, the obtained product comprises at least 50% of CD4⁺Foxp3⁺CD69⁺Treg cells; more preferably, the obtained material comprises at least 55% of CD4⁺Foxp3⁺CD69⁺Treg cells;

preferably, the initial CD4⁺The T cells are derived from at least one of spleen, mesenteric lymph node, inguinal lymph node and other lymph tissue;

preferably, the initial CD4⁺The T cell sorting method includes at least one of magnetic bead or flow sorting.

2. The amplification method according to claim 1, wherein the proteasome inhibitor is at least one selected from the group consisting of MG132 (Z-Leu-CHO), Bortezomib (Bortezomib), Carfilzomib (Carfilzomib); further, MG132 (Z-Leu-Leu-Leu-CHO);

preferably, the pretreatment time of the proteasome inhibitor is 1h to 2.5 h;

preferably, the temperature of the proteasome inhibitor pretreatment is 37 ℃;

preferably, the proteasome inhibitor has a MG132(Z-Leu-Leu-Leu-CHO) concentration of 0.1-20. mu.M, a Bortezomib (Bortezomib) concentration of 0.01-0.5. mu.M, and a Carfilzomib (Carfilzomib) concentration of 0.001-0.01. mu.M;

Preferably, the proteasome inhibitor promotes CD69 expression not only in CD4⁺T cells.

3. The expansion method according to any one of claims 1 or 2, wherein the medium used under traditional mode Treg inducing conditions comprises at least one of a cytokine or an activated antibody;

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

preferably, the cytokine is TGF-beta 1, the concentration is 5-20 ng/mL; more preferably, the concentration of the cytokine TGF-beta 1 is 10 ng/mL; further, preferably, the cytokine is IL-2, and the concentration is 50-100 IU; more preferably, the concentration of cytokine IL-2 is 50 IU; further, preferably, the concentration of the activated antibody anti-CD3/CD28 is 0.5-2 μ g/mL; more preferably, the concentration of the activating antibody anti-CD3/CD28 is 2. mu.g/mL.

4. Treg cells with enhanced immunosuppressive activity in vitro, characterized in that they comprise at least 42% of CD4⁺Foxp3⁺CD69⁺Treg cells; preferably, said CD4⁺Foxp3⁺CD69⁺Treg cells obtained by the expansion method according to any one of claims 1 to 3; preferably, the tregs obtained after MG132 pretreatment comprise at least 55% CD4⁺Foxp3⁺CD69⁺Treg; further, it is preferred that at least 67% of the CD4 be pretreated with MG132 at a concentration of 10. mu.M⁺Foxp3⁺CD69⁺Treg；

Preferably, the tregs secrete higher levels of IL-10 and TGF- β 1 than those induced by the traditional means, and further, preferably, the immunosuppression-associated molecules GITR, ICOS are higher than those induced by the traditional means.

5. An in vitro MG132Treg cell, wherein said MG132Treg cell is MG132 that interacts by activating HSF1 and causing it to bind more to the CD69 promoter region, specifically, by causing HSF1 to bind to HSE sequences, thereby promoting CD4 induced by naive CD4+ T cells⁺Foxp3⁺CD69⁺Treg expression is up-regulated; preferably, the HSE sequence is a GAAnnTTC structure; preferably, the in vitro MG132Treg cells are primary CD4 pretreated in vitro with MG132⁺Induced in traditional manner after T cells and containing high levels of CD69⁺Cells of Tregs.

6. An in vitro MG132Treg cell, wherein the MG132Treg cell is derived from naive CD4⁺T cells, primary CD4⁺The T cells are firstly pretreated by MG132 and then cultured in a traditional Treg induction mode to obtain the T cells;

preferably, the MG132 promotes CD69 expression by HSF1 activationTo promote initial CD4⁺T cell induced CD4⁺Foxp3⁺CD69⁺Treg expression is up-regulated;

preferably, the MG132 tregs are capable of inhibiting the development of autoimmune diseases; preferably, the concentration of MG132Treg cells is 2.5X 10⁵-1×10⁷/mL；

Preferably, the in vitro MG132Treg cell is in vitro MG132CD69⁺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.

7. A Treg cell in-vitro amplification culture medium for promoting the expression of Treg cell membrane protein CD69 is disclosed, wherein the formulation of the Treg cell in-vitro amplification culture medium comprises at least one or more of Hepes, sodium pyruvate, beta-mercaptoethanol, antibodies and cytokines;

preferably, the main body of the Treg cell in-vitro expansion culture medium is a T cell special culture medium containing 1640RPMI with 10% serum; preferably, the Hepes concentration in the culture medium is 10mM, the sodium pyruvate concentration is 1mM, and the beta-mercaptoethanol concentration is 5 μ M;

preferably, the antibody is selected from at least one of CD3 or CD 28; further, preferably, the addition amount of the antibody CD3 and/or CD28 is 0.5-10 mug/mL;

preferably, the cytokine is selected from at least one of TGF-beta 1 or IL-2; further, preferably, the addition amount of the cytokine TGF-beta 1 is 5-20ng/mL, and the addition amount of the cytokine IL-2 is 50-100 IU.

8. A pharmaceutical composition comprising a safe and effective amount of the Treg of claim 4, the MG132Treg of any one of claims 5 or 6, or the CD4 obtained by the expansion method of any one of claims 1 to 3⁺Foxp3⁺CD69⁺At least one of the Tregs is an active ingredient; preferably, the pharmaceutical composition further comprises an acceptable carrier;

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

preferably, the Treg cells or MG132Treg cells are autologous or allogeneic; preferably, the pharmaceutically effective amount of MG132Treg or a salt thereof is 2.5 × 10⁷-5×10⁸D/kg; further, it is preferably 2.5X 10⁸/d/kg；

Preferably, the pharmaceutical composition is administered at least one of intravenously and intraperitoneally; preferably, the pharmaceutical composition is administered intravenously, more preferably, the therapeutic dosage regimen is selected from at least one of a single dose regimen or a multiple dose regimen; preferably, the pharmaceutical composition will be formulated to include at least one of an injectable formulation or a solid form suitable for constitution in solution or suspension, liquid carrier before injection; further, preferably, the carrier refers to a carrier for administration of a therapeutic agent or a pharmaceutically carrier in a therapeutic pharmaceutical composition; preferably, the carrier is one which does not itself induce the production of antibodies harmful to the individual receiving the composition and which is not unduly toxic to the individual upon administration; further, preferably, the acceptable carrier is a liquid; more preferably, the carrier is selected from at least one of PBS or physiological saline;

9. CD4 obtained by the amplification method according to any one of claims 1 to 3⁺Foxp3⁺CD69⁺Use of at least one of Treg cells, MG132Treg cells according to claim 5 or 6, pharmaceutical composition according to claim 8 in the treatment of autoimmune diseases.

10. The autoimmune disease of claim 9, wherein the autoimmune disease is a disease resulting from an immune response of the body to an autoantigen; preferably, the autoimmune disease is selected from inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, diabetes, systemic lupus erythematosus.