KR101996390B1 - Composition for immunopotentiating containing aminoquinoline-based compound, composition for promoting development into regulatory T cell, and method using the same - Google Patents

Abstract

A composition for enhancing immunity comprising an aminoquinoline compound, a stereoisomer, a derivative, a hydrate or a pharmaceutically acceptable salt thereof, and a composition for promoting differentiation into regulatory T cells, a method of differentiating into regulatory T cells using the same, The present invention provides a cell therapy agent for immune enhancement comprising regulatory T cells differentiated by the method. The aminoquinoline-based compound, its stereoisomer, derivative, hydrate or pharmaceutically acceptable salt thereof can be used as a preventive or therapeutic agent for immunological diseases since it has an immunostimulating effect.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for promoting differentiation into regulatory T cells, and a method using the composition for promoting differentiation, comprising the aminoquinoline compound, a composition for promoting differentiation into regulatory T cells, }

An aminoquinoline compound, a composition for promoting differentiation into regulatory T cells, and a method for differentiating into regulatory T cells using the same.

Immunity is a biological self-protection system for all external polymeric substances (antigens) that enter or are injected into living tissue. Normal individuals have the ability to recognize, react, and eliminate many non-self antigens, while they do not respond harmfully to antigenic substances that make up the self. Such a non-response of a living body to a self-antigen is called immunologic unresponsiveness or tolerance. When problems arise in inducing or maintaining such self-tolerance, an immune response to the self-antigen occurs, thereby attacking the self-organization, resulting in various autoimmune diseases. Drugs that suppress immune function are mainly used as the main methods for treating such autoimmune diseases. However, because of the large number of side effects, it is difficult to use continuously, and the treatment is limited because it does not prevent recurrence sufficiently.

On the other hand, amodiaquine is an aminoquinoline compound, which is marketed as a therapeutic agent for malaria and has been reported as a therapeutic agent for Parkinson (Korean 10-2008-0106928 A). Amodia queen has already been proven safe, and the drug reposition is highly likely to be useful and useful.

Therefore, for immunity enhancement, it is necessary to search for a substance which is harmless to the human body, has few side effects, and is excellent in immunomodulating function.

One aspect provides an immunoconjugate composition comprising an aminoquinoline-based compound, a stereoisomer, derivative, hydrate or a pharmaceutically acceptable salt thereof.

Another aspect provides a composition for promoting differentiation from T cells to regulatory T cells.

Another aspect provides a method of differentiating T cells into regulatory T cells.

One aspect provides an immunoconjugate composition comprising an aminoquinoline-based compound, a stereoisomer, derivative, hydrate or a pharmaceutically acceptable salt thereof.

The composition may include an aminoquinoline compound represented by the following formula (1), a stereoisomer, a derivative, a hydrate thereof or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

In the above formula (1), R ₁ may be a substituted C ₆ to C ₁₄ aryl group or a substituted C ₂ to C ₆ aminoalkyl group, and the substitution may be a hydroxy group, a C ₁ to C ₅ alkyl group- An aminomethyl group, a C ₁ to C ₅ alkyl group, or a combination thereof. R ₂ may be hydrogen or a C ₁ to C ₅ alkyl group.

Wherein R ₁ is a substituted C ₆ aryl group, the substitution is a hydroxyl group, an aminomethyl group substituted with a C ₁ to C ₅ alkyl group, or a combination thereof, and R ₂ is hydrogen . In Formula 1, R ₁ is a substituted C ₆ aryl group, and the substitution is a hydroxyl group, a diethylaminomethyl group, or a combination thereof, and R ₂ may be hydrogen.

In Formula 1, R ₁ is a substituted C ₂ to C ₆ aminoalkyl group, the substitution is a C ₁ to C ₅ alkyl group, and R ₂ is hydrogen. In Formula 1, R ₁ is a substituted C ₂ to C ₆ aminoalkyl group, and the substitution is a C ₁ alkyl group, a C ₂ alkyl group, or a combination thereof, and R ₂ may be hydrogen .

In that the "optionally substituted (substituted)" will be replaced with a hydrogen atom of a hydroxy group one included in the functional groups or more, an amino group substituted with an alkyl group of C ₁ to C _5, alkyl group of C ₁ to C _5, or a combination thereof, or transforming it means.

"Alkyl" means a fully saturated branched or unbranched (or linear or linear) hydrocarbon. Non-limiting examples of alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, n-pentyl, isopentyl, neopentyl, Hexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl and the like.

The term "aryl" means a residue obtained by removing one hydrogen atom from the nucleus of an aromatic hydrocarbon, and includes a group in which an aromatic ring is fused to one or more carbon rings. Non-limiting examples of aryl include phenyl, naphthyl, tetrahydronaphthyl, and the like.

The formula (1) may be selected from the group consisting of the compounds listed in the following table.

compound Compound name constitutional formula


One
4 - ([7-chloro-4-quinolinyl] amino) -2 - ([diethylamino] methyl)
(4 - ([7-Chloro-4-quinolinyl] amino) -2 - ([diethylamino] methyl)

2
4-N- (7-chloroquinolin-4-yl) -1-N, 1-N-diethylpentane-
(4-N- (7-chloroquinolin-4-yl) -1-N, 1-N-diethylpentane-

The term "stereoisomer" means a compound in which the molecular formula and the connecting method of the constituents are the same but the spatial arrangement between atoms is not the same. The stereoisomer may be a diasteromer or an enantiomer. An enantiomer is an isomer that does not overlap with its mirror image, such as the relationship between the left and right hands, and is also referred to as an optical isomer. Enantiomers are divided into R (Rectus) and S (sinister) counterclockwise when four or more substituents are different on the chiral center carbon. Diastereomers are stereoisomers that are not mirror images of each other, and the spatial arrangement of atoms is different and can be divided into the cis-trans isomers.

The "derivative" means a compound obtained by substituting a part of the structure of the above compound with another atom or atomic group.

The "salt" means an addition salt of an inorganic acid salt, an organic acid salt, or a metal salt of a compound. The salt may be a pharmaceutically acceptable salt. The pharmaceutically acceptable salts may be salts which do not cause serious irritation to the organism to which the compound is administered or to which the compound is administered and which do not impair the biological activity and properties of the compound. The inorganic acid salt may be a hydrochloride, a bromate, a phosphate, a sulfate, or a disulfide. The organic acid salt may be at least one selected from the group consisting of a formate, a acetate, an acetate, a propionate, a lactate, an oxalate, a tartrate, a malate, a maleate, a citrate, a fumarate, a beateate, a camphateate, an edisleite, trichloroacetic acid, But are not limited to, acetic acid salts, benzoic acid salts, gluconic acid salts, methanesulfonic acid salts, glycolic acid salts, succinic acid salts, 4-toluenesulfonic acid salts, Sulfonic acid, or aspartate. The metal salt may be a calcium salt, a sodium salt, a magnesium salt, a strontium salt, or a potassium salt.

The above-mentioned "hydrate" means formed in combination with water molecules, and may include solvates.

The term "immunopotentiation" means to inhibit the function of abnormally activated immune cells to control the inflammatory response. Immune enhancement may be used in the same sense as immunomodulation or immunostaining.

The composition may be to reduce or inhibit the production of inflammatory cytokines. The inflammatory cytokine may be a cytokine secreted by Th1, Th2, or Th17 cells and may include, for example, IFN-y, IL-17, IL-4, IL-5, . The composition may be one that promotes differentiation into regulatory T cells. The composition may be one that increases the activity of regulatory T cells. The composition may be to increase the ratio of regulatory T cells to effector helper T cells (effector T cells). The composition may be to reduce or inhibit the production of inflammatory cytokines, while increasing the activity of differentiated and regulated T cells to regulatory T cells, thereby inhibiting or alleviating the inflammatory response.

Another aspect provides a composition for promoting differentiation from T cells to regulatory T cells, comprising an aminoquinoline-based compound, a stereoisomer, derivative, hydrate or pharmaceutically acceptable salt thereof. The composition may include an aminoquinoline compound represented by Formula 1, a stereoisomer, a derivative, a hydrate thereof, or a pharmaceutically acceptable salt thereof.

The aminoquinoline-based compounds, their stereoisomers, derivatives, hydrates or pharmaceutically acceptable salts thereof are as described above.

The T cell may be a separate T cell or a T cell present in the body. The T cells may be non-contact T cells, for example, non-contact CD4 ⁺ T cells.

The "non-contact T cell" means a T cell not in contact with an antigen. Dendritic cells are antigen presenting cells (APCs), and their main function is to recognize antigens from external pathogens infection or tumor development in the human body and present them to non-contact T cells. Thus, dendritic cells direct various immune responses by stimulating the differentiation of non-contacted CD4 ⁺ T cells into Th1, Th2, Th17, or regulatory T cells. Non-contact T cells may be used in the same sense as undifferentiated T cells.

The above "differentiation" means that the undifferentiated T cell is converted or developed into an inflammatory reactive Th cell or a regulatory T cell and the phenomenon that the structure or function of the cell is specialized, This can be a change in form or function to accomplish the task given to each. That is, the composition may promote the differentiation of T cells into regulatory T cells. The composition may be one that promotes differentiation of undifferentiated T cells into regulatory T cells. The composition may further promote differentiation from regulatory T cells to regulatory T cells.

The composition may be one that promotes the activity of regulatory T cells in T cells, undifferentiated T cells or regulatory T cells. That is, the composition may reduce or inhibit the production of inflammatory cytokines, while increasing the activity of differentiated and regulated T cells to regulatory T cells, thereby inhibiting or alleviating the inflammatory response.

This "activity" means that all the mechanisms of immune cells are promoted or promoted, and may include, for example, the generation of cytokines secreted by immune cells.

The activity of the regulatory T cells means that all the mechanisms of regulatory T cells, that is, natural regulatory T cells and / or adaptive regulatory T cells, are promoted or promoted in vivo, Controlling the inflammatory response by inhibiting the function of abnormally activated immune cells, for example, promoting or enhancing the immunosuppressive response.

The composition may be one that increases the production of cytokines secreted by regulatory T cells. The cytokine secreted by the regulatory T cells may be IL-10.

The regulatory T cells are composed of the IL-2 receptor alpha chain (CD25), Foxp3 (forkhead box P3), cytotoxic T lymphocyte-associated protein 4 (CTLA4, CD152) Glucocorticoid-induced TNFR-related protein (GITR), or a combination thereof. Expression of the CD25, Foxp3, CTLA4, or GITR gene can modulate the differentiation and activity of regulatory T cells. The regulatory T cells may be those expressing positive surface markers, CD25, Foxp3, CTLA4, GITR, or a combination thereof, against the CD25, Foxp3, CTLA4, and GITR markers expressed on the cell surface. The expression of the marker may be increased by 5%, 10%, 20%, 30%, 50%, 70%, 100%, or 200% as the composition is added.

The term "positive" as used herein, in relation to a regulatory T cell marker, means that the marker is present at higher or higher concentrations when compared to other regulatory T cells or inflammatory reactive Th cells on which the marker is based.

The composition may be one that increases the transcriptional activity, mRNA level or protein level of CD25, Foxp3, CTLA4, or GITR.

Expression of the CD25, Foxp3, CTLA4, or GITR gene can be confirmed by measuring transcription (promoter) activity, mRNA level, or protein level. Methods for detecting transcriptional activity levels include, but are not limited to, reporter gene assays such as luciferase and beta -galactosidase enzyme activity assays . In this study, we used RT-PCR (reverse transcription polymerase chain reaction), competitive RT-PCR, Real-time RT-PCR, RNase But are not limited to, RNase protection assay (RPA), Northern blotting, DNA chip, and the like. Methods for detecting protein levels include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), Protein Chip, and the like are available. However, But is not limited thereto.

The increased level of transcriptional activity, mRNA or protein levels of CD25, Foxp3, CTLA4, or GITR indicates that the composition binds to NR4A located within the target gene of nuclear receptor subfamily 4 (NR4A) It is expected that NR4A can bind to an active region (e.g., the NGFI-B response element: NBRE) and thus increase the transcriptional activity, mRNA level or protein level of the target gene. That is, the composition may increase the transcriptional activity, mRNA level or protein level of NR4A. The NR4A may be NR4A1, NR4A2 or NR4A3.

The composition may be one that inhibits proliferation, activity, or a combination of inflammatory reactive Th (effector helper T) cells.

This "proliferation" means that the cells divide and the homogeneous is blown, and the number of cells may be increased quantitatively. The composition may inhibit cell cycle progression and division of inflammatory Th cells.

The composition may be to decrease the activity of inflammatory < RTI ID = 0.0 > Th < / RTI > cells, thereby reducing or inhibiting the production of inflammatory cytokines. The inflammatory cytokine may be a cytokine secreted by Th1, Th2 or Th17 cells and may include, for example, IFN-y, IL-17, IL-4, IL-5 and IL-13 .

The composition may be one which inhibits the differentiation of T cells into inflammatory reactive Th cells.

Another aspect provides a method of differentiating T cells into regulatory T cells, comprising the step of differentiating T cells into regulatory T cells by adding a composition for promoting differentiation to regulatory T cells from said T cells.

The compositions for promoting the differentiation of T cells into regulatory T cells, aminoquinoline compounds, stereoisomers, derivatives, hydrates or pharmaceutically acceptable salts thereof are as described above.

The T cell may be a separate T cell or a T cell present in the body. The T cells may be non-contact T cells, for example, non-contact CD4 ⁺ T cells.

In this method, the step of differentiating may be performed after separating the T cells from the biological sample separated from the individual. Methods for isolating T cells may be performed according to various methods known in the art.

The above-mentioned "adding a composition for promoting differentiation to T cells" may be a step of dispensing the T cells into a medium containing the composition, or adding the composition to the T cells. The addition of the composition for promoting differentiation to the T cell may be such that the T cell and the composition for promoting differentiation are in contact with each other.

In the above method, the step of differentifying may be performed for 2 hours to 700 hours, 10 hours to 350 hours, 15 hours to 100 hours, or 20 hours to 50 hours.

In the above method, the differentiating step may be performed simultaneously with or simultaneously with the application of a T cell receptor (TCR) stimulus. For example, the aminoquinoline compound, its stereoisomer, derivative, hydrate or pharmaceutically acceptable salt may be added to the separated T cells simultaneously with the TCR stimulation, or after the TCR stimulus is applied, the aminoquinoline compound, A stereoisomer, a derivative, a hydrate or a pharmaceutically acceptable salt thereof. For example, after 6 hours, 12 hours, 15 hours, 18 hours, 21 hours, 27 hours, 30 hours, 33 hours, 36 hours, or 48 hours after TCR stimulation on the separated T cells, the aminoquinoline compound , Its stereoisomers, derivatives, hydrates or pharmaceutically acceptable salts thereof.

In this method, the concentration of the aminoquinoline compound, its stereoisomer, derivative, hydrate or pharmaceutically acceptable salt is about 0.1 to 100 μM, about 0.2 to 50 μM, about 0.5 to 20 μM, about 0.75 to 15 μM, 12 μM, about 5 to 12 μM, or about 10 μM.

In the above method, the step of differentiating may be performed simultaneously or sequentially with the addition of a transforming growth factor beta (TGFβ). For example, the aminoquinoline compound, its stereoisomer, derivative, hydrate or pharmaceutically acceptable salt may be added to the T cell simultaneously with the TGF beta, or after the addition of TGF beta, the aminoquinoline compound, its stereoisomer or derivative , Hydrate or a pharmaceutically acceptable salt thereof.

The concentration of TGF [beta] may be 5 to 500 ng / ml, 10 to 250 ng / ml, 25 to 100 ng / ml, or 40 to 60 ng / ml. The differentiating step may be performed without the addition of IL-6.

The method may be to inhibit the proliferation, activation, or a combination of the inflammatory T cells.

Another aspect provides a cell therapy agent for immune enhancement comprising regulatory T cells differentiated by a method of differentiating T cells into regulatory T cells.

The above-mentioned "cell therapeutic agent" may be used to restore the function of cells or tissues, such as autologous, allogenic, xenogenic cells are grown and selected in vitro or the biological characteristics of the cells are changed by other methods Refers to medicines used for the purpose of treatment, diagnosis and prevention through a series of acts. The cell treatment agent may be an immune cell therapeutic agent for controlling an immune response, such as suppression of an in vivo immune response or enhancement of an immune response.

The method of administering the cell therapeutic agent may be parenteral administration. The method of administration may be, for example, transdermal, subcutaneous, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, or intradermal. The cell therapeutic agent may be formulated into a suitable form together with carriers, excipients, or diluents commonly used in cell therapy.

Another aspect is the use of an immunoconjugate composition comprising an aminoquinoline-based compound, a stereoisomer, derivative, hydrate or pharmaceutically acceptable salt thereof, to a subject in need thereof, / RTI > The composition may include an aminoquinoline compound represented by the following formula (1), a stereoisomer, a derivative, a hydrate thereof or a pharmaceutically acceptable salt thereof.

The subject may be a mammal, including a mouse, rat, dog, pig, cat, cow, horse, monkey, camel, and human.

The preferred dosage of the immunoconjugate composition varies depending on the condition and body weight of the individual, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by a person skilled in the art. The immunoconjugate composition may be administered to the individual by various routes. The method of administration may be oral or parenteral administration. The method of administration may be, for example, oral, transdermal, subcutaneous, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, topical, or intradermal routes. The daily dosage is about 0.0001 to 1000 mg / kg, about 0.001 to 100 mg / kg, about 1 to 100 mg / kg, about 2 to 50 mg / kg, about 5 to 20 mg / kg, May be administered once or several times a day. The administration period may be from 1 day to 1 year, from 1 day to 6 months, from 1 day to 3 months, from 2 days to 40 days, from 4 days to 20 days, or from 5 days to 15 days.

Another aspect provides a pharmaceutical composition for enhancing immunity comprising an aminoquinoline compound, a stereoisomer, a derivative, a hydrate thereof, or a pharmaceutically acceptable salt thereof. The composition comprises the aminoquinoline compound represented by Formula 1, A stereoisomer, a derivative, a hydrate or a pharmaceutically acceptable salt thereof.

The pharmaceutical composition may further comprise a carrier, an excipient or a diluent. The carrier, excipient or diluent may be, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.

The pharmaceutical composition may have an oral or parenteral dosage form. The pharmaceutical compositions can be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods. In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used.

In the above pharmaceutical composition, the solid preparation for oral administration may be a tablet, a pill, a powder, a granule, or a capsule. The solid preparation may further contain an excipient or a lubricant. The excipient may be, for example, starch, calcium carbonate, sucrose, lactose, or gelatin. The lubricant may also be magnesium stearate, or talc. In the above pharmaceutical composition, the liquid preparation for oral administration may be a suspension, a solution, an emulsion, or a syrup. The liquid formulation may comprise water, or liquid paraffin. The liquid preparations may contain excipients such as wetting agents, sweetening agents, perfumes, or preservatives. In the above pharmaceutical composition, the preparation for parenteral administration may be a cream, a lotion, an ointment, a liquid, a patch, a sachet, an injection, a sterilized aqueous solution, a nonaqueous solution, a suspension, an emulsion, have.

The pharmaceutical composition may contain an aminoquinoline compound represented by the general formula (1), a stereoisomer, a derivative, a hydrate thereof or a pharmaceutically acceptable salt thereof in an effective amount. The effective amount will depend on factors such as the severity of the disease, the age, weight, health, sex, sensitivity, time of administration, route of administration and rate of excretion, duration of treatment, drugs used in combination or concurrently with the pharmaceutical composition, Can be determined according to well-known factors. The daily dosage is about 0.0001 to 1000 mg / kg, about 0.001 to 100 mg / kg, about 1 to 100 mg / kg, about 2 to 50 mg / kg, about 5 to 20 mg / kg, May be administered once or several times a day. The administration period may be from 1 day to 1 year, from 1 day to 6 months, from 1 day to 3 months, from 2 days to 40 days, from 4 days to 20 days, or from 5 days to 15 days.

The immunological diseases in which the pharmaceutical composition achieves the immunoenhancing effect and are to be treated or prevented may be a disease caused by a decrease in the activity of regulatory T cells. Such immunological diseases include, for example, osteoarthritis, rheumatoid arthritis, asthma, dermatitis, psoriasis, cystic fibrosis, post-transplantation late post-transplantation late graft-versus-host disease, graft-versus-host disease, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, Hashimoto thyroid gland, (Hashimoto thyroiditis), polymyositis, scleroderma, Addison disease, vitiligo, pernicious anemia, glomerulonephritis and pulmonary fibrosis, inflammatory growth Inflammatory bowel disease, inflammatory bowel disease, Crohns disease, autoimmune diabetes, diabetic retinopathy, rhinitis, ischemia-reperfusion injury (Isc post-angioplasty restenosis, chronic obstructive pulmonary disease (COPD), Graves disease, gastrointestinal allergy, conjunctivitis, hemorrhagic fever, Atherosclerosis, coronary artery disease, angina, cancer metastasis, small artery disease, and mitochondrial disease.

Another aspect provides an immunomodulating food composition comprising an aminoquinoline-based compound, a stereoisomer, derivative, hydrate or pharmaceutically acceptable salt thereof. The composition may include an aminoquinoline compound represented by the general formula (1), a stereoisomer, a derivative, a hydrate thereof or a pharmaceutically acceptable salt thereof.

The aminoquinoline-based compound represented by Formula 1, its stereoisomer, derivative, hydrate or pharmaceutically acceptable salt thereof may be formulated into an encapsulated, powdered, or a suspension to be used as a functional food or added to various foods. The food may be, for example, meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinks, tea, Functional food, and health food.

The aminoquinoline-based compound, its stereoisomer, derivative, hydrate or pharmaceutically acceptable salt thereof has an immunostimulating effect and can be used as a composition for immunostimulating or for the prophylactic or therapeutic treatment of immune diseases.

1A and 1C show the degree of cleavage of T cells when 1 μM, 5 μM, and 10 μM of AQ were added to inflammatory reactive Th cells, respectively. In FIG. 1A, B shows AQ of 1 μM, 5 μM, and 10 μM, respectively, And the degree of cleavage of T cells. FIG. 1B is a graph showing the number of total cells according to cell division of T cells when AQ and CQ are added. FIG.
In FIG. 2, A is a graph showing the level of IL-2 production in inflammatory reactive Th cells and regulatory T cells when AQ is added in a concentration-dependent manner. 2, B is a graph showing IL-2 mRNA levels in inflammatory reactive Th cells and regulatory T cells when AQ is added in a concentration-dependent manner. 2, C is a graph showing the expression levels of IL-2 in inflammatory reactive Th cells and regulatory T cells when AQ is added in a concentration-dependent manner.
3A is a graph showing the expression levels of CD25 and Foxp3 in regulatory T cells when AQ is added in a concentration-dependent manner. 3A, C is a graph showing the expression level of CTLA4 in regulatory T cells when AQ is added in a concentration-dependent manner. 3A and 3B are graphs showing mRNA levels of Foxp3, CTLA4, GITR and IL-10 in regulatory T cells when AQ was added. 3A is a graph showing the level of IL-10 production in regulatory T cells when AQ is added in a concentration-dependent manner. Figure 3b is when cultured in differentiation non the detached condition and the CD4 ⁺ T cell differentiation into Th1 cells, conditions, process the AQ and CQ, a graph showing the production level of IFN-γ.
In FIG. 4, A is a graph showing the expression levels of CD25, CD122 and CD132 in regulatory T cells when AQ is added in a concentration-dependent manner. In FIG. 4, B is a graph showing the expression level of CD25 in regulatory T cells with the addition of AQ and the passage of time. 4, C is a graph showing mRNA levels of CD25, CD122 and CD132 in regulatory T cells when AQ is added in a concentration-dependent manner.
In Figure 5, A is a graph showing the expression levels of CD25 in the presence of anti-IL-2 and anti-CD25 antibodies. 5, B and C are graphs showing mRNA levels of CD25 and CD132 in the presence of anti-IL-2 and anti-CD25 antibodies.
6 is a graph showing the activity of NBRE-luc luciferase enzyme when AQ was added to 293T cells to which NR4A1, NR4A2, and NR4A3 were respectively introduced and expressed. In FIG. 6, B is an image showing the degree of binding of NR4A1, NR4A2, and NR4A3 to NBRE when AQ is added to 293T cells transfected with NR4A1, NR4A2, and NR4A3, respectively. In FIG. 6, 1 of B represents the amount of the protein bound to the NBRE, and 2 represents the amount of the introduced protein. 6, C is a graph showing the luciferase activity of CD25-luc when AQ was added to 293T cells into which NR4A1, NR4A2, and NR4A3 were introduced, respectively. In FIG. 6, D is an image showing the degree of binding of NR4A1, NR4A2, and NR4A3 to NBRE in the promoter region of CD25 when AQ is added to 293T cells into which NR4A1, NR4A2, and NR4A3 have been introduced, respectively. In Fig. 6, 3 of B represents the amount of the protein bound to the NBRE of CD25, and 4 represents the amount of introduced protein.
7, A and B are graphs showing the body weight and the colon length in the DSS-administered experimental group and AQ-treated control group, respectively. In FIG. 7, C is an image stained with H & E and PAS in an experimental group and a control group to which DSS was administered and AQ and vehicle were respectively administered. In FIG. 7, D is an image obtained by administering non-contact T cells and staining an experimental group and a control group, to which AQ and vehicle were respectively administered, with H & E. In Fig. 7, E is an image showing the expression levels of Foxp3, RORγt and T-bet when immunized with AQ by immunoblot analysis. In Fig. 7, F is a graph showing the levels of IL-17 and IFN-y production when AQ was administered.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these embodiments are for illustrative purposes only, and the scope of the present invention is not limited to these embodiments.

Example  One. Aminoquinoline-based  Identification of regulatory T cell differentiation and facilitating activity of compounds

One. Aminoquinoline-based  Preparation of compounds

Aminoquinoline compound amodiaquine (AQ) was purchased from Sigma-Aldrich (St. Louis, Mo.) (amodiaquine hydrochloride, 4 - ([7-Chloro-4-quinolinyl] diethylamino] methyl) phenol, MW = 464.81, purity> 98%) and 100% purity in dimethyl sulfoxide (DMSO). Chloroquine (CQ), an aminoquinoline compound, was purchased from Sigma-Aldrich (St. Louis, Mo.) (Chloroquine phosphate, N4- (7-Chloro-4-quinolinyl) -N, pentanediamine diphosphate salt, Chloroquine diphosphate salt, and phosphate buffered saline (PBS). The aminoquinoline compound stock was frozen and stored.

2. Mouse

Male, C57BL6 mice and 6-week-old male, RAG2 (recombination activation gene 2) knockout (KO) mice at 8-10 weeks of age were raised in an aseptic animal laboratory in Ewha Womans University. All animal handling and testing was approved by the Institutional Animal Care and Use Committee (IACUC No. 2012-01-071, 2014-01-011) and performed in accordance with institutional, national and international laws and guidelines.

3. CD4 ⁺  T cell separation and culture

The lymph nodes were isolated from the mice and CD4 ⁺ T cells were isolated using mini MACS beads (Miltenyi Biotec, Auburn, Calif.) With anti-CD4 antibody according to the manufacturer's instructions. Isolated CD4 ⁺ T cells were plated on plates coated with anti-CD3 (2 g / ml) and anti-CD28 (1 g / ml) IL-2 (10 U / ml) was added and incubated for 24 hours to activate. AQ and CQ were added to the cells and cultured respectively or the cells were treated with differentiation into regulatory T cells containing 5 占 퐂 / ml of anti-IL-4, 5 占 퐂 / ml of anti-IFN-y and 50 ng / ml of TGF? AQ and CQ were added and cultured.

4. AQ TGFβ  On the anti-proliferative activity of regulatory T cells induced by

AQ was confirmed that inhibits cell cycle progression in differentiated conditions to regulatory T cells. The CD4 ⁺ T cells isolated from 3-carboxy fluorescein diacetate succinimidyl ester of 5 μM: a (carboxyfluorescein diacetate succinimidyl ester CSFE) ( Sigma-Aldrich) , and the cover, wherein -CD3 (2 ㎍ / ㎖), and wherein -CD28 (1 [mu] g / ml) was coated on the coated plate and incubated for 24 hours to activate. AQ was then added to the cells and cultured for 24 or 48 hours before being obtained for flow cytometry.

FIG. 1A is a graph showing the degree of cleavage of T cells when AQ is added in a concentration-dependent manner under conditions of differentiation into inflammatory Th cells and regulatory T cells. In FIG. 1, A and C show the degree of division of T cells when 1 μM, 5 μM, and 10 μM of AQ are added to inflammatory reactive Th cells, respectively. In FIG. 1A, B shows AQ of 1 μM, 5 μM and 10 μM, respectively, And the degree of cleavage of T cells. As shown in Fig. 1A, AQ inhibited cell division of inflammatory Th cells in a concentration-dependent manner, and markedly inhibited cell cycle progression of T cells under non-differentiation conditions. In particular, at 48 hours after T cell receptor (TCR) stimulation, the population of cells with a high number of cell divisions among the cell populations differentiating into inflammatory Th cells decreased in a dose dependent manner. On the other hand, AQ suppressed the cell division of T cells somewhat in the condition of differentiation into regulatory T cells, but this inhibitory activity was lower than the inhibition of cell division of inflammatory reactive Th cells.

AQ and CQ were cytotoxic in T cells. 3, AQ and CQ were added to CD4 ⁺ T cells, respectively, and cytotoxicity was confirmed using an EZ-CYTOX assay kit according to the manufacturer's instructions. FIG. 1B is a graph showing the number of total cells according to cell division of T cells when AQ and CQ are added. FIG. As shown in FIG. 1B, the number of CD4 ⁺ T cells decreased when 10 μM of AQ and CQ were added. However, there was no significant cytotoxicity.

5. Effect of AQ on the production of IL-2 in differentiation conditions of regulatory T cells

IL-2 < / RTI > production and expression level, which is a growth factor of T cells.

(5.1) Analysis of cytokine production by enzyme immunoassay (EIA)

AQ on the production of IL-2 in inflammatory Th cells and regulatory T cells. The supernatant of cultured T cells was obtained under differentiation conditions into inflammatory Th cells and regulatory T cells, reacted with an IL-2 antibody on an EIA plate, and reacted with a secondary antibody polymerized with phosphatase. Subsequently, p-nitrophenylphosphate, a substrate of phosphatase, was added to develop color, and the degree of color development was measured using a microplate reader (Molecular Devices, Sunnyvale, Calif.). In FIG. 2, A is a graph showing the level of IL-2 production in inflammatory reactive Th cells and regulatory T cells when AQ is added in a concentration-dependent manner.

(5.2) Reverse transcription  And real-time PCR mRNA  Level analysis

AQ on IL-2 mRNA levels in inflammatory Th cells and regulatory T cells. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, Calif.) And cDNA was synthesized using reverse transcriptase (Promega, Madison, Wis.). Using the synthesized cDNA as a template and using the IL-2 specific primer set described in Table 2 below and a StepOnePlus (Applied Biosystems Inc., Foster City, Calif.), Quantitative real-time PCR Quantitative real-time polymerase chain reaction was performed. Transcription levels of the following genes were normalized with actin. 2, B is a graph showing IL-2 mRNA levels in inflammatory reactive Th cells and regulatory T cells when AQ is added in a concentration-dependent manner.

gene SEQ ID NO: 5 '- > 3' IL-2 SEQ ID NO: 1 5'-cctgagcaggatggagaattaca-3 ' SEQ ID NO: 2 5'-tccagaacatgccgcagag-3 ' CD25 SEQ ID NO: 3 5'-cgttgcttaggaaactcctgga-3 ' SEQ ID NO: 4 5'-gctttctcgatttgtcatggg-3 ' CD122 SEQ ID NO: 5 5'-cgtccatgccaagtcgaac-3 ' SEQ ID NO: 6 5'-gatgcctgcctcacaagactt-3 ' CD132 SEQ ID NO: 7 5'-ggttggaacgaatgcctccaattc-3 ' SEQ ID NO: 8 5'-gcagaaccgttcactgtagtctgg-3 ' CTLA -4 SEQ ID NO: 9 5'-gggctcagttgcataaaccga-3 ' SEQ ID NO: 10 5'-acactgccagcttttggtttcc-3 ' GITR SEQ ID NO: 11 5'-aaggttcagaacggaagtg-3 ' SEQ ID NO: 12 5'-gggtctccacagtggtact-3 ' IL-10 SEQ ID NO: 13 5'-tttgaattccctgggtgagaa-3 ' SEQ ID NO: 14 5'-acaggggagaaatcgatgaca-3 ' NR4A1 SEQ ID NO: 15 5'-gttgatgttcccgcctttg-3 ' SEQ ID NO: 16 5'-cctggagcccgtgtcg-3 ' NR4A2 SEQ ID NO: 17 5'-gctgccctggctatggt-3 ' SEQ ID NO: 18 5'-caggtagttgggtcggtt-3 ' NR4A3 SEQ ID NO: 19 5'-cactgatctccccaaagaag-3 ' SEQ ID NO: 20 5'-gcaggacaagtccattgc-3 ' Actin
(actin) SEQ ID NO: 21 5'-agagggaaatcgtgcgtgac-3 ' SEQ ID NO: 22 5'-caatagtgatgacctggccgt-3 '

(5.3), expression analysis by flow cytometry (flow cytometry)

AQ on the expression level of IL-2 in inflammatory Th cells and regulatory T cells. To stain intracellular cytokines, the cells were incubated for 6 hours with Phorbol myristate acetate (PMA) and ionomycin, or anti-CD3 (1 ug / ml) And cultured with monensin (4 μM) for 2 hours before cells were obtained. The cells were then allowed to permeate and incubated with anti-mIL-2 antibody conjugated with phycoerythrin (PE) followed by flow cytometry and CellQuest analysis (BD Biosciences). 2, C is a graph showing the expression levels of IL-2 in inflammatory reactive Th cells and regulatory T cells when AQ is added in a concentration-dependent manner.

As shown in Fig. 2, A, B, and C, IL-2 production by regulatory T cells was significantly increased in a concentration-dependent manner, even though total cell number decreased. Similarly, transcription of the IL-2 gene was significantly increased by AQ regardless of the introduction of exogenous IL-2. In addition, the number of cells producing IL-2 in the condition of differentiation into regulatory T cells was further increased by adding AQ. On the other hand, IL-2 production by inflammatory Th cells did not show any significant difference when AQ was added.

6. AQ is CD25 ⁺ Foxp3 ⁺ Antigen Control with characteristics  Analysis of effects on differentiation into T cells

AQ on the differentiation into regulatory T cells.

(6.1) Flow cell  Analysis of expression by analysis

AQ on the expression levels of CD25 and Foxp3 in regulatory T cells. CD25 < / RTI > and Foxp3 antibody, and CTLA4 antibody. 3A is a graph showing the expression levels of CD25 and Foxp3 in regulatory T cells when AQ is added in a concentration-dependent manner. 3A, C is a graph showing the expression level of CTLA4 in regulatory T cells when AQ is added in a concentration-dependent manner. As shown in FIGS. 3A and 3C, when AQ was added, expression levels of CD25 and Foxp3, and CTLA4, which are markers of regulatory T cells, were increased.

(6.2) Real-time polymerase chain reaction mRNA  Level analysis

AQ on mRNA levels of Foxp3, CTLA4, GITR and IL-10 in regulatory T cells. The same method as in (5.2) was carried out except that the primers specific for Foxp3, CTLA4, GITR and IL-10 described in Table 2 were used. 3A and 3B are graphs showing mRNA levels of Foxp3, CTLA4, GITR and IL-10 in regulatory T cells when AQ was added. As shown in B and D in Fig. 3A, when AQ was added, the transcription of Foxp3 was significantly and simultaneously increased in a concentration-dependent manner. In addition, mRNA levels of CTLA4, GITR and IL-10, surface markers of regulatory T cells, were increased.

(6.3) Analysis of cytokine production by enzyme immunoassay 1

AQ on the production of IL-10 in regulatory T cells. Was performed in the same manner as in (5.1) except that the cells were incubated with an IL-10 antibody on an EIA plate. 3A is a graph showing the level of IL-10 production in regulatory T cells when AQ is added in a concentration-dependent manner. As shown in Figure 3A, IL-10 production was increased when AQ was added during differentiation into regulatory T cells.

(6.4) Analysis of cytokine by enzyme immunoassay 2

3 ^< / RTI & gt ^; CD4 ^{< +} & gt ^; T cells were added AQ and CQ, and a supernatant of the cultured T cells was obtained, Was performed in the same manner as in (5.1), except that the antibody was incubated with an IFN-? Antibody on an EIA plate. In addition, 3 separate CD4 ⁺ T cells were cultured under differentiation conditions to Th1 cells, and the same procedure as in (5.1) was performed except that AQ and CQ were added and incubated with an IFN-? Antibody on an EIA plate .

Figure 3b is when cultured in differentiation non the detached condition and the CD4 ⁺ T cell differentiation into Th1 cells, conditions, process the AQ and CQ, a graph showing the production level of IFN-γ. As shown in Fig. 3B, when AQ and CQ were added, the production of IFN-y, a cytokine of Th1, decreased.

7. Effect of AQ on the expression of IL-2 receptor CD25 in regulatory T cells

AQ directly affects the differentiation of the IL-2 receptor, CD25 (IL-2R?), CD122 (IL-2R?) And CD132 (? Common chain) in regulatory T cells.

(7.1) Flow cell  Analysis of expression by analysis

AQ on the expression levels of CD25, CD122 and CD132 in regulatory T cells. In order to stain flow cytometry and markers on the cell surface, cells were fixed and incubated with the CD25, CD122, and CD132 antibodies in the same manner as (5.3). In FIG. 4, A is a graph showing the expression levels of CD25, CD122 and CD132 in regulatory T cells when AQ is added in a concentration-dependent manner. In FIG. 4, B is a graph showing the expression level of CD25 in regulatory T cells with the addition of AQ and the passage of time. As shown in A and B in Fig. 4, when AQ was added, expression levels of CD25 were increased on the surface of regulatory T cells. On the other hand, the expression levels of CD122 and CD132 did not show a significant difference. Expression of CD25 was increased by TCR stimulation, but decreased with time in regulatory T cell differentiation, but when AQ was added, expression level was maintained in the late phase of regulatory T cell differentiation.

(7.2) Real-time polymerase chain reaction mRNA  Level analysis

AQ was found to affect CD25, CD122 and CD132 mRNA levels in regulatory T cells. The same procedure as in (5.2) was carried out except that the primers specific for CD25, CD122 and CD132 described in Table 2 were used.

4, C is a graph showing mRNA levels of CD25, CD122 and CD132 in regulatory T cells when AQ is added in a concentration-dependent manner. As shown in FIG. 4C, when AQ was added, transcription of CD25 was significantly increased. On the other hand, there was no significant difference in mRNA levels of CD122 and CD132 when AQ was added.

8. Analysis of Relation between Increased Expression of CD25 and Activation of IL-2 / IL-2R Signal in Regulatory T Cells by Addition of AQ

In the presence of AQ, the increased expression of CD25 was confirmed by activation of the IL-2 / IL-2R signal. In Figure 5, A is a graph showing the expression levels of CD25 in the presence of anti-IL-2 and anti-CD25 antibodies. When AQ was added, the expression level of CD25 was increased on the surface of regulatory T cells with anti-IL-2 antibody added. In addition, regardless of the addition of AQ, the CD25 blocking antibody reduced the intensity of CD25 staining on the surface of regulatory T cells. 5, B and C are graphs showing mRNA levels of CD25 and CD132 in the presence of anti-IL-2 and anti-CD25 antibodies. When AQ was added, mRNA level of CD25 was increased regardless of IL-2 neutralization and CD25 blockade, and CD132 expression level was not significantly different. In other words, AQ increases CD25 expression independent of the IL-2 / IL-2R signal.

9. Mechanism of AQ promoting transcriptional activity of CD25

Since AQ is a ligand of NR4A and is expected to promote the transcription activity of a target gene mediated by NR4A, it was confirmed that AQ promotes the DNA binding activity of NR4A to increase the transcription activity of the target gene of NR4A.

(9.1) Reporter enzyme activity analysis

Expression vectors of NR4A1, NR4A2, or NR4A3 and a reporter vector of NBRE-luc or pCD25-luc containing the NR4A-binding region were transfected into 293T cells to which foreign gene introduction was easy. The pCD25-luc reporter vector was used in which the -600 to +10 region of the mouse CD25 promoter was linked to the pGL2-basic reporter gene (Promega, Madison, Wis.). Luciferase activity was measured using a luciferase assay kit (Promega) and normalized to? -Galactosidase activity. β-Galactosidase activity was measured by transfecting 293T cells with the pCMVβ vector and using a Galacto-light system kit (Thermo Fisher Scientific, Carlsbad, Calif.).

In FIG. 6, A is a graph showing the activity of NBRE-luc luciferase enzyme when AQ is added to 293T cells expressing NR4A1, NR4A2, and NR4A3, respectively. As shown in Fig. 6A, the luciferase enzyme activity of NBRE-luc increased when overexpressing NR4A1, NR4A2, or NR4A3, and further increased when AQ was added. 6, C is a graph showing the luciferase activity of CD25-luc when AQ was added to 293T cells into which NR4A1, NR4A2, and NR4A3 were introduced, respectively. As shown in Fig. 6C, the luciferase enzyme activity of CD25-luc was markedly increased upon overexpression of NR4A1, further increased by addition of AQ.

(9.2) DNA binding pulldown ) analysis

293T cells were transfected with expression vectors of NR4A1, NR4A2, or NR4A3, and cultured with addition of AQ. Proteins were then extracted using HKMG buffer. The HKMG buffer was used containing 10 mM HEPES (pH 7.9), 100 mM KCl, 5 mM MgCl 2, 1 mM DTT, 0.1% NP-40, and 10% glycerol. The extracted proteins were reacted with biotinylated double-stranded DNA containing NBRE, which is presumed in the NBRE or mouse CD25 promoter, for 1 hour. Thereafter, the protein-DNA binding complex was precipitated with streptavidin-agarose beads, proteins in the precipitate were separated by polyacrylamide gel electrophoresis (SDS-PAGE), and polyvinylidene And transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was reacted with the primary antibodies, NR4A1, NR4A2, and NR4A3 antibodies (Cell Signaling Technology, Beverly, Mass.) And sequentially reacted with the secondary antibody, a secondary antibody conjugated with peroxidase. Protein bands were then confirmed using enhanced chemiluminescence (ECL) and autoradiography.

In Fig. 6, B is an image showing the degree of binding of NR4A1, NR4A2, and NR4A3 to NBRE when AQ is added to 293T cells expressing NR4A1, NR4A2, and NR4A3, respectively. As shown in Figure 6, B, the DNA binding activity of NR4A1, NR4A2, or NR4A3 was further increased when AQ was added. 6, D is an image showing the degree of binding of NR4A1, NR4A2, and NR4A3 to NBRE in the promoter region of CD25, respectively, when AQ was added to 293T cells expressing NR4A1, NR4A2, and NR4A3, respectively. As shown in Fig. 6D, NR4A1 specifically binds to NBRE in the CD25 promoter region, and binding is further increased by AQ treatment.

(9.3) Real-time polymerase chain reaction mRNA  Level and protein level analysis

6, E and F are graphs showing mRNA and expression levels of NR4A1, NR4A2, and NR4A3 when AQ is added to CD4 ⁺ T cells of 3. As shown in E and F in Fig. 6, mRNA and expression levels of NR4A1 were further increased when AQ was added.

10. AQ is in the system (in vivo ) On Colitis in Korea

In vivo, we confirmed whether AQ inhibited the inflammatory response.

(10.1) Histological analysis of inflammatory reaction by colitis 1

Male C57BL6 mice (18) between 10 and 12 weeks of age were dosed with water containing 3% DSS (MP Biomedicals, Solon, OH) for 6 days. The experimental group injected AQ (10 mg / kg) daily with injection peritoneal (ip). Control group was injected with intraperitoneal injection of vehicle. The body was monitored daily for the presence of stool and stool, and the length of the colon at the time of dissection was measured. Colonic tissue sections were stained with hematocylin and eosin (H & E) and periodic-acid-Schiff (PAS).

7, A and B are graphs showing the body weight and the colon length in the experimental group administered with DSS and the control group administered with AQ, and the control group administered with vehicle. Referring to FIGS. 7A and 7B, the weight of the mice was decreased with the administration of DSS, and the weight reduction in the experimental group to which AQ was administered was alleviated. FIG. 7 is a graph showing images obtained by DSS-administered, H & E and PAS-stained images in the experimental group and the control group in which AQ and vehicle were respectively administered. As shown in Fig. 7C, AQ decreased immune cell invasion and inhibited epithelial cell damage.

(10.2) Histological analysis of inflammatory reaction by colitis 2

To induce colitis with T cells, lymph nodes were isolated from C57BL6 mice and cultured using a naive T cell enrichment kit (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions, Non-contact T cells were isolated. Isolated, untouched T cells were injected intraperitoneally into RAG KO mice at 6 weeks of age. After 6 weeks of injection, the experimental group was injected with peritoneal injection of AQ (10 mg / kg) daily for 2 weeks. In the control group, the vehicle was injected by intraperitoneal injection. The body weight, stool status, and whether the stool was present were monitored daily, and the length of the colon was measured at the time of dissection. Colonic tissue sections were stained with H & E and PAS. In FIG. 7, D is a graph showing an image of H & E staining of an experimental group and a control group to which non-contact T cells were administered and AQ and vehicle were respectively administered. As shown in Fig. 7D, colitis was induced in RAG KO mice by injected T cells, and the inflammatory response was remarkably inhibited when AQ was administered.

(10.3) Inflammatory reactivity Th  Analysis of expression levels of transcription factors in cells and regulatory T cells

A protein lysate was obtained from the spleen of the mouse of (10.2) using RIPA lysis buffer. The extracted proteins were separated by SDS-PAGE and transferred to a PVDF membrane. The PVDF membrane was incubated with the primary antibody, Foxp3 antibody (Cell Signaling Technology, Beverly, MA), RORγt, T-bet and actin antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.), And incubated with secondary antibody conjugated with peroxidase, an antibody. After that, the protein bands were detected by ECL and radiometric automatic method. In Fig. 7, E is an image showing the expression levels of Foxp3, RORγt and T-bet when immunized with AQ by immunoblot analysis. As shown in Figure 7E, when AQ was administered, the expression of Foxp3, a marker of regulatory T cells, was increased, but the expression level of RORγt and T-bet, which are markers of inflammatory reactive Th cells, Th17 and Th1, Respectively.

(10.4) Inflammatory reactivity Th  Cell cytokine measurement 1

(10.2), the supernatant of the cultured T cells was obtained, and incubated with the IL-17 and IFN-? Antibodies on the EIA plate. . In FIG. 7, F is a graph showing the levels of IL-17 and IFN-y production when AQ was administered. As shown in Fig. 7F, when AQ was administered, the production of IL-17, a cytokine of Th17 cells, and IFN-y, a cytokine of Th1, decreased.

11. Statistical Analysis

All data were expressed as mean ± SD of three or more independent experiments. Statistical significance was calculated using one-way analysis of variance (ANOVA) and Student's t-test. Statistical significance was expressed as follows. *, P <0.05; **, P <0.005; and ***, p < 0.0005.

<110> Ewha University-Industry Collaboration Foundation EWHA WOMANS UNIVERSITY <120> Composition for immunopotentiatingz          aminoquinoline-based compound, composition for promoting          development into regulatory T cell, and method using the same <130> PN117519 <160> 22 <170> Kopatentin 2.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> IL-2 primer <400> 1 cctgagcagg atggagaatt aca 23 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> IL-2 primer <400> 2 tccagaacat gccgcagag 19 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CD25 primer <400> 3 cgttgcttag gaaactcctg ga 22 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CD25 primer <400> 4 gctttctcga tttgtcatgg g 21 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> CD122 primer <400> 5 cgtccatgcc aagtcgaac 19 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CD122 primer <400> 6 gatgcctgcc tcacaagact t 21 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CD132 primer <400> 7 ggttggaacg aatgcctcca attc 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CD132 primer <400> 8 gcagaaccgt tcactgtagt ctgg 24 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CTLA-4 primer <400> 9 gggctcagtt gcataaaccg a 21 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CTLA-4 primer <400> 10 acactgccag cttttggttt cc 22 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GITR primer <400> 11 aaggttcaga acggaagtg 19 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GITR primer <400> 12 gggtctccac agtggtact 19 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL-10 primer <400> 13 tttgaattcc ctgggtgaga a 21 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL-10 primer <400> 14 acaggggaga aatcgatgac a 21 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> NR4A1 primer <400> 15 gttgatgttc ccgcctttg 19 <210> 16 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> NR4A1 primer <400> 16 cctggagccc gtgtcg 16 <210> 17 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> NR4A2 primer <400> 17 gctgccctgg ctatggt 17 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> NR4A2 primer <400> 18 caggtagttg ggtcggtt 18 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NR4A3 primer <400> 19 cactgatctc cccaaagaag 20 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> NR4A3 primer <400> 20 gcaggacaag tccattgc 18 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> actin primer <400> 21 agagggaaat cgtgcgtgac 20 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> actin primer <400> 22 caatagtgat gacctggccg t 21

Claims (12)

1. An immunomodulating composition comprising an aminoquinoline compound represented by the following formula (2), a stereoisomer thereof, a hydrate thereof or a pharmaceutically acceptable salt thereof:
(2)

. delete delete A composition for promoting the differentiation of isolated T cells into regulatory T cells, comprising an aminoquinoline compound represented by the following formula (2), a stereoisomer, a hydrate thereof or a pharmaceutically acceptable salt thereof:
(2)

. delete delete 5. The composition of claim 4, wherein the T cell is a non-contact CD4 ⁺ T cell. 5. The composition of claim 4, wherein said regulatory T cells express CD25, Foxp3, CTLA4, GITR, or a combination thereof. 5. The composition of claim 4, wherein the composition inhibits proliferation, activity, or a combination thereof, of an inflammatory reactive Th (Thorough) T cell. And dividing the T cells into regulatory T cells by adding the composition of claim 4 to the T cells separated from the subject. [Claim 11] The method according to claim 10, wherein the differentiating step is performed simultaneously with or sequentially with the addition of a transforming growth factor beta (TGF [beta]). 11. The method of claim 10, wherein the composition inhibits proliferation, activity, or a combination thereof, of an inflammatory reactive Th cell.

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