JP6815943B2 - Composition for Inducing Foxp3-Positive T Cells and Method for Producing Foxp3-Positive T Cells - Google Patents

Description

The present invention relates to a composition for inducing Foxp3-positive T cells and a method for producing Foxp3-positive T cells.

Allergy refers to "a phenomenon in which symptoms that are detrimental to the living body are caused through an antigen-specific immunological mechanism caused by foreign substances such as mites, dust, pollen, and food" (for example, non-patent documents). See 1). In addition, an autoimmune disease refers to "a symptom that is detrimental to a living body caused by an attack by immunity by regarding one's own cells or hormones as foreign substances". In both cases, the immune system, which is supposed to work against what is harmful to the living body, over-functions against what is harmless, causing detrimental symptoms to the living body.

However, the living body also has a system that suppresses such an overreaction of immunity. For example, oral immune tolerance, which is a mechanism that does not cause an immune response to foreign proteins, is controlled so that food allergies do not occur (see, for example, Non-Patent Document 2). In other words, food allergy is considered to be a condition in which immune tolerance to a specific food does not work well.

Until now, allergy treatment has mainly prevented the onset of allergies by thoroughly removing allergens. However, the life of removing allergens puts a great deal of psychological stress on patients and their families. Moreover, unless oral immune tolerance to the causative allergen is induced in the living body, it is not a radical treatment. With regard to food allergies, oral immunotherapy, which induces the acquisition of resistance to the causative allergen by ingesting small amounts of food containing the causative allergen, has been clinically studied as a therapeutic method aiming at a fundamental treatment. However, since anaphylactic shock often occurs during the course of treatment and serious side reactions may occur, this treatment method is not recommended as general practice at this stage (see, for example, Non-Patent Document 2). ). Currently, coping therapy is the main treatment for autoimmune diseases, and it is difficult to treat it with the aim of complete cure.

Therefore, if it becomes possible to induce immune tolerance efficiently, it is possible to safely treat allergies and autoimmune diseases, and to prevent the onset of people who have not yet developed these diseases. Can be done.

Various immune cells are involved in the immune system in the body. T cells are known as important cells responsible for the immune system, and their name comes from the fact that they are cells made in the thymus. T cells are classified into dozens or more types according to the type and number of cell surface markers expressed, and each plays an individual role.

Regulatory T cells are known as one type of T cells, and among them, regulatory T cells positive for Foxp3 (hereinafter referred to as Foxp3-positive T cells, Treg, etc.) are antigen-specifically abnormal or abnormal. It is known that it can play an important role in immune tolerance, suppressing the action of excess other T cells. For example, it has been reported that indigenous bacteria induce Tregs to maintain immune homeostasis in the human intestine, and that the number and population of Tregs affect the development of allergies and autoimmune diseases (non-). (See Patent Documents 3 and 4).

Regarding the effect of nicotinamide adenine dinucleotide (NAD) on Foxp3-positive T cells, intravenous administration of NAD reduces Foxp3-positive T cells and activates Foxp3-positive T cells in tumor tissue (Non-Patent Document 5). (See), NAD is known to convert Foxp3-positive T cells to Th17 cells (see Non-Patent Document 6). It is also known that NAD can act on helper T cells such as Th1 cells, Th2 cells, and Th17 cells to change the properties of the cells (see Non-Patent Document 7).

"Food Allergy Treatment Guide 2011" 2011 "Food Allergy Practice Guidelines 2012" 2012 Robinson D.S. et al., The Journal of clinical Investigation., 2004, 114, 1389-1397 Fessler, J. et al., BioDrugs., 2013, 27, 281-291 Hubert, S., et al., J. Ex. Med., 2010, pp.2561-2568 Elkhal et al., Scientific reports, 2016, pp.1-12 Tullius et al., Nature Communications, 2014, pp.1-17

As described above, Foxp3-positive T cells have an action of promoting immune tolerance in vivo, and by this action, immune system symptoms such as allergic symptoms and autoimmune symptoms can be improved, alleviated, suppressed and the like. In addition, Non-Patent Documents 5 to 6 describe that the number of Foxp3-positive T cells can be reduced by converting NAD into Th17 cells, and that Foxp3-positive T cells in tumor tissue can be activated. However, it has not been known so far to efficiently increase the proportion of Foxp3-positive T cells.

Therefore, the problem to be solved by the present invention is to provide a composition for inducing differentiation into Foxp3-positive T cells, which contains an active ingredient that acts to increase the proportion of Foxp3-positive T cells, and to provide the composition. An object of the present invention is to provide a method for producing Foxp3-positive T cells.

As a result of intensive research to solve the above problems, the present inventors surprisingly have adenine, nicotinamide, nucleoside phosphate compounds and the structures of these compounds in the molecule, including NAD. It has been found that a compound (hereinafter, also referred to as NAD or the like) exhibits an action of inducing differentiation of Foxp3-negative naive T cells (hereinafter, also referred to as Foxp3-negative T cells) into Foxp3-positive T cells.

So far, it has been known that NAD reduces the number of Foxp3-positive T cells and activates Foxp3-positive T cells at specific sites. In addition, as shown in Figure 10 of Non-Patent Document 7, in the presence of transforming growth factor-β (TGF-β) and interleukin-2 (IL-2), as well as IL-4 and IL-12. It was known that Foxp3-positive T cells could be induced to differentiate from naive CD4-positive T cells in the presence of antibodies against IFN-γ.

However, as an action of NAD and the like, the action of inducing differentiation of Foxp3-negative T cells such as naive CD4-positive T cells into Foxp3-positive T cells and increasing the proportion of Foxp3-positive T cells is the first by the present inventors. It is an action found. Then, based on the action of NAD and the like, the present inventors have a composition for inducing differentiation into Foxp3-positive T cells containing NAD and the like as an active ingredient, and Foxp3-positive T cells using the composition. Succeeded in creating a manufacturing method. The present invention has been completed based on these findings and successful examples.

Therefore, according to the present invention, there is provided a composition for inducing differentiation into Foxp3-positive T cells, which contains at least one compound selected from the group consisting of the following compounds (1) to (4) as an active ingredient. To.
(1) Adenine (2) Nicotinamide (3) Nucleoside phosphate compound (4) A compound having an intramolecular structure of at least one compound selected from the group consisting of the compounds (1) to (3) above.

According to another aspect of the present invention, an immunosuppressive composition, an immune tolerance promoting composition, an immunomodulatory composition or an immunosuppressive composition containing the composition for inducing differentiation into Foxp3-positive T cells of the present invention as an active ingredient. A composition for improving the intestinal environment is provided.

Preferably, in the composition of the present invention, the nucleoside phosphate compound is adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, citidine monophosphate, cytidine diphosphate, cytidine triphosphate, thymidine monophosphate. Acids, thymidine diphosphate, thymidine triphosphate, uridine monophosphate, uridine diphosphate, uridine triphosphate, guanosine monophosphate, guanosine diphosphate or guanosine triphosphate.
Preferably, in the composition of the present invention, the compound (4) is oxidized or reduced nicotinamide adenine dinucleotide, oxidized or reduced nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, nicotin. Amidoguanine dinucleotide, nicotinamide hypoxanthin dinucleotide, nicotinamide 1,N6-ethenoadenin dinucleotide, acetyl coenzyme A or acetoacetyl coenzyme A.
Preferably, the composition of the present invention is a composition for inducing differentiation of Foxp3-negative T cells into Foxp3-positive T cells in the presence of cytokines.
Preferably, the composition of the present invention differentiates Foxp3-negative T cells into Foxp3-positive T cells in the presence of TGF-β at 1.2 times the intensity as compared to the case without the composition. It is a composition for inducing.
Preferably, the composition of the present invention is a composition for suppressing the differentiation of Foxp3-negative T cells into Th17-positive T cells in the presence of cytokines.

According to another aspect of the present invention, there is provided a method for producing Foxp3-positive T cells, which comprises contacting Foxp3-negative T cells collected from a subject with the composition of the present invention in the presence of cytokines. ..

The composition of the present invention has an action of inducing differentiation of Foxp3-negative T cells such as naive CD4-positive T cells by the active ingredient into Foxp3-positive T cells, as well as an immunosuppressive action, an immunotolerance promoting action, an immunomodulatory action and an intestinal tract. Compositions and immunotherapies for improving, alleviating, suppressing, treating or preventing allergic and autoimmune symptoms such as asthma, atopy, pollinosis, food allergies, in oral or parenteral forms through environmental improvement effects Can be used as a composition for.

In particular, for a person who develops a food allergy, ingestion of an allergen-free food is extremely burdensome for the person who develops the allergy and those around him, but the composition of the present invention capable of enhancing immune tolerance is incorporated into foods and drinks, pharmaceuticals, and the like. Alternatively, it can be expected to improve allergic symptoms by using it as such.

Further, according to the production method of the present invention, since Foxp3-positive T cells can be produced in vitro, the above-mentioned allergic symptoms can be obtained by transferring Foxp3-positive T cells produced in vitro into the living body. It can be expected to improve, alleviate, suppress, treat or prevent autoimmune symptoms.

FIG. 1 is a diagram showing the results of the differentiation-inducing action of Foxp3-positive T cells and the reducing action of Th17 cells by NAD, as described in Example 1 of Examples described later. FIG. 2 is a diagram showing the results of NAD not inducing Th17 cell differentiation in the presence of IL-2, as described in Example 2 of Examples described later. FIG. 3 is a diagram showing the results of NAD inducing the differentiation of Foxp3-positive T cells in the presence of IL-2, as described in Example 2 of Examples described later. FIG. 4 shows the result that NAD did not promote the differentiation of Th17 cells in the presence of various antibodies and cytokines for inducing the differentiation into Th17 cells, as described in Example 3 of Examples described later. It is a figure. FIG. 5 shows the results of NAD inducing the differentiation of Foxp3-positive T cells in the presence of various antibodies and cytokines for inducing differentiation into Th17 cells, as described in Example 3 of Examples described later. It is a figure.

The details of the present invention will be described below. The present invention is not limited to the following embodiments, and can be variously modified and implemented within the scope of the gist thereof.

[Composition for Inducing Differentiation into Foxp3-Positive T Cells]
One aspect of the composition of the present invention is a composition for inducing differentiation into Foxp3-positive T cells, which contains at least one compound selected from the group consisting of the following compounds (1) to (4) as an active ingredient. is there.
(1) Adenine (2) Nicotinamide (3) Nucleoside phosphate compound (4) A compound having an intramolecular structure of at least one compound selected from the group consisting of the compounds (1) to (3) above.

Unless otherwise specified, each term in the present specification is used in the meaning commonly used by those skilled in the art. For example, the "composition" is not particularly limited as having a commonly used meaning, but is, for example, a combination of two or more kinds of substances, specifically, an active ingredient and another substance. Examples include those obtained by combining two or more kinds of active ingredients, and more specifically, one or more kinds of active ingredients combined with one or more kinds of solid carriers or solvents. Examples thereof include solid compositions and liquid compositions.

Generally, T cells produced in the bone marrow circulate in the bloodstream as Foxp3-negative T cells after differentiation and maturation in the thymus. Naive T cells differentiate into each subset when stimulated by various cytokines. For example, naive T cells differentiate into Th17 cells and Foxp3-positive T cells by stimulation with TGF-β or IL-6. It has also been suggested that Th17 cells reversibly or irreversibly convert to Foxp3-positive T cells. Differentiation generally means that a cell having no characteristic changes into a cell having various characteristics, and "differentiation" in the present specification means that a cell having one characteristic has another characteristic. In addition to broadly meaning to turn into, it can be used as a broader concept, including reversible or irreversible reciprocal conversion between two types of cells commonly known as differentiated cells.

The compounds (1) to (4) are not particularly limited as long as they have an action of inducing differentiation of Foxp3-negative T cells into Foxp3-positive T cells, that is, an action of inducing differentiation into Foxp3-positive T cells. For example, in addition to those compounds themselves, those which can be the above compounds when applied to a subject and those which can function like the above compounds are included, specifically adenine, nicotine amide and nucleoside phosphate compounds and It has a certain range, including salts such as pharmaceutically acceptable salts of compounds having the structure of adenine, nicotine amide and / or nucleoside phosphate compounds in the molecule, analogs and the like.

The compounds (1) to (4) above can be appropriately changed depending on the form and use of the composition. For example, when used as an oral composition, the compound is given to a subject when orally. On the other hand, it is preferable that the safety is recognized. The compounds (1) to (4) are the compound of the above (1), the compound of the above (2), the compound of the above (3) and the compound of the above (4), respectively, or two or more of them. Can be used in combination.

The nucleoside phosphate compound, which is the compound of (3) above, has a structure having a base, a sugar (glycosyl group) and a phosphate group in the molecule, and has an action of inducing differentiation into Foxp3-positive T cells. If there is, the present invention is not particularly limited, and examples thereof include adenosine diphosphate (ADP) and ADP analogs in which modifications such as substitutions, deletions, and insertions are made in the structure of ADP, and specific examples thereof include adenosine monophosphate (AMP). ), Adenosine diphosphate (ATP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), thymidin monophosphate (TMP), thymidin diphosphate (TDP), Timidin triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine diphosphate Examples thereof include phosphoric acid (GTP), and preferably AMP, ADP, CMP, TMP, UMP and GMP, which have a high effect of inducing differentiation into Foxp3-positive T cells and low cytotoxicity. The nucleoside phosphate compound may be, for example, a cyclic type such as cAMP or a deoxy type such as dAMP. As the compound (3), for example, any one of the nucleoside phosphate compounds may be used alone, or two or more of the nucleoside phosphate compounds may be used in combination.

The compound (4) is not particularly limited as long as it has the structure of adenine, nicotinamide and / or a nucleoside phosphate compound in the molecule and has an action of inducing differentiation into Foxp3-positive T cells. For example, nicotinamide adenine dinucleotides such as oxidized nicotinamide adenine dinucleotide (NAD, NAD +) and reduced nicotinamide adenine dinucleotide (NADH), oxidized nicotinamide adenine dinucleotide phosphate (NADP, NADP +) and reduced nicotin. Nicotinamide adenine dinucleotide phosphate such as amide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), nicotinamide guanine dinucleotide (NGD), nicotinamide mononucleotide (NMN), nicotinamide hypoxanthine dinucleotide, Examples thereof include nicotinamide 1,N6-ethenoadenine dinucleotide, acetyl codenine A (acetyl CoA) or acetoacetyl coadenine A (acetoacetyl CoA), preferably having the structure of ADP in the molecule and Foxp3. NAD, NADH, NADP, NADPH, FAD, acetyl CoA and acetoacetyl CoA, which have a remarkably high effect of inducing differentiation into positive T cells. As the compound of (4), for example, any one of the compounds of (4) can be used alone, or two or more of the compounds of (4) can be used in combination.

Other examples of the compound of (4) above include compounds having nicotinamide as a part of the structure and its analogs, and specifically, partial pyridine and / or carboxylic acid amide constituting nicotinamide. Examples thereof include pyridinecarboxylic acid amide derivatives which have been modified by substitution, deletion, insertion or the like. Examples of the pyridinecarboxylic acid amide derivative include 2-chloronicotinamide, 6-methylnicotinamide, isonicotinamide, 4-pyridinecarboxylic acid amide, timine, cytosine, uracil, niacin, picolin, vinylpyridine and 2-amino-. Examples include, but are not limited to, 3-methylpyridine, 3-aminopyridine, 3-methylpyridine, 3-cyanopyridine, 3-ethylpyridine, 3-pyridinemethanol, 3-vinylpyridine and the like.

The compounds described in (1) to (4) above are not particularly limited in terms of their acquisition method, and are obtained, for example, as commercially available compounds or those produced by a synthetic method usually known to those skilled in the art. be able to. For example, the compounds (1) to (4) above are microorganisms when the production method includes a microbial fermentation step, separately from or in combination with the addition of the compounds (1) to (4). It can be produced by adopting a method for increasing the compounds (1) to (4) present in the body of a bacterium such as lactic acid bacterium. The microbial fermented product thus obtained can itself be used as one aspect of the composition of the present invention.

The composition for inducing differentiation into Foxp3-positive T cells exhibits a differentiation-inducing action on Foxp3-positive T cells by the functions of the compounds (1) to (4) contained above as active ingredients. The action of inducing differentiation into Foxp3-positive T cells is an action of inducing differentiation of Foxp3-negative T cells into Foxp3-positive T cells, and if the action can increase the proportion of Foxp3-positive T cells as a result, the mechanism of action thereof. The introduction, other actions and degrees associated with it are not particularly limited.

Usually, cytokines such as TGF-β and IL-2 are present in the living body. Therefore, when a composition for inducing differentiation into Foxp3-positive T cells is applied in vivo, differentiation of Foxp3-negative T cells into Foxp3-positive T cells is induced in the presence of cytokines. Therefore, a specific aspect of the composition of the present invention is a composition for inducing differentiation of Foxp3-negative T cells into Foxp3-positive T cells in the presence of cytokines. Examples of cytokines include cytokines related to T cell proliferation and differentiation, and specific examples thereof include TGF-β, IL-2, IL-12, IL-4, IL-6, and IL-23. Although mentioned, preferred are TGF-β and IL-2. Cytokines can be used alone or in combination of two or more.

The compounds (1) to (4) above are related to or irrespective of the action of inducing differentiation of Foxp3 negative T cells into Foxp3 positive T cells, and the Foxp3 negative T cells to Th17 positive T cells. It can exhibit an action of suppressing differentiation into Th17-positive T cells, that is, an action of suppressing differentiation into Th17-positive T cells. Therefore, a specific aspect of the composition of the present invention is a composition for suppressing the differentiation of Foxp3-negative T cells into Th17-positive T cells in the presence of cytokines.

IL-17 produced by Th17-positive T cells is an inflammatory cytokine or inflammatory mediator (IL-1, IL-2, IL-6, IL-8, IL-22, G-CSF, MCP-1, TNF-α. , NOS-2, metalloprotease, chemokines, etc.) are known to induce production. Therefore, the compounds (1) to (4) can suppress the production of the above-mentioned inflammatory cytokines and inflammatory mediators through the action of suppressing differentiation into Th17-positive T cells.

The effect of inducing differentiation into Foxp3-positive T cells and the effect of suppressing differentiation into Th17-positive T cells are not particularly limited in terms of the evaluation method, and for example, as described in Examples described later, Foxp3-positive T cells and Th17-positive It can be evaluated by measuring the expression level of genes specifically expressed by T cells and by measuring the number and ratio of these cells.

As a specific example of a method for evaluating the differentiation-inducing action on Foxp3-positive T cells and the differentiation-inducing action on Th17-positive T cells, a Foxp3-negative T cell and a composition for inducing differentiation into Foxp3-positive T cells are co-cultured. Next, RNA was extracted from the cells collected after culturing, and then the extracted RNA was reverse transcribed to obtain a cDNA library, and then Foxp3-positive T cells and Th17-positive T cells were specifically selected based on the obtained cDNA library. It is a method including measuring the expression level of the expressed gene.

Another specific example of the method for evaluating the differentiation-inducing action on Foxp3-positive T cells and the differentiation-inducing action on Th17-positive T cells is that Foxp3-negative T cells and a composition for inducing differentiation into Foxp3-positive T cells are co-cultured. Then, it is a method including measuring the differentiation of Foxp3-positive T cells and Th17-positive T cells by intracellular labeling or extracellular labeling in cultured cells. Population of Foxp3-positive T cells and Th17-positive T cells can be measured by, for example, a method such as flow cytometry, a method utilizing an antigen-antibody reaction such as ELISA or radioimmunoassay (RIA), or the like.

The effect of inducing differentiation into Foxp3-positive T cells is not particularly limited, but for example, in the presence of TGF-β, the effect of increasing the proportion of Foxp3-positive T cells as compared with the case where the composition of the present invention is not used. Yes, preferably, it is an action of increasing the proportion of Foxp3 positive T cells by 1.1 times or more as compared with the case where the composition of the present invention is not used, and more preferably, as compared with the case where the composition of the present invention is not used. It is an action of increasing the proportion of cells 1.2 times or more, more preferably an action of increasing the proportion of Foxp3-positive T cells 1.3 times or more as compared with the case where the composition of the present invention is not used, and even more preferably. Is an action that increases the proportion of Foxp3-positive T cells by 1.4 times or more as compared with the case where the composition of the present invention is not used.

The effect of suppressing the differentiation of Foxp3-negative T cells into Th17-positive T cells is not particularly limited, but for example, it is an effect of lowering the proportion of Th17-positive T cells as compared with the case where the composition of the present invention is not used. It is an action of lowering the proportion of Th17-positive T cells to 0.9 times or less, more preferably than when the composition of the present invention is not used, and more preferably Th17-positive T cells than when the composition of the present invention is not used. It is an action of lowering the ratio of Th17-positive T cells to 0.85 times or less, and more preferably an action of lowering the ratio of Th17-positive T cells to 0.80 times or less as compared with the case where the composition of the present invention is not used.

[Immunosuppressive composition, immune tolerance promoting composition, immunomodulatory composition and intestinal environment improving composition]
A specific aspect of the composition of the present invention is immunity containing the compounds (1) to (4) above as an active ingredient, or a composition for inducing differentiation into Foxp3-positive T cells as an active ingredient. It is a composition for suppressing, a composition for promoting immune tolerance, a composition for immunomodulating, and a composition for improving the intestinal environment.

The immunosuppressive composition is not particularly limited as long as it is generally known, and may be, for example, a composition for suppressing or reducing the immune response of the applied subject.

The composition for promoting immune tolerance is not particularly limited as long as it is as is generally known, and for example, the composition is in a state in which the immune response to the antigen in the applied subject is suppressed (that is, the immune tolerance state). It may be a composition for promoting a state in which immunity that excessively responds to self is suppressed.

The immunomodulatory composition is not particularly limited as long as it is generally known, and for example, to the applied subject, through immunosuppression, promotion of immune tolerance, or regardless of these. It may be a composition for optimizing the immune response by relaxing or improving it.

The composition for improving the intestinal environment is not particularly limited as long as it is as is generally known, and for example, the intestinal immune response of the applied subject is optimized to improve the intestinal environment. It can be a composition for improving or maintaining such.

The mechanism of immune response and immune tolerance is largely unclear, and the technical scope of the present invention is not bound by any theory or speculation, but is a specific embodiment of the composition of the present invention. The suppressive composition, the immune tolerance promoting composition, the immunomodulating composition and the intestinal environment improving composition exert their respective actions by increasing the proportion of Treg in the applied subject, for example. obtain.

The immunosuppressive state and the immune tolerant state can be evaluated without particularly limiting the method known to those skilled in the art as to the evaluation method. For example, a method of evaluating by measuring the reactivity of Treg can be mentioned, and a specific method thereof can be mentioned. Examples of the method for evaluating the differentiation-inducing action on Foxp3-positive T cells and the differentiation-suppressing action on Th17-positive T cells as described above can be mentioned.

Immunosuppressive compositions, immune tolerance-promoting compositions and immunomodulatory compositions may be those used to induce Tregs to enhance immune tolerance, especially oral tolerance. For example, oral immunotolerance can be achieved by applying foods and drinks or pharmaceuticals containing an immunosuppressive composition, an immune tolerance promoting composition and an immunomodulating composition to a subject for whom treatment or prevention of food allergy is desired. It is induced and can improve, alleviate, suppress, treat or prevent allergic symptoms such as food allergies and autoimmune symptoms. The immunosuppressive composition, the immune tolerance promoting composition and the immunomodulating composition may induce parenteral immune tolerance.

In addition, immunosuppressive compositions, immunotolerance-promoting compositions, and immunomodulatory compositions are associated with the production of IL-17 and the production of inflammatory cytokines and inflammatory mediators induced by IL-17, resulting in rheumatoid arthritis and systemic lupus erythematosus. It can be expected to improve, alleviate, suppress, treat or prevent symptoms such as lupus erythematosus, multiple sclerosis, psoriasis, inflammatory colitis, and Crohn's disease.

The composition for inducing differentiation into Foxp3-positive T cells, the composition for immunosuppression, the composition for promoting immune tolerance, the composition for immunomodulating, and the composition for improving the intestinal environment are used alone or in combination thereof. It can take the form of a food or drink composition or a pharmaceutical composition. Therefore, a specific embodiment of the composition of the present invention contains at least one compound selected from the group consisting of the compounds (1) to (4) above as an active ingredient, or to Foxp3-positive T cells. Food and drink compositions, pharmaceutical compositions, quasi-drugs containing a composition for inducing differentiation, a composition for immunosuppression, a composition for promoting immune tolerance, a composition for immunomodulating or a composition for improving the intestinal environment. Compositions, cosmetic compositions, animal feed compositions and the like.

The application target and application method of the composition for inducing differentiation into Foxp3-positive T cells, the composition for immunosuppression, the composition for promoting immune tolerance, the composition for immunomodulating and the composition for improving the intestinal environment are particularly limited. However, for example, the application target includes animals, particularly mammals, and mammals include humans, dogs, cats, cows, horses, etc. Among these, humans are preferable.

[Food and drink composition]
The food and drink composition can take any form of food and drink as long as it can exert at least the action of inducing differentiation into Foxp3-positive T cells after ingestion. For example, fruit juice drink, vegetable juice, fruit vegetable juice, tea drink, coffee. Beverages, sports drinks, frozen desserts, health drinks, etc .; dairy products such as yogurt and cheese; processed foods and drinks such as soups, noodles, puddings, jams; chewing gum, candy, desserts, gummy jelly, chocolate, biscuits, snacks, etc. Confectionery; Frozen desserts such as ice cream, sherbet, and frozen desserts; Seasonings such as soy sauce and miso. As long as the object of the present invention can be achieved, the food and drink composition has an action of inducing differentiation into Foxp3-positive T cells other than the compounds (1) to (4) above, an immunosuppressive action, and an immune tolerance promoting action. Natural products or non-natural products having an immunosuppressive effect and / or an intestinal environment improving effect and having eating experience, or inclusions thereof may be blended. For example, food and drink compositions include lactic acid bacteria and fermented products containing the lactic acid bacteria, which are known to have an effect of enhancing oral immune tolerance; soymilk containing flavonoids, genistein, genistein, etc.; soybean fermented polysaccharides contained in soy sauce, etc.; vitamins. It is preferable to add vitamins such as C.

Examples of the lactic acid bacterium include lactic acid bacteria of the genus Pediococcus, Lactobacillus, and Lactococcus, but lactic acid bacteria of the genus Lactococcus are preferable, Lactococcus lactis is more preferable, and Lactococcus lactis K478 strain (Lactococcus). The Lactococcus lactis K478 strain was received as K478 by the Patent Organism Depositary, Product Evaluation Technology Infrastructure Organization, on January 9, 2014, and has been given the accession number NITE-P01786).

The content of the active ingredient in the food and drink composition is not particularly limited as long as it can exert at least a differentiation-inducing action on Foxp3-positive T cells after ingestion, but for example, a differentiation-inducing action on Foxp3-positive T cells. Can be appropriately set according to the physique, age, degree of desired effect, etc. of the ingestor in view of the dose-dependent tendency. Specifically, when the active ingredient is NAD or NADH, the content of the active ingredient in the food and drink composition is adjusted so that the intake of the active ingredient is, for example, 1 to 1,000 mg / body weight 60 kg / day. It can be set as appropriate, but is not limited to this. The number of intakes is not particularly limited, but is, for example, 1 to 3 times a day, and the number of intakes can be appropriately increased or decreased according to the amount of intake.

As the active ingredient, any one of the above compounds (1) to (4) can be used alone or in combination of two or more of these, or these and the above (1) to (4) can be used. It can be used in combination with other components known to have the same action as the compound.

The food and drink composition can be mixed with other ingredients as long as the object of the present invention can be achieved. Other ingredients include, for example, sugar sweeteners, stabilizers, emulsifiers, starches, processed starch products, starch decomposition products, salt, flavoring agents, coloring agents, acidulants, flavoring ingredients, nutrients, fruit juices, eggs, etc. It can further contain additives used in conventional food processing such as animal and vegetable foodstuffs, excipients, bulking agents, binders, thickeners, perfume oils and the like. The amount of the additive used is not particularly limited as long as it does not interfere with the solution of the problem of the present invention, and can be appropriately set.

The food and drink composition is not particularly limited as long as it is in a commonly used form, and is, for example, solid, liquid, gel, suspension, cream, sheet, stick, powder, granular, granular, or tablet. It can take various forms such as a rod shape, a plate shape, a block shape, a paste shape, a capsule shape, and a caplet shape.

A specific aspect of the food and drink composition is, for example, a functional food and drink which is a food and drink having a certain functionality with respect to a living body. Functional foods and drinks include, for example, foods and drinks for specified health use, foods and drinks with functional claims, foods and drinks with nutritional functions, foods and drinks with health functions, foods and drinks for special purposes, foods and drinks for nutritional supplements, healthy foods and drinks, supplements, beauty foods and drinks. In addition to so-called healthy foods and drinks such as, it includes foods and drinks for specific persons such as foods and drinks for infants, foods and drinks for pregnant women, and foods and drinks for the elderly. Further, functional foods and drinks include healthy foods and drinks to which a health claim based on the food standards of Codex (FAO / WHO Joint Food Standards Committee) is applied.

The packaging form of the food and drink composition is not particularly limited and can be appropriately selected depending on the dosage form and the like. For example, blister packs such as PTP; strip packaging; heat seals; aluminum pouches; film packaging using plastics, synthetic resins, etc. Glass containers such as vials; plastic containers such as ampoules.

[Pharmaceutical composition]
The pharmaceutical composition can take any pharmaceutical form as long as it exerts at least a differentiation-inducing effect on Foxp3-positive T cells after application and can thus treat and / or prevent immune disorders. As long as the object of the present invention can be achieved, the pharmaceutical composition has an action of inducing differentiation into Foxp3-positive T cells other than the compounds (1) to (4) above, an immunosuppressive action, an immune tolerance promoting action, and immunity. A physiologically active substance having a regulating action and / or an intestinal environment improving action may be blended. For example, as an active ingredient of a pharmaceutical composition, the above compounds (1) to (4) and IL-2, T which are important for maintaining the functions of TGF-β and Treg, which play an important role in inducing differentiation of Treg. It is conceivable to use a co-stimulatory molecule anti-CD28 antibody, an antibody-stimulated molecule anti-CD3e antibody, vitamin A and its derivative retinoic acid, which contribute to cell differentiation and activation, in combination.

Immune diseases that can be treated and / or prevented by the pharmaceutical composition are not particularly limited, but for example, various autoimmune diseases, allergic diseases, implant-to-host disease (GVHD), inflammatory diseases, infectious diseases, neoplastic diseases. Specifically, by inducing Treg in vivo, allergic diseases such as pollinosis can be treated.

The pharmaceutical composition may exist in the form of a pharmaceutically acceptable salt in which the active ingredient is dissolved in a water-soluble solvent. Examples of such pharmaceutically acceptable salt forms include physiologically acceptable water-soluble salts, for example, salts buffered at physiological pH in the form of salts such as sodium, potassium, magnesium and calcium. Be done. In addition to the water-soluble solvent, a water-insoluble solvent can be used, and examples of such a water-insoluble solvent include alcohol, ethanol, propylene glycol, and the like.

In addition to the active ingredient, the pharmaceutical composition may contain other ingredients for various purposes, and such other ingredients include, for example, preservatives, buffers and the like. Preservatives include sodium bisulfite, sodium bisulfite, sodium thiosulfite, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercury acetate, phenylmercury nitrate, methylparaben, polyvinyl alcohol, phenylethyl alcohol, ammonia, dithiothreitol, Examples include beta-mercury acetate. In addition, examples of the buffer include sodium carbonate, sodium borate, sodium phosphate, sodium acetate, sodium bicarbonate and the like. These agents can be present in an amount capable of maintaining the pH of the system between 2-9, preferably 4-8.

The dosage form of the pharmaceutical composition is not particularly limited, and examples thereof include injections (muscle, subcutaneous, intradermal), oral preparations, and nasal drops preparations. For example, the pharmaceutical composition can be prepared as a vaccine for treating allergic diseases such as pollinosis. When the pharmaceutical composition is in the form of a vaccine, it may be a mixed cocktail vaccine containing a plurality of active ingredients.

When the pharmaceutical composition is used as a vaccine, it contains an inactive ingredient that is a component other than the pharmaceutical composition, has no activity by itself, and contains an ingredient having an effect of further enhancing the vaccine effect of the pharmaceutical composition. It may be a vaccine. Examples of the Inactive component include adjuvants and toxoids. Examples of adjuvants, but not intended to be limited, include sedimentation types such as aluminum hydroxide, aluminum phosphate, calcium phosphate, and oily types such as Freund complete adjuvant and Freund incomplete adjuvant. Be done.

When the pharmaceutical composition is present in the form of a vaccine, it is preferably injected or injected by intradermal, subcutaneous, intravenous, intramuscular administration, etc., or transdermally, or inhaled through the mucous membranes of the nose, pharynx, etc. in the body. Can be administered to.

When preparing a pharmaceutical composition, since the immunosuppressive effect is dose-dependent, the amount of the active ingredient can be appropriately set according to the age, physique, symptoms and the like of the patient. When NAD or NADH is used as an active ingredient, the dose is, for example, 1 to 1,000 mg / body weight 60 kg / day. Patients who require administration of the pharmaceutical composition include allergic patients, such as food allergic patients with disrupted oral tolerance, and infants with immature gastrointestinal function or oral tolerance.

[Method for producing Foxp3-positive T cells]
As another aspect of the present invention, a step of contacting Foxp3-negative T cells collected from a subject with any one aspect of the composition of the present invention in the presence of cytokines such as TGF-β and IL-2 ( Hereinafter, a method for producing Foxp3-positive T cells, which also includes a contact step), is provided.

Foxp3-negative T cells used in the contact step are not particularly limited, for example, peripheral blood mononuclear cells (PBMC), CD4, CD62L and / or CD45RA from cells isolated from the blood or spleen of a subject. It can be prepared by appropriately selecting cells expressing the above. The subject is not particularly limited, and examples thereof include mammals such as humans, mice, rats, dogs, and cats. The method for sorting the cells is not particularly limited, and for example, any molecule can be used as an index for sorting.

Contact between Foxp3-negative T cells and the composition of the present invention is preferably carried out in a suitable medium. The amount of the composition of the present invention to be used can be appropriately determined by those skilled in the art in view of the tendency that the differentiation-inducing action of the composition of the present invention on Foxp3-positive T cells is dose-dependent. When NAD or NADH is added to the medium, the amount added is, for example, about 2 to 50 μg per 1 ml of the medium. The contact time is, for example, about 2.5 to 3 days. The contact step may be carried out in the presence of other additional components, for example, anti-CD28 antibody, anti-CD3e antibody and the like may be added to the medium.

The method of adding the cytokine is not particularly limited, and for example, it can be added at the same time as the Foxp3-negative T cells and the composition of the present invention are brought into contact with each other, or before and after that time.

The Treg produced by the above method is considered to be used in vivo or ex vivo. For example, immunosuppression can be exerted in a subject by returning the produced Treg to a subject suffering from an immune disease via a blood vessel or the like.

In the production method of the present invention, various steps and operations can be added before or after the contact step or during the step as long as the object of the present invention can be achieved. In addition, it can be carried out continuously or intermittently within and between steps.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples, and the present invention can take various aspects as long as the problems of the present invention can be solved. ..

[Example 1. Evaluation of Foxp3-positive T cell inducibility by NAD]
Using the human T cell line Jurkat, the induction of Foxp3-positive T cells (Tregs) of nicotinamide adenine dinucleotide (NAD) was evaluated by measuring the expression levels of Treg and Th17 cell-specific genes.

Tregs are known as factors that affect immune tolerance. In order to verify that NAD induces Treg, NAD and TGF-β and Jurkat, which is a human CD4 positive cell (T cell) strain, were co-cultured and the Treg-inducing activity by NAD was evaluated. In addition, Th17 cells are known to decrease as Treg increases. Therefore, we also examined how Th17 cells are changed by NAD.

Induction to Treg and Th17 cells was evaluated by quantifying the expression levels of Treg marker gene Foxp3 mRNA and Th17 cell marker gene RORγt (hereinafter, also referred to as PORgt) mRNA. As a positive control against NAD, retinoic acid (RA), which is known to have Treg-inducing activity, was used.

NAD solution and RA solution were prepared by dissolving in 1 × PBS buffer so that NAD was 1,000 μM or RA was 500 μM. The NAD solution and the RA solution were added to the cell suspension described below so that the final concentration was 100 μM (NAD) or 50 μM (RA), respectively.

The medium for culturing Jurkat was prepared by adding a reagent to RPMI 1640 times ground manufactured by Gibco so as to have the following final concentration.
10% (v / v) FBS
1% (w / v) Penicillline / Streptomycin
10 mM HEPES
1 mM Sodium Pyruvate

Jurkat (Clone E6-1, ATCC TIB-152) frozen stock was added to the medium, and the cells were cultured at 37 ° C. and 5% CO ₂ conditions while changing the medium about twice a week, and the fifth passage (P5). At this time, 3 × 10 ⁶ cells / mL working stock was made. The working stock was added to the medium and subcultured twice after culturing, and the cells at P9 were collected, washed once with the medium, and then the cells were collected again. Sampling a portion of the recovered cells, than the value obtained by counting the number of cells using a hemocytometer, it added an appropriate amount of media harvested cells cell suspension (5.7 × 10 ⁵ cells / mL ) Was prepared.

The following reagents and cell suspensions were added to a 96-well flat bottom plate (manufactured by BD FALCON) to the following final concentrations, and the plates were cultured at 37 ° C. and 5% CO ₂ conditions for 3 days. , Jurkat was induced into Foxp3-positive T cells. Here, the NAD group is a group containing Jurkat, TGF-β and NAD; the RA group is a group containing Jurkat, TGF-β and RA; the PBS group is Jurkat, TGF-β and 1 × PBS (NAD and). It is a group containing the same amount as RA).
Jurkat cells 1 × 10 ⁵ / well TGF-β (manufactured by R & D System) 2 ng / mL
NAD 100 μM, RA 50 μM or 1 × PBS

The cells after induction were collected, washed once with 1 × PBS, and then RNA was extracted from the cells using the NucleoSpin RNA kit manufactured by MACHEREY-NAGEL. After reverse transcribing the extracted RNA according to the instructions of the kit, the obtained cDNA was subjected to quantitative PCR using SYBR Premix Ex Taq II manufactured by TAKARA and primers manufactured by TAKARA, and the expression level of Foxp3 mRNA (mFoxp3) was performed. And the expression level of RORgt mRNA (mRORgt) was quantified. The results showing the relative expression levels of mFoxp3 and mRORgt in the NAD group and the RA group with the PBS group as the reference (1) are shown in FIG.

As shown in FIG. 1, the expression of Foxp3 mRNA was significantly increased in the NAD group as in the RA group as compared with the PBS group in which only the solvent 1 × PBS was added and cultured without adding the sample. Induced (risk rate 5%). In addition, the NAD group reduced the expression of RORgt mRNA as in the RA group. From these results, it was confirmed that NAD has an action of inducing differentiation of CD4 positive T cells into Treg.

In addition, by adopting another assay system, the following items were confirmed: NAD and reduced NAD (NADH) are TGF-β, which is a Th17 cell differentiation condition when the addition amount is more than about 20 μM. And to show the effect of inducing CD4-positive CD62L-positive T cells to Treg in a concentration-dependent manner in the presence of IL-6; nicotinamide adenine dinucleotide phosphate (NADP), reduced NADP (NADPH), flavin adenine. Nucleotide (FAD), adenine diphosphate (ADP), nicotinamide guanine dinucleotide (NGD), nicotinamide mononucleotide (NMN), nicotinamide, adenine, citidine monophosphate (CMP), uridine monophosphate (UMP) , Guanosin monophosphate (GMP), adenine monophosphate (AMP), acetyl co-enzyme A (acetyl CoA) and aceto acetyl co-enzyme A (acet acetyl CoA) in the presence of TGF-β and IL-6. Showing the effect of inducing CD4-positive CD62L-positive T cells to Treg; γ-glutamyl tyrosine (γ-Glu-Tyr) transfers CD4-positive CD62L-positive T cells to Treg in the presence of TGF-β and IL-6. No inducing action; of these compounds, NAD, NADH, NADP, NADPH, FAD, acetyl CoA and acetoacetyl CoA and ADP, which have the structure of ADP in the molecule, induce differentiation into Treg, which is particularly remarkable. Show action.

From the above, NAD and compounds having an ADP structure in the molecule, and adenine, nicotinamide, nucleoside monophosphate and nucleoside diphosphate, especially NAD and compounds having an ADP structure in the molecule are undifferentiated T. It was confirmed that it has an action of inducing differentiation of cells into Treg.

[Example 2. Evaluation of Foxp3-positive T cell inducibility by NAD in the presence of IL-2]
Using naive T cells isolated from humans, the induction of NAD Tregs in the presence of IL-2 was evaluated by measuring the expression of Treg and Th17 cell-specific markers.

Blood was collected from the subjects, and peripheral blood mononuclear cells (PBMC) were separated according to a conventional method. That is, a mixed solution in which blood and physiological saline were mixed in equal amounts was added to Ficoll, which is half the amount of the mixed solution, in a layered manner. The obtained solution was centrifuged at 600 g for 30 minutes, and the intermediate layer was recovered to obtain PBMC.

The recovered PBMC was washed with phosphate buffer (PBS) having a pH of 7.2, and then naive T cells were recovered by using human Naive CD4 ⁺ T Cell Isolation Kit II (Milteny Biotech). The recovered naive T cells were coated with anti-human CD3 antibody (Biolegend) 20 μg / ml-PBS (37 ° C., 2 hours) on a 96-well flat-bottomed plate (Japan BD) with 5 μg / ml anti-human CD28 antibody (Biolegend). , And 10% FBS-containing RPMI 1640 medium (Sigma) supplemented with 20 U / ml IL-2 (PeproTech), and seeded at 5 × 10 ⁴ cells / well (culture solution volume 200 μl). .. Here, NAD was added so as to have a final concentration of 50 μM. Moreover, as a control, a system to which NAD was not added was used. 96-well flat-bottomed plates seeded with naive T cells were cultured.

After 7 days of culturing, Phorbol 12-Myristate 13-acetylate (PMA, Sigma), ionomycin (Sigma) and GorgiStop (BD) had final concentrations of 0.1 μg / ml, 0.25 μg / ml and 2 μl / ml, respectively. It was added so as to be. The cells collected 4 hours after the addition of these reagents were stained with PE-Cy7 labeled anti-human CD4 antibody (BioLegend) and PE-labeled anti-human CD25 antibody (BioLegend). For the stained cells, the cells were fixed with Foxp3 Steining Kit (eBioscience), and FITC-labeled anti-human IL-17 antibody (BioLegend), BV421-labeled anti-human IFN-γ antibody (Biolegend) and Alexa Fluor647-labeled anti-human. The cells were stained with Foxp3 antibody (BioLegend), and the expression of each marker was analyzed by a flow cytometer.

From the expression analysis of the marker, the result showing the population of Th17 cells (IL-17-producing cells) is shown in FIG. 2, and the result showing the population of Foxp3-positive T cells is shown in FIG. From the comparison of event Q1 in FIG. 2, it can be confirmed that there was no difference in the proportion of Th17 cells depending on the presence or absence of the addition of NAD. This indicates that NAD did not induce differentiation into Th17 cells.

On the other hand, from the comparison of the event P4 in FIG. 3, it can be confirmed that there was a difference in the proportion of Foxp3-positive T cells depending on the presence or absence of the addition of NAD. This indicates that NAD induced differentiation into Foxp3-positive T cells.

[Example 3. Evaluation of Foxp3-positive T cell inducibility by NAD under Th17 cell-inducing conditions]
Using naive T cells isolated from humans, the induction of NAD Tregs in the presence of various antibodies and cytokines to induce differentiation into Th17 cells, and the expression of Treg and Th17 cell-specific markers were measured. Evaluated by doing.

The naive T cells collected in the same manner as in Example 2 were coated on a 96-well flat bottom plate (BD Japan) coated with 20 μg / ml-PBS of anti-human CD3 antibody (BioLegend) (37 ° C., 2 hours) at 5 μg / ml. Anti-human CD28 antibody, 5 μg / ml and anti-human IFN-γ antibody (BioLegend) and 10 ng / ml human IL-1β, 10 ng / ml human IL-6, 10 ng / ml human IL-23, 1 ng / ml human TGF- Using RPMI1640 medium (Sigma) containing β and 20 U / ml IL-2 (PeproTech), seeding was performed so as to have 5 × 10 ⁴ cells / well (culture solution volume 200 μl). Here, NAD was added so as to have a final concentration of 20 μM and 40 μM. Moreover, as a control, a system to which NAD was not added was used. 96-well flat-bottomed plates seeded with naive T cells were cultured.

After 7 days of culturing, Phorbol 12-Myristate 13-acetylate (PMA, Sigma), ionomycin (Sigma) and GorgiStop (BD) had final concentrations of 0.1 μg / ml, 0.25 μg / ml and 2 μl / ml, respectively. It was added so as to be. The cells collected 4 hours after the addition of these reagents were stained with PE-Cy7 labeled anti-human CD4 antibody (BioLegend). For the stained cells, the cells were fixed with Foxp3 Steining Kit (eBioscience), and FITC-labeled anti-human IL-17 antibody (BioLegend), BV421-labeled anti-human IFN-γ antibody (Biolegend) and Alexa Fluor647-labeled anti-human. The cells were stained with Foxp3 antibody (BioLegend), and the expression of each marker was analyzed by a flow cytometer.

From the expression analysis of the marker, the result showing the population of Th17 cells (IL-17-producing cells) is shown in FIG. 4, and the result showing the population of Foxp3-positive T cells is shown in FIG. From the comparison of each figure in FIG. 4, it can be confirmed that there was no difference in the proportion of Th17 cells regardless of the presence or absence of NAD addition and the addition amount. This indicates that although it was a condition for inducing the differentiation of naive T cells into Th17 cells, the presence of NAD did not induce the differentiation into Th17 cells.

On the other hand, from the comparison of each figure in FIG. 5, it can be confirmed that there was a difference in the proportion of Foxp3-positive T cells depending on the presence or absence of the addition of NAD. Considering the results of FIG. 4 together, it is shown that the differentiation into Foxp3-positive T cells was preferentially induced by NAD even under the condition of inducing the differentiation into Th17 cells.

The composition of the present invention contains an active ingredient applicable in both oral and parenteral aspects, and has an action of inducing differentiation into Foxp3-positive T cells, an immunosuppressive action, an immune tolerance promoting action, and immunity. It is useful for ingestors who expect anti-allergic activity, anti-inflammatory activity, anti-infectious disease activity, anti-tumor activity, etc. through its regulating action and intestinal environment improving action, and contributes to the health and welfare of such ingestors. It can be used as food and drink, pharmaceuticals, quasi-drugs, cosmetics, supplements, etc.

Claims (6)

Oxidized or reduced nicotinamide adenine dinucleotide (NAD and NADH), oxidized or reduced nicotinamide adenine dinucleotide phosphate (NADP and NADPH), flavin adenine dinucleotide (FAD), acetyl coenzyme A (acetyl) It contains at least one compound selected from the group consisting of CoA), acetoacetyl coenzyme A (acetoacetyl CoA) and adenine diphosphate (ADP) as an active ingredient, and contains TGF-β, IL-2 and IL-. A composition for inducing differentiation into Foxp3-positive T cells, which is used to induce differentiation of Foxp3-negative T cells into Foxp3-positive T cells in the presence of at least one cytokine selected from the group consisting of 6 . Excludes immunosuppressive compositions, immunotolerance-promoting compositions, immunomodulatory compositions, or intestinal environment-improving compositions that contain NAD as an active ingredient and are used to reduce Foxp3-positive T cells. ) . The cytokine is TGF-β, and the composition induces differentiation of Foxp3-negative T cells into Foxp3-positive T cells with 1.2 times the intensity as compared with the case where the composition is not used. The composition for inducing differentiation into Foxp3-positive T cells according to claim 1, which is used in 1. The Foxp3-positive T cell according to any one of claims 1 to 2, wherein the composition is a composition for suppressing differentiation of Foxp3-negative T cells into Th17-positive T cells in the presence of the cytokine. Composition for inducing differentiation into. Foxp3 contains the composition according to any one of claims 1 to 3 as an active ingredient and in the presence of at least one cytokine selected from the group consisting of TGF-β, IL-2 and IL-6. An immunosuppressive composition, an immune tolerance-promoting composition, an immunomodulatory composition or an intestinal environment improving composition (provided that NAD is contained as an active ingredient) that induces differentiation of negative T cells into Foxp3-positive T cells. Except for immunosuppressive compositions, immune tolerance-promoting compositions, immunomodulatory compositions, or intestinal environment-improving compositions used to reduce Foxp3-positive T cells) . 6. According to any one of claims 1 to 4, in the presence of at least one cytokine selected from the group consisting of TGF-β, IL-2 and IL-6 in Foxp3-negative T cells collected from a subject. A method for producing Foxp3-positive T cells, which comprises a step of contacting the compositions of the above. The composition according to any one of claims 1 to 4, further containing at least one cytokine selected from the group consisting of TGF-β, IL-2 and IL-6.