Induced human regulatory t cells and method for producing the same

Abstract

To provide induced regulatory T cells with high function and stable immunosuppressive function.SOLUTION: The present invention provides an induced human regulatory T cell having at least one characteristic selected from the group consisting of FoxP3-positive, CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172 g-positive, and CD26-positive, which is induced by obtaining stable inducible T cells induced from human peripheral T cells by stimulation with an anti-CD3 antibody when regulatory T cells are induced from human peripheral T cells, subsequently subjecting the cells to resting culture, stimulating the cells with anti-CD3 antibody again to culture them, and further subjecting the cells to resting culture.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to highly functional human inducible regulatory T cells and a method for producing the same. [Background technology]

[0002] An important feature of CD25+CD4+ regulatory T cells that reside in the immune system is that they specifically express the transcription factor FoxP3, and FoxP3 deficiency or mutation can impair the development, differentiation, and suppressive function of regulatory T cells. Regulatory T cells suppress the immune system by expressing a variety of genes, including FoxP3, CTLA4, and IL-10. It is believed that epigenetic conditions such as DNA demethylation contribute to the comprehensive gene expression control of regulatory T cells, including stable expression of FoxP3, and these conditions are correlated with the functional phenotype of regulatory T cells. Summary of the Invention [Means for solving the problem]

[0003] The present inventors have found for the first time that, when inducing regulatory T cells from human peripheral T cells, stable inducible T cells are induced from human peripheral T cells by stimulation with an anti-CD3 antibody, followed by resting culture, stimulation with anti-CD3 antibody again, culture, and then resting culture again, thereby enabling the induction of regulatory T cells with high expression of inhibitory molecules and high inhibitory function.

[0004] Thus, the present disclosure provides: (Item X1) An inducible human regulatory T cell having at least one characteristic selected from the group consisting of FoxP3 positive, CTLA4 positive, NT5E positive, ITGAE (CD103) positive, AREG positive, CD172g positive, and CD26 positive. (Item X1A) NT5E-positive inducible human regulatory T cells. (Item X1B) ITGAE (CD103) positive inducible human regulatory T cells. (Item X1C) AREG-positive inducible human regulatory T cells. (Item X1D) CD172g positive inducible human regulatory T cells. (Item X1E) CD26 positive inducible human regulatory T cells. (Item X1F) CTLA4-positive inducible human regulatory T cells. (Item X1G1) An inducible human regulatory T cell having at least two characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity. (Item X1G2) An inducible human regulatory T cell having at least three characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity. (Item X1G3) An inducible human regulatory T cell having at least four characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity. (Item X1G4) An inducible human regulatory T cell having at least five characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity. (Item X1G5) Inducible human regulatory T cells that have all the characteristics of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, AREG-positive, CD172g-positive, and CD26-positive. (Item X2) The inducible regulatory human T cell of claim 1, which is at least CTLA4 positive and FoxP3 positive. (Item X3) The inducible regulatory human T cell of any one of the preceding items, which is at least CD172g positive and / or CD26 positive. (Item X4) The inducible regulatory human T cell according to any one of the preceding items, wherein the CNS2 site of the FOXP3 gene is demethylated. (Item X5) The inducible human regulatory T cell according to any one of the preceding items, which is CD4 positive or CD8 positive. (Item X6) The inducible human regulatory T cells according to any one of the preceding items, which are obtained or derived from human peripheral blood T cells or human tissue-derived T cells. (Item X7) (a) stimulating CD4 positive T cells or CD8 positive T cells in human peripheral blood with a first basal medium for about 1 to about 5 days; (b) dormantly culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days; (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days; and (d) dormantly culturing the cells obtained in step (c) in a medium containing IL-2 for at least about 1 to about 3 days. An inducible human regulatory T cell obtained by a method comprising the steps of: (Item X8) A cell population comprising the inducible regulatory human T cells according to any one of the preceding items, wherein the cell population has a percentage of at least one characteristic selected from the group consisting of FoxP3 positivity, CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more. (Item X8A) A cell population comprising an inducible human regulatory T cell, the cell population having an NT5E positive rate of about 50% or more. (Item X8B) A cell population comprising inducible human regulatory T cells, the cell population having an ITGAE (CD103) positive rate of about 50% or more. (Item X8C) A cell population comprising inducible human regulatory T cells, the cell population having an AREG-positive rate of about 50% or more. (Item X8D) A cell population comprising inducible human regulatory T cells, the cell population having a CD172g positive rate of about 50% or more. (Item X8E) A cell population comprising inducible human regulatory T cells, the cell population having a CD26 positive rate of about 50% or more. (Item X8F) A cell population comprising inducible human regulatory T cells, the cell population having a CTLA4 positive rate of about 50% or more. (Item X8G1) A cell population comprising inducible human regulatory T cells, the cell population having a percentage of at least two characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more. (Item X8G2) A cell population comprising inducible human regulatory T cells, the cell population having a percentage of at least three characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more. (Item X8G3) A cell population comprising inducible human regulatory T cells, the cell population having a percentage of at least four characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more. (Item X8G4) A cell population comprising inducible regulatory human T cells, the cell population having a percentage of at least five characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more. (Item X8G5) A cell population comprising inducible human regulatory T cells, wherein the cell population has a percentage of all of the following characteristics: CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of approximately 50% or more. (Item X9) The cell population according to any one of the preceding items, wherein at least the CTLA4 positive and FoxP3 positive rates are each about 50% or more. (Item X10) The cell population according to any one of the preceding items, wherein at least the CD172g positive and / or CD26 positive proportions are each about 50% or more. (Item X11) The cell population according to any one of the preceding claims, wherein the proportion of said at least one characteristic in said cell population is about 60% or more. (Item X12) The cell population according to any one of the preceding items, wherein the proportion of said at least one characteristic in said cell population is about 80% or more. (Item X12a) The cell population according to any one of the preceding items, wherein the rate of strongly FoxP3 positive cells is about 50% or more. (Item X13) The cell population of any one of the preceding items, wherein about 90% or more of the cell population are T cells. (Item X14) A pharmaceutical composition comprising the inducible regulatory human T cell described in any one of the above items or the cell population described in any one of the above items. (Item X15) A regenerative medical material or product comprising the inducible regulatory human T cell described in any one of the above items or the cell population described in any one of the above items. (Item X16) A method for producing inducible human regulatory T cells, comprising: (a) stimulating CD4 positive T cells or CD8 positive T cells in human peripheral blood with a first basal medium for about 1 to about 5 days; (b) dormantly culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days; (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days; and (d) dormantly culturing the cells obtained in step (c) in a medium containing IL-2 for at least about 1 to about 3 days. A method comprising: (Item X17) The method according to any one of the preceding items, wherein the first basal medium contains at least one factor selected from the group consisting of an anti-CD3 antibody, TGF-β1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, a CDK8 / 19 inhibitor, and ascorbic acid. (Item X18) The method according to any one of the preceding items, wherein the first basal medium comprises an anti-CD3 antibody, TGF-β1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, a CDK8 / 19 inhibitor, and ascorbic acid. (Item X19) The method according to any one of the preceding items, wherein the second basal medium contains at least one factor selected from the group consisting of an anti-CD3 antibody, TGF-β1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, and a CDK8 / 19 inhibitor. (Item X20) The method according to any one of the preceding items, wherein the second basal medium comprises an anti-CD3 antibody, TGF-β1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, and a CDK8 / 19 inhibitor. (Item X21) The method according to any one of the preceding items, wherein step (a) stimulates the CD4-positive T cells or CD8-positive T cells with the first basal medium for about 3 days. (Item X22) The method according to any one of the preceding items, wherein the step (b) comprises dormantly culturing the cells obtained in the step (a) in a medium containing the IL-2 for at least about 2 days. (Item X23) 23. The method according to claim 16, wherein in step (c), the cells obtained in step (b) are stimulated with the second basal medium for about 3 days. (Item X24) The method according to any one of the preceding items, wherein the step (d) comprises dormantly culturing the cells obtained in the step (c) in a medium containing the IL-2 for at least about 2 days. (Item X25) An inducible regulatory human T cell or a cell population containing the inducible regulatory human T cell, produced by the method according to any one of the preceding items.

[0005] The present disclosure also provides: (Item 1) An inducible regulatory T cell having at least one characteristic selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, and AREG positivity. (Item 2) The inducible regulatory T cells described in the above item have at least two characteristics selected from the group consisting of CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, and AREG positivity. (Item 3) The inducible regulatory T cell according to any one of the preceding items, which is at least CTLA4 positive. (Item 4) The inducible regulatory T cell of any one of the preceding items, wherein the CNS2 site of the FOXP3 gene is demethylated. (Item 5) The inducible regulatory T cell according to any one of the preceding items, which is CD4 positive or CD8 positive. (Item 6) The inducible regulatory T cells of any one of the preceding items, which are obtained or derived from human peripheral blood T cells or human tissue-derived T cells. (Item 7) (a) stimulating CD4 positive T cells or CD8 positive T cells in peripheral blood with a first basal medium for about 1 to about 5 days; (b) dormantly culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days; (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days; and (d) dormantly culturing the cells obtained in step (c) in a medium containing IL-2 for at least about 1 to about 3 days. An inducible regulatory T cell obtained by a method comprising the steps of: (Item 8) A cell population comprising T cells, wherein about 50% or more of the T cells in the cell population are the inducible regulatory T cells described in any one of the preceding items. (Item 9) The cell population according to claim 8, wherein approximately 80% or more of T cells in the cell population are the inducible regulatory T cells described in any one of the preceding items. (Item 10) The cell population according to any one of the preceding items, wherein the T cells in the cell population are regulatory T cells. (Item 11) The cell population of any one of the preceding items, wherein about 90% or more of the cell population are T cells. (Item 12) A pharmaceutical composition comprising the induced regulatory T cells according to any one of the preceding items or the cell population according to any one of the preceding items. (Item 13) A regenerative medical material or product comprising the induced regulatory T cells according to any one of the above items or the cell population according to any one of the above items. (Item A1) A method for producing an inducible regulatory T cell, comprising: (a) stimulating CD4 positive T cells or CD8 positive T cells in peripheral blood with a first basal medium for about 1 to about 5 days; (b) dormantly culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days; (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days; and (d) dormantly culturing the cells obtained in step (c) in a medium containing IL-2 for at least about 1 to about 3 days. A method comprising: (Item A2) The method according to the preceding item, wherein the first basal medium contains at least one factor selected from the group consisting of an anti-CD3 antibody, TGF-β1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, a CDK8 / 19 inhibitor, and ascorbic acid. (Item A3) The method according to any one of the preceding items, wherein the first basal medium comprises an anti-CD3 antibody, TGF-β1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, a CDK8 / 19 inhibitor, and ascorbic acid. (Item A4) The method according to any one of the preceding items, wherein the second basal medium contains at least one factor selected from the group consisting of an anti-CD3 antibody, TGF-β1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, and a CDK8 / 19 inhibitor. (Item A5) The method according to any one of the preceding items, wherein the second basal medium comprises an anti-CD3 antibody, TGF-β1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, and a CDK8 / 19 inhibitor. (Item A6) The method according to any one of the preceding items, wherein step (a) stimulates the CD4-positive T cells or CD8-positive T cells with the first basal medium for about 3 days. (Item A7) The method according to any one of the preceding items, wherein the step (b) comprises dormantly culturing the cells obtained in the step (a) in a medium containing the IL-2 for at least about 2 days. (Item A8) The method according to any one of the preceding items, wherein step (c) stimulates the cells obtained in step (b) with the second basal medium for about 3 days. (Item A9) The method according to any one of the preceding items, wherein the step (d) comprises dormantly culturing the cells obtained in the step (c) in a medium containing the IL-2 for at least about 2 days. (Item A10) An induced regulatory T cell or a cell population containing the induced regulatory T cell, generated by the method according to any one of the preceding items.

[0006] It is contemplated that one or more of the above features may be provided in combinations other than those specifically described herein.Further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

[0007] Furthermore, features and notable actions and effects of the present disclosure other than those described above will become apparent to those skilled in the art by referring to the following description of the preferred embodiments of the present invention and the drawings. Effect of the Invention

[0008] The present disclosure can provide a method for inducing highly functional regulatory T cells from human peripheral T cells in vitro. The regulatory T cells induced by the method of the present disclosure highly express genes related to regulatory T cells and have strong suppressive ability. Therefore, as regulatory T cells, they can be used for the treatment and prevention of various immune diseases, inflammatory diseases such as autoimmune diseases, etc.

[0009] The method of the present disclosure can induce functional regulatory T cells in vitro from human peripheral T cells. The regulatory T cells obtained by the method of the present disclosure are functional (e.g., have high suppressive function) and are stable as regulatory T cells. That is, the method of the present disclosure can induce highly functional regulatory T cells that stably express FoxP3, the master gene of regulatory T cells, from human peripheral T cells. [Brief description of the drawings]

[0010] [Figure 1]FIG. 1 is a diagram showing a phenotype analysis (flow cytometry) of one embodiment of the inducible regulatory T cells (also called human highly functional stable iTreg (HSF iTreg) cells) of the present disclosure. The inducible regulatory T cells (HSF-iTreg in the figure) of the present disclosure were prepared from human CD4 positive T cells, and the expression of FOXP3, CTLA4, and Helios was analyzed by flow cytometry. A comparison was made between cells stimulated with conventional nTreg (CD4 positive CD25 positive T cells) and conventional iTreg (Conventional iTreg: cells obtained by stimulating CD4 positive T cells with CD3 / CD28 for 3 days in the presence of IL-2 and TGF-β1). [Diagram 2] 2 is a diagram showing epigenome analysis (bisulfite method) of one embodiment of the iTreg (HSF iTreg) cell of the present disclosure. The demethylation state of the FOXP3 CNS2 region of each cell in FIG. 1 was analyzed by the bisulfite method. [Diagram 3] 3 is a diagram showing an analysis of the suppressive ability (in vitro suppression assay) of one embodiment of the iTreg (HSF iTreg) cells of the present disclosure. The in vitro suppressive ability of each cell in FIG. 1 was analyzed. Human CD4-positive T cells were stained with Cell Trace Violet reagent and mixed and cultured with each cell in FIG. 1 at a fixed ratio in the presence of Treg Suppression Inspector (Miltenyi Biotec) (5 μL / well) for 3 days, and the Cell Trace Violet intensity was analyzed by flow cytometry. [Figure 4] 4 is a diagram showing gene expression pattern analysis (RNA-seq method) of one embodiment of the iTreg (HSF iTreg) cells of the present disclosure. Comprehensive gene expression patterns were analyzed by RNA sequencing for each cell in FIG. 1 and FOXP3-negative cells (Tconv) obtained by stimulating CD4-positive T cells in the presence of IL-2. [Diagram 5] 5 is a diagram showing CD103 expression (flow cytometry) in one embodiment of the iTreg (HSF iTreg) cells of the present disclosure. CD103 expression in FOXP3-positive cells of each of the cells in FIG. 1 was analyzed by flow cytometry. [Figure 6]6 is a diagram showing a phenotype analysis (flow cytometry) of one embodiment of the iTreg (HSF iTreg) cells of the present disclosure prepared from human CD8 positive T cells. Regulatory T cells of the present disclosure were prepared from human CD8 positive T cells, and the expression of FOXP3, CTLA4, and Helios was analyzed by flow cytometry. [Figure 7] 7 is a diagram showing CD25 expression and FOXP3 expression (flow cytometry) of one embodiment of the iTreg (HSF iTreg) cells of the present disclosure. The expression intensities of CD25 and FOXP3 were analyzed by flow cytometry using a BD FACSLyric flow cytometer. [Figure 8] 8 is a diagram showing a phenotype analysis of one embodiment of the iTreg (HSF iTreg) cells of the present disclosure. The inducible regulatory T cells (HSF-iTreg) of the present disclosure were generated, and the expression of CD172g, CD26, and the like was analyzed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present disclosure will be described below while showing the best mode. Throughout this specification, the expression of the singular form should be understood to include the concept of the plural form, unless otherwise specified. Therefore, the singular article (for example, in the case of English, "a", "an", "the", etc.) should be understood to include the concept of the plural form, unless otherwise specified. In addition, it should be understood that the terms used in this specification are used in the sense commonly used in the field, unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, the present specification (including definitions) will take precedence.

[0012] The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0013] In this specification, "about" means ±10% of the following numerical value. For example, "about 20" includes "18 to 22". Numerical ranges include all numerical values ​​between and at the endpoints. "About" in relation to a range applies to both endpoints of the range. Thus, for example, "about 20 to 30" includes "18 to 33".

[0014] In this specification, when a gene name and its product are written in all capital letters, unlike the usual usage, it may refer to both the gene and the protein. For example, FOXP3 gene and FOXP3 protein may be used separately, and when written as FoxP3, it refers to both the concept and the entity (whole) of the gene or protein.

[0015] As used herein, "regulatory T cells" refer to T cells positive for FoxP3 expression. As used herein, they may also be referred to as "Tregs." Tregs include naturally occurring regulatory T cells (nTregs) and inducible regulatory T cells (iTregs). Regulatory T cells can usually have various functions (e.g., immunosuppressive functions).

[0016] As used herein, the term "inducible regulatory T cells (iTreg)" refers to regulatory T cells that are negative for IKZF2 (Helios) expression. iTregs are usually obtained by inducing differentiation from naive CD4 positive T cells or the like.

[0017] As used herein, the term "naturally occurring regulatory T cells (nTreg)" refers to regulatory T cells that are positive for the expression of IKZF2 (Helios) and CTLA4. These cells are normally present in the body.

[0018] As used herein, "peripheral T cells" refers to T cells present outside the thymus gland, and can be obtained from peripheral blood, lymph nodes, and other tissues. As used herein, "peripheral T cells" refers only to the cell population containing peripheral T cells, and does not require that the T cells be isolated. In addition to T cells, cell fractions containing various lymphocytes, such as peripheral blood mononuclear cells (PBMC), may also be used.

[0019] In this specification, "flow cytometry" refers to a technique for measuring the number of cells, individuals and other biological particles suspended in a liquid, as well as the physical, chemical and biological properties of each particle. A device using this technique is called a "flow cytometer." In this disclosure, the "positive" and "negative" of cell markers (e.g., FoxP3, CTLA4, Helios, CD103, etc.) are determined by flow cytometry, as is commonly used in the art. More specifically, in flow cytometry, cells are lined up in a line and allowed to flow, and the number of cells is counted by a spectroscopic method. For example, cells labeled with fluorescence or luminescent enzymes are irradiated with laser light, and the fluorescence or luminescence signal emitted from the cells is detected by a detector such as a photodiode, thereby counting the number of target cells. In addition, the detection results from the detector can be input into a computer and a two-dimensional plot can be generated and displayed. This makes it easy to grasp the presence or absence of target cells, as well as their number.

[0020] As used herein, "demethylation" refers to the removal of methylation modifications of typically methylated adenine (e.g., m6A at position 6, m1A at position 1) and cytosine (e.g., m5C at position 5, m3C at position 3). Demethylation can be determined using a method known in the art, and can be measured, for example, using the bisulfite method.

[0021] As used herein, a "cell population" refers to a population containing two or more cells, and may be, for example, a collection of cells in a planar state, or a cell mass formed by cells adhering to each other in a three-dimensional manner. Furthermore, a "cell population" may be formed of a single type of cell, or may contain multiple types of cells.

[0022] As used herein, "stimulation" refers to stimulation via TCR. For example, stimulation of T cells includes stimulation with an anti-CD3 antibody or a complex containing the same, stimulation with a peptide that binds to TCR or a complex containing the peptide, stimulation with antigen-presenting cells, stimulation with an anti-CD3 antibody and antigen-presenting cells simultaneously, stimulation with a peptide or protein and antigen-presenting cells simultaneously, etc.

[0023] As used herein, the term "dormant culture" refers to culturing cells in the absence of the above-mentioned stimuli.

[0024] (Preferred embodiment) Preferred embodiments of the present disclosure are described below. The embodiments provided below are provided for a better understanding of the present disclosure, and the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description in this specification. In addition, the following embodiments of the present disclosure can be used alone or in combination.

[0025] (inducible regulatory T cells) The present disclosure relates to highly functional stable induced regulatory T cells (also referred to as highly functional stable iTreg, HSF iTreg) and uses thereof.

[0026] In one aspect of the present disclosure, an induced regulatory human T cell is provided having at least one characteristic selected from the group consisting of FoxP3 positive, CTLA4 positive, NT5E positive, ITGAE (CD103) positive, AREG positive, CD172g positive, and CD26 positive. In one embodiment of the present disclosure, the induced regulatory T cell of the present disclosure may have at least two characteristics selected from the group consisting of CTLA4 positive, NT5E positive, ITGAE (CD103) positive, and AREG positive. In one embodiment of the present disclosure, the induced regulatory T cell of the present disclosure may be at least CTLA4 positive. Without intending to be limited, the induced regulatory T cell of the present disclosure may be CD4 positive or CD8 positive.

[0027] In one embodiment of the present disclosure, the induced regulatory T cells of the present disclosure may be at least CTLA4 positive and FoxP3 positive. In one embodiment of the present disclosure, the induced regulatory T cells of the present disclosure may be at least CD172g positive and / or CD26 positive.

[0028] The present disclosure provides CD172g-positive inducible human regulatory T cells. CD172g is a tyrosine kinase-related protein that is involved in cell adhesion of neurons and the like (adhesion of cerebellar neurons, neurite outgrowth and glial cell attachment). Therefore, CD172g-positive inducible human regulatory T cells are expected to activate functions related to adhesion and the like and to be highly effective against various diseases associated therewith.

[0029] The present disclosure provides CD26-positive inducible human regulatory T cells. CD26 is a gene also known as DPP4, and is related to glucose metabolism, insulin metabolism, and immune function (notably in infectious diseases), so CD26-positive inducible human regulatory T cells are expected to be highly effective against various diseases related thereto.

[0030] The present disclosure provides NT5E-positive inducible human regulatory T cells. NT5E (CD73) is a cell membrane protein that has a catalytic activity of converting extracellular nucleotides into membrane-permeable nucleosides. The encoded protein is used as a determinant of lymphocyte differentiation. Since defects in this gene can cause calcification of joints and arteries, NT5E-positive inducible human regulatory T cells are expected to be highly effective against various diseases associated with this.

[0031] The present disclosure provides ITGAE (CD103) positive inducible human regulatory T cells. ITGAE (CD103) encodes an α integrin with an I domain that undergoes post-translational cleavage in the extracellular domain to generate disulfide-linked heavy and light chains. This protein binds to β7 integrin to form an E-cadherin-binding integrin known as human mucosal lymphocyte-1 antigen. This protein is preferentially expressed on human intestinal intraepithelial lymphocytes (IELs) and may function as an accessory molecule for IEL activation in addition to its adhesive role. Therefore, ITGAE (CD103) positive inducible human regulatory T cells are expected to be highly effective against various diseases associated with these diseases.

[0032] The present disclosure provides an AREG-positive inducible regulatory human T cell. AREG is a member of the epidermal growth factor family and is related to epidermal growth factor (EGF) and transforming growth factor alpha (TGF-α). This protein interacts with the EGF / TGF-α receptor to promote the growth of normal epithelial cells and inhibit the growth of certain aggressive cancer cell lines. It also functions in the development of mammary glands, oocytes, and bone tissue. This gene is associated with a skin phenotype similar to psoriasis, and is also associated with other pathological diseases, including various types of cancer and inflammatory conditions. Therefore, it is expected that the AREG-positive inducible regulatory human T cell will be highly effective against various diseases related thereto.

[0033] The present disclosure provides CTLA4-positive inducible human regulatory T cells. CTLA4 belongs to the immunoglobulin superfamily and is a protein that transmits an inhibitory signal to T cells. Membrane-bound CTLA4 functions as a homodimer linked by disulfide bonds, while soluble CTLA4 functions as a monomer. Mutations in this gene are said to be associated with insulin-dependent diabetes mellitus, Graves' disease, Hashimoto's thyroiditis, celiac disease, systemic lupus erythematosus, thyroid-associated orbitopathy, and other autoimmune diseases. Therefore, CTLA4-positive inducible human regulatory T cells are expected to be highly effective against various diseases associated with these diseases.

[0034] The expression of FoxP3 in regulatory T cells can be induced in vitro by culturing CD4 positive T cells in the presence of anti-CD3 and anti-CD28 antibodies in a medium containing IL2 and TGFβ, and then culturing them in a medium containing IL2 and TGFβ. Although several methods for inducing regulatory T cells from peripheral T cells are known, the expression of FoxP3 in induced regulatory T cells is unstable, and the expression of many functional molecules other than FoxP3 has not been confirmed.

[0035] In recent years, culture methods have been developed to induce DNA demethylation in induced regulatory T cells by adding ascorbic acid to the medium (Kasahara et al. Int. Immunol. (2017) 29(10):457-469) and by not using CD28 antibody stimulation (Mikami et al. Proc Natl Acad Sci US A. (2020)117(22):12258-12268.). However, the expression of functional molecules has not been confirmed in the induced regulatory T cells produced by this method, and sufficient immunosuppressive activity has not been obtained. One clinical trial using induced regulatory T cells has also been conducted for GVHD, but the proportion of regulatory T cells used was low, and no efficacy has been confirmed for preventing GVHD at this time (MacMillan et al., Blood Adv. (2021) 5(5):1425-1436).

[0036] In the present disclosure, it is possible to provide an inducible regulatory T cell that stably expresses FoxP3 and advantageously stably retains an immunosuppressive effect, and such an inducible regulatory T cell can be produced, for example, by a method for producing an inducible regulatory T cell described elsewhere in the present specification. For example, the present disclosure can provide an inducible regulatory T cell obtained by a method including the steps of: (a) stimulating CD4-positive T cells or CD8-positive T cells in peripheral blood with a first basal medium for about 1 to about 5 days; (b) rest-culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days; (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days; and (d) rest-culturing the cells obtained in step (c) in a medium containing IL-2 for at least about 1 to about 3 days.

[0037] The induced regulatory T cells prepared in the present disclosure have an induced regulatory T cell-specific demethylation state. The obtained regulatory T cells can be confirmed to be in a regulatory T cell-specific demethylation state, for example, by demethylation of the CNS2 site of the FoxP3 gene (FOXP3 (all italics)). Since such a demethylation state can be an indicator of a stable type, the induced regulatory T cells of the present disclosure can be shown to be stable induced regulatory T cells by confirming the demethylation state.

[0038] In the present specification, the immunosuppressive activity or immunosuppressive effect of the induced regulatory T cells of the present disclosure can be confirmed, for example, by measuring the Cell Trace Violet intensity in responder T cells. The induced regulatory T cells of the present disclosure can stably provide immunosuppressive activity or immunosuppressive effect, for example, the induced regulatory T cells of the present disclosure can provide immunosuppressive activity or immunosuppressive effect for at least about 2 weeks. As shown in the examples described below, the induced regulatory T cells of the present disclosure can have a higher immunosuppressive effect than conventional regulatory T cells (including induced and endogenous). Therefore, the induced regulatory T cells of the present disclosure can be said to be functional or highly functional induced regulatory T cells.

[0039] In one embodiment, the induced regulatory T cells of the present disclosure can stably express FoxP3. Therefore, the induced regulatory T cells of the present disclosure can also be called stable induced regulatory T cells. In one aspect, the induced regulatory T cells of the present disclosure are highly functional and stable, and can be called highly functional stable induced regulatory T cells (HSF iTreg).

[0040] In one embodiment of the present disclosure, whether or not the marker in the induced regulatory T cells of the present disclosure is positive can be determined by measuring the positive rate by flow cytometry. For example, the marker can be analyzed by a flow cytometer, and whether or not it is positive can be determined from the ratio of cells that show an antigen expression level equal to or higher than a standard. Depending on the expression intensity of the cell surface marker, it can also be classified as negative, weakly positive, medium positive, strongly positive (weakly positive, medium positive, and strongly positive are collectively referred to as "positive"). For example, depending on the settings of the device, the values ​​of the median fluorescence intensity of each marker / median fluorescence intensity of the negative control (stained with an isotype control antibody) can be negative, weakly positive, medium positive, and strongly positive, respectively. Such a determination can be made as exemplified in (measurement of the positive rate by flow cytometry), but the present disclosure is not limited thereto.

[0041] In one embodiment of the present disclosure, the expression intensity of a cell surface marker in the induced regulatory T cells of the present disclosure is, for example, based on the results of analysis using a BD FACSLyric flow cytometer (BD Biosciences) using a sample prepared as described in the Examples of the present application, and the expression intensity is within the range of 10 2 10 or more is weak positive, 3 10 or more are moderately positive, 4 or more may be considered strongly positive, or 10 3 A score of 10 or higher may be considered strongly positive, and a score of 10 or lower may be considered weakly positive. 2 A value of 0.01 or more may be considered to be strongly positive, and a value of 0.01 or less may be considered to be weakly positive. Such a determination can be appropriately set depending on the experimental conditions, experimental purpose, cell type, device setting conditions, etc., and the present disclosure is not limited thereto. In addition, even when analysis is performed using a flow cytometer device other than the BD FACSLyric flow cytometer, the expression intensity in the other device corresponding to the expression intensity in analysis using the BD FACSLyric flow cytometer can be calculated, and a value indicating the expression intensity according to the device used can be appropriately found.

[0042] In one embodiment of the present disclosure, the induced regulatory T cells produced in the present disclosure can be induced from any cells, but are preferably induced and obtained from human peripheral blood T cells or human tissue-derived T cells.

[0043] In one embodiment of the present disclosure, the induced regulatory T cells or cell population of the present disclosure can be human cells, and can be induced human regulatory T cells or cell populations induced by a predetermined method using T cells obtained from human peripheral blood. The properties of human cells and mouse cells are clearly different, and even if the same cell surface marker is present, the properties of the cells cannot be considered to be the same. For example, in the field of induced regulatory T cells, the applicable techniques are different between mice and humans, and while most lymphocytes in mice are antigen-naïve, in humans, the T cells in peripheral blood have a variety of antigen sensitization and activation levels. Therefore, although highly functional stable induced regulatory T cells can be directly produced from T cells collected from mouse lymphoid tissues in mice, this is difficult to do in humans, and the cells cannot be considered to be similar simply based on the presence of a cell surface marker.

[0044] In one aspect of the present disclosure, there is provided a cell population comprising a cell population containing an inducible regulatory human T cell as described above, wherein the cell population has a percentage of at least one characteristic selected from the group consisting of FoxP3 positivity, CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity of about 50% or more.

[0045] In another aspect of the present disclosure, a cell population is provided that comprises an inducible human regulatory T cell, and the cell population has a CTLA4 positive and FoxP3 positive ratio of about 50% or more. In one embodiment, the cell population of the present disclosure can have a CTLA4 positive and FoxP3 positive ratio of about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more.

[0046] In one embodiment of the present disclosure, the cell population of the present disclosure may have at least the CD172g positive and / or CD26 positive ratios of about 50% or more. In one embodiment, the cell population of the present disclosure may have a CD172g positive and / or CD26 positive ratio of about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more.

[0047] In one embodiment of the present disclosure, the cell population of the present disclosure may have a FoxP3 strongly positive rate of about 50% or more. In one embodiment, the cell population of the present disclosure may have a FoxP3 strongly positive rate of about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more. Without intending to be limited thereto, the expression intensity of cell surface markers in such a cell population may be measured by measuring the positive rate by flow cytometry as described above.

[0048] In another aspect of the present disclosure, a cell population is provided that includes an inducible regulatory human T cell, the cell population having a FoxP3 positive rate of about 50% or more. Without intending to be limited thereto, in one embodiment, such a cell population can include FoxP3 positive inducible regulatory human T cells at a cell number basis of about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more.

[0049] In another aspect of the present disclosure, a cell population is provided that includes an inducible regulatory human T cell, the cell population having a CTLA4 positive rate of about 50% or more. Without intending to be limited thereto, in one embodiment, such a cell population can include CTLA4 positive inducible regulatory human T cells at a cell number basis of about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more.

[0050] In another aspect of the present disclosure, a cell population is provided that contains an inducible regulatory human T cell, the cell population having a NT5E positive rate of about 50% or more. Without intending to be limited thereto, in one embodiment, such a cell population may contain NT5E positive inducible regulatory human T cells at a cell number basis of about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more.

[0051] In another aspect of the present disclosure, a cell population is provided that includes an inducible regulatory human T cell, the cell population having a percentage of ITGAE (CD103) positive cells of about 50% or more. Without intending to be limited thereto, in one embodiment, such a cell population may include ITGAE (CD103) positive inducible regulatory human T cells of about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more based on the number of cells.

[0052] In another aspect of the present disclosure, a cell population is provided that comprises an inducible regulatory human T cell, the cell population having an AREG positive rate of about 50% or more.Without intending to be limited thereto, in one embodiment, such a cell population can comprise AREG positive inducible regulatory human T cells at a cell number standard of about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more.

[0053] In another aspect of the present disclosure, a cell population is provided that includes an inducible regulatory human T cell, the cell population having a CD172g positive rate of about 50% or more. Without intending to be limited thereto, in one embodiment, such a cell population may include about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more of CD172g positive inducible regulatory human T cells based on cell number.

[0054] In another aspect of the present disclosure, a cell population is provided that includes an inducible regulatory human T cell, the cell population having a CD26 positive rate of about 50% or more. Without intending to be limited thereto, in one embodiment, such a cell population may include about 50% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more of CD26 positive inducible regulatory human T cells based on cell number.

[0055] As described above, the properties of human cells and mouse cells are clearly different, and therefore even if the induction conditions are the same, the induction rate of specific cells will differ between mouse cells and human cells. In the present disclosure, when induced by a specified induction method, a cell population in which the positive rate of a specified cell surface marker is about 50% or more can be obtained, and preferably a cell population in which "the positive rates of CTLA4 and FoxP3 are each about 50% or more" can be obtained.

[0056] CTLA4 is a molecule that is localized within cells, and at least it is not at a level that can be used as an indicator for enrichment or purification. In addition, FoxP3 is a transcription factor and is a molecule that is entirely present within cells, so it cannot be detected at all on the surface and cannot be used for purification. In other words, since CTLA4 and FoxP3 are markers that are expressed "inside" T cells, it is not possible to obtain a cell population in which CTLA4-positive and / or FoxP3-positive are about 50% or more by sorting and enriching using CTLA4 and / or FoxP3 as indicators. Therefore, it is also not possible to obtain a cell population in which the proportions of CTLA4-positive and FoxP3-positive are less than about 50% each according to the conventional method, and then use such a cell population as a raw material to enrich using CTLA4 and / or FoxP3 as indicators to obtain a cell population in which the respective cells are about 50% or more.

[0057] In one embodiment of the present disclosure, the inducible regulatory human T cells of the present invention are primarily responsible for the immunosuppressive function of Tregs, and when these cells account for more than half of a cell population, a medically effective immunosuppressive effect can be stably achieved, which is important in terms of technical and medical effects. In other words, when the cell population contains 50% or more T cells that exert a medically effective immunosuppressive effect, a stable cell preparation can be provided, and this effect is an extremely important point in terms of medicine, and is not merely a difference in quantity or numerical value, but is an important point in terms of quality as to whether the preparation can be established.

[0058] In one embodiment of the present disclosure, in the cell population of the present disclosure, the induced regulatory human T cells can further have the characteristics of ITGAE (CD103) positive, NT5E positive, and / or AREG positive. These cell markers are genes that play an important role in immunosuppressive function, and due to the high expression of these markers, the functional stability can be maintained more than that of conventional induced regulatory T cells after administration of the cells. For example, CD103 is important for migration of regulatory T cells to inflammatory sites, NT5E (CD73) is important for the production of adenosine, which has immunosuppressive ability, and AREG is important for tissue regeneration by regulatory T cells. Due to the high expression of these markers, the cell population of the present disclosure can achieve the effect of maintaining functional stability.

[0059] In one embodiment of the present disclosure, the T cells in the cell population of the present disclosure can be regulatory T cells, in which case about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more of the regulatory T cells in the cell population of the present disclosure can be inducible regulatory T cells as described elsewhere herein.

[0060] In one embodiment of the present disclosure, the cell population of the present disclosure may contain cells other than T cells, but preferably, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more of the cell population of the present disclosure may be T cells, and preferably, about 90% or more may be T cells.

[0061] In one aspect of the present disclosure, a pharmaceutical composition comprising the inducible regulatory T cells or cell population of the present disclosure is provided. In another aspect, a regenerative medicine material or product comprising the inducible regulatory T cells or cell population of the present disclosure is provided. This pharmaceutical composition or regenerative medicine material or product can be used for autoimmune diseases, inflammatory diseases, and allergies that can be treated by immunosuppressive action. These medicines, regenerative medicine materials or products can be used together with culture media and any other additives used in the field. As such a medium, a medium obtained by adding necessary factors to a basal medium for animal cell culture used for cell culture can be used. Examples of such media are described in detail elsewhere in this specification. Examples of components added to the medium are also described in detail elsewhere in this specification. When provided as such a product, it may contain DMSO, etc.

[0062] (Production method) The present disclosure provides a method for producing the inducible regulatory T cells of the present disclosure. The method of the present disclosure is a novel method for producing highly functional and stable inducible regulatory T cells, which is described in detail herein below.

[0063] In one aspect of the present disclosure, there is provided a method for producing an inducible regulatory T cell, comprising the steps of (a) stimulating CD4-positive T cells or CD8-positive T cells in peripheral blood with a first basal medium for about 1 to about 5 days, (b) resting and culturing the cells obtained in step (a) in a medium containing IL-2 for at least about 1 to about 3 days, (c) stimulating the cells obtained in step (b) with a second basal medium for about 1 to about 5 days, and (d) resting and culturing the cells obtained in step (c) in a medium containing IL-2 for at least about 1 to about 3 days. In the present disclosure, the inducible regulatory T cell of the present disclosure can be produced using either CD4-positive T cells or CD8-positive T cells as a raw material, and in one embodiment, the inducible regulatory T cell of the present disclosure can also be produced using a mixed cell of CD4-positive T cells and CD8-positive T cells as a raw material.

[0064] In one embodiment, the method of the present disclosure can also be a method for producing regulatory T cells from human peripheral T cells (including CD4-positive T cells or CD8-positive T cells), comprising the steps of culturing human peripheral T cells in a medium containing TGFβ and IL-2 in the presence of anti-CD3 antibody stimulation, culturing them in a medium containing IL-2 in the absence of anti-CD3 antibody, and again culturing them in a medium containing TGFβ and IL-2 in the presence of anti-CD3 antibody stimulation.

[0065] In one embodiment, the stimulation of T cells refers to a stimulation for obtaining Tregs, and is not particularly limited as long as it is a stimulation via TCR. For example, the stimulation of T cells can include stimulation with an anti-CD3 antibody or a complex containing the antibody, stimulation with a peptide that binds to TCR or a complex containing the peptide, stimulation with antigen-presenting cells, stimulation with an anti-CD3 antibody and antigen-presenting cells simultaneously, stimulation with a peptide or protein and antigen-presenting cells simultaneously, etc., but the medium and conditions are not particularly limited as long as Tregs can be obtained.

[0066] In one embodiment, the medium and conditions for dormant culture are not particularly limited as long as the cells are cultured in the absence of the above-mentioned stimuli.

[0067] As used herein, "anti-CD3 antibody stimulation" means providing specific stimulation to the CD3 receptor on a cell. An example of CD3 stimulation is an anti-CD3 agonist antibody. The anti-CD3 agonist antibody may be a commercially available product as a research reagent, or may be prepared by a conventional method. The anti-CD3 antibody may be derived from an animal, such as a mouse, rabbit, goat, or cow, or from a human.

[0068] In one embodiment, the method of the present disclosure involves first inducing iTreg (stimulating T cells to induce demethylation) (step a), then exchanging the medium and resting culture for 1 to 3 days to recover the cells (step b), and stimulating the T cells once more to induce demethylation (step c), thereby obtaining functional and stable inducible regulatory human T cells. Generally, it is common technical knowledge that T cells undergo apoptosis after two stimulations, but the method of the present disclosure has been found to obtain functional and stable inducible regulatory human T cells without apoptosis by performing "resting culture" and "second stimulation".

[0069] That is, in the method of the present disclosure, functional and stable inducible regulatory human T cells can be obtained by performing T cell stimulation, resting culture, and subsequent re-stimulation, and therefore the desired effect of the present disclosure can be achieved with the inducible regulatory human T cells thus obtained or a cell population containing such inducible regulatory human T cells. Thus, in one embodiment of the present disclosure, the medium for culture and the type of stimulus are not particularly limited, and functional and stable inducible regulatory human T cells can be obtained by using a medium of any composition and any type of stimulus.

[0070] In one embodiment, the medium used in each step may further contain retinoic acid and / or ascorbic acid. Preferably, the medium may contain ascorbic acid. In one embodiment, the medium may further contain a CDK8 inhibitor, a CDK19 inhibitor, and / or a CDK8 / 19 inhibitor. Preferably, the medium may contain a CDK8 inhibitor, a CDK19 inhibitor, and / or a CDK8 / 19 inhibitor.

[0071] In one embodiment of the present disclosure, the first basal medium and the second basal medium may each independently contain at least one, at least two, at least three, at least four, at least five, or all of the factors selected from the group consisting of anti-CD3 antibody, TGF-β1, IL-2, retinoic acid, CDK8 inhibitor, CDK19 inhibitor, CDK8 / 19 inhibitor, and ascorbic acid. In another embodiment, the first basal medium and the second basal medium may each independently contain anti-CD3 antibody, TGF-β1, IL-2, retinoic acid, CDK8 inhibitor, CDK19 inhibitor, CDK8 / 19 inhibitor, and ascorbic acid. The concentration of each of these components may be a normal concentration used in the art. In one embodiment, the concentration of the CDK8 inhibitor, CDK19 inhibitor, and / or CDK8 / 19 inhibitor that can be used may be any appropriate concentration that can be used in the art, and for example, when Senexin A is used, it can be about 0.1 μM or more, about 0.5 μM or more, about 1 μM or more, about 2 μM or more, about 3 μM or more, about 4 μM or more, about 5 μM or more, about 6 μM or more, about 7 μM or more, about 8 μM or more, about 9 μM or more, about 10 μM or more, about 12 μM or more, about 14 μM or more, about 16 μM or more, about 18 μM or more, about 20 μM or more, etc., but is not limited to these concentrations, and can be changed as appropriate by a person skilled in the art depending on other medium compositions.

[0072] In one embodiment, the method of the present disclosure produces regulatory T cells from human peripheral T cells. The peripheral T cells include naive regulatory T cells, CD4 positive T cells, CD8 positive T cells, and the like. Regulatory T cells may be induced from a culture containing multiple types of T cells, or specific cells such as CD4 positive T cells or CD8 positive T cells may be isolated from these cells and then the regulatory T cells may be induced. Regulatory T cells may also be induced after isolating T cells specific to a specific antigen. Therefore, the induced regulatory T cells of the present disclosure include CD4 positive ones and CD8 positive ones. In addition, when "CD4 positive" or "CD4+" is used in this specification, it refers to CD4 positive CD8 negative single positive cells unless otherwise specified. In addition, in the production method of the present disclosure, either CD4 positive or CD8 positive T cells can be used as a raw material, and even after the induced regulatory T cells are generated, the CD4 positive or CD8 positive characteristics can be maintained unless a special operation is performed.

[0073] In one embodiment, in the method of the present disclosure, the antibody may be added to the culture medium, or may be immobilized on the inner wall of a culture vessel or the surface of an insoluble carrier. The insoluble carrier may be a material capable of physically or chemically binding an anti-CD3 antibody and insoluble in an aqueous solution. Examples of materials capable of physically adsorbing an anti-CD3 antibody include synthetic resins such as polystyrene, polyethylene terephthalate, polycarbonate, and polypropylene, and glass. The shape of the insoluble carrier is not particularly limited, and may be, for example, a plate shape, a bead shape, or a container shape. The amount of anti-CD3 antibody varies depending on the titer and origin of the antibody used, but may be appropriately set so as to provide sufficient stimulation for the induction of regulatory T cells.

[0074] In one embodiment, in the method of the present disclosure, a medium obtained by adding necessary factors to a basal medium for animal cell culture can be used for cell culture. Examples of basal medium for animal cell culture that can be used in the method of the present disclosure include Iscove's modified Eagle's Medium medium, Ham's F12 medium, MEM Zinc Option medium, IMEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, RPMI1640 medium, Fischer's medium, and mixtures or media with partially modified compositions thereof.

[0075] The basal medium may contain serum (e.g., fetal bovine serum (FBS)) or may be serum-free. The serum-free medium may optionally contain one or more serum replacements, such as, for example, albumin, bovine serum albumin (BSA), transferrin, apotransferrin, KnockOut Serum Replacement (KSR) (a serum replacement for ES cell culture) (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), B27 supplement (Thermo Fisher Scientific), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, and monothioglycerol. The basal medium may also contain one or more substances such as lipids (e.g., a chemically defined lipid concentrate), amino acids, L-glutamine, GlutaMAX (Thermo Fisher Scientific), non-essential amino acids (NEAA), vitamins (e.g., nicotinamide, ascorbic acid), growth factors, antibiotics (e.g., penicillin and streptomycin), antioxidants, pyruvate, buffers, inorganic salts, and the like.

[0076] In one embodiment, an example of the basal medium is RPMI 1640 medium containing serum and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

[0077] In the method of the present disclosure, the cells may be cultured under general animal cell culture conditions. The culture temperature is, but is not limited to, about 30 to 40° C., preferably about 37° C. The culture is preferably performed in an atmosphere of CO2-containing air, and the CO2 concentration is preferably about 2 to 5%.

[0078] In the method of the present disclosure, the anti-CD3 antibody may be added directly to the medium, or may be immobilized on the inner wall of a culture vessel or on the surface of an insoluble carrier. The amount of the anti-CD3 antibody varies depending on the titer and origin of the antibody used, but may be appropriately set so as to provide a sufficient stimulation for the induction of regulatory T cells.

[0079] Examples of TGFβ used include TGFβ1, TGFβ2, and TGFβ3, for example, TGFβ1. The concentration of TGFβ can be determined appropriately by those skilled in the art and is not particularly limited. When TGFβ1 or TGFβ3 is used as TGFβ, the concentration in the medium is not particularly limited, but may be 0.25 to 25 ng / mL, for example, about 10 ng / mL.

[0080] The concentration of IL-2 in the medium used is not limited, but can be about 5 U / mL to about 500 U / mL, for example, about 100 U / mL.

[0081] The medium used in the present disclosure may further contain retinoic acid and / or ascorbic acid. Preferably, the medium contains ascorbic acid. The concentration of ascorbic acid is not limited, but is about 1 to about 100 μg / mL, for example, about 10 μg / mL.

[0082] The medium used in the present disclosure may further contain a CDK8 inhibitor, a CDK19 inhibitor, and / or a CDK8 / 19 inhibitor. Any CDK8 inhibitor, CDK19 inhibitor, and / or CDK8 / 19 inhibitor may be used, and examples of such inhibitors include 4-[1-(2-methyl-1H-benzimidazol-5-yl)-1H-imidazo[4,5-c]pyridin-2-yl]-1,2,5-oxadiazol-3-amine, 3-{1-[1-(4-methoxyphenyl)piperidin-4-yl]-4-methyl-1H-imidazo[4,5-c]pyridin-2-yl}pyrazin-2-amine, or salts, hydrates, solvates thereof, or compounds described in U.S. Patent No. 8,598,344, WO2013 / 001310, WO 2013 / 040153, WO2013 / 116786, WO2014 / 029726, WO2014 / 063778, WO2014 / 072435, WO2014 / 090692, WO2014 / 106606, WO2014 / 123900, WO2014 / 154723, WO2014 / 194245, WO2015 / 049325, WO2015 / 100420, WO2015 / 144290, WO2015 / 159937, WO2015 / 159938, WO2016 / 009076, or WO2018 / 139660. One example is SenexinA or AS2863619, but the present disclosure is not limited thereto. The concentration of the CDK8 inhibitor, CDK19 inhibitor, and / or CDK8 / 19 inhibitor that can be used may be any suitable concentration that can be used in the art, or any suitable concentration described in the above-mentioned documents, and those skilled in the art can appropriately change it according to other medium compositions.

[0083] In the method of the present disclosure, human peripheral T cells are stimulated with an anti-CD3 antibody in a medium containing TGFβ and IL-2 to induce regulatory T cells, and regulatory T cells having a high immunosuppressive function can be induced by stimulating the cells with an anti-CD3 antibody again after performing resting culture in an IL-2-containing medium not containing an anti-CD3 antibody. The high immunosuppressive function of the obtained induced regulatory T cells can be confirmed by, for example, comprehensive gene expression analysis using an RNA sequencing method or an in vitro cell proliferation inhibition test.

[0084] In one embodiment of the present disclosure, the number of days of culture in the step (a) of stimulating CD4-positive T cells or CD8-positive T cells in peripheral blood with a first basal medium for about 1 to about 5 days can be appropriately determined by those skilled in the art and is not particularly limited, but can be, for example, about 3 days.

[0085] In one embodiment, the number of days for culture in the dormant culture step in step (b) can be appropriately set by those skilled in the art and is not particularly limited, but can be, for example, about 2 days.

[0086] In one embodiment, the number of days for the culture step in the second basal medium in step (c) can be appropriately set by a person skilled in the art and is not particularly limited, but can be, for example, about 3 days.

[0087] In one embodiment, the number of days for culture in the resting culture step in step (d) can be appropriately set by those skilled in the art and is not particularly limited, but can be, for example, about 2 days. In one embodiment, regulatory T cells having an induced regulatory T cell-specific demethylation state can be expanded by culturing in an unstimulated state in the presence of IL-2. The medium may further contain ascorbic acid, and a stable regulatory T cell culture of the induced regulatory T cells can be obtained by culturing in a medium further containing IL-2.

[0088] To isolate regulatory T cells from the obtained cell culture containing regulatory T cells, the cells may be isolated by a conventional method based on a cell surface marker specific to regulatory T cells, for example, by extracting a FoxP3-positive fraction using a cell sorter. If desired, regulatory T cells having a specific antigenic property may be isolated.

[0089] The inducible regulatory T cells obtained by the method of the present disclosure are expected to be used in the treatment of human inflammatory diseases, such as autoimmune diseases and allergies.

[0090] (Positive rate measured by flow cytometry) In one embodiment, the positive rate measurement by flow cytometry can be performed as follows.

[0091] preparation ·Fixation / Permeabilization Concentrate (hereinafter referred to as Buffer) (eBio Science, 00-5123-43) Fixation / Permeabilization Diluent (hereinafter, Diluent) (eBio Science, 00-5223-56) ·Permeabilization Buffer(10×)(eBio Science, 00-833-56) ·FOXP3 Monoclonal Antibody (236A / E7), PE (hereinafter referred to as anti-FOXP3 antibody) (eBio Science, 12-4777-42) Mouse IgG1 kappa Isotype Control (P3.6.2.8.1), PE (hereinafter referred to as PE control) (eBio Science) ·BV421, Mouse, Anti-Human, CD152 (hereinafter referred to as anti-CTLA4 antibody) (BD) BV421 Mouse IgG2a, k Isotype Control (hereinafter referred to as BV421 control) (BD) ·CD4 Monoclonal Antibody (RPA-T4), APC (hereinafter referred to as anti-CD4 antibody) (eBio Science) Mouse IgG1 kappa Isotype Control (P3.6.2.8.1), APC (hereafter referred to as APC control) (eBio Science) ·D-PBS (Nacalai Tesque, 14249-95) FBS (HyClone, SH30084.03) 0.5 mol / l-EDTA solution (pH 8.0) (Nacalai Tesque, 06894-14) MilliQ Water ·Centrifuge ·Safety cabinet Micropipettes (P200, P1000) 5mL polystyrene round tube (hereinafter, dedicated tube) (Falcon, 352008) Nylon mesh (65μm) (Kyoshin Ricoh, PP-65N)

[0092] Reagent preparation ※Fixation Buffer (100μL per sample) Mix the buffer and diluent in a 1:3 ratio. ※Perm Buffer Dilute the Permeabilization Buffer (10x) 10 times with MilliQ water. ※FACS Buffer (when preparing 500 mL) D-PBS 489mL FBS 10mL (final concentration 2%) 0.5mol / l EDTA solution 1mL (final concentration 1mM) After preparation, store the above reagents at 4°C or on ice.

[0093] method (1) Add 500 μL of FACS Buffer to a 1.5 mL tube. (2) 1x10 in tube (1) 6Add the final product and gently suspend using a micropipette. (3) Centrifuge at 500 x g and 4°C for 5 minutes. (4) After removing the supernatant with an aspirator, add 100 μL of Fixation Buffer and gently pipette. → Be careful not to create bubbles. (5) Fix by leaving it on ice in the dark for at least 30 minutes. → 45 minutes is also acceptable. (6) After fixation, add 1 mL of Perm Buffer to the tube and gently pipette. (7) Prepare new 1.5 mL tubes for the number of samples. (8) Dispense 500 μL of the sample for control and antibody staining. (9) Centrifuge at 500 x g and 4°C for 5 minutes. (10) During centrifugation, prepare anti-FOXP3 antibody (stock concentration = 0.05 mg / ml), anti-CTLA4 antibody (Lot: 0030269 stock concentration = 0.2 mg / ml), and anti-CD4 antibody (stock concentration = 0.1 mg / ml) at a 100-fold dilution with Perm Buffer (hereinafter referred to as antibody preparation solution). For control samples, prepare PE control (stock concentration = 0.1 mg / ml), BV421 control (stock concentration = 0.2 mg / ml), and APC control (stock concentration = 0.1 mg / ml) at the same concentration as the corresponding dye antibodies (hereinafter referred to as control solution). *CTLA4 staining is not performed in the in-process control test. (11) After removing the supernatant with an aspirator, add 100 μL of antibody preparation solution to the antibody-stained sample and control solution to the control sample, and gently pipette. → Be careful not to create bubbles. (12) Leave on ice in the dark for 60 minutes to fix. (13) Start up the measuring equipment during dyeing. (14) After staining, add 1 mL of FACS Buffer and gently pipette. (15) Centrifuge at 500 x g for 5 minutes at 4°C. → Remove the supernatant using an aspirator, and then repeat steps (14) and (15) to wash once more. (16) After removing the supernatant using an aspirator, add 500 μL of FACS Buffer and gently pipette. (17) Place a nylon mesh on the dedicated tube with tweezers and filter the suspension. (18) Analyze using any flow cytometer. The number of cells collected for data should be 10,000 or more. In calculating the target antigen positivity rate, a reference line is drawn so that the positivity rate of the control sample is 5% or less, and the percentage of cells showing antigen expression levels above the reference is the positivity rate.

[0094] remarks The reagents used for fixation and staining can also be purchased as a set of three bottles called the "Foxp3 / Transcription Factor Staining Buffer Set" (Cat.:00-5523). -As long as the instrument settings are not so far off that it is clearly determined from a general or scientific point of view that normal measurements are not being performed, there is no problem with any settings. Clear deviations include cases where the various signals of the target cell population are below the set fluorescence threshold value, or where the various signal values ​​of the control sample or the sample to be measured are below or above the limit of normal measurement by the instrument, etc.

[0095] (Immunosuppressive activity measurement) The cells of the present disclosure may have immunosuppressive activity. Immunosuppressive activity can be measured in a variety of ways.

[0096] In one embodiment, as described in the Examples, the suppression can be determined by measuring cell proliferation due to an immune response caused by responder T cells. In addition, various kits can be used, including, but not limited to, a Treg immunosuppression assay (horizon discovery; https: / / www.horizondiscoverykk.com / products / research-services / immune-cell-based-assay / immune-suppression-assays / #Treg). The immunosuppressive activity of the cells of the present disclosure may be enhanced compared to conventional iTreg.

[0097] (General technology) The molecular biological, biochemical and microbiological techniques used herein are well known and commonly used in the art, and are described, for example, in Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, FM (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, MA et al. (1995). PCR Strategies, Academic Press; Ausubel, FM (1999).These methods are described in Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press, Special Edition of Experimental Medicine: "Gene Introduction & Expression Analysis Experimental Methods" Yodosha, 1997, etc., and the relevant parts (possibly in their entirety) of these are incorporated herein by reference.

[0098] Regarding DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, gene synthesis and fragment synthesis services such as those of GeneArt, GenScript, Integrated DNA Technologies (IDT) and the like can be used. In addition, for example, Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, RL et al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (I996). Bioconjugate Techniques, Academic Press, etc., the relevant portions of which are incorporated herein by reference.

[0099] In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when it is specified that "within the range of" "two values", the range includes the two values ​​themselves. All references cited herein, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each was specifically set forth.

[0100] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples, but the above description and the following examples are provided for illustrative purposes only and are not provided for the purpose of limiting the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described in this specification, but is limited only by the scope of the claims. EXAMPLES

[0101] In the following examples, glutamine-free RPMI1640 (containing 10% FCS (v / v), 60 μg / mL penicillin G, 100 μg / mL streptomycin, and 10 mM HEPES) was used as the basal medium. If necessary, 30 to 300 mg / L of L-glutamine was added to the medium.

[0102] Bisulfite sequencing Genomic DNA was obtained from the induced cells, treated with the MethylEasy Xceed Rapid DNA Bisulphite Modification Kit (Human Genetic Signatures), and the demethylation of genes characteristic of regulatory T cells was examined. The demethylation of each gene was analyzed using known methods and primers (Floess et al. (2007) PloS Biology Volume 5, Issue 2, e38, and Ohkura et al. (2012) Immunity 37(5) 785-799).

[0103] In addition, in the analysis of the human Foxp3 CNS2 region, 5'-TTGGGTTAAGTTTGTTGTAGGATAG-3' (SEQ ID NO: 1) was used for the forward side, and 5'-ATCTAAACCCTATTATCACAACCCC-3' (SEQ ID NO: 2) was used for the reverse side.

[0104] Obtaining each T cell fraction Human CD4 + T cells were isolated from PBMCs of healthy donors and purified from enriched PBMCs using CliniMACS CD4 GMP MicroBeads according to the manufacturer's protocol.

[0105] (Example 1: Production of regulatory T cells from CD4 positive human peripheral T cells) CD4 positive T cells were stimulated for 3 days with basal medium containing anti-CD3 antibody (100 μL of 10 μg / ml antibody was added per well and allowed to stand at room temperature for 60 minutes to immobilize on the container), hIL-2 (100 U / ml), hTGFβ1 (10 ng / ml), retinoic acid (1 μM), and Senexin A (5 μM). After that, the cells were cultured for 2 days in basal medium containing hIL-2 (100 U / ml). After 2 days, the medium was replaced in the same way, and the cells were cultured for another 2 days. After 2 days, the cells were stimulated again for 2 days with basal medium containing anti-CD3 antibody (100 μL of 10 μg / ml antibody was added per well and allowed to stand at room temperature for 60 minutes to immobilize the antibody on the container), hIL-2 (100 U / ml), h TGFβ1 (10 ng / ml), retinoic acid (1 μM), Senexin A (5 μM), and ascorbic acid (10 μg / ml). The cells were then cultured for 2 days in basal medium containing hIL-2 (100 U / ml). After 2 days, the medium was replaced in the same manner, and the cells were cultured for another 2 days in basal medium. The expression of FoxP3, CTLA4, and Helios in the regulatory T cells obtained after 2 days was analyzed by flow cytometry (Figure 1).

[0106] Conventional induced regulatory T cells (conventional iTreg) have insufficient expression of functional molecules (for example, CTLA4 in this case), and the efficiency of FoxP3 induction is also low. On the other hand, endogenous regulatory T cells (nTreg) express CTLA4 and FoxP3, and express Helios, a representative nTreg marker molecule. It can be confirmed that the highly functional induced regulatory T cells obtained by the preparation method of the present application are Helios negative, and are a population containing many cells expressing FoxP3 and CTLA4. Helios is a marker for regulatory T cells endogenous to the body, and being Helios negative indicates that they are induced regulatory T cells. Inducible regulatory T cells obtained by the conventional method are, for example, negative for CTLA4, so that the obtained induced regulatory T cells are induced regulatory T cells newly induced in vitro, at least in terms of being positive for CTLA4.

[0107] In addition, the demethylation state of cells cultured in the same experimental system was confirmed by bisulfite sequencing (Figure 2). The induced regulatory T cells obtained by the method of the present disclosure show a demethylation state similar to that of nTreg.

[0108] The induced regulatory T cells obtained in the above examples are different from endogenous nTregs and are newly induced regulatory T cells in vitro, but they have been shown to have characteristics not found in existing induced regulatory T cells, such as high induction efficiency, high CTLA4 expression, and demethylation of the FOXP3 CNS2 region.

[0109] Example 2: In vitro suppressive activity of regulatory T cells Regulatory T cells were obtained using the same experimental system as in Example 1. Cell Trace Violet-labeled responder T cells (5 x 10 4 ), and the generated regulatory T cells (5x10 4) were mixed and cultured for 3 days in the presence of Treg Suppression Inspector (Miltenyi Biotec) (5 μL / well), and the Cell Trace Violet intensity was analyzed by flow cytometry. When responder T cells cause an immune response, the Cell Trace Violet intensity shows multiple weak peaks due to cell proliferation, but when this immune response is suppressed, the Cell Trace Violet intensity remains as a single strong peak. It was confirmed that the regulatory T cells obtained by the preparation method of the present disclosure strongly suppress the immune response of responder T cells. Therefore, it was confirmed that the obtained regulatory T cells have a higher suppressive function than existing regulatory T cell populations (Figure 3).

[0110] (Example 3: Comprehensive gene expression analysis of regulatory T cells) The gene expression pattern of the regulatory T cells prepared by the method of Example 1 was confirmed by RNA sequencing (Figure 4). RNA sequencing was performed using a known method (Kitagawa et al. (2017) Nat. Immunol. 18, 173-183). High expression of genes of FOXP3, IL-2RA (CD25), and major inhibitory molecules of Treg (CTLA4, etc.) was confirmed in the induced regulatory T cells and nTreg cells obtained by the preparation method of the present disclosure, compared to existing induced regulatory T cells and activated Tconv cells. In addition, it was confirmed that the expression of IKZF2, a marker of nTreg cells, was low in the induced regulatory T cells. It can be confirmed that the induced regulatory T cells obtained by the preparation method of the present disclosure have high expression of several inhibitory molecules such as ENTPD1, ​​NT5E, and AREG, compared to other regulatory T cell populations. It can also be confirmed that the expression of adhesion molecules such as ITGAE and chemokine receptors such as CCR4 is high (Figure 4).

[0111] For example, when the expression of the CD103 molecule encoded by ITGAE was analyzed by flow cytometry, it was confirmed that the induced regulatory T cells obtained by the production method of the present application had higher CD103 expression than other regulatory T cell populations (Figure 5). As a comprehensive consideration, the induced regulatory T cells obtained by the production method of the present application are expected to exhibit higher suppression ability than existing regulatory T cells.

[0112] (Example 4: Production of regulatory T cells from CD8 positive human peripheral T cells) CD8 positive T cells were stimulated for 3 days with basal medium containing anti-CD3 antibody (100 μL of 10 μg / ml antibody was added per well and allowed to stand at room temperature for 60 minutes to immobilize on the container), hIL-2 (100 U / ml), hTGFβ1 (10 ng / ml), retinoic acid (1 μM), and Senexin A (5 μM). After that, the cells were cultured for 2 days in basal medium containing hIL-2 (100 U / ml). After 2 days, the medium was replaced in the same way, and the cells were cultured for another 2 days. After 2 days, the cells were stimulated again for 2 days with basal medium containing anti-CD3 antibody (100 μL of 10 μg / ml antibody was added per well and allowed to stand at room temperature for 60 minutes to immobilize the antibody on the container), hIL-2 (100 U / ml), hTGFβ1 (10 ng / ml), retinoic acid (1 μM), Senexin A (5 μM), and ascorbic acid (10 μg / ml). The cells were then cultured for 2 days in basal medium containing hIL-2 (100 U / ml). After 2 days, the medium was replaced in the same manner, and the cells were cultured for another 2 days in basal medium. The expression of FoxP3, CTLA4, and Helios in the regulatory T cells obtained after 2 days was analyzed by flow cytometry (Figure 6).

[0113] It was also confirmed that the inducible regulatory T cells of the present disclosure prepared from CD8-positive T cells were Helios-negative and constituted a population rich in cells expressing FoxP3 and CTLA4.

[0114] Example 5: Production of inducible regulatory T cells from human peripheral T cells Using human peripheral T cells, induced regulatory T cells or cell populations thereof are obtained in the same manner as in Example 1. A pharmaceutical product is produced that contains the induced regulatory T cells or cell populations thereof and a carrier. The pharmaceutical product contains about 2 x 10 induced regulatory T cells or cell populations thereof. 5 The drug contains cells, and its effectiveness can be confirmed based on the degree of inflammation in tissue collected one month after administration to a patient with an inflammatory disease.

[0115] Example 6: Formulation Example Inducible regulatory T cells or a cell population thereof are obtained in the same manner as in Example 1. A pharmaceutical comprising the inducible regulatory T cells or a cell population thereof and a carrier is produced.

[0116] (Example 7: Expression intensity analysis) The expression intensities of CD25 and FOXP3 were analyzed by flow cytometry using a BD FACSLyric flow cytometer for the cell population of induced regulatory T cells produced in Example 1. The results are shown in FIG. In all cells tested, FOXP3 Hi It was confirmed that a high proportion of the induced regulatory T cell population was present in the mice.

[0117] (Example 8: Antibody panel analysis of inducible regulatory T cells) Antibody panel analysis was performed to comprehensively analyze the cell surface markers in the induced regulatory T cells produced in Example 1. The induced regulatory T cells produced in Example 1 were stained using BioLegend's LEGENDScreen Human PE Kit, and antibodies reacting with the induced regulatory T cells of the present disclosure were analyzed by flow cytometry. A portion of the results (those in which clear and significant expression was confirmed) are shown in Tables 1 to 4. When stained with an antibody panel containing nearly 400 types, the positive / negative characteristics of cell surface markers in the induced regulatory T cells of the present disclosure could be confirmed by at least 185 types of surface marker molecules.

[0118] [Table 1]

[0119] [Table 2]

[0120] [Table 3]

[0121] [Table 4]

[0122] (Example 9: Analysis of cell surface markers in induced regulatory T cells) Among the results of the antibody screening carried out in Example 8, characteristic results are shown in FIG. The induced regulatory T cells of the present disclosure were found to be positive for specific cell surface markers such as CD172g and CD26.

[0123] (Note) As described above, the present disclosure has been illustrated using preferred embodiments of the present disclosure, but it is understood that the scope of the present disclosure should be interpreted only by the claims. It is understood that the patents, patent applications and other documents cited in this specification should be incorporated by reference to this specification in the same manner as if the contents themselves were specifically described in this specification. This application claims priority to Japanese Patent Application No. 2021-190127 filed on November 24, 2021 with the Japan Patent Office, the contents of which are incorporated by reference in their entirety as if they constitute the contents of this application. [Industrial Applicability]

[0124] The induced regulatory T cells and / or cell population of the present disclosure can be used for the treatment and prevention of various immune diseases, inflammatory diseases such as autoimmune diseases, etc. Furthermore, the method for producing the induced regulatory T cells and / or cell population of the present disclosure makes it possible to stably induce highly functional regulatory T cells from peripheral T cells, and is expected to be applied in the medical field. [Sequence List Free Text]

[0125] SEQ ID NO: 1: Forward primer used in the examples SEQ ID NO: 2: Reverse primer used in the examples

Claims

[Claim 1] The invention described in the specification.

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