Human induced regulatory T cells and method for producing 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 induced regulatory T cells and methods for producing them. [Background technology]

[0002] An important feature of CD25+CD4+ regulatory T cells in the immune system is their specific expression of 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 thought that epigenetic conditions such as DNA demethylation contribute to the global gene expression control of regulatory T cells, including stable FoxP3 expression, and these conditions correlate 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, and then stimulation with an anti-CD3 antibody again, followed by culture, and then again by resting culture, thereby enabling the induction of regulatory T cells with high expression of inhibitory molecules and high suppressive function.

[0004] Thus, the present disclosure provides: (Item X1) Inducible human regulatory T cells 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) Inducible human regulatory T cells 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) Inducible human regulatory T cells 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) Inducible human regulatory T cells 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) Inducible human regulatory T cells 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 are CTLA4-, NT5E-, ITGAE (CD103-, AREG-, CD172g-, and CD26-positive. (Item X2) The inducible human regulatory T cells of claim 1, which are at least CTLA4-positive and FoxP3-positive. (Item X3) The inducible human regulatory T cells according to any one of the preceding items, which are at least CD172g-positive and / or CD26-positive. (Item X4) The inducible human regulatory 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 cells according to any one of the preceding items, which are 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 induced 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) in 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. Inducible human regulatory T cells obtained by a method comprising: (Item X8) A cell population comprising the inducible human regulatory 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 inducible human regulatory T cells, wherein the proportion of NT5E-positive cells in the cell population is about 50% or more. (Item X8B) A cell population comprising inducible human regulatory T cells, wherein the cell population has an ITGAE (CD103) positive rate of about 50% or more. (Item X8C) A cell population comprising inducible human regulatory T cells, wherein the cell population has an AREG-positive rate of about 50% or more. (Item X8D) A cell population comprising inducible human regulatory T cells, wherein the cell population has a CD172g positive rate of about 50% or more. (Item X8E) A cell population comprising inducible human regulatory T cells, wherein the cell population has a CD26-positive rate of about 50% or more. (Item X8F) A cell population comprising inducible human regulatory T cells, wherein the cell population has a CTLA4-positive rate of about 50% or more. (Item X8G1) A cell population comprising inducible human regulatory T cells, wherein the cell population has a proportion 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, wherein the cell population has 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 approximately 50% or more. (Item X8G3) A cell population comprising inducible human regulatory T cells, wherein the cell population has 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 human regulatory T cells, wherein the cell population has 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 percentages of all of the following characteristics are approximately 50% or more: CTLA4 positivity, NT5E positivity, ITGAE (CD103) positivity, AREG positivity, CD172g positivity, and CD26 positivity. (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 proportion of CD172g positive and / or CD26 positive cells is about 50% or more, respectively. (Item X11) The cell population described in any one of the above items, wherein the proportion of the at least one characteristic in the cell population is about 60% or more. (Item X12) The cell population described in any one of the above items, wherein the proportion of the at least one characteristic in the 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 described in any one of the preceding items, wherein approximately 90% or more of the cell population are T cells. (Item X14) A pharmaceutical composition comprising the inducible regulatory human T cells 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 human regulatory T cells described in any one of the above items or the cell population described in any one of the above items. (Item X16) 1. 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) in 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. 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 step (b) involves dormant culture of the cells obtained in 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 step (c) stimulates the cells obtained in step (b) with the second basal medium for about 3 days. (Item X24) The method according to any one of the preceding items, wherein step (d) involves dormant culture of the cells obtained in 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) Inducible regulatory T cells 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 according to the above item, which 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 cells according to any one of the preceding items, which are at least CTLA4-positive. (Item 4) The inducible regulatory T cell according to any one of the preceding items, wherein the CNS2 site of the FOXP3 gene is demethylated. (Item 5) The inducible regulatory T cells according to any one of the preceding items, which are CD4-positive or CD8-positive. (Item 6) The inducible regulatory T cells according to any one of the preceding items, which are obtained or induced 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) in 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. Inducible regulatory T cells obtained by a method comprising: (Item 8) A cell population comprising T cells, wherein approximately 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 the 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 described in any one of the preceding items, wherein approximately 90% or more of the cell population are T cells. (Item 12) A pharmaceutical composition comprising the induced regulatory T cells described in any one of the above items or the cell population described in any one of the above items. (Item 13) A regenerative medical material or product comprising the inducible regulatory T cells described in any one of the above items or the cell population described in any one of the above items. (Item A1) A method for producing inducible regulatory T cells, 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) in 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. 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 step (b) involves dormant culture of the cells obtained in 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 step (d) involves dormant culture of the cells obtained in 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, produced 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 explicitly stated. 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] Note that features and significant 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 invention and the drawings. [Effects of the Invention]

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

[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 explanation of the drawings]

[0010] [Figure 1]Figure 1 shows a phenotype analysis (flow cytometry) of one embodiment of the inducible regulatory T cells (also referred to as 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 generated from human CD4+ T cells, and the expression of FOXP3, CTLA4, and Helios was analyzed by flow cytometry. Comparison was made between cells stimulated with conventional nTreg (CD4+CD25+ T cells) and conventional iTreg (CD4+ T cells stimulated with CD3 / CD28 for 3 days in the presence of IL-2 and TGF-β1). [Figure 2] 2 is a diagram showing epigenome analysis (bisulfite method) of one embodiment of the iTreg (HSF iTreg) cells of the present disclosure. The demethylation status of the FOXP3 CNS2 region of each cell in FIG. 1 was analyzed by the bisulfite method. [Figure 3] Figure 3 shows 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 Figure 1 was analyzed. Human CD4-positive T cells were stained with Cell Trace Violet reagent and co-cultured with each cell in Figure 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] Figure 4 shows a diagram illustrating gene expression pattern analysis (RNA-seq) of one embodiment of the iTreg (HSF iTreg) cells of the present disclosure. The comprehensive gene expression patterns of each cell in Figure 1 and FOXP3-negative cells (Tconv) stimulated with IL-2 were analyzed by RNA sequencing. [Figure 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 shows a diagram illustrating the phenotype analysis (flow cytometry) of one embodiment of 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) in 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 shows a diagram illustrating the phenotype analysis of one embodiment of the iTreg (HSF iTreg) cells of the present disclosure. Inducible regulatory T cells (HSF-iTreg) of the present disclosure were generated and analyzed for the expression of CD172g, CD26, and the like. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present disclosure will now be described, illustrating the best mode thereof. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Thus, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, terms used in this specification should be understood to have the meaning commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the event of conflict, the present specification (including definitions) will prevail.

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

[0013] As used herein, "about" refers to ±10% of the following numerical value. For example, "about 20" includes "18 to 22." A range of numerical values ​​includes all values ​​between and including the endpoints. When "about" refers to a range, it 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, the FOXP3 gene and the FOXP3 protein may be used interchangeably, and the term FoxP3 refers to both the concept and entity (whole) of the gene or protein.

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

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

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

[0018] As used herein, "peripheral T cells" refers to T cells present outside the thymus, and can be obtained from peripheral blood, lymph nodes, and other tissues. The term "peripheral T cells" as used herein simply refers to a cell population containing peripheral T cells, and does not require that the T cells be isolated. Cell fractions containing various lymphocytes other than T cells, such as peripheral blood mononuclear cells (PBMCs), may also be used.

[0019] As used herein, "flow cytometry" refers to a technique for measuring the number of cells, solids, and other biological particles suspended in a liquid, as well as their individual physical, chemical, and biological properties. A device using this technique is called a "flow cytometer." In this disclosure, the "positive" and "negative" results of cell markers (e.g., FoxP3, CTLA4, Helios, CD103, etc.) are determined by flow cytometry, as commonly used in the art. More specifically, in flow cytometry, cells are lined up and flowed, and the number of cells is counted using spectroscopic techniques. For example, target cells are counted by irradiating cells labeled with fluorescent or luminescent enzymes with laser light, and the resulting fluorescent or luminescent signals are detected by a detector such as a photodiode. Furthermore, the detection results from the detector can be input into a computer and displayed as a two-dimensional plot. This allows for easy identification of the presence and number of target cells.

[0020] As used herein, "demethylation" refers to the removal of methylation modifications from typically methylated adenines (e.g., m6A at position 6, m1A at position 1) and cytosines (e.g., m5C at position 5, m3C at position 3). Demethylation can be determined using techniques 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 by 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 antibody, stimulation with a peptide that binds to TCR or a complex containing the peptide, stimulation with antigen-presenting cells, stimulation using an anti-CD3 antibody and antigen-presenting cells simultaneously, and stimulation using a peptide or protein and antigen-presenting cells simultaneously.

[0023] As used herein, "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 those skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description herein. 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 iTregs, or HSF iTregs) and uses thereof.

[0026] In one aspect of the present disclosure, there is provided an induced regulatory human 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. In one embodiment of the present disclosure, the induced regulatory T cell of the present disclosure may also have at least two characteristics selected from the group consisting of CTLA4-positive, NT5E-positive, ITGAE (CD103)-positive, and AREG-positive. In another 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 thereto, 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 other cells (adhesion of cerebellar neurons, neurite outgrowth, and glial cell attachment). Therefore, CD172g-positive inducible human regulatory T cells have activated functions related to adhesiveness and are expected to be highly effective against various diseases associated with these conditions.

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

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

[0031] The present disclosure provides ITGAE (CD103)-positive inducible human regulatory T cells. ITGAE (CD103) encodes an I-domain α integrin 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 IELs.

[0032] The present disclosure provides AREG-positive inducible regulatory human T cells. 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 resembling psoriasis and is also associated with other pathological diseases, including various types of cancer and inflammatory conditions. Therefore, AREG-positive inducible regulatory human T cells are expected to be highly effective against various related diseases.

[0033] The present disclosure provides CTLA4-positive inducible human regulatory T cells. CTLA4 is a protein belonging to the immunoglobulin superfamily that transmits inhibitory signals to T cells. Membrane-bound CTLA4 functions as a disulfide-bonded homodimer, 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 related diseases.

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

[0035] Recently, several methods have been developed to induce DNA demethylation in induced regulatory T cells, including the addition of ascorbic acid to the culture medium (Kasahara et al. Int. Immunol. (2017) 29(10):457-469) and a method without 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 induced regulatory T cells generated using these methods, and sufficient immunosuppressive activity is lacking. One clinical trial using induced regulatory T cells has also been conducted for the treatment of GVHD, but the percentage of regulatory T cells used was low, and no efficacy in preventing GVHD has been confirmed to date (MacMillan et al., Blood Adv. (2021) 5(5):1425-1436).

[0036] The present disclosure can provide inducible regulatory T cells that stably express FoxP3 and advantageously stably retain immunosuppressive activity. Such inducible regulatory T cells can be produced, for example, by the method for producing inducible regulatory T cells described elsewhere in this specification. For example, the present disclosure can provide inducible regulatory T cells obtained by a method comprising the steps of: (a) stimulating CD4+ T cells or CD8+ 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.

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

[0038] Herein, 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 two weeks. As shown in the Examples below, the induced regulatory T cells of the present disclosure can have a stronger immunosuppressive effect than conventional regulatory T cells (including induced and endogenous). Therefore, the induced regulatory T cells of the present disclosure can also be referred to as 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 referred to as 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 referred to as highly functional stable induced regulatory T cells (HSF iTreg).

[0040] In one embodiment of the present disclosure, whether or not a marker in an induced regulatory T cell of the present disclosure is positive can be determined by measuring the positive rate using flow cytometry. For example, analysis can be performed using a flow cytometer, and positive or negative can be determined based on the percentage of cells that express antigens at or above a certain level. Depending on the expression intensity of the cell surface marker, cells can be classified as negative, weakly positive, moderately positive, or strongly positive (weakly positive, moderately positive, and strongly positive are collectively referred to as "positive"). For example, depending on the instrument settings, values ​​of the median fluorescence intensity of each marker / median fluorescence intensity of the negative control (stained with an isotype control antibody) can be determined as negative, weakly positive, moderately positive, or strongly positive, respectively, when the values ​​are less than 5, 5 to less than 10, 10 to less than 30, or 30 or more. Such determinations can be made as exemplified in (measuring the positive rate using 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 determined 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 disclosure, and the expression intensity is 10 2 above weak positive, 10 3 above is moderately positive, 10 4 or more may be considered strongly positive, or 10 3 A value of 10 or more may be considered strongly positive, and a value of 10 or less may be considered weakly positive. 2 A value above this level may be considered strongly positive, and a value below this level may be considered weakly positive. Such a determination can be appropriately set depending on the experimental conditions, experimental purpose, cell type, instrument settings, etc., and the present disclosure is not limited thereto. Furthermore, even when analysis is performed using a flow cytometer instrument other than the BD FACSLyric flow cytometer, the expression intensity on the other instrument corresponding to the expression intensity when analyzed using the BD FACSLyric flow cytometer can be calculated, and a value indicating the expression intensity can be appropriately found depending on the instrument used.

[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 obtained by induction 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 populations thereof of the present disclosure can be human cells, and can be induced human regulatory T cells or cell populations thereof by a predetermined method using T cells obtained from human peripheral blood. Human cells and mouse cells have distinctly different properties, and even if the same cell surface markers are present, the properties of the cells cannot be considered identical. For example, in the field of induced regulatory T cells, the applicable techniques for mice and humans differ. In mice, the majority of lymphocytes are antigen-naïve, while in humans, T cells in peripheral blood vary in the degree of antigen sensitization and activation. Therefore, while it is possible to directly generate highly functional and stable induced regulatory T cells from T cells collected from mouse lymphoid tissues in mice, this is difficult to achieve in humans, and the cells cannot be considered similar simply based on the presence of cell surface markers.

[0044] In one aspect of the present disclosure, there is provided a cell population comprising the above-described inducible regulatory human T cells, 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, there is provided a cell population comprising inducible regulatory human T cells, wherein the cell population has a CTLA4-positive and FoxP3-positive rate of about 50% or more. In one embodiment, the CTLA4-positive and FoxP3-positive rates of the cell population of the present disclosure can be 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 percentage of CD172g-positive and / or CD26-positive cells in the cell population of the present disclosure can be about 50% or more. In one embodiment, the percentage of CD172g-positive and / or CD26-positive cells in the cell population of the present disclosure can be 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 percentage of strongly FoxP3 positive cells in the cell population of the present disclosure can be about 50% or more. In one embodiment, the percentage of strongly FoxP3 positive cells in the cell population of the present disclosure can be 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. While not intended to be limiting, in such a cell population, the expression intensity of cell surface markers can be measured by measuring the positivity rate using flow cytometry as described above.

[0048] In another aspect of the present disclosure, there is provided a cell population comprising inducible regulatory human T cells, wherein the cell population has a FoxP3-positive percentage of about 50% or more. Although not intended to be limiting, in one embodiment, such a cell population may contain 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 FoxP3-positive inducible regulatory human T cells by cell number.

[0049] In another aspect of the present disclosure, there is provided a cell population comprising inducible regulatory human T cells, wherein the cell population has a CTLA4-positive percentage of about 50% or more. Although not intended to be limiting, in one embodiment, such a cell population may contain CTLA4-positive inducible regulatory human T cells in an amount 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.

[0050] In another aspect of the present disclosure, there is provided a cell population comprising inducible regulatory human T cells, wherein the cell population has an NT5E-positive percentage of about 50% or more. Although not intended to be limiting, in one embodiment, such a cell population may contain NT5E-positive inducible regulatory human T cells at a cell number ratio 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, there is provided a cell population comprising inducible regulatory human T cells, wherein the cell population has an ITGAE (CD103)-positive percentage of about 50% or more. Although not intended to be limiting, in one embodiment, such a cell population may contain ITGAE (CD103)-positive inducible regulatory human T cells at a percentage 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 cell number.

[0052] In another aspect of the present disclosure, there is provided a cell population comprising inducible regulatory human T cells, wherein the cell population has an AREG-positive rate of about 50% or more. Without intending to be limiting, in one embodiment, such a cell population can comprise AREG-positive inducible regulatory human T cells at a cell number rate 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, there is provided a cell population comprising inducible regulatory human T cells, wherein the cell population has a CD172g-positive percentage of about 50% or more. Although not intended to be limiting, in one embodiment, such a cell population may contain CD172g-positive inducible regulatory human T cells in an amount 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.

[0054] In another aspect of the present disclosure, there is provided a cell population comprising inducible regulatory human T cells, wherein the cell population has a CD26-positive percentage of about 50% or more. Although not intended to be limiting, in one embodiment, such a cell population may contain, by cell number, 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.

[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 using a specified induction method, a cell population in which the rate of positivity for a specified cell surface marker is approximately 50% or more can be obtained, and preferably a cell population in which "the rate of CTLA4 positivity and FoxP3 positivity is approximately 50% or more each" can be obtained.

[0056] CTLA4 is a molecule localized intracellularly, at least to a level that cannot be used as an indicator for enrichment or purification. Furthermore, FoxP3 is a transcription factor and is entirely intracellular; therefore, it cannot be detected on the surface at all and cannot be used for purification. In other words, because CTLA4 and FoxP3 are markers expressed "inside" T cells, it is impossible to obtain a cell population with CTLA4 and / or FoxP3 positivity of approximately 50% or more by sorting and enriching using CTLA4 and / or FoxP3 as indicators. Therefore, it is also impossible to obtain a cell population with CTLA4 and / or FoxP3 positivity of less than approximately 50% using conventional methods, and then use such a cell population as a starting material to enrich using CTLA4 and / or FoxP3 as indicators to obtain a cell population with CTLA4 and / or FoxP3 positivity of approximately 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 mediated by 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 both technical and medical effects. In other words, a stable cell preparation can be provided by containing 50% or more T cells that exert a medically effective immunosuppressive effect. This effect is extremely important from a medical perspective, and is not merely a matter of quantity or numerical value, but is an important point in terms of quality, i.e., whether the preparation can be established.

[0058] In one embodiment of the present disclosure, the induced regulatory human T cells in the cell population of the present disclosure can further be characterized as ITGAE (CD103)-positive, NT5E-positive, and / or AREG-positive. These cell markers are genes that play important roles in immunosuppressive function, and high expression of these markers allows the cells to maintain greater functional stability after administration than conventional induced regulatory T cells. For example, CD103 is important for regulatory T cell migration to inflammatory sites, NT5E (CD73) is important for the production of adenosine, which has immunosuppressive properties, and AREG is important for tissue regeneration by regulatory T cells. High expression of these markers allows the cell population of the present disclosure to 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 induced regulatory T cells or cell population of the present disclosure is provided. In another aspect, a regenerative medicine material or product comprising the induced 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 activity. These pharmaceuticals, regenerative medicine materials, or products can be used together with culture media and any other additives used in the relevant field. Such media can be a medium prepared by adding necessary factors to a basal animal cell culture medium used for cell culture. Examples of such media are described in detail elsewhere herein. Examples of components added to the medium are also described in detail elsewhere herein. When provided as such a product, it may contain DMSO, etc.

[0062] (Manufacturing 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 capable of producing highly functional and stable inducible regulatory T cells, and is described in detail herein below.

[0063] In one aspect of the present disclosure, there is provided a method for producing inducible regulatory T cells, comprising the steps of: (a) stimulating CD4+ T cells or CD8+ 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 cells of the present disclosure can be produced using either CD4+ T cells or CD8+ T cells as a starting material, and in one embodiment, the inducible regulatory T cells of the present disclosure can also be produced using a mixture of CD4+ T cells and CD8+ T cells as a starting 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 stimulation when obtaining Tregs, and is not particularly limited as long as it is mediated by TCR. For example, 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 using an anti-CD3 antibody and antigen-presenting cells simultaneously, and stimulation using a peptide or protein and antigen-presenting cells simultaneously. However, 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" refers to specific stimulation of the CD3 receptor on a cell. Examples of CD3 stimulation include anti-CD3 agonist antibodies. Anti-CD3 agonist antibodies may be commercially available research reagents or may be prepared by conventional methods. Anti-CD3 antibodies may be derived from animals such as mice, rabbits, goats, and cows, or from humans.

[0068] In one embodiment, the method of the present disclosure involves first inducing iTregs (stimulating T cells to induce demethylation) (step a), then changing the medium and resting culture for 1 to 3 days to recover the cells (step b), and then stimulating the T cells again to induce demethylation (step c), thereby obtaining functional and stable inducible human regulatory T cells. While it is common knowledge that T cells typically undergo apoptosis after two rounds of stimulation, the method of the present disclosure has been found to obtain functional and stable inducible human regulatory T cells without undergoing 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 effects of the present disclosure can be achieved with inducible regulatory human T cells obtained in this manner or a cell population containing such inducible regulatory human T cells. Therefore, 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 can 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 can 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 an anti-CD3 antibody, TGF-β1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, a CDK8 / 19 inhibitor, and ascorbic acid. In another embodiment, the first basal medium and the second basal medium may each independently contain an anti-CD3 antibody, TGF-β1, IL-2, retinoic acid, a CDK8 inhibitor, a CDK19 inhibitor, a CDK8 / 19 inhibitor, and ascorbic acid. The concentrations of each of these components may be those typically 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, 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 those skilled in the art can change them as appropriate depending on other medium compositions.

[0072] In one embodiment, the method of the present disclosure produces regulatory T cells from human peripheral T cells. Peripheral T cells include naive regulatory T cells, CD4+ T cells, CD8+ 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+ T cells or CD8+ T cells, may be isolated from these cells and then induced. Alternatively, regulatory T cells may be induced from T cells specific to a specific antigen. Therefore, the induced regulatory T cells of the present disclosure include both CD4+ and CD8+ cells. Note that, in this specification, "CD4+" or "CD4+" refers to single-positive CD4+CD8-negative cells unless otherwise specified. Furthermore, in the production method of the present disclosure, either CD4+ or CD8+ T cells can be used as the starting material, and even after the generation of induced regulatory T cells, the CD4+ or CD8+ characteristics can be maintained unless special manipulation 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 aqueous solution. Examples of materials capable of physically adsorbing an anti-CD3 antibody include synthetic resins such as polystyrene, polyethylene terephthalate, polycarbonate, and polypropylene, as well as glass. The shape of the insoluble carrier is not particularly limited, and may be, for example, a plate, bead, or container. The amount of anti-CD3 antibody varies depending on the titer and origin of the antibody used, but may be appropriately determined to provide sufficient stimulation for the induction of regulatory T cells.

[0074] In one embodiment, the method of the present disclosure can use a medium prepared by adding necessary factors to a basal animal cell culture medium for cell culture. Examples of basal animal cell culture media 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 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 substitutes, such as albumin, bovine serum albumin (BSA), transferrin, apotransferrin, KnockOut Serum Replacement (KSR) (a serum substitute 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, or 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, the basal medium is exemplified by RPMI 1640 medium containing serum and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

[0077] In the method of the present disclosure, 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 carried out in an atmosphere of CO2-containing air, with the CO2 concentration preferably being 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 anti-CD3 antibody varies depending on the titer and origin of the antibody used, but should be appropriately determined so as to provide sufficient stimulation for the induction of regulatory T cells.

[0079] Examples of TGFβ that can be 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 can be used, and examples thereof 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. Examples include Senexin A and AS2863619, but the present disclosure is not limited thereto. The concentrations 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 can be appropriately changed by a person skilled in the art depending on other medium compositions.

[0083] In the method of the present disclosure, regulatory T cells are induced by stimulating human peripheral T cells with an anti-CD3 antibody in a medium containing TGFβ and IL-2, and regulatory T cells with high immunosuppressive function can be induced by culturing the cells in a resting state in an IL-2-containing medium without the anti-CD3 antibody and then stimulating them again with the anti-CD3 antibody. The high immunosuppressive function of the resulting induced regulatory T cells can be confirmed, for example, by comprehensive gene expression analysis using RNA sequencing or by 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 of step (b) can be appropriately determined 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 culturing in the second basal medium in step (c) can be set appropriately 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 (d) can be appropriately determined 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 induced regulatory T cell-specific demethylation can be expanded by culturing the cells under unstimulated conditions 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 the cells in a medium further containing IL-2.

[0088] Regulatory T cells can be isolated from the resulting cell culture containing regulatory T cells by conventional methods based on cell surface markers specific to regulatory T cells, such as by extracting FoxP3-positive cells using a cell sorter. Furthermore, regulatory T cells with specific antigenic properties may be isolated, if desired.

[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 referred to as 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 referred to as "dedicated tube") (Falcon, 352008) Nylon mesh (65 μm) (Kyoshin Riko, PP-65N)

[0092] Reagent preparation *Fixation Buffer (100 μL 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 (for 500 mL preparation) 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 with a micropipette. (3) Centrifuge at 500 x g for 5 minutes at 4°C. (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 the sample 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 into each well 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) Fix the sample by leaving it on ice for 60 minutes in the dark. (13) Start up the measuring equipment during dyeing. (14) After staining, add 1 mL of FACS Buffer and pipette gently. (15) Centrifuge at 500 x g for 5 minutes at 4°C. Remove the supernatant using an aspirator, then repeat steps (14) and (15) to wash the cells. (16) After removing the supernatant with 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 should be 10,000 or more. When calculating the target antigen positivity rate, a baseline should be 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 baseline should be used as the positivity rate.

[0094] remarks The reagents used for fixation and staining can also be purchased as a set of three, known as the "Foxp3 / Transcription Factor Staining Buffer Set" (Cat.: 00-5523). Any settings of the instrument are acceptable as long as they do not deviate to the extent that it can be determined from a general or scientific standpoint that normal measurements are not being performed. Clear deviations include when various signals from the target cell population fall below the set fluorescence threshold value, or when various signal values ​​from the control sample or the sample to be measured fall below or above the limit of normal measurement by the instrument.

[0095] (Immunosuppressive activity measurement) The cells of the present disclosure may have immunosuppressive activity. Immunosuppressive activity can be measured by various techniques.

[0096] In one embodiment, as described in the Examples, suppression can be determined by measuring cell proliferation due to an immune response initiated by responder T cells. Various kits can also 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 disclosed herein may be enhanced compared to conventional iTregs.

[0097] (General technology) The molecular biological, biochemical, and microbiological techniques used herein are well known and commonly used in the art, and can be found in, for example, 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; and "Experimental Methods for Gene Transfer and Expression Analysis," a special edition of Experimental Medicine, Yodosha, 1997, all of which are incorporated herein by reference in their entirety.

[0098] Regarding DNA synthesis technology and nucleic acid chemistry for producing artificially synthesized genes, gene synthesis and fragment synthesis services such as GeneArt, GenScript, and Integrated DNA Technologies (IDT) can be used. Other examples include 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 (1996). 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 "within a range" of "two values" is specified, the range includes the two values ​​themselves. All references cited herein, including scientific literature, patents, patent applications, and the like, are incorporated by reference in their entirety to the same extent as if each were 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. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the scope of the claims. [Example]

[0101] In the following examples, glutamine-free RPMI 1640 (containing 10% FCS (v / v), 60 μg / mL penicillin G, 100 μg / mL streptomycin, and 10 mM HEPES) was used as the basal medium. 30 to 300 mg / L of L-glutamine was added to the medium as needed.

[0102] Bisulfite sequencing Genomic DNA was isolated from the induced cells and treated with the MethylEasy Xceed Rapid DNA Bisulfite Modification Kit (Human Genetic Signatures). Demethylation of genes characteristic of regulatory T cells was examined using previously published 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+ 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 the antibody), hIL-2 (100 U / ml), hTGFβ1 (10 ng / ml), retinoic acid (1 μM), and Senexin A (5 μM). Then, 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. Two days later, the cells were stimulated again with basal medium containing anti-CD3 antibody (100 μL per well at 10 μg / ml concentration, immobilized at room temperature for 60 minutes), hIL-2 (100 U / ml), hTGFβ1 (10 ng / ml), retinoic acid (1 μM), Senexin A (5 μM), and ascorbic acid (10 μg / ml) for two days. Then, the cells were rested for two days in basal medium containing hIL-2 (100 U / ml). After two days, the medium was replaced in the same way, and the cells were rested for another two days. The expression of FoxP3, CTLA4, and Helios in the regulatory T cells obtained after two days was analyzed by flow cytometry (Figure 1).

[0106] Conventional induced regulatory T cells (conventional iTregs) express functional molecules (e.g., CTLA4 in this case) insufficiently and have low FoxP3 induction efficiency. On the other hand, endogenous regulatory T cells (nTregs) express CTLA4 and FoxP3, as well as Helios, a representative nTreg marker molecule. The highly functional induced regulatory T cells obtained by the present method are Helios-negative, confirming that they are a population containing many cells expressing FoxP3 and CTLA4. Helios is a marker for endogenous regulatory T cells in the body, and Helios-negative results indicate that the cells are induced regulatory T cells. Since induced regulatory T cells obtained by conventional methods are, for example, CTLA4-negative, the fact that the obtained induced regulatory T cells are at least CTLA4-positive indicates that they are newly induced regulatory T cells induced in vitro.

[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 exhibited a demethylation state similar to that of nTregs.

[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 co-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 initiate 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. The regulatory T cells obtained by the production method of the present disclosure were confirmed to strongly suppress the immune response of responder T cells. Therefore, it was confirmed that the obtained regulatory T cells have a stronger suppressive function than existing regulatory T cell populations (Figure 3).

[0110] (Example 3: Comprehensive gene expression analysis of regulatory T cells) The gene expression patterns of regulatory T cells generated by the method of Example 1 were confirmed by RNA sequencing (Figure 4). RNA sequencing was performed using a known method (Kitagawa et al. (2017) Nat. Immunol. 18, 173-183). Higher expression of genes encoding FOXP3, IL-2RA (CD25), and key Treg inhibitory molecules (e.g., CTLA4) was confirmed in induced regulatory T cells and nTreg cells generated by the method of the present disclosure compared with existing induced regulatory T cells and activated Tconv cells. Furthermore, lower expression of IKZF2, a marker for nTreg cells, was confirmed in induced regulatory T cells. Compared with other regulatory T cell populations, induced regulatory T cells generated by the method of the present disclosure were confirmed to have higher expression of several inhibitory molecules, such as ENTPD1, ​​NT5E, and AREG. Furthermore, higher expression of adhesion molecules, such as ITGAE, and chemokine receptors, such as CCR4, was confirmed (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 method of the present application had higher CD103 expression than other regulatory T cell populations (Figure 5). Overall, it is expected that the induced regulatory T cells obtained by the method of the present application will exhibit higher suppressive ability than existing regulatory T cells.

[0112] Example 4: Production of regulatory T cells from CD8-positive human peripheral T cells CD8+ 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 the antibody), hIL-2 (100 U / ml), hTGFβ1 (10 ng / ml), retinoic acid (1 μM), and Senexin A (5 μM). Then, 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. Two days later, the cells were stimulated again with anti-CD3 antibody (100 μL of 10 μg / ml antibody per well was added and allowed to stand at room temperature for 60 minutes to immobilize the antibody on the vessel), hIL-2 (100 U / ml), hTGFβ1 (10 ng / ml), retinoic acid (1 μM), Senexin A (5 μM), and ascorbic acid (10 μg / ml) for two days. Then, the cells were rested in basal medium containing hIL-2 (100 U / ml) for two days. After two days, the medium was replaced in the same way, and the cells were rested for another two days. The expression of FoxP3, CTLA4, and Helios in the regulatory T cells obtained after two 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 containing many cells expressing FoxP3 and CTLA4.

[0114] Example 5: Production of inducible regulatory T cells from human peripheral T cells Inducible regulatory T cells or cell populations thereof are obtained using human peripheral T cells in the same manner as in Example 1. A pharmaceutical product is produced containing the inducible regulatory T cells or cell populations thereof and a carrier. The pharmaceutical product contains approximately 2 x 10 induced regulatory T cells or cell populations thereof. 5 The drug contains 100 cells, and its effectiveness can be confirmed by 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 cell populations thereof are obtained in the same manner as in Example 1. A pharmaceutical product containing the inducible regulatory T cells or cell populations 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 Figure 7. In all cells tested, FOXP3 Hi It was confirmed that a high proportion of induced regulatory T cell populations were present in the mice.

[0117] Example 8: Antibody panel analysis of induced 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. Using BioLegend's LEGENDScreen Human PE Kit, the induced regulatory T cells produced in Example 1 were stained, and antibodies reactive with the induced regulatory T cells of the present disclosure were analyzed by flow cytometry. Some of the results (those in which clear and significant expression was confirmed) are shown in Tables 1 to 4. Staining with an antibody panel containing nearly 400 types confirmed the positive / negative characteristics of cell surface markers in the induced regulatory T cells of the present disclosure using 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 Characteristic results of the antibody screening carried out in Example 8 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) While the present disclosure has been illustrated using preferred embodiments thereof, it is understood that the scope of the present disclosure should be interpreted solely by the claims. It is understood that the patents, patent applications, and other documents cited herein are incorporated by reference into this specification in their entirety as if the contents themselves were specifically set forth herein. 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 populations disclosed herein can be used for the treatment and prevention of various immune diseases, autoimmune diseases, and other inflammatory diseases. Furthermore, the method for producing the induced regulatory T cells and / or cell populations disclosed herein makes it possible to stably induce highly functional regulatory T cells from peripheral T cells, which 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

1. A cell population of inducible human regulatory T cells, in which 50% or more of the inducible human regulatory T cells are FoxP3-positive and CTLA4-positive, but Helios-negative.

2. The cell population of claim 1 , wherein the CNS2 site of the FOXP3 gene is demethylated.

3. The cell population of claim 1 , wherein the induced regulatory human T cells maintain their immunosuppressive activity or effect for at least about two weeks.

4. The cell population of claim 1 , wherein the inducible regulatory human T cells are CD4 positive.

5. A method comprising the steps of: (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) 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, the first basal medium comprises an anti-CD3 antibody, TGF-β, IL-2, retinoic acid, and a CDK8 / 19 inhibitor; A cell population of inducible regulatory human T cells produced by the method, wherein the second basal medium comprises an anti-CD3 antibody, TGF-β, IL-2, retinoic acid, ascorbic acid, and a CDK8 / 19 inhibitor.

6. The cell population of claim 5 , wherein the CDK8 / 19 inhibitor contained in the first basal medium comprises Senexin A or AS2863619.

7. The cell population according to claim 5, wherein the TGF-β contained in the first basal medium and the second basal medium comprises TGF-β1.

8. The cell population of claim 5 , wherein the CDK8 / 19 inhibitor contained in the second basal medium comprises Senexin A or AS2863619.

9. A method comprising the steps of: (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) 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, the first basal medium comprises an anti-CD3 antibody, TGF-β1, IL-2, retinoic acid, and Selexin A; A cell population of inducible human regulatory T cells produced by the method, wherein the second basal medium comprises an anti-CD3 antibody, TGF-β1, IL-2, retinoic acid, Selexin A, and ascorbic acid.

10. The cell population according to claim 5 , wherein step (a) stimulates the CD4-positive T cells or CD8-positive T cells with the first basal medium for about 3 days.

11. The cell population according to claim 10, wherein in step (b), the cells obtained in step (a) are cultured in a medium containing said IL-2 for at least about 2 days until they become dormant.

12. The cell population of claim 11, wherein step (c) stimulates the cells obtained in step (b) with the second basal medium for about 3 days.

13. The cell population according to claim 12, wherein step (d) comprises dormantly culturing the cells obtained in step (c) in a medium containing said IL-2 for at least about 2 days.

14. A pharmaceutical comprising the cell population of claim 1, 5 or 9.

15. A regenerative medical material or product comprising the cell population of claim 1, 5 or 9.

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