Method for preparing T cells

Abstract

The present invention provides an excellent method for preparing CD4 single positive T cells (CD4SP T cells) that are each IL-4 non-secreting and IFN-γ secreting (Th1 type) or IFN-γ non-secreting and IL-4 secreting (Th2 type). The method for preparing Th1 type or Th2 type CD4SP T cells of the present invention comprises the following steps: inducing hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) that are substantially deficient in factors involved in the secretion of IL-4 or factors involved in the secretion of IFN-γ into CD4 single positive T cells.

Description

Technical Field

[0001] The present invention relates to a method for preparing CD4 single-positive T cells that are IL-4 non-secreting and IFN-γ secreting (Th1 type) and IFN-γ non-secreting and IL-4 secreting (Th2 type), respectively, similar to natural Th1 cells and Th2 cells, for example, a method for preparing using artificial thymic organoids (ATO) containing hematopoietic stem cells (HSC) and / or hematopoietic progenitor cells (HPC) (HPC) induced by differentiation from iPS cells. In addition, the present invention relates to a method for regulating the differentiation of hematopoietic stem cells (HSC) and / or hematopoietic progenitor cells (HPC) into Th1 type and / or Th2 type CD4 single-positive T cells. Background Art

[0002] Attempts to differentiate iPS cells into cytotoxic T cells, i.e., CD8 single positive T cells (sometimes referred to as "CD8SP T cells" in this specification), introduce antigen-specific TCR (T cell receptor), CAR (chimeric antigen receptor) and apply them to tumor immunotherapy are being actively carried out. In such tumor immunotherapy, the development is promoted with an eye on the excellent anti-tumor activity of cytotoxic T cells, and the hidden background is that there is no technology for inducing helper T cells, i.e., CD4 single positive T cells (sometimes referred to as "CD4SP T cells" in this specification) from iPS cells. If helper T cells can be induced, it can be used to enhance both cellular immunity and humoral immunity and improve the effect of immune cell therapy. For example, it is known that in CAR-T therapy, when CD4SP T cells are administered together with CD8SP T cells into which CAR is introduced, the therapeutic effect can be enhanced. In addition, if Th1 cells and Th2 cells as subpopulations of helper T cells are specifically prepared or amplified, they can also be applied to fields other than tumor immunotherapy, for example, regulating the Th1 / 2 balance that is regarded as the pathological background of allergic diseases. In addition, it is believed that if suppressor T cells (Treg cells) can be induced, allergies and autoimmune diseases can be treated by suppressing cellular immunity and humoral immunity. The ability to produce Th1 cells, Th2 cells, Treg cells, etc. from iPS cells is important for improving the effect of immune cell therapy or expanding its diversity.

[0003] As a method for preparing CD8SP T cells and / or CD4SP T cells, there is known a method including the step of culturing an artificial thymic organoid (ATO) which is a three-dimensional cell aggregate including stromal cells expressing Notch ligands and hematopoietic progenitor cells, as described in Patent Document 1. The examples of Patent Document 1 disclose that, as a more specific embodiment of the method, ATO is prepared using (primary) human hematopoietic stem cell / progenitor cells (HSPC) separated from umbilical cord blood, etc.; CD8SP T cells, CD4SP T cells, and CD4·CD8 double positive T cells (sometimes referred to as "CD4·CD8DP T cells" in this specification) are obtained from the ATO; and a very small number of cells expressing only IFN-γ and cells expressing only IL-4 are obtained by treating CD4SP T cells with PMA / ionomycin (Patent Document 1). Figure 4 B. Figure 6 B, etc.).

[0004] Non-patent document 1 describes that hematopoietic stem / progenitor cells (CD34 + CD3 - HSPCs) were co-cultured with stromal cells (MS5-hDLL1 cell line) expressing Notch ligands to produce ATOs; in addition to CD4·CD8DP T cells, CD8SP T cells and CD4SP T cells were also generated from the ATOs ( Figure 1 b, 2b, 3b, 4a, 4b, 4f), etc.

[0005] Non-patent document 2 also records that embryonic mesodermal progenitors (EMP) induced by differentiation of human iPS cells were co-cultured with stromal cells (MS5-hDLL1 / 4 cell line) expressing Notch ligand to produce embryonic mesodermal organoids (EMO), which were further induced into hematopoietic lineage to produce ATO; in addition to CD4·CD8DP T cells, CD8SP (CD8αβ + )T cells and a small number of CD4SP (CD4 + )T cells( Figure 2 C); CD8SP T cells were treated with PMA / ionomycin to obtain cells expressing IFN-γ and / or IL-2 ( Figure 3F), etc. It should be noted that when the aforementioned human iPS cells are prepared from fibroblasts, mature naive T cells are obtained, and TCRs have various repertoires. On the other hand, when iPS cells are modified in a manner that expresses TCRs specific to tumor-associated antigens (NY-ESO-1 peptides), T cells expressing such TCRs can be obtained.

[0006] However, none of the above three documents describes a method for effectively producing IFN-γ-secreting (Th1 type) CD4SP T cells and IL-4-secreting (Th2 type) CD4SP T cells, or a method for regulating the differentiation of hematopoietic stem cells (HSC) and / or hematopoietic progenitor cells (HPC) into Th1 type and / or Th2 type CD4 single positive T cells.

[0007] Non-Patent Document 3 describes transcription factors involved in Th1 differentiation (INF-γ gene expression) and their signal transduction, and transcription factors involved in Th2 differentiation (IL-4 gene expression) and their signal transduction.

[0008] Prior art literature

[0009] Patent Literature

[0010] Patent Document 1: WO2017 / 075389

[0011] Non-patent literature

[0012] Non-patent document 1: Seet et al., Nature Methods vol.14 no.5, p521-530 (2017)

[0013] Non-patent document 2: Montel-Hagen et al., Cell Stem Cell. 2019 Mar 7; 24(3): 376-389

[0014] Non-patent document 3: Usui., Jpn. J. Clin. Immunol., 30(6), p419-427 (2007) Summary of the invention

[0015] Problems to be solved by the invention

[0016] As described above, although it is desirable to produce IFN-γ-secreting (Th1 type) CD4SP T cells and IL-4-secreting (Th2 type) CD4SP T cells from iPS cells, means for achieving this have not yet been achieved. In particular, when considering the use in immune cell therapy and production efficiency, it is extremely useful to be able to produce ATO for generating the above-mentioned CD4SP T cells using hematopoietic stem / progenitor cells obtained by differentiation induction from iPS cells prepared from cells collected from patients, established iPS cell lines, iPS cells further modified therefrom, etc., rather than primary hematopoietic stem / progenitor cells.

[0017] It should be noted that primary T cells collected from a living body (peripheral blood) (not T cells produced by ATO) have the specificity to secrete either IFN-γ or IL-4 by stimulation with PMA / ionomycin, so Th1 cells or Th2 cells can be obtained by using primary T cells. In contrast, when ATO is produced using hematopoietic progenitor cells (HPCs) induced by iPS cells, there is a problem that when T cells produced by ATO are stimulated with PMA / ionomycin, there is insufficient specificity for the secretion of IFN-γ and IL-4, and it is not possible to selectively obtain Th1 type and Th2 type CD4SP T cells that secrete either one.

[0018] The present invention aims to provide an excellent method for preparing CD4 single positive T cells (CD4SP T cells) that are non-IL-4 secreting and IFN-γ secreting (Th1 type) or non-IFN-γ secreting and IL-4 secreting (Th2 type). In addition, the present invention aims to provide a method for regulating the differentiation of CD4 single positive T cells into Th1 type or Th2 type.

[0019] Means for solving problems

[0020] The inventors of the present application have discovered that by culturing hematopoietic progenitor cells or hematopoietic stem cells that are substantially deficient in factors involved in the secretion of IL-4 or factors involved in the secretion of IFN-γ, for example, by using hematopoietic progenitor cells obtained from iPS cells in which the IL4 gene or TBX21 gene is knocked out to produce ATO and culturing them, Th1 type CD4SP T cells and Th2 type CD4SP T cells can be effectively generated, respectively.

[0021] It should be noted that high secretion of IL-4 and high expression of T-bet were confirmed during T cell redifferentiation of wild-type iPS cells, so it was thought that knocking out the IL4 gene and TBX21 gene encoding them might inhibit the differentiation itself into mature helper T cells. However, surprisingly, even when the IL4 gene and TBX21 gene were knocked out, the above inhibition did not occur, and Th1 and Th2 CD4SP T cells were successfully produced.

[0022] That is, the present invention includes at least the following matters. [1]

[0024] A method for preparing Th1 or Th2 CD4 single positive T cells, comprising the following steps:

[0025] Inducing hematopoietic stem cells (HSC) and / or hematopoietic progenitor cells (HPC) substantially deficient in the following factors into CD4 single positive T cells:

[0026] A factor involved in the secretion of IL-4; or

[0027] Factor involved in the secretion of IFN-γ. [2]

[0029] The method described in [1] is a method for preparing Th1 type CD4 single positive T cells, which comprises the following steps:

[0030] (B1) Hematopoietic stem cells (HSC) and / or hematopoietic progenitor cells (HPC) substantially deficient in factors involved in IL-4 secretion are induced to become CD4 single-positive T cells. [3]

[0032] The method described in [1] is a method for preparing Th2 type CD4 single positive T cells, which comprises the following steps:

[0033] (B2) Hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) substantially deficient in factors involved in the secretion of IFN-γ are induced into CD4 single-positive T cells. [4]

[0035] The method according to [1], wherein the HSC and / or HPC are (b1) HSC and / or HPC in which a gene involved in IL-4 secretion is knocked out. [5]

[0037] The method according to [1], wherein the HSC and / or HPC is (b2) HSC and / or HPC in which a gene involved in IFN-γ secretion is knocked out. [6]

[0039] The method described in [1] is a method for preparing Th1 type CD4 single positive T cells, which comprises the following steps:

[0040] By culturing artificial thymic organoids (ATO), IL-4 non-secreting and IFN-γ-secreting (Th1 type) CD4 single positive T cells are generated from the ATO, wherein the ATO comprises:

[0041] (a) stromal cells expressing Notch ligands; and

[0042] (b1) Hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) in which a gene involved in IL-4 secretion is knocked out but a gene involved in IFN-γ secretion is not knocked out.

[0043] [6-1]

[0044] A method for preparing Th1 type CD4 single positive T cells, comprising the following steps:

[0045] By culturing artificial thymic organoids (ATO), IL-4 non-secreting and IFN-γ-secreting (Th1 type) CD4 single positive T cells are generated from the ATO, wherein the ATO comprises:

[0046] (a) stromal cells expressing Notch ligands; and

[0047] (b1) Hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) in which the gene involved in the secretion of IL-4 is knocked out but the gene involved in the secretion of IFN-γ is not knocked out.

[0048] [6a]

[0049] The method according to [6] or [6-1], wherein the gene involved in the secretion of IL-4 is the IL4 gene. [7]

[0051] The method described in [1] is a method for preparing Th2 type CD4 single positive T cells, which comprises the following steps:

[0052] By culturing artificial thymic organoids (ATO), IFN-γ non-secreting and IL-4-secreting (Th2 type) CD4 single positive T cells are generated from the ATO, wherein the ATO comprises:

[0053] (a) stromal cells expressing Notch ligands; and

[0054] (b2) Hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) in which a gene involved in the secretion of IFN-γ is knocked out but a gene involved in the secretion of IL-4 is not knocked out.

[0055] [7-1]

[0056] A method for preparing Th2 type CD4 single positive T cells, comprising the following steps:

[0057] By culturing artificial thymic organoids (ATO), IFN-γ non-secreting and IL-4-secreting (Th2 type) CD4 single positive T cells are generated from the ATO, wherein the ATO comprises:

[0058] (a) stromal cells expressing Notch ligands; and

[0059] (b2) Hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) in which the gene involved in the secretion of IFN-γ is knocked out but the gene involved in the secretion of IL-4 is not knocked out.

[0060] [7a]

[0061] The method according to [7] or [7-1], wherein the gene involved in the secretion of IFN-γ is the TBX21 gene. [8]

[0063] The method according to [1], wherein the hematopoietic stem cells (HSC) and / or hematopoietic progenitor cells (HPC) are cells induced to differentiate from iPS cells (iPSC).

[0064] [8-1]

[0065] The method according to [6-1] or [7-1], wherein the hematopoietic stem cell (HSC) or hematopoietic progenitor cell (HPC) of (b1) or (b2) is a cell induced by differentiation from an iPS cell (iPSC). [9]

[0067] The method according to [8] or [8-1], wherein the iPSC is an iPS cell derived from a T cell (T-iPSC).

[10]

[0069] The method as described in [9], wherein the T cells are CD4 single positive T cells.

[11]

[0071] Th1 type CD4 single positive T cells are obtained by the method described in [1].

[12]

[0073] Th2 type CD4 single positive T cells are obtained by the method described in [1].

[13]

[0075] Among Th1-type CD4 single-positive T cells, factors involved in the secretion of IL-4 are substantially deficient.

[14]

[0077] Among Th2-type CD4 single-positive T cells, factors involved in the secretion of IFN-γ are substantially deficient.

[15]

[0079] The CD4 single-positive T cells according to

[13] or

[14] , wherein the Th1 type CD4 single-positive T cells or the Th2 type CD4 single-positive T cells are cells induced to differentiate from iPS cells (iPSCs).

[16]

[0081] The CD4 single-positive T cells as described in

[15] , wherein the iPSCs are T-cell-derived iPS cells (T-iPSCs).

[17]

[0083] A method for preparing Th1 or Th2 CD4 single positive T cells, comprising the following steps:

[0084] iPS cells in which a gene involved in the secretion of IL-4 or a gene involved in the secretion of IFN-γ is knocked out are induced into CD4 single-positive T cells.

[18]

[0086] Hematopoietic stem cells (HSC) and / or hematopoietic progenitor cells (HPC), wherein a factor involved in the secretion of IL-4 or a factor involved in the secretion of IFN-γ is substantially deficient.

[0087] [18a]

[0088] The hematopoietic stem cell (HSC) and / or hematopoietic progenitor cell (HPC) as described in

[18] , wherein a factor involved in the secretion of IL-4 is substantially deficient.

[0089] [18b]

[0090] The hematopoietic stem cell (HSC) and / or hematopoietic progenitor cell (HPC) as described in

[18] , wherein a factor involved in the secretion of IFN-γ is substantially deficient.

[19]

[0092] A method for regulating the differentiation of CD4 single positive T cells into Th1 type or Th2 type, comprising the following steps:

[0093] A factor involved in the secretion of IL-4 or a factor involved in the secretion of IFN-γ in hematopoietic stem cells (HSC) and / or hematopoietic progenitor cells (HPC) is substantially deficient.

[20]

[0095] A pharmaceutical composition comprising the CD4 single-positive T cells described in

[11] ,

[12] ,

[13] or

[14] . [twenty one]

[0097] Th1 type CD4 single positive T cells are obtained by the method described in [6-1]. [twenty two]

[0099] Th2 type CD4 single positive T cells are obtained by the method described in [7-1].

[0100] Effects of the Invention

[0101] According to the present invention, Th1 type CD4SP T cells and Th2 type CD4SP T cells can be efficiently prepared, and a great contribution can be made to the practical application and application of immune cell therapy using helper T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] [ Figure 1 ] Figure 1 The signal transduction pathways involved in the differentiation of Th1 and Th2 and the proteins (T-bet and IL-4) that are substantially deleted by knockout or the like in the present invention are shown in Usui T. (2007). Jpn. J. Clin. Immunol., 30(6), 419-427. Figure 3 Made some modifications.

[0103] [ Figure 2 ] Figure 2 The sequencing results of TBX21KO 654 (upper) and IL4KO 656 (lower) in Example 1 are shown in FIG.

[0104] [ Figure 3 ] Figure 3 The culture conditions (medium components, oxygen concentration) when iPS cell lines confirmed to be homozygous mutants of TBX21 and IL4 (IL4KO and TBX21KO iPS cell lines) were differentiated into hematopoietic progenitor cells (HPCs) by the embryoid body method (EB method) in Example 2 are shown.

[0105] [ Figure 4 ] Figure 4The markers expressed in cells obtained from ATO produced using HPCs derived from IL4 knockout iPS cell line (IL4KO, the same below) and TBX21 knockout iPS cell line (TBX21KO, the same below), peripheral blood mononuclear cells (PBMCs, the same below, sometimes referred to as PBMC) and wild-type iPS cell line (Wild, the same below, sometimes referred to as Wild) in which a specific gene is not knocked out are shown in Example 2. "IL4KO" and "TBX21KO" also contain cells showing an expression pattern of "CD4+CD8β-" and an expression pattern of "CD4+CD8α-" (within the frame of each display area), that is, CD4SP T cells, as in "PBMCs" and "Wild".

[0106] [ Figure 5 ] Figure 5 The chemokine receptor expression patterns of CD4SP T cells obtained from ATO produced using HPCs derived from IL4KO, TBX21KO, PBMC, and wild type, respectively, in Example 2 are shown in FIG.

[0107] [ Figure 6 ] Figure 6 The cytokine secretion patterns of CD4SP T cells obtained from ATO prepared using HPCs derived from IL4KO, TBX21KO, PBMC, and wild type, respectively, in Example 3 are shown in FIG.

[0108] [ Figure 7 ] Figure 7 The IL-13 production of various cells in Example 3 is shown in the figure. Left: Comparison of CD4SP T cells derived from wild-type cells of TKT3V1-7 (TKT-wild-CD4SPs) and primary CD4SP T cells (primary CD4SPs). Right: Comparison of CD4SP T cells derived from wild-type cells of TKT3V1-7 (TKT-wild-CD4SPs) and CD4SP T cells derived from IL4KO cells of TKT3V1-7 (TKT IL4KO641 / 656-CD4SPs). Vertical axis: cell number, horizontal axis: fluorescence intensity. DETAILED DESCRIPTION

[0109] In the following description, “(B1) hematopoietic stem cells (HSC) and / or hematopoietic progenitor cells (HPC) substantially deficient in factors involved in the secretion of IL-4” is sometimes abbreviated as “HSPC (B1)”; “(B2) hematopoietic stem cells (HSC) and / or hematopoietic progenitor cells (HPC) substantially deficient in factors involved in the secretion of IFN-γ” is abbreviated as “HSPC (B2)”; “(a) stromal cells expressing Notch ligands” is abbreviated as “stromal cells” "(b1) hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) in which the genes involved in the secretion of IL-4 are knocked out but the genes involved in the secretion of IFN-γ are not knocked out" is abbreviated as "HSPCs (b1)"; "(b2) hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) in which the genes involved in the secretion of IFN-γ are knocked out but the genes involved in the secretion of IL-4 are not knocked out" is abbreviated as "HSPCs (b2)". HSPCs (b1) and HSPCs (b2) can be HSCs and HPCs in which the genes involved in the secretion of IL-4 are knocked out but the genes involved in the secretion of IFN-γ are not knocked out, and HSCs and HPCs in which the genes involved in the secretion of IFN-γ are knocked out but the genes involved in the secretion of IL-4 are not knocked out, respectively. In addition, "hematopoietic stem cells and / or hematopoietic progenitor cells" (a general term for hematopoietic stem cells and hematopoietic progenitor cells) are sometimes abbreviated as "HSPCs".

[0110] In the following description, "IL-4 non-secreting and IFN-γ-secreting (Th1 type) CD4 single positive T cells" may be referred to as "Th1 type CD4SP T cells"; "IFN-γ non-secreting and IL-4 secreting (Th2 type) CD4 single positive T cells" may be referred to as "Th2 type CD4SP T cells". Th1 type CD4SP T cells obtained by the method for preparing Th1 type CD4SP T cells of the present invention may be referred to as "Th1 type CD4SP T cells of the present invention", and Th2 type CD4SP T cells obtained by the method for preparing Th2 type CD4SP T cells of the present invention may be referred to as "Th2 type CD4SP T cells of the present invention". The Th1 type CD4SP T cells of the present invention and the Th2 type CD4SP T cells of the present invention may be Th1 type CD4 single positive T cells substantially deficient in factors involved in the secretion of IL-4 and Th2 type CD4 single positive T cells substantially deficient in factors involved in the secretion of IFN-γ, respectively, and their preparation methods are not particularly limited. In addition, “CD4 single positive T cells” may be abbreviated as “CD4SP cells.” It should be noted that “CD4SP cells” in the examples correspond to CD4 single positive T cells.

[0111] In this specification, "hematopoietic stem cell" (HSC) is a pluripotent stem cell (multipotent stem cell) that can differentiate into blood cell lineage cells (white blood cells (neutrophils, eosinophils, basophils, lymphocytes, mononuclear cells (monocytes), macrophages), red blood cells, platelets, mast cells and dendritic cells). In addition, in this specification, "hematopoietic progenitor cells" (HPC) are cells that have the ability to differentiate into blood cell lineage cells but do not have the self-renewal ability of stem cells. In humans, HSC and HPC are mainly found in the bone marrow, and are also found in peripheral blood and umbilical cord blood, and can be recovered from various parts. In the present invention, hematopoietic stem cells can be cells isolated from biological tissues such as bone marrow, blood, etc., or can be cells made from ES cells or iPS cells. Hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC) are cells that are CD34 positive and CD3 negative in terms of cell markers (CD34 + CD3 - Cells). A cell can be confirmed as a hematopoietic stem cell based on the following: a cell that has been transplanted into an animal and has engrafted is taken out and then transplanted into another individual and then engrafts, which means that it has the ability to self-renew, that is, it is a "stem cell" rather than a "progenitor cell."

[0112] In the present specification, “induced pluripotent stem cells” (iPSCs) refer to cells obtained by introducing specific factors (nuclear reprogramming factors) into mammalian somatic cells or undifferentiated stem cells and reprogramming them. Currently, there are many types of "induced pluripotent stem cells". In addition to the iPS cells established by Yamanaka et al. by introducing the four factors Oct3 / 4, Sox2, Klf4 and c-Myc into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), iPS cells derived from human cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al., Cell, (2007) 131: 861-872.), Nanog-iPS cells established by introducing the above four factors and sorting them based on Nanog expression as an indicator (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317.), iPS cells produced by a method without c-Myc (Nakagawa M, Yamanaka S., et al., Nature Biotechnology, (2008) 26, 101-106), iPS cells established by introducing 6 factors using a virus-free method (Okita K et al., Nat. Methods 2011 May; 8(5): 409-12, Okita K et al., Stem Cells. 31(3): 458-66.). In addition, induced pluripotent stem cells established by introducing the four factors OCT3 / 4, SOX2, NANOG, and LIN28 produced by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920), induced pluripotent stem cells produced by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), and induced pluripotent stem cells produced by Sakurada et al. (Japanese Patent Application Laid-Open No. 2008-307007) can also be used.In addition, all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol3, Issue 5, 568-574; Kim JB., Scholer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA., et al., Nature Biotechnology, (2008) 26, No. Any of the induced pluripotent stem cells known in the art described in WO2007 / 051274, WO2008 / 069666, WO2008-118220, WO2008-124133, WO2008-151058, WO2009-006930, WO2009-006997, and WO2009-007852. As induced pluripotent stem cells, various iPS cell lines established by NIH, RIKEN, Kyoto University, etc. can be used. For example, in the case of human iPS cell lines, examples include HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, Nips-B2 of RIKEN, 253G1, 201B7, 409B2, 454E2, 606A1, 610B1, 648A1 of Kyoto University, etc. It should be noted that in the present invention, iPS cell lines derived from T cells described later may also be used.

[0113] In this specification, "marker" refers to "marker protein" or "marker gene", which is a protein or gene specifically expressed on the cell surface, in the cytoplasm and / or in the nucleus in a specific cell type. The marker can be a positive screening marker or a negative screening marker. Preferably, the marker is a cell surface marker, and in particular, based on the cell surface positive screening marker, it is possible to implement the concentration, separation and / or detection of viable cells.

[0114] The detection of marker protein can be performed using immunological analysis using specific antibodies of the marker protein, such as ELISA, immunostaining, flow cytometry, etc. As a specific antibody of the marker protein, an antibody that binds to a specific amino acid sequence in the marker protein or a specific sugar chain bound to the marker protein can be used. In addition, the following method can be used: a marker protein (such as a transcription factor or its subunit, a cytokine, etc.) that is expressed in the cell and does not appear on the cell surface (on the cell membrane) or is secreted by the cell is used as the object, and after the cell is fixed, the specific antibody of the marker protein is used to perform fluorescent staining in the cell, or a reporter protein is expressed together with the marker protein. This method is preferably used in the case where a suitable cell surface marker cannot be confirmed. On the other hand, the detection of marker genes can be performed using nucleic acid amplification methods and / or nucleic acid detection methods known in the field, such as RT-PCR (including quantitative PCR), microarrays, biochips, and RNAseq.

[0115] In this specification, a marker, etc. is "positive" (positive) means that the expression amount of a protein or gene such as a marker (reflecting its measured value, signal) exceeds the amount that can be detected by the above-mentioned method known in the field or the prescribed reference value (or above). In this specification, a marker, etc. is "negative" (negative) means that the expression amount of a protein or gene such as a marker is lower than the amount that can be detected by all or any of the above-mentioned methods known in the field or the prescribed reference value (or below). The detectable amount and reference value of the expression of a protein or gene vary depending on the method adopted and the purpose of the analysis. For example, if it is the expression amount (secretion amount) of a protein that becomes a marker, the fluorescent signal in a flow cytometry (represented by fluorescence activated cell sorting: FACS) in which cells are stained with a fluorescently labeled antibody is higher (above the reference) than the prescribed reference set based on the fluorescent signal of non-stained cells, it can be judged as "positive", and lower (below the reference), It can be judged as "negative". Sometimes a "+" sign is used to indicate positive expression, and a "-" sign is used to indicate negative expression (eg, "CD4+CD8-" is synonymous with "CD4 positive CD8 negative").

[0116] "IFN-γ" refers to "interferon γ" (a protein known to be mainly produced by Th1 cells stimulated by antigens and has various functions such as enhancing antiviral ability, anti-tumor effect, and immune response regulation). "IL-4" refers to "interleukin 4" (a protein known to regulate the initial CD4 +"CXCR3" refers to a specific shared receptor for the chemokines CXCL9 (also known as MIG: monokine induced by interferonγ) and CXCL10 (also known as IP-10: interferon-inducible protein 10) that are selectively expressed in Th1 cells. "CCR4" refers to a specific shared receptor for the chemokines CCL17 (also known as TARC: thymus and activation-regulated chemokine) and CCL22 (also known as MDC: macrophage-derived chemokine) that are selectively expressed in Th2 cells.

[0117] In the present invention, "IL-4 is not secreted and IFN-γ is secreted" means that, in the expression of proteins present in cells, which can be measured by, for example, flow cytometry (FACS, etc.), IL-4 is "negative" and IFN-γ is "positive." "IFN-γ is not secreted and IL-4 is secreted" means that, in the expression of proteins present in cells, which can be measured by, for example, flow cytometry (FACS, etc.), IL-4 is "positive" and IFN-γ is "negative."

[0118] In this specification, unless otherwise specified, "secreted" of a protein is synonymous with "positive", and "non-secreted" of a protein is synonymous with "negative".

[0119] In this specification, "stromal cells" are cells that constitute the connective tissue supporting parenchymal cells in biological tissues, and particularly refer to stromal cells that can produce ATO that produces T cells, and are representative of stromal cells contained in hematopoietic tissues such as bone marrow and thymus. Stromal cells can be cell lines such as MS-5, OP9, S17 as mouse bone marrow cell lines, HS-5, HS-27a as human stromal cell lines, or primary cells collected from humans, etc., or cells differentiated and induced by pluripotent stem cells (iPS cells, etc.) of humans, etc.

[0120] - Preparation method -

[0121] The method for preparing Th1 type CD4 single positive T cells (Th1 type CD4SP T cells) or Th2 type CD4 single positive T cells (Th2 type CD4SP T cells) of the present invention comprises the following steps: inducing hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs) substantially lacking factors involved in the secretion of IL-4 or factors involved in the secretion of IFN-γ into CD4 single positive T cells (CD4S PT cells). Specifically, the method for preparing Th1 type CD4SP T cells of the present invention comprises the following steps: inducing HSPCs (B1) substantially lacking factors involved in the secretion of IL-4 (on the other hand, factors involved in the secretion of IFN-γ are not substantially lacking) into CD4SP T cells. The method for preparing Th2 type CD4SP T cells of the present invention comprises the following steps: inducing HSPCs (B2) substantially lacking factors involved in the secretion of IFN-γ (on the other hand, factors involved in the secretion of IL-4 are not substantially lacking) into CD4SP T cells.

[0122] "Factor involved in the secretion of IL-4" refers to a gene involved in the secretion of IL-4, or a protein encoded by the gene. The "gene involved in the secretion of IL-4" is substantially defective for the purpose of making HSPC (B1), and is substantially not defective for the purpose of making HSPC (B2). The "gene involved in the secretion of IL-4" is typically the "IL4 gene", which is a gene that can inhibit the secretion of IL-4 by making it substantially defective. It can also be a gene other than this as long as it does not hinder the preparation of Th1 type CD4SP T cells and is a gene that does not inhibit the secretion of IFN-γ. More specifically, it can be from the gene encoding Figure 1 The "gene involved in the secretion of IL-4" is selected from the genes of the proteins described in the right figure (Th2 signal), namely IL-4, IL-4R, Stat6, GATA3, mel-18, IL-5 and IL-13 (when the protein is a complex, at least a part thereof). For example, the GATA3 gene and the IL4Ra (CD124) gene, which are known as master regulators of Th2 type CD4SP T cells, can also be used as "genes involved in the secretion of IL-4" if they do not hinder differentiation into T cells due to substantial defects and can achieve inhibition of IL-4 expression. HSPCs (B1) can also be prepared in the same manner by replacing the above-mentioned "gene involved in the secretion of IL-4" and substantially deleting the protein encoded by the gene.

[0123] "Factors involved in the secretion of IFN-γ" refers to genes involved in the secretion of IFN-γ, or proteins encoded by the genes. "Genes involved in the secretion of IFN-γ" are substantially missing for the purpose of making HSPC (B2), but are substantially not missing for the purpose of making HSPC (B1). "Genes involved in the secretion of IFN-γ" are typically genes encoding T-bet (see Figure 1 ) is a gene that can inhibit the secretion of IFN-γ by making it substantially defective. As long as it does not hinder the preparation of Th2 type CD4SP T cells and is a gene that does not inhibit the secretion of IL-4, it can also be a gene other than it. More specifically, the "gene involved in the secretion of IFN-γ" can be selected from the genes encoding the proteins recorded in the left figure (Th1 signal), namely IL-12, IFN-γ, IL-12Rβ2, IFNγR, Stat4, T-bet, HLX (when the protein is a complex, at least a part of it). For example, for the IFN-γ gene and IL-12Rβ2 gene, if differentiation into T cells is not hindered by substantial defect, they can also be used as "genes involved in the secretion of IFN-γ". HSPC (B2) can also be prepared in the same way by replacing the above-mentioned "gene involved in the secretion of IFN-γ" and making the protein encoded by the gene substantially defective.

[0124] The "substantially defective" hematopoietic stem cells and / or hematopoietic progenitor cells (HSPC) of the above-mentioned specific factor means an HSPC that does not carry out the same signal transduction as when the factor works normally. It refers to, for example, (i) HSPCs in which the above-mentioned specific gene is modified in a manner that does not express the above-mentioned specific protein, (ii) HSPCs in which the above-mentioned specific (endogenous) protein is functionally defective, (iii) HSPCs in which the expression of the above-mentioned specific (endogenous) protein is reduced, etc., which exert the effect of the present invention, that is, when cultured under appropriate conditions, can be induced into HSPCs of CD4SP T cells as the target. As HSPCs such as (i), for example, HSPCs in which the gene is knocked out, HSPCs in which at least one base is added, inserted, substituted and / or deleted in the gene, etc. can be cited. Examples of HSPCs such as (ii) include HSPCs that express a dominant negative mutant (e.g., STAT6ΔC in STAT6 (FEBS letters 579 (2005) 3953-3959)) to thereby exert a dominant effect on an endogenous protein and weaken the normal function of the endogenous protein. Examples of HSPCs such as (iii) include HSPCs that contain shRNA and / or siRNA targeting the above-mentioned specific protein. Successful production of HSPCs that are "substantially deficient" in the above-mentioned specific factor can also be confirmed based on the following situation: the HSPCs are induced to become CD4SP T cells, and IL-4 or IFN-γ secreted from the obtained CD4SP T cells is measured. In addition, in the above (iii), "reduced expression of a specific protein" means that the expression level of the specific protein in the HSPC is reduced. In one embodiment, the expression level after treatment is reduced by at least about 25%, 40%, 50%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% (e.g., 96%, 97%, 98% or 99%) compared to the expression level in the HSPC before treatment (e.g., introducing shRNA and / or siRNA for the above-mentioned specific (endogenous) protein into the HSPC). Generally, various means (methods, conditions, reagents, devices, etc. for treating) are known to be used to make cells that are "substantially defective" of a gene or protein. If a person skilled in the art is skilled in the art, an HSPC that does not perform the same signal transduction as when the above-mentioned specific factor (gene or protein) works normally, that is, an HSPC that is "substantially defective" of the above-mentioned specific factor, can be produced by selecting appropriate means from known means or designing in a manner suitable for the present invention to achieve the effect of the present invention.

[0125] According to the above matters, in one aspect of the present invention, HSPCs substantially lacking factors involved in the secretion of IL-4 or factors involved in the secretion of IFN-γ (HSPCs substantially lacking factors involved in the secretion of IL-4, HSPCs substantially lacking factors involved in the secretion of IFN-γ, respectively) can be provided. In addition, in another aspect of the present invention, a method for regulating the differentiation of CD4 single positive T cells of Th1 type or Th2 type is provided, which comprises the step of substantially lacking factors involved in the secretion of IL-4 or factors involved in the secretion of IFN-γ in hematopoietic stem cells and / or hematopoietic progenitor cells (HSPC).

[0126] <Knockout>

[0127] In a preferred embodiment of the present invention, the HSPC (B1) with a substantial defect in a factor involved in the secretion of IL-4 is a HSPC (b1) in which a gene involved in the secretion of IL-4 is knocked out. In a preferred embodiment of the present invention, the HSPC (B2) with a substantial defect in a factor involved in the secretion of IFN-γ is a HSPC (b2) in which a gene involved in the secretion of IFN-γ is knocked out.

[0128] HSPC (b1) and HSPC (b2) can be prepared by knocking out each specific gene in HSPC or in the original cells that can be induced to differentiate into HSPC. For example, when HSPC is induced by differentiation from iPSC as described above, it is preferable to perform the knockout treatment at the iPSC stage.

[0129] The means for knocking out a specific gene are not particularly limited, and known various means can be used. For example, the CRISPR / Cas system is one of the suitable means for knocking out a specific gene in the present invention. With the base sequence contained in the specific locus on genomic DNA as the target, when the CRISPR / Cas system is applied, double-strand breaks (DSB) of DNA will occur repeatedly. As a result, repair errors are caused during non-homologous end joining (NHEJ) or homologous recombination repair (HR), and mutations such as deletion and insertion of bases are introduced, so that a specific gene is knocked out by frameshifting.

[0130] As a CRISPR / Cas system, in addition to the representative CRISPR / Cas9 system, various deformations are known, and various systems can also be used in the present invention. The CRISPR / Cas system includes a first class (type I, type III, type IV) involving multiple RNA-induced nucleases (Cas) and a second class (type II, type V, type VI) involved in a single Cas. As a suitable CRISPR / Cas system, for example, a second class II type CRISPR / Cas9 system and a second class V type CRISPR-Cas12a / Cpf1 can be cited.

[0131] In the CRISPR / Cas system, guide RNA (gRNA) and RNA-inducible nuclease are used, but it is known that each of them has various modifications, and various embodiments of gRNA and nuclease can also be used in the present invention.

[0132] gRNA is composed of crRNA and tracrRNA. CrRNA and tracrRNA can be different RNA chains formed into a complex, or they can be a single RNA chain (single guide RNA: sgRNA or chimeric RNA) connected by a spacer group. The base sequence (base sequence adjacent to the PAM sequence) to be targeted in order to knock out each specific gene in HSPC, that is, the base sequence of the crRNA corresponding to the base sequence can be appropriately designed by those skilled in the art. gRNA can be introduced into the cell in the form of RNA or in the form of DNA (expression plasmid, etc.) containing the base sequence of the RNA that can be generated by transcription. The gRNA in RNA form can be a gRNA formed only by natural nucleotides (adenine, guanine, cytosine, uracil), or it can be a gRNA containing natural nucleotides and nucleotide analogs. Examples of nucleotide analogs include sugar-modified nucleotides (2'-O-methyl ribose, 2'-O-propyl ribose, 2'-O-methoxyethoxy ribose, 2'-O-methoxyethyl ribose, 2'-O-[2-(guanidinium)ethyl]ribose, 2'-O-fluororibose, etc.); bridged structure artificial nucleic acids (BNAs) (artificial locked nucleic acids (LNAs), ethylene bridged structure artificial nucleic acids (ENA), etc.); phosphodiester bond-modified nucleotides (phosphodiester bond-substituted nucleotides with phosphorothioate bonds, N3'-P5' phosphoramidate bonds, etc.); In one embodiment of the present invention, a portion of the gRNA (preferably at least one base at each of the 3' and 5' ends of the base sequence, more preferably two dibases or three bases) can be set as a nucleotide analog (preferably a sugar-modified nucleotide and a phosphodiester bond-modified nucleotide, more specifically, a 2'-O-methyl ribose and a phosphorothioate bond-substituted phosphodiester bond).

[0133] RNA-inducible nucleases can be wild-type nucleases in which both nuclease domains are active, or mutant nucleases (nickases) in which only one is active. RNA-inducible nucleases can be introduced into cells in the form of a protein or a nucleic acid (mRNA or DNA such as an expression plasmid) containing a base sequence encoding the amino acid sequence of the protein.

[0134] The means for introducing the specific RNA and RNA-inducible nuclease used in the CRISPR / Cas system into cells (in the present invention, for example, in HSPCs) are not particularly limited. In one embodiment of the present invention, the RNA strand of the gRNA and the protein of the RNA-inducible nuclease (Cas9, etc.) can be introduced into the cell by electroporation.

[0135] The various cells used in the present invention, i.e., cells used to produce HSPC (B1) and (b1), HSPC (B2) and (b2), and iPS cells of HSPC (B1) and HSPC (B2) described later, and cells (T cells, etc.) used to produce the iPS cells, may be cells derived from humans or cells derived from animals other than humans, such as mammals such as mice, rats, dogs, pigs, and monkeys, and may be selected according to the use of the Th1 type CD4SP T cells or Th2 type CD4SP T cells obtained by the present invention. For example, if Th1 type CD4SP T cells or Th2 type CD4SP T cells are administered to humans, it is preferred that the various cells described above are cells derived from humans.

[0136] There is no particular limitation on how HSPCs (B1) (B2) and the like, i.e., cells before substantial loss of specific factors, are obtained or produced. For example, cells may be produced by inducing differentiation of iPS cells, ES cells, or other cells having the ability to differentiate into HSPCs, or HSCs or HPCs collected from cord blood, bone marrow, peripheral blood, or other organisms.

[0137] <Differentiation of iPSCs into HSPCs>

[0138] In a preferred embodiment of the present invention, HSPC is a cell induced by differentiation of iPS cells (iPSC). For example, iPSC is prepared using cells (preferably T cells, more preferably CD4SP T cells) collected from a patient to whom Th1 type CD4SP T cells and Th2 type CD4SP T cells obtained by the preparation method of the present invention are to be administered, and ATO prepared using the iPSC is used, thereby obtaining Th1 type CD4SP T cells and Th2 type CD4SP T cells that are compatible with the patient's HLA type and do not cause immune rejection. In addition, iPSCs that have been generalized (having a specific HLA type that does not cause immune rejection in a large number of patients, or knocking out HLA) can also be used.

[0139] The basic matters and specific embodiments for inducing differentiation from iPSC to HSPC are known and are not particularly limited. The composition of the culture medium, the culture conditions, etc. can be appropriately adjusted and changed according to the differentiation induction method adopted, the number of culture days, etc., preferably in a manner that can improve the differentiation induction efficiency and cell increase rate to HSPC.

[0140] In a preferred embodiment of the present invention, HSPCs produced from iPSCs (preferably T-iPSCs described below, and the same below) can be produced by a feeder-free method. For example, preferably the "embryoid body method (EB method)" described in AE Grigoriadis et al., (2010). Blood, 115 (14): 2769-2776., which forms embryoid bodies to produce HPCs without using feeder cells.

[0141] In addition, for example, iPSC can also be cultured on feeder cells as described in WO2011 / 096482 and WO2013 / 176197 and differentiated into HSPC. From the viewpoint of being easy to induce differentiation to mesoderm lineage, preferably feeder cells are stromal cells. From the viewpoint of being easy to induce differentiation to hematopoietic lineage, preferably stromal cells are OP9 cells, 10T1 / 2 cells (C3H10T1 / 2 cells) etc. that have been treated with radiation irradiation.

[0142] As a culture medium for a feeder-free method (EB method, etc.), for example, StemFit (registered trademark, Ajinomoto Co., Ltd.) and StemPro (registered trademark, Thermo Fisher Scientific) can be cited. As a culture medium for co-culturing iPSC and feeder cells, for example, X-VIVO culture medium, IMDM culture medium (Iscove's modified Dulbecco's medium), α-MEM, and DMEM can be cited. From the viewpoint of high efficiency in forming a bag-like structure (sack) containing HSPC, etc., IMDM culture medium is preferred.

[0143] The culture medium does not necessarily contain cytokines, but preferably contains cytokines and serum (for example, fetal bovine serum (FBS)) as required, insulin, transferrin, sodium selenite, L-glutamine (or GlutaMAX (Gibco)), α-thioglycerol, ascorbic acid, etc. From the viewpoint of efficiently inducing differentiation of iPSC to HSPC, it is preferred that cytokines be at least one cytokine selected from the group consisting of BMP, bFGF, VEGF, SCF, TPO and FLT3L. In the EB method, for example, preferably, the combination of three kinds of BMP4, bFGF and VEGF, the combination of three kinds of bFGF, VEGF and SCF or the combination of five kinds of bFGF, VEGF, SCF, TPO and FLT3L are used while changing according to the number of days in culture. In the co-culture method of iPSC and feeder cells, for example, the combination of three kinds of VEGF, SCF and TPO or the combination of three kinds of VEGF, SCF and FLT3L are more preferred.

[0144] The culture time is set to a time until a sufficient number of HSPCs can be produced (in the co-culture method of iPSCs and feeder cells, a capsule containing them can be formed), for example, preferably 10 to 14 days from the start of culture. The culture environment is not particularly limited, preferably about 5% CO2, about 37 ° C. In addition, from the viewpoint of improving the production efficiency of HSPCs (capsule formation efficiency), it is more preferable to culture under low oxygen concentration conditions (e.g., 5 to 20%).

[0145] The capsule formed by differentiation induction as described above usually contains HSPC and other cells (blood cells, etc.). The cells present in the interior of the capsule can be separated and recovered from the capsule using physical means, such as by a sterilized sieve-like apparatus (cell screen, etc.). HSPC is a cell expressing CD34 as a cell surface marker, and therefore, it can be separated from the cell population obtained by culture using, for example, a fluorescently labeled anti-CD34 antibody and a flow cytometry of a cell sorter. In a preferred embodiment of the present invention, HSPC is HPC.

[0146] <Creating iPSCs from T cells>

[0147] In a preferred embodiment of the present invention, the iPSC used for differentiation induction into HSPC is an iPSC derived from T cells (T-iPSC). The basic matters and specific embodiments for making iPSC from T cells are known, for example, referring to WO2011 / 096482 and WO2013 / 176197. It should be noted that the basic matters and specific embodiments for making iPSC from cells other than T cells are also known.

[0148] "T cells" used to make iPSCs include: cells expressing T cell receptors (TCR: T cell receptor), especially TCR α chain and β chain (TCRαβ) on the surface, their progenitor cells, that is, progenitor T cells (pro-T cells) that do not express TCRαβ, pre-T cells in which TCRβ and pre-TCRα are associated, etc. In addition, "T cells" include: T cells in which both CD4 and CD8 are negative (negative) (CD4·CD8 double negative T cells), T cells in which both CD4 and CD8 are positive (positive) (CD4·CD8DP T cells), T cells in which CD4 is positive and CD8 is negative (CD4SP T cells), and T cells in which CD8 is positive and CD4 is negative (CD8SP T cells). It should be noted that the description of expression as "positive" or "negative" is as shown in the separate description in this specification. In the present invention, in particular, for "CD4SP T cells" generated by ATO, the "predetermined criterion" (threshold) for judging CD4 as "positive" and CD8 as "negative" can refer to the expression levels of CD4 and CD8 of natural helper T cells.

[0149] As T cells used for preparing iPSCs, for example, T cells collected from T cell-containing tissues such as peripheral blood, lymph nodes, bone marrow, thymus, spleen, umbilical cord blood, and lesion tissues can be used.

[0150] In a preferred embodiment of the present invention, the T cells used to prepare iPSCs are CD4 single positive T cells (CD4SP T cells). CD4SP T cells can be separated from a cell population contained in a tissue, etc., using, for example, flow cytometry using a fluorescently labeled anti-CD4 antibody and a cell sorter. iPS cell lines prepared from CD4SP T cells have been established, and, for example, TKT3V1-7 strain, b3a2 strain, etc. can be used in the present invention to prepare iPSCs.

[0151] The collected T cells have antigen specificity, i.e., antigen specific TCR brought by the reconstructed TCR gene. The Th1 type or Th2 type CD4SP T cells of the present invention produced by ATO made using iPSC may have the same or substantially the same antigen specificity (antigen specific TCR) as the T cells used to make the iPSC. It should be noted that T cells with desired antigen specificity can be separated from tissues using, for example, purification treatments such as affinity chromatography columns fixed with desired antigens, and purification treatments such as multimers of MHC (major histocompatibility complex) bound to desired antigens (e.g., "MHC tetramers", "Pro 5 (registered trademark) MHC class I pentamers"), etc.

[0152] In the preparation method of the present invention, the means (method, conditions, device, etc. for culturing) of HSPCs (B1) and (B2) substantially lacking factors involved in the secretion of IL-4 or factors involved in the secretion of IFN-γ into the target CD4SP T cells, i.e., Th1 type CD4SP T cells and Th2 type CD4SP T cells, respectively, are not particularly limited. Those skilled in the art can select appropriate means or design culture conditions (culture medium, period, temperature, atmosphere, etc.) suitable for the present invention to culture the above-mentioned HSPCs, thereby inducing them into the above-mentioned CD4SP T cells. For example, HSPCs (B1) and (B2) can be induced into Th1 type CD4SP T cells and Th2 type CD4SP T cells by culturing them according to the "ATO method". Hereinafter, as a representative example of an induction method (culture method), the ATO method using HSPC (b1) and (b2) in which a specific gene is knocked out is specifically described below, but the induction method (culture method) that can be used in the preparation method of the present invention is not limited thereto, and HSPC (B1) and (B2) other than HSPC (b1) and (b2) can also be used. When the present invention is implemented according to an induction method (culture method) other than the ATO method, it can be within the necessary range (for example, with respect to the culture medium), referring to the following matters recorded about the ATO method.

[0153] <ATO Law>

[0154] In a preferred embodiment, the method for preparing Th1 type CD4SP T cells of the present invention comprises the following steps:

[0155] An artificial thymic organoid (ATO) is cultured to generate IL-4 non-secreting and IFN-γ-secreting (Th1 type) CD4SP T cells from the ATO, wherein the ATO comprises:

[0156] (a) stromal cells expressing Notch ligands; and

[0157] (b1) Hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) in which a gene involved in IL-4 secretion is knocked out but a gene involved in IFN-γ secretion is not knocked out.

[0158] In a preferred embodiment, the method for preparing Th2 type CD4SP T cells of the present invention comprises the following steps: culturing artificial thymic organoids (ATO) to generate IFN-γ non-secreting and IL-4-secreting (Th2 type) CD4SP T cells from the ATO, wherein the ATO comprises:

[0159] (a) stromal cells expressing Notch ligands; and

[0160] (b2) Hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) in which a gene involved in the secretion of IFN-γ is knocked out but a gene involved in the secretion of IL-4 is not knocked out.

[0161] For the ATO method in the preparation method of the present invention, HSPC (B1) or (B2) in which a specific factor is substantially defective is used in advance, preferably HSPC (b1) or (b2) in which a specific gene is knocked out in advance, in addition, ATO can be made and the produced cells can be recovered and implemented in the same manner as before. The basic matters and specific implementation methods of the ATO method are known (for example, see patent document 1: WO2017 / 075389), and the present invention is based on this and changes the necessary parts. As an example, in the method described in patent document 1, embryonic mesodermal organoids are initially formed on an insert culture dish (insert) and cell components equivalent to HPC are obtained to make ATO, but some of them can also be changed, using the "embryonic body method (EB method)" (AE Grigoriadis et al., (2010). Blood, 115 (14): 2769-2776.) After inducing HPC by differentiation from iPSC, ATO is made using it. In the present invention, HSPCs used to produce ATO are replaced with HSPCs (b1) or (b2) in which a specific gene has been knocked out in advance. In addition, in the present invention, among the cells produced by ATO, the cells to be recovered are Th1 type CD4SP T cells and Th2 type CD4SP T cells.

[0162] The medium used for culturing ATO (stromal cells (a) and HSPC (b1) and (b2)) can be selected from the medium used for culturing animal cells, and can be used by adding the required components at appropriate concentrations. The medium and the added components can be changed as the number of culture days passes.

[0163] Culture medium, for example, can include AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL1066, Glasgow MEM, improved MEM zinc selective culture medium (improved MEM zinc option), IMDM, 199 culture medium, Eagle MEM, αMEM, DMEM, Ham, RPMI-1640, and Fisher culture medium. The above culture medium can be used alone or in combination of two or more. As an example of a preferred culture medium, RPMI-1640 can be mentioned.

[0164] The culture medium can be a serum-containing culture medium or a serum-free culture medium, or a Xeno-free culture medium. From the viewpoint of preventing contamination caused by xeno-derived components, serum can be derived from an animal of the same species as the cells being cultured. A serum-free culture medium refers to a culture medium that does not have unprocessed or unpurified serum, and therefore may include a culture medium with purified blood-derived components or animal tissue-derived components (growth factors, etc.). The culture medium may contain any substitute for serum, or may not contain any substitute for serum. Serum substitutes may include: albumin (lipid-rich albumin, bovine albumin, recombinant albumin or humanized albumin, etc., albumin substitutes, plant starch, dextran, and protein hydrolyzates, etc.), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, thioglycerol (α-thioglycerol, MTG), or materials appropriately containing their equivalents. Commercially available materials such as knockout serum replacement (KSR), chemically-defined lipid concentrated (Gibco), and GlutaMAX (Gibco) can also be used. The culture medium can be a serum-free medium (SFM) suitable for cell development. For example, the culture medium can contain B-27 (registered trademark) supplement, XenoB-27 (registered trademark) supplement, NS21 supplement, GS21 (trademark) supplement, or a combination thereof at a concentration effective for generating T cells from 3D cell aggregates.

[0165] The culture medium may contain one or more selected from the group consisting of biotin, DLα-tocopheryl acetate, DLα-tocopherol, vitamins such as vitamin A (acetate), BSA (bovine serum albumin) or human albumin, Fraction V without fatty acids, catalase, human recombinant insulin, human transferrin, proteins such as superoxide dismutase, corticosterone, D-galactose, ethanolamine HCl, glutathione (reduced form), L-carnitine HCl, linoleic acid, linolenic acid, progesterone, putrescine 2HCl, sodium selenite, and T3 (triiodo-L-thyronine), and PSG (penicillin, streptomycin, and L-glutamine). The culture medium may also contain ascorbic acid or its derivatives (e.g., ascorbic acid 2-phosphate: PAA) added from the outside. The culture medium may also contain one or more selected from the group consisting of fatty acids or lipids, amino acids (non-essential amino acids, etc.), vitamins, growth factors, cytokines, antibiotics, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, and inorganic salts added from the outside.

[0166] The culture medium may contain cytokines added from the outside. As cytokines, for example, FLT3 ligand (FLT3L), interleukin 7 (IL-7), stem cell factor (SCF), thrombopoietin ((TPO), IL-2, IL-3, IL-4, IL-6, IL-12, IL-15, IL-21, TNF-α, TGF-β, interferon-γ, interferon-λ, TSLP, thymopentin, pleiotrophin, and midkine. Any one of these cytokines may be used alone, or two or more may be used in combination.

[0167] The 3D cell aggregate may or may not contain an exogenous extracellular matrix or a scaffold. Examples of the exogenous extracellular matrix include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin, and mixtures thereof, such as Matrigel (trademark) and lysed cell membrane preparations.

[0168] Other ATO culture conditions can be set appropriately. The culture temperature can be set to 20 to 40° C., for example, preferably about 37° C. The CO2 concentration can be set to 2% to 10%, for example, preferably 2 to 5%. The oxygen concentration can be set to 1 to 20%, for example, preferably 5 to 20%.

[0169] The stromal cells (a) and HSPCs (b1) or HSPCs (b2) in ATO can be co-cultured at any ratio. For example, the stromal cells (a): HSPCs (b1) or HSPCs (b2) can be set to 100:1 to 1:100, 90:1 to 1:90, 80:1 to 1:80, 70:1 to 1:70, 60:1 to 1:60, 50:1 to 1:50, 40:1 to 1:40, 30:1 to 1:30, 20:1 to 1:20, preferably 20:1 to 1:4. When HSPC (b1) or HSPC (b2) is produced from T cell-derived iPS cells, the above ratio is preferably approximately 1 (e.g., MS5hDLL4):4 (e.g., HPC). On the other hand, when HSPC (b1) or HSPC (b2) is produced from non-T cell-derived iPS cells, the above ratio is preferably approximately 20 (e.g., MS5hDLL4):1 (e.g., HPC).

[0170] The culture time of ATO is, for example, 4 weeks or more, preferably 5 weeks or more, more preferably 6 weeks or more, and more preferably 8 weeks or more or 9 weeks or more. The upper limit of the culture time is not particularly limited, but is preferably 16 weeks or less, more preferably 14 weeks or less, and more preferably 12 weeks or less. Specifically, the culture time is, for example, 4 to 16 weeks, preferably 5 to 16 weeks, more preferably 6 to 14 weeks, and more preferably 8 to 12 weeks or 9 to 12 weeks. Until about 4 to 9 weeks, the longer the culture time of ATO is, the higher the proportion of CD4SP T cells in the obtained cell population tends to be. On the other hand, after a certain period of time, the proportion of CD4SP T cells may not increase easily, and CCR7-positive cells may gradually decrease (differentiate in the direction of effectors). Therefore, the culture time of ATO can be adjusted within the above range (for example, set to about 9 weeks) according to the cell line, culture conditions, etc. in order to obtain a cell population of the desired composition.

[0171] In the production method of the present invention, there is a case where the cells produced by ATO include CD8SP T cells, CD4·CD8DN T cells and CD4·CD8DP T cells in addition to CD4SP T cells. For example, CD4SP T cells can be separated and recovered from the produced cells using flow cytometry (represented by fluorescence activated cell sorting: FACS) using a fluorescently labeled anti-CD4 antibody and a cell sorter. Furthermore, the presence of Th1 type CD4SP T cells and Th2 type CD4SP T cells among the CD4SP T cells can be confirmed using each fluorescently labeled anti-IFN-γ antibody and anti-IL-4 antibody and flow cytometry for intracellular staining. When it is intended to recover Th2 type CD4SP T cells as live cells, for example, flow cytometry can be used to recover cells that are CXCR3 negative, CCR4 positive (consistent with the secretion tendency of IL-4), and CCR6 negative (a marker of Th17 cells).

[0172] <Stromal cells (a)>

[0173] Stromal cells (a) can be prepared by introducing Notch ligand genes into stromal cells and expressing them. Stromal cells (a) can be stromal cells derived from humans or stromal cells derived from animals other than humans, as with the aforementioned HSPCs. The means for introducing Notch ligand genes are not particularly limited, and for example, known suitable means such as viral vectors (retroviral vectors, lentiviral vectors, etc.) can be used. For example, an MS-5 mouse stromal cell strain (MS5-hDLL1 strain and MS5-hDLL4 strain, respectively) has been established in which human DLL1 and human DLL4 have been transduced as Notch ligands, and such cell strains can be used as stromal cells (a) in the present invention.

[0174] The term "Notch ligand" used in this specification is synonymous with the term in Patent Document 1: WO2017 / 075389 (corresponding to Japanese Patent Publication No. 2018-533364), which describes a method for producing ATO, and includes "typical Notch ligands" and "atypical Notch ligands". Typical Notch ligands include, for example, delta-like ligand 4 (DLL4), delta-like ligand 1 (DLL1), jagged protein 1 (JAG1, Jagged 1), jagged protein 2 (JAG2, Jagged 2), delta-like ligand 3 (DLL3), and X-delta2. Examples of atypical Notch ligands include contactin-1, NOV / CCN3, contactin-6, periostin / OSF-2, DLK2 / EGFL9, Pref-1 / DLK1 / FA1, DNER, thrombospondin-2, MAGP-1 / MFAP2, thrombospondin-3, MAGP-2 / MFAP5, thrombospondin-4, and netrin-1.

[0175] -cell-

[0176] The first embodiment of the Th1 type CD4SP T cell of the present invention is a Th1 type CD4SP T cell obtained by the aforementioned preparation method of the present invention, that is, a preparation method comprising the step of inducing hematopoietic stem cells and / or hematopoietic progenitor cells (HSPC) substantially deficient in factors involved in the secretion of IL-4 into CD4 SPT cells. The second embodiment of the Th1 type CD4SP T cell of the present invention is a Th1 type CD4SP T cell substantially deficient in factors involved in the secretion of IL-4. It should be noted that the Th1 type CD4SP T cell of the present invention may also be a cell belonging to both the first embodiment and the second embodiment. As described above, the Th1 type CD4SP T cell of the present invention is preferably a cell induced by differentiation of an iPS cell (iPSC), for example, an iPS cell derived from a T cell (T-iPSC).

[0177] The first embodiment of the Th2 type CD4SP T cell of the present invention is a Th2 type CD4SP T cell obtained by the aforementioned preparation method of the present invention, that is, a preparation method comprising the step of inducing hematopoietic stem cells and / or hematopoietic progenitor cells (HSPC) substantially deficient in factors involved in the secretion of IFN-γ into CD4 SPT cells. The second embodiment of the Th2 type CD4SP T cell of the present invention is a Th2 type CD4SP T cell substantially deficient in factors involved in the secretion of IFN-γ. It should be noted that the Th2 type CD4SP T cell of the present invention may also be a cell belonging to both the first embodiment and the second embodiment. As described above, the Th2 type CD4SP T cell of the present invention is preferably a cell induced by differentiation of an iPS cell (iPSC), for example, an iPS cell derived from a T cell (T-iPSC).

[0178] The first and second embodiments of the Th1 type CD4SP T cells of the present invention can be distinguished from natural Th1 type CD4SP T cells existing in a living body (hereinafter sometimes abbreviated as "natural Th1 cells") and Th1 type CD4SP T cells obtained by a production method other than the present invention. For example, in a preferred embodiment of the production method of the present invention, a gene involved in the secretion of IL-4 of hematopoietic stem cells and / or hematopoietic progenitor cells (HSPC) is knocked out, and a gene involved in the secretion of IL-4 in the Th1 type CD4SP T cells obtained by the production method is also knocked out, so that it can be clearly distinguished from natural Th1 cells and the like (in this case, the Th1 type CD4SP T cells belong to both the first and second embodiments). The fact that a specific gene has been knocked out can be confirmed by investigating the base sequence of genomic DNA according to a conventional method. In addition, in the second embodiment of the Th1 type CD4SP T cells of the present invention, a factor involved in the secretion of IL-4 is substantially deficient, so it can be distinguished from natural Th1 cells and the like that do not have such a feature. The Th1 type CD4SP T cells obtained by other embodiments of the production method of the present invention (first embodiment) can also be distinguished from natural Th1 cells etc. based on traces corresponding to the characteristics of the embodiment of the production method (particularly, means for substantially deficient factors involved in the secretion of IL-4 of HSPC). Similarly, the first and second embodiments of the Th2 type CD4SP T cells of the present invention can also be distinguished from natural Th2 type CD4SP T cells existing in a living body (hereinafter sometimes abbreviated as natural Th2 cells) and Th2 type CD4SP T cells obtained by production methods other than the present invention.

[0179] It should be noted that, generally, the Th1 type CD4SP T cells and Th2 type CD4SP T cells of the present invention are cells that further express CD45RA or CD45RО, CD3ε, TCRαβ, etc., similarly to natural Th1 cells and Th2 cells. Judging from the expression of chemokine receptors, the Th1 type CD4SP T cells of the present invention are CXCR3 positive and CCR4 negative, similarly to natural Th1 cells, and the Th2 type CD4SP T cells of the present invention are CXCR3 negative and CCR4 positive, similarly to natural Th2 cells. In addition, the Th1 type CD4SP T cells and Th2 type CD4SP T cells of the present invention also strongly express CD5 (strongly positive), that is, they become mature Th1 type CD4SP T cells and mature Th2 type CD4SP T cells.

[0180] In addition to the above characteristics, the Th1 type CD4SP T cells and Th2 type CD4SP T cells of the present invention have the advantage of containing components expressing CD28 and CCR7 representing the phenotype of central memory T cells and having high self-replication ability.

[0181] <Application>

[0182] The use of the Th1 type CD4SP T cells and Th2 type CD4SP T cells of the present invention is not particularly limited, and they can be used, for example, to treat or prevent tumors, infections (eg, chronic infections), allergic diseases, autoimmune diseases, and the like.

[0183] Therefore, in one aspect of the present invention, Th1 type CD4SP T cells and Th2 type CD4SP T cells obtained by the production method of the present invention, and pharmaceutical compositions (cell preparations) containing the cells are provided for treating or preventing the above diseases.

[0184] In another aspect, the present invention provides a method (immune cell therapy) of administering Th1 CD4SP T cells and Th2 CD4SP T cells obtained by the production method of the present invention, or a pharmaceutical composition (cell preparation) containing the cells, for the treatment or prevention of the above diseases.

[0185] The pharmaceutical composition (cell preparation) in the above embodiment (hereinafter referred to as "the pharmaceutical composition (cell preparation) of the present invention") may contain either Th1 type CD4SP T cells or Th2 type CD4SP T cells, or may contain both at any mixing ratio.

[0186] The pharmaceutical composition (cell preparation) of the present invention can be formulated using known pharmaceutical methods according to the administration route, for example, it can be prepared into an injection (intravenous injection, drip, etc.), a liquid preparation, a suspension, an emulsion, etc. In such a formulation, a pharmaceutically acceptable carrier (medium), an additive can be appropriately combined as needed, specifically, sterile water, physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, stabilizer, carrier (vehicle), preservative, binder, diluent, isotonic agent, analgesic, filler, disintegrant, buffer, coating agent, lubricant, colorant, solubilizer, etc. In addition, it can also contain other active ingredients (other drugs, cells, etc., such as CD8SP T cells introduced with CAR) corresponding to the purpose of treatment or prevention together with Th1 type CD4SP T cells and / or Th2 type CD4SP T cells, immunostimulants, etc.

[0187] The administration method of the pharmaceutical composition (cell preparation) of the present invention is not particularly limited, and is preferably parenteral administration, such as intravenous, intraperitoneal, subcutaneous or intramuscular administration, or local administration to the affected area, and more preferably intravenous administration or local administration to the affected area. The administration amount of the pharmaceutical composition (cell preparation) of the present invention can be appropriately adjusted according to the age, weight, symptoms, health status, dosage form, administration method, etc. of the subject.

[0188] The product of the pharmaceutical composition (cell preparation) of the composition of the present invention may be labeled with information that it can be used to treat or prevent diseases, etc. For example, information that it can be used to treat or prevent diseases, etc. may be recorded on the product body, container, packaging, etc., or on the product manual, accompanying manual, promotional materials, other printed materials, etc.

[0189] As used in this specification and claims, unless otherwise required by the context, terms in the singular include the plural and terms in the plural include the singular. Therefore, unless otherwise indicated, it should be understood that articles in the singular (e.g., in the case of English, "a", "an", "the", etc.) also include the concept of their plural forms.

[0190] Example

[0191] [Example 1] Preparation of iPS cells (TBX21 KO cell line, IL4 KO cell line)

[0192] As iPS cells, the TKT3V1-7 strain provided by Kyoto University (CiRA) was used. iPS cells that were passaged and maintained in AK03N medium (Ajinomoto Co., Ltd.) and were in a subconfluent state in the wells were recovered as single cells using TripLESelect (Thermo Fisher Scientific). The recovered cells were divided into 1.0×10 cells per cuvette. 6 The cells were resuspended in Opti-MEM (manufactured by ThermoFisher Scientific) at 100 μL / cell.

[0193] Guide RNAs were designed for the TBX21 gene encoding the transcription factor T-bet and the IL4 gene encoding the cytokine IL-4, and guide RNAs were formed using Alt-R CRISPR-Cas9 crRNA (manufactured by Integrated DNA Technologies) and Alt-R CRISPR-Cas9 tracrRNA (manufactured by the same company) containing the recognition sequences. The guide RNA suspension was reacted with Alt-RS.p.Cas9 Nuclease 3NLS (manufactured by the same company) to form an RNP complex.

[0194] The RNP complex suspension was mixed with 1.0×10 6 iPS cells of 100 μg / mL were mixed, and the mixture was electroporated using NEPA21 electroporator (manufactured by Nepa Gene).

[0195] It should be noted that during electroporation, the Puro (puromycin resistance gene) plasmid was electroporated at the same time, and the cells were cultured in a medium supplemented with puromycin, thereby indirectly selecting cells into which the Cas9 protein was introduced. This utilizes the correlation between the introduction of Cas9 protein into cells and the introduction of plasmids.

[0196] The electroporated iPS cells were seeded on a 10 cm culture dish coated with iMatrix-511 (Matrixome), and colonies generated after 12 days of culture were picked and passaged. Cell lines derived from each colony were established and stored, and the knockout and mutation forms of the target gene were confirmed by sequencing.

[0197] The PCR product obtained by using primers designed to sandwich the target sequence of the guide RNA was sequenced. An actual example of the sequencing result is shown in Figure 2Even with knockout (KO) treatment using the same guide RNA, the resulting mutations differed depending on the strain, and the cell lines that were sequenced and confirmed for traits are listed in Table 1. It should be noted that, regardless of the form of mutation, it was later confirmed that the target trait was equally expressed among these cell lines.

[0198] [Table 1]

[0199] Table 1: List of obtained KO strains

[0200]

[0201] *1 The base sequence within the IL4 gene targeted by gRNA4 (SEQ ID NO. 1)

[0202] 5'-GTGTCCGTGGACAAAGTTGC-3'

[0203] *2 The base sequence within the IL4 gene targeted by gRNA5 (SEQ ID NO. 2)

[0204] 5'-CAAGTGCGATATCACCTTAC-3'

[0205] *3 The base sequence within the TBX21 gene targeted by gRNA2 (SEQ ID NO. 3)

[0206] 5'-GCGGTACCAGAGCGGCAAGT-3'

[0207] [Example 2] Preparation of Th1 cells and Th2 cells from iPS cells (TBX21KO cell line, IL4KO cell line) (iPSC→HPC / HPC→Th1 cells or Th2 cells)

[0208] The iPS cell line with homozygous mutants of TBX21 or IL4, which is the target gene, confirmed by sequencing, was differentiated into hematopoietic progenitor cells (HPCs) using the embryoid body method (EB method). The EB method refers to a method for forming embryoid bodies and producing hematopoietic progenitor cells without using feeder cells as described in AE Grigoriadis et al., (2010). Blood, 115(14): 2769-2776. In this example, Figure 3 That's how the changes were made.

[0209] In TKT3V1-7 or its KO cell line, the day when differentiation induction was started for undifferentiated cells was recorded as day 0, and it was used for ATO production on day 9. EB blocks contained in the culture medium were removed using a 40 μm cell screen (manufactured by Fisher Scientific) to recover only the suspended cells. It is believed that the recovered suspended cells contain components equivalent to hematopoietic progenitor cells. In this example, they were not sorted and used as ATO materials in the form of a group (bulk).

[0210] The artificial thymic organoid method is an organoid culture method that originally differentiates primary hematopoietic stem cells into αβ-type T cells, reported by CS Seet et al. (2017). Nat Methods. 14(5): 521-530. In this original method, CD34-positive CD3-negative cells are sorted and used using a cell sorter, and it is believed that hematopoietic progenitor cells can also be sorted.

[0211] In this example, iPS cell-derived hematopoietic progenitor cells of the unsorted recovered population were mixed with MS5hDLL4 cells at a cell number ratio of 4:1, and a drop of the suspension was placed on a culture insert (MILLICELL INSERT 30MM ORGANOTYPIC PTFE 0.4UM 50 / PK, manufactured by Merck (Millipore) Co., Ltd.), and the culture insert was suspended in a culture medium, thereby starting the ATO differentiation culture method. The culture medium was prepared as follows: B-27 was added to RPMI-1640 (manufactured by Wako Co., Ltd.) in a 25-fold dilution. TM Supplement (50X), serum-free (ThermoFisher Scientific), PSG (penicillin, streptomycin, and L-glutamine) was added in a 100-fold dilution, GlutaMAX was added in a 100-fold dilution, IL-7 (final concentration 5 ng / mL) was added to the resulting medium, and Flt3L (5 ng / mL) and PAA (50 μg / mL) were added in the same manner. It should be noted that "MS5hDLL4 cells" are cells produced by introducing the hDLL4 gene into the mouse bone marrow cell line "MS-5" using a lentiviral vector.

[0212] After 9 to 12 weeks of culturing a mixture of iPS cell-derived HPCs and MS5hDLL4 on an insert suspended in this medium, CD4 single positive (SP) cells and CD8 single positive (SP) cells differentiated. Figure 4In the wild-type and PBMCs, a strong IL-4 secretion tendency was observed, and a high T-bet expression was also observed. Therefore, it is assumed that IL4KO and TBX21KO may inhibit CD4SP, CD8SP differentiation, or T cell differentiation itself. However, Figure 4 As shown, all KO strains were able to achieve CD4SP differentiation.

[0213] Based on the analysis of primary helper T cells, the common understanding is that, as the expression pattern of chemokine receptors, Th1 cells responsible for cellular immunity are CXCR3-positive and CCR4-negative, and Th2 cells responsible for humoral immunity are CXCR3-negative and CCR4-positive. It is assumed that in IL4KO, in which the secretion of IL-4, a functional cytokine of Th2, disappears, the cells exhibit a Th1-type expression pattern, and in the KO of TBX21, which encodes T-bet, a master regulator of Th1 (a transcription factor that mainly induces differentiation), a Th2-type expression pattern is exhibited. This was verified by flow cytometry-based analysis using anti-CXCR3 antibodies (manufactured by BD, clone: ​​1C6 / CXCR3) and anti-CCR4 antibodies (manufactured by BD, clone: ​​1G1). The results are as follows. Figure 5 As shown, the CD4SP cells induced by wild-type differentiation without KO treatment and the primary CD4SP cells in PBMCs contain both CXCR3-positive CCR4-negative cells and CXCR3-negative CCR4-positive cells. In contrast, the CD4SP cells induced by differentiation of IL4KO cells consist only of CXCR3-positive CCR4-negative cells, which is consistent with the expression pattern of Th1 cells. In addition, the CD4SP cells induced by differentiation of TBX21KO cells consist only of CXCR3-negative cells, most of which are CCR4-positive. This is consistent with the expression pattern of Th2 cells.

[0214] [Example 3] Confirmation of IL-4 and IFN-γ secretion by obtained Th1 cells and Th2 cells

[0215] Next, the cytokine production capacity of CD4SP cells induced by differentiation from IL4KO cells and CD4SP cells induced by differentiation from TBX21KO cells was evaluated by intracellular staining flow cytometry using anti-IFN-γ antibody (manufactured by BD, clone: ​​B27) and anti-IL-4 antibody (manufactured by Biolegend, clone: ​​8D4-8). The cells were stimulated with PMA (40 ng / mL) and ionomycin (4 μg / mL) for 3 hours with the addition of monensin (2 μM). Figure 6As shown, among the CD4SP cells derived from wild-type iPS cells, 55.2% produced IL-4 and 5.67% produced IFN-γ. 26.6% showed the production of both. In contrast, among the CD4SP cells differentiated and induced by IL4KO cells, 89.3% showed the secretion of only IFN-γ. This is consistent with the nature of Th1 cells. In addition, among the CD4SP cells derived from TBX21KO cells, 35.7% showed the secretion of only IL-4, which is consistent with the nature of Th2 cells. On the other hand, the cells that showed the secretion of only IFN-γ were 0.34%, the cells that showed the secretion of both were 0%, and the cells that showed the secretion of IFN-γ almost disappeared. Therefore, it was confirmed that Th1 cells that showed the secretion of only IFN-γ can be obtained by IL4KO, and Th2 cells that showed the secretion of only IL-4 can be obtained by TBX21KO.

[0216] For CD4SP cells derived from IL4KO cells, there is a possibility that the disappearance of IL-4 production was observed only because the gene of cytokine IL-4, which is the final product of Th2 cells, was KO. However, as seen in Example 2, the chemokine receptor expression pattern also changed to the chemokine receptor expression pattern of CXCR3-positive CCR4-negative Th1 cells due to IL4KO. In addition, the production of IL-13, which is considered to be a representative functional cytokine of Th2 cells together with IL-4, was verified by intracellular staining flow cytometry using an anti-IL-13 antibody (manufactured by Biolegend, clone: ​​JES10-5A2). The results are as follows Figure 7 As shown, CD4SP cells derived from wild-type iPS cells contain components that produce more IL-13 than primary CD4SP cells, while components that secrete high levels of IL-13 disappear in CD4SP cells derived from multiple IL4KO cell lines. The reduction in IL-13 secretion by cells whose genes are not directly knocked out by IL4KO can be considered as one of the evidences that IL4KO hinders the phenotypic expression of Th2 cells.

Claims

1. A method for preparing Th1 or Th2 CD4 single positive T cells, comprising the following steps: Inducing hematopoietic stem cells (HSC) and / or hematopoietic progenitor cells (HPC) substantially deficient in the following factors into CD4 single positive T cells: A factor involved in the secretion of IL-4, which is a gene involved in the secretion of IL-4 or a protein encoded by the gene involved in the secretion of IL-4, wherein: The gene involved in the secretion of IL-4 is selected from the group consisting of IL4, IL-4R, Stat6, GATA3, mel-18, IL-5 and IL-13 genes; or A factor involved in the secretion of IFN-γ, which is a gene involved in the secretion of IFN-γ or a protein encoded by the gene involved in the secretion of IFN-γ, wherein the gene involved in the secretion of IFN-γ is selected from the group consisting of TBX21, IL-12, IFN-γ, IL-12Rβ2, IFNγR, Stat4, T-bet and HLX genes.

2. The method according to claim 1, which is a method for preparing Th1 type CD4 single positive T cells, comprising the following steps: (B1) Hematopoietic stem cells (HSC) and / or hematopoietic progenitor cells (HPC) substantially deficient in factors involved in IL-4 secretion are induced to become CD4 single-positive T cells.

3. The method according to claim 1, which is a method for preparing Th2 type CD4 single positive T cells, comprising the following steps: (B2) Hematopoietic stem cells (HSC) and / or hematopoietic progenitor cells (HPC) substantially deficient in factors involved in the secretion of IFN-γ are induced into CD4 single-positive T cells.

4. The method of claim 1, wherein: The HSC and / or HPC are (b1) HSC and / or HPC in which the gene involved in the secretion of IL-4 is knocked out but the gene involved in the secretion of IFN-γ is not knocked out.

5. The method of claim 1, wherein: The HSC and / or HPC are (b2) HSC and / or HPC in which the gene involved in the secretion of IFN-γ is knocked out but the gene involved in the secretion of IL-4 is not knocked out.

6. The method according to claim 1, which is a method for preparing Th1 type CD4 single positive T cells, comprising the following steps: Artificial thymic organoids (ATO) are cultured to generate Th1 type CD4 single positive T cells that do not secrete IL-4 but secrete IFN-γ from the ATO, wherein the ATO comprises: (a) stromal cells expressing Notch ligands; and (b1) Hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) in which a gene involved in IL-4 secretion is knocked out but a gene involved in IFN-γ secretion is not knocked out.

7. The method according to claim 1, which is a method for preparing Th2 type CD4 single positive T cells, comprising the following steps: Artificial thymic organoids (ATO) are cultured to generate IFN-γ non-secreting and IL-4-secreting Th2-type CD4 single-positive T cells from the ATO, wherein the ATO comprises: (a) stromal cells expressing Notch ligands; and (b2) Hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) in which a gene involved in the secretion of IFN-γ is knocked out but a gene involved in the secretion of IL-4 is not knocked out.

8. The method of claim 1, wherein: The hematopoietic stem cells (HSC) and / or hematopoietic progenitor cells (HPC) are cells induced by differentiation of iPS cells (iPSC).

9. The method of claim 8, wherein: The iPSCs are iPS cells derived from T cells (T-iPSCs).

10. The method of claim 9, wherein: The T cells are CD4 single positive T cells.

11. Th1 type CD4 single positive T cells, among which, A factor involved in the secretion of IL-4 is substantially defective, wherein the factor involved in the secretion of IL-4 is a gene involved in the secretion of IL-4 or a protein encoded by the gene involved in the secretion of IL-4, wherein the gene involved in the secretion of IL-4 is selected from the group consisting of IL4, IL-4R, Stat6, GATA3, mel-18, IL-5 and IL-13 genes, Wherein said substantial defect is carried out by: (i) modifying the gene involved in the secretion of IL-4 in such a way that the protein encoded by the gene involved in the secretion of IL-4 is not expressed; (ii) a functional defect in the protein encoded by the gene involved in the secretion of IL-4; or (iii) The expression of the protein encoded by the gene involved in the secretion of IL-4 is reduced.

12. Th2 type CD4 single positive T cells, among which, A factor involved in the secretion of IFN-γ is substantially defective, wherein the factor involved in the secretion of IFN-γ is a gene involved in the secretion of IFN-γ or a protein encoded by the gene involved in the secretion of IFN-γ, wherein the gene involved in the secretion of IFN-γ is selected from the group consisting of TBX21, IL-12, IFN-γ, IL-12Rβ2, IFNγR, Stat4, T-bet and HLX genes, Wherein said substantial defect is carried out by: (i) modifying the gene involved in the secretion of IFN-γ in such a way that the protein encoded by the gene involved in the secretion of IFN-γ is not expressed; (ii) a functional defect in the protein encoded by the gene involved in the secretion of IFN-γ; or (iii) The expression of the protein encoded by the gene involved in the secretion of IFN-γ is reduced.

13. The CD4 single-positive T cell according to claim 11 or 12, wherein The Th1 type CD4 single positive T cells or the Th2 type CD4 single positive T cells are cells induced by differentiation from iPS cells (iPSCs).

14. The CD4 single positive T cell according to claim 13, wherein The iPSCs are iPS cells derived from T cells (T-iPSCs).

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