How T cells are produced

Abstract

The present invention provides an excellent method for producing an IL-4-non-secreting and IFN-γ-secreting (Th1-type) or IFN-γ-non-secreting and IL-4-secreting (Th2-type) CD4 single-positive T cell (CD4SP T cell). The method for producing a Th1-type or Th2-type CD4SP T cell according to the present invention comprises a step of inducing a CD4 single-positive T cell from a hematopoietic stem cell (HSC) and / or a hematopoietic progenitor cell (HPC) in each of which a factor associated with the secretion of IL-4 or a factor associated with the secretion of IFN-γ is substantially damaged.

Description

[Technical Field]

[0001] The present invention relates to a method for producing IL-4 non-secreting, IFN-γ-secreting (Th1 type) and IFN-γ non-secreting, IL-4-secreting (Th2 type) CD4 single-positive T cells similar to natural Th1 cells and Th2 cells, respectively, for example, a method for producing them using artificial thymic organoids (ATO) containing hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) differentiated from iPS cells.The present invention also relates to a method for controlling the differentiation of hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) into Th1 type and / or Th2 type CD4 single-positive T cells.

[0002] [Background of the invention] Active research is currently being conducted into the differentiation of cytotoxic T cells, i.e., CD8 single-positive T cells (hereinafter referred to as "CD8SP T cells") from iPS cells, transduced with antigen-specific TCRs (T cell receptors) or CARs (chimeric antigen receptors), and their application in tumor immunotherapy. While development of such tumor immunotherapy has focused on the superior antitumor activity of cytotoxic T cells, the lack of technology to induce helper T cells, i.e., CD4 single-positive T cells (hereinafter referred to as "CD4SP T cells") from iPS cells, is a key factor behind this. If helper T cells could be induced, they could enhance both cellular and humoral immunity, thereby enhancing the efficacy of immunotherapy. For example, in CAR-T therapy, administering CD4SP T cells together with CAR-transduced CD8SP T cells has been shown to enhance therapeutic efficacy. Furthermore, if we could specifically produce or amplify Th1 and Th2 cells, which are subsets of helper T cells, it would be possible to apply them to applications other than tumor immunotherapy, such as modulating the Th1 / 2 balance, which is believed to be the underlying pathology of allergic diseases. Furthermore, if we could induce them to become inhibitory T cells (Treg cells), it would be possible to treat allergies and autoimmune diseases by suppressing cellular and humoral immunity. The ability to generate Th1, Th2, and Treg cells from iPS cells is important for improving the effectiveness and expanding the variety of immune cell therapy.

[0003] A known method for producing CD8SP T cells and / or CD4SP T cells includes culturing three-dimensional cell aggregates containing stromal cells expressing Notch ligands and hematopoietic progenitor cells, i.e., artificial thymic organoids (ATOs), as described in Patent Document 1. The Examples in Patent Document 1 disclose, as a more specific embodiment of the method, the production of ATOs using (primary) human hematopoietic stem / progenitor cells (HSPCs) isolated from umbilical cord blood or other sources, the generation of CD8SP T cells, CD4SP T cells, and CD4·CD8 double-positive T cells (hereinafter sometimes referred to as "CD4·CD8DP T cells") from the ATOs, and the generation of extremely low numbers of cells expressing only IFN-γ and cells expressing only IL-4 by treating CD4SP T cells with PMA / ionomycin (see, e.g., Figures 4B and 6B of Patent Document 1).

[0004] Non-patent document 1 describes a method for the production of hematopoietic stem / progenitor cells (CD34) collected from umbilical cord blood, bone marrow, peripheral blood, etc. + CD3 - They reported that ATOs were generated by co-culturing CD4+CD8DP T cells with stromal cells (MS5-hDLL1 cell line) that express Notch ligands, and that these ATOs produced CD8SP T cells and CD4SP T cells in addition to CD4+CD8DP T cells (Figures 1b, 2b, 3b, 4a, 4b, 4f).

[0005] Non-Patent Document 2 reports that embryonic mesodermal progenitors (EMPs) differentiated from human iPS cells were co-cultured with stromal cells (MS5-hDLL1 / 4 cell line) that express Notch ligands to produce embryonic mesodermal organoids (EMOs), which were then further induced to develop hematopoietic systems to produce ATOs. From these ATOs, CD4·CD8DP T cells as well as CD8SP (CD8αβ + ) T cells and a small number of CD4SP (CD4+ (Figure 2C), and that treatment of CD8SP T cells with PMA / ionomycin resulted in cells expressing IFN-γ and / or IL-2 (Figure 3F). Furthermore, when the human iPS cells were generated from fibroblasts, mature naive T cells with a diverse repertoire of TCRs were obtained, whereas when iPS cells were modified to express a TCR specific for a tumor-associated antigen (NY-ESO-1 peptide), T cells expressing such a TCR were obtained.

[0006] However, none of the above three publications describes a method for efficiently generating IFN-γ-secreting (Th1 type) CD4SP T cells and IL-4-secreting (Th2 type) CD4SP T cells, nor a method for controlling the differentiation of hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) 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, as well as transcription factors involved in Th2 differentiation (IL-4 gene expression) and their signal transduction. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2017 / 075389 [Non-patent literature]

[0009] [Non-Patent Document 1] Seet et al., Nature Methods vol.14 no.5, p521-530 (2017) [Non-patent document 2] Montel-Hagen et al., Cell Stem Cell. 2019 Mar 7;24(3):376-389 [Non-patent document 3] Usui., Jpn.J.Clin.Immunol., 30(6), p419-427 (2007) Summary of the Invention [Problem to be solved by the invention]

[0010] As mentioned above, despite the demand for the generation of IFN-γ-secreting (Th1 type) CD4SP T cells and IL-4-secreting (Th2 type) CD4SP T cells from iPS cells, a means to achieve this has not yet been achieved. In particular, in order to generate ATO for producing the above-mentioned CD4SP T cells, it would be extremely useful to be able to use hematopoietic stem / progenitor cells induced to differentiate from iPS cells prepared from cells collected from patients, established iPS cell lines, or iPS cells modified from these, rather than primary hematopoietic stem / progenitor cells, in terms of applications in immune cell therapy and production efficiency.

[0011] Primary T cells collected from the living body (peripheral blood), rather than T cells produced from ATO, have the specificity to secrete either IFN-γ or IL-4 when stimulated with PMA / ionomycin, so Th1 or Th2 cells can be obtained using primary T cells. However, when ATO is produced using hematopoietic progenitor cells (HPCs) induced from iPS cells, the T cells generated from the ATO lack sufficient specificity for the secretion of IFN-γ and IL-4 when stimulated with PMA / ionomycin, making it difficult to selectively obtain Th1- or Th2-type CD4SP T cells that secrete either one of them.

[0012] An objective of the present invention is to provide an excellent method for producing IL-4 non-secreting, IFN-γ-secreting (Th1 type) or IFN-γ non-secreting, IL-4-secreting (Th2 type) CD4 single positive T cells (CD4SP T cells).Another objective of the present invention is to provide a method for controlling differentiation into Th1 type or Th2 type CD4 single positive T cells. [Means for solving the problem]

[0013] The present inventors have found that Th1-type CD4SP T cells and Th2-type CD4SP T cells can be efficiently produced by culturing hematopoietic progenitor cells or hematopoietic stem cells in which factors involved in IL-4 secretion or factors involved in IFN-γ secretion are substantially impaired, for example, by producing and culturing ATO using hematopoietic progenitor cells obtained from iPS cells in which the IL4 gene or the TBX21 gene has been knocked out, respectively.

[0014] Furthermore, because high levels of IL-4 secretion and T-bet expression were observed during T cell redifferentiation from wild-type iPS cells, it was thought that knocking out the IL4 and TBX21 genes that encode these genes might inhibit differentiation into mature helper T cells. However, knocking out the IL4 and TBX21 genes did not result in such inhibition, and it was surprising to find that Th1- and Th2-type CD4SP T cells could be generated.

[0015] That is, the present invention includes at least the following items. [1] Factors involved in the secretion of IL-4, or Factors involved in IFN-γ secretion Inducing substantially impaired hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) into CD4 single-positive T cells, A method for producing CD4 single-positive T cells of the Th1 or Th2 type. [2] A method for producing Th1 type CD4 single positive T cells, comprising: (B1) inducing hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) in which factors related to IL-4 secretion are substantially impaired into CD4 single-positive T cells; [1] The method described in [1]. [3] A method for producing Th2 type CD4 single positive T cells, comprising: (B2) inducing hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) in which factors related to IFN-γ secretion are substantially impaired into CD4 single-positive T cells; [1] The method described in [1]. [4] The method according to [1], wherein the HSCs and / or HPCs are (b1) HSCs and / or HPCs in which a gene involved in IL-4 secretion has been knocked out. [5] The method according to [1], wherein the HSCs and / or HPCs are (b2) HSCs and / or HPCs in which a gene involved in IFN-γ secretion has been knocked out. [6] A method for producing Th1 type CD4 single positive T cells, comprising: (a) stromal cells expressing Notch ligands; (b1) Hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) in which genes involved in IL-4 secretion have been knocked out, but genes involved in IFN-γ secretion have not been knocked out. By culturing artificial thymus organoids (ATOs) containing producing IL-4 non-secreting, IFN-γ secreting (Th1 type) CD4 single-positive T cells from the ATO; [1] The method described in [1]. [6-1] (a) stromal cells expressing Notch ligands; (b1) Hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) in which the genes involved in IL-4 secretion have been knocked out, but the genes involved in IFN-γ secretion have not. By culturing artificial thymus organoids (ATOs) containing producing IL-4 non-secreting, IFN-γ secreting (Th1 type) CD4 single-positive T cells from the ATO; A method for producing Th1 type CD4 single positive T cells. [6a] The method according to [6] or [6-1], wherein the gene involved in IL-4 secretion is the IL4 gene. [7] A method for producing Th2 type CD4 single positive T cells, comprising: (a) stromal cells expressing Notch ligands; (b2) Hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) in which genes involved in the secretion of IFN-γ, but not IL-4, have been knocked out. By culturing artificial thymus organoids (ATOs) containing producing IFN-γ non-secreting, IL-4 secreting (Th2 type) CD4 single-positive T cells from the ATO; [1] The method described in [1]. [7-1] (a) stromal cells expressing Notch ligands; (b2) Hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) in which genes involved in IFN-γ secretion have been knocked out, but genes involved in IL-4 secretion have not been knocked out. By culturing artificial thymus organoids (ATOs) containing producing IFN-γ non-secreting, IL-4 secreting (Th2 type) CD4 single-positive T cells from the ATO; A method for producing Th2 type CD4 single positive T cells. [7a] The method according to [7] or [7-1], wherein the gene involved in the secretion of IFN-γ is the TBX21 gene. [8] The method according to [1], wherein the hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) are cells induced to differentiate from iPS cells (iPSCs). [8-1] The method according to [6-1] or [7-1], wherein the hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) of (b1) or (b2) are cells induced to differentiate from iPS cells (iPSCs). [9] The method according to [8] or [8-1], wherein the iPSCs are T-cell-derived iPS cells (T-iPSCs).

[10] The method according to [9], wherein the T cells are CD4 single-positive T cells.

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

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

[13] CD4 single-positive T cells of Th1 type with substantial impairment of factors involved in IL-4 secretion.

[14] CD4 single-positive T cells of the Th2 type that were substantially impaired in factors involved in the secretion of IFN-γ.

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

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

[17] genes involved in the secretion of IL-4, or Genes involved in IFN-γ secretion A method for producing CD4 single-positive T cells of Th1 type or Th2 type, comprising the step of inducing knockout iPS cells into CD4 single-positive T cells.

[18] Hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) that are substantially impaired in factors involved in the secretion of IL-4 or factors involved in the secretion of IFN-γ. [18a]

[18] Hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) according to

[18] , in which factors involved in the secretion of IL-4 are substantially impaired. [18b] Hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) according to

[18] , in which factors involved in the secretion of IFN-γ are substantially impaired.

[19] A method for controlling differentiation of hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) into Th1 or Th2 type CD4 single-positive T cells, comprising a step of substantially impairing factors involved in IL-4 secretion or factors involved in IFN-γ secretion in hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs).

[20] A pharmaceutical composition comprising the CD4 single-positive T cells according to

[11] ,

[12] ,

[13] or

[14] . [twenty one] Th1 type CD4 single positive T cells obtained by the method described in [6-1]. [twenty two] Th2 type CD4 single positive T cells obtained by the method described in [7-1]. [Effects of the Invention]

[0016] The present invention makes it possible to efficiently produce Th1-type CD4SP T cells and Th2-type CD4SP T cells, which will greatly contribute to the practical application and application of immune cell therapy that utilizes helper T cells. [Brief explanation of the drawings]

[0017] [Figure 1]Figure 1 shows the signaling pathways involved in Th1 and Th2 differentiation and the proteins (T-bet and IL-4) targeted for substantial disruption by knockout or other methods in the present invention. Figure 1 is a partial modification of Figure 3 in Usui T. (2007). Jpn. J. Clin. Immunol., 30(6), 419-427. [Figure 2] FIG. 2 shows the sequencing results of TBX21KO 654 (top) and IL4KO 656 (bottom) in Example 1. [Figure 3] FIG. 3 shows the culture conditions (medium components, oxygen concentration) when iPS cell lines confirmed to be homozygous mutants of TBX21 and IL4, respectively (IL4KO and TBX21KO iPS cell lines) in Example 2 were differentiated into hematopoietic progenitor cells (HPCs) by the Embryonic Body method (EB method). [Figure 4] Figure 4 shows the markers expressed in cells obtained from ATOs prepared using HPCs derived from an IL4 gene knockout iPS cell line (IL4KO, hereinafter the same) and a TBX21 gene knockout iPS cell line (TBX21KO, hereinafter the same), as well as from peripheral blood mononuclear cells (PBMCs, hereinafter also referred to as PBMCs), and a wild-type iPS cell line in which a specific gene has not been knocked out (Wild, hereinafter also referred to as Wild-type) in Example 2. Like "PBMCs" and "Wild," "IL4KO" and "TBX21KO" also contain cells showing the "CD4+CD8β-" and "CD4+CD8α-" expression patterns (within the boxes adjacent to each label), i.e., CD4SP T cells. [Figure 5] FIG. 5 shows the chemokine receptor expression patterns of CD4SP T cells obtained from ATOs prepared using HPCs derived from IL4KO, TBX21KO, PBMCs, and wild-type mice in Example 2. [Figure 6]FIG. 6 shows the cytokine secretion patterns of CD4SP T cells obtained from ATOs prepared using HPCs derived from IL4KO, TBX21KO, PBMCs, and wild-type mice in Example 3. [Figure 7] Figure 7 shows IL-13 production by various cells in Example 3. Left: Comparison of CD4SP T cells derived from TKT3V1-7 wild-type cells (TKT-Wild-CD4SPs) and primary CD4SP T cells (primary CD4SPs). Right: Comparison of CD4SP T cells derived from TKT3V1-7 wild-type cells (TKT-Wild-CD4SPs) and CD4SP T cells derived from TKT3V1-7 IL4KO cells (TKT IL4KO641 / 656-CD4SPs). Vertical axis: cell number, horizontal axis: fluorescence intensity. DETAILED DESCRIPTION OF THE INVENTION

[0018] In the following description, "(B1) hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) in which factors related to the secretion of IL-4 have been substantially impaired" will be referred to as "HSPC(B1)", "(B2) hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) in which factors related to the secretion of IFN-γ have been substantially impaired" will be referred to as "HSPC(B2)", "(a) stromal cells expressing a Notch ligand" will be referred to as "stromal cells(a)", and "(b1) hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) in which factors related to the secretion of IL-4 have been substantially impaired" will be referred to as "HSPC(B1)". "Hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) in which genes involved in IFN-γ secretion have been knocked out but genes involved in IFN-γ secretion have not been knocked out" are sometimes abbreviated as "HSPC(b1)," and "(b2) hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) in which genes involved in IFN-γ secretion have been knocked out but genes involved in IL-4 secretion have not been knocked out" are sometimes abbreviated as "HSPC(b2)." HSPC(b1) and HSPC(b2) may be HSCs and HPCs in which genes involved in IL-4 secretion have been knocked out but genes involved in IFN-γ secretion have not been knocked out, respectively, and HSCs and HPCs in which genes involved in IFN-γ secretion have been knocked out but genes involved in IL-4 secretion have not been knocked out. Furthermore, "hematopoietic stem cells and / or hematopoietic progenitor cells" (a collective term for hematopoietic stem cells and hematopoietic progenitor cells) are sometimes abbreviated as "HSPC."

[0019] In the following description, "IL-4 non-secreting, IFN-γ-secreting (Th1 type) CD4 single-positive T cells" may be abbreviated as "Th1 type CD4SP T cells," and "IFN-γ non-secreting, IL-4-secreting (Th2 type) CD4 single-positive T cells" may be abbreviated as "Th1 type CD4SP T cells," and "IFN-γ non-secreting, IL-4-secreting (Th2 type) CD4 single-positive T cells" may be abbreviated as "Th2 type CD4SP T cells." Th1 type CD4SP T cells obtained by the method for producing Th1 type CD4SP T cells of the present invention may be simply referred to as "Th1 type CD4SP T cells of the present invention," and Th2 type CD4SP T cells obtained by the method for producing Th2 type CD4SP T cells of the present invention may be simply 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 in which factors related to IL-4 secretion are substantially impaired, and Th2 type CD4 single-positive T cells in which factors related to IFN-γ secretion are substantially impaired, respectively, and their production methods are not particularly limited. Furthermore, "CD4 single positive T cells" may be abbreviated as "CD4SP cells." In the examples, "CD4SP cells" correspond to CD4 single positive T cells.

[0020] As used herein, "hematopoietic stem cells" (HSCs) are multipotent stem cells that can differentiate into blood cells (white blood cells (neutrophils, eosinophils, basophils, lymphocytes, monocytes, macrophages), red blood cells, platelets, mast cells, and dendritic cells). Furthermore, as used herein, "hematopoietic progenitor cells" (HPCs) are cells that have the ability to differentiate into blood cells but do not have the self-renewal ability of stem cells. In humans, HSCs and HPCs are primarily present in the bone marrow, but are also present in peripheral blood and umbilical cord blood, and can be collected from these sites. In the present invention, hematopoietic stem cells may be cells isolated from biological tissues such as bone marrow, blood, or the like, or may be cells prepared from ES cells or iPS cells. Both hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) are CD34-positive and CD3-negative cells (CD34+CD3-cells). A cell can be confirmed to be a hematopoietic stem cell if it is transplanted into an animal, survives, is extracted, and then transplanted into another individual, and also has the ability to self-renew; in other words, it is a "stem cell" rather than a "progenitor cell."

[0021] As used herein, "induced pluripotent stem cells" (iPSCs) refer to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of "induced pluripotent stem cells," including iPS cells established by Yamanaka et al. by introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676); human-derived iPS 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 selecting cells using Nanog expression as an indicator after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317); and iPS cells created using a method that does not include c-Myc (Nakagawa M, Yamanaka S., et al. al. Nature Biotechnology, (2008) 26, 101-106), and iPS cells established by introducing six 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.) can also be used. Other examples that can be used include induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, as developed by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920), induced pluripotent stem cells developed by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), and induced pluripotent stem cells developed by Sakurada et al. (JP Patent Publication No. 2008-307007).In addition, all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol. 3, 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. 7, Any of the induced pluripotent stem cells known in the art and described in the literature (e.g., JP 2008-307007 A, JP 2008-283972 A, US 2008-2336610 A, US 2009-047263 A, WO 2007-069666 A, WO 2008-118220 A, WO 2008-124133 A, WO 2008-151058 A, WO 2009-006930 A, WO 2009-006997 A, WO 2009-007852 A) can be used. Various iPS cell lines established by the NIH, RIKEN, Kyoto University, etc. can be used as induced pluripotent cell lines. Examples of human iPS cell lines include RIKEN's HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, and Nips-B2 strain, and Kyoto University's 253G1 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, and 648A1 strain. Note that, in the present invention, T cell-derived iPS cell lines, as described below, can also be used.

[0022] As used herein, the term "marker" refers to a "marker protein" or a "marker gene," which refers to a protein or its gene that is specifically expressed on the cell surface, in the cytoplasm, and / or in the nucleus, etc., of a given cell type. A marker may be a positive selection marker or a negative selection marker. Preferably, the marker is a cell surface marker, and in particular, a cell surface positive selection marker enables enrichment, isolation, and / or detection of viable cells.

[0023] Marker proteins can be detected by immunological assays using antibodies specific to the marker protein, such as ELISA, immunostaining, and flow cytometry. Examples of antibodies specific to the marker protein include antibodies that bind to specific amino acid sequences in the marker protein or specific sugar chains bound to the marker protein. Furthermore, for marker proteins (e.g., transcription factors or their subunits, cytokines, etc.) that are expressed intracellularly but not on the cell surface (on the cell membrane) or that are secreted from cells, methods can be used in which cells are fixed and then fluorescently stained intracellularly using antibodies specific to the marker protein, or a reporter protein is expressed together with the marker protein. This method is preferably used when no suitable cell surface marker is identified. Marker genes can be detected using nucleic acid amplification and / or nucleic acid detection methods known in the art, such as RT-PCR (including quantitative PCR), microarrays, biochips, and RNA sequencing.

[0024] As used herein, a marker or the like being "positive" means that the expression level of a protein or gene of the marker or the like (a measurement value or signal reflecting the expression level) exceeds (or is equal to or greater than) the detectable level by a method known in the art, such as those described above, or exceeds a predetermined reference value. As used herein, a marker or the like being "negative" means that the expression level of a protein or gene of the marker or the like is less than (or equal to or less than) the detectable level by all or any of the methods known in the art, such as those described above, or is equal to or less than a predetermined reference value. The detectable level and reference value for protein or gene expression may vary depending on the method employed and the purpose of the analysis. For example, the expression level (secretion level) of a marker protein can be determined as "positive" if the fluorescent signal obtained by flow cytometry (typically, fluorescence-activated cell sorting: FACS), in which cells are stained with a fluorescently labeled antibody, is higher than (or equal to) a predetermined reference value established based on the fluorescent signal of unstained cells, and as "negative" if the fluorescent signal is lower than (or equal to) a predetermined reference value established based on the fluorescent signal of unstained cells. Positive expression may be indicated by a "+" symbol, and negative expression by a "-" symbol (e.g., "CD4+CD8-" is synonymous with "CD4 positive CD8 negative").

[0025] "IFN-γ" refers to "interferon gamma" (a protein produced mainly by Th1 cells stimulated by antigens and known to have various functions such as enhancing antiviral activity, antitumor activity, and regulating immune responses). "IL-4" refers to "interleukin 4" (a protein produced by naive CD4 cells stimulated by antigens). +"CXCR3" refers to a shared receptor specific for the chemokines CXCL9 (also known as MIG: monokine-induced by interferon gamma) and CXCL10 (also known as IP-10; interferon-inducible protein 10) that is selectively expressed on Th1 cells. "CCR4" refers to a shared receptor specific for the chemokines CCL17 (also known as TARC: thymus and activation-regulated chemokine) and CCL22 (also known as MDC: macrophage-derived chemokine) that is selectively expressed on Th2 cells.

[0026] In the present invention, "non-secreting IL-4 and secreting IFN-γ" means that the expression of proteins present in the cells, as measured by, for example, flow cytometry (FACS, etc.), is "negative" for IL-4 and "positive" for IFN-γ. "non-secreting IFN-γ and secreting IL-4" means that the expression of proteins present in the cells, as measured by, for example, flow cytometry (FACS, etc.), is "positive" for IL-4 and "negative" for IFN-γ.

[0027] In this specification, unless otherwise stated, "secreted" of a protein is synonymous with "positive" and "non-secreted" of a protein is synonymous with "negative".

[0028] As used herein, the term "stromal cells" refers to cells that constitute connective tissue that supports parenchymal cells in biological tissues, and in particular to stromal cells that can produce ATO that produces T cells, typically stromal cells contained in hematopoietic tissues such as bone marrow and thymus. Stromal cells may be established cell lines such as mouse bone marrow cell lines MS-5, OP9, and S17, or human stromal cell lines HS-5 and HS-27a, or may be primary cells collected from humans or the like, or cells induced to differentiate from pluripotent stem cells (such as iPS cells) from humans or the like.

[0029] -Manufacturing method- The method for producing 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 step of inducing hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs) in which factors related to IL-4 secretion or factors related to IFN-γ secretion are substantially impaired to become CD4 single-positive T cells (CD4S T cells). Specifically, the method for producing Th1-type CD4SP T cells of the present invention comprises the step of inducing HSPCs (B1) in which factors related to IL-4 secretion are substantially impaired (while factors related to IFN-γ secretion are substantially intact) to become CD4SP T cells. The method for producing Th2-type CD4SP T cells of the present invention comprises the step of inducing HSPCs (B2) in which factors related to IFN-γ secretion are substantially impaired (while factors related to IL-4 secretion are substantially intact) to become CD4SP T cells.

[0030] The term "factors involved in IL-4 secretion" refers to genes involved in IL-4 secretion or the proteins encoded by those genes. The "genes involved in IL-4 secretion" are substantially disrupted to generate HSPC(B1) but are not substantially disrupted to generate HSPC(B2). The "genes involved in IL-4 secretion" are typically the "IL4 gene," but may be other genes as long as their substantial disruption suppresses IL-4 secretion, does not interfere with the production of Th1 CD4SP T cells, and does not suppress IFN-γ secretion. More specifically, "genes involved in IL-4 secretion" can be selected from the genes encoding the proteins shown in the right panel of Figure 1 (Th2 signals), i.e., IL-4, IL-4R, Stat6, GATA3, mel-18, IL-5, and IL-13 (or at least a portion of the protein complex). For example, the GATA3 gene, known as a master regulator of Th2-type CD4SP T cells, and the IL4Ra (CD124) gene can also be used as "genes involved in IL-4 secretion" if their substantial disruption does not prevent differentiation into T cells and suppresses IL-4 expression. HSPC(B1) can also be produced by substantially disrupting the proteins encoded by these genes instead of the "genes involved in IL-4 secretion" described above.

[0031] "Factors involved in IFN-γ secretion" refer to genes involved in IFN-γ secretion or the proteins encoded by those genes. "Genes involved in IFN-γ secretion" are substantially disrupted to generate HSPC(B2) but not substantially disrupted to generate HSPC(B1). "Genes involved in IFN-γ secretion" are typically the "TBX21 gene" encoding T-bet (see Figure 1). However, other genes may be used as long as their substantial disruption suppresses IFN-γ secretion, does not interfere with the production of Th2 CD4SP T cells, and does not suppress IL-4 secretion. More specifically, "genes involved in IFN-γ secretion" can be selected from the genes encoding the proteins shown in the left panel of Figure 1 (Th1 signals), namely, IL-12, IFN-γ, IL-12Rβ2, IFNγR, Stat4, T-bet, and HLX (or at least a portion of the protein complex, if present). For example, the IFN-γ gene and the IL-12Rβ2 gene can also be used as "genes involved in IFN-γ secretion" as long as their substantial disruption does not prevent differentiation into T cells. HSPC(B2) can also be produced by substantially disrupting the proteins encoded by these genes, instead of the "genes involved in IFN-γ secretion" described above.

[0032] Hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs) with a "substantially impaired" factor refer to HSPCs that do not undergo signal transduction equivalent to that occurring when the factor functions normally. Examples of such HSPCs include (i) HSPCs in which the gene has been modified to suppress expression of the protein, (ii) HSPCs in which the endogenous protein has been functionally impaired, and (iii) HSPCs in which expression of the endogenous protein has been reduced. These HSPCs exhibit the effects of the present invention, i.e., can be induced to become CD4SP T cells of interest when cultured under appropriate conditions. Examples of HSPCs in category (i) include HSPCs in which the gene has been knocked out and HSPCs in which at least one base has been added, inserted, substituted, and / or deleted in the gene. Examples of HSPCs in the category (ii) include HSPCs in which expression of a dominant-negative mutant (e.g., STAT6ΔC in STAT6 (FEBS letters 579 (2005) 3953-3959)) has been shown to dominate over endogenous proteins, thereby attenuating the normal function of the endogenous protein. Examples of HSPCs in the category (iii) include HSPCs containing shRNA and / or siRNA against the above-mentioned specific proteins. The success of generating HSPCs in which the above-mentioned specific factors are "substantially impaired" can also be confirmed by inducing the HSPCs to become CD4SP T cells and measuring the secretion of IL-4 or IFN-γ from the resulting CD4SP T cells. Furthermore, in the above category (iii), "decreased expression of a specific protein" refers to a decrease in the expression level of the specific protein in HSPCs. 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 (e.g., 96%, 97%, 98%, or 99%) compared to the expression level in HSPCs before treatment (e.g., introducing shRNA and / or siRNA against the above-mentioned (endogenous) protein into HSPCs).In general, various means (treatment methods, conditions, reagents, devices, etc.) for producing cells in which a certain gene or protein is "substantially impaired" are known. By selecting an appropriate means from among these or designing it to suit the present invention, a person skilled in the art can produce HSPCs in which signaling equivalent to that occurring when the specified factor (gene or protein) functions normally is not induced, i.e., HSPCs in which the specified factor is "substantially impaired," so as to achieve the effects of the present invention.

[0033] In view of the above, one aspect of the present invention provides HSPCs in which factors involved in IL-4 secretion or factors involved in IFN-γ secretion are substantially impaired (HSPCs in which factors involved in IL-4 secretion are substantially impaired, and HSPCs in which factors involved in IFN-γ secretion are substantially impaired, respectively). Furthermore, another aspect of the present invention provides a method for controlling differentiation of hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs) into Th1-type or Th2-type CD4 single-positive T cells, the method comprising the step of substantially impairing factors involved in IL-4 secretion or factors involved in IFN-γ secretion.

[0034] <Knockout> In a preferred embodiment of the present invention, the HSPCs (B1) substantially impaired in a factor involved in IL-4 secretion are HSPCs (b1) in which a gene involved in IL-4 secretion has been knocked out. In a preferred embodiment of the present invention, the HSPCs (B2) substantially impaired in a factor involved in IFN-γ secretion are HSPCs (b2) in which a gene involved in IFN-γ secretion has been knocked out.

[0035] HSPC(b1) and HSPC(b2) can be generated by knocking out specific genes in HSPCs or in the cells from which they are differentiated. For example, when inducing differentiation of HSPCs from iPSCs as described above, knockout treatment is preferably performed at the iPSC stage.

[0036] The means for knocking out a specific gene are not particularly limited, and various known means can be used. For example, the CRISPR / Cas system is one suitable means for knocking out a specific gene in the present invention. When the CRISPR / Cas system is applied to target a base sequence contained in a specific gene locus on genomic DNA, repeated double-strand breaks (DSBs) in DNA are generated. As a result, repair errors are induced in the non-homologous end joining (NHEJ) or homology-directed repair (HR) pathway, introducing mutations such as base deletions or insertions, resulting in frameshift knockout of the specific gene.

[0037] Various variations of the CRISPR / Cas system are known, including the representative CRISPR / Cas9 system, and these various systems can be used in the present invention. CRISPR / Cas systems include Class 1 (Type I, Type III, and Type IV) systems involving multiple RNA-guided nucleases (Cas) and Class 2 (Type II, Type V, and Type VI) systems involving a single Cas. Suitable CRISPR / Cas systems include, for example, the Class 2 Type II CRISPR / Cas9 system and the Class 2 Type V CRISPR-Cas12a / Cpf1 system.

[0038] The CRISPR / Cas system uses guide RNA (gRNA) and RNA-guided nucleases, and various variations of each are known, and various embodiments can be used in the present invention.

[0039] The gRNA is composed of a crRNA and a tracrRNA. The crRNA and tracrRNA may be separate RNA strands that form a complex, or they may be a single RNA strand (single guide RNA: sgRNA, or chimeric RNA) linked via a spacer. Those skilled in the art can appropriately design the base sequence (the base sequence adjacent to the PAM sequence) to be targeted to knock out a specific gene in HSPCs, i.e., the base sequence of the crRNA corresponding to that base sequence. The gRNA can be introduced into cells in the form of RNA or in the form of DNA (e.g., an expression plasmid) containing a base sequence that can produce the RNA by transcription. The gRNA in the form of RNA may consist only of natural nucleotides (adenine, guanine, cytosine, and uracil) or may contain natural nucleotides and nucleotide analogs. Examples of nucleotide analogs include sugar-modified nucleotides (2'-O-methylribose, 2'-O-propylribose, 2'-O-methoxyethoxyribose, 2'-O-methoxyethylribose, 2'-O-[2-(guanidinium)ethyl]ribose, 2'-O-fluororibose, etc.); bridged artificial nucleic acids (BNAs) (locked artificial nucleic acids (LNAs), ethylene-bridged artificial nucleic acids (ENAs), etc.); and phosphodiester-linked nucleotides (phosphodiester-linked nucleotides with phosphorothioate bonds, N3'-P5' phosphoamidate-linked nucleotides, 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 or three bases at each end) can be a nucleotide analog (preferably a sugar-modified nucleotide and a phosphodiester-linked nucleotide, more specifically, 2'-O-methylribose and a phosphodiester-linked nucleotide with phosphorothioate bonds).

[0040] The RNA-guided nuclease may be a wild-type nuclease in which both nuclease domains are active, or a mutant nuclease (nickase) in which only one of the nuclease domains is active. The RNA-guided nuclease can be introduced into cells in the form of a protein or in the form of a nucleic acid (e.g., mRNA or DNA such as an expression plasmid) containing a base sequence encoding the amino acid sequence of the protein.

[0041] The means for introducing the specific RNA and RNA-guided nuclease used in the CRISPR / Cas system into cells (e.g., into HSPCs in the present invention) is not particularly limited. In one embodiment of the present invention, the RNA strand of the gRNA and the RNA-guided nuclease protein (e.g., Cas9) can be introduced into cells by electroporation.

[0042] The various cells used in the present invention, i.e., HSPC(B1) and (b1), HSPC(B2) and (b2), and iPS cells for producing HSPC(B1) and HSPC(B2), as described below, and cells for producing the iPS cells (e.g., T cells), may each be derived from humans or non-human animals, such as mammals like mice, rats, dogs, pigs, and monkeys, and can be selected depending on the intended use of the Th1 CD4SP T cells or Th2 CD4SP T cells obtained by the present invention. For example, when the Th1 CD4SP T cells or Th2 CD4SP T cells are intended for administration to humans, the various cells described above are preferably derived from humans.

[0043] There are no particular limitations on how HSPCs for producing HSPCs (B1) (B2), etc., i.e., cells before the specific factors are substantially lost, are obtained or produced. For example, they may be produced by inducing differentiation from iPS cells, ES cells, or other cells capable of differentiating into HSPCs, or they may be HSCs or HPCs collected from umbilical cord blood, bone marrow, peripheral blood, or other living organisms.

[0044] <Inducing differentiation from iPSCs to HSPCs> In a preferred embodiment of the present invention, the HSPCs are cells differentiated from induced pluripotent stem cells (iPSCs). For example, iPSCs are prepared using cells (preferably T cells, more preferably CD4SP T cells) collected from a patient who is scheduled to receive administration of Th1-type CD4SP T cells and Th2-type CD4SP T cells obtained by the production method of the present invention, and by using ATO prepared using the iPSCs, Th1-type CD4SP T cells and Th2-type CD4SP T cells that are HLA-matched to the patient and do not cause immune rejection can be obtained. Also, universalized iPSCs (having a specific HLA type that does not cause immune rejection in a number of patients, or having HLA knocked out) may be used.

[0045] The basic matters and specific embodiments for inducing differentiation from iPSCs to HSPCs are known and are not particularly limited. Depending on the differentiation induction method employed, the number of culture days, etc., the medium composition, culture conditions, etc. can be appropriately adjusted and changed so as to increase the differentiation induction efficiency to HSPCs and the cell growth rate.

[0046] In a preferred embodiment of the present invention, the HSPCs prepared from iPSCs (preferably T-iPSCs described later, the same applies hereinafter) are prepared by a feeder-free method. For example, the "Embryonic body method (EB method)" described in AE Grigoriadis et al. (2010). Blood, 115(14):2769-2776., which forms embryonic bodies without using feeder cells to produce HPCs, is preferred.

[0047] Alternatively, as described in WO2011 / 096482 and WO2013 / 176197, iPSCs can be cultured on feeder cells to induce differentiation into HSPCs. The feeder cells are preferably stromal cells, which facilitate induction of differentiation into mesodermal lineages. The stromal cells are preferably irradiated OP9 cells, 10T1 / 2 cells (C3H10T1 / 2 cells), or the like, which facilitate induction of differentiation into hematopoietic lineages.

[0048] Examples of media for feeder-free methods (such as the EB method) include StemFit (registered trademark, Ajinomoto Co., Inc.) and StemPro (registered trademark, Thermo Fisher Scientific). Examples of media for co-culturing iPSCs and feeder cells include X-VIVO medium, Iscove's modified Dulbecco's medium (IMDM medium), α-MEM, and DMEM. However, IMDM medium is preferred from the viewpoint of high efficiency in forming sac-like structures (sacs) containing HSPCs.

[0049] Although the medium does not necessarily contain cytokines, it is preferable that it further contains cytokines and, if necessary, serum (e.g., fetal bovine serum (FBS)), insulin, transferrin, sodium selenite, L-glutamine (or GlutaMAX (Gibco)), α-monothioglycerol, ascorbic acid, etc. From the viewpoint of efficiently inducing iPSC differentiation into HSPCs, at least one cytokine selected from the group consisting of BMP, bFGF, VEGF, SCF, TPO, and FLT3L is preferred. In the EB method, for example, a combination of three cytokines (BMP4, bFGF, and VEGF), a combination of three cytokines (bFGF, VEGF, and SCF), or a combination of five cytokines (bFGF, VEGF, SCF, TPO, and FLT3L) is preferably used, changing the combination depending on the number of days of culture. In the co-culture method of iPSCs and feeder cells, for example, a combination of three cytokines (VEGF, SCF, and TPO), or a combination of three cytokines (VEGF, SCF, and FLT3L) is more preferred.

[0050] The culture period may be the period until a sufficient number of HSPCs are produced (in the co-culture method of iPSCs and feeder cells, a sac containing them is formed). For example, it is preferably 10 to 14 days from the start of culture. The culture environment is not particularly limited, but preferably, it is under the conditions of about 5% CO2 and about 37 °C. Also, from the viewpoint of increasing the production efficiency of HSPCs (the formation efficiency of the sac), it is more preferable to culture under conditions of a low oxygen concentration (for example, 5 to 20%).

[0051] The sac formed by the above-described differentiation induction usually contains HSPCs and other cells (such as blood cells). The cells present inside the sac can be separated and recovered from the sac by physical means, for example, by passing them through a sterilized sieve-like instrument (such as a cell strainer). Since HSPCs are cells that express CD34 as a cell surface marker, they can be isolated from the cell population obtained by culture by, for example, flow cytometry using a fluorescently labeled anti-CD34 antibody and a cell sorter. In a preferred embodiment of the present invention, the HSPC is a HPC.

[0052] <Production of iPSCs from T cells> In a preferred embodiment of the present invention, the iPSCs for inducing differentiation into HSPCs are iPSCs derived from T cells (T-iPSCs). The basic matters and specific embodiments for producing iPSCs from T cells are known. For example, reference can be made to WO2011 / 096482 and WO2013 / 176197. In addition, the basic matters and specific embodiments for producing iPSCs from other cells than T cells are also known.

[0053] "T cells" for generating iPSCs include cells expressing T cell receptors (TCRs), particularly the α and β chains of TCR (TCRαβ), on their surface, as well as their precursor cells, i.e., pro-T cells that do not express TCRαβ and pre-T cells in which TCRβ and pre-TCRα are associated. Furthermore, "T cells" also include T cells negative for both CD4 and CD8 (CD4·CD8 double-negative T cells), T cells positive for both CD4 and CD8 (CD4·CD8DP T cells), T cells positive for CD4 but negative for CD8 (CD4SP T cells), and T cells positive for CD8 but negative for CD4 (CD8SP T cells). The terms "positive" and "negative" are explained elsewhere in this specification. In the present invention, the "predetermined standard" (threshold) for determining whether CD4 is "positive" and CD8 is "negative," particularly for "CD4SP T cells" produced from ATO, can refer to the expression levels of CD4 and CD8 on natural helper T cells.

[0054] T cells used to produce iPSCs can be collected from tissues containing T cells, such as peripheral blood, lymph nodes, bone marrow, thymus, spleen, umbilical cord blood, and diseased tissue.

[0055] In a preferred embodiment of the present invention, the T cells used to generate iPSCs are CD4 single-positive T cells (CD4SP T cells). CD4SP T cells can be isolated from cell populations contained in tissues, for example, by flow cytometry using a fluorescently labeled anti-CD4 antibody and a cell sorter. iPS cell lines generated from CD4SP T cells have been established, and the TKT3V1-7 and b3a2 lines, for example, can be used to generate iPSCs in the present invention.

[0056] The collected T cells have antigen specificity, i.e., an antigen-specific TCR conferred by the rearranged TCR gene. The Th1 or Th2 CD4SP T cells of the present invention produced from ATOs generated using iPSCs can have the same or substantially the same antigen specificity (antigen-specific TCR) as that of the T cells used to generate the iPSCs. T cells with the desired antigen specificity can be isolated from tissues by, for example, a purification process using an affinity column immobilized with the desired antigen, or by a purification process using an MHC (major histocompatibility complex) multimer (e.g., "MHC tetramer" or "Pro5 (registered trademark) MHC class I pentamer") bound to the desired antigen.

[0057] In the production methods of the present invention, the means (culture method, conditions, apparatus, etc.) for inducing HSPCs in which factors related to IL-4 secretion or factors related to IFN-γ secretion are substantially impaired, i.e., HSPCs (B1) and (B2), into the desired CD4SP T cells, i.e., Th1 CD4SP T cells and Th2 CD4SP T cells, respectively, are not particularly limited. Those skilled in the art can select an appropriate means or design culture conditions (medium, duration, temperature, atmosphere, etc.) compatible with the present invention to culture the HSPCs and induce them into the CD4SP T cells. For example, HSPCs (B1) and (B2) can be induced into Th1 CD4SP T cells and Th2 CD4SP T cells by culturing them according to the "ATO method." As a representative example of an induction method (culture method), the ATO method, which targets HSPCs (b1) and (b2) in which a specific gene has been knocked out, is specifically described below. However, induction methods (culture methods) that can be used in the production method of the present invention are not limited to this, and HSPCs (B1) and (B2) other than HSPCs (b1) and (b2) can also be used. When practicing the present invention using an induction method (culture method) other than the ATO method, the following information regarding the ATO method can be referenced to the extent necessary (e.g., regarding the culture medium).

[0058] <ATO method> In a preferred embodiment, the method for producing Th1-type CD4SP T cells of the present invention comprises (a) stromal cells expressing a Notch ligand, and (b1) hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) in which genes related to the secretion of IL-4 are knocked out, but genes related to the secretion of IFN-γ are not knocked out, and culturing an artificial thymic organoid (ATO) containing the above to produce IL-4 non-secreting and IFN-γ secreting (Th1-type) CD4SP T cells from the ATO.

[0059] In a preferred embodiment, the method for producing Th2-type CD4SP T cells of the present invention comprises (a) stromal cells expressing a Notch ligand, and (b2) hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) in which genes related to the secretion of IFN-γ are knocked out, but genes related to the secretion of IL-4 are not knocked out, and culturing an artificial thymic organoid (ATO) containing the above to produce IFN-γ non-secreting and IL-4 secreting (Th2-type) CD4SP T cells from the ATO.

[0060] The ATO method of the present invention can be carried out essentially in the same manner as conventional methods, except that it uses HSPCs (B1) or (B2) in which a specific factor has been substantially impaired, preferably HSPCs (b1) or (b2) in which a specific gene has been knocked out. The ATO method can be produced and the resulting cells recovered in a manner similar to conventional methods. The basic principles and specific embodiments of the ATO method are publicly known (see, for example, Patent Document 1: WO2017 / 075389). The present invention may also be practiced in accordance with these methods, with modifications made as necessary. For example, the method described in Patent Document 1 initially forms embryonic mesodermal organoids on inserts to obtain cellular components corresponding to HPCs, and then ATOs are produced. However, this portion may be modified to induce differentiation of iPSCs into HPCs using the "embryonic body method (EB method)" (A.E. Grigoriadis et al. (2010). Blood, 115(14):2769-2776), and then the resulting HPCs can be used to produce ATOs. In the present invention, HSPCs used to generate ATO are replaced with HSPCs (b1) or (b2) in which a specific gene has been knocked out in advance. Furthermore, in the present invention, the cells to be collected from ATO are Th1-type CD4SP T cells and Th2-type CD4SP T cells.

[0061] The culture medium for ATO (stromal cells (a) and HSPCs (b1) and (b2)) can be selected from media used for culturing animal cells, and components can be added at appropriate concentrations as needed. The medium and added components can be changed over the course of culture.

[0062] Examples of media include AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, 199 Medium, Eagle's MEM, αMEM, DMEM, Ham, RPMI-1640, and Fisher's Medium. These media may be used alone or in combination. An example of a preferred medium is RPMI-1640.

[0063] The medium may be serum-containing, serum-free, or xeno-free. To prevent contamination with xenogeneic components, the serum may be derived from the same animal as the cells being cultured. Serum-free medium refers to a medium that does not contain raw or unpurified serum and therefore may include a medium with purified blood-derived components or animal tissue-derived components (e.g., growth factors). The medium may or may not contain any substitute for serum. Serum substitutes may include materials that appropriately contain albumin (e.g., albumin substitutes such as lipid-rich albumin, bovine albumin, recombinant albumin, or humanized albumin, plant starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3′-thioglycerol (α-monothioglycerol, MTG), or equivalents thereof. Commercially available materials, such as Knockout Serum Replacement (KSR), Chemically-defined Lipid Concentrated (Gibco), and GlutaMAX (Gibco), can also be used. The medium may be a serum-free medium (SFM) suitable for cell development. For example, the medium may contain B-27® supplement, Xeno-Free B-27® supplement, NS21 supplement, GS21™ supplement, or a combination thereof, at a concentration effective for producing T cells from 3D cell aggregates.

[0064] The medium may contain one or more components selected from the group consisting of biotin, DL-alpha-tocopherol acetate, DL-alpha-tocopherol, vitamins such as vitamin A (acetate), bovine serum albumin (BSA) or human albumin, fatty acid-free fraction V, catalase, human recombinant insulin, human transferrin, proteins such as superoxide dismutase, corticosterone, D-galactose, ethanolamine HCl, glutathione (reduced), L-carnitine HCl, linoleic acid, linolenic acid, progesterone, putrescine 2HCl, sodium selenite, and T3 (triiodo-L-thyronine); PSG (penicillin, streptomycin, and L-glutamine). The medium may also contain exogenously added ascorbic acid or a derivative thereof (e.g., ascorbic acid 2-phosphate: PAA). The medium may contain one or more substances selected from the group consisting of fatty acids or lipids, amino acids (such as non-essential amino acids), vitamins, growth factors, cytokines, antibiotics, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, and inorganic salts, each of which is added from the outside.

[0065] The medium may contain exogenously added cytokines. Examples of cytokines include 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-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleotrophin, and midkine. These cytokines may be used alone or in combination.

[0066] The 3D cell aggregates may or may not include exogenous extracellular matrices or scaffolds, such as collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin, as well as mixtures thereof, such as Matrigel™, and lysed cell membrane preparations.

[0067] Other culture conditions for ATO can be set appropriately. The culture temperature can be, for example, 20 to 40°C, preferably about 37°C. The CO2 concentration can be, for example, 2% to 10%, preferably 2 to 5%. The oxygen concentration can be, for example, 1 to 20%, preferably 5 to 20%.

[0068] Stromal cells (a) and HSPCs (b1) or HSPCs (b2) in ATO can be co-cultured at any ratio. For example, the stromal cell (a):HSPCs (b1) or HSPCs (b2) ratio can be 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, and preferably 20:1 to 1:4. When HSPCs (b1) or HSPCs (b2) are prepared from T cell-derived iPS cells, the ratio is preferably approximately 1 (e.g., MS5hDLL4):4 (e.g., HPCs), whereas when HSPCs are prepared from non-T cell-derived iPS cells, the ratio is preferably approximately 20 (e.g., MS5hDLL4):1 (e.g., HPCs).

[0069] The culture period for ATO can be, for example, 4 weeks or more, preferably 5 weeks or more, more preferably 6 weeks or more, and even more preferably 8 weeks or more or 9 weeks or more. The upper limit of the culture period is not particularly limited, but it is preferably 16 weeks or less, more preferably 14 weeks or less, and even more preferably 12 weeks or less. Specifically, the culture period can be, for example, 4 to 16 weeks, preferably 5 to 16 weeks, more preferably 6 to 14 weeks, and even more preferably 8 to 12 weeks or 9 to 12 weeks. The longer the culture period for ATO up to about 4 to 9 weeks, the higher the proportion of CD4SP T cells in the resulting cell population tends to be. However, beyond a certain period, the proportion of CD4SP T cells becomes difficult to increase, and there is a risk of a decrease in CCR7-positive cells (progression of differentiation toward effector cells). Therefore, the culture period for ATO can be adjusted within the above-mentioned range (for example, set to about 9 weeks) depending on the cell line and culture conditions, etc., to obtain a cell population with the desired composition.

[0070] In addition to CD4SP T cells, cells produced from ATO in the production method of the present invention may contain CD8SP T cells, CD4·CD8DN T cells, and CD4·CD8DP T cells. For example, CD4SP T cells can be isolated and recovered from the produced cells by flow cytometry (typically, fluorescence-activated cell sorting: FACS) using a fluorescently labeled anti-CD4 antibody and a cell sorter. Furthermore, the presence of Th1 CD4SP T cells and Th2 CD4SP T cells can be confirmed among the CD4SP T cells by using fluorescently labeled anti-IFN-γ and anti-IL-4 antibodies, respectively, for intracellular staining and flow cytometry. When Th2 CD4SP T cells are to be recovered as live cells, they can be recovered by, for example, flow cytometry as CXCR3-negative, CCR4-positive (consistent with the tendency to secrete IL-4), and CCR6-negative (a marker for Th17 cells) cells.

[0071] <Stroma cells (a)> Stromal cells (a) can be prepared by introducing a Notch ligand gene into stromal cells and expressing it. Like the HSPCs described above, stromal cells (a) may be derived from humans or non-human animals. The means for introducing the Notch ligand gene is not particularly limited, and any known appropriate means, such as a viral vector (e.g., retroviral vector, lentiviral vector), can be used. For example, the MS-5 mouse stromal cell lines (MS5-hDLL1 and MS5-hDLL4 lines, respectively) transduced with human DLL1 or human DLL4 as Notch ligands have been established, and these cell lines can be used as stromal cells (a) in the present invention.

[0072] The term "Notch ligand" as used herein is synonymous with the term in Patent Document 1: WO2017 / 075389 (corresponding to JP 2018-533364 A), which describes a method for producing ATO, and includes "canonical Notch ligands" and "non-canonical Notch ligands." Canonical Notch ligands include, for example, delta-like ligand 4 (DLL4), delta-like ligand 1 (DLL1), Jagged 1 (JAG1), Jagged 2 (JAG2), delta-like ligand 3 (DLL3), and X-delta 2. Non-canonical Notch ligands include, for example, 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.

[0073] -cell- A first embodiment of the Th1 CD4SP T cells of the present invention are Th1 CD4SP T cells obtained by the aforementioned production method of the present invention, i.e., a production method comprising a step of inducing hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs) in which factors related to IL-4 secretion are substantially impaired into CD4 SPT cells. A second embodiment of the Th1 CD4SP T cells of the present invention are Th1 CD4SP T cells in which factors related to IL-4 secretion are substantially impaired. Note that the Th1 CD4SP T cells of the present invention may correspond to both the first and second embodiments. As described above, the Th1 CD4SP T cells of the present invention are preferably cells induced to differentiate from iPS cells (iPSCs), for example, T cell-derived iPS cells (T-iPSCs).

[0074] A first embodiment of the Th2-type CD4SP T cells of the present invention are Th2-type CD4SP T cells obtained by the aforementioned production method of the present invention, i.e., a production method comprising a step of inducing hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs) in which factors related to IFN-γ secretion are substantially impaired to become CD4 SPT cells. A second embodiment of the Th2-type CD4SP T cells of the present invention are Th2-type CD4SP T cells in which factors related to IFN-γ secretion are substantially impaired. Note that the Th2-type CD4SP T cells of the present invention may correspond to both the first and second embodiments. As described above, the Th2-type CD4SP T cells of the present invention are preferably cells induced to differentiate from iPS cells (iPSCs), for example, T-cell-derived iPS cells (T-iPSCs).

[0075] The Th1 CD4SP T cells of the first and second embodiments of the present invention can be distinguished from natural Th1 CD4SP T cells present in living organisms (hereinafter sometimes abbreviated as "natural Th1 cells") and from Th1 CD4SP T cells obtained by production methods other than those of the present invention. For example, in a preferred embodiment of the production method of the present invention, a gene involved in IL-4 secretion in hematopoietic stem cells and / or hematopoietic progenitor cells (HSPCs) is knocked out. Since the Th1 CD4SP T cells obtained by this production method also have a gene involved in IL-4 secretion knocked out, they can be clearly distinguished from natural Th1 cells and the like (in this case, the Th1 CD4SP T cells correspond to both the first and second embodiments). The knockout of a specific gene can be confirmed by analyzing the nucleotide sequence of the genomic DNA using standard methods. Furthermore, since the Th1 CD4SP T cells of the second embodiment of the present invention have substantially impaired factors involved in IL-4 secretion, they can be distinguished from natural Th1 cells and the like that do not have such characteristics. Th1-type CD4SP T cells (first embodiment) obtained by other embodiments of the production method of the present invention can also be distinguished from natural Th1 cells, etc., based on traces, etc., that correspond to the features of the embodiment of the production method (particularly, the means for substantially impairing factors related to IL-4 secretion by HSPCs). Similarly, Th2-type CD4SP T cells of the first and second embodiments of the present invention can also be distinguished from natural Th2-type CD4SP T cells present in living organisms (hereinafter sometimes abbreviated as natural Th2 cells) and Th2-type CD4SP T cells obtained by production methods other than those of the present invention.

[0076] The Th1 CD4SP T cells and Th2 CD4SP T cells of the present invention typically express CD45RA or CD45RO, CD3ε, TCRαβ, and the like, similar to natural Th1 cells and Th2 cells, respectively. With regard to chemokine receptor expression, the Th1 CD4SP T cells of the present invention are CXCR3-positive and CCR4-negative, similar to natural Th1 cells, while the Th2 CD4SP T cells of the present invention are CXCR3-negative and CCR4-positive, similar to natural Th2 cells. Furthermore, the Th1 CD4SP T cells and Th2 CD4SP T cells of the present invention also strongly express CD5 (strongly positive), i.e., they also become mature Th1 CD4SP T cells and mature Th2 CD4SP T cells.

[0077] In addition to the above characteristics, the Th1-type CD4SP T cells and Th2-type CD4SP T cells of the present invention are characterized by containing fractions expressing CD28 and CCR7, which are representative of the phenotype of central memory T cells, and by having high self-renewal ability.

[0078] <Application> The uses of the Th1-type CD4SP T cells and Th2-type CD4SP T cells of the present invention are not particularly limited, but they can be used, for example, to treat or prevent tumors, infectious diseases (e.g., chronic infectious diseases), allergic diseases, autoimmune diseases, etc.

[0079] Therefore, in one aspect, the present invention provides Th1-type CD4SP T cells and Th2-type CD4SP T cells obtained by the production method of the present invention, as well as pharmaceutical compositions (cell preparations) containing these cells, for use in treating or preventing the above-mentioned diseases and the like.

[0080] In another aspect, the present invention provides a method (immune cell therapy) for administering Th1-type CD4SP T cells and Th2-type CD4SP T cells obtained by the production method of the present invention, or a pharmaceutical composition (cell preparation) containing these cells, for the treatment or prevention of the above-mentioned diseases and the like.

[0081] 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 in any mixture ratio.

[0082] The pharmaceutical compositions (cell preparations) of the present invention can be formulated by known pharmaceutical methods depending on the route of administration, etc., and can be prepared, for example, as injections (intravenous injections, drip infusion injections, etc.), liquids, suspensions, emulsions, etc. In such formulations, pharmacologically acceptable carriers (vehicles) and additives, specifically, sterile water, physiological saline, vegetable oils, solvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, vehicles, preservatives, binders, diluents, isotonic agents, soothing agents, bulking agents, disintegrants, buffers, coating agents, lubricants, colorants, solubilizers, and the like, can be used in appropriate combinations as needed. Furthermore, other active ingredients (other drugs, cells, etc., e.g., CAR-transduced CD8SP T cells), immunostimulants, and the like, depending on the therapeutic or prophylactic purpose, can also be contained together with the Th1 CD4SP T cells and / or Th2 CD4SP T cells.

[0083] The method of administration of the pharmaceutical composition (cell preparation) of the present invention is not particularly limited, but is preferably parenteral administration, for example, intravenous, intraperitoneal, subcutaneous, or intramuscular administration, or local administration to the affected area, more preferably intravenous administration or local administration to the affected area. The dosage of the pharmaceutical composition (cell preparation) of the present invention can be adjusted appropriately depending on the age, weight, symptoms, health condition, dosage form, administration method, etc. of the subject.

[0084] The pharmaceutical composition (cell preparation) product of the composition of the present invention may be labeled to indicate that it is used for the treatment or prevention of a disease, etc. For example, information to the effect that it is used for the treatment or prevention of a disease, etc. may be written on the product itself, container, packaging, etc., or in the product's instruction manual, package insert, promotional materials, other printed materials, etc.

[0085] As used in this specification and claims, singular terms shall include pluralities and plural terms shall include the singular, unless the context otherwise requires. Thus, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural reference, unless otherwise specified. [Example]

[0086] [Example 1] Production of iPS cells (TBX21 KO cell line, IL4 KO cell line) The iPS cells used were the TKT3V1-7 strain provided by Kyoto University (CiRA). Undifferentiated iPS cells were passaged and maintained in AK03N medium (Ajinomoto Co.), and when they reached subconfluence in the wells, they were harvested as single cells using TripLESelect (Thermo Fisher Scientific). The harvested cells were collected at a concentration of 1.0 x 10 per cuvette. 6 The cells were resuspended in Opti-MEM (Thermo Fisher Scientific) to a concentration of 100 μL.

[0087] Guide RNAs were designed for the TBX21 gene, which encodes the transcription factor T-bet, and the IL4 gene, which encodes the cytokine IL-4. Guide RNAs containing these recognition sequences were then generated using Alt-R CRISPR-Cas9 crRNA (Integrated DNA Technologies) and Alt-R CRISPR-Cas9 tracrRNA (Integrated DNA Technologies). The guide RNA suspension was reacted with Alt-R Sp Cas9 Nuclease 3NLS (Integrated DNA Technologies) to form an RNP complex.

[0088] The RNP complex suspension and 1.0 x 10 ATP suspended in Opti-MEM were mixed to give a final RNP complex concentration of 1.6 µM. 6 The mixture was mixed with iPS cells, and electroporation was performed on the mixture using a NEPA21 electroporator (manufactured by Nepa Gene).

[0089] During electroporation, a Puro (puromycin resistance gene) plasmid was also electroporated, and the cells were cultured in a puromycin-containing medium to indirectly select for cells transfected with the Cas9 protein. This takes advantage of the correlation between the introduction of the Cas9 protein and the introduction of the plasmid into the cells.

[0090] Electroporated iPS cells were seeded onto a 10cm dish coated with iMatrix-511 (Matrixome) and cultured for 12 days. The resulting colonies were picked and passaged. Cells derived from each colony were established and stocked, and the knockout and mutation patterns of the target gene were confirmed by sequencing.

[0091] Sequencing was performed on PCR products using primers designed to flank the target sequence of the guide RNA. An example of the sequencing results is shown in Figure 2. Even when the same guide RNA was used for knockout (KO) treatment, the mutations that occurred varied depending on the strain. Table 1 lists the strains for which sequencing and trait confirmation were performed. It was later confirmed that the desired traits were expressed equally among these strains, regardless of the type of mutation.

[0092] [Table 1]

[0093] [Example 2] Production of Th1 cells and Th2 cells from iPS cells (TBX21KO cell line, IL4KO cell line) (iPSC → HPC / HPC → Th1 cells or Th2 cells) iPS cell lines confirmed by sequencing to be homozygous mutants of the target genes TBX21 or IL4 were differentiated into hematopoietic progenitor cells (HPCs) using the embryonic body method (EB method). The EB method, as described by A.E. Grigoriadis et al. (2010). Blood, 115(14):2769-2776, is a method for producing HPCs by forming embryonic bodies without using feeder cells. In this example, the cells were modified as shown in Figure 3.

[0094] For TKT3V1-7 or its knockout cell line, the day differentiation induction of undifferentiated cells was initiated was designated day 0, and ATO cells were used on day 9. EB clumps contained in the culture medium were removed using a 40 μm cell strainer (Fisher Scientific), and only floating cells were collected. The collected floating cells are thought to contain a fraction corresponding to hematopoietic progenitor cells, and in this example, they were used in bulk as the material for ATO without sorting.

[0095] The artificial thymic organoid method, originally reported by C.S. Seet et al. (2017). Nat Methods. 14(5):521-530, is an organoid culture method for differentiating primary hematopoietic stem cells into αβ T cells. This original method uses CD34-positive CD3-negative cells sorted using a cell sorter, and it is thought that it may also be used to sort hematopoietic progenitor cells.

[0096] In this example, bulk iPS cell-derived hematopoietic progenitor cells recovered without sorting and MS5hDLL4 cells were mixed at a cell number ratio of 4:1. A droplet of the suspension was placed on an insert (Merck (Millipore), MILLICELL INSERT 30MM ORGANOTYPIC PTFE 0.4UM 50 / PK) and the insert was suspended in the medium to initiate the ATO differentiation culture method. The medium was RPMI-1640 (Wako) diluted 25-fold with B-27. TMThe cells were prepared by adding 50X serum-free supplement (Thermo Fisher Scientific), PSG (penicillin, streptomycin, and L-glutamine) diluted 100-fold, GlutaMAX diluted 100-fold, IL-7 (final concentration 5 ng / mL), Flt3L (5 ng / mL), and PAA (50 μg / mL). The "MS5hDLL4 cells" were prepared by introducing the hDLL4 gene into the mouse myeloid cell line "MS-5" using a lentiviral vector.

[0097] When a mixture of iPS cell-derived HPCs and MS5hDLL4 cells was cultured on inserts suspended in this medium for 9 to 12 weeks, they differentiated into CD4 single-positive (SP) cells and CD8 single-positive (SP) cells. The CD4SP and peripheral blood mononuclear cells ("Wild" and "PBMCs" in Figure 4) derived from non-KO iPSCs showed a tendency to actively secrete IL-4 and also showed high levels of T-bet expression. Therefore, we anticipated that IL4KO and TBX21KO might inhibit CD4SP, CD8SP differentiation, or even T cell differentiation itself. However, as shown in Figure 4, CD4SP differentiation was observed from all KO lines.

[0098] Analysis of primary helper T cells has led to consensus that Th1 cells, which control cellular immunity, have a chemokine receptor expression pattern of CXCR3-positive and CCR4-negative, while Th2 cells, which control humoral immunity, have a CXCR3-negative and CCR4-positive pattern. IL4 KO, which abolishes secretion of the functional Th2 cytokine IL-4, results in cells exhibiting a Th1-type expression pattern. KO of TBX21, which encodes T-bet, a master regulator of Th1 (a transcription factor that primarily induces differentiation), is predicted to result in a Th2-type expression pattern. This was verified by flow cytometric analysis using anti-CXCR3 antibody (BD, clone: 1C6 / CXCR3) and anti-CCR4 antibody (BD, clone: 1G1). As shown in Figure 5, CD4SP cells induced to differentiate from wild-type cells without KO treatment contained both CXCR3-positive and CXCR3-negative CCR4-positive cells, similar to primary CD4SP cells in PBMCs. In contrast, CD4SP cells induced to differentiate from IL4KO cells consisted exclusively of CXCR3-positive, CCR4-negative cells, consistent with the expression pattern of Th1 cells, whereas CD4SP cells induced to differentiate from TBX21KO cells consisted exclusively of CXCR3-negative cells, many of which were CCR4-positive, consistent with the expression pattern of Th2 cells.

[0099] [Example 3] Confirmation of IL-4 and IFN-γ secretion from the obtained Th1 cells and Th2 cells Next, the cytokine production of CD4SP cells differentiated from IL4KO cells and CD4SP cells differentiated from TBX21KO cells was assessed by intracellular flow cytometry using anti-IFN-γ antibody (BD, clone B27) and anti-IL-4 antibody (Biolegend, clone 8D4-8). After 3 hours of stimulation with PMA (40 ng / mL) and ionomycin (4 μg / mL) in the presence of monensin (2 μM), as shown in Figure 6, 55.2% of CD4SP cells derived from wild-type iPS cells produced IL-4, 5.67% produced IFN-γ, and 26.6% produced both. In contrast, 89.3% of CD4SP cells differentiated from IL4KO cells secreted only IFN-γ, consistent with Th1 cell characteristics. Furthermore, 35.7% of CD4SP cells derived from TBX21KO cells secreted only IL-4, consistent with the properties of Th2 cells, while 0.34% secreted only IFN-γ and 0% secreted both. IFN-γ secretion was almost nonexistent. Therefore, it was confirmed that IL4KO generates Th1 cells that secrete only IFN-γ, and TBX21KO generates Th2 cells that secrete only IL-4.

[0100] It was possible that the CD4SP cells derived from IL4KO cells were merely a result of the loss of IL-4 production due to the knockout of the gene encoding the cytokine IL-4, the end product of Th2 cells. However, as shown in Example 2, IL4KO also altered the chemokine receptor expression pattern to that of CXCR3-positive, CCR4-negative Th1 cells. Furthermore, we examined the production of IL-13, which, along with IL-4, is considered a representative functional cytokine of Th2 cells, using intracellular staining flow cytometry with an anti-IL-13 antibody (Biolegend, clone: JES10-5A2). As shown in Figure 7, CD4SP cells derived from wild-type iPS cells contained a fraction that produced high levels of IL-13 compared to primary CD4SP cells, while the high IL-13-secreting fraction was lost in CD4SP cells derived from multiple IL4KO cell lines. IL-13 secretion, which is not directly knocked out, was reduced by IL4KO, providing further evidence that IL4KO prevented Th2 cell phenotype expression.

Claims

1. A method for producing Th1 type CD4 single positive T cells, comprising: (B1) inducing hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) in which the IL-4 gene has been knocked out into CD4 single-positive T cells; The hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) are cells induced to differentiate from iPS cells (iPSCs). method.

2. A method for producing Th2 type CD4 single positive T cells, comprising: (B2) inducing hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) in which the T-bet gene has been knocked out into CD4 single-positive T cells; The hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) are cells induced to differentiate from iPS cells (iPSCs). method.

3. A method for producing Th1 type CD4 single positive T cells, comprising: (a) stromal cells expressing a Notch ligand; (b1) hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) in which the IL-4 gene has been knocked out but the T-bet gene has not been knocked out; By culturing artificial thymus organoids (ATO) containing producing IL-4 non-secreting and IFN-γ secreting (Th1 type) CD4 single positive T cells from the ATO; The method of claim 1.

4. A method for producing Th2 type CD4 single positive T cells, comprising: (a) stromal cells expressing a Notch ligand; (b2) hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs) in which the T-bet gene has been knocked out but the IL-4 gene has not been knocked out; By culturing artificial thymus organoids (ATO) containing producing IFN-γ non-secreting, IL-4 secreting (Th2 type) CD4 single positive T cells from the ATO; The method of claim 2.

5. The method according to claim 1 or 2, wherein the iPSCs are T-cell-derived iPSCs (T-iPSCs).

6. The method of claim 5, wherein the T cells are CD4 single-positive T cells.

7. A Th1 type CD4 single positive T cell in which the IL-4 gene has been knocked out, Cells induced to differentiate from iPS cells (iPSCs), Th1 type CD4 single positive T cells.

8. A Th2 type CD4 single positive T cell in which the T-bet gene has been knocked out, Cells induced to differentiate from iPS cells (iPSCs), Th2 type CD4 single positive T cells.

9. The CD4 single-positive T cells according to claim 7 or 8, wherein the iPSCs are T cell-derived iPSCs (T-iPSCs).

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