Method for producing type II innate lymphocytes

Abstract

The present invention addresses the problem of establishing a technique for producing type II innate lymphocytes. Type II innate lymphocytes can be obtained by culturing hematopoietic precursor cells in the presence of a CDK8 inhibitor.

Description

Technical Field

[0001] This invention relates to a technique for obtaining type II innate lymphocytes. More particularly, this invention relates to a technique for obtaining type II innate lymphocytes from pluripotent stem cells. Background Technology

[0002] Innate lymphoid cells (ILCs) are a relatively new population of cells in the innate immune system that lack antigen receptors and differentiate from common precursor cells. ILCs are responsible for initiating and controlling the direction of both innate and adaptive immune responses by responding to stimuli from infected or damaged tissues and rapidly producing cytokines characteristic of their respective subsets. ILCs are typically classified into five groups based on the cytokines they produce and the transcription factors that regulate their production and function: NK cells, ILC1, ILC2, ILC3, and LTi cells (Lymphoid tissue inducer cells).

[0003] Group 2 innate lymphoid cells (ILC2), a type of ILC, are resident in peripheral tissues such as adipose tissue, lung, intestine, and skin. ILC2 is activated by alarming signals such as IL-25, IL-33, and TSLP released during epithelial injury, producing large amounts of type II cytokines such as IL-5 and IL-13, and activating helper T2 cells (Th2 cells) and eosinophils. ILC2 plays an important role in the initial defense against parasitic infections and in tissue repair. Furthermore, its association with allergic diseases such as bronchial asthma and chronic sinusitis with nasal polyps (CRSwNP), as well as various diseases including inflammatory diseases and autoimmune diseases, is gradually being elucidated. In addition, the transcription factor GATA3 is essential for the production and functional maintenance of ILC2 (Non-Patent Literature 1-2).

[0004] As a novel subset of ILC2, it has been reported that ILCreg, ILC2reg, and ILC2 exist in human and mouse tissues associated with ILC2 activation. 10 IL-10-producing ILC2s, such as IL-10+ILC2 (Non-Patent Literature 3-6). Such ILC2 subgroups are also suggested to be associated with human allergic diseases (Non-Patent Literature 7). On the other hand, it is known that the activation of ILC2s also helps to reduce inflammatory responses and repair damaged tissues.

[0005] In addition to the tissue repair and regeneration functions of ILC2, IL-10 also has anti-inflammatory and immunosuppressive effects. Therefore, through the adoptive transfer of IL-10-producing ILC2 cells and the induction of IL-10-producing ILC2 cells in vivo, it is expected that treatment methods for allergic diseases, immune diseases and inflammatory diseases can be established.

[0006] In addition, it was reported that all ILC subsets, including NK cells, ILC1, ILC2, and ILC3, can be generated in vitro from CD34-positive hematopoietic progenitor cells derived from human umbilical cord blood and bone marrow, and ILC progenitor cells from human adult peripheral blood (Non-Patent Literature 8-9).

[0007] Existing technical documents

[0008] Non-patent literature

[0009] Non-patent literature 1: Cell. 2018 Aug 23; 174(5): 1054-1066.

[0010] Non-patent literature 2: Nat Med. 2015 Jul; 21(7): 698-708.

[0011] Non-patent literature 3: Nat Commun. 2017 Dec 1; 8(1): 1900.

[0012] Non-patent literature 4: J Allergy Clin Immunol. 2019 Jun; 143(6): 2190-2201.e9.

[0013] Non-patent literature 5: J Allergy Clin Immunol. 2021 Apr; 147(4): 1281-1295.e5.

[0014] Non-patent literature 6: J Exp Med. 2020 Feb 3; 217(2): e20191520.

[0015] Non-patent literature 7: Immunity 2021;54:291-307.

[0016] Non-patent literature 8: Cell 2017; 168(6): 1086-1100.

[0017] Non-patent literature 9: Immunity 2021;54:2417-2432.e5 Summary of the Invention

[0018] The problem that the invention aims to solve

[0019] The quantity of ILC2 and ILC precursor cells that can be isolated and cultured on a large scale from human peripheral blood is limited, and they are not expected to be used as a cell supply source for cell therapy.

[0020] Embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), which are pluripotent cells obtained by introducing reprogramming factors into somatic cells, have been reported as examples of pluripotent cells. These cells can proliferate almost indefinitely while maintaining pluripotency. If a method for inducing ILC2 from pluripotent stem cells can be developed, it could become an effective means in the manufacture of cell therapy drugs. In addition, the induced ILC2 can also be used to explore and evaluate agents that modulate the function of disease-related ILC2, and agents that induce therapeutic subpopulation changes in ILC2 in the body, such as IL-10-producing ILC2.

[0021] To date, regarding ILC, for the purpose of cell immunotherapy that induces antitumor immune responses, methods for inducing NK cells / ILC from iPS cells via CD34-positive hematopoietic progenitor cells have been reported (Cancer Sci. 2020May;111(5):1478-1490).

[0022] However, no method for inducing ILC2 from iPS cells has been reported to date.

[0023] In view of this situation, the subject of this invention is to develop a technology for manufacturing ILC2.

[0024] Methods for solving problems

[0025] In order to solve the above-mentioned problems, the inventors conducted in-depth research and found that ILC2 can be produced by culturing hematopoietic progenitor cells in the presence of CDK8 inhibitors.

[0026] The present invention includes the following embodiments, but is not limited thereto.

[0027] [1] A method for producing type II innate lymphocytes, comprising the step of culturing hematopoietic progenitor cells in the presence of a CDK8 inhibitor to obtain type II innate lymphocytes.

[0028] [2] According to the method of [1], wherein the type II innate lymphocytes include type II innate lymphocytes that produce IL-10.

[0029] [3] The method described in [1] or [2] is performed in vitro.

[0030] [4] According to the method of [1] or [2], in which hematopoietic progenitor cells are cultured in the presence of CDK8 inhibitors, IL-2 and IL-33 to obtain type II innate lymphocytes.

[0031] [5] The method according to [1] or [2], wherein the CDK8 inhibitor comprises Senexin A, CCT-251921, MSC2530818, CCT251545, SEL120-34A or BRD6989.

[0032] [6] According to the method described in [1] or [2], the hematopoietic progenitor cells are hematopoietic progenitor cells differentiated from human induced pluripotent stem cells (human iPS cells).

[0033] [7] According to the method described in [1] or [2], wherein the hematopoietic precursor cells are hematopoietic precursor cells derived from the human body.

[0034] [8] The method according to [1] or [2] further includes the step of inducing differentiation from pluripotent stem cells into hematopoietic progenitor cells.

[0035] [9] According to the method described in [8], wherein the pluripotent stem cells are induced pluripotent stem cells (iPS cells).

[0036]

[10] According to the method described in [8], wherein the pluripotent stem cells are human iPS cells.

[0037] Invention Effects

[0038] According to the present invention, ILC2 can be artificially obtained from hematopoietic progenitor cells. In this invention, hematopoietic progenitor cells derived from pluripotent stem cells such as iPS cells can also be used, and ILC2 can be induced from iPS cells through differentiation using this invention. Furthermore, according to the present invention, ILC2 that produces IL-10 (IL-10-producing ILC2) can be obtained, which is useful in drug development. Attached Figure Description

[0039] Figure 1-1This is a representative example (Experiment 2) confirming the induction of differentiation from hematopoietic progenitor cells (derived from iPS cell line 1231A2) into ILC2 and IL-10-producing ILC2 cells. Differentiation into ILC2 was confirmed by flow cytometry using GATA3 expression and the production capacity of IL-13 and IL-10 as indicators. The upper graph shows the expression levels of IL-13 and GATA3 in lineage marker (CD3, CD14, CD16, CD19, CD20, CD94) negative cells (vertical axis: IL-13 expression level, horizontal axis: GATA3 expression level). Similarly, the lower graph shows the expression levels of IL-13 and IL-10 in lineage marker negative cells (vertical axis: IL-13 expression level, horizontal axis: IL-10 expression level).

[0040] Figure 1-2 It shows that by and Figure 1-1 A graph showing the proportions of ILC2 and IL-10-producing ILC2 obtained from the same analysis (n=3, triplet) (Experiment 2). The left graph shows the proportion of ILC2 in live cells (cells that are lineage marker negative, IL-13 positive, and GATA3 positive), and the right graph shows the proportion of IL-10-producing ILC2 in live cells (cells that are lineage marker negative, IL-13 positive, and IL-10 positive). Error bars represent the standard error of the mean ± standard error.

[0041] Figure 2 This is a representative example (Experiment 3) confirming the induction of differentiation from hematopoietic progenitor cells (derived from the iPS cell line Ff-I01s04) into ILC2 and IL-10-producing ILC2 cells. Differentiation into ILC2 cells was confirmed by flow cytometry using GATA3 expression and IL-13 and IL-10 production capacity as indicators. The upper graph shows the expression levels of IL-13 and GATA3 in lineage marker (CD3, CD14, CD16, CD19, CD20, CD94) negative cells (vertical axis: IL-13 expression level, horizontal axis: GATA3 expression level). Similarly, the lower graph also shows the expression levels of IL-13 and IL-10 in lineage marker negative cells (vertical axis: IL-13 expression level, horizontal axis: IL-10 expression level).

[0042] Figure 3-1This is a representative example (Experiment 4) of the induction of differentiation into ILC2 and IL-10-producing ILC2 cells from hematopoietic progenitor cells (derived from iPS cell line 1231A2) using multiple structurally different CDK8 inhibitors. Differentiation into ILC2 cells was confirmed by flow cytometry using GATA3 expression and IL-13 and IL-10 production capacity as indicators. The upper graph shows the expression levels of IL-13 and GATA3 in lineage marker (CD3, CD14, CD16, CD19, CD20, CD94) negative cells (vertical axis: IL-13 expression level, horizontal axis: GATA3 expression level). Similarly, the lower graph shows the expression levels of IL-13 and IL-10 in lineage marker negative cells (vertical axis: IL-13 expression level, horizontal axis: IL-10 expression level).

[0043] Figure 3-2 It shows that by and Figure 3-1 The same analysis (n=3, repeated three times) yielded a graph showing the ratio of ILC2 to IL-10-producing ILC2 (Experiment 4). The left graph shows the proportion of ILC2 in live cells (cells that are lineage marker negative, IL-13 positive, and GATA3 positive), and the right graph shows the proportion of IL-10-producing ILC2 in live cells (cells that are lineage marker negative, IL-13 positive, and IL-10 positive). Error bars represent the standard error of the mean ± standard error.

[0044] Figure 4 This is a representative example (Experiment 5) confirming the induction of differentiation into ILC2 and IL-10-producing ILC2 cells from CD34-positive cells derived from human umbilical cord blood. Differentiation into ILC2 cells was confirmed by flow cytometry using GATA3 expression and IL-13 and IL-10 production capacity as indicators. The upper graph shows the expression levels of IL-13 and GATA3 in lineage marker (CD3, CD14, CD16, CD19, CD20, CD94)-negative cells (vertical axis: IL-13 expression level, horizontal axis: GATA3 expression level). Similarly, the lower graph shows the expression levels of IL-13 and IL-10 in lineage marker-negative cells (vertical axis: IL-13 expression level, horizontal axis: IL-10 expression level).

[0045] Figure 5 This is the result of a quantitative evaluation of cytokine concentrations in the supernatant of ILC2 differentiation cultures derived from iPS cells (Experiment 6). From left to right, the concentrations of bimodalin (pg / mL), IL-10 (ng / mL), and IL-5 (ng / mL) are shown. The graphs for each condition represent the cytokine concentrations in the supernatant of ILC2 differentiation cultures derived from iPS cells from different batches. Error bars represent the standard error ± mean.

[0046] Figure 6 This is the result of a quantitative evaluation of the inhibitory effect of THP-1 cells on TNFα production shown in differentiated cells containing IL-10-producing ILC2 (Experiment 7). The vertical axis represents the TNFα concentration (pg / mL) in the supernatant derived from THP-1 cells. Conditions 1 and 3 represent values ​​from 3 wells, while the THP-1 group alone represents values ​​from 9 wells.

[0047] Figure 7 This is the result of quantitatively evaluating the inhibitory effect of differentiated cells containing IL-10-producing ILC2s on TNFα in THP-1 cells, which were separated into CD25-CD30- and CD25+CD30+ groups. The vertical axis of the top figure represents the TNFα concentration (pg / mL) in the supernatant derived from THP-1 cells. The vertical axis of the bottom figure represents the IL-10 concentration (pg / mL) produced by differentiated cells derived from iPS cells. The CD25-CD30- and CD25+CD30+ groups represent values ​​from 3 wells, while only the THP-1 group represents values ​​from 6 wells.

[0048] Figure 8 This is a quantitative evaluation of the inhibitory effect of macrophages derived from human peripheral blood mononuclear cells (PBMCs) on TNFα production, as shown in differentiated cells containing IL-10-producing ILC2 (Experiment 9). The vertical axis represents the TNFα concentration (pg / mL) in the supernatant derived from macrophages from human PBMCs. Conditions 1 and 3 represent values ​​from 3 wells, while the macrophage group derived solely from PBMCs represents values ​​from 6 wells. Detailed Implementation

[0049] This invention relates to a method for obtaining type II innate lymphocytes from hematopoietic progenitor cells. Furthermore, this invention includes type II innate lymphocytes produced by the method of this invention and their uses (application).

[0050] Specifically, the present invention includes the following method: a method for producing type II innate lymphocytes, comprising the step of culturing hematopoietic progenitor cells in the presence of a CDK8 inhibitor to obtain type II innate lymphocytes.

[0051] Previously, the technology for artificially obtaining type II innate lymphocytes (ILC2) from pluripotent stem cells had not been established, but this invention has established the technology for manufacturing ILC2. It has been discovered that ILC2, as an innate immune cell, is activated without antigen recognition mechanisms, producing large amounts of type II cytokines, which are not only associated with allergic inflammation but also with the condition of chronic inflammation such as obesity and fibrosis, as well as autoimmune diseases such as rheumatism.

[0052] The ILC2 in this invention is not particularly limited, but since the transcription factor GATA3 is essential for the production and functional maintenance of ILC2, in this invention, the expression of GATA3 confirms the acquisition of ILC2 from hematopoietic progenitor cells. Furthermore, ILC2 is known to produce large amounts of IL-13 and IL-5, so the presence of ILC2 can be confirmed by verifying its ability to produce IL-13 and IL-5. Moreover, the differentiation from hematopoietic progenitor cells to ILC2 can also be confirmed by the absence of lineage markers such as CD3, CD14, CD16, CD19, CD20, and CD94.

[0053] ILC2 can be identified using various well-known methods. This can be achieved through cell staining with antibodies against these proteins, quantitative PCR targeting these mRNAs, or expression analysis using scRNA-seq.

[0054] As a subset of ILC2, the existence of IL-10-producing ILC2s, referred to as ILCreg, has been reported. IL-10 is known as a cytokine with anti-inflammatory effects, and IL-10-producing ILC2s are considered particularly preferred for pharmaceutical use. In one embodiment, the ILC2s manufactured in this invention comprise an IL-10-producing ILC2 subset. The presence of IL-10-producing ILC2s can be confirmed by verifying their ability to produce IL-10. In one embodiment, the method of this invention further includes the step of screening for IL-10-producing ILC2s from the obtained type II innate lymphocytes (ILC2).

[0055] When screening ILC2 cells using biomarkers, various known methods can be employed. For example, when using antibodies that specifically bind to the biomarker, methods include using fluorescently labeled antibodies and a cell sorter (e.g., FACS (registered trademark, BD Biosciences), using antibody-labeled magnetic beads and screening cells by magnetism (e.g., MACS (registered trademark, Miltenyi Biotechnology), and using carriers with immobilized antibodies (e.g., cell concentration columns). Additionally, commercially available antibodies that specifically bind to antigens can be used appropriately.

[0056] In this invention, hematopoietic progenitor cells refer to cells capable of differentiating into hematopoietic cell lines such as lymphocytes, eosinophils, neutrophils, basophils, erythrocytes, and megakaryocytes. These cells can be any of the following: hematopoietic progenitor cells directly collected from an individual organism; primary cultured cells obtained by culturing and proliferating these hematopoietic progenitor cells in vitro; passaged cells; hematopoietic progenitor cells induced from pluripotent stem cells; or ungraded cell populations containing these cells capable of hematopoietic cell differentiation. In one embodiment, the hematopoietic progenitor cells used in this invention are hematopoietic progenitor cells induced from pluripotent stem cells (preferably induced pluripotent stem cells (iPS cells), more preferably human iPS cells). In this invention, pluripotent stem cells refer to stem cells possessing the ability to differentiate into various types of cells with different properties and morphologies present in an organism, and also possessing proliferative capacity, including any cell capable of being induced into hematopoietic progenitor cells. Pluripotent stem cells are not specifically limited, but can include, for example, embryonic stem cells (ES cells), nuclear transfer embryonic stem cells (ntES cells) produced using nuclear transfer technology, sperm line stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), and multi-lineage differentiating stress enduring cells (Muse cells) derived from cultured fibroblasts or bone marrow stem cells. Because they can be obtained without destroying the embryo or egg, iPS cells are preferred, and human iPS cells are more preferred.

[0057] The method for manufacturing iPS cells is well known and can be achieved by introducing reprogramming factors into any somatic cell. Examples of reprogramming factors include, for instance, genes or gene products such as OCT3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, β-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, or Glis1. These reprogramming factors can be used alone or in combination.

[0058] Somatic cells include, without limitation, any of the somatic cells of newborns (infants) and any of the somatic cells of healthy individuals or patients, as well as any of the primary cultured cells, passaged cells, and lineage cells derived from them. Specifically, examples of somatic cells include: (1) tissue stem cells (adult stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells; (2) tissue precursor cells; (3) blood cells (peripheral blood cells, umbilical cord blood cells, etc.), muscle cells, skin cells, hair cells, liver cells, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells, brain cells, lung cells, kidney cells, and adipocytes, and other differentiated cells existing in organs and tissues.

[0059] Furthermore, when using iPS cells as the material for transplantation, from the viewpoint of avoiding rejection, it is preferable to use somatic cells with the same or substantially the same human leukocyte antigen genotype as the individual to whom the transplant is intended. Here, "substantially the same" means that the HLA genotype is identical to the degree to which the transplanted cells can suppress the immune response by immunosuppressants, for example, somatic cells with the same HLA type having 3 loci (HLA-A, HLA-B, HLA-DR) or 4 loci (HLA-C) added to them. Pluripotent stem cells used as the material for inducing hematopoietic progenitor cells can also be used, for example, pluripotent stem cells that do not cause rejection in allogeneic transplantation, prepared by the methods described in Nat. Biotechnol., (2017), 35:765-772, Nat Biomed Eng. 2021 May;5(5):429-440. As the aforementioned pluripotent stem cells that do not cause rejection in allogeneic transplantation, ES cells or iPS cells that do not cause rejection in allogeneic transplantation are preferred, and human ES cells or human iPS cells that do not cause rejection in allogeneic transplantation are more preferred.

[0060] In one embodiment, the hematopoietic progenitor cells used in this invention are hematopoietic progenitor cells derived from the human body. Hematopoietic progenitor cells derived from the human body include any of the following: hematopoietic progenitor cells collected directly from a human body; primary cultured cells obtained by culturing and proliferating hematopoietic progenitor cells collected directly from a human body in vitro; passaged cells; or an ungraded cell population containing them capable of hematopoietic cell differentiation. Human hematopoietic progenitor cells can be collected, for example, from human umbilical cord blood or bone marrow. In one embodiment, the hematopoietic progenitor cells derived from the human body are hematopoietic progenitor cells derived from human umbilical cord blood or bone marrow.

[0061] The cultivation method used in this invention is not particularly limited, and can be carried out by suspension culture, adhesion culture, etc.

[0062] Suspension culture refers to culturing cells in a state where they do not adhere to the culture dish. There are no particular limitations. To improve cell adhesion, culture dishes that have not been artificially treated (e.g., coated with extracellular matrix) or those that have been artificially treated to inhibit adhesion can be used for culturing.

[0063] Adhesion culture refers to culturing cells in a state where they adhere to a culture dish. There are no particular limitations; culturing can also be carried out in coated culture dishes. Examples of coating agents include matrix gelatin, collagen, gelatin, laminin, heparan sulfate proteoglycans, and nestin.

[0064] In this invention, the culture temperature is not particularly limited, but is, for example, about 30-40°C, preferably about 37°C, and the culture is carried out in an atmosphere containing CO2. The CO2 concentration is about 2-5%, preferably about 5%. The culture time is not particularly limited, but is, for example, 0.5-30 days, 1-20 days, or 3-10 days.

[0065] The preparation of culture media can follow well-known methods. For example, one or more substances can be appropriately added to a basal culture medium used for culturing animal cells. Examples of basal culture media include IMDM (Iscove's Modified Dulbecco's Medium), Medium 199, EMEM (Eagle's Minimum Essential Medium), αMEM (alpha Modified Eagle Minimum Essential Medium), DMEM (Dulbecco's Modified Eagle's Medium), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. The culture medium may contain serum, such as fetal bovine serum (FBS), or it may be serum-free. Depending on the needs, one or more serum substitutes such as albumin may be added, or it may contain one or more substances such as transferrin, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, and inorganic salts.

[0066] Differentiation induction from pluripotent stem cells to hematopoietic progenitor cells

[0067] In this invention, hematopoietic progenitor cells derived from pluripotent stem cells such as iPS cells can be used as hematopoietic progenitor cells. The method for inducing differentiation into hematopoietic progenitor cells is not particularly limited in this invention; hematopoietic progenitor cells can be obtained by any method, such as those described in publications like Cancer Sci. 2020 May; 111(5): 1478-1490, Cells. 2023 Jan; 12(2): 321, Blood (2008) 111(11): 5298-5306, and PLoS One. 2011; 6(7): e22261. In one embodiment, the method for producing type II innate lymphocytes of this invention further includes the step of inducing differentiation from pluripotent stem cells, preferably induced pluripotent stem cells (iPS cells), and more preferably human iPS cells into hematopoietic progenitor cells. In one approach, the steps for inducing differentiation of hematopoietic progenitor cells from pluripotent stem cells such as iPS cells include culturing pluripotent stem cells in a culture medium containing one or more proteins selected from the group consisting of BMP4, SCF, VEGF, FGF-2, TPO (thrombopoietin), and FLt3L. In another approach, when inducing differentiation of hematopoietic progenitor cells from pluripotent stem cells such as iPS cells, a method for preparing hematopoietic progenitor cells can be cited as follows: hematopoietic progenitor cells are prepared by co-culturing pluripotent stem cells with C3H10T1 / 2 cells and then co-culturing them with C3H10T1 / 2 cells in the presence of VEGF, FLT3L, and SCF. In this case, vitamin C can be further added for culturing. Here, "network structure" refers to a three-dimensional sac-like (with internal space) structure derived from pluripotent stem cells, formed by endothelial cell groups, etc., and containing hematopoietic progenitor cells internally. In addition, hematopoietic progenitor cells can be prepared from a network structure obtained by culturing pluripotent stem cells on C3H10T1 / 2 cells in the presence of VEGF, as described in Takayama N., et al. J Exp Med. 2817-2830 (2010).In addition, methods for producing hematopoietic progenitor cells from pluripotent stem cells include the formation of embryoids and the addition of cytokines (Chadwick et al. Blood 2003, 102:906-15, Vijayaragavan et al. Cell Stem Cell 2009, 4:248-62, Saeki et al. Stem Cells 2009, 27:59-67), co-culture with xenogeneic stromal cells (Niwa A et al. J Cell Physiol. 2009 Nov;221(2):367-77.), and the combination of the addition of cytokines and coating agents (stromalin or laminin fragments) (WO2011 / 115308).

[0068] Here, hematopoietic precursor cells refer to cells that can differentiate into blood cell lines such as lymphocytes, eosinophils, neutrophils, basophils, erythrocytes, and megakaryocytes. For example, they can be identified by being positive for CD34 as a surface antigen, or by being positive for both CD34 and CD43.

[0069] Differentiation induction from hematopoietic progenitor cells to type II intrinsic lymphocytes

[0070] In this invention, ILC2 is obtained by culturing hematopoietic progenitor cells in the presence of a CDK8 inhibitor. The CDK8 inhibitor is not particularly limited as long as it can inhibit CDK8; examples include Senexin A, CCT-251921, MSC2530818, CCT251545, SEL120-34A, or BRD6989. The CDK8 inhibitor of this invention can inhibit CDK19, a homolog of CDK8. When using Senexin A as a CDK8 inhibitor, its concentration is preferably 0.01–1000 μM, more preferably 0.1–100 μM, and even more preferably 1–10 μM. Similarly, when using CCT-251921 as a CDK8 inhibitor, its concentration is preferably 0.01 nM–100 μM, more preferably 0.01 nM–10 μM, and even more preferably 10 nM–1 μM. When using MSC2530818, CCT251545, and SEL120-34A, the concentration is preferably 0.01 nM to 100 μM, more preferably 0.1 to 100 μM, and even more preferably 0.3 to 10 μM. When using BRD6989, the concentration is preferably 0.01 nM to 100 μM, more preferably 0.1 to 100 μM, and even more preferably 1 to 20 μM.

[0071] There are no particular restrictions on the duration of culturing hematopoietic progenitor cells in the presence of a CDK8 inhibitor; for example, it can be set to 0.5–30 days, 1–20 days, or 2–10 days. Furthermore, there are no particular restrictions on the culture temperature; it can be set to approximately 30–40°C, preferably approximately 37°C. In this invention, it is preferred to culture the cells in an atmosphere containing CO2 air, with a CO2 concentration of approximately 2–5%, preferably approximately 5%.

[0072] In this invention, the culture method is not particularly limited. For example, cell isolation can be performed using methods known in the art, such as suspension culture or adhesion culture. In the case of adhesion culture, a culture container can be used, or co-culture with feeder cells can be performed. As feeder cells for co-culture, stromal cells are preferred, specifically, bone marrow mesenchymal cell line OP9 cells (available from Riken Biotech Center) and MS5 cells can be examples. The feeder cells are preferably cells that stably express Delta-like 1 (Dll1) or cells that stably express Delta-like 4 (Dll4) (Non-Patent Literature 8, 9). If feeder cells are not used, a culture container coated with Dll1, Dll4, or a fusion protein of Dll1 or Dll4 with Fc can be used, preferably a culture container coated with 1 to 10 μg / mL Fc-DLL4 solution.

[0073] In this invention, in a preferred embodiment, IL-2 and / or IL-33 are added to the culture medium; in a more preferred embodiment, IL-2 and IL-33 are added. IL-2 and / or IL-33 can be any known substance; for example, IL-2 can be obtained from BioLegend (589108), PeproTech (200-02), etc., and IL-33 can be obtained from Fujifilm and Koden Pharmaceuticals (099-05611), BioLegend (581806), etc. The concentration of IL-2 can be appropriately selected by those skilled in the art, and its concentration is preferably 1 to 10,000 U / mL, more preferably 10 to 1,000 U / mL, and even more preferably 100 to 300 U / mL. Similarly, the concentration of IL-33 can also be appropriately selected by those skilled in the art, and its concentration is preferably 0.1 to 10,000 ng / mL, more preferably 1 to 1,000 ng / mL, and even more preferably 10 to 100 ng / mL. In one embodiment, the culture medium used for inducing ILC2 differentiation may further contain one or more proteins selected from the group consisting of SCF, TPO (thrombopoietin), FLt3L, SDF-1α, and IL-7. Regarding their concentrations, for example, SCF is 10 ng / mL to 100 ng / mL, TPO is 10 ng / mL to 200 ng / mL, FLt3L is 10 ng / mL to 100 ng / mL, SDF-1α is 100 ng / mL to 500 ng / mL, and IL-7 is 10 ng / mL to 100 ng / mL.

[0074] During this culture process, the culture medium can be changed as appropriate, and the types and concentrations of additives in each culture medium can be adjusted accordingly.

[0075] Uses of Type II Intrinsic Lymphocytes Obtained Through This Invention

[0076] Type II innate lymphocytes (ILC2) obtained by the method of the present invention, and in one embodiment IL-10-producing ILC2, are also included in the present invention. These ILC2s can be used without limitation for various purposes. The ILC2 obtained by the present invention can, of course, be used in laboratories in experiments, etc., and can also be used as a therapeutic agent for diseases. In particular, according to the present invention, IL-10-producing ILC2 can be obtained, therefore the ILC2 of the present invention is useful, for example, for the treatment and prevention of inflammatory diseases. In one embodiment, the present invention provides a therapeutic agent for inflammatory diseases comprising IL-10-producing ILC2 obtained by the method of the present invention, and a method for treating inflammatory diseases including the step of administering the IL-10-producing ILC2 to a subject.

[0077] Examples of diseases to which this invention is applicable include inflammatory diseases such as ulcerative colitis and Crohn's disease, rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, multiple sclerosis, pernicious anemia, pemphigus, autoimmune diseases such as vasculitis, allergic diseases, cancer, acute respiratory distress syndrome (ARDS), and chronic obstructive pulmonary disease (COPD).

[0078] When treating a disease using the ILC2 obtained by the present invention, the ILC2 of the present invention is administered to the subject as a therapeutic agent. There are no particular limitations on the method of administration; for example, it can generally be administered to the subject in the form of a pharmaceutical composition comprising a carrier, excipients, and other additives for formulation. Examples of methods for administering the ILC2 to the subject include: suspending the manufactured ILC2 together with the carrier, excipients, and other additives for formulation in physiological saline or the like and directly transplanting it into the subject's tissue; or suspending the manufactured ILC2 together with the carrier, excipients, and other additives for formulation in physiological saline or the like and intravenously injecting it.

[0079] When administering the pharmaceutical composition containing ILC2 of the present invention to a subject, the number of ILC2 cells administered may be adjusted appropriately according to the severity of the disease, the affected area, the size of the body, etc.

[0080] The present invention will now be described in more detail with reference to specific examples, but the present invention is not limited to the specific examples described below. It should be noted that, unless otherwise specified, in this specification, concentration, etc., are based on weight, and numerical ranges are described in a form including their endpoints.

[0081] Example

[0082] Experiment 1: Induction of differentiation from iPS cells to hematopoietic progenitor cells (HPCs)

[0083] On a 6-well plate (Corning, 3471) after ultra-low adhesion treatment, at 3.0 × 10⁻⁶ 5iPS cells were seeded per well (Day 0). ROCK (Rho-associated coiled-coil forming kinase) inhibitor and GSK-3 (Glycogen synthase kinase 3) inhibitor were added to the iPS maintenance medium (Ajinomoto, StemFit AK02N). The cells were cultured at 37°C in a 5% CO2 atmosphere for 1 day to obtain embryoid bodies (EB). In this experiment, either the 1231A2 strain or the Ff-I01s04 strain established at the iPS Cell Research Institute (CiRA) of Kyoto University was used as the iPS cells. Additionally, Y-27632 (Fujifilm and Koichi Pharmaceutical, 036-24023 or 034-24024) was used as a ROCK inhibitor, and CHIR99021 (Tocris, 4423) was used as a GSK-3 inhibitor, both added to iPS maintenance medium at 10 μM.

[0084] Next, the iPS maintenance medium was changed to embryoid medium (EB medium), and 50 ng / mL BMP4 (R&D, 314-BP), 50 ng / mL VEGF (R&D, 293-VE-500MG / CF), and 50 ng / mL FGF-2 (Peprotech, 100-18B) were added (Day 1). The next day, SB431542 (CAYMAN, 13031) was added to bring the final concentration to 6 μM (Day 2). After two more days of culture, the medium was changed to fresh EB medium, and 50 ng / mL SCF (R&D, 255-SC-200 or Fujifilm and Koden Chemical, 193-15513), 50 ng / mL VEGF, and 50 ng / mL FGF-2 were added (Day 4). After culturing for 3 days, the medium was replaced with fresh EB medium, supplemented with 50 ng / mL SCF, 50 ng / mL VEGF, 50 ng / mL FGF-2, 30 ng / mL TPO (Peprotech, 300-18 or Fujifilm and Hikari Pure Chemicals, 204-16474), and 10 ng / mL Flt3L (Peprotech, 300-19 or Fujifilm and Hikari Pure Chemicals, 067-05393) (day 7). After a total of 9-11 days of culture from day 0, the culture medium containing hematopoietic progenitor cells was recovered and passed through a 40 μm filter to form single cells. These cells were then centrifuged at 400 g, 4°C, for 5 minutes. After removing the supernatant, the HPCs were resuspended in cell cryopreservation medium (KAC, TC protector) and cryopreserved at -80°C (these cryopreserved cells were used as HPC reserve cells).

[0085] It should be noted that the composition of the EB culture medium used in this experiment is as follows.

[0086] [EB medium]

[0087] StemPro-34 SFM (trademark, Gibco, 10639011)

[0088] • 1% GlutaMAX (trademark, Gibco, 35050061)

[0089] • 1% Insulin-Transferrin-Selenium Mixture (ITS-G, Gibco, 41400045)

[0090] • 0.2% L-glutamine-penicillin-streptomycin solution (Sigma Aldrich, G1146)

[0091] 0.4 mM α-monothioglycerol (Sigma Aldrich, M1753)

[0092] • 50 μg / mL ascorbic acid 2-phosphate (Sigma Aldrich, A8960)

[0093] Experiment 2: Differentiation induction from HPC derived from iPS cells to ILC2 (1)

[0094] HPC cells derived from iPS cells in Experiment 1 were used for differentiation induction culture into ILC2. Specifically, 2,500–20,000 HPC reserve cells derived from strain 1231A2 were seeded in 96-well plates (Corning, 3799 or Iwaki, 3860-096) coated with 2.5 μg / mL Fc-DLL4 and 2.5 μg / mL RetroNectin (trademark) (day 0), and differentiation induction culture was performed for a total of 12 days at 37°C and 5% CO2 atmosphere under the culture conditions shown in the table below.

[0095] During the 12-day differentiation induction culture, 100 μL of medium was added on Day 3, and the medium was changed on Day 6 and Day 9. After the medium changes on Day 6 and Day 9, once the cells in the wells had reached confluence, the cells were resuspended and reseeded in new 96-well plates at a dilution of ×1 / 2. It should be noted that in conditions 2–4, IL-2 (BioLegend, 589108 or PeproTech, 200-02) was added to medium A at a concentration of 200 U / mL. In addition, in condition 3, Senexin A (Senexin A, Selleck, S8520), a CDK8 inhibitor, was added to culture medium B at a concentration of 3 μM, and in condition 4, CCT-251921 (MedChemExpress, HY-19984), a CDK8 inhibitor, was added to culture medium B at a concentration of 100 nM.

[0096] [Table 1]

[0097]

[0098] Here, in Cancer Sci. 2020 May;111(5):1478-1490, we describe how to induce NK / ILC1-like cells from iPS cells through the following three steps.

[0099] Step 1: Induction of hematopoietic precursor cells from iPS cells (14 days)

[0100] Step 2: Induction of lymphocyte precursor cells from iPS-derived hematopoietic precursor cells on FcDLL4 (21 days)

[0101] Step 3: Steps to induce the generation and proliferation of NK / ILC1-like cells

[0102] In the inventors' preliminary studies, the presence of ILC1-like cells producing IFN-γ was confirmed, albeit in very small numbers, on day 12 of "Step 2". Therefore, considering the possibility of NK cells and pluripotent ILC precursor cells capable of differentiating into all ILC subsets appearing in this "Step 2", various factors were investigated. "Condition 1" in this experiment corresponds to the first 12 days of "Step 2" in the aforementioned NK / ILC1-like cell differentiation induction method.

[0103] It should be noted that culture medium A and culture medium B used in this experiment were prepared as shown in the table below.

[0104] [Table 2]

[0105] [Table 3]

[0106] For differentiated cells cultured as described above, stimulate them for 4–6 hours at 37°C in a 5% CO2 atmosphere.

[0107] ·IL-1β (Peprotech, 200-01B), final concentration 10 ng / mL

[0108] ·IL-2, final concentration 20 ng / mL

[0109] IL-7, final concentration 50 ng / mL

[0110] ·IL-23 (BioLegend, 574106), final concentration 20 ng / mL

[0111] ·IL-25 (IL-17E) (Peprotech, 200-24), final concentration 20 ng / mL

[0112] eBioscience (trademark) Cell Stimulation Mixture (plus Protein Transport Inhibitor) (Thermo Fisher Scientific, 00-4975-03)

[0113] Next, the stimulated cells were stained and analyzed by flow cytometry using an LSR Tortessa (trademark) Cell Analyzer (BDBiosciences). Cell staining was performed using FlowJo (trademark) (BDBiosciences), as described below.

[0114] • LIVE / DEAD (trademark) Fixed Aqua Dead Cell Staining Kit (Thermo Fisher Scientific, L-34957)

[0115] • Anti-CD3 antibody (BioLegend, UCHT1)

[0116] • Anti-CD14 antibody (BioLegend, M5E2)

[0117] • Anti-CD16 antibody (BioLegend, 3G8)

[0118] • Anti-CD19 antibody (BioLegend, HIB19)

[0119] • Anti-CD20 antibody (BioLegend, 2H7)

[0120] • Anti-CD94 antibody (BD Pharmingen, HP-3D9)

[0121] • Anti-GATA3 antibody (Thermo Fischer Scientific, TWAJ)

[0122] • Anti-IL-13 antibody (BioLegend, JES-5A2)

[0123] • Anti-IL-10 antibody (BioLegend, JES-9D7)

[0124] Representative examples of flow cytometry results are shown in Figure 1-1 Additionally, the percentage of lineage marker-negative GATA3-positive IL-13-positive cells (%ILC2) and the percentage of lineage marker-negative IL-13-positive and IL-10-positive cells (%IL-10+ILC2) in live cells are shown in the figure. Figure 1-2 .

[0125] According to flow cytometry results, under "Condition 3" with the addition of Senexin A as a CDK8 inhibitor, the induction of an ILC2-like cell population that does not express lineage markers (CD3, CD14, CD16, CD19, CD20, CD94), expresses the transcription factor GATA3, and has the ability to produce IL-13 was confirmed. A subset of the ILC2-like cells induced under "Condition 3" also had the ability to produce IL-10. Furthermore, under "Condition 4" with the addition of CCT-251921, which has a different structure than Senexin A, as a CDK8 inhibitor, the induction of an ILC2-like cell population was also confirmed in the same way as under "Condition 3".

[0126] Based on these results, it is believed that culturing HPCs in the presence of a CDK8 inhibitor can induce differentiation into ILC2.

[0127] Experiment 3: Differentiation induction from HPC derived from iPS cells to ILC2 (2)

[0128] In this experiment, to confirm the universality of the differentiation induction method in Experiment 2, HPC cells derived from different iPS cell lines (Ff-I01s04) were used to confirm the differentiation induction to ILC2. Specifically, HPC reserve cells prepared from iPS cells (Ff-I01s04) using the method described in Experiment 1 were cultured to induce differentiation to ILC2 using the method described in Experiment 2.

[0129] Representative examples of the results analyzed using flow cytometry as described in Experiment 2 are shown below. Figure 2 In the case of HPC derived from the Ff-I01s04 strain, similar to the case of HPC derived from the 1231A2 strain, differentiation induction into ILC2-like cells containing IL-10-producing cells was confirmed under "Condition 3" and "Condition 4" with the use of the CDK8 inhibitor.

[0130] That is, ILC2 can be induced by using any HPC made from different iPS cell lines, which confirms that the differentiation induction method of the present invention is universal.

[0131] Experiment 4 Differentiation induction from HPC derived from iPS cells to ILC2 (3)

[0132] In this experiment, several CDK8 inhibitors with different structures were used to confirm the induction of differentiation into ILC2. Specifically, HPC reserve cells prepared from iPS cells (1231A2 strain) using the method described in Experiment 1 were cultured under condition 2 of Experiment 2 with CDK8 inhibitors (compounds 1-4) (see table below) added to induce differentiation into ILC2.

[0133] The results of flow cytometry analysis as described in Experiment 2 are shown in Figure 3. With the addition of any of compounds 1–4, differentiation into ILC2-like cells containing IL-10-producing cells was confirmed, similar to the addition of Senexin A and CCT-251921.

[0134] Based on these results, it is believed that culturing HPCs in the presence of a CDK8 inhibitor can induce differentiation into ILC2.

[0135] [Table 4]

[0136]

[0137] Experiment 5: Differentiation induction from CD34-positive HPC derived from human umbilical cord blood to ILC2

[0138] In this experiment, to confirm the universality of the differentiation induction method in Experiment 2, the differentiation induction from human-derived HPC to ILC2 was verified. Specifically, CD34-positive cells (Lonza, 2C-101) derived from human umbilical cord blood were used for differentiation induction into ILC2 cells using the methods described in Experiment 2 under conditions 1, 2, and 3. It should be noted that in this experiment, the same culture medium as on days 6–12 was used for culture on days 12 and 15, and the cells were reseeded at a dilution of ×1 / 2 once confluence had been achieved in the culture wells.

[0139] Representative examples of results analyzed by flow cytometry on day 19 of culture, as in Experiment 2 on day 12, are shown below. Figure 4 In the case of HPC derived from purchased human umbilical cord blood, similar to the case of HPC derived from iPS cells, differentiation into ILC2-like cells containing IL-10-producing cells was induced under "Condition 3" with the use of a CDK8 inhibitor.

[0140] That is, it can be confirmed that the differentiation induction method of the present invention can be applied not only to HPC derived from iPS cells, but also to HPC isolated from the human body.

[0141] Experiment 6: Cytokine concentrations in the supernatant of differentiated cell culture containing ILC2 derived from iPS cells

[0142] The culture supernatant from differentiation-induced cultures under conditions "1", "2", and "3" described in Experiment 2 was recovered on day 12. The concentrations of IL-10, IL-5, and bimodalin in the culture supernatant were quantified using the kits described below. Here, bimodalin is considered a core cytokine of the tissue repair capabilities of ILC2; a higher concentration of bimodalin indicates the presence of ILC2 with tissue repair capabilities.

[0143] • IL-10: Human IL-10 Immunoassay Kit (PerkinElmer, AL218C)

[0144] • IL-5: Human IL-5 Immunoassay Kit (PerkinElmer, AL267C)

[0145] • Bimodal proteins: Human bimodal protein Quantikine ELISA kit (R&D Systems, DAR00)

[0146] IL-10 and IL-5 were measured using Envision (Revvity). Bismoglobin was measured using Powerscan4 (DSPharma Biomedical). Figure 5 The results show that, under "condition 3" for ILC2 induction, the production of IL-10, IL-5 and bimodal proteins in the culture supernatant was increased in Experiment 2.

[0147] Experiment 7: Inhibition of TNFα production in THP-1 cells by IL-10-producing ILC2 For differentiated cells induced under "condition 1" and "condition 3" as described in Experiment 2, cultured for 12 days (frozen at -80°C), the inhibitory effect on TNFα production in THP-1 cells was confirmed.

[0148] 7-1. THP-1 cells

[0149] 1×10 7 One THP-1 cell (ATCC) was suspended in 10 mL of THP-1 proliferation medium. Then, PMA (phorbol ester, Sigma Aldrich, P-1585) was added as a cell stimulant to a final concentration of 2 μg / mL. The cells were cultured in 100 mm culture dishes (Iwaki, 3020-100) at 37°C under a 5% CO2 atmosphere for 2 days to induce differentiation into macrophages. On the second day of culture, THP-1 cells were recovered and centrifuged at 300 g, room temperature, for 5 minutes. The supernatant was removed to obtain 1 × 10⁶ cells / mL. 6 THP-1 proliferation medium was added at a rate of 1 / mL.

[0150] 7-2. Differentiated Cells

[0151] Differentialized cells obtained under "Condition 1" and "Condition 3" in Experiment 2, which were cryopreserved, were resuspended in the culture medium shown in the table below, as in Experiment 2, and cultured in 96-well plates (Iwaki, 3860-096) at 37°C and 5% CO2 for 2 days. After culture, the differentiated cells obtained under "Condition 1" and "Condition 3" were recovered and centrifuged at 300g, room temperature, for 5 minutes. The supernatant was removed to obtain 1×10⁻⁶ cells. 5 cells / mL or 2×10 4 ILC basal medium is added at a rate of 1 per mL.

[0152] [Table 5]

[0153]

[0154] 7-3. Inhibition of TNFα production by ILC2-like cells

[0155] Next, lipopolysaccharide (LPS) and IFN-γ were introduced into THP-1 cells differentiated into macrophages to induce inflammation, and the secretion of TNFα, an inflammatory cytokine, was confirmed. Specifically, in 96-well plates, using functional evaluation medium supplemented with LPS and IFN-γ, the following (a)–(c) were cultured at 37°C under a 5% CO2 atmosphere for 2 days.

[0156] (a) Differentiated cells obtained under “Condition 1” (5 × 10⁻⁶) 3 One or 1×10 3 (number) and THP-1 cells (ATCC, 5×10) 4 indivual)

[0157] (b) Differentiated cells obtained under “Condition 3” (5 × 10 3 One or 1×10 3 (number) and THP-1 cells (ATCC, 5×10) 4 indivual)

[0158] (c) THP-1 cells only (ATCC, 5×10 4 (THP-1 only)

[0159] On day 2 of culture, the culture supernatant was recovered, and the concentration of TNFα in the recovered culture supernatant was determined using an immunoassay kit (Human TNFα immunoassay kit, PerkinElmer, AL208C). The concentration of TNFα was determined according to the protocol provided with the kit. The assay was performed using Envision.

[0160] The results of the determination of TNFα concentration in the culture supernatant are shown below. Figure 6The results of this assay indicate that differentiated cells under "condition 3" inhibited the production of TNFα in THP-1 cells.

[0161] Here, the composition of THP-1 proliferation medium, ILC basal medium, and THP-1 functional evaluation medium is as follows.

[0162] [THP-1 proliferation medium]

[0163] • RPMI medium (Fujifilm and Kodenpaste, 189-02025)

[0164] ·10% FBS

[0165] ·1% L-glutamine-penicillin-streptomycin mixed solution

[0166] [ILC basal medium]

[0167] α-MEM

[0168] ·15% FBS

[0169] ·1% L-glutamine-penicillin-streptomycin mixed solution

[0170] [Functional Evaluation Culture Medium]

[0171] 44% RPMI medium

[0172] 42% α-MEM

[0173] ·12% FBS

[0174] ·1% L-glutamine-penicillin-streptomycin mixed solution

[0175] • 1% Insulin-Transferrin-Selenium Mixture

[0176] 55 μM 2-mercaptoethanol

[0177] 50 μg / mL ascorbic acid 2-phosphate

[0178] 50 ng / mL IL-7

[0179] 240 ng / mL SDF-1α

[0180] 200 U / mL IL-2

[0181] 20 ng / mL IL-33

[0182] ·100ng / mL LPS (Sigma Aldrich, L6511)

[0183] ·20ng / mL IFN-γ (BioLegend, 570204)

[0184] Experiment 8: TNFα production in THP-1 cells showing CD25 and CD30 expression in IL-10-producing ILC2 cells Inhibition

[0185] For differentiated cells induced under “condition 3” as described in Experiment 2 and cultured for 12 days (frozen at -80°C), the inhibitory effects of CD25+CD30+ and CD25-CD30- groups on TNFα production in THP-1 cells were investigated.

[0186] 8-1. THP-1 cells

[0187] 1×10 7 One THP-1 cell (ATCC) was suspended in 15 mL of THP-1 proliferation medium. Then, PMA was added as a cell stimulant to a final concentration of 2 μg / mL. The cells were cultured in 100 mm culture dishes at 37°C under a 5% CO2 atmosphere for 2 days to induce differentiation into macrophages. On the second day of culture, THP-1 cells were recovered and centrifuged at 300 g, room temperature, for 5 minutes. The supernatant was removed to obtain 1 × 10⁶ cells / mL. 6 THP-1 proliferation medium was added at a rate of 1 / mL.

[0188] 8-2. ILC2 differentiated cells

[0189] Differentialized cells obtained under "Condition 3" in Experiment 2 were suspended in the culture medium shown in the table below, just as in Experiment 2, and cultured in 96-well plates at 37°C and 5% CO2 for 2 days. After culture, the differentiated cells obtained under "Condition 3" were recovered and centrifuged at 300g, room temperature, for 5 minutes. The supernatant was removed, and 100 μL of PBS(-), 4 μL of anti-CD25 antibody (Miltenyi, REA570), 40 μL of anti-CD30 antibody (BD Biosciences, BerH8), and 1 μL of LIVE / DEAD (trademark) Fixed Aqua dead cell staining kit were added. The cells were incubated on ice for 20 minutes. Then, the cells were washed twice with PBS(-), and CD25-CD30- and CD25+CD30+ groups were separated using FACS Aria4 (BD Biosciences). For each cell, the cells were divided into groups of 2 × 10⁻⁶ cells. 5 cells / mL, 1×10 5 cells / mL or 2×10 4 The sample was prepared using ILC basal medium at a rate of 1 sample per mL.

[0190] [Table 6]

[0191]

[0192] 8-3. Inhibition of TNFα production by ILC2-like cells

[0193] Next, inflammation was induced by contacting lipopolysaccharide (LPS) and IFN-γ with THP-1 cells differentiated into macrophages, and the secretion of TNFα, an inflammatory cytokine, was confirmed. Specifically, in 96-well plates, THP-1 functional evaluation medium supplemented with LPS and IFN-γ was used, and the following (a)–(c) were cultured at 37°C under a 5% CO2 atmosphere for 2 days.

[0194] (a) CD25-CD30- cells (1×10⁻⁶) obtained under “Condition 3”. 4 5×10 3 One or 1×10 3 (number) and THP-1 cells (ATCC, 5×10) 4 indivual)

[0195] (b) CD25+CD30+ cells obtained under “Condition 3” (1×10⁻⁶) 4 5×10 3 One or 1×10 3 (number) and THP-1 cells (ATCC, 5×10) 4 indivual)

[0196] (c) THP-1 cells only (ATCC, 5×10 4 (THP-1 only)

[0197] On day 2 of culture, the culture supernatant was recovered, and the concentrations of TNFα and IL-10 in the recovered supernatant were determined using an immunoassay kit. The concentrations of each cytokine were measured according to the protocol provided with the kit. The assays were performed using Envision.

[0198] The results of the determination of TNFα concentration in the culture supernatant are shown below. Figure 7 The results show that, under "condition 3," CD25+CD30+ cells inhibited TNFα production in THP-1 cells, while CD25-CD30- cells did not inhibit TNFα production. Additionally, the results regarding the concentration of IL-10 in the culture supernatant are presented below. Figure 7 Lower part. CD25+CD30+ cells showed IL-10 production, while CD25-CD30- cells did not show IL-10 production.

[0199] Experiment 9: Inhibition of TNFα production by IL-10-producing ILC2-derived macrophages from PBMCs

[0200] For differentiated cells induced on day 12 of culture under “condition 3” as described in Experiment 2 (cryopreserved at -80°C), the inhibitory effect of IL-10-producing ILC2 on TNFα production from macrophages derived from PBMCs was investigated.

[0201] 9-1. Macrophages derived from PBMCs

[0202] PBMC stock solution thawed at 37°C was resuspended in macrophage differentiation medium containing 20 U / mL DNAase (Merck, D5025) and centrifuged at 300g, room temperature, for 5 minutes. The supernatant was removed, and 1 mL of macrophage differentiation medium was added. CD14-positive cells were separated from the PBMC suspension using CD14 microbeads (Miltenyi, 130-050-201). The obtained CD14-positive cells were centrifuged at 300g, room temperature, for 5 minutes and resuspended in 3 mL of macrophage differentiation medium containing 100 ng / mL GM-CSF (Peprotech, 300-03). 1 mL of the medium was added to each of three wells in a 24-well plate (Iwaki, 3820-024) and cultured at 37°C under a 5% CO2 atmosphere. On days 3 and 6 of culture, 800 μL of supernatant was removed, and 1 mL of macrophage differentiation medium containing 100 ng / mL GM-CSF was added. On day 8 of culture, the cell supernatant was removed, and 300 μL of PBS was added, followed by another removal of the supernatant. Then, 200 μL of Accutase (InnovativeCell Technologies, AT104) was added, and the cells were incubated at 37°C for 10 minutes. Next, 800 μL of macrophage differentiation medium was added, and the cells were recovered. Centrifugation was performed at 300 g, room temperature, for 5 minutes to obtain 4 × 10⁶ cells / mL. 5 Macrophage differentiation medium was added at a rate of 1 cell / mL.

[0203] 9-2. ILC2 differentiated cells

[0204] Cryopreserved differentiated cells obtained under "Condition 1" and "Condition 3" in Experiment 2 were suspended in the culture medium shown in the table below, as in Experiment 2, and cultured in 96-well plates at 37°C and 5% CO2 atmosphere for 2 days. After culture, the differentiated cells obtained under "Condition 1" and "Condition 3" were recovered and centrifuged at 300g, room temperature, for 5 minutes. The supernatant was removed to obtain 4×10⁻⁶ cells. 5 cells / mL or 8×10 4 ILC basal medium is added at a rate of 1 per mL.

[0205] [Table 7]

[0206]

[0207] 9-3. Inhibition of TNFα production by ILC2-like cells

[0208] Next, inflammation was induced by contacting lipopolysaccharide (LPS) and IFN-γ with macrophages derived from PBMCs to confirm the secretion of TNFα, an inflammatory cytokine. Specifically, in 96-well plates, macrophages derived from PBMCs were cultured for 2 days at 37°C under a 5% CO2 atmosphere using a macrophage function evaluation medium supplemented with LPS and IFN-γ. (a)–(c)

[0209] (a) Differentiated cells obtained under “Condition 1” (2 × 10⁻⁶) 4 One or 4×10 3 ( ) and macrophages derived from PBMCs (2 × 10 ) 4 indivual)

[0210] (b) Differentiated cells obtained under “Condition 3” (2 × 10⁻⁶) 4 One or 4×10 3 ( ) and macrophages derived from PBMCs (2 × 10 ) 4 indivual)

[0211] (c) Macrophages derived solely from PBMCs (2 × 10⁻⁶) 4 indivual)

[0212] On day 2 of culture, the culture supernatant was recovered, and the concentration of TNFα in the recovered culture supernatant was determined using an immunoassay kit. The concentration of TNFα was determined according to the protocol provided with the kit. The assay was performed using Envision.

[0213] The results of the determination of TNFα concentration in the culture supernatant are shown below. Figure 8 The results of this assay indicate that differentiated cells under "condition 3" inhibited the production of TNFα by macrophages derived from PBMCs.

[0214] Macrophage differentiation culture medium

[0215] RPMI medium

[0216] ·10% FBS

[0217] ·1% L-glutamine-penicillin-streptomycin mixed solution

[0218] • 1% sodium pyruvate solution (Nacalai Tesque, 06977-034)

[0219] • 1% MEM non-essential amino acid solution (Fujifilm and Koichi Pharmaceutical, 139-15651)

[0220] [Macrophage Function Evaluation Medium Derived from PBMCs]

[0221] 44% RPMI medium

[0222] 41% α-MEM

[0223] ·12% FBS

[0224] ·1% L-glutamine-penicillin-streptomycin mixed solution

[0225] • 1% Insulin-Transferrin-Selenium Mixture

[0226] 0.5% sodium pyruvate solution

[0227] · 0.5% MEM non-essential amino acid solution

[0228] 55 μM 2-mercaptoethanol

[0229] 50 μg / mL ascorbic acid 2-phosphate

[0230] 50 ng / mL IL-7

[0231] 240 ng / mL SDF-1α

[0232] 200 U / mL IL-2

[0233] 20 ng / mL IL-33

[0234] 100 ng / mL LPS

[0235] 20 ng / mL IFN-γ

[0236] Industrial availability

[0237] As detailed above, this invention provides a method for differentiating and inducing the production of ILC2 or IL-10-producing ILC2 from hematopoietic progenitor cells derived from pluripotent stem cells. The ILC2 or IL-10-producing ILC2 obtained by this method can be used in drug development.

Claims

1. A method of manufacturing type II innate lymphoid cells, comprising the step of culturing hematopoietic precursor cells in the presence of a CDK8 inhibitor to obtain type II innate lymphoid cells.

2. The method of claim 1, wherein, The type II innate lymphoid cells comprise IL-10 producing type II innate lymphoid cells.

3. The method of claim 1 or 2, which is performed in vitro.

4. The method of claim 1 or 2, wherein, The hematopoietic precursor cells are cultured in the presence of a CDK8 inhibitor, IL-2, and IL-33 to obtain type II innate lymphoid cells.

5. The method of claim 1 or 2, wherein, The CDK8 inhibitor comprises Senexin A, CCT-251921, MSC2530818, CCT251545, SEL120-34A, or BRD6989.

6. The method of claim 1 or 2, wherein, The hematopoietic precursor cells are hematopoietic precursor cells differentiated from human induced pluripotent stem cells (human iPS cells).

7. The method of claim 1 or 2, wherein, The hematopoietic precursor cells are hematopoietic precursor cells derived from a human body.

8. The method of claim 1 or 2, further comprising the step of inducing differentiation from pluripotent stem cells to hematopoietic precursor cells.

9. The method of claim 8, wherein, The pluripotent stem cells are induced pluripotent stem cells (iPS cells).

10. The method of claim 8, wherein, The pluripotent stem cells are human iPS cells.

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