Method for inducing the expression of regulatory T cell inducers in skeletal muscle-derived cells

Abstract

This invention provides a novel method for avoiding or mitigating immune rejection in a patient, even when a cell preparation for regenerative medicine is prepared using allogeneic cells as raw materials and administered to the patient. [Solution] Surface antigen expression in SkM is induced by culturing Skeletal muscle-derived cells (SkM), in which more than 90% are CD56-positive cells, in a culture medium containing interferon-γ (IFN-γ), or by co-culturing them with peripheral blood mononuclear cells (PBMCs).

Description

[Technical Field]

[0001] This disclosure relates to a method for inducing the expression of regulatory T cell-inducing factors in skeletal muscle-derived cells, and to pharmaceutical products. [Background technology]

[0002] In recent years, regenerative medicine technologies to treat heart failure and tissue loss have been rapidly developing. For example, in the field of heart failure treatment, "cell sheet technology" is being researched and developed, which involves collecting the patient's cells (autologous cells), culturing and differentiating them, processing them into a sheet, and attaching it to the affected area to promote tissue repair. An example of such cell sheet technology is the cardiac patch, which has been transplanted to restore function lost due to myocardial infarction, and good results have been reported. In addition to cardiac patches, research is also being conducted on cell transplantation techniques using sheet-formed cells for various tissues and organs, such as skin wound treatment, the liver, and the cornea.

[0003] Incidentally, cell sheets using autologous cells have the advantage of virtually eliminating the problem of immune rejection because they use cells collected from the patient themselves, which are then proliferated and differentiated. However, there are problems with mass production, such as the significant surgical burden involved in collecting cells from the patient and the need to collect and culture cells individually for each patient. Furthermore, the time required from collection to culture and differentiation induction makes it difficult to use in acute treatments.

[0004] One approach to address these challenges is to use cell sheets derived from allogeneic cells. Using allogeneic cells allows for the pre-culturing and stocking of a sufficient quantity of cells, enabling rapid product delivery to patients in need. However, when allogeneic cells are transplanted, the patient's (receptor's) immune system may recognize the transplanted cells as foreign, making immune rejection highly likely. Therefore, in practice, when using allogeneic cells, methods such as administering immunosuppressants or using genetic modification techniques to suppress or remove the expression of the major histocompatibility complex (HLA) in the cells are employed. For example, Patent Document 1 discloses a technique to reduce rejection reactions when allogeneic cells are transplanted into a recipient by using cells that do not express HLA-A and HLA-B proteins, but express at least one HLA-C protein, and in which at least one of the HLA alleles (HLA-A, HLA-B, and HLA-C) is homozygous, achieved by disrupting the HLA-A and HLA-B alleles of donor cells through genome editing. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2023-126897 [Overview of the project] [Problems that the invention aims to solve]

[0006] This disclosure aims to provide a novel means of avoiding or mitigating immune rejection in a target patient, even when a cell preparation for regenerative medicine is prepared using allogeneic cells as raw materials and administered to the patient. [Means for solving the problem]

[0007] The present inventors have conducted intensive studies to solve the above problems. As a result, the inventors have found that the above problems can be solved by co-culturing skeletal muscle-derived cells (SkM) with peripheral blood mononuclear cells (PBMC) to induce the expression of regulatory T cell (Treg) inducing factors in SkM.

[0008] That is, the above objects can be achieved by the present disclosure having the following configuration, and the present disclosure includes the following aspects and forms. More specifically, the present disclosure provides the following subjects A to D.

[0009] Subject A of the present disclosure is based on the new finding that co-culturing skeletal muscle-derived cells (SkM) with peripheral blood mononuclear cells (PBMC) induces expression of regulatory T cell (Treg) inducing factors in SkM. Subject A includes the following aspects A1 to A4.

[0010] Aspect A1 of the present disclosure is 1. A method for inducing expression of a regulatory T cell (Treg) inducing factor in SkM, comprising co-culturing skeletal muscle-derived cells (SkM), 90% or more of which are CD56-positive cells, with peripheral blood mononuclear cells (PBMC): 2. In the method according to the above 1., the Treg inducing factor preferably comprises one or more selected from the group consisting of IDO1, IL-10 and TGF-β: 3. In the method according to the above 1. or 2., the Treg inducing factor is preferably secreted into the supernatant of the co-culture medium: 4. In the method according to any one of the above 1. to 3., the Treg inducing factor comprises IDO1 and / or IL-10, when the Treg inducing factor comprises IDO1, expression of IDO1 is preferably first confirmed when SkM is co-cultured with the PBMC, when the Treg inducing factor comprises IL-10, the expression level of IL-10 is preferably increased 10 times or more as compared with the case where SkM is cultured alone: Aspect A2 of the present disclosure is 5. A method for suppressing cellular immunity in the SkM, comprising performing any of the methods described in 1. to 4. above.

[0011] Aspect A3 of this disclosure is This method involves co-culturing skeletal muscle-derived cells (SkM), in which 6.90% or more are CD56-positive cells, with peripheral blood mononuclear cells (PBMCs), thereby inducing the expression of regulatory T cell (Treg)-inducing factors in SkM cells: 7. In the manufacturing method described in 6. above, it is preferable that the Treg-inducing factor includes one or more selected from the group consisting of IDO1, IL-10, and TGF-β.

[0012] Aspect A4 of this disclosure is 8. This pharmaceutical product contains skeletal muscle-derived cells (SkM), which are CD56-positive and exhibit increased expression of regulatory T cell (Treg) inducers compared to cells cultured alone, as its active ingredient: 9. In the pharmaceutical product described in 8. above, it is preferable that the Treg-inducing factor includes one or more selected from the group consisting of IDO1, IL-10, and TGF-β: 10. The pharmaceutical product described in item 8 or 9 above is preferably a cell preparation for regenerative medicine; 11. In the pharmaceutical product described in any of items 8 to 10 above, it is preferable that the SkM is an allogeneic cell of the target of the pharmaceutical product administration.

[0013] Subject B of this disclosure is based on the novel finding that surface antigen expression in skeletal muscle-derived cells (SkM) can be induced by culturing SkM in a medium containing interferon-γ (IFN-γ) or by co-culturing SkM with peripheral blood mononuclear cells (PBMCs). Subject B encompasses the following embodiments B1 to B4.

[0014] Aspect B1 of this disclosure is, This method involves inducing the expression of surface antigens in skeletal muscle-derived cells (SkM), in which 1.90% or more are CD56-positive cells, by culturing them in a medium containing interferon-γ (IFN-γ) or by co-culturing them with peripheral blood mononuclear cells (PBMCs); 2. In the method described in 1. above, it is preferable that the SkM is obtained by adherent culture; 3. In the method described in 1. or 2. above, the surface antigen preferably comprises one or more selected from the group consisting of costimulatory molecules, programmed cell death ligands, cell adhesion molecules, TIGIT ligands, and non-classical HLA class I antigens; 4. In any of the methods described in 1. to 3. above, the surface antigen preferably comprises one or more selected from the group consisting of PD-L1, PD-L2, ICAM-1, CD155, and HLA-E; 5. The method described in any of 1. to 4. above comprises culturing the SkM in a medium containing the interferon-γ (IFN-γ), wherein the concentration of IFN-γ in the medium is preferably 50 ng / mL or higher.

[0015] 6. In any of the methods described in 1. to 5. above, it is preferable that the expression rate of the surface antigen in the cultured SkM is 1.02 or higher as a relative value, with the expression rate of the surface antigen in SkM cultured alone being set to 1; Aspect B2 of this disclosure is, 7. A method for suppressing cellular immunity in the SkM, comprising performing any of the methods described in 1. to 6. above.

[0016] Aspect B3 of this disclosure is, A method for producing SkM cells in which surface antigen expression is induced, comprising culturing SkM cells, in which 8.90% or more are CD56-positive cells, in a medium containing interferon-γ (IFN-γ), or co-culturing SkM cells with peripheral blood mononuclear cells (PBMCs); 9. In the method for producing SkM described in 8. above, it is preferable that the SkM is obtained by adherent culture; 10. In the method for producing SkM described in 8. or 9. above, it is preferable that the surface antigen includes one or more selected from the group consisting of PD-L1, PD-L2, ICAM-1, and HLA-E.

[0017] Aspect B4 of this disclosure is, 11. This is a pharmaceutical product containing skeletal muscle-derived cells (SkM) as an active ingredient, in which more than 90% are CD56-positive cells and the expression rate of surface antigens is increased compared to the control; 12. In the pharmaceutical product described in 11. above, it is preferable that the surface antigen comprises one or more selected from the group consisting of PD-L1, PD-L2, ICAM-1, and HLA-E; 13. The pharmaceutical product described in item 11 or 12 above is preferably a cell preparation for regenerative medicine.

[0018] 14. In the pharmaceutical product described in any of items 11 to 13 above, it is preferable that the SkM is an allogeneic cell of the target of the pharmaceutical product administration.

[0019] Subject C of this disclosure is based on the novel finding that the expression of surface antigens in skeletal muscle-derived cells (SkM) is suppressed by four or more passages. Subject C encompasses the following embodiments C1 to C4.

[0020] Aspect C1 of this disclosure is, This method involves subculturing skeletal muscle-derived cells (SkM) in which 1.90% or more are CD56-positive cells at least four times to suppress the expression of surface antigens in SkM; 2. In the method described in 1. above, it is preferable that the SkM is obtained by adherent culture; 3. In the method described in 1. or 2. above, the surface antigen preferably includes HLA-ABC; 4. The method described in any of 1. to 3. above preferably includes subculturing the SkM four to seven times; Aspect C2 of this disclosure is, 5. A method for suppressing cellular immunity in the SkM, comprising performing any of the methods described in 1. to 4. above.

[0021] Aspect C3 of this disclosure is, A method for producing SkM cells in which surface antigen expression is suppressed, comprising subculturing SkM cells in which 6.90% or more are CD56-positive cells four or more times; 7. In the method for producing SkM described in 6. above, it is preferable that the SkM is obtained by adherent culture; 8. In the method for producing SkM described in 6. or 7. above, the surface antigen preferably includes HLA-ABC.

[0022] Aspect C4 of this disclosure is, 9. A pharmaceutical product containing skeletal muscle-derived cells (SkM) as an active ingredient, in which 90% or more are CD56-positive cells, and in which the expression level of surface antigens is reduced compared to cells that have not been subcultured or have been subcultured three times or less; 10. In the pharmaceutical product described in 9 above, it is preferable that the surface antigen comprises at least one of HLA-A antigen, HLA-B antigen, and HLA-C antigen; 11. The pharmaceutical product described in item 9 or 10 above is preferably a cell preparation for regenerative medicine; 12. In the pharmaceutical product described in any of items 9 to 11 above, it is preferable that the SkM is an allogeneic cell of the target of the pharmaceutical product administration.

[0023] Subject D of this disclosure is based on the novel finding that co-culturing skeletal muscle-derived cells (SkM) with peripheral blood mononuclear cells (PBMCs) induces the expression of inflammatory cytokines in SkM. Subject D encompasses the following aspects D1 to D4.

[0024] Aspect D1 of this disclosure is, A method for inducing the expression of inflammatory cytokines in skeletal muscle-derived cells (SkM), in which 1.90% or more of the cells are CD56-positive, including co-culturing them with peripheral blood mononuclear cells (PBMCs); 2. In the method described in 1. above, the inflammatory cytokine preferably comprises one or more selected from the group consisting of interleukins, interferons, chemokines, and tumor necrosis factors; 3. In the method described in 1. or 2. above, the inflammatory cytokine preferably comprises one or more selected from the group consisting of interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-15 (IL-15), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ); 4. The method according to any of the methods described in 1. to 3. above, wherein the expression level of the induced inflammatory cytokine is increased compared to when SkM is cultured alone; 5. In any of the methods described in 1. to 4. above, When the inflammatory cytokine is IL-2, it is preferable that IL-2 is expressed only when the SkM is co-cultured with the PBMC. When the inflammatory cytokine is IL-6, it is preferable that the expression level of IL-6 increases by 1.0 times or more compared to when SkM is cultured alone. When the inflammatory cytokine is IL-15, it is preferable that the expression level of IL-15 increases by 1.5 times or more compared to when SkM is cultured alone. When the inflammatory cytokine is TNF-α, it is preferable that the expression level of TNF-α increases by 10.0 times or more compared to when SkM is cultured alone. When the inflammatory cytokine is IFN-γ, it is preferable that the expression level of IFN-γ increases by 3.0 times or more compared to when SkM is cultured alone; Aspect D2 of this disclosure is, 6. A method for promoting the repair of biological tissue by SkM, comprising carrying out the method described in any one of items 1 to 5 above.

[0025] Aspect D3 of this disclosure is A method for producing SkM cells in which inflammatory cytokine expression is induced, comprising co-culturing SkM cells, in which 7.90% or more are CD56-positive cells, with peripheral blood mononuclear cells (PBMCs); 8. In the method for producing SkM described in 7 above, it is preferable that the inflammatory cytokines include one or more selected from the group consisting of interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-15 (IL-15), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ); Aspect D4 of this disclosure is 9. A pharmaceutical product containing skeletal muscle-derived cells (SkM) as an active ingredient, in which 90% or more are CD56-positive cells and the expression level of inflammatory cytokines is increased compared to when cultured alone; 10. In the pharmaceutical product described in 9 above, it is preferable that the inflammatory cytokine comprises one or more selected from the group consisting of interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-15 (IL-15), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ); 11. The pharmaceutical product described in item 9 or 10 above is preferably a cell preparation for regenerative medicine; 12. In the pharmaceutical product described in any of items 9 to 11 above, it is preferable that the SkM is an allogeneic cell of the target of the pharmaceutical product administration. [Effects of the Invention]

[0026] According to this disclosure, a novel means is provided that can avoid or mitigate immune rejection in a patient even when a cell preparation for regenerative medicine is prepared using allogeneic cells as raw material and administered to the patient. [Brief explanation of the drawing]

[0027] [Figure 1] Figure 1 is a graph showing the results of an investigation into the effect of co-cultured SkM with PBMCs on T cell proliferation in the T cell proliferation inhibition test described later in the examples. The upper left shows the results for PBMC alone, the upper right shows the results for co-culture of PBMC with SkM (2.5 × 10⁴ cells / well), the lower left shows the results for co-culture of PBMC with SkM (5.0 × 10⁴ cells / well), and the lower right shows the results for co-culture of PBMC with SkM (1.0 × 10⁵ cells / well). The horizontal axis (FarRed APC-A) shows the fluorescence intensity, and the vertical axis shows the number of cells (Count) showing each fluorescence intensity. The percentage in the graph shows the proportion of T cells that have undergone one or more cell divisions among the single T cells contained in the PBMC. [Figure 2] Figure 2 is a graph showing the changes in the expression levels of the IL-10 gene, Ido-1 gene, and TGF-β gene in skeletal muscle-derived cells (SkM) when co-cultured with peripheral blood mononuclear cells (PBMCs) in Test Examples 1-1 to 1-4 of the Example (Test 1) described later, as confirmed by qPCR. "-PBMC" represents SkM cultured alone, and "+PBMC" represents co-culture of SkM and PBMCs. The vertical axis RQ represents Relative Quantification. [Figure 3] Figure 3 is a graph showing the results of measuring the expression rates of the evaluated surface antigens in each of the following examples (Test 2) described later: SkM cultured alone (Test Example 2-3), SkM activated by IFN-γ (Test Example 2-2), and SkM co-cultured with PBMCs (Test Example 2-1). The horizontal axis shows the surface antigens of each SkM, and the vertical axis shows the relative expression rates by IFN-γ activation or co-culture with PBMCs, with the expression rate of the surface antigen in SkM cultured alone set to 1. [Figure 4]Figure 4 is a graph showing the results of evaluating the expression rate of HLA-ABC antigens on the cell surface when the number of passages of SkM was changed in the example described later (Test 3). The horizontal axis shows, from left to right, passages 4 (P4), 5 (P5), 7 (P7), 9 (P9), and 12 (P12). The vertical axis shows the expression rate of HLA-ABC antigens on the cell surface of SkM at each passage. [Figure 5] Figure 5 is a graph showing the results of evaluating the changes in the expression levels of the inflammatory cytokine (IL-2 gene) in the culture supernatants of PBMCs co-cultured with SkM, SkM co-cultured with PBMCs, and SkM and PBMCs co-cultured, as evaluated by qPCR and array analysis in the example described later (Test 4). Left: Shows the expression level of IL-2 in PBMCs (RQ: Relative Quantification). Center: Shows the expression level of IL-2 in SkM (RQ: Relative Quantification). Right: Shows the increased IL-2 concentration (pg / mL) due to co-culture of SkM and PBMCs. [Figure 6] Figure 6 is a graph showing the results of evaluating the changes in the expression levels of the inflammatory cytokine (IL-6 gene) in the culture supernatants of PBMCs co-cultured with SkM, SkM co-cultured with PBMCs, and SkM and PBMCs co-cultured, as evaluated by qPCR and array analysis in the example described later (Test 4). Left: Shows the expression level of IL-6 in PBMCs. Center: Shows the expression level of IL-6 in SkM. Right: Shows the increased IL-6 concentration (pg / mL) due to co-culture of SkM and PBMCs. [Figure 7] Figure 7 is a graph showing the results of evaluating the changes in the expression levels of the inflammatory cytokine (IL-15 gene) in the culture supernatants of PBMCs co-cultured with SkM, SkM co-cultured with PBMCs, and SkM and PBMCs co-cultured, as evaluated by qPCR and array analysis in the example described later (Test 4). Left: Shows the expression level of IL-15 in PBMCs. Center: Shows the expression level of IL-15 in SkM. Right: Shows the increased IL-15 concentration (pg / mL) due to co-culture of SkM and PBMCs. [Figure 8] Figure 8 is a graph showing the results of evaluating the changes in the expression levels of the inflammatory cytokine (IFN-γ gene) in the culture supernatants of PBMCs co-cultured with SkM, SkM co-cultured with PBMCs, and SkM and PBMCs co-cultured, as evaluated by qPCR and array analysis in the example described later (Test 4). Left: Shows the expression level of IFN-γ in PBMCs. Center: Shows the expression level of IFN-γ in SkM. Right: Shows the increased IFN-γ concentration (pg / mL) due to co-culture of SkM and PBMCs. [Figure 9] Figure 9 is a graph showing the results of evaluating the changes in the expression levels of the inflammatory cytokine (TNF-α gene) in the culture supernatants of PBMCs co-cultured with SkM, SkM co-cultured with PBMCs, and SkM and PBMCs co-cultured, as evaluated by qPCR and array analysis in the example described later (Test 4). Left: Shows the expression level of TNF-α in PBMCs. Center: Shows the expression level of TNF-α in SkM. Right: Shows the increased TNF-α concentration (pg / mL) due to co-culture of SkM and PBMCs. [Modes for carrying out the invention]

[0028] The subject matter, aspects, and embodiments of this disclosure are described below. However, this disclosure is not limited to the embodiments described below. In addition, unless otherwise specified in this specification, operations and measurements of physical properties, etc., are performed under conditions of room temperature (20°C to 25°C) and relative humidity of 40%RH to 50%RH.

[0029] "term" Several terms used in this specification are explained here.

[0030] "Immune rejection" refers to the immune response in which a transplanted organ or tissue is recognized as a foreign substance by the recipient's immune system and rejected. This phenomenon is mainly caused by a mismatch in major histocompatibility complex (MHC) or human leukocyte antigen (HLA) between the graft and the recipient. There are three main forms of immune rejection: hyperacute rejection, acute rejection, and chronic rejection. Hyperacute rejection occurs immediately after transplantation and is caused by antibodies against the graft that the recipient already possesses (e.g., HLA antibodies or ABO blood group antibodies). Acute rejection occurs between one week and several months after transplantation and is mainly attributed to a cellular immune response mediated by T cells. Chronic rejection progresses over several months to several years and is characterized by damage to the vascular endothelium and fibrosis of the graft. Immune rejection is one of the most important challenges in organ transplant medicine, and its control directly impacts patient survival rates and quality of life.

[0031] CD56, also known as a neural cell adhesion molecule (NCAM), is a membrane protein involved in cell-cell adhesion and signal transduction. In the human immune system, CD56-positive cells are an important component of innate immunity. CD56-positive cells play a wide range of roles in the immune system and are a group of cells that are attracting attention in various fields, from basic research to clinical applications. In this specification, "CD56-positive cells" means cells that express the CD56 molecule at a detectable level on their cell surface.

[0032] "Skeletal muscle-derived cells (SkM)" is a general term for a group of cells obtained from skeletal muscle tissue, and includes skeletal myoblasts and, in the case of striated muscle-derived cells, fibroblasts, as cells involved in the formation and regeneration of muscle cells. These cells play an important role in the processes of skeletal muscle growth, repair, and regeneration. In this specification, SkM means a cell population in which 90% or more (preferably 93% or more, more preferably 95% or more, even more preferably 96% or more, particularly preferably 97% or more, and most preferably 98% or more) are CD56 positive. Skeletal myoblasts are well known in the art and can be prepared from skeletal muscle by any known method (for example, a method of separating skeletal myoblasts by enzymatic treatment in which skeletal muscle is immersed in a proteolytic enzyme solution for a predetermined time, discarding the resulting enzyme treatment solution, and then immersing the skeletal muscle in a proteolytic enzyme solution at least once more, and recovering the cells contained in the enzyme treatment solution obtained after each enzyme treatment), or they can be commercially available. Skeletal myoblasts can be identified by markers such as CD56, α7 integrin, myosin heavy chain IIa, myosin heavy chain IIb, myosin heavy chain IId (IIx), MyoD, Myf5, Myf6, myogenin, desmin, and PAX3, although these are not limited to these. In one embodiment, skeletal myoblasts are desmin-positive.

[0033] Peripheral blood mononuclear cells (PBMCs) are mononuclear white blood cells isolated from peripheral blood, and refer to a group of cells including lymphocytes (T cells, B cells, NK cells, etc.) and monocytes. These cells share the common characteristic of having a single nucleus and play a central role in immune responses and inflammatory responses. PBMCs are isolated from red blood cells and polymorphonuclear leukocytes (neutrophils, etc.) among the cellular components of blood using density gradient centrifugation. As a result, they are used as important experimental materials in immunological studies of human and animal models. Furthermore, PBMCs are attracting attention in the fields of regenerative medicine and immunotherapy. In this specification, PBMCs may be autologous or allogeneic cells relative to SkM, but allogeneic cells are preferred.

[0034] Cytokines are small protein molecules that are responsible for intercellular communication and regulate various physiological functions such as immune responses, inflammatory responses, cell proliferation, differentiation, and regeneration. They are mainly produced by immune cells (T cells, B cells, macrophages, etc.) and non-immune cells, and exert their effects through autocrine secretion, paracrine secretion to neighboring cells, and systemic effects (endocrine). Cytokines are classified into interleukins (IL), interferons (IFN), tumor necrosis factor (TNF), growth factors, chemokines, etc., based on their function and structure. Each binds to a specific receptor and activates signaling pathways such as the JAK-STAT pathway and the MAPK pathway to exert its effect on target cells.

[0035] Inflammatory cytokines are a group of cytokines that play a role in promoting immune responses and inflammatory reactions, and are central molecules that induce activation of the immune system during infection and tissue damage. These cytokines are produced by immune cells such as macrophages, dendritic cells, T cells, and neutrophils. Inflammatory cytokines are important as part of the defense response against infection and injury.

[0036] Interleukins are a group of cytokines primarily responsible for information transmission between white blood cells (immune cells), playing crucial roles in immune responses, inflammatory responses, hematopoiesis, and intercellular communication. There are numerous types of interleukins, each with different structures and functions; currently, interleukins ranging from IL-1 to IL-40 and above have been reported. While the roles of interleukins are diverse, they primarily include immune activation, anti-inflammatory effects, inflammation induction, and hematopoietic promotion. Interleukins exert their effects through specific receptors. These receptors are located on the surface of target cells, and signal transduction is initiated when interleukins bind to them. Typical signaling pathways include the JAK-STAT pathway and the MAPK pathway, which regulate gene expression and lead to cell proliferation, differentiation, migration, and functional activation.

[0037] Interferons are important cytokines that suppress viral infections and tumor growth, and regulate the immune system. Produced by infected cells and immune cells, they primarily exert antiviral, immunomodulatory, and tumor-suppressive effects. As a result, they play a central role in both innate and adaptive immunity. Interferons are classified into three classes: type I, type II, and type III, each with different functions.

[0038] Tumor necrosis factor (TNF) is a multifunctional cytokine that plays a central role in inflammatory responses, immunomodulation, and induction of cell death. Its name derives from its initial discovery of its ability to induce necrosis in tumor cells. TNF primarily consists of two main molecules, TNF-α (tumor necrosis factor α) and TNF-β (lymphotoxin), but in this specification, the concept encompasses both. TNF-α is primarily produced by immune cells such as macrophages, T cells, and neutrophils. It is released in response to infection, inflammation, or tissue injury, regulating the activation of the entire immune system. TNF-α binds to specific receptors, the TNF receptor (TNFR1 and TNFR2), activating various intracellular signaling pathways. This signaling induces the expression of inflammatory genes via the NF-κB and MAPK pathways, triggering inflammatory responses. It also generates signals that regulate cell death (apoptosis and necroptosis) and cell survival. The main biological functions of TNF are infection defense and promotion of tumor immunity. The action of TNF-α at the site of inflammation increases the expression of adhesion molecules in vascular endothelial cells, allowing immune cells to be efficiently recruited to the site of injury.

[0039] Chemokines are small cytokines that control the movement (migration) of various cells, including white blood cells, and play an important role in inflammatory and immune responses. The name chemokine comes from "chemotaxis," meaning they have the ability to guide target cells in a specific direction. Chemokines are classified into four families based on their structure: CC, CXC, CX3C, and C. Each classification is based on differences in the arrangement of cysteine ​​residues and the disulfide bonds connecting them. These chemokines exert their function by binding to specific chemokine receptors (G protein-coupled receptors). Chemokines and their receptors have a very specific relationship, finely controlling cell migration and activation. One of the main roles of chemokines is the recruitment of immune cells. When infection or injury occurs, inflammatory chemokines are produced, attracting neutrophils, monocytes, T cells, and other cells to the site of inflammation. On the other hand, homeostatic chemokines maintain immune homeostasis by controlling the migration of lymphocytes to lymph nodes and their placement within tissues. This enables an appropriate immune response.

[0040] Regulatory T cells (Tregs) are a special subset of T cells that play a role in suppressing the immune response. They primarily play an important role in preventing autoimmune diseases and maintaining homeostasis of the immune system. Tregs are classified as CD4-positive T cells, and they highly express CD25 (IL-2 receptor α chain) as a characteristic surface marker, with the expression of the transcription factor Foxp3 playing a central role in their function. Tregs are mainly classified into two types: spontaneously occurring Tregs (nTregs) that differentiate in the thymus, and induced Tregs (iTregs) that are induced from normal CD4-positive T cells in the periphery. Although these develop through different pathways, they share the function of immunosuppression. Mechanisms of suppression include anti-inflammatory effects through the secretion of cytokines (e.g., IL-10 and TGF-β), and direct effects on effector T cells and dendritic cells.

[0041] "Treg-inducing factors" refer to molecules and signaling pathways that promote the differentiation of undifferentiated CD4-positive T cells (naive T cells) into Tregs. These factors play an important role in maintaining immune homeostasis and suppressing autoimmune diseases. Major inducing factors include IDO1, IL-10, TGF-β, IL-2, retinoic acid, and signals from dendritic cells and macrophages.

[0042] IDO (indoleamine 2,3-dioxygenase) is an enzyme responsible for the initial stages of tryptophan metabolism and plays a crucial role in regulating the immune response. It is primarily an enzyme that contributes to immunosuppression and is expressed in dendritic cells, macrophages, and some tumor cells. IDO1 breaks down tryptophan to produce metabolites such as kynurenine, significantly impacting the function of immune cells. IDO1 has the following effects in creating an immunosuppressive environment: First, it inhibits the proliferation of effector T cells (especially Th1 cells) and induces apoptosis by depleting tryptophan. Meanwhile, tryptophan metabolites such as kynurenine promote the induction and activation of regulatory T cells (Tregs). Furthermore, it suppresses the overall immune response by reducing the antigen-presenting ability of innate immune cells (dendritic cells and macrophages). Dendritic cells expressing IDO1 have the ability to differentiate naive T cells into Tregs. They also further promote Treg induction by secreting TGF-β and retinoic acid simultaneously with antigen presentation. Furthermore, IDO1 expression is often induced by inflammatory cytokines (especially IFN-γ). IDO2 is also known as a homolog, and its expression is particularly high in the liver, pancreas, and kidneys. In other words, in this specification, "IDO" is a concept that encompasses both IDO1 and IDO2.

[0043] IL-10 (interleukin-10) is an anti-inflammatory cytokine primarily involved in suppressing immune responses and maintaining immune system homeostasis. IL-10 is produced by many immune cells, including T cells (especially regulatory T cells, Tregs), macrophages, dendritic cells, and B cells. The main effects of IL-10 are the suppression of inflammatory cytokine production and the inhibition of antigen-presenting cell function. In particular, it suppresses the production of pro-inflammatory cytokines (e.g., IL-1, IL-6, TNF-α) by macrophages and dendritic cells, thereby controlling excessive inflammatory responses. Furthermore, by reducing the antigen-presenting ability of these cells, it suppresses the activation of effector T cells. IL-10 signaling occurs via the IL-10 receptor (IL-10R). When IL-10 binds to IL-10R, the JAK-STAT pathway is activated, inducing the expression of anti-inflammatory genes. This signaling pathway is a crucial mechanism for realizing the biological effects of IL-10. IL-10 is an anti-inflammatory cytokine that helps create an immunosuppressive environment. While IL-10 itself does not directly induce Treg cells, it plays a role in indirectly assisting Treg induction by suppressing effector T cells.

[0044] TGF-β (Transforming Growth Factor-β) is a multifunctional cytokine that regulates a wide range of physiological functions, including cell proliferation, differentiation, immune response, and tissue repair. TGF-β is a central factor in Treg induction and promotes the expression of Foxp3 (a Treg-specific transcription factor) in naive T cells. TGF-β signaling also plays a role in maintaining Treg differentiation and stability, and is particularly involved in the formation of peripherally induced Tregs (iTregs). TGF-β has three isoforms: TGF-β1, TGF-β2, and TGF-β3, with TGF-β1 playing a major role in the immune system. An important characteristic of TGF-β is its immunosuppressive effect. TGF-β functions as a central factor in promoting the differentiation of regulatory T cells (Tregs) and suppressing the immune response. In the presence of TGF-β, naive CD4-positive T cells express Foxp3 and differentiate into Tregs. It also prevents excessive inflammation and autoimmune reactions by suppressing the activity of effector T cells. TGF-β is involved not only in the immune system but also in tissue repair and fibrosis. During the wound healing process, it activates fibroblasts to induce collagen production and promotes tissue reconstruction.

[0045] IL-2 (interleukin-2) is a cytokine that primarily promotes the proliferation and differentiation of T cells and plays a crucial role in regulating the immune response. IL-2 is mainly produced by activated CD4-positive T cells and acts on self and surrounding cells via the high-affinity IL-2 receptor (IL-2R). IL-2 signaling occurs via the IL-2 receptor subunits CD25 (α chain), CD122 (β chain), and γc (common γ chain). When IL-2 binds to the receptor, the JAK-STAT pathway is activated, leading to the expression of genes that control T cell proliferation and differentiation. Thus, IL-2 is known as a T cell growth factor, but it is also essential for Treg differentiation and function. IL-2 signaling enhances Foxp3 expression through the activation of STAT5 (Signal Transducer and Activator of Transcription 5), promoting Treg differentiation. Furthermore, IL-2 contributes to Treg survival and proliferation.

[0046] Retinoic acid is an active derivative metabolized from vitamin A (retinol) and is a molecule with a wide range of physiological functions, including cell proliferation, differentiation, development, and immunomodulation. In particular, it plays an important role in embryonic development and the regulation of immune responses. Retinoic acid has two main isomers: all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis retinoic acid). These bind to nuclear receptors (RAR and RXR) within cells and regulate the expression of target genes, thereby influencing cell growth and differentiation. In the immune system, retinoic acid is involved in maintaining immune homeostasis by inducing regulatory T cells (Tregs) and suppressing Th17 cells. In the intestinal tract in particular, retinoic acid produced by dendritic cells and macrophages differentiates naive T cells into Tregs, thereby suppressing inflammation and regulating intestinal immunity.

[0047] IL-6 is a multifunctional cytokine involved in immune responses, inflammatory responses, hematopoiesis, and metabolic regulation, and is mainly produced by macrophages, dendritic cells, T cells, fibroblasts, and endothelial cells. Due to its diverse actions, IL-6 is involved in various physiological and pathological processes, including acute and chronic inflammation, infection defense, autoimmune diseases, and tumor formation. One of the important functions of IL-6 is to promote the acute phase response. IL-6 acts on liver cells to enhance the inflammatory response by inducing the production of acute phase proteins (such as C-reactive protein (CRP), ceryloplasmin, and fibrinogen). It also promotes hematopoiesis in the bone marrow and aids in neutrophil proliferation. In immunomodulation, IL-6 plays an important role in the differentiation of T cells and B cells. In particular, it differentiates naive CD4-positive T cells into Th17 cells, while suppressing the differentiation of regulatory T cells (Tregs) in combination with TGF-β. It also promotes B cell activation and antibody production, thereby enhancing the adaptive immune response.

[0048] IL-15 is a cytokine primarily responsible for regulating the innate and adaptive immune systems, playing a crucial role in the survival, proliferation, and activation of natural killer (NK) cells and CD8-positive T cells. While IL-15 is produced by many cells, including macrophages, dendritic cells, fibroblasts, and endothelial cells, it generally functions through a unique mechanism called "transpresentation." The primary function of IL-15 is the maintenance and activation of NK cells and memory CD8-positive T cells. IL-15 promotes NK cell proliferation and enhances their cytotoxic activity. It also facilitates the differentiation of effector T cells into memory T cells, forming long-term immunological memory. This ensures the persistence of immune responses to infections and tumors.

[0049] IFN-γ is a type I interferon. Its primary function is to exert potent antibacterial and antitumor effects through the activation of macrophages. When macrophages receive IFN-γ, their antigen-presenting ability is enhanced, promoting the elimination of pathogens and tumor cells. Furthermore, IFN-γ increases the expression of MHC class I and class II molecules, enhancing antigen-presenting capacity and thereby strengthening the pathogen-specific immune response by T cells. IFN-γ is also important in T cell differentiation and function. It promotes the differentiation of Th1 cells, leading cellular immunity, while suppressing Th2 cells and their associated humoral immune responses. In addition, IFN-γ strengthens the coordination between innate and adaptive immunity, directly eliminating virus-infected cells and tumor cells through the activation of NK cells and cytotoxic T cells (CTLs).

[0050] Surface antigens are molecules present on the surface of cells that indicate specific properties or functions of the cell. These antigens have structures such as proteins, glycoproteins, and glycolipids, and function as markers for the immune system to recognize specific cells or pathogens. Because cell surface antigens differ depending on the cell type, maturation stage, and activation state, they are an important subject of study in immunology and cell biology. Surface antigens mainly have the following roles: Firstly, they function as recognition molecules in the immune response. For example, the major histocompatibility complex (MHC) is a molecule that presents antigens to T cells and plays a central role in the immune system. Also, a group of surface antigens called CD (cluster of differentiation) molecules are widely used as markers to characterize the classification and function of immune cells. For example, CD4 is specifically expressed on helper T cells, and CD8 is expressed on cytotoxic T cells. Secondly, surface antigens are involved in cell-cell interactions and signal transduction. ICAM-1, VCAM-1, and cell adhesion molecules mediate adhesion between immune cells and vascular endothelial cells, and help in the recruitment of cells to the site of inflammation. Furthermore, many surface antigens act as receptors or co-molecules, regulating the signaling pathways of cytokines and growth factors. Thirdly, surface antigens can sometimes function as specific antigens for pathogens or tumor cells. Techniques such as flow cytometry and immunohistochemistry are used to detect and analyze surface antigens. This makes it possible to identify the type, state, and disease-related nature of the cells.

[0051] "Costimulatory molecules" are cell surface molecules that play a role in regulating the immune response by complementing or regulating antigen recognition. They primarily act on T cells and B cells and are essential for the activation or suppression of the immune system. Costimulatory molecules assist in signal transduction from antigen-presenting cells (APCs) to immune cells in adaptive immune responses, determining the specificity, intensity, and persistence of the immune response. Costimulatory molecules play a crucial role in T cell activation. T cells first recognize antigens by binding to MHC molecules on antigen-presenting cells via the T cell receptor (TCR). However, this first signal alone does not lead to complete activation. The addition of a second signal provided by a costimulatory molecule activates T cells, enabling proliferation, cytokine production, and the expression of effector functions (two-signal model). On the other hand, there are also costimulatory molecules involved in the suppression of the immune response. Costimulatory molecules are involved in the activation of not only T cells but also B cells. CD40 ligand (CD40L) is expressed by T cells and, by binding to CD40 on B cells, promotes B cell proliferation and antibody production. Furthermore, this interaction is essential for class switching and the formation of immunological memory.

[0052] Programmed cell death ligands (PD-Ls) are molecules involved in the suppression of the immune system, contributing to the maintenance of immune homeostasis and the prevention of autoimmune reactions by regulating T cell activity. Two main types of PD-Ls are known: PD-L1 (Programmed Death-Ligand 1) and PD-L2 (Programmed Death-Ligand 2). These function by binding to the programmed cell death receptor (PD-1, Programmed Death-1). PD-L1 is expressed in a wide range of cell types (macrophages, dendritic cells, endothelial cells, tumor cells, etc.) and suppresses T cell activity through binding to PD-1. Specifically, when PD-L1 binds to PD-1 on T cells, T cell receptor (TCR) signaling is inhibited, reducing T cell proliferation, cytokine production, and cytotoxic activity. This suppressive mechanism plays a role in suppressing excessive immune responses and preventing tissue damage and the development of autoimmune diseases. On the other hand, PD-L2 is mainly expressed in dendritic cells and macrophages and has an immunosuppressive function similar to PD-L1, but its expression pattern and role are different from PD-L1.

[0053] Cell adhesion molecules are transmembrane proteins that connect cells to each other and to the extracellular matrix (ECM), playing a crucial role in tissue construction, cell-cell interactions, and signal transduction. These molecules regulate cell adhesion and are involved in diverse physiological and pathological processes, including development, immune responses, wound healing, and cancer progression. Cell adhesion molecules are classified into several major families based on their structure and function. Representative examples include cadherins, integrins, selectins, and the immunoglobulin superfamily (IgSF). Cadherins play a role in connecting cells, and are particularly important between epithelial cells and nerve cells. These are calcium-dependent adhesion molecules that contribute to maintaining cell polarity and tissue structure formation. Integrins are major adhesion molecules that connect cells to the extracellular matrix and are important in cell migration and signal transduction. Integrins bind to components in the extracellular matrix, such as collagen and fibronectin, and regulate cell morphology, migration, and proliferation. They are also involved in the recruitment of immune cells to inflammatory sites. Selectins are expressed on vascular endothelial cells and leukocytes and regulate the recruitment of leukocytes in inflammatory responses. Selectins form weak adhesions to other cells via glycans, which are responsible for the initial stages of immune cell migration to inflammatory sites. The immunoglobulin superfamily (IgSF) is involved in immune responses and nervous system development through cell-cell adhesion. For example, ICAM (Intercellular Adhesion Molecule) and VCAM (Vascular Cell Adhesion Molecule) play important roles in inflammatory responses and immune cell migration. The function of cell adhesion factors extends beyond mere physical cell adhesion; they influence cell fate determination and responsiveness through the initiation and regulation of intracellular signaling. These factors are also involved in many diseases, playing a particularly important role in cancer progression, metastasis, chronic inflammatory diseases, and autoimmune diseases.

[0054] ICAM-1 is a type of cell adhesion molecule that plays a crucial role in cell-cell interactions, immune responses, and inflammatory responses. ICAM-1 is mainly expressed in endothelial cells, immune cells (macrophages, lymphocytes, etc.), and some epithelial cells, and regulates cell-cell adhesion and recruitment of the immune system. ICAM-1 belongs to the immunoglobulin superfamily (IgSF), and its structure consists of an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain is responsible for interactions with ligands (especially integrins), which forms the basis of ICAM-1's function. In particular, β2 integrins (e.g., LFA-1, CD11a / CD18) are major ligands for ICAM-1 and mediate the adhesion, migration, and activation of T cells and neutrophils. ICAM-1 expression is usually low, but it increases significantly when induced by inflammatory cytokines (e.g., IL-1, TNF-α, IFN-γ). At the site of inflammation, ICAM-1 is expressed on the surface of vascular endothelial cells and helps in the process of recruiting these cells to the site of inflammation through interaction with leukocytes. Specifically, leukocytes bind to ICAM-1, causing them to adhere firmly to the vascular endothelium, and then migrate through the blood vessel wall to the surrounding tissue.

[0055] A "TIGIT ligand" is a ligand that binds to the immunosuppressive receptor TIGIT (T cell immunoreceptor with Ig and ITIM domains), and is primarily involved in the suppression and regulation of the immune response. TIGIT is expressed on T cells and natural killer (NK) cells, and through interaction with its ligand, it plays a role in suppressing excessive immune responses. The main TIGIT ligands known are CD155 (Poliovirus Receptor, PVR) and CD112 (Nectin-2). These ligands are expressed on antigen-presenting cells (APCs), tumor cells, and endothelial cells, and play a central role in immunomodulation. When TIGIT binds to its ligand, the activation of T cells and NK cells is suppressed, and an immunosuppressive environment is created. In particular, the interaction between TIGIT and CD155 suppresses cytokine production by effector T cells (Teff) and enhances the immunosuppressive function of regulatory T cells (Treg). This process controls excessive immune responses in autoimmune and inflammatory diseases, while in the tumor microenvironment, tumor cells utilize this pathway to promote immune evasion. Furthermore, CD155 and CD112 can interact with receptors other than TIGIT. For example, the immunoactivating receptor CD226 (DNAM-1) also binds to CD155 and CD112, promoting the activation of immune cells. Since TIGIT and CD226 compete for the same ligand, immunosuppression is emphasized when TIGIT expression is dominant. This competitive relationship plays a crucial role in regulating the immune response.

[0056] Classical HLA class I antigens are molecules that play a central role in adaptive immunity, contributing to the induction of immune responses by presenting intracellular peptide antigens to T cells. These antigens are encoded at three gene loci: HLA-A, HLA-B, and HLA-C, and are known for their high diversity. Classical HLA class I antigens are expressed in all nucleated cells and are important in distinguishing between self and non-self. Their structure is a trimer consisting of a heavy chain (α chain) embedded in the cell membrane, a co-molecule β2-microglobulin, and a short antigenic peptide (usually 8-10 amino acids). This allows HLA class I molecules to provide a platform for recognizing intracellular abnormalities and infections. The main role of HLA class I antigens is to load endogenous antigenic peptides (e.g., viral peptides or tumor antigens) onto MHC class I molecules and present them to CD8-positive T cells (cytotoxic T cells, CTLs). Antigen peptides are fragmented by the proteasome within the cell, transported to the endoplasmic reticulum via TAP (Transporter Associated with Antigen Processing), and then transported to the cell surface after binding to HLA molecules. Through this process, CTLs can recognize and attack infected cells and tumor cells.

[0057] HLA-A, HLA-B, and HLA-C are encoded at different gene loci and are major components of HLA class I molecules. These molecules have a trimer structure consisting of a heavy chain (α chain) present in the cell membrane, a non-covalently bound β2-microglobulin as an accessory molecule, and a bound peptide antigen. HLA-A, HLA-B, and HLA-C are expressed in almost all nucleated cells, and their expression is enhanced by inflammatory cytokines (e.g., IFN-γ). The main function of HLA-A, HLA-B, and HLA-C is to collect endogenous peptides from within the cell and present them on the cell surface to CD8-positive T cells. The peptides presented on HLA molecules are usually short fragments of 8-10 amino acids, allowing T cells to rapidly detect abnormalities such as virus-infected cells and tumor cells. Due to this function, HLA-A, HLA-B, and HLA-C play an important role in immune surveillance. HLA-A, HLA-B, and HLA-C are the most diverse genes in humans, characterized by a high degree of polymorphism (allele variants). This diversity provides an evolutionary advantage by increasing the variety of peptides presented between individuals, enabling adaptation to pathogens. However, this diversity is also a major cause of HLA mismatch in transplant medicine and a risk factor for rejection.

[0058] Non-classical HLA class I antigens are parts of the human leukocyte antigen (HLA) class I molecule and, unlike classical HLA class I antigens (HLA-A, HLA-B, HLA-C), are involved in specific regulation of the immune response and specific physiological functions. Major non-classical HLA class I antigens include HLA-E, HLA-F, and HLA-G. These non-classical HLA class I antigens have a trimer structure (heavy chain, β2-microglobulin, peptide) similar to classical HLA class I antigens, but their function and expression patterns are specific. They primarily play a regulatory role in the immune system, participating in the control of innate and adaptive immunity, fetal-maternal immune tolerance, and tumor immune evasion. HLA-E is known as a regulatory molecule that mainly acts on NK cells and some T cells. HLA-E presents a leader peptide derived from classical HLA class I molecules and suppresses NK cell activity by binding to CD94 / NKG2A, an inhibitory receptor for NK cells. This function prevents self-cells from being mistakenly attacked by NK cells. On the other hand, binding to the NK cell activating receptor (CD94 / NKG2C) can also activate the immune response. HLA-F is normally present inside cells, but can be expressed on the cell surface under certain conditions. Its role is not fully understood, but it is thought to be involved in the regulation of NK cells and T cells. HLA-F interacts with specific immune receptors (e.g., KIR, LILR) to regulate the function of immune cells. HLA-G is a molecule that plays a particularly important role in immune tolerance and is mainly expressed in placental trophoblast cells. HLA-G maintains fetal-maternal immune tolerance by suppressing NK cells, T cells, and antigen-presenting cells (APCs), contributing to the establishment and maintenance of pregnancy. HLA-G may also be expressed in tumor cells and grafts, functioning as a mechanism for immune evasion.

[0059] "Co-culture" is a technique for culturing two or more different types of cells in the same culture system, reproducing intercellular interactions in vivo and utilizing them for research and applications. This technique makes it possible to analyze complex intercellular communication, such as physical contact between cells, interactions mediated by secreted factors, and exchange of metabolites. Co-culture may involve direct contact between cells or the exchange of secreted factors while avoiding physical contact using a diaphragm such as a Transwell. In a preferred embodiment of this disclosure, co-culture is performed while the cells are in direct contact with each other.

[0060] "Adherent culture" is a cell culture method in which cells are grown by attaching them to a solid substrate (usually the surface of a culture dish or flask). Since many cells cannot proliferate or differentiate normally without a suitable adhesion surface, this method is widely used in the culture of mammalian and progenitor cells. In adherent culture, cells attach to the substrate surface, spread, and change their morphology. This process is controlled by interactions mediated by extracellular matrix (ECM) components and cell adhesion molecules. Common substrates include untreated plastic surfaces, as well as surfaces coated with ECM proteins such as collagen and laminin. This improves cell adhesion and viability.

[0061] "Subculturing" refers to the process of dividing cells and transferring them to new culture vessels to prevent excessive cell density and waste accumulation during long-term cell culture. This technique is essential for maintaining cell proliferation and ensuring the necessary number of cells for experiments. In the subculturing process, the cells attached to the culture vessel are first treated to loosen cell-cell adhesion and adhesion to the substrate in order to collect the cells. Generally, treatment using trypsin or EDTA is employed. After collecting the cells, they are seeded in a certain ratio into new culture vessels, and cultured again with fresh culture medium. In the case of suspension cells, centrifugation is performed to remove excess medium, and then the cells are resuspended in fresh medium. The number of subculturing cycles is an important indicator of the cell culture history, and is especially important when using cell lines or stem cells. Some cells are known to change morphologically and functionally with repeated subculturing, and their differentiation and proliferation abilities are reduced. Therefore, it is important to maintain appropriate subculturing conditions and record the number of subculturing cycles.

[0062] Cellular immunity is a type of immune response that directly eliminates abnormal cells such as infected cells and tumor cells, and is a crucial component of adaptive immunity, particularly centered on T cells. This immune mechanism protects against infectious diseases and tumors by detecting and responding to abnormalities in the body. Cellular immunity is especially effective in protecting against pathogens inside cells that cannot be reached by antibodies (humoral immunity). In cellular immunity, the immune response is initiated when abnormal cells present self or non-self antigens. Specifically, virus-infected cells and tumor cells present peptides produced within the cell to HLA class I molecules on their cell surface. CD8-positive T cells (cytotoxic T cells, CTLs) specifically recognize these presented antigens. CTLs adhere to target cells and induce apoptosis in the target cells by releasing cytotoxic molecules such as granzymes and perforins. On the other hand, antigen-presenting cells such as macrophages and dendritic cells present exogenous antigens to HLA class II molecules, activating CD4-positive T cells (helper T cells). These activated helper T cells secrete cytokines, which promote the recruitment and activation of other immune cells. Furthermore, natural killer (NK) cells are also involved in cellular immunity. NK cells have the ability to recognize and directly destroy cells with reduced expression of HLA class I molecules. This function prevents virus-infected cells and some tumor cells from evading immune surveillance. In addition to eliminating pathogens and abnormal cells, cellular immunity also contributes to the formation of immunological memory. Some activated T cells remain as memory T cells, responding rapidly and efficiently in the event of reinfection with the same pathogen. This characteristic makes cellular immunity the foundation of long-term immune defense.

[0063] "Cell therapy products for regenerative medicine" are pharmaceuticals or therapeutic preparations developed and manufactured for cell-based regenerative medicine, and are used for the purpose of regenerating, repairing, and restoring the function of damaged tissues and organs. These preparations exert their therapeutic effect by being transplanted into the body using the patient's own cells (autologous cells) or cells donated by another person (allogeneic cells). Cell therapy products for regenerative medicine preferably meet requirements such as safety (ensuring the origin of the cells used, ensuring sterility during processing, and ensuring genetic stability), efficacy (demonstrating the effect of restoring or regenerating function at the site of injury), consistency of quality (thorough quality control during the manufacturing process and consistent characteristics of the preparation), and biocompatibility (exerting effects in the body while minimizing immune rejection and inflammatory reactions). A variety of cells are used in cell therapy products for regenerative medicine, and their applications differ depending on their characteristics. The following cells are mainly used. In this specification, it is preferable that cell therapy products for regenerative medicine contain skeletal muscle-derived cells related to subjects A to D of this disclosure. Cell therapy products for regenerative medicine are applied to the treatment of a wide range of diseases and injuries. Specific examples include the treatment of bone and cartilage damage in orthopedics (e.g., osteoarthritis, bone defects), the treatment of cardiovascular diseases (e.g., myocardial regeneration after myocardial infarction), the treatment of neurological diseases (e.g., nerve regeneration for spinal cord injury and Parkinson's disease), the treatment of diabetes (e.g., improved blood glucose control through islet cell transplantation), the treatment of liver diseases (e.g., support for liver function through hepatocyte transplantation), and cancer treatments such as immunotherapy. On the other hand, recent technological advancements have expanded the possibilities of patient-specific treatment using iPS cells and combination therapies using cell preparations and biomaterials. Furthermore, the introduction of more efficient cell culture technologies and automated manufacturing systems is progressing, and the widespread use of cell preparations for regenerative medicine is expected.

[0064] Cell-based regenerative medicine preparations can be in sheet form. These preparations involve culturing cells in a sheet-like structure, promoting regeneration and repair by directly attaching them to damaged tissues or organs. This form is noteworthy because it allows cells to form a three-dimensional structure, easily maintaining the cell density and interactions necessary for tissue function. Sheet-type preparations are primarily applied to regenerative medicine for tissues such as myocardium, cornea, skin, and cartilage. These preparations may utilize scaffold materials that mimic the extracellular matrix (ECM), or they may involve densely culturing cells without any materials. For example, using a temperature-responsive culture dish allows for the direct removal of the cell-adhered sheet. This method preserves intercellular junctions and the extracellular matrix, improving engraftment and functionality after transplantation. The advantage of sheet-type preparations lies in their ability to easily replicate in vivo functions due to the three-dimensional structure of the cells. They are also convenient for transplantation and can be customized to suit specific tissue locations. Currently, sheet cell therapies are being researched and applied at the forefront of regenerative medicine in many countries, including Japan, and clinical results have been reported for the treatment of myocardium, cornea, and skin. Further advancements in manufacturing process standardization and cost reduction technologies are expected to lead to applications for a wider range of diseases.

[0065] Furthermore, cell therapies for regenerative medicine may also be in the form of injectable preparations. Cell therapies for regenerative medicine in the form of injectable preparations are designed to treat by directly injecting cells into specific sites in the body, and are attracting attention as a technology that enables minimally invasive and efficient treatment. Preparations in this form are used as cells alone or as a complex with biomaterials to protect and support the cells. In particular, efforts are made to give the cells shapes and properties that make it easier for them to reach the site of injury and exert their effects. In this specification, it is preferable that the cell therapies for injectable preparations also contain skeletal muscle-derived cells related to the themes A to D of this disclosure. These may be injected alone as a suspension, or they may be used encapsulated in hydrogels, nanoparticles, microcapsules, etc. Biomaterials such as hydrogels solidify in the body after injection, making it easier for cells to remain locally, and playing a role in improving cell viability and regenerative capacity by providing an appropriate environment. This technology can be applied to the treatment of a wide range of diseases, including articular cartilage damage, myocardial infarction, spinal cord injury, diabetic ulcers, and neurological diseases. For example, in the treatment of myocardial infarction, an injectable preparation containing cardiomyocytes is directly administered to the damaged myocardium to support myocardial regeneration and functional recovery. The injectable form offers significant advantages, including being less invasive and allowing for efficient delivery of cells to specific sites, thus reducing the burden on the patient while achieving high therapeutic efficacy.

[0066] Biomaterials are materials designed to support the repair and regeneration of damaged tissues and organs, and are important for their biocompatibility and biodegradability. These materials are used in combination with cell and tissue engineering technologies, acting as scaffolds for cells and promoting cell adhesion, proliferation, and differentiation. Naturally derived biomaterials include collagen, hyaluronic acid, and alginic acid, which are widely used in skin, cartilage, and ophthalmic surgery due to their high biosimilarity. On the other hand, synthetic materials such as polylactic acid (PLA), polyglycolic acid (PGA), and polycaprolactone (PCL) allow for adjustment of degradation rate and mechanical strength, making them suitable for bone and cartilage regeneration. Furthermore, new biomaterials using hydrogels and nanofibers have the ability to deliver drugs locally, protect cells, and even form three-dimensional structures, leading to increasing applications in more complex tissue regeneration. In addition, combining them with 3D printing technology enables personalized treatment with patient-specific shapes and characteristics.

[0067] "Autologous cells and allogeneic cells" is a classification based on the source and immunological characteristics of cells, and is an important concept in the fields of cell transplantation, regenerative medicine, and immune response research. Autologous cells refer to cells used for transplantation or treatment that originate from the same individual. For example, this applies when a patient's own cells are collected, processed, cultured, and then returned to the same patient. The main advantage of autologous cells is that there is almost no risk of immune rejection. Because the immune system recognizes self-antigens as "self," immunosuppressants are not required, and post-transplant complications can be minimized. In the field of regenerative medicine, autologous stem cells (e.g., mesenchymal stem cells and hematopoietic stem cells) are widely used. These are collected from bone marrow, adipose tissue, blood, etc., and are applied to repair damaged tissue and treat diseases. On the other hand, the use of autologous cells can be affected by the quality of the autologous cells and the patient's health condition, and the reduced function of cells is a particular challenge in elderly patients and patients with diseases. Allogeneic cells refer to cells used for transplantation that originate from a different individual. This includes cases where cells collected from a donor are transplanted into a recipient. The advantage of allogeneic cells is that high-quality cells can be used by selecting healthy and optimal donor cells. Also, because cells can be supplied quickly and in large quantities, they are suitable for specific diseases or situations requiring acute treatment. In regenerative medicine, "off-the-shelf" cell therapy using allogeneic cells is attracting attention. This eliminates the need to prepare cells for each patient, enabling rapid and efficient treatment, but requires measures to address immune rejection and ethical issues. Autologous cells and allogeneic cells each have their own unique advantages and challenges, and are appropriately selected depending on the treatment method and the patient's condition.

[0068] 《Theme A》 Subject A of this disclosure is based on the novel finding that co-culturing skeletal muscle-derived cells (SkM) with peripheral blood mononuclear cells (PBMCs) induces the expression of regulatory T cell (Treg) inducers in SkM. More specifically, the inventors conducted a study on inhibiting the proliferation of PBMCs into T cells using SkM and confirmed that SkM inhibits T cell proliferation and suppresses PBMC division in a cell number-dependent manner (see "T cell proliferation inhibition study" below). From this, it was found that SkM inhibits T cell proliferation when co-culturing with PBMCs (Figure 1).

[0069] Based on these findings, the inventors further investigated whether co-culturing SkM with PBMCs would induce the expression of several immunosuppressive factors in SkM (see "Experiment 1" below). As a result, it was confirmed that the expression level of the IL-10 gene in the culture supernatant increased with co-culturing with PBMCs compared to when SkM was cultured alone. Furthermore, while the expression of the Ido1 gene was not observed when SkM was cultured alone, the expression of the Ido1 gene in the culture supernatant was observed for the first time when SkM was co-culturified with PBMCs. On the other hand, the expression of the TGF-β gene in the culture supernatant remained almost unchanged before and after co-culturing (Figure 2).

[0070] In Experiment 1, it is known that all immunosuppressive factors whose expression was induced by co-culturing SkM with PBMCs were Treg inducers. Therefore, the suppression of T cell proliferation by co-culturing SkM with PBMCs is thought to be due to the action of Tregs induced via the Treg inducers whose expression was induced by co-culturing. Furthermore, it is thought that SkM co-culturing with PBMCs is less susceptible to rejection reactions by cellular immunity, etc., even when transplanted into an allogeneic population as, for example, a regenerative medicine product, through the induction of Tregs. As a result of this investigation, the inventors have completed Subject A of this disclosure.

[0071] One aspect of Subject A (Aspect A1) is a method for inducing the expression of regulatory T cell (Treg) inducers in SkM, which includes co-culturing SkM, in which more than 90% of cells are CD56-positive, with peripheral blood mononuclear cells (PBMCs).

[0072] The culture conditions for co-culturing SkM and PBMC are not particularly limited. The culture medium is not particularly limited and can be used as long as it is a medium that can maintain cell viability, such as a basal medium based on various cell culture media. Such basal media include, but are not limited to, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM / F12, etc. Many of these basal media are commercially available and their compositions are publicly known. The basal medium may be used as is (e.g., in its commercially available form), or its composition may be appropriately changed depending on the cell type and cell conditions. The medium may usually contain additives such as serum (e.g., bovine serum such as fetal bovine serum (FBS), horse serum, human serum, etc.) and various growth factors (e.g., FGF, EGF, VEGF, HGF, etc.).

[0073] Co-culture can be performed regardless of the prior or subsequent morphology, as long as SkM and PBMCs are cultured together. Alternatively, each cell type can be cultured separately, and the resulting culture from one type can be added to the culture medium containing the other. Co-culture can be performed under conditions commonly used in this art. For example, typical culture conditions include 37°C and 5% CO2. The culture time is not particularly limited as long as the expression level of the target Treg-inducing factor is sufficiently increased, and can range from approximately 1 hour to 10 days, 1 hour to 5 days, 1.5 hours to 3 days (72 hours), 2 hours to 48 hours, or 2 hours to 24 hours. Similarly, the culture temperature can be any temperature commonly used in this art. Furthermore, a step of washing the cell population after culture may be included. Here, it is preferable that the SkM used for co-culture is obtained by adherent culture.

[0074] The Treg-inducing factors whose expression is induced in SkM by the co-culture described above are not particularly limited as long as they promote the process by which naive T cells differentiate into Tregs in the presence of undifferentiated CD4-positive T cells (naive T cells), and examples include IDO1, IL-10, TGF-β, IL-2, retinoic acid, and dendritic cells. In a preferred embodiment of this model, the Treg-inducing factors include one or more selected from the group consisting of IDO1, IL-10, and TGF-β, and more preferably include IDO1 and / or IL-10. The Treg-inducing factors whose expression is induced in SkM by co-culture may be those in which gene (mRNA) expression is induced, or those in which protein expression is induced. Preferably, both are induced. The induction of gene (mRNA) expression can be determined by quantitative PCR (qPCR). The induction of protein expression can be determined by ELISA.

[0075] The Treg-inducing factor whose expression is induced in SkM by co-culture may be induced intracellularly within SkM or secreted into the supernatant of the co-culture medium. Preferably, it is secreted into the supernatant of the co-culture medium, and more preferably both.

[0076] If the Treg-inducing factor whose expression is induced in SkM by co-culturing includes IDO1, it is preferable that the expression of IDO1 is first confirmed when the SkM is co-culturified with the PBMC. Furthermore, if the Treg-inducing factor whose expression is induced in SkM by co-culturing includes IL-10, it is preferable that the expression level of IL-10 increases by 10 times or more compared to when SkM is cultured alone, more preferably by 13 times or more, even more preferably by 15 times or more, and particularly preferably by 18 times or more.

[0077] As described above, the co-cultured SkM according to this embodiment exhibits induced expression of Treg-inducing factors, and is therefore thought to promote differentiation into Tregs in the presence of naive T cells. Since Tregs function to suppress cellular immunity mediated by cytotoxic T cells (killer T cells), this subject also provides a method for suppressing cellular immunity in SkM, including the method for inducing Treg-inducing factors in SkM as described above (Aspect A2). Furthermore, viewing the above embodiments from the perspective of a method for producing SkM, this subject also provides a method for producing SkM in which the expression of regulatory T cell (Treg)-inducing factors is induced, including co-culturing skeletal muscle-derived cells (SkM), in which more than 90% are CD56-positive cells, with peripheral blood mononuclear cells (PBMCs) (Aspect A3).

[0078] This subject also provides information on pharmaceuticals. Specifically, the pharmaceuticals provided by this subject contain skeletal muscle-derived cells (SkM), of which more than 90% are CD56-positive cells, as an active ingredient, in which the expression level of regulatory T cell (Treg) inducers is increased compared to when cultured alone (Aspect A4). In the SkM contained in this pharmaceutical, the expression level of Treg inducers is increased, and as mentioned above, it is thought that differentiation into Tregs is promoted in the presence of naive T cells, thereby suppressing cellular immunity.

[0079] Therefore, the pharmaceutical product according to this embodiment is preferably a cell preparation for regenerative medicine. As described above, cell preparations for regenerative medicine can take various forms, such as a sheet containing cells, an injectable preparation containing cells, or a form in which cells are compounded with a biomaterial, and all of these can be preferably adopted, but a sheet form (cell sheet) is particularly preferred. A "cell sheet" refers to a sheet formed by cells being linked together, and typically consists of one cell layer, but also includes those composed of two or more cell layers. The cells may be linked to each other directly and / or via an intervening substance. The intervening substance is not particularly limited as long as it can at least mechanically link the cells together, but examples include the extracellular matrix. The intervening substance is preferably cell-derived, in particular from the cells that constitute the cell sheet. The cells are linked at least mechanically, but may also be functionally linked, for example, chemically or electrically. It is preferable that the cell sheet does not contain a scaffold (support). Scaffolds are sometimes used in the art to adhere cells to their surface and / or interior and maintain the physical integrity of the cell sheet. For example, membranes made of polyvinylidene fluoride (PVDF) are known, but cell sheets can maintain their physical integrity even without such scaffolds. Furthermore, the cell sheet of the present invention preferably consists only of cell-derived materials that constitute the cell sheet and does not contain any other materials.

[0080] As described above, the SkM related to this subject can suppress cellular immunity in the presence of T cells. For this reason, in the pharmaceutical according to this embodiment, it is preferable that the SkM is an allogeneic cell of the target recipient of the pharmaceutical. With this configuration, even when allogeneic-derived SkM is used as a cell preparation for regenerative medicine, it is possible to avoid or mitigate immune rejection due to cellular immunity, etc.

[0081] According to Subject A of the present disclosure described above, a novel means is provided that can avoid or mitigate immune rejection in a recipient even when a cell preparation for regenerative medicine is prepared using allogeneic cells as raw materials and administered to the recipient.

[0082] 《Theme B》 Subject B of this disclosure is based on the novel finding that the expression of surface antigens in skeletal muscle-derived cells (SkM) is induced by culturing them in a medium containing interferon-γ (IFN-γ) or by co-culturing them with peripheral blood mononuclear cells (PBMCs). More specifically, the inventors investigated how the expression profile of surface antigens in SkM changes when SkM is co-culturned with PBMCs. The same investigation was also conducted when SkM was cultured in a medium containing interferon-γ (IFN-γ) ("Test 2" described later). As a result, it was confirmed that the expression rates of PD-L1, PD-L2, ICAM-1, CD155, and HLA-E increased compared to the control in both IFN-γ activation and co-culturing with PBMCs (Figure 3).

[0083] These results suggest that SkM activated by IFN-γ and SkM co-cultured with PBMCs exhibit an inhibitory effect on T cell proliferation through the expression of the surface antigens mentioned above. Therefore, it is considered that IFN-γ activated SkM and SkM co-cultured with PBMCs are less likely to be rejected by cellular immunity, etc., even when transplanted into allogeneic cells as, for example, a regenerative medicine product. As a result of this investigation, the inventors have completed Subject B of this disclosure.

[0084] One embodiment of Subject B (embodiment B1) is a method for inducing the expression of surface antigens in skeletal muscle-derived cells (SkM), in which more than 90% are CD56-positive cells, by culturing them in a medium containing interferon-γ (IFN-γ) or by co-culturing them with peripheral blood mononuclear cells (PBMCs).

[0085] The culture conditions for culturing SkM in a medium containing IFN-γ, and the culture conditions for co-culturing SkM and PBMCs, are not particularly limited. The culture medium is not particularly limited and can be used as long as it is a medium that can maintain cell viability, such as a basal medium based on various cell culture media. Such basal media include, but are not limited to, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM / F12, etc. Many of these basal media are commercially available and their compositions are publicly known. The basal media may be used in their standard composition (e.g., as commercially available), or its composition may be appropriately changed depending on the cell type and cell conditions. The medium may typically contain serum (e.g., bovine serum such as fetal bovine serum (FBS), horse serum, human serum, etc.) and various growth factors (e.g., FGF, EGF, VEGF, HGF, etc.) as additives.

[0086] Co-culture can be performed regardless of the prior or subsequent morphology, as long as SkM and PBMCs are cultured together. Alternatively, each cell type can be cultured separately, and the resulting culture from one type can be added to the culture medium containing the other. Co-culture can be performed under conditions commonly used in this art. For example, typical culture conditions include 37°C and 5% CO2. The culture time is not particularly limited as long as the expression level of the target Treg-inducing factor is sufficiently increased, and can range from approximately 1 hour to 10 days, 1 hour to 5 days, 1.5 hours to 3 days (72 hours), 2 hours to 48 hours, or 2 hours to 24 hours. Similarly, the culture temperature can be any temperature commonly used in this art. Furthermore, a step of washing the cell population after culture may be included. Here, it is preferable that the SkM used for co-culture is obtained by adherent culture.

[0087] When culturing SkM in a medium containing IFN-γ, the concentration of IFN-γ in the medium is not particularly limited and should be any concentration that can induce the expression of surface antigens in SkM. For example, the IFN-γ concentration in the medium is preferably 50 ng / mL or higher, more preferably 60 to 400 ng / mL, even more preferably 80 to 350 ng / mL, particularly preferably 100 to 300 ng / mL, and most preferably 150 to 250 ng / mL. If the IFN-γ concentration in the medium is within these ranges, the induction of surface antigens in SkM can be suitably achieved.

[0088] The surface antigens whose expression is induced in SkM by the culture described above are not particularly limited as long as they can suppress the proliferation and activity of T cells. For example, it is preferable that they include one or more selected from the group consisting of costimulatory molecules, programmed cell death ligands, cell adhesion molecules, TIGIT ligands, and non-classical HLA class I antigens. In particular, it is more preferable that the surface antigens include one or more selected from the group consisting of PD-L1, PD-L2, ICAM-1, CD155, and HLA-E. The induction of surface antigen expression can be determined using FACS (fluorescence-activated cell sorting), a type of flow cytometry. Flow cytometry is a technique in which cells or particles are arranged in a line on a liquid flow and their characteristics are analyzed by irradiating them with laser light. It can measure cell size, internal structure, and the luminescence of fluorescently labeled antibodies, and identify specific cell populations. In particular, FACS has the function of selecting and collecting cells with specific fluorescence characteristics.

[0089] In cultured SkM, the increase in the percentage of cells expressing the induced surface antigen (expression rate) is preferably 1.02 or higher, more preferably 1.10 or higher, even more preferably 1.50 or higher, even more preferably 2.00 or higher (more than 2 times), even more preferably 3.00 or higher (more than 3 times), particularly preferably 5.00 or higher (more than 5 times), and most preferably 10.00 or higher (more than 10 times).

[0090] As described above, in the co-cultured SkM according to this embodiment, the expression of a surface antigen is induced, and since this surface antigen exhibits a proliferation inhibitory effect on T cells, this subject also provides a method for suppressing cellular immunity in SkM, which includes carrying out the method for inducing the expression of a surface antigen in SkM as described above (Embodiment B2). Furthermore, by considering the above embodiments from the perspective of a method for producing SkM, this subject also provides a method for producing SkM in which the expression of a surface antigen is induced, which includes culturing skeletal muscle-derived cells (SkM) in which more than 90% are CD56-positive cells in a medium containing interferon-γ (IFN-γ), or co-culturing the SkM with peripheral blood mononuclear cells (PBMCs) (Embodiment B3).

[0091] This subject also provides information on pharmaceuticals. Specifically, the pharmaceuticals provided by this subject contain skeletal muscle-derived cells (SkM) as an active ingredient, in which more than 90% are CD56-positive cells, and in which the expression rate of surface antigens is increased compared to the control (Aspect B4). Since the expression rate of surface antigens is increased in the SkM contained in this pharmaceutical, it is thought that in the presence of T cells, T cell proliferation is suppressed and cellular immunity is suppressed.

[0092] Therefore, the pharmaceutical product according to this embodiment is preferably a cell preparation for regenerative medicine. As described above, cell preparations for regenerative medicine can take various forms, such as a sheet containing cells, an injectable preparation containing cells, or a form in which cells are compounded with a biomaterial, and all of these can be preferably adopted, but a sheet form (cell sheet) is particularly preferred. A "cell sheet" refers to a sheet formed by cells being linked together, and typically consists of one cell layer, but also includes those composed of two or more cell layers. The cells may be linked to each other directly and / or via an intervening substance. The intervening substance is not particularly limited as long as it can at least mechanically link the cells together, but examples include the extracellular matrix. The intervening substance is preferably cell-derived, in particular from the cells that constitute the cell sheet. The cells are linked at least mechanically, but may also be functionally linked, for example, chemically or electrically. It is preferable that the cell sheet does not contain a scaffold (support). Scaffolds are sometimes used in the art to adhere cells to their surface and / or interior and maintain the physical integrity of the cell sheet. For example, membranes made of polyvinylidene fluoride (PVDF) are known, but cell sheets can maintain their physical integrity even without such scaffolds. Furthermore, the cell sheet of the present invention preferably consists only of cell-derived materials that constitute the cell sheet and does not contain any other materials.

[0093] As described above, the SkM related to this subject can suppress cellular immunity in the presence of T cells. For this reason, in the pharmaceutical according to this embodiment, it is preferable that the SkM is an allogeneic cell of the target recipient of the pharmaceutical. With this configuration, even when allogeneic-derived SkM is used as a cell preparation for regenerative medicine, it is possible to avoid or mitigate immune rejection due to cellular immunity, etc.

[0094] According to Subject B of the present disclosure described above, a novel means is provided that can avoid or mitigate immune rejection in a recipient even when a cell preparation for regenerative medicine is prepared using allogeneic cells as raw materials and administered to the recipient.

[0095] 《Theme C》 Subject C of this disclosure is based on the novel finding that the expression of surface antigens in skeletal muscle-derived cells (SkM) is suppressed by passaging SkM four or more times. More specifically, the inventors performed passaging of SkM and measured the expression status of HLA-ABC antigens at each passage (Test 3 described later). This confirmed that the expression rate of HLA-ABC in SkM decreased with increasing passage (Table 4 and Figure 4).

[0096] Here, HLA-ABC are three major molecules belonging to MHC class I of HLA, and they are involved in cellular immunity by killer T cells by presenting endogenous antigens. As shown in Table 4 and Figure 4, the expression of HLA-ABC decreases when SkM is passaged four or more times. Therefore, it is thought that SkM that has been passaged four or more times is less likely to be rejected by cellular immunity, etc., even when transplanted into an allogene as, for example, a regenerative medicine product. As a result of this study, the inventors have completed Subject C of this disclosure.

[0097] One embodiment (embodiment C1) provided by Subject C is a method for suppressing the expression of surface antigens in skeletal muscle-derived cells (SkM), in which more than 90% are CD56-positive cells, by subculturing SkM four or more times.

[0098] There are no particular restrictions on the culture conditions for subculturing SkM. The culture medium is not particularly limited and can be used as long as it is a medium that can maintain cell viability, such as a basal medium based on various cell culture media. Such basal media include, but are not limited to, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM / F12, etc. Many of these basal media are commercially available and their compositions are publicly known. The basal medium may be used as is (e.g., in its commercially available form), or its composition may be appropriately changed depending on the cell type and cell conditions. The medium may usually contain additives such as serum (e.g., bovine serum such as fetal bovine serum (FBS), horse serum, human serum, etc.) and various growth factors (e.g., FGF, EGF, VEGF, HGF, etc.). In this case, it is preferable that the SkM used for subculturing is obtained by adherent culture.

[0099] The number of subculturing cycles should be four or more, as this sufficiently reduces HLA-ABC expression. From the viewpoint of cell sheet formation ability, the number of subculturing cycles should preferably be between four and seven, for example, between four and six, or between four and five.

[0100] The surface antigens whose expression in SkM is suppressed by the aforementioned subculturing are not particularly limited as long as they are related to the activation or enhancement of cellular immunity, and it is preferable that they include, for example, HLA-ABC antigens. In particular, it is preferable that the surface antigens include at least one of the HLA-ABC antigens (HLA-A antigen, HLA-B antigen, HLA-C antigen), more preferably at least two of them, and especially preferably all of them. The suppression of surface antigen expression can be determined using FACS (fluorescence-activated cell sorting).

[0101] In subcultured SkM, the decrease in the percentage of cells expressing the suppressed surface antigen (expression rate) is preferably 10% or more (0.9 times or less), more preferably 20% or more (0.8 times or less), even more preferably 30% or more (0.7 times or less), even more preferably 40% or more (0.6 times or less), even more preferably 50% or more (0.5 times or less), particularly preferably 60% or more (0.4 times or less), and most preferably 70% or more (0.3 times or less), as a relative decrease when the total number of cells in each subculture is set to 100%. In one embodiment, the above decrease is preferably 10 to 30% (0.7 to 0.9 times).

[0102] As described above, in the subcultured SkM according to this embodiment, the expression of surface antigens is suppressed, and since these surface antigens are related to the expression and enhancement of cellular immunity, this subject also provides a method for suppressing cellular immunity in SkM, which includes implementing the method for suppressing the expression of surface antigens in SkM described above (Embodiment C2). Furthermore, by considering the above embodiment from the perspective of a method for producing SkM, this subject also provides a method for producing SkM in which the expression of surface antigens is suppressed, which includes subculturing skeletal muscle-derived cells (SkM) in which 90% or more are CD56-positive cells four or more times (Embodiment C3).

[0103] This subject also provides information on pharmaceuticals. Specifically, the pharmaceuticals provided by this subject contain skeletal muscle-derived cells (SkM) as an active ingredient, in which more than 90% are CD56-positive cells, and in which the expression level of surface antigens is reduced compared to cells that have not been passaged or have been passaged three times or less (Aspect C4). In the SkM contained in this pharmaceutical, the reduced expression rate of surface antigens suggests that cellular immunity is suppressed.

[0104] Therefore, the pharmaceutical product according to this embodiment is preferably a cell preparation for regenerative medicine. As described above, cell preparations for regenerative medicine can take various forms, such as a sheet containing cells, an injectable preparation containing cells, or a form in which cells are compounded with a biomaterial, and all of these can be preferably adopted, but a sheet form (cell sheet) is particularly preferred. A "cell sheet" refers to a sheet formed by cells being linked together, and typically consists of one cell layer, but also includes those composed of two or more cell layers. The cells may be linked to each other directly and / or via an intervening substance. The intervening substance is not particularly limited as long as it can at least mechanically link the cells together, but examples include the extracellular matrix. The intervening substance is preferably cell-derived, in particular from the cells that constitute the cell sheet. The cells are linked at least mechanically, but may also be functionally linked, for example, chemically or electrically. It is preferable that the cell sheet does not contain a scaffold (support). Scaffolds are sometimes used in the art to adhere cells to their surface and / or interior and maintain the physical integrity of the cell sheet. For example, membranes made of polyvinylidene fluoride (PVDF) are known, but cell sheets can maintain their physical integrity even without such scaffolds. Furthermore, the cell sheet of the present invention preferably consists only of cell-derived materials that constitute the cell sheet and does not contain any other materials.

[0105] As described above, the SkM related to this subject can suppress cellular immunity in the presence of T cells. For this reason, in the pharmaceutical according to this embodiment, it is preferable that the SkM is an allogeneic cell of the target recipient of the pharmaceutical. With this configuration, even when allogeneic-derived SkM is used as a cell preparation for regenerative medicine, it is possible to avoid or mitigate immune rejection due to cellular immunity, etc.

[0106] According to Subject C of the present disclosure described above, a novel means is provided that can avoid or mitigate immune rejection in a recipient even when a cell preparation for regenerative medicine is prepared using allogeneic cells as raw materials and administered to the recipient.

[0107] 《Theme D》 Subject D of this disclosure is based on the novel finding that co-culturing skeletal muscle-derived cells (SkM) with peripheral blood mononuclear cells (PBMCs) induces the expression of inflammatory cytokines in SkM. More specifically, the inventors confirmed changes in the expression of several inflammatory cytokines in SkM by co-culturing SkM with PBMCs (see "Experiment 4" below). As a result, it was confirmed that the expression levels of IL-2, IL-6, IL-15, IFN-γ, and TNF-α were improved in SkM co-culturned with PBMCs (Figures 5 to 9).

[0108] In Experiment 4, the inflammatory cytokines whose expression was induced by co-culturing SkM with PBMCs all played important roles in the inflammatory, proliferative, and maturation phases of tissue repair at the site of inflammation. Therefore, it is thought that SkM co-culturing with PBMCs can mitigate the effects of immune rejection, even if it is transplanted into an allogeneic organism as, for example, a regenerative medicine product and experiences immune rejection, by promoting tissue repair at the site of the rejection. As a result of this study, the inventors have completed Subject D of this disclosure.

[0109] One embodiment (embodiment D1) provided by Subject D is a method for inducing the expression of inflammatory cytokines in skeletal muscle-derived cells (SkM), in which more than 90% are CD56-positive cells, by co-culturing them with peripheral blood mononuclear cells (PBMCs).

[0110] The culture conditions for co-culturing SkM and PBMC are not particularly limited. The culture medium is not particularly limited and can be used as long as it is a medium that can maintain cell viability, such as a basal medium based on various cell culture media. Such basal media include, but are not limited to, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM / F12, etc. Many of these basal media are commercially available and their compositions are publicly known. The basal medium may be used as is (e.g., in its commercially available form), or its composition may be appropriately changed depending on the cell type and cell conditions. The medium may usually contain additives such as serum (e.g., bovine serum such as fetal bovine serum (FBS), horse serum, human serum, etc.) and various growth factors (e.g., FGF, EGF, VEGF, HGF, etc.).

[0111] Co-culture can be performed regardless of the prior or subsequent morphology, as long as SkM and PBMCs are cultured together. Alternatively, each cell type can be cultured separately, and the resulting culture from one type can be added to the culture medium containing the other. Co-culture can be performed under conditions commonly used in this art. For example, typical culture conditions include 37°C and 5% CO2. The culture time is not particularly limited as long as the expression level of the target Treg-inducing factor is sufficiently increased, and can range from approximately 1 hour to 10 days, 1 hour to 5 days, 1.5 hours to 3 days (72 hours), 2 hours to 48 hours, or 2 hours to 24 hours. Similarly, the culture temperature can be any temperature commonly used in this art. Furthermore, a step of washing the cell population after culture may be included. Here, it is preferable that the SkM used for co-culture is obtained by adherent culture.

[0112] The inflammatory cytokines whose expression is induced in SkM by the co-culture described above are not particularly limited as long as they can promote tissue repair at the site of inflammatory response where rejection has occurred, for example, and preferably include one or more selected from the group consisting of interleukins, interferons, chemokines, and tumor necrosis factor. In a preferred embodiment of this specification, the inflammatory cytokines include one or more selected from the group consisting of interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-15 (IL-15), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). The inflammatory cytokines whose expression is induced in SkM by co-culture may be those in which gene (mRNA) expression is induced, or those in which protein expression is induced. Preferably, both are induced. The induction of gene (mRNA) expression can be determined by quantitative PCR (qPCR). The induction of protein expression can be determined by array analysis.

[0113] The inflammatory cytokines whose expression is induced in SkM by co-culture may be those whose expression is induced within SkM cells, or those that are secreted into the supernatant of the co-culture medium. Preferably, they are secreted into the supernatant of the co-culture medium, and more preferably, both.

[0114] Here, it is preferable that the expression level of inflammatory cytokines induced in SkM by co-culture is increased compared to when SkM is cultured alone. When the inflammatory cytokine induced in SkM by co-culture is IL-2, it is preferable that IL-2 is expressed only when SkM is co-cultured with the PBMC. When the induced inflammatory cytokine is IL-6, it is preferable that the expression level of IL-6 increases by 1.0 times or more compared to when SkM is cultured alone. When the induced inflammatory cytokine is IL-15, it is preferable that the expression level of IL-15 increases by 1.5 times or more compared to when SkM is cultured alone. When the induced inflammatory cytokine is TNF-α, it is preferable that the expression level of TNF-α increases by 10.0 times or more compared to when SkM is cultured alone. When the induced inflammatory cytokine is IFN-γ, it is preferable that the expression level of IFN-γ increases by 3.0 times or more compared to when SkM is cultured alone.

[0115] As described above, the co-cultured SkM according to this embodiment induces the expression of inflammatory cytokines, and therefore, it is thought to contribute to mitigating the effects of immune rejection by promoting tissue repair in inflammatory sites where rejection reactions occur, for example. Therefore, this subject also provides a method for promoting the repair of biological tissue by SkM, including the method for inducing the expression of inflammatory cytokines in SkM as described above (Embodiment D2). Furthermore, viewing the above embodiment from the perspective of a method for producing SkM, this subject also provides a method for producing SkM in which the expression of inflammatory cytokines has been induced, including co-culturing skeletal muscle-derived cells (SkM), in which more than 90% are CD56-positive cells, with peripheral blood mononuclear cells (PBMCs) (Embodiment D3).

[0116] This subject also provides pharmaceuticals. Specifically, the pharmaceuticals provided by this subject contain skeletal muscle-derived cells (SkM), in which more than 90% are CD56-positive cells, as an active ingredient, which show increased expression of inflammatory cytokines compared to when cultured alone (Aspect D4). Since the expression of inflammatory cytokines is increased in the SkM contained in this pharmaceutical, as mentioned above, it is thought to contribute to mitigating the effects of immune rejection by promoting tissue repair in inflammatory sites where rejection reactions occur.

[0117] Therefore, the pharmaceutical product according to this embodiment is preferably a cell preparation for regenerative medicine. As described above, cell preparations for regenerative medicine can take various forms, such as a sheet containing cells, an injectable preparation containing cells, or a form in which cells are compounded with a biomaterial, and all of these can be preferably adopted, but a sheet form (cell sheet) is particularly preferred. A "cell sheet" refers to a sheet formed by cells being linked together, and typically consists of one cell layer, but also includes those composed of two or more cell layers. The cells may be linked to each other directly and / or via an intervening substance. The intervening substance is not particularly limited as long as it can at least mechanically link the cells together, but examples include the extracellular matrix. The intervening substance is preferably cell-derived, in particular from the cells that constitute the cell sheet. The cells are linked at least mechanically, but may also be functionally linked, for example, chemically or electrically. It is preferable that the cell sheet does not contain a scaffold (support). Scaffolds are sometimes used in the art to adhere cells to their surface and / or interior and maintain the physical integrity of the cell sheet. For example, membranes made of polyvinylidene fluoride (PVDF) are known, but cell sheets can maintain their physical integrity even without such scaffolds. Furthermore, the cell sheet of the present invention preferably consists only of cell-derived materials that constitute the cell sheet and does not contain any other materials.

[0118] As described above, the SkM related to this subject can mitigate the effects of immune rejection. For this reason, in the pharmaceutical according to this embodiment, it is preferable that the SkM is allogeneic cells for the target of the pharmaceutical administration. With this configuration, even when allogeneic-derived SkM is used as a cell preparation for regenerative medicine, it is possible to avoid or mitigate immune rejection due to cellular immunity, etc.

[0119] According to Subject D of the present disclosure described above, a novel means is provided that can avoid or mitigate immune rejection in a recipient even when a cell preparation for regenerative medicine is prepared using allogeneic cells as raw materials and administered to the recipient.

[0120] While embodiments of the present disclosure have been described in detail above, this is descriptive and illustrative, and not limiting, and it is clear that the scope of the present disclosure should be interpreted by the attached claims. [Examples]

[0121] The effects of this disclosure will be explained using the following examples and comparative examples. The materials, amounts used, proportions, processing content, and processing procedures shown in the following manufacturing examples and examples can be modified as appropriate, as long as they do not deviate from the spirit of this disclosure. Therefore, the technical scope of this disclosure is not limited to the following examples. The quantitative ratios (units: "parts by mass", "mass%") of the raw materials used as solutions or dispersions are all based on solid content. Unless otherwise specified, each operation was carried out at room temperature (hereinafter, room temperature is defined as 25°C).

[0122] <Culture of skeletal muscle-derived cells> First, a myoblast growth medium (0.08% orgadron, 1 μg / mL EGF, 4% L-glutamine, 20% FBS, MCDB131) was prepared.

[0123] Next, skeletal muscle-derived cells at passage 3, isolated from human semitendinosus muscle and cryopreserved, were thawed by warming them in a 37°C bath for 3 minutes.

[0124] After thawing, skeletal muscle-derived cells were suspended in 40 mL of myoblast proliferation medium and centrifuged at 240 × g for 7 minutes at 4°C.

[0125] After removing the supernatant, the cells were resuspended in myoblast proliferation medium, counted, and additional myoblast proliferation medium was added to obtain 1×10 5 cells / mL.

[0126] Subsequently, the cell suspension was seeded at 1 mL / well into a 24-well plate for adherent cells (trade name: Microplate for Tissue Cells (for adherent cells, manufactured by IWAKI), that is, at a seeding density of 5.3×10 4 cells / cm 2 , and cultured for 48 hours in a 37°C, 5% CO2 incubator. This culture corresponds to passage number 4. Skeletal muscle-derived cells are cultured while adhered to the bottom surface of the culture substrate. Note that in all lots of SkM1, SkM2, and SkM3, the CD56 positive rate was 95% or higher in skeletal muscle-derived cells after freeze-thawing (not shown).

[0127] <Culture of PBMC> First, PBMC (trade name: PB009C-1, manufactured by HemaCare) and media (washing medium (PBS containing 10% FBS), PBMC culture medium (RPMI containing 30 U / mL (5 μL / mL) IL-2 and 10% FBS)) were prepared.

[0128] PBMC were thawed in a 37°C water bath, and one vial of PBMC (1.5×10 6 cells) was suspended using 10 mL of washing medium pre-cooled to 4°C.

[0129] After suspension, centrifugation was performed at 400 × g for 10 minutes at 4°C, and the supernatant was removed.

[0130] After removing the supernatant, the cells were suspended in PBMC culture medium and seeded at 1 mL / well into a 24-well plate for suspension cells (trade name: Microplate for Suspension Culture (no surface treatment), manufactured by IWAKI), that is, at a seeding density of approximately 1.0×10 5 cells / cm 2The cells were seeded to achieve the desired concentration and left standing overnight in a 37°C incubator.

[0131] Thereafter, cell counting was performed, followed by centrifugation at 400×g for 10 min at 4°C, and the supernatant was removed.

[0132] <T Cell Proliferation Inhibition Test> (PBMC Staining·Activation) <PBMC Staining> 10 mL of pre-warmed PBS was added to the PBMC pellet obtained in the above <PBMC Culture> step, and the pellet was suspended.

[0133] After suspension, the suspension was aliquoted into 1 mL portions and centrifuged at 400×g for 10 minutes at 4°C.

[0134] As the T cell staining reagent, a FarRed solution (trade name: CellTrace TM Cell Proliferation kit, manufactured by Invitrogen) was used.

[0135] After removing the supernatant, the cells were suspended using a 1000-fold diluted FarRed solution to a concentration of 1.0×10 6 cells / mL, wrapped in aluminum foil, and left standing for 20 minutes in a 37°C incubator.

[0136] Thereafter, PBMC culture medium in an amount 5 times that of the 1000-fold diluted FarRed solution was added, and incubation was performed for 5 minutes at room temperature. This sample is designated as FarRed(+).

[0137] For the control (not using FarRed), after removing the supernatant, the cells were suspended in myoblast proliferation medium (from TFS) to a concentration of 1.0×10 5 cells / mL.

[0138] <PBMC Activation> A portion of FarRed(+) PBMC prepared in the above <PBMC Staining> step was collected, and cell counting was performed.

[0139] FarRed(+) PBMCs were centrifuged together with the culture medium at 400×g for 10 minutes at 4°C.

[0140] After removing supernatant, 1.0×10 5 FarRed(+) cell suspensions were prepared by suspending the cells in myoblast growth medium to a concentration of cells / mL.

[0141] Subsequently, 2 mL of the cell suspension was dispensed and the cells were left in an inactivated state (resting) (the remainder was left in an activated state).

[0142] To activate the cells, 1 mL of PBS was added to 1.5 mL tubes equal to the number of wells to be seeded. Then, DynaBeads (product name: Dynabeads Human T-Activator CD3 / CD28, Gibco) were added to the 1.5 mL tubes, and the 1.5 mL tubes were placed on a DynaMag stand. The PBS was removed from the opposite side of the magnetic surface. After that, the 1.5 mL tubes were removed from the stand and suspended in cell suspension.

[0143] (Co-culture of PBMCs and skeletal muscle-derived cells) In the above <culture of skeletal muscle blasts>, the seeding density was set to 5.3 × 10⁻⁶. 4 cells / cm 2 In addition to this, the seeding density is 2.6 × 10 4 cells / cm 2 , 1.3 × 10 4 cells / cm 2 We prepared the following: We removed the culture supernatant from a 24-well plate to which skeletal muscle-derived cells were attached, and washed each well with PBS.

[0144] Next, 1 mL / well each, i.e., a seeding density of 5.3 × 10 4 cells / cm 2 PBMC suspensions (inactivated and activated states of FarRed(+) and a control) were seeded in such a manner.

[0145] When IFN-γ was added, it was added to a final concentration of 200 ng / mL.

[0146] After sowing, the seeds were cultured in an incubator for 5 days under conditions of 37°C and 5% CO2.

[0147] <Measurement by recovery and flow cytometry> The PBMC suspension was collected from the 24-well plate into a sample tube for FACS.

[0148] After adding 3 mL of 0.5% BSA-PBS solution and mixing thoroughly, the mixture was centrifuged at 1300 rpm for 3 minutes at 4°C, and the supernatant was removed by decantation. This washing procedure was repeated twice.

[0149] 500 μL of 0.5% BSA-PBS solution was added and mixed, and the samples were subjected to analysis. A FACS (fluorescence-activated cell sorting) instrument, CytoFLEX S (manufactured by Beckman Coulter, Inc.), was used for the analysis. The percentage of T cells that had undergone one or more cell divisions among the single T cells contained in PBMCs was measured.

[0150] The results are shown in Figure 1. From the results shown in Figure 1, it was confirmed that SkM suppresses PBMC division in a cell number-dependent manner. This indicates that SkM suppresses T cell proliferation when co-cultured with PBMCs. Compared to PBMCs alone without co-culture with SkM, co-culturing SkM with PBMCs reduced the percentage of T cells that had divided at least once (72.05% for PBMCs alone). Furthermore, the percentage of T cells that had divided at least once decreased in a SkM seeding density-dependent manner. The SkM seeding conditions were 2.5 × 10⁻⁶. 4 cells / well(1.3×10 4 cells / cm 2 ) at 68.59%, 5.0 × 10 4 cells / well(2.6×10 4 cells / cm 2 ) at 45.83%, 1.0 × 10 5cells / well (5.3×10 4 cells / cm 2 ), it was 32.00%.

[0151] [Test 1: Evaluation of immunosuppressive factor expression] Based on the above results, skeletal muscle-derived cells and PBMCs were co-cultured according to the following procedure, and whether this induces the expression of immunosuppressive factors (IL-10, Ido-1, TGF-β) was evaluated. In the procedure described below, two types of skeletal muscle-derived cells, SkM1 and SkM2, which are cell lines derived from different donors, were used. Further, SkM1 and SkM2 co-cultured with PBMCs according to the following procedure were designated as Test Example 1-1 and Test Example 1-2, respectively. In addition, SkM1 and SkM2 not subjected to co-culture with PBMCs, that is, cultured alone, were designated as Test Example 1-3 and Test Example 1-4, respectively.

[0152] <Co-culture with PBMCs> Skeletal muscle-derived cells were obtained in accordance with the above <Culture of Skeletal Muscle-Derived Cells>, and PBMCs were obtained in accordance with <Culture of PBMCs>. The culture supernatant in the 24-well plate to which the skeletal muscle-derived cells were adhered was removed, and each well was washed with PBS.

[0153] Subsequently, 6.6×10 5 cells / mL, that is, a PBMC suspension was seeded so that the seeding density was 3.5×10 4 cells / cm 2 . After seeding, the cells were cultured for 48 hours in an incubator under conditions of 37°C and 5% CO₂. In co-culture, the skeletal muscle-derived cells are in a state adhered to the culture substrate, and the PBMCs are cultured in a state where they contact the adhered skeletal muscle-derived cells but do not adhere to the culture substrate.

[0154] <qPCR and Antibody Array Analysis> qPCR was performed on skeletal muscle-derived cells obtained in accordance with the above <Culture of Skeletal Muscle-Derived Cells>, PBMC obtained in accordance with <Culture of PBMC>, and the cell population after 48 hours of co-culture. In addition, antibody array analysis was performed on culture supernatants after monoculture and co-culture.

[0155] (Preparation of Lysate) The culture medium of PBMC obtained in accordance with the above <Culture of PBMC> was collected, 15 mL thereof was dispensed into a centrifuge tube, and centrifugation was performed under conditions of 400 × g, 10 minutes, and 4°C. The supernatant after centrifugation was collected and stored at -80°C. The pellet after centrifugation was washed with PBS, treated by adding a lysis solution (Lysis Buffer (trade name: Purelink RNA Mini kit, manufactured by thermo fisher) supplemented with 2-mercaptoethanol), and stored as Cell Lysate of PBMC.

[0156] The culture supernatant of skeletal muscle-derived cells obtained in accordance with <Culture of Skeletal Muscle-Derived Cells> was collected, the supernatant after centrifugation was collected, and stored at -80°C. The skeletal muscle-derived cells adhered to the culture substrate were washed with PBS, treated by adding a lysis solution, and stored as Cell Lysate of skeletal muscle-derived cells.

[0157] The culture supernatant of the co-culture of PBMC and skeletal muscle-derived cells was collected, the supernatant after centrifugation was collected, and stored at -80°C. The pellet after centrifugation was washed with PBS, treated by adding a lysis solution, and stored as Cell Lysate of PBMC after co-culture.

[0158] The skeletal muscle-derived cells adhered to the culture substrate were washed with PBS, treated by adding a lysis solution, and stored as Cell Lysate of skeletal muscle-derived cells after co-culture.

[0159] Total RNA was extracted from the lysates of co-cultured skeletal muscle-derived cells and skeletal muscle-derived cells cultured alone, obtained as described above (Lysate preparation), using the Purelink RNA Mini kit (thermo fisher). Total RNA was also extracted from the lysates of co-cultured PBMCs and PBMCs cultured alone, using the riboPure-blood kit (thermo fisher), and the RNA concentration was measured using NanoDrop One (thermo fisher). Based on the obtained total RNA, cDNA was synthesized using SuperScript IV VILO Master Mix (thermo fisher), and the expression levels of extracellular matrix components were quantified by real-time PCR using TaqMan(R) Gene Expression Assays and TaqMan(R) Fast Advanced Master Mix (both from Life Technologies). The Assay IDs and detected genes for the TaqMan(R) Gene Expression Assays are shown in Table 1 below.

[0160] [Table 1]

[0161] The results are shown in Figure 2. In Figure 2, the results of co-culture with PBMCs are shown as "+PBMC," and the results of solitary culture of skeletal muscle-derived cells without co-culture are shown as "-PBMC." Specifically, SkM1 from "+PBMC" is represented as Test Example 1-1, SkM2 from "+PBMC" as Test Example 1-2, SkM1 from "-PBMC" as Test Example 1-3, and SkM2 from "-PBMC" as Test Example 1-4.

[0162] As shown in Figure 2, in Test Examples 1-1 and 1-2, the expression level of the IL-10 gene in skeletal muscle-derived cells increased more than 10 times compared to Test Examples 1-3 and 1-4, respectively. Furthermore, while Ido1 gene expression was not observed in Test Examples 1-3 and 1-4, it was observed in Test Examples 1-1 and 1-2. On the other hand, the expression of the TGF-β gene was almost the same in all Test Examples 1-1 to 1-4.

[0163] (Antibody array analysis) The concentration of cytokine (IL-10) in the culture supernatants of skeletal muscle-derived cells cultured alone, PBMCs cultured alone, and co-cultured skeletal muscle-derived cells and PBMCs, obtained as described above (Lysate preparation), was measured by ELISA. The measurements were performed using the Quanibody Human Cytokine Array (QAH-CAA-4000) Full Testing Service (Raybiotech, Inc. (USA) / Funakoshi Co., Ltd.). The results are shown in Table 2 below.

[0164] [Table 2]

[0165] As shown in Table 2, antibody array analysis also showed that, similar to the above, the expression level of IL-10 protein in the culture supernatant increased in Test Example 1-1 and Test Example 1-2 compared to Test Example 1-3 and Test Example 1-4, respectively.

[0166] Here, it is known that all immunosuppressive factors whose expression is induced by co-culturing SkM with PBMC in Test 1 are Treg-inducing factors. Therefore, the suppression of T cell proliferation by co-culturing SkM with PBMC is considered to be caused by the action of Treg induced via the Treg-inducing factors whose expression is induced by the co-culture. In addition, it is considered that SkM co-cultured with PBMC is less likely to suffer rejection due to cellular immunity or the like via induction of Treg, even when transplanted to an allogeneic recipient as, for example, a product for regenerative medicine.

[0167] [Test 2: Evaluation of immunosuppression by skeletal muscle-derived cells] <Co-culture with PBMC> The thawed skeletal muscle-derived cells obtained in the above <Culture of skeletal muscle-derived cells> were adjusted to 4.0 × 10 5 cells / mL to prepare a suspension of skeletal muscle-derived cells.

[0168] For single culture of skeletal muscle-derived cells, 10 mL of the suspension was seeded into a T-175 flask (162 cm 2 ) and cultured at 37°C for 4 days.

[0169] For co-culture of PBMC and skeletal muscle-derived cells, after culturing the skeletal muscle-derived cells for 1 day as described above, the culture supernatant was removed, and 15 mL of the PBMC suspension adjusted to 2.67 × 10 5 cells / mL in the above <Culture of PBMC> was seeded, and co-cultured at 37°C for 4 days.

[0170] (Analysis of IFN-γ expression level) The concentration of cytokine (IFN-γ) contained in the culture supernatant of the co-culture was measured by an antibody array method. As a result, the expression level of IFN-γ in the co-culture of SkM and PBMC was 2.6 pg / mL.

[0171] <Activation by IFN-γ> IFN-γ was added during the culture of SkM according to the following procedure.

[0172] 4.0×10 5 A suspension of skeletal muscle-derived cells, prepared to a concentration of cells / mL, is placed in a 10cm dish (55cm) 2 10 mL was seeded in a container, and IFN-γ was added to achieve a final concentration of 200 ng / mL. The culture was then incubated at 37°C for 4 days.

[0173] <Surface antigen analysis (flow cytometry)> Surface antigen analysis using flow cytometry was performed on the SkM co-cultured with PBMCs (Test Example 2-1), SkM activated by IFN-γ (Test Example 2-2), and SkM cultured alone (Test Example 2-3) as described above, according to the following procedure.

[0174] During cell culture, the flasks and dishes were removed from the 37°C / 5% CO2 incubator, the culture supernatant was removed, and the mixture was rinsed with HBSS(-).

[0175] After rinsing, HBSS(-) (product name: HBSS, no calcium, no magnesium, no phenol red, 500 mL, Gibco) was removed.

[0176] TrypLE Select (Product name: TrypLE) TM Select Enzyme (1X), no phenol red, 500 mL (Gibco) was added, and the cells were dissociated for approximately 7 minutes.

[0177] The detached cells were collected, mixed with HBSS(-), centrifuged, and then transferred to 2.0 × 10⁶ FACS tubes. 5Cells were added one by one. 0.5% BSA-PBS solution was added and mixed, then centrifuged. The supernatant was removed, and 20 μL of various fluorescently labeled antibodies, diluted in 0.5% BSA-PBS solution, was added and mixed. The mixture was then incubated under refrigeration and protected from light for 30 to 60 minutes. Table 2 shows the fluorescently labeled antibodies used and their dilutions. After the reaction, 0.5% BSA-PBS solution was added, centrifuged, the supernatant was removed, and the mixture was re-mixed with 0.5% BSA-PBS solution. The mixture was then analyzed using a flow cytometry (fluorescence-activated cell sorting) instrument, CytoFLEX S (manufactured by Beckman Coulter, Inc.).

[0178] The results are shown in Figure 3. In the graph shown in Figure 3, the horizontal axis represents the surface antigens of various SkM cells, and the vertical axis represents the relative expression rates by IFN-γ activation or co-culture with PBMCs, with the expression rate of the surface antigen in SkM monoculture (the percentage of cells expressing each surface antigen relative to the total number of SkM cells counted by FACS) set to 1.

[0179] As shown in Figure 3, in both IFN-γ activation (Test Example 2-1) and co-culture with PBMCs (Test Example 2-2), the expression rates of PD-L1, PD-L2, ICAM-1, CD155, and HLA-E were higher than in the control group (Test Example 2-3). Specifically, in IFN-γ activation (Test Example 2-1), the relative expression rates of PD-L1, PD-L2, ICAM-1, CD155, and HLA-E were 1.62, 14.112, 23.55, 1.23, and 39.66, respectively. In co-culture with PBMCs (Test Example 2-2), the relative expression rates of PD-L1, PD-L2, ICAM-1, CD155, and HLA-E were 1.06, 1.27, 3.54, 1.03, and 1.30, respectively.

[0180] These results suggest that SkM activated by IFN-γ and SkM co-cultured with PBMCs exhibit a proliferation-inhibiting effect on T cells through the expression of each surface antigen shown in Table 3 above. Therefore, it is considered that IFN-γ activated SkM and SkM co-cultured with PBMCs are less likely to be rejected by cellular immunity, etc., even when transplanted into an allogeneic population as, for example, a regenerative medicine product.

[0181] [Table 3]

[0182] [Exam 3] <Subculture> Freeze-thawed passage 3 (P3) skeletal muscle-derived cells, 700-1000 cells / cm² 2 To achieve the seeding density, use a T-500 flask (500 cm³) 2 The seeds were sown in a container and cultured in a 37°C / 5%CO2 incubator for about one week. This resulted in passage number 4 (P4). When the bottom of the flask reached about 70% confluence, it was removed from the 37°C / 5%CO2 incubator, the culture supernatant was removed, and the mixture was rinsed with HBSS(-).

[0183] Removes HBSS(-) after rinsing, TrypLE Select (product name: TrypLE TM Select Enzyme (1X), no phenol red, 500 mL (Gibco) was added, and the cells were dissociated for approximately 7 minutes.

[0184] The detached cells were collected in a 50 mL tube and rinsed with HBSS(-).

[0185] After rinsing, the cells were centrifuged at 240 × g for 7 minutes at 4°C. After removing the supernatant, the cells were resuspended in 3 mL of medium and counted. Again, the count was 700-1000 cells / cm². 2 Seeds were seeded in T-500 flasks to achieve the seeding density, and subculturing was repeated.

[0186] The above passages were repeated, and cells from the 4th passage (P4, Test Example 3-1), 5th passage (P5, Test Example 3-2), 7th passage (P7, Test Example 3-3), 9th passage (P9, Test Example 3-4), and 12th passage (P12, Test Example 3-5) were collected, cryopreserved, and subjected to surface antigen analysis (flow cytometry) as described below.

[0187] <Surface antigen analysis (flow cytometry)> Except for using the fluorescently labeled antibodies shown in Table 4 below at the dilution ratios also shown in Table 4, surface antigen analysis (flow cytometry) was performed in the same manner as described above to evaluate the expression rate of HLA-ABC antigens. The results are shown in Table 5 and Figure 4 below.

[0188] [Table 4]

[0189] [Table 5]

[0190] The results shown in Table 5 and Figure 4 confirm that the expression rate of HLA-ABC in SkM decreases with increasing passage number.

[0191] Furthermore, the recovery rate, viability, CD56 expression rate, and sheet-forming ability of cells for each passage are also shown. Here, the recovery rate is the number of cells after thawing (%), with the number of cells at the time of freezing set to 100. Viability is the cell viability (%) after thawing, when the cells are stained with trypan blue and counted. In addition, the CD56 expression rate was measured according to the method of surface antigen analysis (flow cytometry), except that a CD56 antibody (product name: PE anti-human CD56, manufactured by Becton Dickinson, Inc.) was used on the thawed cells.

[0192] Then, the presence or absence of sheet-forming ability was determined by the following procedure. First, UpCell (registered trademark) (manufactured by Cellseed Inc.) 24-well plate, which had been coated the previous day with a medium containing serum (FBS), was prepared. To this plate, 6 × 10 6 cells / mL × 350 μL (that is, 1.1 × 10 6 cells / cm 2 ) were seeded and incubated at 37°C for up to 26 hours. Thereafter, when the plate was rinsed with cooled HBSS(+) and the cells were detached from the plate, if the cell population was in a sheet form, it was determined to have sheet-forming ability (○). On the other hand, if the cell population was not in a sheet form, it was determined to have no sheet-forming ability (×).

[0193] Here, HLA-ABC are three major molecules belonging to MHC class I among HLAs, which present endogenous antigens and are involved in cell-mediated immunity by killer T cells. As shown in Table 5 and Figure 4, subculturing SkM for 4 or more passages reduces the expression of HLA-ABC, so it is considered that SkM subcultured for 4 or more passages is less likely to undergo rejection via cell-mediated immunity even when transplanted to another individual as, for example, a product for regenerative medicine. It is also found that from the viewpoint of cell sheet-forming ability, the number of subculturing passages is preferably 4 or more and 7 or less. Note that the relative decrease when the total number of cells in each passage is defined as 100% was within the range of 10% to 30% (0.7-fold to 0.9-fold) from P4 to P7 where sheet formation was observed.

[0194] [Test 4: Evaluation of inflammatory cytokine expression] <Co-culture with PBMC> Following the same procedure as <Co-culture of SkM and PBMC> in Test 1, for SkM, three cell lines SkM1, SkM2, and SkM3 derived from different donors were each co-cultured with PBMC. Except for not performing co-culture with PBMC, SkM (SkM1, SkM2) cultured alone and PBMC obtained by the same method as that for the co-culture group were used as controls. Here, PBMC cultured alone was designated as Test Example 4-1, PBMC co-cultured with SkM1 was designated as Test Example 4-2, PBMC co-cultured with SkM2 was designated as Test Example 4-3, and PBMC co-cultured with SkM3 was designated as Test Example 4-4. Further, SkM1 cultured alone was designated as Test Example 4-5, SkM1 co-cultured with PBMC was designated as Test Example 4-6, SkM2 cultured alone was designated as Test Example 4-7, and SkM2 co-cultured with PBMC was designated as Test Example 4-8. Further, the culture supernatant obtained from co-culture of PBMC and SkM1 was designated as Test Example 4-9, and the culture supernatant obtained from co-culture of PBMC and SkM2 was designated as Test Example 4-10.

[0195] <qPCR and antibody array analysis> For each of the test examples described above, the expression of inflammatory cytokines after 48 hours of culture was evaluated.

[0196] Specifically, using PBMC, skeletal muscle-derived cells, and culture supernatant (the above-mentioned Test Examples 4-1 to 4-10) after 48 hours of culture, qPCR and antibody array analysis were performed in accordance with the same procedure as in Test 1, and evaluation was carried out. The inflammatory cytokines evaluated were interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-15 (IL-15), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α). The fluorescent probes (trade name TaqMan TM Gene Expression Assay, manufactured by Thermo Fisher) are shown in Table 6 below.

[0197]

Table 6

[0198] Furthermore, the evaluation results are shown in Figures 5 to 9. In Figures 5 to 9, the PBMC graphs show the results for Test Examples 4-1 to 4-4 in order from left to right, the skeletal muscle-derived cell graphs show the results for Test Examples 4-5 to 4-8 in order from left to right, and the antibody array analysis results show the results for Test Examples 4-9 and 4-10 from left to right.

[0199] Note that the RQ values ​​(relative quantitative values ​​of the expression level of each gene) in the qPCR results for each graph are relative values ​​to the sample with an RQ value of 1. Furthermore, the antibody array analysis results show the increase due to co-culture (increased mass per unit volume).

[0200] As shown in Figures 5 to 9, PBMCs co-cultured with SkM (Test Examples 4-2 and 4-4) showed improved expression levels of IL-2 and IL-6. Furthermore, SkM co-cultured with PBMCs (Test Examples 4-6 and 4-8) showed improved expression levels of IL-2, IL-6, IL-15, IFN-γ, and TNF-α. In qPCR, IL-6 expression increased by more than 1.2 times compared to SkM cultured alone. IL-15 expression increased by more than 1.9 times compared to SkM cultured alone. TNF-α expression increased by more than 36 times compared to SkM cultured alone. IFN-γ expression increased by more than 11 times compared to SkM cultured alone. Furthermore, increased concentrations of IL-2, IL-6, IL-15, IFN-γ, and TNF-α were observed in the culture supernatant (Test Examples 4-9 and 4-10) when PBMCs and SkM were co-cultured. These inflammatory cytokines play important roles in the inflammatory, proliferative, and maturation phases of tissue repair at the site of inflammation. Therefore, it is thought that SkM co-cultured with PBMCs can mitigate the effects of immune rejection, for example, when transplanted into an allogeneic organism as a regenerative medicine product and subjected to immune rejection, by promoting tissue repair at the site of rejection.

Claims

1. A method for inducing the expression of surface antigens in skeletal muscle-derived cells (SkM), in which more than 90% are CD56-positive cells, comprising culturing them in a medium containing interferon-γ (IFN-γ) or co-culturing them with peripheral blood mononuclear cells (PBMCs).

2. The method according to claim 1, wherein the SkM is obtained by adhesive culture.

3. The method according to claim 1, wherein the surface antigen comprises one or more selected from the group consisting of a costimulatory molecule, a programmed cell death ligand, a cell adhesion molecule, a TIGIT ligand, and a non-classical HLA class I antigen.

4. The method according to claim 3, wherein the surface antigen comprises one or more selected from the group consisting of PD-L1, PD-L2, ICAM-1, CD155, and HLA-E.

5. The method according to claim 1, comprising culturing the SkM in a culture medium containing the interferon-γ (IFN-γ), wherein the concentration of IFN-γ in the culture medium is 50 ng / mL or more.

6. The method according to claim 1, wherein the expression rate of the surface antigen in the cultured SkM is 1.02 or higher as a relative value when the expression rate of the surface antigen in SkM cultured alone is set to 1.

7. A method for suppressing cellular immunity in the SkM, comprising carrying out the method according to any one of claims 1 to 6.

8. A method for producing SkM in which surface antigen expression is induced, comprising culturing SkM, in which more than 90% are CD56-positive cells, in a medium containing interferon-γ (IFN-γ), or co-culturing SkM with peripheral blood mononuclear cells (PBMCs).

9. The method for producing SkM according to claim 8, wherein the SkM is obtained by adhesive culture.

10. The method for producing SkM according to claim 8 or 9, wherein the surface antigen comprises one or more selected from the group consisting of PD-L1, PD-L2, ICAM-1, CD155, and HLA-E.

11. A pharmaceutical product containing skeletal muscle-derived cells (SkM) as an active ingredient, in which more than 90% are CD56-positive cells and the expression rate of surface antigens is increased compared to the control.

12. The pharmaceutical product according to claim 11, wherein the surface antigen comprises one or more selected from the group consisting of PD-L1, PD-L2, ICAM-1, CD155, and HLA-E.

13. The pharmaceutical product according to claim 12, which is a cell preparation for regenerative medicine.

14. The pharmaceutical product according to any one of claims 11 to 13, wherein the SkM is an allogeneic cell to the target of the pharmaceutical product administration.

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