Group 2 innate lymphocyte proliferation inhibitors

Abstract

Provided is a group 2 innate lymphoid cell growth inhibitor containing MAIT cells.

Description

[Technical Field]

[0001] The present invention relates to an inhibitor of proliferation of group 2 innate lymphocytes, including MAIT cells. ILC2 proliferation is a cause of various inflammatory diseases, including asthma, and therefore, controlling ILC2 proliferation opens the way to the prevention and treatment of inflammatory diseases. [Background technology]

[0002] Innate lymphoid cells (ILCs) are cells involved in innate immunity and have been discovered as a new type of lymphocyte distinct from T and B lymphocytes. Innate lymphoid cells have the ability to produce cytokines involved in various types of inflammation and are classified into three groups based on their production patterns: group 1 innate lymphoid cells (ILC1s), group 2 innate lymphoid cells (ILC2s), and group 3 innate lymphoid cells (ILC3s). ILC2s, among others, are key players in airway inflammatory diseases, producing type 2 cytokines such as IL-5, IL-9, and IL-13 and inducing eosinophilia. Artificial control of ILC2 proliferation could lead to the development of novel treatments for diseases such as asthma.

[0003] One known invention uses the compound disulfiram as an inhibitor of ILC2 activity (Patent Document 1: JP 2020-40924 A). This invention uses disulfiram to suppress ILC2 proliferation and cytokine production.

[0004] On the other hand, MAIT cells (mucosal-associated invariant T cells) are a type of innate immune T cell that regulates the immune response of individuals and act as a bridge between innate and adaptive immunity through the production of various cytokines. MAIT cells are abundant in humans, accounting for 20-50% of T cells in the liver and 1-10% of T cells in intestinal lamina propria lymphocytes (LPL) and peripheral blood mononuclear cells (PBMC). However, they are rare in mice (Non-Patent Document 2: Dusseaux et al., 2011; Non-Patent Document 3: Le Bourhis et al., 2011).

[0005] MAIT cells have been suggested to be involved in the development and progression of autoimmune and inflammatory diseases, including multiple sclerosis, as well as cancer. + / CD161 high T cells accumulate at sites of inflammation such as the liver and joints, and are considered to be a factor in the development of multiple sclerosis. However, CD8 + / CD161 high More than 90% of T cells have been shown to express the MAIT cell-specific T cell receptor (TCR) α chain, Vα7.2 (Non-Patent Document 4: Walker et al., 2012). Furthermore, it has been reported that MAIT cells accumulate in the lesions of multiple sclerosis patients (Non-Patent Document 5: Illes et al., 2004; Non-Patent Document 6: Miyazaki et al., 2011). MAIT cell accumulation has also been reported in kidney cancer, brain tumors (Non-Patent Document 7: Peterfalvi et al., 2008), and chronic inflammatory demyelinating polyneuropathy (Non-Patent Document 5: Illes et al., 2004). Furthermore, it has been reported that transferred MAIT cells have a protective effect against drug-induced inflammatory tissue damage in inflammatory bowel diseases, such as ulcerative colitis and Crohn's disease (Non-Patent Document 8: Xiao Ruijing et al., 2012).

[0006] It is known that the proliferation and cytokine production of ILC2s are suppressed by interferon beta / gamma, E-cadherin, lipoxin A4, etc., and that their cytokine production is suppressed by MAIT cells (Non-Patent Document 1: Ye et al., MAIT cells restrict allergic airway inflammation, J Allergy Clin Immunol, 145, 1469-1473 (2020)). However, there have been no reports of MAIT cells suppressing ILC2 proliferation. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2020-40924 [Patent Document 2] Patent No. 6275646 [Patent Document 3] WO / 2021 / 085450 [Non-patent literature]

[0008] [Non-Patent Document 1] Ye et al MAIT cells restrict allergic airway inflammation J Allergy Clin Immunol 145, 1469-1473(2020) [Non-patent document 2] Dusseaux et al.,Blood vol.117, page1250-9, 2011 [Non-patent document 3] Le Bourhis et al., Tends Immunol vol.32, page 212-8, 2011 [Non-patent document 4] Walker et al.,Blood vol.119, page422-33, 2012 [Non-patent document 5] Illes et al., Int Immunol vol.16, page223-30, 2004 [Non-patent document 6] Miyazaki et al., Int Immunol vol.23, page529-35, 2011 [Non-Patent Document 7] Peterfalvi et al., Int Immunol vol.12, page1517-25, 2008 [Non-patent document 8] Ruijing et al., Hepatogastroenterology vol.115, page 762-7, 2012 Summary of the Invention [Problem to be solved by the invention]

[0009] Given the above background, there was a need to develop substances other than disulfiram that could inhibit the proliferation of ILC2. [Means for solving the problem]

[0010] As a result of intensive research conducted by the present inventors to solve the above problems, they discovered that MAIT cells suppress the proliferation of ILC2s and are useful for inflammatory diseases, leading to the completion of the present invention. That is, the present invention is as follows. (1) An inhibitor of LC2 proliferation, including MAIT cells. (2) The inhibitor described in (1), wherein the proliferation of eosinophils or mast cells is inhibited by inhibiting the proliferation of ILC2. (3) A drug for suppressing eosinophilia or eosinophilic inflammation, comprising the inhibitor according to (1) or (2). (4) A composition for treating or preventing an ILC2 proliferative disease, comprising MAIT cells. (5) The composition according to (4), wherein the ILC2 proliferative disorder is selected from the group consisting of asthma, asthma-like disorders, allergic rhinitis, eosinophilic sinusitis, atopic dermatitis, contact dermatitis, cirrhosis, biliary atresia, viral hepatitis, parasitic infection, viral infection, eosinophilic pneumonia, eosinophilic pleural effusion, interstitial pneumonia, systemic lupus erythematosus (SLE), food allergy, Helicobacter pylori-associated gastric disease, arteriosclerosis, recurrent cancer, GVHD, and chronic cough associated with eosinophilic inflammation. (6) The composition according to (4), wherein the ILC2 proliferative disease is Th2-type asthma or non-Th2-type asthma, cough-variant asthma, atopic cough, non-atopic cough, or non-asthmatic eosinophilic bronchitis. (7) A composition for treating or preventing an inflammatory disease, comprising MAIT cells. (8) The inflammatory disease is rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, ulcerative colitis, or the like. Enteritis, pharyngitis, cystitis, hepatitis, pneumonia, pancreatitis, enteritis, antiphospholipid syndrome, polymyositis , dermatomyositis, scleroderma, Sjogren's syndrome, IgG4-related disease, vasculitis syndrome, mixed connective tissue disease, adult Still's disease, Crohn's disease, primary biliary cirrhosis, multiple sclerosis, myasthenia gravis, Guillain-Barré syndrome, rapidly progressive glomerulonephritis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura (immune thrombocytopenia), Graves' disease, pemphigus, and pemphigoid. (9) An agent for suppressing the production of interleukin 5, interleukin 6, interleukin 9, or interleukin 13, including MAIT cells. (10) The inhibitor according to (9), for at least one selected from the group consisting of asthma, chronic obstructive pulmonary disease (COPD), SLE, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, rheumatoid arthritis, psoriasis, graft-versus-host disease (GVHD), parasitic disease, chronic sinusitis, allergic dermatitis, peanut allergy, severe combined immunodeficiency, and acute pancreatitis. (11) A method for screening drugs for ILC2 proliferative disorders or inflammatory disorders, comprising contacting a candidate substance with a mouse enriched in MAIT cells or its tissue. (12) A method for screening drugs for ILC2 proliferation disorders or inflammatory disorders, comprising co-culturing MAIT cells collected from mice enriched for MAIT cells with ILC2s collected from wild-type mice in the presence of a candidate substance. (13) A method for screening drugs for ILC2 proliferation disorders or inflammatory disorders, comprising the steps of transferring MAIT cells collected from mice enriched for MAIT cells and ILC2s collected from wild-type mice into severely immunodeficient mice, and then inoculating or administering a candidate substance. [Effects of the Invention]

[0011] The present invention provides MAIT cells that suppress ILC2 proliferation. The MAIT cells of the present invention are useful as ILC2 proliferation inhibitors for the prevention or treatment of various inflammatory diseases (e.g., airway inflammatory diseases). [Brief explanation of the drawings]

[0012] [Figure 1] These figures show the results of a study in which wild-type B6 mice and Vα19 mice were challenged with Alternaria. The left side of Figure 1 shows HE and PAS staining of lungs from wild-type B6 and Vα19 mice in which airway inflammation was induced by nasal inoculation with Alternaria. Hematoxylin-eosin (HE) staining revealed that endobronchial goblet cell hyperplasia (black arrows) and peribronchial inflammation (white arrows) observed in wild-type B6 mice were attenuated in Vα19 mice, which have approximately 20-fold more MAIT cells than B6 mice. Periodic acid-sciff (PAS) staining revealed that mucin production from goblet cells in the bronchi (dark purple) was reduced in Vα19 mice compared with wild-type B6 mice (black arrows) (X400). The right side of Figure 1 shows the number of ILC2s in bronchoalveolar lavage fluid (BALF) 8 days after nasal inoculation with Alternaria between wild-type B6 and Vα19 mice. Although there was no significant difference between the control (PBS) and Alternaria-inoculated (AA) mice, wild-type B6 mice showed a significant increase in ILC2 numbers, whereas Vα19 mice showed a significant suppression of this increase. [Figure 2]This figure shows the number of each cell population in bronchoalveolar lavage fluid from severely immunodeficient mice in which airway inflammation was induced with IL-33. ILC2s alone or ILC2s plus murine MAIT cells were adoptively transferred into severely immunodeficient mice (NOG mice), and airway inflammation was induced with IL-33. Bronchoalveolar lavage fluid was collected on day 8 after IL-33 stimulation, and the number of each cell population was calculated by flow cytometry analysis. Co-transfer of MAIT cells significantly reduced eosinophils and suppressed ILC2 proliferation. [Figure 3] This figure shows the suppression of ILC2 proliferation by non-contact culture of ILC2s and MAIT cells. ILC2s were cultured with or without IL-33 stimulation, and proliferation was monitored. Under these conditions, unstimulated MAIT cells, MAIT cells stimulated with IL-12 / IL-15 / IL-18 (cytokines), and MAIT cells stimulated with TCR using 5-OP-RU (MAIT cell agonist) were added to transwell inserts, and ILC2 proliferation was measured under culture conditions without contact between the two cells. The results showed that cytokine-stimulated MAIT cells significantly suppressed ILC2 proliferation. [Figure 4A] FIG. 1 shows that MAIT cells stimulated with IL-12 / IL-15 / IL-18 (cytokines) produce IFN-γ, but that this production is not affected by co-culture with ILC2s. [Figure 4B] This figure shows the ability of cytokine-stimulated MAIT cells to suppress cytokine production from IL-33-stimulated ILC2 in non-contact culture. [Figure 5] This figure shows that the suppression of ILC2 proliferation by MAIT cells stimulated with IL-12 / IL-15 / IL-18 (cytokines), as observed in Figure 3, is due to IFNγ production by MAIT cells. Addition of isotype IgG to cocultures of cytokine-stimulated MAIT cells and ILC2s in the presence of IL-33 did not relieve the suppression of ILC2 proliferation, but addition of an IFNγ-neutralizing antibody did. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention relates to MAIT cells that negatively regulate the proliferation and cytokine production of ILC2. 1.MAIT cells The MAIT cells used in the present invention are present in large amounts in peripheral blood and can therefore be collected from these tissues. Mononuclear cells were isolated from human peripheral blood, umbilical cord blood, etc. using Ficoll or other methods, and then stained with anti-CD3 or anti-TCRβ antibodies, anti-Vα7.2 antibodies (Biolegend) that recognize MAIT cells, as well as anti-CD161 and anti-IL-18 receptor α (IL-18Ra) antibodies (Biolegend). MAIT cells were identified as Vα7.2. + TCRβ + CD161 + IL-18Rα + These are then separated and purified using a flow cytometer. Similarly, the above mononuclear cells were stained with human MR1 tetramer (hMR1-tet, available from the NIH tetramer core facility) loaded with 5-OP-RU, a reagent that recognizes MAIT cells, and anti-CD3 or anti-TCRβ antibodies to identify hMR1-tet. + CD3 + (TCRβ + ) and can be isolated and purified using a flow cytometer. Mouse MAIT cells can be identified, isolated, and purified from Vα19 mice by the method described in this application, using a combination of 5-OP-RU-loaded mouse MR1 tetramer (hereinafter referred to as mMR1-tet, available from the NIH tetramer core facility) and an antibody that recognizes mouse TCRβ. In this case, the starting materials are the peripheral blood, spleen, liver, lungs, intestines, bone marrow, etc. of Vα19 mice (WO / 2021 / 085450), which have a higher abundance of MAIT cells than wild-type mice.

[0014] MAIT cells can also be differentiated from human iPS cells, following the steps below. iPS cell generation from MAIT cells: One example of this method is described in Patent No. 6275646. Patent No. 6275646 states that the T cell receptor α chain (TCRα chain) of human MAIT cells is Vα7.2-Jα33, but here we are referring to T cells recognized by hMR1-tet, i.e., hMR1-tet + CD3 + (TCRβ + MAIT cells are defined as cells with TCRα chains that are not particularly limited. + CD3 + (TCRβ + MAIT cells, which are human MAIT-iPS cells, are isolated and purified using a flow cytometer. The purified MAIT cells are then converted into iPS cells (human MAIT-iPS cells) using a Sendai virus vector or the like described in WO / 2021 / 085450 and Japanese Patent No. 6275646.

[0015] Differentiation of human MAIT-iPSCs into MAIT-like cells: hMR1-tet + CD3 + (TCRβ + ) cells, iPS cells can be induced to differentiate into MAIT-like cells using OP9, as described in Japanese Patent No. 6275646, and OP9 / DLL1 (OP9 with forced expression of delta-like 1, a Notch ligand) (a publicly known, non-patented method). As described above, mouse MAIT cells can be prepared from Vα19 mice, but they can also be expanded by inducing differentiation of mouse MAIT cell-derived iPS cells using OP9 / DLL1, as described in WO / 2021 / 085450. Furthermore, while WO / 2021 / 085450 specifically illustrates the collection of mouse MAIT cells from the lungs of wild-type mice, MAIT cells can be obtained from any tissue, including bone marrow, intestine, thymus, and lymph nodes.

[0016] The cells obtained in this manner can be confirmed to be MAIT cells by measuring by flow cytometry whether they are stained with hMR1-tet and anti-human CD3 or TCRβ antibody. The obtained MAIT cells can also be frozen and stored until use (storage conditions: frozen in a deep freezer at -80°C using a cell preservation solution such as BAMBANKER (GC lymphotec), and then stored in liquid nitrogen).

[0017] 2. ILC2 proliferation / cytokine production inhibitors and pharmaceutical compositions for ILC2-related diseases ILC2s are activated by cytokines such as IL-25, IL-33, and TSLP. Activated ILC2s produce cytokines such as IL-5, IL-9, and IL-13. Furthermore, ILC2s induce and activate eosinophils and induce goblet cell hyperplasia through the action of these cytokines.

[0018] Therefore, the MAIT cells of the present invention can be used as an inhibitor of ILC2 proliferation and type 2 cytokine production from ILC2. The inhibitor of ILC2 proliferation suppresses ILC2-mediated eosinophil proliferation and can be used to treat or prevent eosinophilic inflammatory diseases. Another aspect of the present invention relates to a pharmaceutical composition comprising MAIT cells for treating or preventing an inflammatory disease. Yet another aspect of the present invention relates to a pharmaceutical composition comprising MAIT cells for treating or preventing an ILC2-associated disease. In the present invention, cytokines that are the target of the type 2 cytokine production inhibitor include interleukin (IL)-5 (IL-5), IL-6, IL-9, IL-13, and the like.

[0019] Examples of inflammatory diseases that can be treated or prevented by the compositions of the present invention include rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, ulcerative colitis, pharyngitis, cystitis, hepatitis, pneumonia, pancreatitis, enteritis, antiphospholipid syndrome, polymyositis, dermatomyositis, scleroderma, Sjogren's syndrome, IgG4-related disease, vasculitis syndrome, mixed connective tissue disease, adult Still's disease, Crohn's disease, primary biliary cirrhosis, multiple sclerosis, myasthenia gravis, Guillain-Barré syndrome, rapidly progressive glomerulonephritis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura (immune thrombocytopenia), Graves' disease, pemphigus, and pemphigoid.

[0020] In the present invention, among inflammatory diseases, diseases associated with ILC2 are particularly preferred. ILC2s have been reported to be present in various tissues, such as the respiratory tract, skin, blood, digestive tract, eyes, and nose, and are involved in diseases of these tissues, particularly allergic diseases. ILC2-mediated allergies are called innate allergies and are distinct from Th2-mediated acquired allergies. However, ILC2-mediated IL-13 and IL-5 can also be produced by Th2 cells, and these can sometimes be indistinguishable from the resulting goblet cell hyperplasia and eosinophil induction. Examples of ILC2-related diseases include allergic inflammation of the skin, respiratory tract, and gastrointestinal tract.

[0021] Respiratory ILC2-associated diseases include asthma, allergic rhinitis, eosinophilic sinusitis, eosinophilic pneumonia (e.g., Loeffler syndrome, chronic eosinophilic pneumonia), exercise-induced bronchoconstriction, chronic obstructive pulmonary disease (COPD), acute bronchitis, chronic bronchitis, emphysema, nasal polyps, idiopathic pulmonary fibrosis, allergic bronchitis, alveolitis, bronchiectasis, eosinophilic pleural effusion, and interstitial pneumonia. Skin ILC2-associated diseases include atopic dermatitis, contact dermatitis, scleroderma, psoriasis, urticaria, and blisters. Other ILC2-associated diseases include liver cirrhosis, biliary atresia, viral hepatitis, parasitic infection, influenza virus infection, systemic lupus erythematosus (SLE), food allergies, Helicobacter pylori-associated gastroenteropathy, atherosclerosis, bladder cancer recurrence, and lower gastrointestinal graft-versus-host disease (GVHD). ILC2-associated diseases include, among others, asthma, allergic rhinitis, eosinophilic sinusitis, atopic dermatitis, chronic cough with eosinophilic inflammation, contact dermatitis, liver cirrhosis, biliary atresia, viral hepatitis, parasitic infection, influenza virus infection, eosinophilic pneumonia, eosinophilic pleural effusion, interstitial pneumonia, systemic lupus erythematosus (SLE), food allergy, Helicobacter pylori-associated gastrointestinal disease, atherosclerosis, bladder cancer recurrence, lower gastrointestinal GVHD, and high-fat diet-induced obesity.

[0022] Diseases for which the inhibitor or composition of the present invention can be used include asthma and asthma-like diseases. Asthma is a chronic airway inflammation. While eosinophilic inflammation is typical, phenotypes in which inflammatory cells other than eosinophils are predominant also exist. Asthma for which the inhibitor or composition of the present invention is effective includes Th2-type asthma and non-Th2-type asthma. Th2-type asthma includes early-onset allergic asthma, aspirin asthma, late-onset eosinophil-predominant asthma, and allergic bronchopulmonary mycosis (ABPM). Non-Th2-type asthma includes late-onset non-allergic asthma, neutrophil-predominant asthma, and obesity-related asthma. From the perspective of the relationship with ILC2, asthma for which the inhibitor or composition of the present invention is effective includes Th2-type asthma, particularly eosinophil-predominant asthma. Asthma-like diseases include chronic cough accompanied by eosinophilic inflammation. Specifically, cough variant asthma, atopic cough, and non-asthmatic eosinophilic bronchitis are known to be associated with ILC2, and the inhibitors or compositions of the present invention are effective in treating these diseases in light of their association with ILC2. Furthermore, some asthma patients experience severe symptoms that are difficult to control even with high-dose inhaled corticosteroids. The involvement of ILC2 in such steroid-resistant asthma has been suggested. Therefore, the inhibitors or compositions of the present invention are useful for treating or preventing steroid-resistant allergic diseases, particularly steroid-resistant asthma. Furthermore, because ILC2 induces high-fat diet-induced obesity, the inhibitors or compositions of the present invention are effective against obesity or diabetes.

[0023] Furthermore, in the present invention, the IL-5, IL-6, IL-9 or IL-13 production inhibitor can be used for at least one selected from the group consisting of asthma, chronic obstructive pulmonary disease (COPD), SLE, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, rheumatoid arthritis, psoriasis, graft-versus-host disease (GVHD), parasitic diseases, chronic sinusitis, allergic dermatitis, peanut allergy, severe combined immunodeficiency, and acute pancreatitis.

[0024] In addition to the MAIT cells, the pharmaceutical compositions of the present invention may contain other cells as long as the effects of the present invention are not impaired. Depending on the intended use and form, pharmaceutically acceptable carriers and additives may be added in accordance with standard methods. Examples of such carriers and additives include, but are not limited to, isotonicity agents, thickeners, sugars, sugar alcohols, preservatives, bactericides or antibacterial agents, pH adjusters, stabilizers, chelating agents, oily bases, gel bases, surfactants, suspending agents, binders, excipients, lubricants, disintegrants, foaming agents, fluidizing agents, dispersants, emulsifiers, buffers, solubilizers, antioxidants, sweeteners, acidulants, colorants, flavoring agents, fragrances, and refreshing agents.

[0025] In the present invention, when MAIT cells are used as the above-mentioned therapeutic or preventive pharmaceutical composition, the dosage can be selected arbitrarily depending on the condition, sex, weight, or age of the patient. The preferred administration route is intravascular administration (preferably intravenous administration), intraperitoneal administration, intraintestinal administration, subcutaneous administration, etc., with intravascular administration being more preferred. The usual dosage (number of cells) for administration to an adult is 5 x 10 7 ~5×10 8 Pieces / dose, preferably 1 x 10 8 ~1×10 9 pieces / time, more preferably 5 x 10 8 ~5×10 9 This dose may be administered multiple times as a single dose, or may be administered in multiple divided doses. In addition, when administered to an adult, the number of cells per body weight is usually 1 x 10 6 ~1×10 7 pieces / kg, preferably 2 x 10 6 ~2×10 7cells / kg (Guo H, Su Y, Deng F. Effects of Mesenchymal Stromal Cell-Derived Extracellular Vesicles in Lung Diseases: Current Status and Future Perspectives. Stem Cell Rev Rep. 2021 Apr;17(2):440-458. doi: 10.1007 / s12015-020-10085-8. Epub 2020 Nov 19. PMID: 33211245; PMCID: PMC7675022.).

[0026] 3. Analysis using mice with abundant MAIT cells (mice of the present invention) The mouse of the present invention can be used as a model mouse for research into allergic diseases and the like. Therefore, the present invention provides a method for screening for a drug for an ILC2 proliferative disorder or an inflammatory disorder, which comprises contacting a candidate substance with a mouse enriched in MAIT cells or its tissue. The present invention also provides a screening method for a drug for an ILC2 proliferative disorder or an inflammatory disorder, which comprises co-culturing MAIT cells collected from a mouse enriched for MAIT cells with ILC2s collected from a wild-type mouse in the presence of a candidate substance. Furthermore, the present invention provides a method for screening for a drug for an ILC2 proliferative disorder or an inflammatory disorder, which comprises the steps of transferring MAIT cells collected from a mouse enriched in MAIT cells and ILC2s collected from a wild-type mouse into a severely immunodeficient mouse, and then inoculating or administering a candidate substance to the mouse.

[0027] MAIT cells are thought to be involved in the pathological control of human infectious diseases, obesity, type II diabetes, allergies, asthma, and various autoimmune diseases. Therefore, by analyzing these disease models using the mouse of the present invention (Vα19 mouse), new knowledge about the onset and pathology of these diseases that cannot be obtained using conventional mouse models can be obtained.

[0028] Specifically, the present invention includes the following steps. (a) contacting a candidate substance with the mouse of the present invention or its tissues (b) examining the ILC2 proliferation or inflammation of the contacted mice or tissues. The candidate substance is not particularly limited and may be an existing drug, or may be in any form such as a peptide, a low molecular weight compound, a high molecular weight compound, or a salt or precursor thereof. Furthermore, mouse tissues are not particularly limited, and examples include lung, bronchi, spleen, skin, small intestine, large intestine, throat, bladder, liver, pancreas, etc. Tissues also include tissue slices and cells isolated from tissues.

[0029] In the present invention, "contact" refers to administering a candidate substance to a mouse. Administration may be oral or parenteral. The route of administration of the candidate substance is not particularly limited as long as it is a route commonly used for drug administration, and examples include oral, sublingual, pulmonary, gastrointestinal, transdermal, ophthalmic, intravenous, subcutaneous, intramuscular, intraperitoneal, local injection, and surgical implantation. Oral or nasal administration is preferred. When tissue is used, the candidate substance may be added to a container containing a tissue slice or a container containing cells separated from the tissue (including culture medium, buffer solution, etc.).

[0030] The present invention also includes the following step (c) or (d): (c) A step of collecting MAIT cells from the mice of the present invention in which MAIT cells have been enriched, collecting ILC2s from wild-type mice, and co-culturing these MAIT cells and ILC2s in the presence of a candidate substance. In this case, a candidate substance is contacted with the co-culture medium, and the production of IL-5, IL-6, IL-9, or IL-13 by ILC2s or ILC2 proliferation is used as an indicator. In this case, the MAIT cells may be stimulated with IL-12 / IL-15 / IL-18 (cytokine-stimulated MAIT cells) as shown in the present invention (Figure 5), or may be in a naive state without stimulation. Furthermore, the ILC2s used in this step may be stimulated with IL-33 as shown in the present invention, or stimulated with a substance that, like IL-33, promotes type 2 cytokine production and proliferation. (d) adoptively transferring the MAIT cells and ILC2s described in (c) into severely immunodeficient mice and inoculating or administering a candidate substance.

[0031] As shown in the present invention (Figure 4b), the indicators are MAIT cell-mediated suppression of cytokine production from ILC2s and ILC2 proliferation. Furthermore, in screening methods using severely immunodeficient mice, the stimulating substance for inflammation induction is not limited to IL-33, as long as it stimulates ILC2s. It may also be various cytokines (including interleukins), bacteria, or antigens derived from house dust mites, cockroaches, pollen, etc. The method also includes a step of inoculating or administering (by injection, oral administration, topical application, etc.) a candidate substance to severely immunodeficient mice sensitized with the stimulating substance.

[0032] The items to be inspected in steps (b)-(d) are at least one of the following: (i) ILC2 proliferation (ii) Eosinophil proliferation (iii) The concentration of type 2 cytokines, such as IL-5, IL-6, IL-9, and IL-13, produced by ILC2s, or the percentage of ILC2s producing these cytokines. (iv) Goblet cell hyperplasia in the airways (v) Mucin production (vi) Airway smooth muscle hyperplasia (vii) Airway subepithelial basement membrane thickening (viii) Bronchial mucous gland hyperplasia (ix) Airway resistance (x) Enlargement of the intercellular spaces of the respiratory epithelium (xi) Desquamation of airway epithelium (xii) Mast cell proliferation (xiii) Neutrophil proliferation If at least one of the above items is improved by contact with the candidate substance compared to the above items before contact or compared to a control, the candidate substance can be selected as a drug for preventing or treating an ILC2 proliferative disorder or an inflammatory disease.

[0033] Example The present invention will be explained in more detail below with reference to examples, although the scope of the present invention is not limited to these examples. [Example]

[0034] Materials and Methods (1) Alternaria arternata (AA) suppresses eosinophilic airway inflammation in Vα19 mice To induce airway inflammation in wild-type B6 mice (C57BL / 6, 6-week-old female, CLEA Japan) and Vα19 mice (6-week-old female), 10 μg of Alternaria arternata (AA) extract (ITEA Corporation, code number 10117) was dissolved in 50 μl of PBS and administered intranasally using a pipette equipped with a 200 μl tip on days 0, 3, and 6. Nasal administration was performed under isoflurane anesthesia. Control mice received intranasal administration of PBS alone on days 0, 3, and 6. Lungs were then removed on day 8, and 2 μm-thick paraffin sections were prepared and stained with hematoxylin-eosin (HE) and periodic acid-Schiff (PAS). Morphological observations of eosinophil infiltration and goblet cells in lung tissue were performed using a microscope (Olympus BX51 equipped with a DP73 microscope camera). Eosinophils were observed as cells larger than neutrophils, with homogeneous, coarse granules staining orange-yellow with eosin affinity filling the cytoplasm and bilobed nuclei. Goblet cells were observed in the airway epithelium as cells scattered among pseudostratified ciliated epithelium, with triangular nuclei at the base that stained intensely with PAS.

[0035] For cell analysis of bronchoalveolar lavage fluid, mice were intubated into the main bronchus with a 20-gauge Surflow needle on day 8, and saline was lavaged twice at the same site using a 1-ml syringe (1-ml syringe, Terumo, SS-01T) at 0.6 ml per lavage. The recovery rate of the lavage fluid was 1 ml / 1.2 ml. The lavage fluid was centrifuged at 1600 rpm for 15 minutes, the supernatant was removed, and the cells were suspended in 100 μl of FACS buffer (PBS containing 5% FBS and 0.02% NaN3) to prepare bronchoalveolar lavage fluid.

[0036] Ten microliters of cells were dispensed into a 96-well plate (Coaster, Cat. No. 2797) and incubated with lineage markers: anti-F4 / 80 (BioLegend, clone: BM8), anti-CD8a (BioLegend, clone: 53-6.7), anti-Ter119 (BioLegend, clone: Ter-119), anti-CD5 (BioLegend, clone: 53-7.3), anti-CD3e (BioLegend, clone: 145-2C11), anti-CD19 (BioLegend, clone: 6D5), anti-NK1.1 (BioLegend, clone: PK136), anti-CD11c (BioLegend, clone: N418), anti-CD11b (BioLegend, clone: M1 / 70), and Anti-FcεRIa (BioLegend, clone: MAR-1) and anti-Gr-1 (BioLegend, clone: RB6-8C5) (both biotin-labeled) were added, and the mixture was incubated at 4°C in the dark for 20 minutes. After washing with FACS buffer, APC-Cy7-conjugated anti-CD45 (BD Pharmingen, clone 104), PE-conjugated anti-ST2 (BioLegend, clone DIH9), Pacific Blue-conjugated anti-CD90.2 (BioLegend, clone 53-2.1), and streptavidin FITC (BD Pharmingen) were added and incubated for 20 minutes in the dark at 4°C. After washing with FACS buffer, the cells were counted using a flow cytometer (MACSQuant, Miltenyi Biotech) as lineage marker-negative, CD45-positive, ST2-positive, and CD90.2-positive ILC2s.

[0037] (2) Adoptive transfer of MAIT cells suppresses ILC2-induced eosinophilic airway inflammation in NOG mice (2-1) Preparation of lung and spleen cells Naive ILC2s were isolated from the lungs of 40 wild-type B6 mice (C57BL / 6, 8-week-old females). MAIT cells were also isolated from the lungs and spleens of 20 Vα19 mice (8-week-old females). After euthanasia, the lungs were removed and washed with RPMI 1640. They were then minced into 2-3 mm cubes using surgical scissors in a C tube (Miltenyi BioTec, Cat. No. 130-096-334). 10 mL of tissue dissociation solution (RPMI 1640 containing 50 μg / mL Liberase™ (Roche, Cat. No. 5401119001) and 10 μg / mL DNase I (Roche, Cat. No. 1284932)) was added per 10 mice and incubated at 37°C for 45 minutes with shaking.

[0038] The tissue was pulverized and lysed using GentleMACS (Miltenyi Biotec) program 2 for mouse lung cell preparation. The cells were passed through a 100 μm cell strainer and centrifuged to pellet. 3 mL of ACK Lysing Buffer was added for 2 minutes for hemolysis, followed by dilution with 30 mL of Hank's Buffer (HBSS containing 2% FBS and 0.01% NaHCO3) and centrifugation to pellet the cells. The pelleted cells were suspended in 30 mL of 30% (v / v) Percoll solution (GE Healthcare, Cat. No. 17544502) and centrifuged at 2000 rpm for 30 minutes. The pelleted cells were then collected to obtain lung cells.

[0039] The spleen tissue was mashed on a 40 μm cell strainer using a 2.5 mL syringe top, and the cells were collected while suspending them in RPMI 1640. The cells were then centrifuged to pellet. The pelleted cells were hemolyzed and washed as described above, and then centrifuged to obtain spleen cells.

[0040] (2-2) ILC2 isolation The lung cells were incubated with the lineage markers listed above for 20 minutes at 4°C in the dark, washed with MACS Buffer (PBS containing 0.5% BSA and 2mM EDTA), and then lineage-negative cells (not expressing cell markers known to be expressed in mature immune cells) were collected using Streptavidin MicroBeads (Miltenyi Biotech, Cat. No. 130-048-102) and LS Columns (Miltenyi Biotech, Cat. No. 130-042-401). Subsequently, the CD45, ST2, and CD90.2 antibodies were added and incubated for 20 minutes at 4°C in the dark, washed with MACS Buffer, and then lineage-negative cells were collected. - CD45 + ST2 + CD90.2 + These were isolated and purified as ILC2 using a cell sorter (BD Bioscience, FACS Aria).

[0041] Purified ILC2s were cultured for 2 weeks in RPMI1640 Complete Medium (RPMI1640 containing 10% FBS, 100 U / mL penicillin-streptomycin, 10 mM HEPES buffer solution, 1x MEM, nonessential amino acids, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, and 50 μg / mL gentamicin sulfate) supplemented with 10 ng / mL IL-2 (R&D Systems, Cat. No. 402-ML-020 / CF) and 10 ng / mL IL-7 (R&D Systems, Cat. No. 407-ML-025 / CF).

[0042] (2-3) MAIT cell isolation Mononuclear cells obtained from the lungs and spleens of Vα19 mice were added to APC-labeled 5-OP-RU-loaded murine MR1 tetramer reagent (provided by the NIH tetramer core facility; hereafter referred to as mMR1-Tet) (0.6 μl / 1.0 x 10 7 The cells were incubated at room temperature in the dark for 60 minutes, washed with MACS Buffer (PBS containing 0.5% BSA and 2 mM EDTA), and the positive cell population was recovered using Anti-APC MicroBeads (Miltenyi Biotech, Cat. No. 130-090-855) and LS Columns (Miltenyi Biotech, Cat. No. 130-042-401). Next, APC-Cy7-labeled anti-TCRβ (BioLegend, clone: H57-597), FITC-labeled anti-CD44 (BioLegend, clone: IM7), PE-labeled anti-B220 (BD Pharmingen, clone: RA3-6B2), and PE-labeled anti-F4 / 80 (BD Pharmingen, clone: T45-2342) were added and incubated at room temperature in the dark for 20 minutes. After washing with MACS Buffer, the cells were incubated with mMR1-Tet + TCRβ + B220 - Gr1 - MAIT cells were isolated and purified using a cell sorter (BD Bioscience, FACS Aria and FACS Jazz).

[0043] The purified MAIT cells were cultured for one day in RPMI1640 Complete Medium supplemented with 10 ng / mL IL-2 and 10 ng / mL IL-7 and then used in experiments.

[0044] (2-4) Induction of eosinophilic airway inflammation by adoptive transfer into severely immunodeficient mice The ILC2 and MAIT cells prepared as described above were injected at 1x10 into NOD / Shi-scid, IL-2RγKO Jic mice (NOG mice, 7-week-old females, In-Vivo Science) (severely immunodeficient mice lacking ILC2 and NK cells in addition to T and B cells). 6 100 μg of recombinant IL-33 (R&D Systems, 3626-ML-010 / CF) was administered intranasally 3 hours and 3 days after intravenous administration of ILC2s alone or ILC2s and MAIT cells to induce airway inflammation. Six days later, alveolar lavage was performed. The leukocyte differential count and ILC2 cell count in the bronchoalveolar lavage fluid were measured by flow cytometry as described in Figure 1. IL-33 is a cytokine induced by AA and has ILC2-activating properties.

[0045] (3) Non-contact co-culture of ILC2s with MAIT cells inhibits ILC2 proliferation ILC2s were isolated from the lungs of wild-type B6 mice (C57BL / 6, 8-week-old females, CLEA Japan) using the method described above (2) and cultured for 2 weeks. MAIT cells were isolated from Vα19 mice (6-week-old females) using the method described above (2) and cultured for 1 day. The isolated MAIT cells were cultured at 2 × 10 per well in a 12-well plate (Corning, Cat. No. 353043) before co-culture. 5 Cells were cultured in RPMI complete medium and stimulated with PBS or a cytokine cocktail consisting of IL-12 (Fuji Film Wako, 095-05331, 10 ng / ml), IL-15 (BioLegend, 566302, 10 ng / ml), and IL-18 (R&D Systems, MBL B002-5, 10 ng / ml) at 37°C for 18 hours (cytokine-activated MAIT cells, denoted as cyt act MAIT in the figures).

[0046] 5-OP-RU was synthesized by mixing 3.6 mM 5-A-RU (Toronto Research Chemicals, A629245) and 1 mM methylglyoxal (Sigma, M0252) at a 1:3 ratio and incubating at 37°C for 30 minutes. The resulting 5-OP-RU (900 μM) was diluted with medium to a final concentration of 10 nM and stimulated for 18 hours at 37°C. These cells were designated T cell receptor activated MAIT cells (referred to as TCR act MAIT in the figure). The cells were then washed twice with PBS and used for coculture with ILC2s. Co-culture of ILC2 and MAIT cells was performed in a 96-well culture dish (each well is separated into upper and lower wells, and the upper well (insert) has a membrane with 0.4 μm pores, allowing only liquid factors to pass through (Corning, HTS transwell-96 permeable support 0.4 μm, Cat. No. 7369).

[0047] The culture medium used was RPMI complete medium. ILC2s were seeded in the lower row (5,000 cells / 235 μl RPMI complete medium / well), and the inserts were then placed in the wells and incubated at 37°C for 18 hours in a CO2 incubator to equilibrate the membrane. Then, for ILC2 monoculture, inserts were filled with culture medium alone, while for ILC2 and MAIT cell coculture, inserts were filled with MAIT cells (5,000 cells / 75 μl RPMI complete medium / well). At the initiation of coculture (day 0) and day 2, IL-33 (R&D Systems, 3626-ML-010 / CF) was added (final concentration 10 ng / ml) to wells containing ILC2 alone, ILC2 + MAIT, ILC2 + cyt act MAIT, or ILC2 + TCR act MAIT. Control wells contained ILC2 alone without IL-33 stimulation, and coculture wells contained ILC2 + MAIT, ILC2 + cyt act MAIT, or ILC2 + TCR act MAIT.

[0048] ILC2 were collected on day 5 of culture, and the CD45, ST2, and CD90.2 antibodies were added and incubated at 4°C in the dark for 20 minutes. After washing with FACS buffer, CD45 + ST2 + CD90.2 + The cells were designated as ILC2s, and their numbers were measured by flow cytometry (MACSQuant, Miltenyi Biotech).

[0049] (4) Cytokines in the supernatant of non-contact co-cultures of ILC2 and MAIT cells ILC2 and MAIT cells were cocultured using the same method as in the coculture experiment described in (3). MAIT cells alone, cyt act MAIT alone, ILC2 alone, ILC2 + MAIT, and ILC2 + cyt act MAIT were prepared. IL-33 (R&D Systems, 3626-ML-010 / CF) was added to all groups at a final concentration of 10 ng / ml (day 0). On day 3 after the start of coculture, 100 μl of culture supernatant was pipetted from the collection port of each well and temporarily stored in a -80°C ultra-low temperature freezer before use in the assay. Cytokine concentrations in the supernatants were measured by flow cytometry (Thermo Fisher Scientific, Attune NxT Acoustic Focusing Cytometer) using the LEGENDplex panel bead-based assay (BioLegend, Mouse Th Cytokine Panel Cat. No. 741044) (BioLegend, Mouse Cytokine Panel 2 Cat. No. 740134).

[0050] (5) MAIT cell-derived IFNγ is an inhibitor of ILC2 proliferation ILC2 and MAIT cells were cocultured using the same method as in the coculture experiment described in (3). All cultures were performed in the presence of IL-33 (R&D Systems, 3626-ML-010 / CF) (final concentration 10 ng / ml). ILC2 alone, ILC2 + MAIT, ILC2 + TCR act MAIT, and ILC2 + cyt act MAIT cells were supplemented with isotype IgG (eBioscience, 16-4301-81, Rat IgG1 kappa Isotype Control) at a final concentration of 10 μg / ml and IFNγ neutralizing antibody (eBioscience, 16-7311-81, IFN gamma Monoclonal Antibody) at 1.0 or 10 μg / ml. ILC2 cell numbers were counted on day 5 according to the method described in Figure 3.

[0051] <Result> Differences in lung inflammation between Vα19 mice (genetically enriched for MAIT cells) and wild-type mice (C57BL / 6) stimulated with A. Alternaria When we examined lung inflammation (a model of ILC2-mediated airway inflammatory disease) after intranasal administration of the fungus Alternaria, we found that Vα19 mice showed significantly reduced inflammation compared to wild-type mice (Fig. 1). Furthermore, when we measured the number of ILC2s in bronchoalveolar lavage fluid, we observed a similar significant decrease in Vα19 mice.

[0052] The results in A suggest that MAIT cells may suppress ILC2-mediated inflammation. Therefore, we transferred ILC2s into severely immunodeficient mice (NOG mice) that lack immune cells (deficient in ILC2s and NK cells in addition to T and B cells) and induced airway inflammation with IL-33 instead of Alternaria. In severely immunodeficient mice adoptively transferred MAIT cells isolated and purified from Vα19 mice together with ILC2s, the degree of inflammation was attenuated compared to mice transferred with ILC2s alone (Figure 2).

[0053] This experiment revealed that MAIT cells suppress ILC2 proliferation and cytokine production in an airway inflammation model (Insight B). To further solidify the findings in B, we investigated whether MAIT cells directly suppress ILC2 proliferation and cytokine production by co-culturing ILC2s alone or with MAIT cells in vitro. This co-culture was performed in a transwell system to prevent cell contact (assuming soluble factors derived from ILC2s or MAIT cells). The results revealed that MAIT cells stimulated with IL-12 / IL-15 / IL-18 most effectively suppressed ILC2 proliferation (Figures 3 and 4). Cytokines produced in the culture supernatant under the same culture conditions as in Figure 3 were quantified using LegendPlex (Biolegend). IL-5, IL-6, IL-9, and IL-13 production by ILC2s were suppressed. However, there was no effect on IFN-γ production by cytokine-stimulated MAIT cells, which is thought to suppress ILC2 proliferation.

[0054] We identified MAIT cells as immune cells that suppress ILC2 proliferation and cytokine production. This study demonstrated that MAIT cells are potential targets for cell therapy or drug discovery for airway inflammatory diseases, including asthma.

Claims

1. An inhibitor of proliferation of group 2 innate lymphocytes, including MAIT cells stimulated with a cytokine cocktail including interleukin-12, interleukin-15 and interleukin-18.

2. The inhibitor according to claim 1, wherein the inhibition of proliferation of Group 2 innate lymphocytes inhibits proliferation of eosinophils or mast cells.

3. A drug for suppressing eosinophilia or eosinophilic inflammation, comprising the inhibitor according to claim 1 or 2.

4. A composition for treating or preventing Group 2 spontaneous lymphoproliferative diseases, comprising MAIT cells stimulated with a cytokine cocktail including interleukin-12, interleukin-15, and interleukin-18, wherein the Group 2 spontaneous lymphoproliferative diseases are selected from the group consisting of asthma, eosinophilic sinusitis, eosinophilic pneumonia, Th2 asthma, non-Th2 asthma, cough-variant asthma, atopic cough, non-atopic cough, and non-asthmatic eosinophilic bronchitis.

5. An inhibitor of the production of interleukin-5, interleukin-6, interleukin-9 or interleukin-13, comprising MAIT cells stimulated with a cytokine cocktail including interleukin-12, interleukin-15 and interleukin-18.

6. The inhibitor described in claim 5 for at least one selected from the group consisting of asthma, eosinophilic sinusitis, eosinophilic pneumonia, Th2 asthma, non-Th2 asthma, cough-variant asthma, atopic cough, non-atopic cough, and non-asthmatic eosinophilic bronchitis.

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