Non-human autoimmune disease model animal, myositis model animal, method for producing same, and use thereof

Abstract

Provided is a non-human autoimmune disease model animal derived from an immunodeficient non-human animal as a pathologic model animal in which symptoms of an autoimmune disease are more faithfully reproduced, wherein peripheral blood mononuclear cells (PBMCs) derived from a heterologous animal subjected to CD8-positive T cell removal treatment are transfected, and symptoms of an autoimmune disease are exhibited.

Description

Non-human autoimmune disease model animals, myositis model animals, methods for producing them, and uses thereof

[0001] The present invention relates to a non-human autoimmune disease model animal, a method for producing the same, and uses thereof. The present invention also relates to a myositis model animal, a method for producing the same, and uses thereof.

[0002] To analyze the mechanisms of human diseases and evaluate the efficacy of preventive and therapeutic drugs against these diseases, animal models have been used in which human cells have been introduced into non-human mammals such as mice to reproduce human diseases. In particular, mouse models transplanted with human peripheral blood mononuclear cells (PBMCs) have been used to analyze the human immune system. Furthermore, attempts have been made to transfer human PBMCs into immunodeficient mice such as NOG-MHC KO (NOG-ΔMHC) mice to avoid the onset of graft-versus-host disease (GVHD) (Non-Patent Documents 1 and 2).

[0003] Improved engraftment of human peripheral blood mononuclear cells in NOG MHC double knockout mice generated using CRISPR / Cas9 Yuyo Ka et al. Immunol Let. 2021 Jan;229:55-61.Human PBMC-transferred murine MHC class I / II-deficient NOG mice enable long-term evaluation of human immune responses. Yaguchi T et al. Cell Mol Immunol. 2018 Nov;15(11):953-962.

[0004] As mentioned above, non-human autoimmune disease animal models in which human PBMCs were transferred to immunodeficient mice were known, but the engraftment rate of T cells and B cells contained in human PBMCs was low, making it difficult to obtain model animals that adequately reflect the human immune system.

[0005] Therefore, an objective of the present invention is to provide a pathological model animal that more faithfully reproduces the symptoms of autoimmune diseases in xenogenic animals such as humans by efficiently engrafting immune cells, particularly follicular helper T (Tfh) cells, peripheral helper T (Tph) cells, and B cells, from xenogenic animals such as humans into immunodeficient animals. Another objective of the present invention is to provide a pathological model animal that more faithfully reproduces the symptoms of myositis in xenogenic animals such as humans.

[0006] The present inventors conducted extensive research to solve the above-mentioned problems. As a result, they found that by transferring PBMCs that had been subjected to a process to remove CD8+ T cells into immunodeficient mice, human-derived Tfh cells, Tph cells, and B cells could be efficiently engrafted, and these mice could be used as non-human animal models of autoimmune diseases that exhibit symptoms of autoimmune diseases such as Sjögren's syndrome. Furthermore, they found that by transferring PBMCs that had not been subjected to a process to remove CD8+ T cells into immunodeficient mice, they could produce non-human animal models of myositis that exhibit symptoms of myositis. Based on these findings, the present invention was completed.

[0007] The present invention includes the following aspects: [1] A non-human autoimmune disease model animal derived from an immunodeficient non-human animal, into which xenogeneic animal-derived peripheral blood mononuclear cells (PBMCs) that have been subjected to a CD8+ T cell depletion treatment have been transferred, and the non-human autoimmune disease model animal exhibits symptoms of an autoimmune disease. [2] The non-human autoimmune disease model animal according to [1], wherein the immunodeficient non-human animal is an immunodeficient non-human mammal. [3] The non-human autoimmune disease model animal according to [2], wherein the immunodeficient non-human mammal is an immunodeficient mouse. [4] The non-human autoimmune disease model animal according to any of [1] to [3], wherein the xenogeneic animal-derived PBMCs are human-derived PBMCs. [5] The non-human autoimmune disease model animal according to [4], wherein the human-derived PBMCs are PBMCs derived from a human autoimmune disease patient or a healthy human subject. [6] The non-human autoimmune disease model animal according to any of [1] to [5], wherein the CD8+ T cell depletion treatment is a treatment using an anti-CD8 antibody. [7] The non-human autoimmune disease model animal according to any one of [1] to [6], wherein the autoimmune disease is one or more selected from the group consisting of Sjögren's syndrome, rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, myasthenia gravis, inflammatory bowel disease, Graves' disease, autoimmune hepatitis, and IgG4-related disease. [8] The non-human autoimmune disease model animal according to any one of [1] to [6], wherein the xenogeneic animal-derived Tph cells, xenogeneic animal-derived Tfh cells, and xenogeneic animal-derived B cells are each 1×10 per 200 mg of spleen. ７ More than cells, 1 x 10 ６ cells or more and 1 x 10 ７[9] A method for producing a non-human autoimmune disease model animal according to any one of [1] to [7], comprising at least one CD8+ T cell-depleted PBMC. [9] A method for producing a non-human autoimmune disease model animal, comprising: an administration step of administering PBMCs derived from a xenogeneic animal that have been subjected to a CD8+ T cell depletion treatment to an immunodeficient non-human animal; and a selection step of selecting an individual exhibiting symptoms of an autoimmune disease.

[10] The method according to [9], further comprising a step of administering a cytokine before the selection step.

[11] The method according to

[10] , wherein the cytokine is one or more selected from the group consisting of IL-12, TGF-β, and IFN-α.

[12] The method according to any one of [9] to

[11] , further comprising a step of administering an adjuvant together with the antigen before the selection step.

[13] The method for producing a non-human autoimmune disease model animal according to any one of [9] to

[12] , wherein the non-human autoimmune disease model animal is a Sjögren's syndrome model animal, and the selection step comprises measuring one or more of the following: (i) the amount of saliva of the non-human autoimmune disease model animal, (ii) the amount of T cells in the salivary glands of the non-human autoimmune disease model animal, (iii) the amount of cytokines in the salivary glands of the non-human autoimmune disease model animal, and (iv) the amount of Tph cells or Tfh cells in the kidneys or lungs of the non-human autoimmune disease model animal.

[14] The method for producing a non-human autoimmune disease model animal according to any of [9] to

[12] , wherein the non-human autoimmune disease model animal is a Sjögren's syndrome model animal, and the selection step comprises measuring any one or more of the following: (i) the amount of saliva of the non-human autoimmune disease model animal, (ii) the amount of T cells in the salivary glands of the non-human autoimmune disease model animal, (iii) the amount of cytokines in the salivary glands of the non-human autoimmune disease model animal, (iv) the amount of Tph cells or Tfh cells in the kidneys or lungs of the non-human autoimmune disease model animal, (v) the amount of cytokines in the kidneys or lungs of the non-human autoimmune disease model animal, and (vi) the amount of cytokines in the blood of the non-human autoimmune disease model animal.

[15] The method for producing a non-human autoimmune disease model animal according to

[13] or

[14] , wherein the cytokines in the steps (iii), (v), and (vi) are inflammatory cytokines.

[16] The method for producing the non-human autoimmune disease model animal according to

[15] , wherein the inflammatory cytokine is one or more selected from CXCL13, IFN-γ, and IL-21.

[17] A method for screening or evaluating a candidate compound for a therapeutic and / or prophylactic agent for an autoimmune disease, comprising the steps of administering a test compound to the non-human autoimmune disease model animal according to any of [1] to [8], measuring an indicator of autoimmune disease in the animal, and comparing the indicator of autoimmune disease with a control group that has not been administered the compound.

[18] The method for screening or evaluating the method according to

[17] , wherein the autoimmune disease is Sjogren's syndrome, and the step of measuring an indicator of autoimmune disease comprises measuring one or more of the following: (i) the amount of saliva in the non-human autoimmune disease model animal; (ii) the amount of T cells in the salivary glands of the non-human autoimmune disease model animal; (iii) the amount of cytokines in the salivary glands of the non-human autoimmune disease model animal; and (iv) the amount of Tph cells or Tfh cells in the kidneys or lungs of the non-human autoimmune disease model animal.

[19] The screening method or evaluation method according to

[17] , wherein the autoimmune disease is Sjögren's syndrome, and the step of measuring an indicator of the autoimmune disease comprises measuring one or more of the following: (i) the amount of saliva of the non-human autoimmune disease model animal, (ii) the amount of T cells in the salivary glands of the non-human autoimmune disease model animal, (iii) the amount of cytokines in the salivary glands of the non-human autoimmune disease model animal, (iv) the amount of Tph cells or Tfh cells in the kidneys or lungs of the non-human autoimmune disease model animal, (v) the amount of cytokines in the kidneys or lungs of the non-human autoimmune disease model animal, or (vi) the amount of cytokines in the blood of the non-human autoimmune disease model animal.

[20] The screening method or evaluation method according to

[18] or

[19] , wherein the cytokines in the steps (iii), (v), and (vi) are inflammatory cytokines.

[21] The screening method or evaluation method according to

[20] , wherein the inflammatory cytokines are one or more selected from CXCL13, IFN-γ, and IL-21.

[22] A method for producing Tph cells, Tfh cells, and / or B cells, comprising a step of recovering Tph cells, Tfh cells, and / or B cells from the non-human autoimmune disease model animal according to any one of [1] to [8].

[23] A non-human myositis model animal derived from an immunodeficient non-human animal, into which CD8-positive T cells derived from a heterologous animal or peripheral blood mononuclear cells (PBMCs) containing said CD8-positive T cells have been transferred, and which exhibits symptoms of myositis.

[24] A method for producing a non-human myositis model animal, comprising a step of administering CD8-positive T cells derived from a heterologous animal or PBMCs containing said CD8-positive T cells to an immunodeficient non-human animal, and a step of selecting an individual exhibiting symptoms of myositis.

[25] A method for screening or evaluating a candidate compound for a therapeutic and / or preventive agent for myositis, comprising the steps of administering a test compound to the non-human myositis model animal of

[23] or tissues or cells thereof, measuring an indicator of myositis in the animal, and comparing the indicator of myositis with a group not administered the compound.

[26] A method for screening or evaluating a candidate compound for a therapeutic and / or preventive agent for pulmonary symptoms associated with myositis, comprising the steps of administering a test compound to the non-human myositis model animal of

[23] or tissues or cells thereof, measuring an indicator of myositis in the animal, and comparing the indicator of myositis with a group not administered the compound.

[0008] According to the present invention, Tfh cells, Tph cells, and B cells derived from xenogeneic PBMCs (e.g., human) can be efficiently engrafted, thereby providing a non-human autoimmune disease model animal that exhibits symptoms of autoimmune disease while suppressing GVHD. The non-human autoimmune disease model animal can be used to elucidate the physiological functions of immune cells in autoimmune diseases, as well as for drug discovery screening and drug efficacy evaluation. Furthermore, by transferring PBMCs from patients with autoimmune diseases such as Sjögren's syndrome, systemic lupus erythematosus, and IgG4-related disease, B cells producing patient-specific autoantibodies can be engrafted together with Tfh cells and Tph cells, enabling disease-specific functional analysis and drug discovery screening. Furthermore, according to the present invention, a non-human myositis model animal that exhibits symptoms of myositis can be provided. The non-human myositis model animal can be used for drug discovery screening and drug efficacy evaluation for myositis.

[0009] Figure 1 shows the frequency of GVHD symptoms and ALT concentrations in mice injected with PBMCs and mice injected with CD8+ T cell-depleted PBMCs. "PBMC" refers to the condition in which human blood-derived PBMCs were injected, and "PBMCΔCD8T" refers to the condition in which PBMCs from which CD8+ T cells were removed using an anti-CD8 antibody were injected. The "% of GVHD" on the vertical axis indicates the incidence of GVHD. "ALT U / L" is a measured value of alanine aminotransferase activity in the blood and indicates the degree of liver damage. Figure 2 shows the percentage and number of Tph and Tfh cells in the spleens of mice injected with PBMCs and mice injected with CD8+ T cell-depleted PBMCs. The "% of CD4+ T cells in spleen" on the vertical axis in the upper graph indicates the percentage of CD4+ T cells in the spleen. The "Count in spleen" on the vertical axis in the lower graph indicates the number of cells in the spleen. Figure 3 shows the numbers of CD45+ cells, T cells, CD4+ T cells, CD8+ T cells, and B cells in the spleens of PBMC-transferred mice and CD8+ T cell-depleted PBMC-transferred mice. The B cell counts in the spleens of CD4+ T cell-depleted PBMC-transferred mice (PBMCΔCD4T) are also shown. Figure 4 shows the blood CXCL13 levels in untransferred mice and CD8+ T cell-depleted PBMC-transferred mice. Figure 5A shows the saliva volume in untransferred mice and CD8+ T cell-depleted PBMC-transferred mice (drug-naïve, tacrolimus-treated, cytokine-treated). The vertical axis (g / min / g) (20 min) indicates the amount of saliva collected from mice over 20 min, corrected for time (min) and mouse body weight (g). For cytokine administration, mice were given 1 μg each of recombinant human IL-12, recombinant human TGF-β, and recombinant human IFN-α subcutaneously three times a week (Figures 5B, 5C, and 6). Figure 5B shows the pathological evaluation of the salivary glands of uninjected mice (control) and mice injected with CD8+ T cell-depleted PBMCs (no drug administration, tacrolimus administration, cytokine administration). Pathological evaluation was performed based on the following criteria:-: No remarkable (no findings), + / -: Minimal (very slight findings), + / -: Slight (slight findings), 2+: Moderate (severe findings), 3+: Severe (severe findings). Figure 5C shows the number of IL-21-positive Tph cells and IFN-γ-positive Tph cells in the salivary glands of mice (untreated, tacrolimus-treated, or cytokine-treated) that received CD8+ T cell-depleted PBMCs. "CD8-dep" indicates the condition in which CD8+ T cells were depleted from PBMCs using an anti-CD8 antibody. Figure 6 shows micrographs showing the pathological findings of salivary glands of untreated mice and mice (untreated, tacrolimus-treated, or cytokine-treated) that received CD8+ T cell-depleted PBMCs. The bold borders indicate areas of pathology (cell infiltration and acinar cell atrophy). The cell infiltration and atrophic areas of acinar cells observed in the non-treated group were reduced by tacrolimus administration and expanded by cytokine administration. The scale bar indicates 500 μm. Figure 7 is a graph showing the number of Tfh and Tph cells in the salivary glands, lungs, and kidneys of mice transferred with CD8+ T cell-depleted PBMCs. Figure 8 is a graph showing the expression levels of CXCL13, IFN-γ, and IL-21 in the salivary glands, lungs, and kidneys of untransferred mice and mice transferred with CD8+ T cell-depleted PBMCs. Figure 9 is a graph showing the number of Tph and Tfh cells in the spleen of mice transferred with CD8+ T cell-depleted PBMCs (untreated or antigen / adjuvant-treated). Figure 10 is a graph showing blood ALT, AST, and CK activities in untransferred mice, PBMC-transferred mice, and CD8+ T cell-depleted PBMC-transferred mice. "NT" refers to the condition without any treatment, "hPBMC" refers to the condition in which human blood-derived PBMCs were transferred, and "hPBMCΔCD8T" refers to the condition in which PBMCs from which CD8-positive T cells were removed using an anti-CD8 antibody were transferred. "ALT U / L" is a measurement of the blood activity of alanine aminotransferase and indicates the degree of liver damage. "AST U / L" is a measurement of the blood activity of aspartate aminotransferase and indicates the degree of muscle, liver, and heart damage."CK U / L" is a measurement of creatine kinase activity in the blood and indicates the degree of muscle damage. Figure 11 is a graph showing the gene expression levels of Vcam1, Icam1, and Saa1 in muscles of untransfected mice, PBMC-transfected mice, and CD8+ T cell-depleted PBMC-transfected mice. "Vcam1 / Gapdh," "Icam1 / Gapdh," and "Saa1 / Gapdh" are values ​​of Vcam1, Icam1, and Saa1 gene expression in muscles corrected with the internal standard Gapdh, respectively, and indicate the degree of muscle damage. Figure 12 is a micrograph showing the pathological images of muscles of untransfected mice, PBMC-transfected mice, and CD8+ T cell-depleted PBMC-transfected mice. HE indicates hematoxylin-eosin stained pathological sections of each pathological image. Purple-stained cells indicate immune cells infiltrating muscle tissue. CD8 refers to a pathological section immunohistochemically stained with anti-CD8a antibody. Brown stained cells indicate CD8-positive T cells infiltrating muscle tissue. The scale bar indicates 100 μm. Figure 13 shows graphs showing blood ALT, AST, and CK activity in PBMC-transfected mice and PBMC-transfected mice treated with steroids or tacrolimus. "hPBMC" indicates the condition in which human blood-derived PBMCs were transfected and vehicle was administered. "PSL" indicates the condition in which steroids (prednisolone) were administered at 1 or 10 mg / kg, and "TAC" indicates the condition in which tacrolimus was administered at 1 or 10 mg / kg. "ALT U / L" is a measurement of blood alanine aminotransferase activity and indicates the degree of liver damage. "AST U / L" is a measurement of blood aspartate aminotransferase activity and indicates the degree of muscle, liver, and heart damage. "CK U / L" is a measurement of creatine kinase activity in the blood and indicates the degree of muscle damage. Figure 14 is a graph showing the expression levels of Vcam1, Icam1, and Saa1 genes in the muscles of PBMC-transferred mice and PBMC-transferred mice administered steroids or tacrolimus. "Vcam1 / Gapdh," "Icam1 / Gapdh," and "Saa1 / Gapdh" are the gene expression levels of Vcam1, Icam1, and Saa1 in the muscles, respectively, corrected with the internal standard Gapdh, and indicate the degree of muscle damage.Figure 15 shows micrographs of muscle pathology in PBMC-transfected mice and PBMC-transfected mice treated with steroids or tacrolimus. HE refers to hematoxylin-eosin stained sections of each pathology image; purple stained cells indicate immune cells infiltrating muscle tissue. CD8 refers to pathology sections immunohistochemically stained with anti-CD8a antibody; brown stained cells indicate CD8-positive T cells infiltrating muscle tissue. The scale bar represents 100 μm. Figure 16 shows micrographs of lung pathology in non-transfected mice (NT) and PBMC-transfected mice (hPBMC). HE refers to hematoxylin-eosin stained sections of each pathology image; purple stained cells indicate immune cells infiltrating lung tissue. The scale bar represents 200 μm. Figure 17 is a graph showing the blood SP-D levels in non-transfected mice (NT) and PBMC-transfected mice (hPBMC). Figure 18 is a graph showing the percentages of Tph cells, Tfh cells, and B cells in the spleens of humanized mice transfused with CD8 T cell-depleted PBMCs derived from three healthy donors. CD8TΔPBMC refers to humanized mice transfused with CD8 T cell-depleted PBMCs. Different lots indicate different donors. Figure 19 is a graph showing the blood CXCL13 concentrations of humanized mice transfused with CD8 T cell-depleted PBMCs derived from three healthy donors. NT indicates untreated mice not transfused with cells. CXCL13 indicates the level of Sjögren's syndrome. Figure 20 is a graph showing the saliva volume of humanized mice transfused with CD8 T cell-depleted PBMCs derived from three healthy donors. (g / min / g) (15 min) indicates the drug-induced saliva volume over 15 minutes. A decrease in saliva volume indicates the level of Sjögren's syndrome. Figure 21 is a graph showing the blood IgG, anti-SS-A antibody, and anti-SS-B antibody concentrations or antibody titers. IgG indicates the level of autoimmune disease, while anti-SS-A and anti-SS-B antibodies indicate the level of Sjögren's syndrome. Figure 22 is a graph showing blood antinuclear antibody titers. Antinuclear antibodies indicate the level of autoimmune disease. ANA indicates antinuclear (anti-ds-DNA) antibodies. Ab index indicates the level of antibody titers. Figure 23-1 is a microscopic photograph showing pathological images of the salivary glands in non-transfected mice and mice transfected with CD8-positive T cell-depleted PBMCs.These are pathological sections stained with hematoxylin and eosin. The → symbol indicates blood vessels and ducts, and the ⇒ symbol indicates cellular infiltration. The scale bar indicates 500 μm. Figure 23-2 shows micrographs of muscle pathology in untransfected mice and mice transfected with CD8+ T cell-depleted PBMCs. These are pathological sections stained with hematoxylin and eosin. The → symbol indicates blood vessels, and the ⇒ symbol indicates cellular infiltration. The scale bar indicates 500 μm. Figure 23-3 shows micrographs of skin pathology in untransfected mice and mice transfected with CD8+ T cell-depleted PBMCs. These are pathological sections stained with hematoxylin and eosin. The} symbol indicates subcutaneous fat, and the ⇒ symbol indicates cellular infiltration. The scale bar indicates 500 μm. Figure 23-4 shows micrographs of kidney pathology in untransfected mice and mice transfected with CD8+ T cell-depleted PBMCs. These are hematoxylin-eosin stained pathological sections. The arrow indicates blood vessels, and the arrow indicates tubular degeneration. The scale bar represents 500 μm. Figure 23-5 is a micrograph showing the pathology of the liver in a non-transfected mouse and a mouse transfected with CD8+ T cell-depleted PBMCs. The arrow indicates cellular infiltration. The scale bar represents 500 μm. Figure 23-6 is a micrograph showing the pathology of the lung in a non-transfected mouse and a mouse transfected with CD8+ T cell-depleted PBMCs. The arrow indicates blood vessels and ducts, and the arrow indicates cellular infiltration. The scale bar represents 500 μm. Figure 23-7 is a micrograph showing the pathology of the pancreas in a non-transfected mouse and a mouse transfected with CD8+ T cell-depleted PBMCs. These are pathological sections stained with hematoxylin and eosin, with → indicating blood vessels and pancreatic ducts and → indicating cellular infiltration. The scale bar indicates 500 μm. Figure 24 is a graph showing blood SP-D levels. The SP-D level indicates the degree of lung lesions. Figure 25 is a graph showing blood glucose levels. The blood glucose level indicates the degree of pancreatic lesions. Figure 26 is a graph showing proteinuria levels. The proteinuria level indicates the degree of kidney lesions.

[0010] <Non-human autoimmune disease model animal> One aspect of the present invention relates to a non-human autoimmune disease model animal derived from a non-human immunodeficient animal, into which PBMCs derived from a xenogeneic animal that have been subjected to a CD8-positive T cell depletion treatment are transferred, and which exhibits symptoms of an autoimmune disease.

[0011] Here, "derived from a non-human immunodeficient animal" means that the non-human autoimmune disease model animal was produced using a non-human immunodeficient animal.

[0012] The non-human immunodeficient animal is preferably a non-human mammal, more preferably a mouse or a rat, and even more preferably a mouse.

[0013] Known non-human immunodeficient animals can be used as non-human immunodeficient animals. Examples include, but are not limited to, the following. For example, immunodeficient mice can have immune system defects due to defects in MHC class I, II, or both; defects in B cells, T cells, or both; or defects in genes encoding proteins selected from cytokines, cytokine receptors, TLR receptors, and various transducers and transcription factors of signal transduction pathways. Immunodeficient mice include nude mouse (nu) strains, severe combined immunodeficient (Scid, Scid / Beigh) strains, non-obese diabetic (NOD, NOD / Scid, NOG, NSG, NOJ) strains, single gene mutant models such as the RAG strain resulting from targeted gene deletion (RAG1KO, RAG2KO, BRG, BRJ), double and triple mutant mouse strains with additional defects, and various hybrids produced by crossbreeding them.

[0014] Non-limiting examples of immunodeficient mouse models include the following mouse strains: nude (nu) [Flanagan SP. Genet Res 1966; 8:295-309; and Nehls M et al. Nature 1994; 372: 103-7]; Scid (scid) [Bosma GC et al. Nature 1983; 301:527-30; Mosier DE et al. Nature 1988; 335: 256-9; and Greiner DL et al. StemCells 1998; 16: 166-77]; NOD [Kikutani H et al. Adv Immunol 1992; 51: 285-322; and Anderson MS et al. Ann Rev Immunol 2005; 23: 447-85]; RAG1 and RAG2 (rag) [Mombaerts P et al. Cell1992; 68: 869-77; Shinkai U et al. Cell 1992; 68: 855-67]; NOD-scid [Greiner DL et al. 1998; ShultzLD et al. J Immunol 1995; 154: 180-91; Melkus MW et al. Nature Med 2006; 12:1316-22; and Denton PW et al. PLoS Med 2008; 4(12): e357]; IL2rg null [DiSanto JP et al. Proc Natl AcadSci USA 1995; 92: 377-81]; B2m null [Christianson SW et al. J Immunol1997; 158: 3578-86]; NOD-scid IL2rγ null [Shultz LD et al. NatRev Immunol 2007; 7: 118-30; Ito M et al. Blood 2002; 100: 3175-82; Ishikawa I etal. Blood 2005; 106: 1565-73; and Macchiarini F et al. J Exp Med 2005; 202:1307-11]; NOD-scid B2m null [Shultz et al.2007; ShultzLD et al. Transplantation 2003;76:1036-42; Islas-OhlmayerMA et al. J Virol2004;78:13891-900; and Macchiarini et al. 2005]; and HLA transgenic mice [Grusby MJ et al. ProcNatl Acad Sci USA 1993;90(9):3913-7; and Roy CJ et al. Infect Immun 2005;73(4):2452-60]. See, for example, Belizario JE The Open Immunology Journal, 2009;2:79-85. NSG-HLA-A2 / HHD (Leonard D. Shultz et al. Proc Natl Acad Sci USA 2010; 107(29): 13022-27). NSG-Tg (Hu-IL15).

[0015] The non-human model animal is transferred with PBMCs derived from a xenogeneic animal that has been subjected to a CD8+ T cell depletion treatment. Here, "xenogeneic animal" means a species that is different from the non-human model animal. For example, if the non-human model animal is a mouse, it means an animal other than a mouse, but preferably a human. That is, the non-human model animal is preferably transferred with PBMCs derived from a human that has been subjected to a CD8+ T cell depletion treatment.

[0016] PBMCs are mononuclear cells, including monocytes and lymphocytes, isolated from peripheral blood. PBMCs can contain a variety of blood cells, such as T cells, B cells, NK cells, monocytes, and dendritic cells. PBMCs can be obtained from peripheral blood by known separation methods, such as density gradient centrifugation. PBMCs from xenogeneic animals can be obtained from healthy subjects or from subjects suffering from autoimmune diseases. That is, when the xenogeneic animal is human, PBMCs can be obtained from healthy subjects or from patients with autoimmune diseases. Using PBMCs from patients with autoimmune diseases allows for the sustained production of patient-derived autoantibodies in vivo in mice. For example, using PBMCs from patients with systemic lupus erythematosus may potentially induce systemic lupus erythematosus pathology by in vivo induction of pathogenic autoantibodies. The applicable diseases include any disease in which autoantibodies contribute to the worsening of the pathology, and are not limited to Sjögren's syndrome and systemic lupus erythematosus.

[0017] The PBMCs to be transferred into the non-human immunodeficient animals have been subjected to a CD8-positive T cell depletion treatment, which will be described later.

[0018] The non-human autoimmune disease model animal contains xenogeneic animal-derived PBMCs that have been subjected to a CD8+ T cell depletion treatment, resulting in the xenogeneic animal-derived Tfh cells, Tph cells, and B cells. Tfh cells are T cells that interact with B cells within lymphoid follicles in secondary lymphoid tissues induced by antigen sensitization, leading to their differentiation and germinal center formation. These cells can be identified as PD-1-positive and CXCR5-positive. Tph cells are helper T cells involved in the induction of B cell differentiation outside the follicles and can be identified as PD-1-positive and CXCR5-negative.

[0019] In one embodiment, the non-human autoimmune disease model animal comprises xenogeneic animal-derived Tph cells, Tfh cells, and B cells. The xenogeneic animal-derived Tph cells, Tfh cells, and B cells are preferably contained in the spleen of the non-human autoimmune disease model animal, but may also be contained in other tissues such as the salivary gland, kidney, and lung. The amounts of xenogeneic animal-derived Tph cells, Tfh cells, and B cells are not particularly limited. For example, 1 x 10 xenogeneic animal-derived Tph cells can be contained per 200 mg of spleen of the non-human autoimmune disease model mouse. ７ Preferably, the xenogeneic animal-derived Tfh cells are contained in an amount of 1×10 ６ Preferably, the xenogeneic animal-derived B cells are contained in an amount of 1×10 ７ It is preferable that the number of cells or more is included.

[0020] The non-human autoimmune disease model animal exhibits symptoms of autoimmune diseases, including the following diseases, more preferably Sjögren's syndrome, systemic lupus erythematosus, and IgG4-related disease.

[0021] <Autoimmune diseases> Chronic thyroiditis, Graves' disease, primary mucosal edema, thyrotoxicosis, pernicious anemia, Goodpasture's syndrome, acute progressive glomerulonephritis, myasthenia gravis, pemphigus vulgaris, bullous pemphigoid, insulin-resistant diabetes, juvenile diabetes, type I diabetes, type II diabetes, Addison's disease, atrophic gastritis, male infertility, premature menopause, lens-induced uveitis, Behçet's disease, sympathetic phlebitis, multiple sclerosis , inflammatory bowel disease, ulcerative colitis, Crohn's disease, primary biliary cholangitis, primary sclerosing cholangitis, autoimmune hepatitis, metabolic disorder-related steatohepatitis, chronic active hepatitis, autoimmune hemolytic anemia, paroxysmal hemoglobinuria, idiopathic thrombocytopenic purpura, IgA nephropathy, lupus nephritis, dermatomyositis, inclusion body myositis, immune-mediated myopathy, celiac disease, autoimmune pancreatitis, and Sjögren's syndrome. Rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, adult-onset Still's disease, juvenile idiopathic arthritis, systemic lupus erythematosus, discoid lupus erythematosus, IgG4-related disease, polymyositis, scleroderma, plaque psoriasis, cystic psoriasis, relapsing polychondritis, vasculitis, microscopic polyangiitis, granulomatosis with polyangiitis, eosinophilic granulomatosis with polyangiitis, polyarteritis nodosa, Takayasu's arteritis, giant cell arteritis, and mixed connective tissue disease.

[0022] Symptoms of autoimmune diseases may be one or more of the symptoms defined as clinical symptoms of each autoimmune disease. Examples of symptoms of autoimmune diseases include inflammation, pain, tissue damage, joint deformation, itching, edema, increased cytokines (preferably inflammatory cytokines (CXCL13, IFN-γ, IL-12, IL-21, CCL4, GZMA, GZMB, GZMH, GZMK, etc.)), increased autoantibodies, excessive hormone secretion, and infiltration / increase of immune cells (Tph cells, Tfh cells, B cells) in tissues / lymph nodes. However, symptoms are not particularly limited to these and can be appropriately selected depending on the type of disease.

[0023] For example, when the autoimmune disease is Sjogren's syndrome, symptoms include a decrease in saliva flow and an increase in the amount of immune cells, such as lymphocytes, including Tph cells and Tfh cells, in the salivary glands.

[0024] <Method for Producing Non-Human Autoimmune Disease Model Animal> Next, an example of a method for producing a non-human autoimmune disease model animal will be described.

[0025] First, an administration step (hereinafter, sometimes referred to as a PBMC administration step) is performed in which PBMCs derived from a different animal species and subjected to a CD8+ T cell depletion treatment are administered to a non-human immunodeficient animal. Preferably, an administration step is performed in which human PBMCs subjected to a CD8+ T cell depletion treatment are administered to a non-human immunodeficient animal such as a mouse.

[0026] Examples of treatments for removing CD8-positive T cells include treatments using anti-CD8 antibodies, such as a method in which PBMCs are subjected to flow cytometry using anti-CD8 antibodies to remove CD8-positive T cells, or a method in which PBMCs are adsorbed onto beads or the like carrying anti-CD8 antibodies to remove CD8-positive T cells. Note that CD8-positive T cells are also CD3-positive and CD4-negative.

[0027] In PBMCs with a reduced amount of CD8-positive T cells, the amount of CD8-positive T cells is reduced to, for example, 1 / 10 or less, preferably 1 / 100 or less, and more preferably 1 / 1000 or less, compared to PBMCs not subjected to CD8-positive T cell depletion treatment. It is even more preferable that the amount of CD8-positive T cells is below the detection limit.

[0028] The method for transferring PBMCs into a non-human immunodeficient animal is not particularly limited as long as it allows the cells contained in the PBMCs to reach immune system tissues such as the spleen. Any common cell administration method can be used, but intravenous injection is preferred.

[0029] The amount of PBMC administered may be any amount that allows the cells contained in the PBMC to be sufficiently engrafted in immune system tissues such as the spleen. For example, 1 × 10 5 Cell ~1×10 9 It is a cell.

[0030] After the PBMC administration step, a selection step is carried out to select individuals who will develop an autoimmune disease. The selection step is preferably carried out after the administration step, when the cells contained in the PBMC have sufficiently engrafted, and is preferably carried out one day or more after the end of the administration step.

[0031] Symptoms of autoimmune diseases may be any symptoms observed in each autoimmune disease, including those described above, and can be selected appropriately depending on the type of disease. In Sjögren's syndrome, a decrease in saliva volume, an increase in the amount of Tph cells and Tfh cells in the salivary glands, and an increase in cytokines (preferably inflammatory cytokines, particularly IL-21, IFN-γ, CXCL13, etc.) in Tph cells are observed. Therefore, if the autoimmune disease is Sjögren's syndrome, one or more of these can be used as indicators in the selection process. Increases in the amount of Tph cells and Tfh cells and increases in cytokines (preferably inflammatory cytokines, particularly IL-21, IFN-γ, CXCL13, etc.) are also observed in organs such as the lungs and kidneys, and increases in blood levels of cytokines, particularly CXCL13, are also observed. These can also be used as indicators in the selection process.

[0032] After the PBMC administration step and before the selection step, a step of administering a cytokine (preferably, an inflammatory cytokine) can be performed. By performing this step, symptoms of an autoimmune disease can be efficiently and / or significantly induced. The cytokine may be any cytokine that enhances the condition of an autoimmune disease, and examples thereof include one or more cytokines (preferably, inflammatory cytokines) selected from IL (Interleukin)-12, TGF (Transforming growth factor)-β, and IFN (Interferon)-α. Two or three types of IL-12, TGF-β, and IFN-α may be administered.

[0033] The method of administration of cytokines (preferably, inflammatory cytokines) is not particularly limited, and may be, for example, intravenous injection. The dose of cytokines (preferably, inflammatory cytokines) may be any amount sufficient to enhance the symptoms of autoimmune disease and can be adjusted appropriately. For example, the dose is preferably 0.01 μg to 100 μg for IL-12, 0.01 μg to 100 μg for TGF-β, and 0.01 μg to 100 μg for IFN-α. The cytokines (preferably, inflammatory cytokines) may be administered multiple times.

[0034] Furthermore, after the PBMC administration step and before the selection step, a step of administering an adjuvant together with the antigen can be carried out, which can efficiently and / or significantly induce symptoms of an autoimmune disease.

[0035] The antigen is not particularly limited as long as it induces an antigen-antibody reaction in a non-human autoimmune disease model animal, and examples thereof include chicken gamma globulin, keyhole limpet hemocyanin, and bovine serum albumin, with ovalbumin being preferred. Furthermore, the antigen may be modified with a hapten, such as a nitrophenyl compound (e.g., trinitrophenyl or dinitrophenyl).

[0036] Adjuvants can be substances that enhance the immunogenicity of antigens, and can be selected appropriately depending on the type of antigen. Examples of adjuvants include aluminum compounds such as aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate.

[0037] The antigen and adjuvant may be administered sequentially, but are preferably administered simultaneously. The amount of antigen administered may be any amount that can induce an antigen-antibody reaction in a non-human model animal, and can be adjusted appropriately, for example, from 1 μg to 10 mg. The amount of adjuvant can be selected appropriately depending on the amount of antigen.

[0038] Either or both of the step of administering a cytokine (preferably an inflammatory cytokine) and the step of administering an adjuvant together with an antigen may be performed. When both are performed, either one may be performed first.

[0039] Furthermore, after the PBMC administration step and before the selection step, a step of administering an agent that induces or enhances an autoimmune disease can be performed. By performing this step, symptoms of an autoimmune disease can be efficiently and / or significantly induced. For example, since it is known that administration of a stimulant such as pristane to C57BL / 6 mice causes systemic lupus erythematosus-like symptoms, it is possible that systemic lupus erythematosus pathology can also be induced in this non-human autoimmune disease model by administering pristane.

[0040] <Method for screening or evaluating candidate compounds for therapeutic and / or prophylactic agents for autoimmune diseases> One aspect of the present invention relates to a method for screening candidate compounds for therapeutic and / or prophylactic agents for autoimmune diseases. The screening method according to one aspect of the present invention comprises the steps of administering a test compound to the non-human autoimmune disease model animal or tissues or cells thereof, measuring an indicator of autoimmune disease in the animal, and comparing the indicator of autoimmune disease with that in a group not administered the test compound.

[0041] Any substance can be used as the test compound, and examples thereof include one or more compounds selected from proteins, peptides, low-molecular-weight compounds, high-molecular-weight compounds, and natural compounds, as well as one or more compounds selected from cell extracts, cell culture supernatants, microbial fermentation products, extracts derived from marine organisms, and plant extracts, each containing one or more of these compounds. A library containing multiple test compounds can also be used.

[0042] The test compound may be administered to the model animal, or may be added to cells or tissues obtained from the model animal. The method of administering the test compound to the model animal is not particularly limited, and examples thereof include oral administration, intravenous injection, intraperitoneal administration, and local administration such as intramuscular injection.

[0043] Indicators of autoimmune disease vary depending on the type of autoimmune disease, but examples include the symptoms of the autoimmune disease as described above, the expression levels of marker proteins or marker genes for autoimmune disease, and the production levels of autoantibodies. Examples of autoantibodies include antinuclear antibodies, anti-SS-A antibodies, anti-SS-B antibodies, and IgG-type autoantibodies. Since the non-human autoimmune disease models show elevated levels of antinuclear antibodies and IgG, which are general pathological markers for autoimmune diseases, these markers can be used as indicators to evaluate the effects of drugs on autoimmune diseases in general.

[0044] For example, when the autoimmune disease is Sjögren's syndrome, one or more of the following can be used as indicators of autoimmune disease: (i) the saliva volume of the non-human autoimmune disease model animal; (ii) the amount of T cells, preferably Tph cells and / or Tfh cells, in the salivary glands; (iii) the amount of cytokines (preferably inflammatory cytokines, particularly IL-21, IFNγ, CXCL13, etc.) in the salivary glands (preferably Tph cells in the salivary glands); (iv) the amount of Tph cells or Tfh cells in the kidneys or lungs of the non-human autoimmune disease model animal; (v) the amount of cytokines (preferably inflammatory cytokines, particularly IL-21, IFNγ, CXCL13, etc.) in the kidneys or lungs of the non-human autoimmune disease model animal; and (vi) the amount of cytokines (preferably inflammatory cytokines, more preferably CXCL13) in the blood of the non-human autoimmune disease model animal. Furthermore, (vii) proteinuria in the urine of the non-human autoimmune disease model animal can also be used as an indicator of autoimmune disease.

[0045] After the above measurement, the index of autoimmune disease is compared with that of the test compound non-administered group. If the index of autoimmune disease is significantly improved in the test compound administered group compared to the test compound non-administered group, the test compound can be selected as a candidate substance for a therapeutic or preventive drug for autoimmune disease. For example, if the index of autoimmune disease is an index that shows a high value in autoimmune disease, the test compound can be selected as a candidate substance for a therapeutic or preventive drug for autoimmune disease if the index in the test compound administered group is reduced to 50% or less, 20% or less, or 10% or less compared to the non-administered group. On the other hand, if the index of autoimmune disease is an index that shows a low value in autoimmune disease, the test compound can be selected as a candidate substance for a therapeutic or preventive drug for autoimmune disease if the index in the test compound administered group is restored to 1.2 times or more, 1.5 times or more, or 2 times or more compared to the non-administered group.

[0046] Furthermore, not only screening but also evaluation of a single compound can be performed in a similar manner. That is, another aspect of the present invention relates to a method for evaluating a candidate compound for a therapeutic and / or prophylactic agent for an autoimmune disease. The evaluation method according to one aspect of the present invention includes the steps of administering a test compound to the non-human autoimmune disease model animal or its tissues or cells, measuring an indicator of autoimmune disease in the animal, and comparing the indicator of autoimmune disease with that in a compound-unadministered group. If the indicator of autoimmune disease is significantly improved in the test compound-administered group compared to the test compound-unadministered group, the test compound can be evaluated as having a therapeutic or prophylactic effect for an autoimmune disease.

[0047] <Method for Producing Tph Cells, Tfh Cells, and / or B Cells> As described above, a non-human autoimmune disease model animal according to one embodiment of the present invention contains xenogeneic Tph cells, Tfh cells, and B cells by transferring peripheral blood mononuclear cells derived from the xenogeneic animal that have been subjected to a CD8+ T cell depletion treatment. Therefore, one embodiment of the present invention relates to a method for producing Tph cells, Tfh cells, and / or B cells, which includes a step of recovering Tph cells, Tfh cells, and / or B cells from the non-human autoimmune disease model animal. The method may also include a step of administering PBMCs derived from the xenogeneic animal that have been subjected to a CD8+ T cell depletion treatment to an immunodeficient non-human animal to obtain the non-human autoimmune disease model animal. Another embodiment of the present invention relates to Tph cells, Tfh cells, and / or B cells produced by the above-described production method. Tph cells, Tfh cells, and / or B cells can be recovered from, but are not limited to, the spleen of the non-human autoimmune disease model animal. The method for recovering Tph cells, Tfh cells, and / or B cells is not particularly limited, and examples thereof include cell sorting using cell surface markers specific to these cells, such as CD4, PD-1, and CXCR5 for Tph cells and Tfh cells, and CD20 and CD19 for B cells.

[0048] <Myositis Model Animal> One aspect of the present invention relates to a non-human myositis model animal derived from a non-human immunodeficient animal, into which CD8-positive T cells derived from a heterologous animal or PBMCs containing the CD8-positive T cells have been transferred, and which exhibits symptoms of myositis.

[0049] Here, "derived from a non-human immunodeficient animal" means that the non-human myositis model animal was produced using a non-human immunodeficient animal.

[0050] The non-human immunodeficient animal is preferably a non-human mammal, more preferably a mouse or a rat, and even more preferably a mouse.

[0051] The non-human immunodeficient animal can be a known non-human immunodeficient animal such as those exemplified above in <Non-human autoimmune disease model animal>.

[0052] The non-human model animal is transferred with xenogeneic animal-derived CD8-positive T cells or PBMCs containing the CD8-positive T cells, for example, PBMCs derived from xenogeneic animals that have not been subjected to CD8-positive T cell depletion treatment. Here, "xenogeneic animal" means a species that is different from the non-human model animal. For example, if the non-human model animal is a mouse, it means an animal other than a mouse, but preferably a human. That is, the non-human model animal is preferably transferred with human-derived CD8-positive T cells or human-derived PBMCs containing CD8-positive T cells.

[0053] The CD8-positive T cells to be transferred into the non-human immunodeficient animal may be separated from PBMCs and transferred, or CD8-positive T cell-containing PBMCs, i.e., PBMCs that have not been subjected to a CD8-positive T cell depletion treatment, may be transferred.

[0054] Here, the PBMCs derived from a heterologous animal may be PBMCs derived from a healthy subject or from a subject suffering from myositis. That is, when the heterologous animal is a human, PBMCs derived from a healthy subject or from a myositis patient may be used.

[0055] Non-human myositis model animals exhibit myositis symptoms, including polymyositis, dermatomyositis, inclusion body myositis, immune-mediated necrotizing myopathy, and GVHD-associated myositis. Myositis symptoms include elevated serum aspartate aminotransferase (AST) and creatine kinase (CK), increased expression of markers such as vascular cell adhesion molecule-1 (Vcam1), intercellular adhesion molecule 1 (Icam1), and serum amyloid A1 (Saa1) in muscle tissue, and CD8+ T cell infiltration into muscle tissue. Non-human myositis model animals may also exhibit myositis-associated pulmonary symptoms. These symptoms include pulmonary inflammation due to elevated serum surfactant protein D (SP-D) and IL-6, as well as immune cell infiltration into lung tissue.

[0056] <Method for Producing Myositis Model Animal> Next, an example of a method for producing a non-human myositis model animal will be described.

[0057] First, an administration step (hereinafter sometimes referred to as a CD8-positive T cell administration step) is performed in which CD8-positive T cells derived from a heterologous animal or PBMCs containing the CD8-positive T cells are administered to a non-human immunodeficient animal. Preferably, an administration step is performed in which human CD8-positive T cells or human PBMCs containing CD8-positive T cells are administered to a non-human immunodeficient animal such as a mouse.

[0058] The method for transferring CD8-positive T cells or PBMCs containing CD8-positive T cells into a non-human immunodeficient animal is not particularly limited as long as it allows the CD8-positive T cells to reach immune system tissues such as the spleen. Any common cell administration method can be used, with intravenous injection being preferred.

[0059] The amount of CD8-positive T cells administered may be any amount that allows the CD8-positive T cells to be sufficiently engrafted in immune system tissues such as the spleen. For example, 1×10 5 Cell ~1×10 9 The dose of PBMCs containing CD8-positive T cells to be administered may be any amount that allows the CD8-positive T cells to be sufficiently engrafted in immune system tissues such as the spleen. For example, 1 × 105 Cell ~1×10 9 It is a cell.

[0060] After the CD8-positive T cell administration step, a selection step is carried out to select individuals that will develop myositis. The selection step is preferably carried out after the administration step, when the cells contained in the PBMCs have sufficiently engrafted, and is preferably carried out one day or more after the end of the administration step.

[0061] As described above, symptoms of myositis include increased blood AST and CK, increased expression of markers such as Vcam1, Icam1, and Saa1 in muscle tissue, and infiltration of CD8+ T cells into muscle tissue. These can be used as indicators to select individuals that will develop myositis. Additionally, symptoms of lung lesions, such as increased blood SP-D and IL-6 associated with myositis and infiltration of immune cells into lung tissue, can also be used as indicators.

[0062] <Method for screening or evaluating candidate compounds for therapeutic and / or preventive agents for myositis> One aspect of the present invention relates to a method for screening candidate compounds for therapeutic and / or preventive agents for myositis. The screening method according to one aspect of the present invention comprises the steps of administering a test compound to the non-human myositis model animal or tissues or cells thereof, measuring an indicator of myositis in the animal, and comparing the indicator of myositis with that in a group not administered the test compound.

[0063] Any substance can be used as the test compound, and examples thereof include one or more compounds selected from proteins, peptides, low-molecular-weight compounds, high-molecular-weight compounds, and natural compounds, as well as one or more compounds selected from cell extracts, cell culture supernatants, microbial fermentation products, extracts derived from marine organisms, and plant extracts, each containing one or more of these compounds. A library containing multiple test compounds can also be used.

[0064] The test compound may be administered to the model animal, or may be added to cells or tissues obtained from the model animal. The method of administering the test compound to the model animal is not particularly limited, and examples thereof include oral administration, intravenous injection, intraperitoneal administration, and local administration such as intramuscular injection.

[0065] Examples of indicators of myositis include the above-mentioned symptoms of myositis, the expression levels of myositis marker proteins or marker genes, infiltration of CD8-positive T cells into muscle tissue, etc. In addition, symptoms of lung lesions, such as increases in blood SP-D and IL-6 associated with myositis, and infiltration of immune cells into lung tissue, may also be used as indicators.

[0066] After the above measurement, the myositis index is compared with that of the test compound non-administered group. If the myositis index is significantly improved in the test compound administered group compared to the test compound non-administered group, the test compound can be selected as a candidate substance for a myositis treatment or prevention drug. For example, if the myositis index is an index that indicates a high value in myositis, the test compound can be selected as a candidate substance for a myositis treatment or prevention drug if the index in the test compound administered group is reduced to 50% or less, 20% or less, or 10% or less compared to the non-administered group. On the other hand, if the myositis index is an index that indicates a low value in myositis, the test compound can be selected as a candidate substance for a myositis treatment or prevention drug if the index in the test compound administered group is restored to 1.2 times or more, 1.5 times or more, or 2 times or more compared to the non-administered group.

[0067] Furthermore, in the screening method of the present invention described above, by changing the indicator for myositis to an indicator for pulmonary symptoms associated with myositis and proceeding in the same manner, it is also possible to screen for candidate compounds for therapeutic and / or preventive drugs for pulmonary symptoms associated with myositis.

[0068] Furthermore, not only screening but also evaluation of a single compound can be performed in a similar manner. That is, another aspect of the present invention relates to a method for evaluating a candidate compound for a therapeutic and / or preventive agent for myositis. The evaluation method according to one aspect of the present invention includes the steps of administering a test compound to the non-human myositis model animal or its tissues or cells, measuring an index of myositis in the animal, and comparing the index of myositis with that in a compound-unadministered group. If the index of myositis is significantly improved in the test compound-administered group compared to the test compound-unadministered group, the test compound can be evaluated as having a therapeutic or preventive effect on myositis.

[0069] In the above-described evaluation method of the present invention, candidate compounds for therapeutic and / or preventive agents for pulmonary symptoms associated with myositis can also be evaluated by changing the indicator for myositis to an indicator for pulmonary symptoms associated with myositis and performing the same procedure.

[0070] The present invention will be specifically described below with reference to examples, but the embodiments of the present invention are not limited to the following examples.

[0071] Example 1: Human Peripheral Blood Mononuclear Cells, Frozen: 1 x 10^8 cells (PBMC, STEMCELL Technologies) were thawed and washed with PBS. Negatively selected CD8+ T cell-depleted PBMC were obtained using CD8 MicroBeads (Miltenyi Biotec Inc.). The fraction used was analyzed by flow cytometry to confirm depletion efficiency. The cell suspension was resuspended in PBS, and 1 x 10^7 cells (0.2 mL) per mouse were transferred via the tail vein of NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ (NSG) mice (Day 0). Each mouse was euthanized, and exsanguinated from the caudal vena cava under isoflurane anesthesia at the time of GVHD-like symptoms or 31 days after PBMC transfer. Blood and spleens were collected and subjected to various analyses. The collected blood was centrifuged at 2000 × g at 4°C for 10 minutes to recover plasma. The GVHD score was calculated based on the results of body weight measurement and general symptom observation using the criteria shown in Table 1 (Reference 1), with each item scored from 0 to 2, with a maximum of 10. Blood ALT levels were measured using a Fuji DryChem Slide GPT / ALT-PIII (Fujifilm) with an FDC7000i (Fujifilm) according to the protocol.

[0072] As a result, as shown in Figure 1, it was found that the transfer of PBMCs from which the CD8-positive T cells had been removed suppressed the GVHD symptoms and the increase in ALT levels that were caused by the transfer of PBMCs from which CD8-positive T cells had not been removed.

[0073]

[0074] Example 2 Immune cells were isolated from the spleen collected in Example 1. 3 mL of RPMI was added to a GentleMACS C tube (Miltenyi Biotec), and the spleen, cut into quarters with scissors, was placed in the tube. The spleen was then homogenized using a gentleMACS Octo Dissociator (Miltenyi Biotec). The cell suspension was centrifuged (340 × g, 4°C, 5 minutes), the supernatant was removed, and 1 mL of HLB solution was added. The tube was then left to stand at room temperature for 2 minutes to perform hemolysis. 10 mL of RPMI-1640 Medium (RPMI, Sigma-Aldrich) was added to terminate the hemolysis reaction. After centrifugation, the supernatant was removed, and the cells were suspended in flow cytometry staining buffer (FACS buffer) and used for flow cytometry analysis. The cell suspension in FACS buffer was transferred to a round-bottom 96-well plate and treated with Fc block for 5 minutes. After treatment, Flow-Count (Becman Coulter) and fluorescently labeled antibodies, Fixable Viability Dye eFluor 780 (FVD, Thermo Fisher Scientific), were added to stain cell surface antigens. After washing with FACS buffer, the cells were centrifuged and resuspended in FACS buffer. The percentage and number of various immune cells were then identified using a flow cytometer (BD Sciences, LSR Fortessa X-20). The definitions of the various immune cells are listed in Table 2, and the antibodies used are listed in Table 3.

[0075]

[0076]

[0077] As a result, as shown in Figures 2 and 3, it was found that Tfh, Tph, and B cells engrafted in the spleen at a higher rate in the model where CD8+ T cell-depleted PBMCs were injected than in the model where CD8+ T cell-undepleted PBMCs were injected. It was confirmed that this phenomenon did not occur in the P12 model where CD4+ T cells were depleted.

[0078] Example 3 Using the blood collected in Example 1, blood CXCL13 concentrations were measured using an Envision plate reader (PerkinElmer) according to the protocol for the AlphaLISA Human CXCL13 Detection Kit. As a result, as shown in Figure 4, an increase in CXCL13, a pathological biomarker for Sjögren's syndrome, was confirmed in humanized mice transfected with CD8 T cell-depleted PBMCs, suggesting that these mice may be a pathological model for Sjögren's syndrome. It also suggests that drug efficacy evaluation in this pathological model is possible using CXCL13 as an indicator.

[0079] Example 4: Human peripheral blood mononuclear cells (PBMCs; suspended in PBS) or PBMCs depleted of CD8+ T cells were administered via the tail vein of immunodeficient NSG mice to generate humanized mice. CD8+ T cells were depleted using a commercially available kit, CD8 Microbeads, human (Miltenyi), according to the manufacturer's recommended protocol in a sterile environment. Twelve days later, mice were immunized intraperitoneally with a TNP-KLH solution prepared by mixing equal volumes of TNP-KLH prepared in PBS with Imject Alum Adjuvant (Thermo Scientific) in a sterile environment. The non-immunized group consisted of unimmunized animals. After TNP-KLH immunization, blood was collected weekly from the tail vein and antibody titers were measured to confirm the induction of an antigen-specific immune response. Blood samples were collected when an increase in antibody titer was observed (maximum 3 weeks after immunization).

[0080] Frozen human peripheral blood mononuclear cells (PBMCs: 1 x 10^8 cells) were thawed and washed with PBS. PBMCs were negatively selected using CD8 MicroBeads and depleted of CD8+ T cells. The fraction used was analyzed by flow cytometry to confirm depletion efficiency. The cell suspension to be used was resuspended in PBS, and 1 x 10^7 cells (0.2 mL) per mouse were transferred into the tail vein of NSG mice (Day 0). To remove any remaining CD8+ T cells, anti-CD8 antibodies were administered via the tail vein on days 10 and 20 after PBMC transfer to remove CD8+ T cells in vivo (performed only in Example 4).

[0081] Drug Administration The day of transfer of CD8-positive T cell-depleted human PBMCs was defined as Day 0, and from Day 15 onwards, Tacrolimus (Astellas Pharma, 10 mg / kg) diluted with 0.5% methylcellulose solution was orally administered once daily.

[0082] Cytokine Administration: From Day 3, 1 μg of recombinant human IL-12 p70 (Peprotech), recombinant human TGF-b1 (Peprotech), and recombinant human IFN-α (PBL assay science) were dissolved in PBS to a total of 3 μg, and 0.15 mL was administered subcutaneously three times a week.

[0083] On days 14 and 29, mice were subcutaneously administered a triple anesthetic mixture. After confirming sufficient anesthesia, the secretory stimulant pilocarpine hydrochloride (a muscarinic acetylcholine receptor agonist, Wako Pure Chemical Industries) was administered intraperitoneally to stimulate saliva secretion. Saliva was collected using a pipette tip and weight was used as an indicator for up to 20 minutes per mouse. After saliva collection, mice were awakened by intraperitoneal administration of atipamezole (an α2-adrenergic receptor antagonist, Kyoritsu Pharmaceutical Co., Ltd.). On day 31 after PBMC transfer, mice were euthanized and exsanguinated via the caudal vena cava under isoflurane anesthesia. Salivary glands were then collected and subjected to histopathological and flow cytometric analysis. For histopathological analysis, formalin-fixed paraffin-embedded sections were prepared from salivary gland tissue fixed in 10% neutral buffered formalin solution, stained with hematoxylin eosin (HE), and graded according to the following criteria: -: No remarkable, + / -: Minimal, +: Slight, 2+: Moderate, 3+: Severe.

[0084] Isolation of salivary gland immune cells: Digest buffer (RPMI containing 10% FBS, 1 mg / mL Collagenase, and 0.1 mg / mL DNase) was added to a GentleMACS C tube, and the tissue was placed in the tube. The tissue was finely minced with scissors and then homogenized using a GentleMACS Octo Dissociator. The tube was then incubated at 37°C for 30-60 minutes with rotation, after which it was homogenized again using a GentleMACS Octo Dissociator. The tissue was suspended in RPMI, transferred to a 15 mL tube, and centrifuged (340 × g, 4°C, 5 minutes). The supernatant was removed, and the tissue was suspended in 5 mL of 40% diluted Percoll (GE Healthcare). 2 mL of 75% diluted Percoll was layered on top using a Long Pasteur filter. After centrifugation at 700-800 × g for 20 minutes at 20°C, the upper layer of debris was removed using an aspirator, and the mononuclear cells in the middle layer were collected in a separate 15 mL tube, diluted 5-fold with RPMI, and centrifuged. The supernatant was removed and suspended in FACS buffer for flow cytometry analysis.

[0085] Flow cytometry analysis. Isolated immune cells were transferred to a round-bottom 96-well plate, stimulated with PMA / Ionomycin (Sigma-Aldrich), and cultured for 4 hours. After incubation, the cells were washed by centrifugation with FACS buffer. Flow-Count diluted 1:10 in PBS was added and centrifuged at 400 × g for 2 minutes at 4°C. After that, Fc block (anti-CD16 / 32 antibody) and Zombie NIR die (BioLegend) were added and incubated for 15 minutes at 4°C. After centrifugation with FACS buffer, the cells were treated with Fc block (anti-CD16 / 32 antibody) for 5 minutes. Primary antibody for cell surface staining was added and incubated for 20 minutes at 37°C in the dark. After centrifugation with FACS buffer, secondary antibody for cell surface staining was added and incubated for 20 minutes at 37°C in the dark. After washing by centrifugation with FACS buffer, the cells were fixed by adding Fixation / Permeabilization Concentrate Diluent (Thermo Fisher Scientific), diluted 4-fold with Fixation / Permeabilization Diluent (Thermo Fisher Scientific), and incubated at room temperature for 20 minutes in the dark. Then, Permeabilization buffer Diluent (Thermo Fisher Scientific), diluted 10-fold with Milli-Q water, was added and the cells were centrifuged. Antibodies for intracellular staining were added and incubated at room temperature for 40 minutes, followed by centrifugation and washing with Permeabilization buffer. Finally, 80-150 μL of Permeabilization buffer was added, the cells were suspended, and the cells were measured using an ID7000 (SONY). The definition of Tph cells is shown in Table 2, and the antibodies used are listed in Table 4.

[0086]

[0087] As shown in Figure 5A, humanized mice injected with CD8+ T cell-depleted PBMCs showed decreased saliva flow and worsening salivary gland pathology, which improved with drug administration, demonstrating the utility of this model for drug evaluation of Sjögren's syndrome. Furthermore, as shown in Figures 5B and 5C, administration of the cytokines (IL-12, TGF-β1, and IFN-α) further worsened salivary gland pathology and activated salivary gland Tph cells, demonstrating that this method can induce more severe pathology. Furthermore, as shown in Figure 6, cytokine administration further worsened salivary gland pathology in humanized mice injected with CD8+ T cell-depleted PBMCs.

[0088] Example 5: Human Peripheral Blood Mononuclear Cells, Frozen: 1 x 10^8 cells (PBMCs) were thawed and washed with PBS. PBMCs were negatively selected using CD8 MicroBeads to remove CD8+ T cells. The fraction used was confirmed for depletion efficiency by FACS. The cell suspension to be used was resuspended in PBS, and 1 x 10^7 cells (0.2 mL) per mouse were transferred via the tail vein of NSG mice (Day 0). When GVHD-like symptoms were observed in each mouse or 34 days after PBMC transfer, exsanguination was performed under isoflurane anesthesia, and the mice were euthanized. Salivary glands, lungs, and kidneys were then collected.

[0089] Isolation of salivary gland and kidney immune cells: Digest buffer (RPMI containing 10% FBS, 1 mg / mL Collagenase, and 0.1 mg / mL DNase) was added to a GentleMACS C tube, and the tissue was placed in the tube. The tissue was finely minced with scissors and then homogenized using a GentleMACS Octo Dissociator. The tube was then incubated at 37°C for 30-60 minutes with rotation, after which it was homogenized again using a GentleMACS Octo Dissociator. The tissue was suspended in RPMI, transferred to a 15 mL tube, and centrifuged (340 × g, 4°C, 5 minutes). The supernatant was removed, and the tissue was suspended in 5 mL of 40% diluted Percoll. Approximately 2 mL of 75% diluted Percoll was layered on top using a Long Pasteur tube. After centrifugation at 700-800 × g for 20 minutes at 20°C, the upper layer of debris was removed using an aspirator, and the mononuclear cells in the middle layer were collected in a separate 15 mL tube, diluted approximately 5-fold with RPMI, and centrifuged. The supernatant was removed and suspended in FACS buffer for flow cytometry analysis.

[0090] Isolation of lung immune cells. A 100 μm cell strainer (Corning) was placed on a dish, 5 mL of RPMI was added, and the tissue was placed on top. The tissue was mashed with the handle of a 1 mL syringe (TERUMO), and the suspension was collected in a 15 mL tube. Another 5 mL of RPMI was added, and the tissue was mashed again. The suspension was collected in a 15 mL tube and centrifuged (340 × g, 4°C, 5 minutes). The supernatant was removed, and the tissue was suspended in FACS buffer and used for flow cytometry analysis.

[0091] Flow cytometry analysis. Immune cells isolated from each tissue were transferred to a round-bottom 96-well plate, and Flow-Count diluted 1:10 with PBS was added. The plate was then centrifuged (400 × g, 4°C, 2 min). The supernatant was removed, and Zombie NIR die (BioLegend) was added and incubated for 15 min at 4°C. After centrifugation and washing with FACS buffer, the plate was treated with an Fc block (anti-CD16 / 32 antibody) for 5 min. A primary antibody for cell surface staining was added and incubated for 20 min at 4°C in the dark. After centrifugation and washing with FACS buffer, a secondary antibody for cell surface staining was added and incubated for 20 min at 4°C in the dark. After centrifugation and washing with FACS buffer, Fixation / Permeabilization Concentrate diluted 4:1 with Fixation / Permeabilization Diluent was added and incubated for 20 min at room temperature in the dark to fix the cells. Then, Permeabilization buffer diluted 1:10 with Milli-Q water was added and the plate was centrifuged for washing. After adding antibodies for intracellular staining, the cells were incubated at 4°C for 40 minutes and then centrifuged and washed with permeabilization buffer. Finally, 80-150 μL of permeabilization buffer was added to suspend the cells, and they were measured using an ID7000 (SONY). The definitions of Tph and Tfh cells are shown in Table 2, and the antibodies used are shown in Table 4.

[0092] As a result, as shown in Figure 7, in humanized mice injected with CD8+ T cell-depleted PBMCs, infiltration of human Tfh and Tph cells was observed not only in the salivary glands, which are organs that manifest glandular symptoms of Sjögren's syndrome, but also in the kidneys and lungs, which are organs that manifest extraglandular symptoms, demonstrating that organ infiltration of these cells can be used as an indicator to evaluate glandular and extraglandular symptoms.

[0093] Example 6: 1000 μL of RNAlater was added to the salivary gland, lung, and kidney fragments collected in Example 5 and homogenized twice in a Tissue Lyzer LT (Qiagen) for 2 minutes at 50 (1 / s). 150 μL of chloroform was added, followed by centrifugation. 150 μL of Buffer RLT was added to 200 μL of the supernatant, and RNA was extracted using a QIAcube (Qiagen). cDNA was prepared by reverse transcription from the RNA using a High-Capacity RNA to cDNA kit. mRNA expression levels were measured using real-time PCR with a Taqman probe on a 7500 Fast RealTime PCR system (Applied Biosystems). As shown in Figure 8, increased expression of inflammatory cytokine genes (Tph-related genes) was observed in the salivary glands, lungs, and kidneys of humanized mice injected with CD8+ T cell-depleted PBMCs. This demonstrated that measuring Tph-related genes in each tissue can be used to measure the level of Tph infiltration and evaluate drug efficacy.

[0094] Example 7: Human Peripheral Blood Mononuclear Cells, Frozen: 1 x 10^8 cells (PBMC) were thawed and washed with PBS. Negatively selected CD8-positive T cell-depleted PBMCs were obtained using CD8 MicroBeads (Miltenyi Biotec Inc.). The fraction to be used was confirmed for depletion efficiency by FACS. The cell suspension to be used was resuspended in PBS, and 1 x 10^7 cells (0.2 mL) per mouse were transferred via the tail vein of NSG mice (Day 0).

[0095] NP-OVA / Alum Administration. On day 12 after PBMC transfer, mice were immunized by intraperitoneal administration of 0.2 mL of NP-OVA solution, which was prepared by mixing equal volumes of nitrophenyl-conjugated ovalbumin (NP-OVA; NP19-OVA, Biosearch Technologies) adjusted to 1 mg / mL in PBS with aluminum hydroxide (Alum; Alhydrogel™ 2%, InvivoGen) under a sterile environment. On day 33 after PBMC transfer, each mouse was euthanized after exsanguination from the caudal vena cava under isoflurane anesthesia, and the spleen was collected.

[0096] Isolation of splenic immune cells. Immune cells were isolated from the harvested spleen. 3 mL of RPMI was added to a GentleMACS C tube, and the spleen, divided into quarters with scissors, was placed in the tube. The spleen was then homogenized using a gentleMACS Octo Dissociator. The cell suspension was centrifuged (340 × g, 4°C, 5 minutes), the supernatant was removed, and 1 mL of HLB Solution was added. The tube was then left to stand at room temperature for 2 minutes for hemolysis. 10 mL of RPMI was added to stop the hemolysis. After centrifugation, the supernatant was removed, and the cells were suspended in FACS buffer and used for flow cytometry analysis. The cell suspension in FACS buffer was treated with an Fc block (anti-CD16 / 32 antibody) for 5 minutes, followed by addition of Flow-Count and fluorescently labeled antibody, Fixable Viability dye, to stain cell surface antigens. After washing with FACS buffer, the tube was centrifuged and resuspended in FACS buffer. The percentage and number of various immune cells were then identified using a flow cytometer (BD Sciences, LSR SORP). The definitions of Tph cells and Tfh cells are shown in Table 2, and the antibodies used are shown in Table 5.

[0097]

[0098] As shown in Figure 9, administration of NP-OVA / Alum to humanized mice transfected with CD8+ T cell-depleted PBMCs increased the number of Tfh and Tph cells in the spleen, demonstrating that this method can be used to induce Tfh and Tph cells in vivo.

[0099] Example 8: Human peripheral blood mononuclear cells, frozen: 1 x 10^8 cells (PBMC, STEMCELL Technologies) were thawed and washed with PBS to obtain PBMC for transfer to mice. CD8+ T cell-depleted PBMC were prepared by negatively selecting CD8+ T cells from PBMC using CD8 MicroBeads. 1 x 10^7 cells (0.2 mL) of PBMC or CD8+ T cell-depleted PBMC per mouse were transferred via the tail vein of NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ (NSG) mice (Day 0). On Day 20 after transfer, each mouse was exsanguinated from the caudal vena cava under isoflurane anesthesia, euthanized, and blood and spleens were collected for various analyses. The collected blood was centrifuged at 2000 x g for 10 minutes at 4°C, and plasma was collected. Blood ALT, AST, and CK levels were measured using a Fuji DryChem Slide GPT / ALT-PIII (Fujifilm) according to the protocol and an FDC7000i (Fujifilm). Total RNA was extracted from muscle tissue from mouse thigh muscles using TRIzol (Thermo Fisher Scientific) according to the protocol. Total RNA was reverse transcribed using a High Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific) to obtain cDNA, and muscle tissue Vcam1, Icam1, and Saa1 expression was measured using TaqMan probes and TaqMan Universal Master Mix. Gene expression values ​​were normalized using Gapdh as an internal control. Immune cell infiltration in muscle tissue was examined by histopathological analysis using H&E staining and immunostaining. As a result, in the humanized mice injected with PBMCs, not only the liver damage marker ALT but also the muscle damage markers AST and CK were found to be increased in the blood, and in the muscle tissue, Vcam1, Icam1, and Saa1 gene expression and infiltration of immune cells, including CD8+ T cells, were found to be enhanced (Figures 10, 11, and 12).On the other hand, when PBMCs depleted of CD8+ T cells were transferred, the onset of these muscle symptoms was alleviated (Figs. 10, 11, and 12), demonstrating that these symptoms are caused by CD8+ T cells, suggesting that these mice may be useful as a myositis model.

[0100] Example 9: Human peripheral blood mononuclear cells, frozen: 1 x 10^8 cells (PBMC, STEMCELL Technologies) were thawed and washed with PBS to obtain PBMC for mouse transfer. 1 x 10^7 cells (0.2 mL) per mouse were transferred via the tail vein of NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ (NSG) mice (Day 0). Immunosuppressants, steroids (prednisolone, PSL) and tacrolimus (TAC), were administered from Day 7 until the day of sampling. On Day 31 after transfer, each mouse was exsanguinated from the caudal vena cava under isoflurane anesthesia, euthanized, and blood and spleens were collected for various analyses. The collected blood was centrifuged at 2000 x g for 10 minutes at 4°C, and plasma was collected. Blood ALT, AST, and CK levels were measured using a Fuji DryChem Slide GPT / ALT-PIII (Fujifilm) according to the protocol on an FDC7000i (Fujifilm). Total RNA was extracted from muscle tissue from mouse thigh muscles using TRIzol (Thermo Fisher Scientific) according to the protocol. Total RNA was reverse transcribed using a High Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific) to obtain cDNA. Expression of Vcam1, Icam1, and Saa1 in muscle tissue was measured using TaqMan probes and TaqMan Universal Master Mix. Gene expression values ​​were normalized using Gapdh as an internal control. Immune cell infiltration in muscle tissue was examined by histopathological analysis using H&E staining and immunostaining. The results showed that tacrolimus and steroid administration improved the increased muscle damage markers and immune cell infiltration, including CD8+ T cells, in PBMC-transfected humanized mice (Figures 13, 14, and 15). This demonstrated that this model can be used as a myositis model to evaluate the efficacy of drugs.

[0101] Example 10: Human peripheral blood mononuclear cells, frozen: 1 x 10^8 cells (PBMC, STEMCELL Technologies) were thawed and washed with PBS to obtain PBMC for mouse transfer. 1 x 10^7 cells (0.2 mL) per mouse were transferred via the tail vein of NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ (NSG) mice (Day 0). On Day 22 after transfer, each mouse was euthanized by exsanguination from the caudal vena cava under isoflurane anesthesia, and blood and lung tissue were collected for various analyses. Immune cell infiltration in the lung tissue was examined by histopathological analysis using H&E staining. The collected blood was centrifuged at 2000 x g at 4°C for 10 minutes, and plasma was collected. Serum SP-D levels were calculated by measuring absorbance using a SpectraMAX (Molecular Devices) kit for rat / mouse SP-D EIA according to the protocol. The results demonstrated immune cell infiltration into lung tissue (Figure 16) and elevated serum SP-D levels (Figure 17) in PBMC-transfected humanized mice, demonstrating that this model exhibits pulmonary lesions associated with myositis. Therefore, this model may be useful for evaluating the efficacy of drugs against myositis-related pulmonary symptoms.

[0102] Example 11: Human peripheral blood mononuclear cells (PBMCs) derived from three healthy donors (1 x 10^8 frozen cells) were thawed and washed with PBS. Then, CD8 T cell-depleted PBMCs were obtained by negative selection using CD8 MicroBeads. The fraction to be used was confirmed for depletion efficiency by FACS. The cell suspension to be used was resuspended in PBS, and 1 x 10^7 cells (0.2 mL) per mouse were transferred into the tail vein of NSG mice (Day 0).

[0103] On day 20, mice were subcutaneously administered a triple anesthetic mixture. After confirming sufficient anesthesia, the secretory stimulant pilocarpine hydrochloride (a muscarinic acetylcholine receptor agonist, Wako Pure Chemical Industries) was administered intraperitoneally to stimulate saliva secretion. Saliva was collected using a pipette tip and weight was used as an indicator for up to 20 minutes per mouse. After saliva collection, mice were awakened by intraperitoneal administration of atipamezole (an α2-adrenergic receptor antagonist, Kyoritsu Pharmaceutical Co., Ltd.). On day 26 after PBMC transfer, mice were euthanized by exsanguination via the caudal vena cava under isoflurane anesthesia, and blood and spleens were collected for various analyses.

[0104] Measurement of blood CXCL13 concentration Plasma was isolated from the collected blood and measured using an Envision plate reader (PerkinElmer) according to the protocol for the AlphaLISA Human CXCL13 Detection Kit.

[0105] Spleen cell isolation: 3 mL of RPMI was added to a GentleMACS C tube (Miltenyi Biotec), and the spleen, cut into quarters with scissors, was placed in the tube. The spleen was then homogenized using a gentleMACS Octo Dissociator (Miltenyi Biotec). The cell suspension was centrifuged (340 × g, 4°C, 5 minutes), the supernatant was removed, and 1 mL of HLB solution was added and the tube was left at room temperature for 2 minutes to hemolyze the cells. The hemolysis was stopped by adding 10 mL of RPMI-1640 Medium (RPMI, Sigma-Aldrich). After centrifugation, the supernatant was removed, and the cells were suspended in flow cytometry staining buffer (FACS buffer) and used for flow cytometry analysis.

[0106] Flow cytometry analysis. Immune cells isolated from each tissue were transferred to a round-bottom 96-well plate, and Flow-Count diluted 1:10 with PBS was added. The plate was centrifuged at 400 × g for 2 minutes at 4°C. The supernatant was removed, and Zombie NIR die (BioLegend) was added and incubated for 15 minutes at 4°C. After centrifugation with FACS buffer, the plate was treated with an Fc block (anti-CD16 / 32 antibody) for 5 minutes. A primary antibody for cell surface staining was added and incubated for 20 minutes at 4°C in the dark. After centrifugation with FACS buffer, a secondary antibody for cell surface staining was added and incubated for 20 minutes at 4°C in the dark. After centrifugation with FACS buffer, Fixation / Permeabilization Concentrate diluted 4:1 with Fixation / Permeabilization Diluent was added and incubated for 20 minutes at room temperature in the dark to fix the cells. Then, Permeabilization buffer diluted 1:10 with Milli-Q water was added and the plate was centrifuged. After adding an antibody for intracellular staining and incubating at 4°C for 40 minutes, the cells were centrifuged and washed with permeabilization buffer. Finally, the cells were suspended in permeabilization buffer and measured using an ID7000 (SONY). The definitions of Tph and Tfh cells are shown in Table 2, and the antibodies used are shown in Table 6. Using cells from all three donors, we observed high engraftment of Tph / Tfh / B cells in the spleen (Figure 18), as well as elevated blood CXCL13 levels (Figure 19) and decreased saliva production (Figure 20), which are symptoms of Sjögren's syndrome, demonstrating that pathological conditions can be developed without any differences between donors.

[0107]

[0108] Example 12: Frozen human peripheral blood mononuclear cells (PBMC: 1 x 10^8 cells) were thawed and washed with PBS. Negatively selected CD8 T cell-depleted PBMCs were obtained using CD8 MicroBeads. The fraction to be used was confirmed for depletion efficiency by flow cytometry analysis. The cell suspension to be used was resuspended in PBS, and 1 x 10^7 cells (0.2 mL) per mouse were transferred into the tail vein of NSG mice (Day 0).

[0109] Drug Administration: The day of CD8 T cell-depleted human PBMC transfer was designated Day 0, and Tacrolimus (Astellas Pharma, 10 mg / kg) diluted with 0.5% methylcellulose solution was orally administered once daily from Day 15. On Day 30 after PBMC transfer, exsanguination was performed under isoflurane anesthesia via the caudal vena cava, and the mice were euthanized. Blood samples were collected and used to measure IgG, anti-SS-A antibody, and anti-SS-B antibody levels.

[0110] Measurement of blood IgG, anti-SS-A antibody, and anti-SS-B antibody concentrations. Plasma was isolated from the collected blood and measured using the Human Anti-dsDNA IgG High Sensitivity ELISA KIT (M-3100-HS, Alpha Diagnostic), the Anti-Sjogren's syndrome type A antigen (SSA / Ro) IgG ELISA KIT (3210-SSA, Alpha Diagnostic), and the Anti-Sjogren's syndrome type B antigen (SSB / La) IgG ELISA KIT (3220-SSB, Alpha Diagnostic) protocols using an Envision plate reader (PerkinElmer) and a SpectraMax ABS Plus (Molecular Devices). The results showed elevated IgG, a marker for autoimmune diseases in general, including Sjogren's syndrome, as well as anti-SS-A and anti-SS-B antibodies, which are markers for Sjogren's syndrome (Figure 21). This suggests that this model may be a useful pathological model for Sjogren's syndrome and other autoimmune diseases in general.

[0111] Example 13: Human Peripheral Blood Mononuclear Cells, Frozen: 1 x 10^8 cells (PBMC) were thawed and washed with PBS. CD8 T cell-depleted PBMCs were obtained by negative selection using CD8 MicroBeads. The fraction to be used was confirmed for depletion efficiency by FACS. The cell suspension to be used was resuspended in PBS, and 1 x 10^7 cells (0.2 mL) per mouse were transferred via the tail vein of NSG mice (Day 0). Thirty days after PBMC transfer, exsanguination was performed under isoflurane anesthesia from the posterior vena cava, and the mice were euthanized. Blood and spleens were collected and subjected to various analyses.

[0112] Measurement of blood antinuclear antibody concentrations Plasma was isolated from the collected blood and measured using a SpectraMax ABS Plus (Molecular Devices) according to the protocol for the ANA Screen ELISA Kit (KA 0939, Abnova). As a result, an increase in antinuclear antibodies, a marker for autoimmune diseases in general, including Sjögren's syndrome, was observed in CD8 T cell-depleted PBMC-transferred humanized mice (Figure 22), suggesting the possibility that this model may serve as a pathological model for Sjögren's syndrome and autoimmune diseases in general.

[0113] Example 14: Human Peripheral Blood Mononuclear Cells, Frozen: 1 x 10^8 cells (PBMCs) were thawed and washed with PBS to obtain CD8 T cell-depleted PBMCs negatively selected using CD8 MicroBeads. The fraction to be used was confirmed for depletion efficiency by FACS. The cell suspension to be used was resuspended in PBS, and 1 x 10^7 cells (0.2 mL) per mouse were transferred via the tail vein of NSG mice (Day 0). When GVHD-like symptoms were observed in each mouse or 34 days after PBMC transfer, exsanguination was performed under isoflurane anesthesia, and the mice were euthanized. Salivary glands, muscle, skin, kidneys, liver, lungs, and pancreas were then collected. For histopathological analysis, formalin-fixed paraffin-embedded sections were prepared from salivary gland tissue fixed in 10% neutral buffered formalin and stained with hematoxylin eosin (HE). The results were graded according to the following criteria: - (No remarkable), + / - (Minimal), + (Slight), 2+ (Moderate), 3+ (Severe). As shown in Figures 23-1 to 23-7 and Table 7, in CD8 T cell-depleted PBMC-transferred humanized mice, cellular infiltration was observed not only in the salivary glands, which are the organs that manifest glandular symptoms of Sjögren's syndrome, but also in the kidneys, lungs, and other organs that manifest extraglandular symptoms. Histopathological degeneration was also observed in the kidneys and liver. These findings demonstrate that pathological evaluation of CD8 T cell-depleted PBMC-transferred humanized mice is possible for the evaluation of not only glandular symptoms but also extraglandular symptoms.

[0114]

[0115] Example 15: Frozen human peripheral blood mononuclear cells (PBMC: 1 x 10^8 cells) were thawed and washed with PBS. Negatively selected CD8 T cell-depleted PBMCs were obtained using CD8 MicroBeads. The fraction to be used was confirmed for depletion efficiency by flow cytometry analysis. The cell suspension to be used was resuspended in PBS, and 1 x 10^7 cells (0.2 mL) per mouse were transferred into the tail vein of NSG mice (Day 0).

[0116] Drug Administration: The day of CD8 T cell-depleted human PBMC transfer was designated Day 0, and Tacrolimus (Astellas Pharma, 10 mg / kg) diluted with 0.5% methylcellulose solution was orally administered once daily from Day 15. On Day 30 after PBMC transfer, exsanguination was performed under isoflurane anesthesia via the caudal vena cava, and the mice were euthanized. Blood samples were collected for SP-D and blood glucose measurements.

[0117] Measurement of blood SP-D and blood glucose levels. Plasma was isolated from the collected blood and measured using a SpectraMax ABS Plus (Molecular Devices) according to the protocol for the Rat / Mouse SP-D kit YAMASA EIA (96 Reactions / 1 Kit) (Yamasa Shoyu) and the Glucose colorimetric assay kit (Cayman Chemical Company). Results showed that SP-D, a marker of lung damage, an extraglandular manifestation of Sjögren's syndrome, was significantly elevated in CD8 T cell-depleted PBMC-transferred humanized mice (Figure 24). Furthermore, administration of tacrolimus improved the elevated SP-D levels. However, blood glucose levels, an indicator of pancreatic lesions, remained unchanged (Figure 25). These results demonstrate that this model can be used to evaluate the effects of drugs on lung lesions, an extraglandular manifestation of Sjögren's syndrome, using SP-D as an indicator.

[0118] Example 16: Frozen human peripheral blood mononuclear cells (PBMC: 1 x 10^8 cells) were thawed and washed with PBS. Negatively selected CD8 T cell-depleted PBMCs were obtained using CD8 MicroBeads. The fraction to be used was confirmed for depletion efficiency by flow cytometry analysis. The cell suspension to be used was resuspended in PBS, and 1 x 10^7 cells (0.2 mL) per mouse were transferred into the tail vein of NSG mice (Day 0).

[0119] The day of CD8 T cell-depleted human PBMC transfer was designated Day 0, and Tacrolimus (Astellas Pharma, 10 mg / kg) diluted with 0.5% methylcellulose solution was orally administered once daily from Day 15. On Day 28 after PBMC transfer, urine was collected by abdominal compression and used to measure urinary glucose and proteinuria.

[0120] Measurement of urinary glycoproteinemia was measured using a SpectraMax ABS Plus (Molecular Devices) according to the protocols of the Glucose colorimetric assay kit (Cayman Chemical Company) and Pierce Coomassie Plus (Bradford) Assay Kit 1KIT (Thermo Fisher Scientific).

[0121]

[0122] As a result, no change was observed in urinary glucose levels, an indicator of pancreatic lesions, in CD8 T cell-depleted PBMC-transferred humanized mice (Table 8). On the other hand, an increase in proteinuria levels, an indicator of renal lesions, was observed (Figure 26). These results suggest that this model may be useful for evaluating the effects of drugs on renal lesions, an extraglandular symptom of Sjögren's syndrome, using proteinuria as an indicator.

[0123] Example 17: In a non-human autoimmune disease model, systemic lupus erythematosus pathology can be induced by administering pristane as a stimulant. Furthermore, by using PBMCs from systemic lupus erythematosus patients, pathogenic autoantibodies can be induced in vivo, thereby inducing systemic lupus erythematosus pathology.

Claims

1. A non-human autoimmune disease model animal derived from an immunodeficient non-human animal, into which peripheral blood mononuclear cells (PBMCs) derived from a xenogeneic animal that have been treated to remove CD8+ T cells are transferred, and which exhibits symptoms of an autoimmune disease.

2. The non-human autoimmune disease model animal according to claim 1, wherein the immunodeficient non-human animal is an immunodeficient non-human mammal.

3. The non-human autoimmune disease model animal according to claim 2, wherein the immunodeficient non-human mammal is an immunodeficient mouse.

4. The non-human autoimmune disease model animal according to claim 1, wherein the PBMCs derived from a heterologous animal are human-derived PBMCs.

5. The non-human autoimmune disease model animal according to claim 4, wherein the human-derived PBMCs are PBMCs derived from a human autoimmune disease patient or PBMCs derived from a healthy human subject.

6. The non-human autoimmune disease model animal according to claim 1, wherein the CD8-positive T cell depletion treatment is a treatment using an anti-CD8 antibody.

7. The non-human autoimmune disease model animal according to claim 1, wherein the autoimmune disease is one or more selected from the group consisting of Sjogren's syndrome, rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, myasthenia gravis, inflammatory bowel disease, Graves' disease, autoimmune hepatitis, and IgG4-related disease.

8. 1 x 10 x xenogeneic Tph cells, xenogeneic Tfh cells, and xenogeneic B cells per 200 mg of spleen. ７ More than cells, 1 x 10 ６ cells or more and 1 x 10 ７ The non-human autoimmune disease model animal according to claim 1, comprising at least one cell.

9. A method for producing a non-human autoimmune disease model animal, comprising: an administration step of administering PBMCs derived from a xenogeneic animal that have been subjected to a CD8+ T cell depletion treatment to an immunodeficient non-human animal; and a selection step of selecting an individual exhibiting symptoms of an autoimmune disease.

10. The method of claim 9, further comprising the step of administering a cytokine prior to said selecting step.

11. The method according to claim 10, wherein the cytokine is one or more selected from the group consisting of IL-12, TGF-β, and IFN-α.

12. The method of claim 9, further comprising administering an adjuvant together with the antigen prior to said selection step.

13. The method for producing a non-human autoimmune disease model animal according to claim 9, wherein the non-human autoimmune disease model animal is a Sjögren's syndrome model animal, and the selection step comprises measuring one or more of the following: (i) the amount of saliva of the non-human autoimmune disease model animal, (ii) the amount of T cells in the salivary glands of the non-human autoimmune disease model animal, (iii) the amount of cytokines in the salivary glands of the non-human autoimmune disease model animal, and (iv) the amount of Tph cells or Tfh cells in the kidneys or lungs of the non-human autoimmune disease model animal.

14. The method for producing a non-human autoimmune disease model animal according to claim 9, wherein the non-human autoimmune disease model animal is a Sjögren's syndrome model animal, and the selection step comprises measuring one or more of the following: (i) the amount of saliva of the non-human autoimmune disease model animal, (ii) the amount of T cells in the salivary glands of the non-human autoimmune disease model animal, (iii) the amount of cytokines in the salivary glands of the non-human autoimmune disease model animal, (iv) the amount of Tph cells or Tfh cells in the kidneys or lungs of the non-human autoimmune disease model animal, (v) the amount of cytokines in the kidneys or lungs of the non-human autoimmune disease model animal, and (vi) the amount of cytokines in the blood of the non-human autoimmune disease model animal.

15. The method for producing a non-human autoimmune disease model animal according to claim 13, wherein the cytokine is an inflammatory cytokine.

16. The method for producing a non-human autoimmune disease model animal according to claim 15, wherein the inflammatory cytokine is one or more selected from CXCL13, IFN-γ, and IL-21.

17. A method for screening or evaluating candidate compounds for therapeutic and / or prophylactic agents for autoimmune diseases, comprising the steps of: administering a test compound to the non-human autoimmune disease model animal or its tissues or cells according to any one of claims 1 to 8; measuring an indicator of autoimmune disease in said animal; and comparing said indicator of autoimmune disease with that in a group not administered with the compound.

18. The screening method or evaluation method according to claim 17, wherein the autoimmune disease is Sjögren's syndrome, and the step of measuring an indicator of the autoimmune disease comprises measuring one or more of the following: (i) the amount of saliva in the non-human autoimmune disease model animal, (ii) the amount of T cells in the salivary glands of the non-human autoimmune disease model animal, (iii) the amount of cytokines in the salivary glands of the non-human autoimmune disease model animal, or (iv) the amount of Tph cells or Tfh cells in the kidneys or lungs of the non-human autoimmune disease model animal.

19. The screening method or evaluation method according to claim 17, wherein the autoimmune disease is Sjögren's syndrome, and the step of measuring an indicator of the autoimmune disease comprises measuring one or more of the following: (i) the amount of saliva in the non-human autoimmune disease model animal, (ii) the amount of T cells in the salivary glands of the non-human autoimmune disease model animal, (iii) the amount of cytokines in the salivary glands of the non-human autoimmune disease model animal, (iv) the amount of Tph cells or Tfh cells in the kidneys or lungs of the non-human autoimmune disease model animal, (v) the amount of cytokines in the kidneys or lungs of the non-human autoimmune disease model animal, or (vi) the amount of cytokines in the blood of the non-human autoimmune disease model animal.

20. The screening method or evaluation method according to claim 18, wherein the cytokines in steps (iii), (v), and (vi) are inflammatory cytokines.

21. The screening method or evaluation method according to claim 20, wherein the inflammatory cytokine is one or more selected from CXCL13, IFN-γ, and IL-21.

22. A method for producing Tph cells, Tfh cells and / or B cells, comprising a step of recovering Tph cells, Tfh cells and / or B cells from a non-human autoimmune disease model animal described in any one of claims 1 to 8.

23. A non-human myositis model animal derived from an immunodeficient non-human animal, into which CD8-positive T cells derived from a heterologous animal or peripheral blood mononuclear cells (PBMCs) containing said CD8-positive T cells have been transferred, and which exhibits symptoms of myositis.

24. A method for producing a non-human myositis model animal, comprising: an administration step of administering CD8-positive T cells derived from a heterologous animal or PBMCs containing the CD8-positive T cells to an immunodeficient non-human animal; and a selection step of selecting an individual exhibiting symptoms of myositis.

25. A method for screening or evaluating candidate compounds for therapeutic and / or preventive agents for myositis, comprising the steps of administering a test compound to the non-human myositis model animal or tissues or cells thereof according to claim 23, measuring an indicator of myositis in the animal, and comparing the indicator of myositis with that in a group not administered the compound.

26. A method for screening or evaluating candidate compounds for therapeutic and / or preventive agents for pulmonary symptoms associated with myositis, comprising the steps of administering a test compound to the non-human myositis model animal or tissues or cells thereof according to claim 23, measuring an index of pulmonary symptoms associated with myositis in said animal, and comparing the index of pulmonary symptoms associated with myositis with that in a group not administered the compound.

Images