WO2007144974A1 - Autoimmune nonhuman animal and screening method using the same - Google Patents

Abstract

A mouse in which a part or the whole of a BTLA gene on chromosome is deleted and the function of expressing BTLA to be expressed in a wild type is lost and the responsiveness to a ligand recognized by BTLA is specifically impaired is used as a disease model of systemic autoimmune disease, particularly autoimmune hepatitis. This mouse with spontaneous autoimmune disease/autoimmune hepatitis is used for a method for diagnosing an autoimmune disease or screening a pathogenic autoantigen, a substance promoting or inhibiting autoimmune response.

Description

 Specification

 Autoimmune non-human animal and screening method using the same

 Technical field

 [0001] The present invention relates to an autoimmune non-human animal and a screening method using the same.

 Background art

 [0002] In addition to an antigen receptor that recognizes an antigen, an auxiliary receptor that binds to various ligands on an antigen-presenting cell and transmits a signal for promoting or suppressing activity is present on lymphocytes. These are called costimulatory molecules, and are roughly classified into promoting costimulatory molecules and inhibitory costimulatory molecules. For example, T lymphocytes (T cells) respond strongly to foreign antigens, do not respond to self antigens and maintain immune tolerance, but T cells after such antigen recognition In addition to antigen-specific signals via the T cell receptor (TCR), the appropriate immune response is known to be associated with costimulatory signals of costimulatory molecular forces on the T cell surface ( For example, see Non-Patent Documents 1 and 2 below).

 [0003] Inhibitory costimulatory molecules negatively regulate the immune response by suppressing the proliferation of lymphocytes and the production of cytodynamic force. Inhibitory costimulatory molecules have already been identified as CTLA-4 and PD-1 and functional analysis is underway (for example, see Non-Patent Documents 3 to 5 below). The ability of these inhibitory costimulatory molecules to cause knockout (deficient) mice to be impaired in tolerance and the onset of autoimmune diseases in vivo has also revealed the ability to set lymphocyte activation thresholds and excessively. It is thought that it plays a very important role in stopping the immune response (see, for example, Non-Patent Documents 6 to 8 below).

[0004] An autoimmune disease is an excess of the immune system that is originally responsible for recognizing and eliminating foreign substances such as bacteria, viruses, and tumors that are different from self. It is a general term for diseases that cause symptoms by responding to and attacking. Autoimmune diseases can be divided into two types: systemic autoimmune diseases that affect the entire body and organ-specific diseases that affect only specific organs. For example, rheumatoid arthritis and systemic lupus erythematosus (SLE) Collagen diseases such as sidalen syndrome are systemic autoimmune diseases. In patients with autoimmune disease, self reacts with components of the body (self-antigen) Antibodies are often found and it is clear that the normal immune control system for self is not functioning. That is, even when the antigen is recognized as “self”, antigen-specific helper T cells are activated and activate B cells and cytotoxic T cells (self-reactive lymphocytes). As a result, autoantibodies against self-antigens are produced and self-reactive cytopathic T cells damage self-targeting cells. At that time, various autoimmune diseases are caused by different organ lesions due to differences in autoantigens recognized by autoreactive lymphocytes. Even in healthy individuals, there are a few autoreactive lymphocytes, but various immunosuppressive factors including inhibitory side-stimulatory signals do not cause autoimmune diseases.

 [0005] Autoimmune hepatitis is a chronically occurring hepatitis that is thought to involve an autoimmune mechanism in the onset and persistence of hepatocellular injury, and is characterized by frequent occurrence after middle age. It is a disease. Persistent or repetitive serum transaminase (GOT, GPT) elevation, hypergammaglobulinemia, serum autoantibody positive (antinuclear antibody, anti-DNA antibody, anti-smooth muscle antibody, hepatic kidney microsomal antibody, etc.) It is characterized by. Complications of other autoimmune diseases are seen in approximately 1Z3 cases, with the most common being chronic thyroiditis, rheumatoid arthritis, and Siren-Dallen syndrome. In addition, immunosuppressants and corticosteroids are effective for treatment.

 [0006] The present inventors screened a difference in gene expression between Thl cells and Th2 cells, which are two subsets of mouse-derived active CD4 positive T cells, by using a DNA chip, thereby providing a novel inhibitory accessory gene. A stimulating molecule, BTLA (B and T Lymphocyte Atte nuator), was identified (see Non-Patent Document 9 below). Figure 1 shows a comparison of the amino acid sequences of BTLA.

[0007] BTLA is an inhibitory costimulatory molecule expressed on lymphocytes, and TBT proliferation and production of site force-in such as IL-2 are suppressed by BTLA stimulation. It is presumed that the response is negatively controlled and functions to maintain homeostasis of the immune system, that is, tolerance (see, for example, Non-Patent Document 9 below). There is also a report that BTLA ligand, HVEM, is relatively expressed in the liver in humans (see Non-Patent Document 10 below), and BTLA may play an important role in tolerance in the liver. Has been suggested. [0008] Non-Patent Document l: Annu. Rev. Immunol., 2001, 19, 565-594

 Non-Patent Document 2: Annu. Rev. Immunol., 2001, 19, 225-252

 Non-Patent Document 3: Annu. Rev. Immunol., 1996, 14, 233-258

 Non-Patent Document 4: Curr. Opin. Cell Biol., 1999, 11, 203-210

 Non-Patent Document 5: Trends. Immunol., 2001, 22, 265-268

 Non-Patent Document 6: Science, 1995, 270, 985-988

 Non-Patent Document 7: Immunity, 1999, 11, 141-151

 Non-Patent Document 8: Science, 2001, 291, 319-322

 Non-Patent Document 9: Nat. Immunol., 2003, 4, 670-679

 Non-Patent Document 10: Biol. Chem., 1997, 272, 14029-14032

 Disclosure of the invention

 Problems to be solved by the invention

[0009] Certainly, according to the reports described in Non-Patent Documents 9 and 10 above, immune responses are likely to be activated due to functional deficiencies or declines in BTLA, and immune tolerance is broken, that is, autoimmune diseases develop, especially autoimmune hepatitis. The possibility of causing However, Non-Patent Document 9 does not report nor investigate the onset of disease after aging in living organisms deficient in BTLA.

 Accordingly, an object of the present invention is to provide an autoimmune disease, particularly an autoimmune hepatitis model non-human animal, an organ damage analysis method using them, a screening method for therapeutic drugs, and the like.

 Means for solving the problem

 [0011] A non-human animal according to an embodiment of the present invention that solves the above problems is characterized in that a part or all of BTLA on a chromosome is missing. Here, the gene sequence of BTLA is shown in the sequence listing.

 [0012] Although not limited, it is desirable that the non-human animal exhibits a pathological image of autoimmune hepatitis or a mouse.

[0013] In addition, a screening method for an autoimmune disease diagnostic method according to one embodiment of the present invention uses a cell or a biological tissue to increase or decrease self-antigen, self-reactivity, or self-antigen. Measure genes that increase or decrease the expression of self-antibodies, cell surface markers, serum proteins, enzymes, and inorganic substances! Using immunocompetent cells or blood immunoglobulins and analytes Measure the response to the test substance in the immunocompetent cells and the binding of the immunoglobulin and the test substance, measure the response to the test substance in a non-human animal, and use the immunocompetent cells and the test substance Thus, it is desirable to measure the response to self-antigen in the immunocompetent cells and to measure the response to self-antigen. The invention's effect

 As described above, the present invention can provide an autoimmune disease, particularly an autoimmune hepatitis model non-human animal, an organ damage analysis method using them, a screening method for a therapeutic drug, and the like.

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0015] The non-human animal according to one embodiment of the present invention is deficient in part or all of the BTLA gene, loses the function of expressing BTLA expressed in the wild type, and is a ligand recognized by BTLA. This is not particularly limited if it is a model non-human animal that specifically impairs responsiveness to BTLA. As a ligand recognized by BTLA, HVEM has been identified as a ligand. In addition, unidentified BTLA-binding proteins and carriers carrying these ligands are also included in the ligands recognized by BTLA (hereinafter referred to as “the present ligands”).

 [0016] Autoimmune disease onset refers to a decrease in the reactivity of the immunocompetent cells to the stimulation by the present ligands, resulting in an excessive immune response due to a loss or a substantial loss of the immune response. It means exhibiting abnormalities and organ damage. Therefore, in the present invention, the autoimmune disease naturally occurring model non-human animal is almost lost in response to stimulation by the present ligands due to a decrease in the response of immunocompetent cells via BTLA. A non-human animal such as a mouse, rat, or rabbit. Stimulations that induce an autoimmune response include natural exposure to self-antigens in vivo, self-antigens in cells separated from the living body, and stimulation at the in vitro port where self-derived cells are contacted. Can be mentioned.

[0017] The non-human animals according to one embodiment of the present invention include non-human animals that are unresponsive to the present ligands and are hyper-responsive to various self-antigens. Can Specific examples include non-human animals in which the function of the BTLA gene is deficient on the chromosome.

 Examples of the animal deficient in the function of the BTLA gene include rodent animals such as rats lacking the expression function of the BTLA gene in addition to the BTLA deficient mouse. Non-human animals deficient in the expression function of these BTLA genes should be obtained with BTLA —Z—non-human animals that are born according to Mendel's law, with wild-type littermates that can be used for accurate comparative experiments. It is preferable in that it can. The method for producing an animal deficient in the expression function of the earning BTLA gene is described below using BTLA-deficient mice as an example.

 [0019] The BTLA gene can be screened using a probe derived from the mouse BTLA gene after a mouse gene library is amplified by PCR or the like. The screened BTLA gene can be subcloned using a plasmid vector or the like, and can be identified by restriction enzyme mapping and DNA sequencing. Next, all or part of this BTLA-encoding gene is replaced with a neomycin-resistant gene force set, etc. 3, and the diphtheria toxin A fragment (DT-A) gene or a simple herpesvirus thymidine is placed on the terminal side. A target vector is prepared by introducing a gene such as a kinase (HSV-tk) gene.

[0020] The produced targeting vector is linearized and introduced into ES cells by the electro-poration method (electroporation), etc., and homologous recombination is performed. Select ES cells that have undergone homologous recombination with antibiotics such as G418 or ganciclovir (GANC). In addition, it is preferable to confirm whether the selected ES cell is the target recombinant by Southern blotting or the like. The confirmed ES cell clone is microinjected into the blastocyst of the mouse, and the blastocyst that returns is returned to the temporary parent mouse to produce a chimeric mouse. When this chimeric mouse is crossed with a wild-type mouse, a heterozygous mouse (F1 mouse: + Z-) can be obtained, and by crossing this heterozygous mouse, BTLA- / -(BTLA deficient) mice can be produced. In addition, as a method for confirming that the BTLA gene is not expressed in a BTLA-deficient mouse, for example, RNA is isolated from the mouse obtained by the above method and analyzed by the Northern blot method or the like. Western blot for BTLA expression in There is a method of examining by a method, a flow cytometry method using an anti-BTLA antibody or soluble HVEM.

 [0021] In addition, the BTLA-deficient mice produced are unresponsive to the ligands. For example, antigen-presenting cells forcibly expressing HVEM and antigens can be expressed as antigen-specific CD4-positive T cells in BTLA-deficient mice. This can be confirmed by contacting a cell in vitro and measuring the proliferation reaction in such a cell or the production of cytodynamic ins such as IL-2.

 [0022] The produced BTLA-deficient mice gradually develop an autoimmune state when kept in a SPF-environmental mouse breeding facility for a long period of time, and autoantibodies such as hypergammaglobulinemia and antinuclear antibodies from about 6 months of age. At 12 months of age, autoimmune organ damage occurs in multiple organs such as salivary glands, lungs, joints, and liver. The liver pathology at this time is a chronic hepatitis with hepatocyte necrosis and piecemeal necrosis in the Darrison sheath region, which is similar to so-called autoimmune hepatitis.

 [0023] Immune cells and blood proteins derived from non-human animals and model animals according to an embodiment of the present invention include elucidation of the action mechanism of BTLA and screening for autoantigens that induce autoimmunity, as well as autoimmune responses. It can be used for screening of substances that suppress or promote substances. As a screening method for a substance that promotes or suppresses an autoimmune response, a screening method that uses a immune cell derived from a model non-human animal and a test substance to measure and evaluate the response of the immune cell to the test substance is evaluated. Or a screening method that uses a model non-human animal individual and a test substance to measure and evaluate the response of the model non-human animal individual to the test substance. Embodiments will be described below with examples.

 For example, an immune cell such as a spleen cell or a lymphocyte capable of obtaining model non-human animal power according to an embodiment of the present invention and a test substance are cultured together, and the degree of cell activity in the immune cell is measured. 'Screening method to be evaluated or test substance is administered to a model non-human animal in advance, and then the immune cells obtained from the non-human animal force are cultured and the degree of cellular activity in the immune cells is measured' A screening method can be mentioned.

[0025] Measurement of degree of cellular activity in immune cells such as spleen cells or lymphocytes [Assessment method] As an evaluation method, measure the amount of production of cytodynamic ins such as IL 2, IFN y and IL 4 Examples of methods and methods for measuring and evaluating changes in the degree of cell proliferation and surface markers can be given. In addition, when measuring 'evaluation of the degree of cellular activity in immune cells', it is possible to evaluate' comparison with measured values of wild-type non-human animals of model non-human animals, particularly wild-type non-human animals of the same litter as a control. It is preferable because errors due to individual differences can be eliminated.

 [0026] The promoter or suppressor obtained by the method for screening a promoter or suppressor for spontaneous development of autoimmune disease according to one embodiment of the present invention is useful for elucidating the mechanism in BTLA signaling in vivo, In particular, inhibitors may be used as preventive and therapeutic agents for autoimmune diseases. Prevention and treatment of autoimmune diseases When using the above-mentioned accelerators that can be expected to be effective as pharmaceuticals, pharmaceutically acceptable ordinary carriers, binders, stabilizers, excipients, diluents, pH buffering agents, disintegration Various compounding ingredients for preparations such as an agent, a solubilizer, a solubilizer, and an isotonic agent can be added. In the preventive or therapeutic methods using these pharmaceuticals, an appropriate dose corresponding to the gender's body weight and symptoms can be administered orally or parenterally. That is, it can be administered orally in a commonly used dosage form such as a powder, granule, capsule, syrup, suspension, or the like, or in a dosage form such as a solution, emulsion, suspension, etc. This can be administered parenterally in the form of injections or by intranasal administration in the form of sprays.

 Hereinafter, an embodiment of the invention will be described with reference to the drawings. However, the technical scope of the present invention is not limited to these examples.

 (Example)

[0028] In order to elucidate the physiological role of BTLA, the present inventors created BTLA-deficient mice by gene targeting. The mouse BTLA gene consists of six etasons. The present inventors constructed a targeting vector and replaced exon 3 to exon 6 with a neomycin resistant gene cassette. Two ES clones that had been accurately targeted were microinjected into C57BL / 6 blastocysts to produce chimeric mice. Chimera mouse pups were crossed with C57BLZ6 female mice, and transmission of the mutant-antigenic gene was monitored by Southern plot analysis. Heterozygous mice are mated and BTLA gene Progeny carrying the non-expressing mutation were produced. BTLA-deficient mice were born at the expected ratio of menstrual forces and were not affected by developmental abnormalities. To confirm the deficiency of the BTLA gene, Northern blot analysis was performed on total RNA obtained from wild-type spleen cells and BTLA-deficient spleen cells. BTLA transcripts were not observed in BTLA-deficient mice.

 [0029] The amino acid sequence and structure of BTLA are shown in FIG. Fig. 1 (A) is a comparison of the amino acid sequences of mouse BTLA and human BTLA, and the leader sequence and the transmembrane portion are shown by solid lines. In the figure, (-) is the N-glycosylation site in the extracellular region, and (is the cysteine residue essential for Ig domain formation. Figure 1 (B) shows the structure of the BTLA gene. The square part shows the area where amino acid is coded, and the white square part shows the 5 'and 3' untranslated areas. 1 (C) shows the domain structure of mouse BTLA, the leader sequence is exon 1, the Ig domain is exon 2, and the transmembrane site is exon. The mouse BTLA cDNA encodes an approximately 38 kDa, 306 amino acid protein with an Ig domain at the N-terminus, and human BTLA consists of 289 amino acids, which is equivalent to the mouse BTLA. BTLA is a naïve T cell in mice. It is weakly expressed and increases with activity, and when it differentiates into Th2 cells, the expression disappears and becomes Thl cell specific, and also expressed in spleen naive B cells. In mice, there are reports that NK cells, macrophages, and rod cells are also expressed, and at the tissue level, BTLA is strongly expressed in spleen and lymph nodes. However, when CD3 and BTLA are simultaneously cross-linked with an antibody, BTLA is tyrosine phosphorylated and, along with tyrosine phosphorylation, SHP-2 'SHP-2, an intracytoplasmic phosphatase, is bound. When CD3 and BTLA are simultaneously stimulated with an antibody in which a hybridoma is immobilized, IL_2 production is reduced as compared with anti-CD3 antibody single stimulation.

[0030] FIG. 2 shows the creation of a BTLA knockout (deficient) mouse and confirmation of the gene deficiency. (A) shows the structure of the BTLA gene, the targeting vector, and the predicted disrupted gene in order of strength. The black squares indicate the encoded exoons. For restriction enzymes: Bg represents Bglll, X represents Xbal, B represents BamHI, E represents EcoRI, Sal represents Sail, Xh represents Xhol, TK represents the thymidine kinase gene, and neo represents the neomycin resistance gene. When BTLA exons 3 to 6 were deleted by homologous recombination, cleavage with Bglll resulted in a 14.2 Kb wild-type DNA fragment and 8.4 Kb in a knockout mouse. (B) is a Southern blot analysis of offspring generated by heterozygous intercrossing. Genomic DNA was extracted from the tail of the mouse, cut with Bglll, and electrophoresed using the radiolabeled probe shown in (A) for hybridization. Southern blots showed that a single 14.2 kb band force was obtained for wild-type mice (+ Z +), a 8.4 kb band force for homozygous mice (one Z-), and a heterozygous mouse (+ Z-). Both bands were obtained. (C) shows a Northern blot analysis of total RNA extracted from the spleen cells of the offspring generated by the heterozygote junction. When hybridization was performed using the full-length mouse BTLA cDNA as a probe, BTLA gene mRNA was detected in wild-type mice (+ Z +), but BTLA gene mRNA was detected in heterozygous mice (+ Z). In the homozygous mouse (one Z), BTLA gene mRNA was not detected.

BTLA-deficient mice developed normally. * Matured and young adult mice were apparently strong without any abnormalities. The ability of young adults to examine the number of cells in the blood cell line with a single cytometry. The number of lymphocytes, granulocytes, macrophages, mast cells, NK cells, and surface markers. I got it. When lymphocyte proliferative responses were studied, T cells from BTLA-deficient mice showed excessive proliferation compared to wild-type T cells in response to stimulation with anti-CD3 antibodies. Sarakuko BTLA-deficient mice were mated with OVA-specific TCR transgenic mice (DO-Tg mice), and Thl cells were prepared from spleen naive T cells derived from these mice, and the lymphocyte proliferation response to antigen stimulation was examined. did. As a result, BTL A

Deficient Thl cells clearly showed increased proliferation compared to wild-type Thl cells. BTLA-deficient naive T cells, on the other hand, stimulate Th1 and Th2 cells when antigen-stimulated under Thl differentiation conditions (IL-12 + anti-IL-4 antibody) or Th2 differentiation conditions (4+ anti-12 antibody), respectively. From the analysis, it is considered that the BTLA signal itself is not involved in ThlZTh2 analysis. [0032] In order to examine the role of BTLA in vivo, BTLA-deficient mice were immunized with an antigen (NP-KLH), and the increase in the specific antibody (anti-NP antibody) titer in blood was measured by ELISA. As a result, the anti-NP antibody titer at 4 weeks after immunization was about 3 times higher than that of the wild type in the IgGlZ G2aZlgG2b subclass. In addition, Expenmental autoimmune encephalomyelitis (also known as Thl-dependent immune response) (7), compared to the wild type of the 29-series genetic background, BTLAKO mice The clinical score increased, the onset time was early, and the symptoms were prolonged.

 [0033] FIG. 3 shows the functional analysis of the immune system in BTLA-deficient mice, and shows the onset of autoimmune disease, hypergammaglobulinemia, and antinuclear antibodies. (A) shows that when BTLA-deficient mice were bred for a long time, they began to die around the age of 6 months and died earlier than wild-type mice. The BTLA-deficient mice bred for a long time did not show any apparent abnormality, and there was no evidence of infection or malignant neoplasm. (B) shows hypergammaglobulinemia in BTLA-deficient mice. Serum gamma globulin in 7 month old mice was measured by ELI SA method. BTLA-deficient mice showed a significant increase in IgGl and IgG2a subclasses compared to the wild type, and developed hypergammaglobulinemia. (C) shows the appearance of antinuclear antibodies in BTLA-deficient mice. Blood was collected from mice aged 6-9 months and 12 months, and serum antinuclear antibodies were measured by ELISA. Relative antinuclear antibody titers were measured using 6-month-old normal mice as the standard serum. . BTLA-deficient mice showed a significant increase in antinuclear antibody titer at 6-9 months of age compared to the wild type, and higher antinuclear antibody titer at 12 months of age. BTLA-deficient mice died earlier compared to wild-type mice when bred for a long time in a SPF environment mouse breeding facility. Long-term BTLA-deficient mice show no apparent abnormalities, no evidence of infection or malignant neoplasm, and the cause of death develops an autoimmune disease as described below, resulting in death due to systemic organ damage It became clear that this led to (Figure 3A).

[0034] When 7-month-old BTLA-deficient mice were measured for serum immunoglobulins by ELISA, it became clear that they exhibited significantly higher gamma globulinemia than wild-type mice. In particular, IgGl and IgG2a subclass gamma globulin increased (Fig. 3Β). In addition, the presence or absence of autoantibodies in the serum was examined. Autoantibodies such as nuclear antibodies (Fig. 3C), anti-double-stranded DNA antibodies, and anti-mitochondrial antibodies were significantly increased in the blood. These means that BTLA-deficient mice are in a systemic autoimmune state.

 [0035] When the lymphoid tissue of 12-month-old BTLA-deficient mice was analyzed by the flow cytometry method, the number of T cells, B cells, CD4 positive T cells, CD8 positive T cells, etc. in the spleen was compared with the wild type And the change was strong. However, when the surface force of CD4 positive T cells in spleen of BTLA-deficient mice was analyzed, the activity of CD25 and CD69 increased and CD62L decreased compared to wild type splenic CD4 + T cells. The change which shows chemistry was recognized. This indicates that CD4-positive T cells recognized and activated some antigens (probably autoantigens) in vivo in BTL A-deficient mice.

 [0036] Analyzes of changes in various organs of 12-month-old BTLA-deficient mice showed normal findings in wild-type mice in salivary glands, whereas in BTLA-deficient mice, there was a marked focus around the gland duct of salivary glands. Lymphocyte infiltration was observed. In the lung, wild-type mice showed normal findings, while in BTLA-deficient mice, lung bronchial and blood vessel perilymphatic lymphocyte cell infiltration was observed. These findings are similar to the pathological findings of Sidaren syndrome, a systemic autoimmune disease in humans. Also, mild inflammatory cell infiltration was observed in the joint.

[0037] FIG. 4 shows the appearance of abnormal liver function in BTLA-deficient mice. Serum was collected from 6-, 9- and 12-month-old wild-type and BTLA-deficient mice, and serum GOT, GPT, γ-GTP, ALP, and T-Bil were measured over time. In 12-month-old BTLA-deficient mice, liver function abnormalities (increased serum GOT and GPT) gradually began around the age of 6 months, and at 12-month-old, GOT and GPT were significantly higher than wild type It was. When hepatic pathology was observed, hepatocellular necrosis in the hepatocyte region, piecemeal necrosis in the Darrison sheath region, and endothelialitis in the portal vein were observed. Characteristic findings were observed. FIG. 5 shows the state of organ damage in BTLA-deficient mice. (A) is a drawing of organs taken from 12-month-old BTLA-deficient mice and wild-type mice, pathological sections were prepared, and hematoxylin-eosin staining was performed. The left two rows are wild-type, and the right two rows are BTLA-deficient mice. The left side is a slightly enlarged (100 times) image and the right side is a strongly enlarged (400 times) image. a and b indicate lung tissue. Trachea in BTLA-deficient mice Significant inflammatory cell infiltration mainly consisting of lymphocytes is observed in the parenchyma and lung parenchyma. c and d are salivary glands (submandibular glands). In BTLA-deficient mice, inflammatory cell infiltration, mainly lymphocytes, is observed in salivary gland tissue. e and f indicate the liver. None of the central venous region, hepatocyte region, or Darrison sheath region showed abnormal findings in wild-type mice, but in BTLA-deficient mice, small round cell infiltration that appears to be lymphocytes was seen around the Darisson sheath. Necrosis (piecemeal necrosis) and solitary hepatocyte necrosis E (spotty necrosis;

 Admit. Endothelial cytopathic findings (endothelialitis) in portal veins and bile ducts have also been observed, which are characteristic of autoimmune reactions. g and h represent kidneys. There are no obvious abnormal findings in either wild-type or BTLA-deficient mice. (B) shows autoimmune hepatitis in BTLA-deficient mice. Liver samples were collected from 12-month-old wild-type mice (a, b) and BTLA-deficient mice (c, d), and hematoxylin 'eosin staining (left) and silver chloride staining (right) were performed. . All show strong magnification (400 times) images. The white arrow indicates the portal vein (PV), the black triangle indicates the bile duct (BD), and the white dotted line and the red triangle indicate the boundary between the Darrison sheath region and the liver cell region. In BTLA knockout mice, the findings of erythrocyte infiltration and hepatocellular necrosis (piece meal necrosis) mainly consisting of lymphocytes and neutrophils straddling the force of the Darison sheath region and hepatocyte region were observed. Match.

[0038] As described above, BTLA-deficient mice spontaneously developed systemic autoimmune disease, and in particular, autoimmune hepatitis developed as a unique lesion not found in mice lacking other costimulatory molecules. These results indicate that BTLA suppresses an abnormal immune response to self in vivo and is an important costimulatory molecule especially for the establishment of immune tolerance in the liver.

 Industrial applicability

[0039] Since the non-human animal of the present invention causes autoimmune organ damage in systemic organs such as chronic hepatitis, screening of new test methods and diagnostic methods for human collagen disease, autoimmune hepatitis, etc. Available for Jung. In addition, it is possible to screen for self-antigens that induce inflammation in each organ that has been previously unknown in detail. In addition, screening for substances that suppress or promote autoimmune organ damage is possible, and collagen disease and autoimmune hepatitis Information useful for the development of drugs for human diseases such as the above can be obtained. BTLA knockout mice are young, individuals appear to be normal, and reproductive functions are maintained. However, with aging, various autoimmune diseases develop. This is similar to the onset of collagen disease and autoimmune hepatitis in humans, and can be said to be closer to a more physiological state as a spontaneous disease model of collagen disease 'autoimmune hepatitis and provide an animal model.

Brief Description of Drawings

FIG. 1 shows the amino acid sequence and structure of BTLA.

 FIG. 2 is a diagram showing the generation of a BTLA knockout mouse and confirmation of gene deletion.

 FIG. 3 is a diagram showing the onset of autoimmune disease, hypergammaglobulinemia, and the appearance of antinuclear antibodies in BTLA-deficient mice.

 FIG. 4 is a diagram showing the appearance of abnormal liver function in BTLA-deficient mice.

FIG. 5 is a diagram showing the state of organ damage in BTLA-deficient mice.

Claims

The scope of the claims  [1] A non-human animal that lacks some or all of the BTLA on the chromosome. [2] The non-human animal according to claim 1, wherein the non-human animal develops an autoimmune disease.  [3] The non-human animal according to claim 1 or 2, which exhibits a pathological image of autoimmune hepatitis [4] The non-human animal according to any one of claims 1 to 3, wherein the non-human animal is a mouse.  [5] A self-antigen, autoantibody, or cell surface that is increased or decreased compared to a wild type using the cell or biological tissue derived from the non-human animal according to any one of claims 1 to 4. A screening method for a self-immune disease diagnostic method, characterized by measuring markers, serum proteins, enzymes, inorganic substances, and genes.  [6] A test substance in the immunocompetent cell using the immunocompetent cell or blood immunoglobulin derived from the non-human animal according to any one of claims 1 to 4 and the test substance. A method for screening an autoantigen comprising measuring and evaluating a response and binding between the immunoglobulin and a test substance.  [7] A self-antigen screening characterized in that a response to the test substance in the non-human animal is measured using the non-human animal according to any one of claims 1 to 4 and the test substance. Method.  [8] Using the immunocompetent cell derived from the non-human animal according to any one of claims 1 to 4 and a test substance, a response to the self antigen in the immunocompetent cell is measured. A screening method for an autoimmune response promoter or inhibitor.  [9] Using the non-human animal according to any one of claims 1 to 4 and a test substance, promotion of an autoimmune state, wherein a response to the self-antigen in the non-human animal is measured. A screening method for substances or inhibitors.