JP2006288382A - Autoimmune disease-inducing agent and its utilization - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To establish a technique for treating an autoimmune disease, to provide a judgment method by which the crisis of the autoimmune disease can objectively and accurately be diagnosed, and to provide a method for treating an autoimmune disease. <P>SOLUTION: The autoimmune disease-inducing agent, and a method for making an autoimmune disease crisis model animal with the autoimmune disease-inducing agent are provided. The method for judging the crisis of the autoimmune disease, and an agent for treating the autoimmune disease are provided. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an autoimmune disease inducer that causes autoimmune disease and use thereof, and more particularly, to an autoimmune disease inducer that causes autoimmune disease by repeated administration and use thereof.

  Autoimmune diseases are systemic diseases, but can be broadly classified into two categories: organ-specific diseases and non-specific diseases. Organ-specific autoimmune diseases include chronic thyroiditis, primary mucosal edema, thyroid poisoning, pernicious anemia, Goodpasture syndrome, acute progressive glomerulonephritis, myasthenia gravis, pemphigus vulgaris, bullous celestial Pemphigus, insulin resistant diabetes, juvenile diabetes, Addison's disease, atrophic gastritis, male infertility, premature menopause, phakogenic uveitis, sympathetic vasculitis, multiple sclerosis, ulcerative colitis, primary If you are a patient with biliary cirrhosis, chronic active hepatitis, autoimmune hemolytic anemia, paroxysmal hemoglobinuria, idiopathic thrombocytopenic purpura, or Sjogren's syndrome. Non-organ-specific autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, discoid lupus erythematosus, polymyositis, scleroderma, and mixed connective tissue disease.

  Among these autoimmune diseases, for example, in systemic lupus eryhtematosus (SLE), an autoantibody, particularly an anti-DNA antibody, is excessively produced and binds to DNA as an antigen to form an immune complex. . SLE is a disease characterized by systemic inflammatory lesions caused by activation of the complement system resulting from tissue deposition of such immune complexes.

  The symptoms of autoimmune diseases are very diverse, and their course is completely different depending on the severity of the symptoms. In addition, the prognosis of autoimmune diseases varies depending on the severity of symptoms. For example, the prognosis of SLE has a 5-year survival rate of 95% or more, but the prognosis is not good when symptoms such as lupus nephritis, central nervous lupus and pulmonary hypertension are observed. In addition, SLE has a high recurrence rate and half of SLE patients experience one or more recurrences. Therefore, patients with autoimmune diseases suffer from great physical and mental pain.

  The diagnostic criteria of the American College of Rheumatology are used for the diagnosis of autoimmune diseases (see Non-Patent Document 1). As described above, since SLE has a high recurrence rate, it is important to correctly evaluate disease activity and determine the possibility of recurrence. As an evaluation standard for disease activity, SLEDAI (SLE disease activity index) published by the University of Toronto, Canada is useful (see Non-Patent Document 2).

  As a method for treating autoimmune diseases, corticosteroids that correct immune abnormalities, nonsteroidal anti-inflammatory analgesics that reduce fever and / or arthritis, and immunosuppressants are used. For example, for treatment of SLE, azathioprine, cyclophosphamide and the like are used as immunosuppressants.

  Although the cause of the development of autoimmune diseases is hardly elucidated, for example, the agreement rate of SLE in monozygotic twins is about 25%, so environmental factors such as infection, sex hormones, It is speculated that the disease will develop with the addition of ultraviolet rays, drugs, etc.

By the way, if an experimental animal is continuously immunized with the same antigen, the immune response will be at its extreme and will eventually become exhausted. It is known that T cell anergy breakdown, rheumanoid factor (RF) increase in serum, and Th2 immune response are induced during this period, resulting in various autoimmune pathologies such as SLE.
ACR Committee, Arthritis Rheum 42: 1785 (1999) Bombardier C et al., Arthritis Rheum 35: 630 (1992) Porcelli S et al., Nature 360: 593 (1992) Yoshimoto T et al., Science 270: 1845 (1995) Meiza MA et al., Journal of Immunology 156: 4035 (1996) Sumida T et al., Journal of Experimental Medicine 182: 1163 (1995) Yanagihara Y et al., Clinical Experimental Immunology 118: 131 (1999) Sumida Takayuki et al., Contemporary Medicine 33: 1030 (2000)

  As described above, many positive research results have been reported on various autoimmune diseases. However, little is known about molecular biology regarding the onset mechanism of autoimmune diseases.

  As described above, if an experimental animal continues to be immunized repeatedly with the same antigen, an autoimmune disease state occurs in the experimental animal. What kind of cells are involved in the process is described in detail. Is unclear. A technique for objectively and accurately diagnosing the onset of an autoimmune disease and a technique for reliably treating an autoimmune disease have not yet been established. Therefore, establishment of such a technique is strongly desired.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide an autoimmune disease inducer that develops an autoimmune disease that can be used to establish a technique for treating an autoimmune disease, and the self An object of the present invention is to provide a method for producing an autoimmune disease model animal using an immune disease inducer. It is another object of the present invention to provide a determination method for enabling an objective and accurate diagnosis of the onset of an autoimmune disease, a therapeutic agent for an autoimmune disease, and a screening method thereof.

  As a result of intensive studies in view of the above problems, the present inventors have found that the occurrence of autoimmune pathology caused by repeated administration (ie, prolonged sensitization) of the same antigen to experimental animals is (1) prolonged feeling. In the initial stage of production, it was found that it is essential that natural killer T cells (NKT cells) do not induce anergy, or (2) no inhibitory mechanism is induced, and the present invention has been completed. .

  That is, the autoimmune disease inducer according to the present invention is characterized by containing a natural killer T cell activator.

  In the autoimmune disease inducer according to the present invention, the natural killer T cell activator is preferably ovalbumin (OVA), Staphylococcal enterotoxin B (SEB) or α-galactosylceramide (α-GC).

  A method for producing an autoimmune disease model animal according to the present invention includes a step of subjecting a subject to prolonged sensitization with the above-mentioned autoimmune disease inducer.

  The method for determining an autoimmune disease according to the present invention includes a step of detecting cells that do not fall into anergy in a subject sample.

  The method for screening a therapeutic agent for an autoimmune disease according to the present invention comprises a step of activating natural killer T cells in a subject sample with a natural killer T cell activator, and the natural killer T cells together with a candidate factor. Comparing the activation level of the natural killer T cells before and after incubation. Examples of therapeutic agents for autoimmune diseases screened by this method include antagonistic inhibitors.

  The method for screening a therapeutic agent for an autoimmune disease according to the present invention comprises a step of incubating a natural killer T cell in a subject sample in the presence or absence of a candidate factor, and the natural killer T cell in a natural manner. Comparing the activation level of the natural killer T cell before and after being activated by a killer T cell activator.

  The screening method according to the present invention includes a step of administering a candidate factor to the model animal, a step of collecting natural killer T cells from the model animal, and the natural killer T in order to screen for a therapeutic agent for autoimmune disease. A step of measuring the activation level of the cell.

  The therapeutic agent for autoimmune disease according to the present invention is characterized by being screened by the above method.

  In addition, the autoimmune disease inducer according to the present invention is characterized by containing a CD8-positive T cell differentiation abnormality inducing factor.

  In the autoimmune disease-inducing agent according to the present invention, the CD8-positive T cell differentiation abnormality inducing factor is preferably ovalbumin (OVA).

  A method for producing an autoimmune disease model animal according to the present invention includes a step of subjecting a subject to prolonged sensitization with the above-mentioned autoimmune disease inducer.

  The method for determining an autoimmune disease according to the present invention is characterized by including a step of detecting abnormal differentiation of CD8-positive T cells in a subject sample.

  The kit for determining an autoimmune disease according to the present invention is characterized by comprising an antibody against naive CD8-positive T cells and an antibody against effector CD8-positive T cells and / or an antibody against memory CD8-positive T cells.

  The screening method according to the present invention includes a step of administering a candidate factor to the model animal, a step of collecting CD8 positive T cells from the model animal, and the CD8 positive T in order to screen for a therapeutic agent for an autoimmune disease. And a step of detecting abnormal cell differentiation.

  The therapeutic agent for autoimmune disease according to the present invention is characterized by being screened by the above method.

  As described above, according to the present invention, using an autoimmune disease inducer that induces abnormal activation of NKT cells in the initial stage of prolonged sensitization, or an autoimmune disease inducer that does not induce an inhibitory mechanism, Since the autoimmune disease can be developed by prolonged sensitization of the model animal, there is an effect that an autoimmune disease onset model animal can be produced. Furthermore, by using the model animal, it is possible to provide a therapeutic agent for autoimmune disease and a screening method thereof.

  Further, according to the present invention, since the onset of autoimmune disease can be determined by detecting abnormal activation of NKT cells or abnormal differentiation of CD8 positive T cells, autoimmune diseases can be diagnosed. There is an effect.

As described above, the present inventors have found that the onset of autoimmune disease by prolonged sensitization of experimental animals with the same antigen is associated with abnormal activation of NKT cells or abnormal differentiation of CD8 ⁺ T cells. As a result, the present invention has been completed.

That is, the present invention is an autoimmune disease inducer that induces abnormal activation of NKT cells and causes autoimmune disease in the early stage of prolonged sensitization, or develops autoimmune disease without inducing an inhibitory mechanism. An autoimmune disease inducer to be produced, and a method for producing an autoimmune disease onset model animal using these autoimmune disease inducers are provided. In addition, in order to objectively determine the onset of autoimmune disease, abnormal activation of NKT cells or abnormal differentiation of CD8 positive T cells (CD8 ⁺ T cells) is detected, and these abnormalities are used as indicators to detect autoimmune diseases. A method for determining an autoimmune disease for determining the onset of the disease is provided. Furthermore, a screening method for a substance that corrects the factors causing these abnormalities, and a therapeutic agent for an autoimmune disease containing the substance as a main component are provided.

  Hereinafter, the autoimmune disease inducing agent according to the present invention, a method for producing an autoimmune disease onset model animal, an autoimmune disease onset determination method and determination kit, and an autoimmune disease therapeutic agent and screening method will be described in detail below. To do.

[1: Autoimmune disease inducer that causes autoimmune disease by prolonged sensitization]
As described above, it has been known that when experimental animals are prolonged sensitized with the same antigen, the experimental animals develop autoimmune diseases such as SLE. The detailed mechanism (for example, what kind of cells are involved) was unknown.

  Therefore, the present inventors examined in detail the causes of phenomena induced by prolonged sensitization of experimental animals with the same antigen (ie, T cell anergy breakdown and serum RF elevation).

  As a result, as shown in Examples described later, the present inventors have found that NKT cells are not effective for T cell anergy failure and serum RF elevation, which are indicators of autoimmune diseases that develop by prolonged sensitization with the same antigen. Involvement of abnormal activation, induction of these phenomena, NKT cells do not fall into anergy at the initial stage of prolonged sensitization (repeated stimulation) (for example, second to third administration in 8 repeated antigen administrations) It was clarified that this is essential.

  It is known that the amount of NKT cells is decreased in autoimmune disease model mice (eg, MRL-lpr / lpr, C3H gld / gld, etc.) and human scleroderma, rheumatoid arthritis, Sjogren's syndrome, etc. (See Non-Patent Documents 5 to 8). NKT cells are activated when their TCR is stimulated by an antigen such as Staphylococcal enterotoxin B (SEB) to produce interleukin 4 (IL-4) and / or interferon gamma (IFN-γ). The produced IL-4 induces differentiation of Th2 cells (see Non-Patent Documents 3 and 4). However, no specific knowledge has been obtained about how NKT cells are related to the onset of autoimmune diseases. That is, no attempt has been made to diagnose and / or treat autoimmune diseases by targeting NKT cells.

  That is, the present invention provides an autoimmune disease inducer that induces abnormal activation of NKT cells and develops an autoimmune disease at an early stage of prolonged sensitization. The autoimmune disease inducing agent according to the present invention is characterized by containing an antigen that causes autoimmune disease, and the antigen induces abnormal activation of natural killer T cells at the initial stage of prolonged sensitization. The biological properties and physicochemical properties are not particularly limited.

  As used herein, “activation of an NKT cell” is intended to mean that the NKT cell expresses a function specific to the NKT cell in response to a signal from inside or outside the cell. It is intended that NTK cells promote the production, metabolism, secretion or absorption of substances. Examples of factors that activate NKT cells include OVA, SEB, α-galactosylceramide (α-GC), and NKT cell receptor stimulators. In addition, as used herein, “factor that activates NKT cells” is used interchangeably with “factor that induces activation of natural killer T cells”.

  As used herein, an “autoimmune disease” refers to an anti-DNA antibody and / or autoantibody (eg, RF, etc.) against DNA and / or self components (eg, immunoglobulins) that is detected by self Diseases where immunity is involved in the pathology are contemplated. Autoimmune diseases include organ-specific autoimmune diseases (eg, chronic thyroiditis, primary mucosal edema, thyroid poisoning, pernicious anemia, Goodpasture syndrome, acute progressive glomerulonephritis, myasthenia gravis, vulgaris Pemphigoid, bullous pemphigoid, insulin resistant diabetes, juvenile diabetes, Addison's disease, atrophic gastritis, male infertility, premature menopause, lensogenic uveitis, sympathetic vasculitis, multiple sclerosis, Ulcerative colitis, primary biliary cirrhosis, chronic active hepatitis, autoimmune hemolytic anemia, paroxysmal hemoglobinuria, idiopathic thrombocytopenic purpura, Sjogren's syndrome, etc.), and non-organ-specific autoimmune diseases (For example, rheumatoid arthritis, SLE, discoid lupus erythematosus, polymyositis, scleroderma, mixed connective tissue disease, etc.). The autoimmune disease as an application target of the present invention is particularly preferably SLE.

  As used herein, “antigen” is intended to be a substance that produces antibodies in vivo, and examples of antigens include polynucleotides (DNA, RNA, etc.), polypeptides (peptides, proteins), saccharides. (Oligosaccharides, polysaccharides, sugar chain derivatives, etc.), and other high molecular compounds or low molecular compounds.

  As used herein, “initial stage of prolonged sensitization” refers to the total number of sensitizations (antigen administration) required to develop autoimmune disease with prolonged sensitization (multiple antigen administration). Of these, the stage of sensitization for the first approximately 30 to 30 times is intended. Conversely, the “last stage of prolonged sensitization” is intended to be the last sensitization when developing autoimmune disease by prolonged sensitization. When SEB is used as an antigen (for example, when 25 μg of SEB is administered intraperitoneally to a mouse once every 5 days, the mouse develops an autoimmune disease after 8 administrations). The “initial stage of prolonged sensitization” is the stage where SEB was administered 2 to 3 times, and the “final stage of prolonged sensitization” is the stage where SEB was administered 8 times.

  As used herein, “NKT cell activation abnormality” intends a state in which the amount of a substance produced, metabolized, secreted, or absorbed by NKT cells differs from that of a healthy person. Abnormal activation of NKT cells includes (1) no anergy induction, (2) abnormal production of cytokines (eg, IL-4 and INF-γ), and (3) changes in cell surface antigens. Examples include abnormalities. Examples of abnormal cytokine production include abnormal Th1 / Th2 balance.

  On the other hand, the present inventors have found that SLE, which is one of typical autoimmune diseases, develops in mice sensitized with ovalbumin (OVA). Therefore, the present inventors analyzed in detail the onset mechanism of an autoimmune disease that develops by prolonged sensitization, using SLE-onset mice that were sensitized by OVA.

  As a result, in the SLE-onset mice, lesions characteristic of SLE and an increase in autoantibodies in the serum were observed on the pathological tissues of animals that had been sensitized with the same antigen. Moreover, production of inhibitory cytokines was not induced in the SLE-onset mice.

Furthermore, the present inventors have found that the in SLE onset mice, the total CD8 ⁺ T effector occupied cells CD8 ⁺ T cells and memory CD8 ⁺ T the proportion of cells is increased, as well as CD8 ⁺ T cells producing IFN-gamma The CD8 ⁺ T cell differentiation abnormality (fractional variation) and IFN-γ production contributed to the development of autoimmune diseases.

  That is, the present invention provides an autoimmune disease inducer comprising an antigen that causes an autoimmune disease without inducing an inhibitory mechanism. In the autoimmune disease inducer according to the present invention, the antigen may be any antigen that causes an autoimmune disease without inducing an inhibitory mechanism, and its biological and physicochemical properties are particularly limited. Not. Preferably, the autoimmune disease pathogenic antigen according to the present invention may be OVA.

  As used herein, “suppressive mechanism” includes inhibitory cytokine production and the like. The “suppressive cytokine” is a cytokine produced from Th2 cells, and is intended to be a cytokine that suppresses the proliferation of Th1 cells or a cytokine that suppresses cytokine production from Th1 cells. And interleukin 10 (IL-10).

It is preferable that the onset antigen of the autoimmune disease according to the present invention induces abnormal differentiation of CD8 ⁺ T cells by prolonged sensitization. As used herein, “CD8 positive T cell” is used interchangeably with “CD8 ⁺ T cell” and is intended to be “T cell with CD8”.

As used herein, “cell differentiation” is intended to specialize a cell morphologically and / or functionally. For example, “T cell differentiation” is described as follows:
(1) As for T cells, lymphoid stem cells derived from bone marrow enter the thymus and differentiate into pro T cells. Next, the α-chain and β-chain genes of the T cell receptor are rearranged to differentiate into immature T cells, and further differentiate into mature T cells.
(2) When the antigen is sensitized to T cells that have never been stimulated by the antigen (naive T cells), the naive T cells differentiate into effector T cells. Furthermore, some of the effector T cells differentiate into memory T cells.

  As used herein, “abnormal cell differentiation” refers to a state in which the morphology and / or function of a differentiated cell is different from that of a healthy person, or the number or proportion of specialized cells is different from that of a healthy person. Different states are intended.

Examples of the induction of “abnormal differentiation of CD8 ⁺ T cell” include induction of abnormal differentiation from naive T cells to effector T cells and memory T cells, and / or induction of an increase in IFN-γ producing cells, Abnormal differentiation in CD8 ⁺ T cells can be detected by using as an index an abnormality induced in the abundance ratio of these cells. For example, OVA can cause CD8 ⁺ T cell differentiation abnormality (fractional variation) by prolonged sensitization (repeated administration).

[2: Method for producing animal model of autoimmune disease development]
The method for producing an autoimmune disease onset model animal according to the present invention only needs to include a step of prolonged sensitization of the model animal with the above-mentioned autoimmune disease inducer, and other specific steps, materials, conditions, There are no particular limitations on the equipment used, equipment used, and the like.

  That is, in one aspect, the present invention provides a method for producing an autoimmune disease model animal comprising the step of subjecting a subject to prolonged sensitization with the autoimmune disease inducer according to the present invention. In the method for producing an autoimmune disease model animal according to the present invention, the autoimmune disease inducer preferably contains a natural killer T cell activator, and the natural killer T cell activator is Staphylococcal enterotoxin B (SEB). Or it is more preferable that it is (alpha) -galactosylceramide ((alpha) -GC).

  That is, in another aspect, the present invention provides a method for producing an autoimmune disease model animal comprising the step of subjecting a subject to prolonged sensitization with the autoimmune disease inducer according to the present invention. In the method for producing an autoimmune disease model animal according to the present invention, the autoimmune disease inducing agent preferably contains a CD8-positive T cell differentiation-inducing factor, and the CD8-positive T cell differentiation-inducing factor is ovalbumin ( OVA) is more preferable.

  As used herein, a “model animal” is intended to be an experimental animal used to develop preventive or therapeutic methods for human diseases, and the model animal includes a non-human mammal (eg, Mouse, rat, rabbit, monkey, goat, sheep, cow, dog, etc.), and other vertebrates.

  In the method for producing an autoimmune disease onset model animal according to the present invention, a method for administering the autoimmune disease inducing agent to the model animal is not particularly limited. Preferred administration methods include, for example, transdermal, intranasal, transbronchial, intramuscular, intraperitoneal, intravenous, intraarticular, subcutaneous, intrathecal, intraventricular, or oral administration. However, it is not limited to these.

  As described above, according to the method for producing an autoimmune disease onset model animal according to the present invention, a mouse that has developed an autoimmune disease (for example, SLE) can be produced. That is, the present invention can be used for SLE research (for example, screening for therapeutic agents for autoimmune diseases) using autoimmune disease onset model mice.

[3: Determination method of autoimmune disease]
The method for determining the onset of an autoimmune disease according to the present invention only needs to include at least the step of detecting abnormal activation of NKT cells or abnormal differentiation of CD8 ⁺ T cells in a sample (sample) isolated from a living body. Other specific processes, materials, conditions, equipment used, equipment used, etc. are not particularly limited.

  That is, in one aspect, the present invention provides a method for determining an autoimmune disease including a step of detecting abnormal activation of natural killer T cells in a subject sample.

  As used herein, “subject sample” is used interchangeably with “sample separated from a living body” and can be easily obtained from a human body by a conventional method. Examples of the subject sample include hair, peripheral blood, organs, peripheral lymphocytes, and synovial cells. In addition, those obtained by culturing and proliferating the obtained cells may be used. The subject sample includes a sample (sample) separated from a living body by a doctor or a person who has specialized knowledge under the supervision of a doctor.

  The above-described method for detecting abnormal activation of NKT cells is based on components that are specifically present in activated NKT cells or non-activated cells (for example, high molecular compounds such as DNA, RNA, protein, and the like). Molecular compounds, etc.), their characteristic morphology (eg, size, color, shape, etc.) and physiological activity (eg, substance production ability, substance secretion ability, substance absorption ability, substance resolution, etc.), and these There is no particular limitation as long as it is a method for detecting abnormal activation of the NKT cells using a conventionally known identification marker used for identifying cells as an index.

  In one embodiment, the method for determining an autoimmune disease according to the present invention preferably includes a step of detecting cells that do not fall into anergy in a subject sample.

  In the method for determining the onset of an autoimmune disease according to the present invention, the method for collecting the NKT cells is not particularly limited, and those skilled in the art can recognize peripheral blood, spleen, lymph nodes, liver, skin, intestinal mucosa, female organs. NKT cells can be easily collected from each living organ or living tissue according to a known method.

  That is, in another aspect, the present invention provides a method for determining an autoimmune disease comprising a step of detecting abnormal differentiation of CD8-positive T cells in a subject sample.

In the present invention, "a step of detecting an abnormal differentiation of CD8 + ^T cells" may be any step of detecting "abnormal differentiation of CD8 + ^T cells" described above is not particularly limited. Examples of the step of detecting an abnormal differentiation of CD8 ⁺ T cells include a step of detecting an abnormal ratio of naive T cells, effector T cells and memory T cells in CD8 ⁺ T cells, or the above-mentioned interferon in T cells. A step of detecting an abnormality in the abundance ratio of the γ-producing cells is included.

The above-described method for detecting abnormal differentiation of CD8 positive T cells is a component that specifically exists in each differentiated CD8 ⁺ T cell (for example, a high molecular compound such as DNA, RNA, protein, and a low molecular compound). Distinguishing these cells from their characteristic morphology (eg, size, color, shape, etc.) and physiological activity (eg, substance-producing ability, substance-secreting ability, substance-absorbing ability, substance-resolving ability, etc.) The method is not particularly limited as long as it is a method for detecting abnormal differentiation of the CD8 ⁺ T cells using a conventionally known identification marker used for this purpose as an index.

Examples of the method for detecting abnormal differentiation of CD8 positive T cells include a method for measuring the abundance ratio of naive CD8 ⁺ T cells, effector CD8 ⁺ T cells, and memory CD8 ⁺ T cells, as shown in the Examples described later. Is mentioned. In this case, using CD44 and CD62L as identification markers and using antibodies against them, the CD8 ⁺ T cells in the subject sample are the above three types (ie, naive CD8 ⁺ T cells, effector CD8 ⁺ T cells, and memory CD8 ⁺ T cells). by classifying the cells), to calculate the respective proportions, then the calculated proportions of the three types of CD8 + ^T cells, compared with the proportions of the three types of CD8 + ^T cells in healthy individuals , Abnormal differentiation of CD8 ⁺ T cells can be detected.

Other methods for detecting abnormal differentiation of CD8 positive T cells include a method for detecting the presence of IFN-γ producing cells in CD8 ⁺ T cells. In this case, by comparing the ratio of CD8 ⁺ T cells producing IFN-γ to the total CD8 ⁺ T cells using IFN-γ as an identification marker with the above-mentioned ratio in healthy subjects, abnormal differentiation of CD8 ⁺ T cells is observed. Can be detected.

  The identification markers may be used alone or in combination. The method for detecting the identification marker can be appropriately selected according to each identification marker, and is not particularly limited. As a method for detecting the identification marker, for example, PCR method, gene introduction method, Southern blotting method, Northern blotting method, Western blotting method, radioimmunoassay, enzyme immunoassay, morphological detection method (for example, tissue immunostaining, Cell immunostaining, flow cytometry (eg, fluorescence-activated cell sorter (FACS), etc.), chromatography (eg, liquid chromatography, gas chromatography, etc.), mass spectrometry, etc. These can be used alone or in combination.

Further, in the method for determining the onset of autoimmune disease according to the present invention, the method for collecting the CD8 ⁺ T cells is not particularly limited, and those skilled in the art can recognize peripheral blood, spleen, lymph nodes, liver, skin, intestinal mucosa, According to a known method, CD8 ⁺ T cells can be easily collected from each living organ such as a female organ or living tissue.

[4: Autoimmune disease determination kit]
If the determination kit for the onset of autoimmune disease according to the present invention is equipped with a reagent for carrying out the method for determining the onset of autoimmune disease according to the present invention (eg, antibody, primer, probe, etc.) The configuration is not particularly limited, and other reagents can be appropriately combined.

  That is, in one aspect, the present invention provides an autoimmune disease determination kit comprising a reagent for detecting abnormal activation of natural killer T cells in a subject sample.

  In addition, the autoimmune disease determination kit according to the present invention is characterized by components that are specifically present in NKT cells (for example, high molecular compounds such as DNA, RNA, protein, and low molecular compounds), and these cells. Traditional forms (eg, size, color, shape, etc.) and physiological activities (eg, substance-producing ability, substance-secreting ability, substance-absorbing ability, substance-resolving ability, etc.), and conventionally known to be used to distinguish these cells A reagent for detecting the identification marker may be provided.

  In addition, as a kit for detecting a gene that is specifically expressed in an NKT cell, a kit designed to amplify a gene that is specifically expressed in the cell is provided so that the gene can be detected. And a kit further provided with reagents necessary for detecting the above-mentioned genes, such as probes designed in the above, reagents used in nucleotide sequencing such as the Maxam Gilbert method and the Sanger method.

  In addition, although the said reagent can be suitably selected according to the detection method employ | adopted, As a preferable reagent, dATP, dUTP, dTTP, dGTP, DNA synthetase, RNA synthetase etc. are mentioned, for example. Furthermore, the kit according to the present invention may contain an appropriate buffer solution and washing solution that do not interfere with the detection of the gene.

  In another aspect, the present invention provides an autoimmune disease determination kit comprising a reagent for detecting abnormal differentiation of CD8-positive T cells in a subject sample.

As described above, when determining an autoimmune disease based on the abundance ratios of naive T cells, effector T cells, and memory T cells in CD8 ⁺ T cells, the autoimmune disease determination kit according to the present invention is naive. Reagents for detecting T cells, effector T cells, and memory T cells are provided. Reagents for detecting naive T cells, effector T cells, and memory T cells include antibodies to naive T cells, effector T cells, and memory T cells (eg, anti-CD44 antibody and anti-CD62L antibody).

When determining an autoimmune disease based on the ratio of IFN-γ producing cells in CD8 ⁺ T cells, the autoimmune disease determination kit according to the present invention is a reagent for detecting interferon γ producing cells. It has. Examples of the reagent for detecting interferon γ-producing cells include antibodies against interferon γ-producing cells.

In addition, the autoimmune disease determination kit according to the present invention includes components that are specifically present in specific CD8 ⁺ T cells (for example, high molecular compounds such as DNA, RNA, protein, and low molecular compounds), these To identify the cell's characteristic morphology (eg, size, color, shape, etc.) and physiological activity (eg, substance-producing ability, substance-secreting ability, substance-absorbing ability, substance-resolution, etc.) and these cells You may provide the reagent which detects the conventionally well-known identification marker used.

A kit for detecting a gene that is specifically expressed in a specific CD8 ⁺ T cell comprises a primer designed to amplify a gene that is specifically expressed in the cell. Examples include kits further equipped with reagents necessary for detecting the above genes, such as probes designed for detection, reagents used in base sequencing methods such as restriction enzymes, Maxam Gilbert method and Sanger method. .

  In addition, although the said reagent can be suitably selected according to the detection method employ | adopted, As a preferable reagent, dATP, dUTP, dTTP, dGTP, DNA synthetase, RNA synthetase etc. are mentioned, for example. Furthermore, the kit according to the present invention may contain an appropriate buffer solution and washing solution that do not interfere with the detection of the gene.

  As used herein, “detecting a cell” is intended to detect the cell itself but also includes classifying the cell based on the results after detecting the cell. .

  As used herein, “determination of onset” is intended to determine the likelihood of onset (risk of onset), and the method of determining onset may be used as a disease diagnosis method or disease prevention method. Is available.

[5: Autoimmune disease therapeutic agent and screening method thereof]
In one aspect, the present invention provides a therapeutic agent for an autoimmune disease comprising as a main component a substance that corrects a factor causing abnormal activation of NKT cells. The therapeutic agent for an autoimmune disease according to the present invention may be a therapeutic agent whose main component is a substance that corrects a factor causing abnormal activation of NKT cells, and other components, manufacturing methods, manufacturing apparatuses, etc. Other configurations are not particularly limited. The therapeutic agent for autoimmune diseases according to the present invention can be used for the treatment of patients with autoimmune diseases in which abnormal activation of NKT cells is observed. As used herein, “correcting a factor” is intended to inhibit or enhance the function of the factor.

  As used herein, the “factor” refers to a polymer such as a polynucleotide (DNA, RNA, etc.), a peptide (polypeptide, protein), a saccharide (oligosaccharide, polysaccharide, sugar chain derivative, etc.). Compounds, and small molecule compounds are contemplated. In addition, the above factor may be directly involved in the activation of NKT cells or indirectly through other factors. Moreover, what promotes the activation of a NKT cell is intended for the said factor. Examples of factors that activate NKT cells include SEB, α-galactosylceramide (α-GC), OVA, NKT cell receptor stimulators, and the like.

In another aspect, the present invention provides a therapeutic agent for an autoimmune disease comprising as a main component a substance that corrects a factor causing abnormal differentiation of CD8 ⁺ T cells. The therapeutic agent for autoimmune diseases according to the present invention may be a therapeutic agent whose main component is a substance that corrects a factor causing abnormal differentiation of CD8 ⁺ T cells. Other components, manufacturing method, and manufacturing apparatus Other configurations are not particularly limited. The therapeutic agent for autoimmune diseases according to the present invention can be used for the treatment of patients with autoimmune diseases in which abnormal differentiation of CD8 ⁺ T cells is observed.

As described above, the “factor” includes a high molecular compound such as a polynucleotide (DNA, RNA, etc.), a peptide (polypeptide, protein), a saccharide (oligosaccharide, polysaccharide, sugar chain derivative, etc.), and a low molecule. Compounds are also contemplated, and the factors may be directly involved in CD8 ⁺ T cell differentiation or indirectly through other factors. Further, the agent may be one which abnormal differentiation involved in CD8 + ^T cells, may also be to promote differentiation of CD8 + ^T cells, but may be suppressed. For example, OVA can cause abnormalities in the differentiation of CD8 ⁺ T cells by repeated administration.

  In either aspect, the administration conditions of the therapeutic agent for clinical application can be determined using the model animal system for autoimmune disease described herein. That is, by using the model animal, administration conditions such as dosage, administration interval, administration route and the like can be examined, and conditions for obtaining an appropriate preventive effect or therapeutic effect can be determined. Such a therapeutic agent becomes a medicament for prevention or treatment of autoimmune diseases.

  The therapeutic agent can also be a composition further comprising any pharmaceutically acceptable carrier. Examples of carriers include sterilized water, physiological saline, buffering agents, vegetable oils, emulsifiers, suspension agents, salts, stabilizers, preservatives, surfactants, sustained-release agents, other proteins (such as BSA), and transfection reagents. (Including lipofection reagents and liposomes). Furthermore, examples of carriers that can be used in the present invention include glucose, lactose, gum arabic, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea and the like.

  The dosage form when the therapeutic agent according to the present invention is formulated is not particularly limited, and may be, for example, a solution (injection), powder, microcapsule, tablet, or the like. For example, by combining the therapeutic agent of the present invention with a sustained-release agent or storing it in a sustained-release container (for example, a capsule), it is possible to perform drug delivery targeting a disease site exhibiting an autoimmune disease Thus, effective treatment can be performed.

  The route of administration of the therapeutic agent according to the present invention to the patient is appropriately selected according to the nature of the active ingredient, for example, percutaneous, intranasal, transbronchial, intramuscular, intraperitoneal, intravenous, joint. It can be performed internally, subcutaneously, intrathecally, intraventricularly, or orally, but is not limited thereto. The therapeutic agent according to the present invention can be administered systemically or locally, but when side effects due to systemic administration become a problem, local administration to the lesion site is preferred. The dose and administration method vary depending on the tissue transferability of the active ingredient of the therapeutic agent, the purpose of treatment, the patient's weight, age, symptoms, etc., but those skilled in the art can appropriately select them.

  Further, the therapeutic agent of the present invention contains the target substance in an amount of 0.1 to 90% by weight of the total composition. The dosage of the target substance contained in the therapeutic agent of the present invention is 0.0001 mg to 1000 mg, preferably 0.001 mg to 300 mg, more preferably 0.01 mg to 100 mg per kg body weight per day for parenteral administration. is there. However, depending on the disease state, body weight, individual patient response to treatment, the type of composition to which the therapeutic agent is administered, the mode of administration, the stage of the disease course, or the interval between doses, these dosing frequencies may be adjusted accordingly obtain. The administration can be performed once to several times, and can be administered 1 to 5 times per day.

  Examples of individuals to be treated include human and non-human mammals (eg, mice, rats, rabbits, monkeys, etc.), and other vertebrates. Application to non-human mammals is also useful for developing preventive or therapeutic methods for human autoimmune diseases. For example, by using a model animal produced using a non-human mammal, a new treatment protocol for preventing the onset of an autoimmune disease can be developed.

  In another aspect, the present invention provides a screening method for screening for therapeutic agents for autoimmune diseases.

  In one embodiment, the screening method according to the present invention comprises a step of providing a candidate substance to a model animal of autoimmune disease, a step of collecting natural killer T cells from the model animal, and an activation level of the natural killer T cells. The step of measuring is included. In another embodiment, the screening method according to the present invention comprises a step of providing a candidate substance to a model animal of autoimmune disease, a step of collecting CD8 positive T cells from the model animal, and abnormal differentiation of the CD8 positive T cells. And a step of detecting.

  In a further embodiment, the screening method according to the present invention comprises a step of activating natural killer T cells in a subject sample with a natural killer T cell activator, and before and after incubating the natural killer T cells with a candidate factor. Comparing the activation level of said natural killer T cells at In a still further embodiment, the screening method according to the present invention comprises incubating natural killer T cells in a subject sample in the presence or absence of a candidate factor, and the natural killer T cells are natural killer T. A step of comparing the activation level of the natural killer T cell before and after being activated by a cell activator.

  By using the screening method according to the present invention described above, the therapeutic agent for the autoimmune disease described above can be obtained.

  The present invention is not limited to the configurations described above, and various modifications can be made within the scope of the claims, and the technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention.

  Although this invention is demonstrated more concretely based on an Example and FIGS. 1-11, this invention is not limited to this. Those skilled in the art can make various changes, modifications, and alterations without departing from the scope of the present invention.

[Example 1: T cell anergy failure due to prolonged sensitization]
The BALB / c mouse was subjected to any of the following treatments (1) to (6), and an anergy analysis was performed using the mice after the treatment. In anergy analysis, anergy failed in cell proliferation by measuring the production amount of interleukin 2 (IL-2) in SEB-responsive T cells in the spleen cells of each treatment-treated mouse and the number of cell divisions. It was used as an indicator of whether or not it was maintained. The production amount of IL-2 was quantified by an ELISA method using a mouse IL-2 ELISA kit (BIOSOURCE). The number of cell divisions was measured by detecting CFSE (Molecular Probes) incorporated into the cells with a flow cytometer:
Treatment (1): SEB administered twice (25 μg / time)
Treatment (2): SEB administered 8 times (25 μg / time)
Treatment (3): Staphylococcal enterotoxin A (SEA) administered twice (25 μg / time)
-Treatment (4): SEA administered 8 times (25 μg / time)
Treatment (5): SEA and α-galactosylceramide (α-GC) administered simultaneously 8 times (SEA: 25 μg / time, α-GC: 5 μg / time)
Treatment (6): vehicle alone was administered 8 times, and it was administered by intraperitoneal administration once every 5 days or once every 15 days.

  The result of measuring the production amount of IL-2 from T cells is shown in FIG. The vertical axis in FIG. 1 indicates the ratio of the amount of IL-2 production in the spleen cells of each treated mouse when the amount of IL-2 production in the spleen cells of the mouse treated (6) is 100. Indicates.

  In the mouse subjected to the treatment (2), the production amount of IL-2 that had been reduced due to the generation of anergy was recovered to about 60%, and it was confirmed that the anergy failed. In contrast, in the mice that had been treated (4), the production amount of IL-2 was low and it was found that anergy persisted. However, in the mice treated (5), the production amount of IL-2 was recovered to about 40%, and the anergy failed as in the mice treated (2).

  Further, when cell proliferation (number of cell divisions) was examined, as in the result of the production amount of IL-2, in the mice subjected to treatment (4), cell proliferation was not observed, but treatment (2) Alternatively, in the mice subjected to (5), cells that once fell into anergy grew again (data not shown).

  The administration interval was every 5 days and every 15 days, and in both cases, the failure of anergy was observed in the mice treated (2) or (5) by repeated immunization up to the 8th time. As a result, it was found that repeated administration (ie, prolonged sensitization) was not necessary for the failure of anergy, but an administration interval.

[Example 2: Increase in serum RF level due to prolonged sensitization]
In mice subjected to the treatment (2), (4), (5), (6), and the treatment (7) shown below in Example 1, blood was collected on the second day from the last administration of each treatment, Serum was collected and the production of RF, which is an autoantibody against the Fc portion of activated IgG, was measured by ELISA using Levis rheumatoid factor IgG mouse ELISA kit (Shibayagi):
Treatment (7): α-GC administered 8 times (5 μg / time)
The administration method was intraperitoneal administration once every 5 days or once every 15 days.

  The results of measuring the amount of RF production in mouse serum are shown in FIG. Compared with the mouse | mouth which received the process (6), the production amount of RF was increasing significantly in the mouse | mouth which received the process (2). On the other hand, in the mice treated (4), the amount of RF produced was not significantly different from the mice treated (6). However, the amount of RF production was significantly increased in the mice that received simultaneous administration of SEA and α-GC (treatment (5)), compared with the mice that received treatment (6).

  In addition, although the administration interval was performed every 5 days and every 15 days, production of RF was observed in the mice subjected to the treatment (2) or (5) by repeated administration up to the 8th time in either case. Thus, it was found that it is not a dose interval but a repeated dose that is necessary for the production of RF.

[Example 3: Anergy induction and failure of NKT cells by prolonged sensitization]
In mice subjected to the treatments (1) and (2) in Example 1 and the treatment (8) shown below, anergic analysis in NKT cells was performed. In the anergy analysis, whether anergy has failed in cell proliferation by measuring the production amount of interleukin 2 (IL-2) and the number of cell divisions in NKT cells collected from spleen cells of mice treated with each treatment, or It was used as an indicator of whether it was maintained. The production amount of IL-2 was quantified by an ELISA method using a mouse IL-2 ELISA kit (BIOSOURCE). The number of cell divisions was measured by detecting CFSE (Molecular Probes) incorporated into the cells with a flow cytometer:
Treatment (8): PBS was administered 8 times The administration method was intraperitoneal administration once every 5 days. The results of measuring the amount of IL-2 produced from NKT cells are shown in FIG.

  As shown in Example 1, normal T cells fall into anergy by two SEB stimulations (treatment (1)). IL-2 production from anergic T cells is 1% or less of control (treatment (8)), but IL-2 production is up to 40-60% after 8 doses (treatment (2)) Recover. However, in NKT cells, IL-2 production from NKT cells after two SEB administrations (treatment (1)) was about 40% of control (treatment (8)), and eight administrations (treatment (2)). ) Mice recovered to almost 100%.

  Further, when cell proliferation (number of cell divisions) was examined, NKT cells with cell proliferation were confirmed in the mice treated (1), as in the case of IL-2 production amount (data is Not shown). This indicates that NKT cells are less susceptible to anergy than normal T cells.

[Example 4: T cell anergy failure by prolonged sensitization with α-GC]
The BALB / c mouse was subjected to any of the following treatments (I) to (IV). Two days after the final administration, blood was collected from the mice and serum was collected. In addition, spleen cells were isolated from mice sacrificed 9 days after the final administration, and the isolated spleen cells were cultured with stimulation with SEB for 24 hours, and the amount of interleukin 2 (IL-2) produced in the culture supernatant was determined. Quantified by ELISA using mouse IL-2 ELISA kit (BIOSOURCE):
Treatment (I): PBS twice + vehicle 8 times treatment (II) SEB 2 times + vehicle 8 times (SEB; 25 μg / time)
Treatment (III): SEB twice + α-GC twice (SEB; 25 μg / time, α-GC; 5 μg / time)
Treatment (IV): SEB 2 times + α-GC 8 times (SEB; 25 μg / time, α-GC; 5 μg / time)
The administration was carried out by intraperitoneal administration once every 5 days.

  The results of measuring the amount of IL-2 produced from SEB-responsive T cells are shown in FIG. Group I is a control group in which SEB-responsive T cells are not anergic, and Group II is a control group in which SEB-responsive T cells are anergic. In group III, which received α-GC twice, IL-2 production was low, but in group IV, which received α-GC eight times, IL-2 production recovered to about 80% of group I did.

  CFSE (Molecular Probes) was taken up into the isolated spleen cells, the cells were cultured for 72 hours while being stimulated with SEB, and the number of cell divisions was measured by detecting the fluorescence intensity of CFSE in CD4T cells with a flow cytometer. .

  The results are shown in FIG. In the group III where the IL-2 production amount was low, cell proliferation was not observed, but in the group IV, the same number of cell divisions as in the group I were observed, confirming that the cells were proliferating.

  From the above, it was shown that the T cell anergy produced by SEB administration is broken by repeatedly stimulating NKT cells with α-GC.

[Example 5: Increase in serum RF level due to prolonged sensitization with α-GC]
The amount of RF in the serum collected in Example 4 was measured by an ELISA method using Levis rheumatoid factor IgG mouse ELISA kit (Shibagogi).

  The results are shown in FIG. Individuals producing RF were observed in the group IV where anergy breakdown was observed in SEB-responsive T cells. This indicates that RF is produced by the breakdown of T cell anergy.

[Example 6: SLE onset due to prolonged sensitization]
The BALB / c mouse was subjected to any of the following treatments (8) to (11). Urine was collected from the mice 2 days after the final administration, and the kidneys were removed from the mice sacrificed 9 days after the final administration, and examined for the occurrence of SLE-like nephritis. Proteinuria was detected by a color reaction with tetrabromphenol blue using Albus Sticks (Bayer):
Treatment (8): PBS administered 8 times Treatment (9): SEB administered 8 times (25 μg / time)
Treatment (10): PBS administered 12 times Treatment (11): OVA administered 12 times (500 μg / time)
The administration was carried out by intraperitoneal administration once every 5 days.

  The results are shown in FIG. Proteinuria was not detected in the mice treated with any of the treatments (8) to (10), but proteinuria was detected in the mice treated (11) (see FIG. 8 (a)). ).

  FIG. 8 (b) shows IgG deposition in the kidneys of mice subjected to the above treatment. Kidney tissue sections were stained with fluorescently labeled anti-mouse IgG antibody, and IgG deposits were observed with a fluorescence microscope. As a result, IgG deposition was not observed in the mice treated with any of the treatments (8) to (10), but in the mice treated with the treatment (11), lesions characteristic of systemic lupus erythematosus (SLE) were observed. An IgG deposit was observed.

  From the above, it was found that SLE-like nephritis has developed due to prolonged sensitization with OVA.

[Example 7: Production of autoantibodies by prolonged sensitization]
Autoantibodies in the sera of mice subjected to the treatment shown in Example 6 were measured. The measured autoantibodies are rheumatoid factor (RF), anti-Sm antibody (autoantibody against snRNP which is a complex of small nuclear RNA and protein), anti-ssDNA antibody (autoantibody against single-stranded DNA), and anti-dsDNA. Antibody (autoantibody against double-stranded DNA).

  FIGS. 9A to 9D show the results of measuring the production amounts of RF, anti-Sm antibody, anti-ssDNA antibody, and anti-dsDNA antibody by ELISA, respectively. For the measurement of the amount of RF production, Levis rheumatoid factor IgG type mouse ELISA kit (Shibagogi) was used. In addition, the serum of disease model mouse MRL / lpr was used as a positive control for the measurement of the production amount of anti-Sm antibody, anti-ssDNA antibody and anti-dsDNA antibody.

  As shown in FIGS. 9 (a) and (b), in 60% of the mice subjected to the treatment (9), an increase in the production amount of RF and anti-Sm antibody was observed. On the other hand, in the mice subjected to the treatment (11), the production amounts of RF, anti-Sm antibody, anti-ssDNA antibody and anti-dsDNA antibody increased in all individuals.

[Example 8: Measurement of CD8 ⁺ T cell differentiation abnormality]
The spleen CD8 ⁺ T cells of the mice subjected to the treatment shown in Example 6 were detected by detecting the expression of CD44 and CD62L on the cell surface with a flow cytometer, and subjected to naive CD8 ⁺ T cells and effector CD8 ⁺ T cells. And memory CD8 ⁺ T cells.

The results are shown in FIG. In the figure, (a) shows CD8 ⁺ T cells as naive CD8 ⁺ T cells (“native CD8 ⁺ T” in the figure), effector CD8 ⁺ T cells (“effector CD8 ⁺ T” in the figure) and memory CD8 ^+. The result of classification into T cells (in the figure, “memory CD8 ⁺ T”) is shown.
(B) represents the result of (a) as a bar graph.

As shown in FIG. 10, in the mice subjected to the treatments (8) to (10), there was no difference in the CD8 ⁺ T cell fractionation, but in the mice subjected to the treatment (11), the effector CD8 ⁺ The proportion of T cells and memory CD8 ⁺ T cells was significantly increased.

Moreover, the CD8 + ^T cells of mice of the treated intracellular staining was detected CD8 + ^T cells producing IFN-gamma by flow cytometer.

The results are shown in FIG. In addition, (a) in a figure shows the result of having measured the ratio of the CD8 ^<+> T cell which produces IFN-gamma after repeated administration of an antigen. Also,
(B) represents the result of (a) as a bar graph.

As shown in FIG. 11, the proportion of CD8 ⁺ T cells producing IFN-γ was significantly higher in the mice treated (11).

From the above, it was found that abnormal differentiation of CD8 ⁺ T cells contributed to autoimmune disease in mice sensitized with OVA.

  Note that the present invention is not limited to the configurations described above, and various modifications are possible within the scope of the claims, and technical means disclosed in different embodiments and examples respectively. Embodiments and examples obtained by appropriately combining them are also included in the technical scope of the present invention.

As described above, in the present invention, an autoimmune disease inducer that does not induce an inhibitory mechanism or an autoimmune disease inducer that induces abnormal activation of NKT cells in the early stage of prolonged sensitization, the model animal is prolonged. By making an autoimmune disease, an autoimmune disease onset model animal can be produced. Therefore, the present invention can be suitably used for pharmaceuticals for diagnosis of autoimmune diseases, screening tests for pharmaceuticals for prevention and / or treatment of autoimmune diseases, and the like. Moreover, since the onset of autoimmune diseases can be determined by detecting abnormal differentiation of CD8 ⁺ T cells or abnormal activation of NKT cells, it is represented by a method and a determination kit for determining the onset of autoimmune diseases. It can be used in the field of diagnostic medicine.

In the present Example, it is a graph which shows the result of having measured the production amount of IL-2 from a SEB responsive T cell or a SEA responsive T cell in the BALB / c mouse which repeatedly administered the antigen. In a present Example, it is a graph which shows the result of having measured the RF production amount in the BALB / c mouse which repeatedly administered the antigen. In a present Example, it is a graph which shows the result of having measured the production amount of IL-2 from the NKT cell in the BALB / c mouse which repeatedly administered the antigen. In a present Example, it is a graph which shows the result of having measured the production amount of IL-2 from the SEB response T cell in the BALB / c mouse which repeatedly administered the antigen. In the present Example, it is a graph which shows the result of having measured the cell division frequency of the CD4T cell extract | collected from the BALB / c mouse which administered the antigen repeatedly. In the present Example, it is a graph which shows the result of having measured the amount of RF in the serum collect | recovered from the BALB / c mouse which administered the antigen repeatedly. FIGS. 7 (a) and (b) are diagrams showing the results of examining the onset of SLE-like nephritis in BALB / c mice to which the antigen was repeatedly administered in this example. FIGS. 8A to 8D are graphs showing the results of examining the production of autoantibodies in BALB / c mice repeatedly administered with an antigen in this example. FIGS. 9A and 9B are graphs showing the results of examining changes in the kinetics of each fraction of CD8 ⁺ T cells in BALB / c mice to which antigens were repeatedly administered in this Example. FIGS. 10 (a) and (b) are graphs showing the results of examining changes in IFN-γ producing cells in CD8 ⁺ T cells in BALB / c mice repeatedly administered with an antigen in this Example.

Claims (16)

  An autoimmune disease inducer comprising a natural killer T cell activator.   The autoimmune disease inducer according to claim 1, wherein the natural killer T cell activator is ovalbumin (OVA), Staphylococcal enterotoxin B (SEB), or α-galactosylceramide (α-GC).   A method for producing an autoimmune disease model animal, comprising the step of subjecting a subject to prolonged sensitization with the autoimmune disease inducer according to claim 1.   A method for determining an autoimmune disease, comprising a step of detecting cells that do not fall into anergy in a subject sample. A method for screening for a therapeutic agent for an autoimmune disease, comprising:
Activating a natural killer T cell in a subject sample with a natural killer T cell activator, and comparing the level of activation of the natural killer T cell before and after incubating the natural killer T cell with a candidate factor A method comprising the steps. A method for screening for a therapeutic agent for an autoimmune disease, comprising:
Incubating a natural killer T cell in a subject sample in the presence or absence of a candidate factor, and the natural killer T before and after the natural killer T cell is activated by a natural killer T cell activator Comparing the level of activation of the cells. A method for screening for a therapeutic agent for an autoimmune disease, comprising:
Administering a candidate factor to a model animal produced by the method according to claim 3,
A method comprising collecting natural killer T cells from the model animal, and measuring an activation level of the natural killer T cells.   The therapeutic agent of an autoimmune disease screened by the method of any one of Claims 5-7.   An autoimmune disease inducing agent comprising a CD8-positive T cell differentiation-inducing factor.   The autoimmune disease inducer according to claim 9, wherein the CD8-positive T cell differentiation-inducing factor is ovalbumin (OVA).   A method for producing an autoimmune disease model animal, comprising the step of subjecting a subject to prolonged sensitization with the autoimmune disease inducer according to claim 9 or 10.   A method for determining an autoimmune disease, comprising a step of detecting abnormal differentiation of CD8-positive T cells in a subject sample.   An autoimmune disease determination kit comprising an anti-CD44 antibody and an anti-CD62L antibody.   A kit for determining an autoimmune disease, comprising an anti-IFN-γ antibody. A method for screening for a therapeutic agent for an autoimmune disease, comprising:
Administering a candidate factor to a model animal produced by the method of claim 11;
A method comprising collecting CD8 positive T cells from the model animal, and detecting abnormal differentiation of the CD8 positive T cells. The therapeutic agent of an autoimmune disease screened by the method of Claim 15.