JP2010004750A - Method for diagnosing onset or onset possibility of tissue disorder by autoimmune disease, and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an objective and simple method for diagnosing the onset or the onset possibility of tissue disorder by autoimmune disease, and to provide the utilization thereof. <P>SOLUTION: The method for diagnosing the onset or the onset possibility of the tissue disorder by the autoimmune disease includes the step of detecting the cross presentation of an antigen-presenting cell in a sample collected from a living body. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for diagnosing the onset or possibility of onset of tissue injury due to an autoimmune disease, and use thereof. More specifically, the present invention relates to a method for diagnosing the onset or possibility of onset of tissue injury due to an autoimmune disease using cross presentation of antigen presenting cells as an index, and use thereof.

  The immune system exists as a defense mechanism of a living body against invasion of harmful foreign substances from the outside world. However, sometimes the action of this immune system can be harmful to the living body as a result. One of them is allergy. A living body is known to cause a kind of allergic reaction not only to foreign substances from the outside world but also to its own components, and is called an autoimmune phenomenon. Diseases in which certain pathological conditions are caused by autoimmunity are called autoimmune diseases.

  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 There are primary biliary cirrhosis, chronic active hepatitis, autoimmune hemolytic anemia, paroxysmal hemoglobinuria, idiopathic thrombocytopenic purpura, and Sjogren's syndrome. Non-organ-specific autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, discoid lupus erythematosus, polymyositis, scleroderma, and mixed connective tissue disease.

  Many of these autoimmune diseases involve lesions in tissues and are accompanied by tissue injury.

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. As a result, it is known that various autoimmune pathologies accompanied by tissue injury occur (for example, see Patent Document 1).
JP 2006-288382 A (released on October 26, 2006)

  When an experimental animal is continuously immunized with the same antigen, an autoimmune pathological condition accompanied by tissue injury occurs in the experimental animal. However, what kind of cells are involved in the process of onset of tissue injury has not been specifically clarified. For this reason, the technology for preventing and treating immune tissue injury is not well established, and the establishment of a new technology is strongly desired.

  Therefore, the present invention has been made in view of the above problems, and an object of the present invention is to provide an objective and simple method for diagnosing the onset or possibility of onset of tissue injury due to an autoimmune disease.

As a result of intensive studies in view of the above problems, the present inventors have (1) that cross-presentation is enhanced by repeated administration of the same antigen to experimental animals (ie, prolonged sensitization), and (2) in antigen-presenting cells. By cross-presentation, it was found that CD8 ⁺ T cells are activated, and (3) tissue injury due to autoimmune disease caused by prolonged sensitization is induced by CD8 ⁺ T cells, and the present invention has been completed. It was.

  That is, the method for diagnosing the onset or possibility of onset of tissue injury due to an autoimmune disease according to the present invention includes a step of detecting a cross presentation of antigen presenting cells in a sample collected from a living body.

  The diagnostic method according to the present invention preferably detects the cross presentation by detecting a change in expression of a gene involved in the cross presentation in the antigen-presenting cell.

The diagnostic method according to the present invention preferably detects the cross presentation by detecting activation of CD8 ⁺ T cells.

  The present invention also provides a diagnostic kit for carrying out the method for diagnosing the onset or possibility of onset of tissue injury due to the autoimmune disease, comprising a primer, a probe, a microarray, and A diagnostic kit for the onset or possibility of onset of tissue injury due to an autoimmune disease, comprising at least one of antibodies, is included.

  The present invention also provides a tissue injury inhibitor that suppresses tissue injury caused by autoimmune disease, including a factor that inhibits cross-presentation of antigen-presenting cells, wherein the factor is chloroquine, primaquine, brefeldin A, lactacystin, MG132, Tissue injury inhibitors characterized by US6 or ICP47 are included.

  The present invention also provides a method for identifying a factor involved in cross-presentation in antigen-presenting cells, comprising a step of prolonged sensitization of a subject with an antigen, and co-culture of antigen-presenting cells collected from the subject with the antigen. And a method of detecting a factor having an abnormality in the abundance in the antigen-presenting cells co-cultured.

  In addition, the present invention includes a method for producing a tissue injury onset model animal that develops tissue injury due to autoimmune disease, including the step of promoting cross-presentation of antigen-presenting cells.

  In the production method of the present invention, it is preferable to promote the cross presentation by prolonged sensitization of the antigen.

  In addition, the present invention is a method for screening a therapeutic agent for tissue injury caused by autoimmune disease, wherein the antigen-presenting cell derived from a living body sensitized with a tissue injury inducer that induces tissue injury caused by autoimmune disease is obtained. A screening method comprising a culture step of culturing in the presence of a candidate factor and a detection step of detecting cross-presentation in the antigen-presenting cells after the culture step is included.

  As described above, the method for diagnosing the onset or possibility of onset of tissue injury due to an autoimmune disease according to the present invention includes a step of detecting a cross-presentation of antigen-presenting cells in a sample collected from a living body. Therefore, the onset or possibility of onset of tissue injury due to autoimmune disease can be objectively and simply diagnosed using the cross-presentation of antigen-presenting cells as an index.

  Hereinafter, a method for diagnosing the onset or possibility of onset of tissue injury due to an autoimmune disease according to the present invention, a diagnostic kit, a tissue injury inhibitor, a method for identifying factors involved in cross-presentation of antigen-presenting cells, and production of a tissue injury onset model animal The method and the method for screening a therapeutic agent for tissue injury will be described in detail, but the present invention is not limited thereto.

[1. (Diagnosis method of onset or possibility of tissue injury due to autoimmune disease)
The method for diagnosing the onset or possibility of onset of tissue injury due to an autoimmune disease according to the present invention (hereinafter, also simply referred to as “diagnostic method of the present invention”) detects the cross-presentation of antigen-presenting cells in a sample collected from a living body. Any other process may be used as long as it includes a process, and other specific processes, conditions, instruments used, and apparatuses used are not particularly limited.

  In the present specification, the term “autoimmune disease” is intended to refer to a disease in which autoimmunity (eg, rheumatoid factor) against a self component (eg, immunoglobulin) is detected and autoimmunity is involved in the disease state. . 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, and Sjogren's syndrome), and organ-specific autoimmunity Diseases such as rheumatoid arthritis, systemic lupus erythematosus, discoid lupus erythematosus, polymyositis, scleroderma, and mixed connective tissue disease. Rheumatoid arthritis, systemic lupus erythematosus, discoid lupus erythematosus, polymyositis, scleroderma and mixed connective tissue disease are known as collagen diseases. That is, collagen disease is included in systemic autoimmune diseases. The autoimmune disease as an application target of the present invention is preferably collagen disease.

  In this specification, “tissue injury” is intended to mean a state in which at least one of the form and physiological function of a tissue in a living body is different from that of a healthy person. For example, in the case of tissue injury in the kidney, the onset of proliferative glomerulonephritis and abnormal proteinuria positivity can be mentioned.

  In the present specification, “tissue injury due to autoimmune disease” is intended to be caused by the above-described tissue injury caused by an autoimmune phenomenon caused by an abnormal immune response. The autoimmune phenomenon is a phenomenon that causes an immune reaction against self components, not against foreign substances from the outside world.

  Examples of tissues that may cause tissue damage due to autoimmune diseases include kidney, liver, thyroid gland, salivary gland (submandibular gland), auricle and skin.

  In the present specification, “cross presentation” refers to an antigen presentation mechanism in which an antigen-presenting cell takes in a foreign antigen, processes the foreign antigen with a proteasome, etc., and then presents the antigen with an MHC class I molecule. Normally, MHC class I molecules present antigens produced and processed by bacteria and viruses that have entered cells (eg, peptide fragments generated by degradation by the proteasome). On the other hand, when foreign antigens such as bacteria and toxins that have entered the body are taken into the antigen-presenting cells by endocytosis, they are processed and decomposed by intracellular enzymes. This degradation product (eg, peptide fragment) is presented as an antigen by MHC class II molecules. That is, foreign antigens taken up by antigen-presenting cells are originally presented by MHC class II molecules. However, according to cross-presentation, antigens originally presented by MHC class II molecules are presented by MHC class I molecules.

  As used herein, “cross-presentation” also intends a phenomenon or state in which an antigen originally presented by an MHC class II molecule is presented by an MHC class I molecule. It is.

  In the present specification, “enhancement of cross-presentation” means that the frequency of occurrence of cross-presentation in an antigen-presenting cell is compared with a state in which no treatment is performed on a living body or a cell or a state in which a control treatment is performed. Intended to be high. As used herein, “enhancement of cross presentation” is used interchangeably with “promotion of cross presentation”.

  In this specification, “probability of onset” refers to an index indicating the risk of tissue injury due to autoimmune disease. If the probability of onset is high, tissue injury due to autoimmune disease is more likely to occur, and conversely onset If the possibility is low, it indicates that it is difficult to suffer from tissue injury due to autoimmune disease.

  In the diagnostic method of the present invention, the sample collected from the living body only needs to contain antigen-presenting cells. Such a sample may be collected from each biological organ or tissue such as peripheral blood, spleen, lymph node, liver, and skin according to a known method.

  The step of detecting the cross-presentation of antigen-presenting cells in the diagnostic method of the present invention is not particularly limited as long as it detects the cross-presentation of antigen-presenting cells. Preferably, the cross presentation is detected and the enhancement of the cross presentation is evaluated.

Examples of the method for detecting the cross-presentation of antigen-presenting cells include a method for detecting activation or activity of CD8 ⁺ T cells, which will be described later, and a method for detecting abnormal expression of genes involved in cross-presentation. There is no particular limitation. The gene involved in cross presentation can be obtained by the identification method described later.

  The antigen-presenting cell that detects cross-presentation is preferably a dendritic cell.

In one embodiment of the diagnostic method of the present invention, cross-presentation of antigen-presenting cells can be detected by detecting activation of CD8 ⁺ T cells.

As shown in Examples described later, CD8 ⁺ T cells are activated by cross-presentation of antigen-presenting cells.

In this specification, “activation of CD8 ⁺ T cells” means that CD8 ⁺ T cells (CD8 positive T cells) express a function specific to CD8 ⁺ T cells in response to signals from inside and outside the cells. It is intended to be in a state to do. Specifically, major histocompatibility (MHC) antigen class I molecules on the surface of target cells and foreign antigen peptides presented on MHC class I molecules are recognized and cytotoxic against target cells presenting the antigen peptides. It is intended to become functional.

Since CD8 expressed in CD8 ⁺ T cells is an auxiliary receptor that recognizes MHC class I molecules, CD8 ⁺ T cells cannot recognize antigens presented on MHC class II molecules. Thus, CD8 ⁺ T cells are not activated in the presence of such antigens. However, when cross-presentation of antigen-presenting cells is triggered, antigens originally presented by MHC class II molecules will be presented by MHC class I molecules. Thereby, CD8 ⁺ T cells can recognize this antigen, and CD8 ⁺ T cells are activated. That is, CD8 ⁺ T cells are activated when cross presentation is triggered.

Methods for detecting activation of CD8 ⁺ T cells activated components that are specifically present in activated CD8 ⁺ T cells (eg, high molecular compounds such as DNA, RNA and proteins, and low molecular weight compounds) Morphology and physiological activity characteristic of CD8 ⁺ T cells (eg, substance production ability, substance secretion ability, substance absorption ability and substance resolution), and conventionally known ones used to distinguish activated CD8 ⁺ T cells The method is not particularly limited as long as it is a method for detecting activation of CD8 ⁺ T cells using an identification marker as an index.

Examples of the method for detecting the activation of CD8 ⁺ T cells include a method for detecting the expression of CD69 in CD8 ⁺ T cells, as shown in Examples described later. In this case, using an antibody against CD69 as an identification marker, by measuring the abundance ratio of CD69-positive CD8 ⁺ T cells in CD8 ⁺ T cells, compared with the existing ratio in a control CD8 ⁺ T cells, CD8 ⁺ T cells Activation can be detected.

Other methods of detecting the activation of CD8 ⁺ T cells, and a method of detecting the presence of interferon gamma (IFN [gamma]) producing cells in the CD8 ⁺ T cells. In this case, as the identification marker IFN [gamma], the existence ratio of CD8 ⁺ T cells producing IFN [gamma] in the total CD8 ⁺ T cells was determined by comparing the abundance ratio in the control CD8 ⁺ T cells, CD8 ⁺ T cell activity Can be detected.

As another method for detecting the activation of CD8 ⁺ T cells, and a method of detecting the CD8 ⁺ T cells interferon produced gamma (IFN [gamma]) or interleukin-2 (IL-2). In this case, measuring the amount of IFNγ or IL-2 in the culture supernatant CD8 ⁺ T cells produced, by comparing the production of a control CD8 ⁺ T cells, detecting the activation of CD8 ⁺ T cells can do.

  The identification markers may be used singly or in combination. The method of detecting the identification marker can be appropriately selected according to each identification marker, and is not particularly limited. Examples of the method for detecting the identification marker include PCR method, gene introduction method, DNA array, protein array, Southern blotting method, Northern blotting method, Western blotting method, radioimmunoassay method, enzyme immunoassay method, morphological detection Methods (eg, tissue immunostaining and cell immunostaining), flow cytometry such as fluorescence activated cell sorter (FACS), chromatography (eg, liquid chromatography, and gas chromatography), And mass spectrometry. Moreover, these methods can be used alone or in combination.

In the step of detecting the cross presentation of antigen presenting cells in an embodiment of the present invention, to measure the abundance ratio of the CD8 + ^T cells activity or activation of CD8 + ^T cells in a subject by the method described above. Thereafter, the presence of CD8 ⁺ T cells the abundance ratio of the activity or activated CD8 ⁺ T cells of CD8 ⁺ T cells were activated or activation of CD8 ⁺ T cells in healthy individuals that were measured in the same manner to obtain the ratio Compare with If there is a significant difference between the two, the subject is diagnosed as having developed tissue damage due to autoimmune disease or having the potential to develop tissue damage due to autoimmune disease.

More specifically, if the rise CD8 + ^T cell activity or abundance ratio of activated CD8 + ^T cells, compared with normal subjects, either to develop tissue damage due to autoimmune diseases, Alternatively, it can be diagnosed that tissue injury due to autoimmune disease may occur.

  In another embodiment of the diagnostic method of the present invention, a cross presentation can be detected by detecting a change in expression of a gene related to the cross presentation.

In the present specification, “detecting a change in gene expression” includes any of the following (i) to (iii).
(I) The expression level of mRNA that is a gene transcription product is measured.
(Ii) The expression level of the protein that is the translation product of the gene is measured.
(Iii) The amount of activity derived from the protein that is the translation product of the gene is measured.

  In the present specification, the “gene expression level” is used in the meaning including both the transcription level of the gene, that is, the mRNA level of the gene and the protein level that is the translation product of the gene.

  When a method for measuring the expression level of the target gene is used as a method for detecting the expression of the target gene, the measurement method is not particularly limited. Specifically, the method for measuring the gene expression level may be a method for measuring the gene expression level at the mRNA level, a method for measuring the gene expression level at the protein level, or a method for measuring both. . More specifically, (A) a method for measuring the expression level of a gene by quantifying mRNA (cDNA) of the gene of interest, and (B) quantifying a protein that is a translation product of the gene of interest. Thus, any method for measuring the expression level of a gene can be used. In addition, the expression level of the gene may be measured by both methods (A) and (B). By doing so, the expression level of the target gene can be measured more accurately. Hereinafter, the methods (A) and (B) will be described.

(A) Method using mRNA (cDNA) When using mRNA, for example, Northern hybridization method, dot blot method, DNA microarray method, etc. using mRNA or total RNA extracted from a sample collected from a subject Thus, mRNA of the gene of interest can be quantified. Furthermore, gene amplification techniques such as RT-PCR can be used. In the RT-PCR method, it is possible to perform more quantitative analysis by using the PCR amplification monitoring method in the gene amplification process.

  In the PCR gene amplification monitoring method, primers labeled with different fluorescent dyes that can cancel each other's fluorescence are used at both ends, and hybridized to a detection target (DNA or reverse transcription product of RNA). As the PCR proceeds and the 5'-3 'exonuclease activity of Taq polymerase degrades this primer, the two fluorescent dyes are separated and fluorescence is detected. This fluorescence is detected in real time. By simultaneously measuring a standard sample having a clear copy number with respect to the detection target, the copy number of the detection target in the target sample is determined by the number of cycles with linearity of PCR amplification (Holland, PM et al., 1991). , Proc. Natl. Acad. Sci. USA 88: 7276-7280; Livak, KJet al., 1995, PCR Methods and Applications 4 (6): 357-362; Heid, CAet al., Genome Research 6: 986 -994; Gibson, EMU et al., 1996, Genome Research 6: 995-1001). Thereby, a detection target can be quantified.

  The primer is not particularly limited, and may be designed by a conventionally known method using the base sequence of cDNA of the target gene.

  The method for extracting mRNA or total RNA from the collected sample is not particularly limited, and mRNA or total RNA can be extracted using a conventionally known method or a commercially available kit.

  In addition, when the primers used for RT-PCR and the probes used for the Northern hybridization method and the dot blot method are labeled with a fluorescent dye, the labeling method is not particularly limited. For example, nick translation method using DNA polymerase I, end labeling method using polynucleotide kinase, fill-in end labeling method using Klenow fragment (Berger SL, Kimmel AR. (1987) Guide to Molecular Cloning Techniques, Method in Enzymology, Academic Press; Hames BD, Higgins SJ (1985) Genes Probes: A Practical Approach. IRL Press; Sambrook J, Fritsch EF, Maniatis T. (1989) Molecular Cloning: a Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press) , Labeling by transcription using RNA polymerase (see Melton DA, Krieg, PA, Rebagkiati MR, Maniatis T, Zinn K, Green MR. (1984) Nucleic Acid Res., 12, 7035-7056), radioisotopes A method of incorporating modified nucleotides that are not used into DNA (see Krick a LJ. (1992) Nonisotopic DNA Probing Techniques. Academic Press) and the like can be used. It is also possible to label the probe by binding an enzyme such as alkaline phosphatase directly to the nucleic acid by chemical crosslinking.

(B) Method using protein When using a protein extracted from a sample collected from a subject, an antibody that specifically binds to a protein that is a translation product of the gene to be analyzed is used, such as Western blotting and ELISA. Proteins can be quantified by conventionally known immunochemical techniques. Further, the protein can be quantified by measuring the activity of the protein to be analyzed. Furthermore, the present invention is not limited to this, and proteins can be quantified using any conventionally known technique. Thus, the expression level of a gene can be measured by quantifying the protein which is the translation product of the target gene.

  In addition, the method for extracting the protein from the collected sample is not particularly limited, and the protein is not decomposed from a biological sample containing the protein (for example, cells and tissues). Proteins can be extracted using conventionally known buffers, devices, kits, and the like.

  The antibody may be a polyclonal antibody or a monoclonal antibody. Moreover, the acquisition method of an antibody is not specifically limited, A conventionally well-known method can be used. For example, a polyclonal antibody can be obtained by taking blood of a mammal sensitized with an antigen and separating serum from this blood by a known method. Moreover, as a polyclonal antibody, the serum containing a polyclonal antibody can be used. Furthermore, if necessary, a fraction containing a polyclonal antibody may be further isolated from this serum.

  Monoclonal antibodies can also be obtained by, for example, cloning a hybridoma obtained by removing immune cells from a mammal sensitized with an antigen and fusing them with myeloma cells and recovering the antibody from the culture. can do.

  In the step of detecting the cross-presentation of antigen-presenting cells in one embodiment of the present invention, the expression level of the target gene in the subject is measured by the method described above. Then, the expression level is compared with the expression level in a healthy person measured by the same method. If there is a significant difference between the two, the subject is diagnosed as having developed or potentially developing tissue damage due to autoimmune disease.

  More specifically, in the case of a gene whose expression increases when cross-presentation is enhanced, if the gene is increased compared to healthy individuals, does it cause tissue damage due to autoimmune disease? Alternatively, it can be diagnosed that there is a possibility of developing tissue injury due to autoimmune disease. On the other hand, in the case of a gene whose expression decreases when cross-presentation is enhanced, if the gene is decreased as compared to a healthy subject, tissue damage due to autoimmune disease has occurred, or autoimmune disease It can be diagnosed that there is a possibility of developing tissue injury due to the above.

[2. (Diagnostic kit)
A diagnostic kit according to the present invention is a diagnostic kit used for carrying out the above-described method for diagnosing the onset or likelihood of tissue injury due to an autoimmune disease, comprising a primer, a probe, a microarray, and a cross-presentation It is sufficient that at least one of the antibodies is provided.

Examples of the primer, probe, microarray, and antibody for detecting cross-presentation include those used for detecting an identification marker of activated CD8 ⁺ T cells. As described above, CD8 ⁺ T cells are activated by cross presentation. CD8 ⁺ T cells produce CD69, IL-2 and IFNγ when activated. Therefore, the enhancement of cross-presentation can be determined by detecting activated CD8 ⁺ T cells using these primers, probes, microarrays or antibodies.

  In addition to the above-described primers, probes, microarrays and antibodies for detecting cross-presentation, primers, probes, microarrays and antibodies for detecting the expression of genes involved in cross-presentation can be mentioned. As described above, enhancement of cross presentation can be detected by detecting changes in the expression of genes involved in cross presentation. Therefore, by using these primers, probes, microarrays, or antibodies, it is possible to determine the enhancement of cross-presentation by detecting changes in gene expression in antigen-presenting cells.

  For the explanation of these primers, probes, microarrays and antibodies, see [1. The description in the section “Method for diagnosing onset or possibility of onset of tissue injury due to autoimmune disease” can be applied mutatis mutandis.

  Further, the configuration of the kit is not limited to that described above, and may include other enzymes, reagents, culture instruments, measuring instruments, and the like.

  By using the determination kit according to the present invention, it is possible to objectively and easily diagnose the onset or possibility of onset of tissue injury due to an autoimmune disease using cross-presentation in antigen-presenting cells as an index.

[3. Tissue injury inhibitor that suppresses tissue injury caused by autoimmune diseases)
The tissue injury inhibitor according to the present invention includes a molecule that suppresses tissue injury due to autoimmune disease. The molecule may be any agent that inhibits cross-presentation, and the biological and physical properties thereof are not particularly limited.

  As used herein, “factor” refers to polynucleotides (such as DNA and RNA), peptides (such as polypeptides and proteins), saccharides (such as oligosaccharides, polysaccharides, and sugar chain derivatives). High molecular compounds such as, and low molecular compounds are contemplated. In addition, this factor may be directly involved in the inhibition of cross-presentation or indirectly through other factors.

In this specification, “inhibition of cross-presentation” includes: (i) the frequency of occurrence of cross-presentation in an antigen-presenting cell is a state in which no inhibition treatment is performed on a living body or a cell, or a state in which a control treatment is performed. (Ii) even when processing that promotes cross-presentation is performed, the frequency of occurrence of cross-presentation is compared with a state where only processing that promotes cross-presentation is performed without performing inhibition processing. And (iii) preventing CD8 ⁺ T cells from recognizing antigens presented by cross-presentation and MHC class I molecules presenting antigens.

  The inhibitor is not particularly limited as long as it inhibits cross presentation as described above. The factor that inhibits cross-presentation of antigen-presenting cells is preferably a factor that inhibits the process by which antigens are presented on MHC class I molecules. Examples of such factors include chloroquine (CQ), primaquine, brefeldin A, lactacystin, MG132, US6 and ICP47.

  By administering the tissue injury inhibitor according to the present invention to a subject, the onset of tissue injury due to an autoimmune disease in the subject can be suppressed.

  The method for administering the tissue injury inhibitor to the subject is not particularly limited, and those skilled in the art can appropriately determine the method according to the tissue injury inhibitor used. Preferred administration methods include, for example, transdermal, intranasal, transbronchial, intramuscular, intraperitoneal, intravenous, intraarticular, subcutaneous, intrathecal, intraventricular and oral administration.

  According to the tissue injury inhibitor according to the present invention, it is possible to suppress the onset of tissue injury due to an autoimmune disease resulting from cross presentation. Therefore, the present invention can be used for the prevention and treatment of tissue injury due to autoimmune diseases.

[4. (Method for identifying factors related to cross-presentation)
The identification method according to the present invention is a method for identifying a factor involved in promoting cross-presentation in an antigen-presenting cell, wherein the subject is subjected to prolonged sensitization with the antigen, and the antigen-presenting cell collected from the subject is treated as the antigen. And a step of detecting a factor having an abnormality in the abundance in the antigen-presenting cells that have been co-cultured, and other specific steps, conditions, equipment used, and equipment used are as follows: It is not particularly limited.

  In the present specification, “prolonged sensitization of antigen” is intended to repeatedly administer the same antigen to a subject to induce antibody production or a T cell response.

  The method for administering the antigen to the subject is not particularly limited. Preferred administration methods include, for example, transdermal, intranasal, transbronchial, intramuscular, intraperitoneal, intravenous, intraarticular, subcutaneous, intrathecal, intraventricular and oral administration. However, it is not limited to these.

  In the repeated administration, the dose per administration, the number of repetitions, the interval and the period are not particularly limited, and the antigen used, the tissue causing the injury, the degree and mode of the injury, the type of the subject, the body weight It may be determined appropriately according to the state and the like. For example, the number of repetitions is preferably 8 to 24 times, the interval between each administration is 5 to 15 days, and the period of repeated administration is preferably 40 to 20 weeks.

  The antigen used for the prolonged sensitization may be any substance that can be finally presented by the MHC antigen. For example, polynucleotides such as RNA and DNA, polypeptides such as peptides and proteins, oligosaccharides, polysaccharides and saccharide derivatives, etc. And other high molecular compounds and low molecular compounds.

  Ovalbumin (OVA) is preferable as an antigen used for prolonged sensitization.

  The method for co-culturing antigen-presenting cells with an antigen is not particularly limited as long as the antigen-presenting cells are cultured in the presence of the antigen. Those skilled in the art can appropriately determine depending on the cells to be cultured and the antigen to be used.

  As used herein, “abnormality in abundance” means co-cultured antigen-presenting cells of a subject that has been prolonged sensitized and antigen presentation of a subject that has not been prolonged sensitized. It is contemplated that there is a change in abundance in antigen presenting cells compared to when the cells are co-cultured with the antigen. Moreover, that there is a change in the abundance means that both the abundance is increasing and the abundance is decreasing.

  The method for detecting a factor having an abnormality in the abundance is not particularly limited as long as it can detect a change in the abundance in the antigen-presenting cell. As a method for detecting a factor having an abnormal abundance, a conventionally known transcriptome analysis method, proteome analysis method, and metabolome analysis method such as microarray, differential display method, two-dimensional electrophoresis, and mass spectrometry may be used. it can.

  The method for extracting mRNA, total RNA, protein, saccharide, or metabolite from antigen-presenting cells is not particularly limited, and can be extracted using a conventionally known method. It can also be extracted using a commercially available kit.

  In one embodiment of the identification method according to the present invention, the method for detecting a factor having an abnormal abundance is preferably a microarray analysis using RNA extracted from antigen-presenting cells. According to the microarray analysis, information on a large number of genes can be easily obtained.

  According to the identification method of the present invention, it is possible to identify a factor whose abundance changes when cross-presentation is promoted. By measuring the amount of these identified factors in the antigen-presenting cell, it is possible to detect the presence or absence of enhanced cross presentation. Therefore, by detecting the factor obtained by the identification method according to the present invention, it is possible to diagnose the onset or possibility of onset of tissue injury due to autoimmune disease. In addition, by adjusting the amount of the factor obtained by the identification method according to the present invention, it is possible to regulate the enhancement of cross presentation and to induce or suppress the onset of tissue injury due to autoimmune disease. .

[5. (Method for producing tissue injury onset model animal causing tissue injury due to autoimmune disease)
The method for producing a tissue injury onset model animal according to the present invention (hereinafter also simply referred to as “the production method of the present invention”) may include a step of promoting the cross-presentation of antigen-presenting cells. There are no particular limitations on the various processes, conditions, equipment used, and equipment used.

  The method of promoting the cross-presentation of antigen-presenting cells is not particularly limited as long as the frequency of cross-presentation in the antigen-presenting cells subjected to the promoting treatment is higher than the frequency of cross-presentation of control antigen-presenting cells. Absent. Examples of the method for promoting the cross presentation of antigen-presenting cells include prolonged sensitization of an antigen to a subject.

  In one embodiment of the production method of the present invention, cross-presentation of antigen-presenting cells can be promoted by prolonged sensitization of a non-human mammal with an antigen.

  As a method of prolonged sensitization of a subject, for example, the above [4. The method described in “Method for identifying factor related to cross presentation” can be used.

  The subject non-human mammal is not limited as long as it is a mammal other than a human, but it is an experimental animal used for developing a preventive or therapeutic method for tissue injury due to human autoimmune disease. Is preferred. Specific examples include mice, rats, rabbits, monkeys, goats, pigs, sheep, cows and dogs.

In another embodiment of the production method of the present invention, activated CD8 ⁺ T cells are collected from a living body in which cross-presentation of antigen-presenting cells is promoted, and the collected activated CD8 ⁺ T cells are used as another living body. By transferring, a model animal that develops tissue injury due to autoimmune disease can be produced.

The method for transferring CD8 ⁺ T cells into a living body is not particularly limited, and a conventionally known method may be used. For example, a method of injecting subcutaneously, intradermally, vein, artery and abdominal cavity can be mentioned. When populating the activated CD8 + ^T cells in vivo, CD8 + ^T cells to transfer is preferably a biological homologous or syngeneic animal is a transfer destination.

Activated CD8 ⁺ T cells may be collected from each biological organ or tissue such as peripheral blood, spleen, lymph node, liver and skin according to a known method.

  According to the production method of the present invention, a non-human model animal that causes immune tissue injury can be produced. Therefore, the present invention is a study of tissue injury caused by autoimmune disease using a tissue injury onset model animal that develops tissue injury caused by autoimmune disease (for example, screening for preventive and therapeutic agents for tissue injury caused by autoimmune disease). Can be used.

  It can be diagnosed by the above-described diagnostic method of the present invention whether or not the tissue injury onset model animal has developed tissue injury due to autoimmune disease. In addition, other methods for detecting whether or not a tissue injury onset model animal has developed tissue injury due to autoimmune disease include detection of the presence or absence of abnormal gene expression in tissues, physiological methods (for example, substance production, Detection of substance secretion, substance absorption, substance decomposition, etc.), histopathological techniques such as tissue immunostaining, and techniques using previously known identification markers used for identifying tissue injury as indices. It is not limited to. These techniques may be used alone or in combination of two or more.

[6. (Screening method for therapeutic agent for tissue injury due to autoimmune disease)
The screening method according to the present invention comprises a culture step of culturing a living body-derived antigen-presenting cell sensitized with a tissue injury inducer that induces tissue injury due to an autoimmune disease in the presence of a candidate factor, And a detection step of detecting a cross-presentation in the antigen-presenting cell.

  The prolonged sensitization in the present invention is not particularly limited as long as the subject can be prolonged sensitized. For example, [4. The method described in the section “Method for identifying factors involved in cross-presentation” can be used.

  Candidate factors are not particularly limited, polynucleotides (such as DNA and RNA), peptides (such as polypeptides and proteins), which are expected to directly or indirectly inhibit cross-presentation, High molecular compounds such as saccharides (oligosaccharides, polysaccharides, sugar chain derivatives, etc.) and low molecular compounds are preferred.

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

  Moreover, the therapeutic agent obtained by this invention can be used for the treatment of the patient who is causing the tissue injury by the autoimmune disease resulting from a cross presentation.

  The administration conditions of the therapeutic agent for clinical application can be determined using the tissue injury onset model animal system described herein. That is, by using this 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 the onset of tissue injury due to autoimmune disease.

  The therapeutic agent can also be a composition further comprising any pharmaceutically acceptable carrier. Carriers include, for example, sterile water, saline, buffering agents, vegetable oils, emulsifiers, suspensions, salts, stabilizers, preservatives, surfactants, sustained release agents, other proteins (such as BSA) and transfection Examples include reagents (including lipofection reagents and liposomes). Further, 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 and urea.

  The dosage form when the therapeutic agent obtained by the screening method according to the present invention is formulated is not particularly limited, and may be, for example, a solution (injection), powder, microcapsule or tablet. For example, a drug that targets a tissue that has developed tissue damage due to autoimmune disease 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) Delivery will be possible. Thereby, an effective treatment can be performed.

  The route of administration of the therapeutic agent obtained by the screening method according to the present invention to a patient is appropriately selected according to the nature of the active ingredient. For example, percutaneous, intranasal, transbronchial, intramuscular, abdominal cavity It can be performed internally, intravenously, intraarticularly, subcutaneously, intrathecally, intraventricularly or orally, but is not limited thereto. The therapeutic agent 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 dosage 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.

  The therapeutic agent obtained by the screening method according to 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 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. 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.

  Individuals to be treated include, for example, 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 tissue injury diseases. For example, by using a model animal produced using a non-human mammal, a new treatment protocol for preventing the onset of tissue injury due to an autoimmune disease can be developed.

  Examples will be shown below, and the embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention is also applied to the embodiments obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention. Moreover, all the literatures described in this specification are used as reference.

[Example 1: Induction of tissue injury by CD8 ⁺ T cells]
BALB / c mice were administered 0.5 mg ovalbumin (OVA) or PBS once every 5 days for a total of 12 doses by intraperitoneal administration. B cells (B220 positive cells), T cells (CD3 positive cells), CD4 ⁺ T cells (CD4 positive cells) and CD8 ⁺ T cells were isolated from the spleens of mice after administration using magnetic beads.

These cells were adoptively transferred by intravenous injection into 2.5 × 10 ⁷ cells each in another naïve BALB / c mouse. 24 hours after cell transfer, 0.5 mg of OVA was administered once intraperitoneally. Proteinuria was detected 2 weeks after cell transfer. Furthermore, after slaughtering these mice, pathological evaluation of the kidney was performed by hematoxylin and eosin staining (HE staining, H & E staining).

The detection result of proteinuria is shown in FIG. As shown in FIG. 1, proteinuria was detected in the T cell transfer group. Further, when comparing the CD4 ⁺ T cell transfer group and the CD8 + T cell transfer group, the positive degree of proteinuria was higher in the CD8 ⁺ T cell transfer group.

The result of HE staining of kidney is shown in FIG. As shown in FIG. 2, as a result of pathological evaluation of the kidney, proliferative glomerulonephritis developed in the CD8 ⁺ T cell transfer group.

From the above, it was found that tissue injury was induced by transferring CD8 ⁺ T cells activated by prolonged sensitization of OVA.

[Example 2: Suppression of tissue injury due to CD8 ⁺ T cell deficiency]
BALB / c mice (wild type mice) and CD8 ⁺ T cell-deficient mice, β ₂ -microglobulin (β ₂ m) -deficient mice (genetic background is BALB / c mice), 0.5 mg of OVA or PBS It was administered by intraperitoneal administration once a day for a total of 12 times.

  Proteinuria was detected on the 9th day after the last administration. In addition, these mice were sacrificed and immune complex deposition of IgG and C3 in the kidney was detected by immunofluorescent staining.

The result by immunofluorescence staining is shown in FIG. As shown in FIG. 3, the immune complex deposition in the kidneys of β ₂ m-deficient mice administered 12 times with OVA was similar to that in the kidneys of wild-type mice.

The detection result of proteinuria is shown in FIG. As shown in FIG. 4, the positive degree of proteinuria in β ₂ m-deficient mice was lower than that in wild-type mice.

Compared to wild-type mice, β ₂ m-deficient mice have the same degree of immune complex deposition, but proteinuria is less positive, and nephritis is suppressed in CD8 ⁺ T-cell-deficient mice. Was shown to be.

From the above, it was found that CD8 ⁺ T cells are required for tissue injury due to autoimmune diseases.

[Example 3: Activation of CD8 ⁺ T cells by cross-presentation]
BALB / c mice were administered 0.5 mg OVA or PBS once every 5 days for a total of 12 times by intraperitoneal administration. Dendritic cells (dendritic cells; DC, CD11c positive cells) were isolated from the spleens of the mice after administration by magnetic beads and cultured in vitro with OVA (1 mg / ml) for 3 hours. DCs pulsed with OVA were cultured with OVA-responsive CD8 ⁺ T cells isolated from a transgenic mouse (DO11.10 TCR TG mouse) into which an OVA-specific T cell receptor (TCR) was introduced.

24 hours after the start of co-culture with OVA-responsive CD8 ⁺ T cells, the expression of CD69 (activation marker) on CD8 ⁺ T cells was detected by FACS. Furthermore, IL-2 and IFNγ in the culture supernatant were measured by ELISA to examine activation of OVA-responsive CD8 ⁺ T cells.

It should be noted that when OVA is pulsed to DC, if the treated OVA peptide is not presented on MHC class I, OVA-responsive CD8 ⁺ T cells will not be activated. When cross-presentation occurs and OVA peptides are presented on MHC class I, OVA-responsive CD8 ⁺ T cells are activated. Therefore, by analyzing the activation of OVA-responsive CD8 ⁺ T cells after co-culture with DC, the presence or absence of antigen cross-presentation in DC can be examined.

The detection results of CD69 expression in OVA-responsive CD8 ⁺ T cells are shown in FIG. In the figure, (a) shows the results of measuring the proportion of OVA-responsive CD8 ⁺ T cells expressing CD69, and (b) shows the results of (a) as a bar graph. As shown in FIG. 5, co-culture was performed using the OVA administration group (“OVA DC” in the figure) compared to the case of using the PBS administration group DC (“PBS DC” in the figure). In some cases, the expression of CD69 in OVA-responsive CD8 ⁺ T cells (DO11.10 CD8 ⁺ T cell) was significantly increased (P <0.001).

  The detection results of IL-2 and IFNγ expression in the culture supernatant are shown in FIG. As shown in FIG. 6, when IL-2 and IFNγ in the culture supernatant were also co-cultured using DC of the OVA administration group, their production was significantly (IL-2: P <0.001, IFNγ: P <0.005) increased.

From the above, it was found that cross-presentation in DC was enhanced by repeated administration of OVA, thereby activating CD8 ⁺ T cells.

[Example 4: Inhibition of cross-presentation in vitro]
BALB / c mice were administered 0.5 mg OVA or PBS once every 5 days for a total of 12 times by intraperitoneal administration. DCs (CD11c positive cells) were isolated from the spleens of mice after administration by magnetic beads, cultured in vitro with chloroquine (CQ) (20 μg / ml) for 2 hours, and then in a culture solution without chloroquine. Incubated with OVA (1 mg / ml) for 3 hours.

DCs pulsed with CQ and OVA were co-cultured with OVA-responsive CD8 ⁺ T cells and CD4 ⁺ T cells isolated from DO11.10 TCR TG mice introduced with OVA-specific TCR, respectively.

24 hours after the start of co-culture with OVA-responsive CD8 ⁺ T cells and CD4 ⁺ T cells, CD69 expression on CD8 + T cells and CD4 + T cells was detected by FACS, and activation of OVA-responsive T cells was examined. did.

FIG. 7 shows the detection results of CD69 expression in OVA-responsive T cells. As shown in FIG. 7, as shown in Example 3, when only OVA was pulsed to DC, if OVA administration group DC (“OVA DC” in the figure) was used for co-culture, OVA was performed. The expression of CD69 was increased in responsive CD8 ⁺ T cells (“DO11.10CD8 ⁺ T cell” in the figure).

On the other hand, when DC was pulsed with CQ, the expression of CD69 on the OVA-responsive CD8 ⁺ T cells was the DC of the PBS-administered group (“PBS DC” in the figure) even when the DC of the OVA-administered group was used. It was suppressed to the same extent as the case.

When DC pulsed with only OVA was co-cultured with OVA-responsive CD4 ⁺ T cells (DO11.10CD4 ⁺ T cell), DCs in the OVA-administered group and the PBS-administered group had OVA-responsive CD4 ⁺ T cells to the same extent. Was activated. Furthermore, even when DC was pulsed with CQ, OVA-responsive CD4 ⁺ T cells were fully activated.

From the above, it was found that CQ does not affect the activation of CD4 ⁺ T cells but suppresses only the activation of CD8 ⁺ T cells, that is, inhibits cross presentation.

[Example 5: Suppression of tissue injury by inhibition of cross presentation]
BALB / c mice were administered 250 μg of CQ by intraperitoneal administration, and 3 hours later, 0.5 mg of OVA or PBS was administered by intraperitoneal administration. This was repeated once every 5 days for a total of 12 times.

Proteinuria was detected on the 9th day after the last administration. Furthermore, this mouse was sacrificed, spleen cells were stained intracellularly, and IFNγ-producing CD8 ⁺ T cells were detected by FACS.

The detection result of IFNγ-producing CD8 ⁺ T cells is shown in FIG. As shown in FIG. 8, when OVA is repeatedly administered alone, the proportion of IFNγ-producing CD8 ⁺ T cells increases. On the other hand, when CQ and OVA were repeatedly administered (“CQ + OVA” in the figure), the proportion of IFNγ-producing CD8 ⁺ T cells did not increase.

  The detection result of proteinuria is shown in FIG. As shown in FIG. 9, proteinuria detected when CQ and OVA were repeatedly administered had a lower degree of positivity than proteinuria when OVA alone was repeatedly administered.

By inhibiting the cross-presentation by administration of CQ, activation of CD8 ⁺ T cells was suppressed and nephritis was suppressed.

[Example 6: Search for genes involved in cross-presentation]
BALB / c mice were administered 0.5 mg OVA or PBS once every 5 days for a total of 12 intraperitoneal administrations. DCs (CD11c positive cells) were isolated from the spleens of these mice by magnetic beads. DCs were cultured with OVA (1 mg / ml) for 3 hours to induce cross presentation, and then RNA was extracted. Using this RNA, microarray analysis was performed using the Whole Mouse Genome Oligo Microarray of Agilent, and the gene expression in DCs of the PBS administration group and the OVA administration group was compared.

  Table 1 shows genes in which mRNA expression in DCs in the OVA administration group was increased by a factor of 5 or more compared to expression in DCs in the PBS administration group.

  As shown in Table 1, genes involved in cross-presentation could be identified.

  As described above, according to the present invention, the onset or possibility of onset of tissue injury due to autoimmune disease can be diagnosed using cross-presentation as an index. In addition, the present invention can be suitably used for pharmaceutical screening tests for the prevention or treatment of tissue injury due to autoimmune diseases. Therefore, the present invention can be widely used in industries in the life science field including the medical field, pharmaceutical field, and health medical field.

In a present Example, it is a graph which shows the result of having measured the proteinuria of the mouse | mouth into which the cell of the BALB / c mouse | mouth which repeatedly administered the antigen was transferred. In a present Example, it is a figure which shows the result of having investigated the onset of nephritis in the mouse | mouth which the cell of the BALB / c mouse | mouth which repeatedly administered the antigen was transferred. In the present Example, it is a figure which shows the result of having detected the deposit of the immune complex in the kidney of the CD8 ^<+> T cell defect | deletion mouse | mouth which repeatedly administered the antigen by immunofluorescence staining. In the present Example, it is a graph which shows the result of having measured the proteinuria of the CD8 ^<+> T cell defect | deletion mouse | mouth which administered the antigen repeatedly. (A) And (b) is a figure which shows the result of having investigated the fluctuation | variation of the CD69 positive cell in the OVA responsive CD8 ^<+> T cell cultured with DC of the BALB / c mouse | mouth which administered the antigen repeatedly in this Example. is there. In the present Example, it is a figure which shows the result of having investigated the fluctuation | variation of the quantity of IL-2 and IFN (gamma) which the OVA response CD8 ^<+> T cell culture | cultivated with DC of the BALB / c mouse | mouth which administered the antigen repeatedly. In the present Example, it is a figure which shows the result of having investigated the fluctuation | variation of the CD69 positive cell in the OVA response CD8 ^<+> T cell co-cultured in presence of CQ with DC of the BALB / c mouse | mouth which repeatedly administered the antigen. In the present Example, it is a figure which shows the result of having investigated the fluctuation | variation of the production cell of IFN (gamma) in the CD8 ^<+> T cell of the BALB / c mouse which administered antigen and CQ repeatedly. In a present Example, it is a figure which shows the result of having measured the proteinuria of the BALB / c mouse which administered the antigen and CQ repeatedly.

Claims (9)

  A method for diagnosing the onset or possibility of onset of tissue injury due to an autoimmune disease, comprising a step of detecting a cross-presentation of antigen-presenting cells in a sample collected from a living body.   The diagnostic method according to claim 1, wherein the cross-presentation is detected by detecting a change in expression of a gene involved in the cross-presentation in the antigen-presenting cell. The diagnostic method according to claim 1, wherein the cross presentation is detected by detecting activation of CD8 ⁺ T cells. A diagnostic kit for carrying out the method for diagnosing the onset or possibility of onset of tissue injury due to the autoimmune disease according to any one of claims 1 to 3,
A diagnostic kit for the onset or possibility of onset of tissue injury due to an autoimmune disease, comprising at least one of a primer, a probe, a microarray and an antibody for detecting the cross presentation.   A tissue injury inhibitor that suppresses tissue injury due to autoimmune disease, including a factor that inhibits cross-presentation of antigen-presenting cells, wherein the factor is chloroquine, primaquine, brefeldin A, lactacystin, MG132, US6 or ICP47 A tissue injury inhibitor characterized by. A method for identifying factors involved in cross-presentation in antigen-presenting cells,
Prolonged sensitization of a subject with an antigen; and
Co-culturing antigen-presenting cells collected from the subject with the antigen;
Detecting a factor having an abnormality in the abundance in the antigen-presenting cells co-cultured.   A method for producing a tissue injury onset model animal that develops tissue injury due to an autoimmune disease, comprising a step of promoting cross-presentation of antigen-presenting cells.   The manufacturing method according to claim 7, wherein the cross presentation is promoted by prolonged sensitization. A method for screening a therapeutic agent for tissue injury caused by autoimmune disease, comprising:
A culture step of culturing an antigen-presenting cell derived from a living body sensitized with a tissue injury inducer that induces tissue injury due to autoimmune disease in the presence of a candidate factor;
And a detection step of detecting a cross-presentation in the antigen-presenting cell after the culturing step.