Detection method of autoreactive t cell

Abstract

To provide a detection method of autoreactive T cells activated in an organ damaged by an autoimmune disease.SOLUTION: A detection method of autoreactive T cells includes: a T cell hybridoma producing step for producing T cell hybridomas by fusing T cells and tumor cells derived from a mammal having an autoimmune disease with damage to a tissue / organ; a T cell hybridoma transfer step for transferring the produced T cell hybridomas to the tissue / organ damaged by the autoimmune disease in an immunocompromised non-human mammal; a T cell hybridoma recovering step for recovering the T cell hybridomas from the tissue / organ; and a T cell hybridoma detection step for quantifying an interleukin-2 gene expression amount of the recovered T cell hybridomas.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for detecting autoreactive T cells. [Background technology]

[0002] Sjogren's syndrome (SS) is an intractable autoimmune disease in which chronic inflammation of the exocrine glands causes functional disorders such as dry eyes and dry mouth, and can also lead to systemic autoimmune diseases such as systemic lupus erythematosus (SLE) and malignant lymphoma. There are approximately 100,000 SS patients in Japan, but no specific treatment for SS has been established.

[0003] It is well known that T cells play a central role in the pathogenesis of SS. Non-Patent Document 1 discloses that mice lacking special AT-rich sequence binding protein-1 (SATB1), a transcription factor that regulates gene expression required for T cell differentiation, in blood cell-specific manner (blood cell-specific SATB1-deficient mice, SATB1 conditional knockout mice) exhibit pathological conditions similar to Sjogren's syndrome from an early age (4 weeks of age), and that T cells accumulating in cervical lymph nodes are important for the reduction in saliva in blood cell-specific SATB1-deficient mice.

[0004] Patent Document 1 describes that multiple sclerosis (abbreviated as MS), which is an autoimmune disease, is a disease triggered by autoreactive T cells. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2018-088887 A [Non-patent literature]

[0006] [Non-Patent Document 1] Tanaka, Y., et al., “SATB1 Conditional Knockout Results in Sjogren's Syndrome in Mice”, The Journal of Immunology, 2017, Vol. 199, No. 12, pp. 4016-4022 Summary of the Invention [Problem to be solved by the invention]

[0007] It is unclear what self antigens pathogenic T cells (also called "autoreactive T cells") react with. Generally, the T cell receptor expressed by one T cell recognizes only one type of antigen peptide, so the cervical lymph nodes of blood cell-specific SATB1-deficient mice may contain autoreactive T cells that react with self antigens as well as T cells that do not. Therefore, a means of detecting autoreactive T cells from T cells in the cervical lymph nodes of blood cell-specific SATB1-deficient mice is needed.

[0008] The present invention has been made in view of the above circumstances, and an objective of the present invention is to provide a method for detecting autoreactive T cells activated in damaged organs caused by autoimmune diseases. [Means for solving the problem]

[0009] The inventors have conducted intensive research to solve the above-mentioned problems, and have found that when T cell hybridomas established by cell fusion between lymph node T cells derived from Sjogren's syndrome model mice and a thymoma cell line capable of autonomous proliferation were directly injected into the salivary glands of immunodeficient mice, and the increased expression of interleukin-2 gene in T cell hybridomas recovered from the salivary glands several days later was examined, it was possible to detect autoreactive T cell hybridomas that become activated in response to salivary gland tissue, thereby completing the present invention. That is, the present invention includes the following aspects.

[0010] [1] A T cell hybridoma production process in which a T cell derived from a mammal suffering from an autoimmune disease accompanied by tissue and cell damage is fused with a tumor cell to produce a T cell hybridoma; a T cell hybridoma transfer step of transferring the prepared T cell hybridoma into a tissue or organ damaged by the autoimmune disease of an immunodeficient non-human mammal; a T cell hybridoma recovery step of recovering T cell hybridomas from the tissue or organ; and a T cell hybridoma detection step for quantifying the expression level of interleukin 2 gene in the collected T cell hybridomas; A method for detecting autoreactive T cells, comprising: [2] The method for detecting autoreactive T cells described in [1], wherein the immunodeficient non-human mammal is a non-human mammal homozygously deficient in one or both of the Rag1 gene and the Rag2 gene. [3] The method for detecting autoreactive T cells described in [1] or [2], wherein the T cells derived from a mammal suffering from an autoimmune disease accompanied by tissue and cell damage are cervical lymph node T cells. [4] The method for detecting autoreactive T cells according to any one of [1] to [3], wherein the autoimmune disease accompanied by tissue / cell damage is Sjogren's syndrome. [5] The method for detecting autoreactive T cells according to any one of [1] to [4], wherein the tissue or organ damaged by the autoimmune disease is an exocrine gland tissue. [6] The method for detecting autoreactive T cells according to any one of [1] to [5], wherein the tissue or organ damaged by the autoimmune disease of the immunodeficient non-human mammal is an organoid. [7] The method for detecting autoreactive T cells according to any one of [1] to [6], wherein the immunodeficient non-human mammal is an immunodeficient xenogeneic chimeric mammal. Autoreactive T cell hybridomas detected by any of the methods for detecting autoreactive T cells listed in [8][1]-[7]. Effect of the Invention

[0011] According to the present invention, a method for detecting autoreactive T cells activated in damaged organs caused by autoimmune diseases can be provided. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a schematic diagram illustrating a conventional method for detecting autoreactive T cells. [Diagram 2] FIG. 2 is a schematic diagram illustrating the method for detecting autoreactive T cells of the present invention. [Diagram 3] FIG. 3 is a schematic diagram illustrating an example of a method for producing a T cell hybridoma. [Figure 4] FIG. 4 is a graph showing the results of quantitatively determining IL-2 mRNA by quantitative RT-PCR in Experimental Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] RAG2 knockout mice (Rag2 - / - The mouse model is based on the NKT (natural killer T) cell line, which is a type of immunodeficient mouse that lacks Rag2 (recombination activating gene 2) and is therefore unable to reconstitute T and B cell receptors.

[0014] Although the embodiments of the present invention will be described in detail below, the present invention is not limited to the embodiments described below, and various modifications are possible without departing from the gist of the present invention.

[0015] [Methods for detecting autoreactive T cells] The method for detecting autoreactive T cells according to the present embodiment, which is described in FIG. - / - The mouse 106 was injected into the salivary gland 101, and several days later (six days later in FIG. 2), the salivary gland 101 was excised, and a T cell hybridoma 104 was isolated and Rag2 - / - It detects activated T cell hybridomas in the salivary glands 101 of mice 106.

[0016] One embodiment of the method for detecting autoreactive T cells of the present invention includes a T cell hybridoma production step, a T cell hybridoma transfer step, a T cell hybridoma recovery step, and a T cell hybridoma detection step.

[0017] <T cell hybridoma production step> The T cell hybridoma production step is a step of producing a T cell hybridoma by fusing T cells derived from a mammal suffering from an autoimmune disease accompanied by tissue / cell damage with tumor cells.

[0018] The autoimmune disease accompanied by tissue / cell damage is not particularly limited, and examples include Sjögren's syndrome, multiple sclerosis, systemic lupus erythematosus, Addison's disease, polymyositis, systemic scleroderma, glomerulonephritis, Graves' disease, rheumatoid arthritis, Hashimoto's thyroiditis, type 1 diabetes, and vasculitis. Among these, Sjögren's syndrome, multiple sclerosis, and systemic lupus erythematosus are preferable as the autoimmune disease accompanied by tissue / cell damage, and Sjögren's syndrome is more preferable.

[0019] The tissue or cell in which the tissue / cell damage occurs is not particularly limited. In Sjögren's syndrome, for example, exocrine glands such as salivary glands and lacrimal glands can be mentioned. In multiple sclerosis, for example, the central nervous system can be mentioned. In systemic lupus erythematosus, for example, the skin, joints, blood vessels, and kidneys can be mentioned. In type 1 diabetes, for example, pancreatic β cells can be mentioned. In rheumatoid arthritis, for example, joints can be mentioned.

[0020] The mammal is not particularly limited, and examples thereof include humans and non-human mammals. The non-human mammal is not particularly limited, and examples thereof include non-human primates such as tarsiers, long-tailed monkeys, gibbons, spider monkeys, capuchin monkeys, saki, lemurs, pygmy lemurs, indri, ferret lemurs, aye-ayes, galagos, lorises, chimpanzees, gorillas, and orangutans, rodents such as mice, rats, and guinea pigs, carnivores such as dogs, cats, bears, raccoons, weasels, and sea lions, perissodactyls such as horses, tapirs, and rhinos, cetaceans such as camels, wild boars, deer, cows, giraffes, hippos, blue whales, sperm whales, and dolphins, and lagomorphs such as rabbits and pikas.

[0021] The T cells are not particularly limited, but are preferably lymph node T cells, because there are many pathogenic T cells that induce tissue / cell damage in lymph nodes in the vicinity of tissues where tissue / cell damage occurs.

[0022] The T cells may be directly fused with tumor cells to produce hybridomas, or may be transferred into an organ or tissue of an immunodeficient non-human mammal and concentrated therein, and then fused with tumor cells to produce hybridomas.

[0023] The tumor cells are not particularly limited, but thymoma cells are preferred because of their ease of fusion with T cells.

[0024] The method for fusing T cells and tumor cells is not particularly limited, and any conventionally known method, such as the polyethylene glycol method (PEG method) using polyethylene glycol or the electric fusion method using an electric current, can be used. If the cells before fusion are diploid, the hybridoma immediately after fusion will be tetraploid, but will be stabilized during culture and become a single cell with the properties of the two types of cells before fusion.

[0025] That the prepared T cell hybridoma has the properties of T cells can be confirmed, for example, by the expression of CD4, CD8, and T cell receptor β (TCRβ). More specifically, for example, it is preferable to confirm that it is TCRβ-positive and at least one of CD4 and CD8 is positive.

[0026] An example of the T cell hybridoma preparation process will be described with reference to FIG. 3. In this example, as the mammal suffering from an autoimmune disease accompanied by tissue / cell damage, a hematopoietic cell-specific SATB1-deficient mouse is used as the T cells, and lymph node T cells are used as the tumor cells, thymoma cells. The cervical lymph nodes 108 are collected from the hematopoietic cell-specific SATB1-deficient mouse 107, and lymph node T cells are separated from the cervical lymph nodes 108. The cervical lymph node T cells + thymoma cells 109 are fused by the PEG method to obtain T cell hybridomas 104.

[0027] <T cell hybridoma transplantation step> The T cell hybridoma transplantation step is a step of transplanting the prepared T cell hybridoma into the tissue / organs damaged by the autoimmune disease of an immunodeficient non-human mammal.

[0028] The immunodeficient non-human mammal is not particularly limited, but a non-human animal in which one or both of the Rag1 gene and the Rag2 gene are homozygously deficient is preferred. Here, the non-human mammal is not particularly limited, but non-human primates such as Macaca mulatta, Macaca fascicularis, Macaca nemestrina, Callithrix jacchus, Macaca arctoides, Saki, Cynomolgus monkey, Callimico goeldii, Indri, Nycticebus coucang, Aye-aye, Galago, Loris, Chimpanzee, Gorilla, and Orangutan, rodents such as mice, rats, and guinea pigs, carnivores such as dogs, cats, bears, raccoons, weasels, and seals, odd-toed ungulates such as horses, donkeys, and zebras, cetartiodactyls such as camels, wild boars, deer, cows, giraffes, hippos, blue whales, sperm whales, and dolphins, and lagomorphs such as rabbits and pikas are exemplified. The immunodeficient non-human mammal may also be an immunodeficient xenogeneic chimeric mammal.

[0029] The tissue or organ affected by the autoimmune disease in the immunodeficient non-human mammal may be, for example, an organoid of a tissue or an organ.

[0030] <T cell hybridoma recovery step> The T cell hybridoma recovery step is a step of recovering T cell hybridomas from the tissue or organ. The method for recovering T cell hybridomas from the tissue or organ is not particularly limited. For example, after dissociating the cells of the tissue or organ by enzymatic treatment of the tissue or organ, a method of recovering T cell hybridomas by means such as density gradient centrifugation or flow cytometry can be mentioned.

[0031] <T cell hybridoma detection step> The T cell hybridoma detection step is a step of quantifying the interleukin-2 (IL-2) gene expression level of the recovered T cell hybridomas and detecting T cell hybridomas with a significantly high IL-2 gene expression level as autoreactive T cell hybridomas.

[0032] The IL-2 gene expression level of T cell hybridomas is preferably quantified, for example, by quantitative reverse transcription PCR (RT-qPCR). As a control, it is preferable to use unstimulated T cell hybridomas not injected into the tissue or organ. Furthermore, it is also preferable to use T cell hybridomas stimulated by a general stimulus, for example, phorbol 12-myristate 13-acetate (PMA) and ionomycin, for reference.

[0033] [Autoreactive T cell hybridoma] Another embodiment of the present invention is an autoreactive T cell hybridoma detected by the method for detecting autoreactive T cells described above.

[0034] [Function and effect] The conventional method for detecting autoreactive T cells shown in Figure 1 involved preparing a salivary gland extract 102 from a salivary gland 101, contacting it with antigen-presenting cells 103, culturing T cell hybridomas 104 in the presence of the antigen-presenting cells 103, and detecting the activated T cell hybridomas. Conventional methods for detecting autoreactive T cells have determined the reactivity of pathogenic T cells in a two-dimensional state, but the method for detecting autoreactive T cells of this embodiment can examine the reactivity of pathogenic T cells in a three-dimensional state in vivo while maintaining the shape of the organ. EXAMPLES

[0035] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the experimental examples described below.

[0036] [Example 1] Creation of hematopoietic cell-specific SATB1-deficient mice (SATB1 cKO mice) SATB1 was isolated using the method described in Reference 1 (Tanaka et al., 2016). fl / fl Vav-Cre + Mice were generated and used as SATB1 cKO mice.

[0037] [Experimental Example 1] Detection of autoreactive T cells 1. Generation of Pathogenic T Cell Hybridomas (1) Preparation of T cells a) Cervical lymph nodes were harvested from blood cell-specific SATB1-deficient mice, and cervical lymph node T cells isolated from the harvested cervical lymph nodes were labeled with Thy1.2 antibody (an antibody that recognizes a T cell surface antigen) and separated using a BD FACSAria Fusion cell sorter (BD Biosciences). b) Isolated cervical lymph node T cells were subjected to Rag2 - / - The virus was transferred intravenously to mice, causing salivary gland inflammation. c) Rag2 of b - / -The cervical lymph nodes of the mice were harvested and the whole lymph node cells were ground up to prepare T cells (cervical lymph node T cells) for cell fusion. To enrich for pathogenic T cells, Rag2 - / - was transferred to mice.

[0038] 2. Generation of T cell hybridomas (1) Experimental procedure on the first day a) The above cervical lymph node T cells (1 × 10 7 cells) and mouse lymphoma BW5147 cells (1 × 10 7 Each of the pieces (pieces) was transferred to a FALCON 50 mL conical centrifugation tube (Corning, #352098; hereinafter, also simply referred to as "FALCON tube"). b) Each FALCON tube was centrifuged at 1,500 rpm for 5 minutes at room temperature. c) After centrifugation, the supernatant in each FALCON tube was discarded. d) After discarding the supernatant, 5 mL of RPMI1640 medium (FCS-) was added to each FALCON tube and mixed. e) The cell number was counted. f) Equal numbers of mouse lymphoma BW5147 cells and cervical lymph node T cells were placed in one FALCON tube. g) The FALCON tube was centrifuged at 1,500 rpm for 5 minutes at room temperature. h) After centrifugation, the supernatant in the FALCON tube was discarded. i) 1 mL of 50% w / v PEG (50% w / v polyethylene glycol 6,000 (Hampton Research, #HR2-533)) was added, and the mixture was incubated in a 37°C water bath for 1 minute. j) The mixture was stirred with a 1 mL glass pipette for an additional minute. k) 2 mL of RPMI1640 medium (FCS-) was added to the above FALCON tube and mixed with a 10 mL pipette for 2 minutes. l) 7 mL of RPMI1640 medium (FCS-) was added to the above FALCON tube and mixed with a 10 mL pipette for 2 minutes. m) 30 mL of RPMI1640 medium (FCS-) was added to the above FALCON tube and mixed with a 10 mL pipette (total 40 mL). n) The FALCON tube was centrifuged at 1,500 rpm at room temperature for 5 minutes. o) The number of cells is 1 x 10 6 RPMI1640 medium (containing 10% FCS) was added to the FALCON tube so that the cell concentration became 50 / mL. p) The cell dispersion was plated in a 96-well microplate (round bottom, Corning, #3799) at 100 μL / well. q) The cells were incubated in a CO2 incubator at 5% CO2 concentration at 37°C for 24 hours.

[0039] (2) Experimental procedures from the second day onwards a) 2xHAT medium was prepared by mixing 2 mL of 50xHAT supplement (Thermo Fischer Scientific, #21060017) and 48 mL of RPMI1640 medium (containing 10% FCS). b) After incubation, the above 2×HAT medium was added to the 96-well microplate at 100 μL / well. c) The cells were incubated at 5% CO2 and 37°C using a CO2 incubator. d) During the incubation, 100 μL of the medium was removed from 200 μL per well every 3 days, and 100 μL of fresh 2×HAT medium was added. This was continued for 1 week. e) 2xHT medium was prepared by mixing 1 mL of 100xHT supplement (Thermo Fischer Scientific, #11067030) and 49 mL of RPMI1640 medium (containing 10% FCS). f) The medium in the 96-well microplate was replaced from 2×HAT medium to 2×HT medium.

[0040] 3. Screening of T cell hybridomas a) A portion of the T cell hybridomas produced was collected, and the expression of CD4, CD8, and T cell receptor β (TCRβ) was examined by flow cytometry. c) T cell hybridomas that were TCRβ positive and at least one of CD4 and CD8 positive, that is, T cell hybridomas that met any of the following criteria, were selected. TCRβ + CD4 + TCRβ + CD8 + TCRβ + CD4 + CD8 + d) The selected T cell hybridomas were cloned and preserved as monoclonal cells.

[0041] 4. Analysis of autoantigen reactivity of T cell hybridomas (1) Activation of T cell hybridomas a) Selected and stored T cell hybridomas were analyzed using Rag2 - / - The mice were administered the drug directly into the left and right salivary glands using a syringe. - / - After anesthetizing the mouse, it was placed on its back and the local area was disinfected with povidone-iodine solution. The midline of the throat was incised with scissors to confirm the location of the left and right salivary glands (submandibular glands). A dispersion (8 × 10 5 A single dose (20 μL) of the solution was injected into each of the left and right salivary glands using a syringe equipped with a 30 G needle, and the incision was sutured. b) Six days after injection of the T cell hybridomas into the salivary glands, the mice were sacrificed and the salivary glands were removed. c) The salivary glands were treated with enzymes to disaggregate the tissue cells, and the lymphocyte fraction in the salivary gland tissue was separated by density gradient centrifugation using Percoll II PLUS (GE HealthCare). This fraction contained the injected T cell hybridoma.

[0042] (2) IL-2 gene expression analysis of T cell hybridomas a) Total RNA was extracted from isolated T cell hybridomas and interleukin-2 (IL-2) expression was analyzed at the mRNA level by quantitative reverse transcription PCR (RT-qPCR, reverse transcription quantitative PCR). d) For comparison, IL-2 expression was also analyzed by RT-qPCR for unstimulated T cell hybridomas and T cell hybridomas stimulated with general stimulation (T cell hybridomas stimulated with 10 ng / ml PMA (Phorbol 12-myristate 13-acetate, Sigma-Aldrich #P1585) and 1 μM Ionomysin (Sigma-Aldrich #I9657) in a 48-well flat bottom plate (FALCON, #353078) at 37°C for 4 hours). Equipment: ABI 7500 fast system (Applied Biosystems) Primers used: IL-2: TaqMan gene expression assay primer Mm00434256_m1 (Applied Biosystems), endogenous control HPRT: TaqMan gene expression assay primer Mm00446968_m1 (Applied Biosystems) Reaction conditions: TaqMan gene expression assay kit used

[0043] 5.Results FIG. 4 shows the amount of IL-2 mRNA (IL-2 gene expression level) in unstimulated T cell hybridomas, salivary gland-transferred T cell hybridomas, and general stimulatory T cell hybridomas. The results shown in Fig. 4 show that T cell hybridomas derived from lymph node T cells from blood cell-specific SATB1-deficient mice express Rag2 - / - This suggests that it was activated in response to self-antigens in the mouse salivary glands.

[0044] [References] Reference 1: Kondo, M., Y. Tanaka, T. Kuwabara, T. Naito, T. Kohwi-Shigematsu, and A. Watanabe. 2016. SATB1 plays a critical role in establishment of immune tolerance. J. Immunol. 196: 563-572.

[0045] [Registered trademark] "FALCON" and "Falcon" are registered trademarks of Corning Incorporated in Japan (Trademark Registration No. 1458331, Trademark Registration No. 1458332). "EASYSEP" is a registered trademark of StemCell Technologies Canada Inc. in Japan (Trademark Registration No. 5540296). "GIBCO" is a registered trademark of Life Technologies Corporation in Japan (Trademark Registration No. 4891372). "FACSAria" is a registered trademark of Becton, Dickinson & Company in Japan (Trademark Registration No. 4703652). [Industrial Applicability]

[0046] The method of the present invention for identifying T cells activated in damaged organs of autoimmune diseases is a technique that is likely to lead to the development of new diagnostic and therapeutic methods for autoimmune diseases. More specifically, the autoreactive T cell hybridomas detected by the method of the present invention can be used to develop therapeutic, therapeutic, or diagnostic methods for autoimmune diseases. [Explanation of symbols]

[0047] 101…Salivary gland 102…Salivary gland extract 103...antigen presenting cells 104…T cell hybridoma 105…Petrile dish 106…Rag2 - / - mouse 107…Hematopoietic cell-specific SATB1 knockout mice 108…Cervical lymph nodes 109…cervical lymph node T cells + thymoma cells

Claims

1. a T cell hybridoma preparation step of fusing a T cell derived from a mammalian subject suffering from an autoimmune disease accompanied by tissue / cell damage with a tumor cell to prepare a T cell hybridoma; a T cell hybridoma transfer step of transferring the prepared T cell hybridoma into a tissue or organ damaged by the autoimmune disease of an immunodeficient non-human mammal; A T cell hybridoma recovery step of recovering T cell hybridomas from the tissue or organ; and A T cell hybridoma detection step of quantifying the expression level of interleukin 2 gene in the recovered T cell hybridoma. A method for detecting autoreactive T cells, comprising:

2. The method for detecting autoreactive T cells according to claim 1 , wherein the immunodeficient non-human mammal is a non-human mammal homozygously deficient in one or both of the Rag1 gene and the Rag2 gene.

3. The method for detecting autoreactive T cells according to claim 1 or 2, wherein the T cells derived from a mammal suffering from an autoimmune disease accompanied by tissue / cell damage are cervical lymph node T cells.

4. The method for detecting autoreactive T cells according to claim 1 or 2, wherein the autoimmune disease accompanied by tissue / cell damage is Sjogren's syndrome.

5. The method for detecting autoreactive T cells according to claim 1 or 2, wherein the tissue or organ damaged by the autoimmune disease is an exocrine gland tissue.

6. The method for detecting autoreactive T cells according to claim 1 or 2, wherein the tissue or organ damaged by the autoimmune disease of the immunodeficient non-human mammal is an organoid.

7. The method for detecting autoreactive T cells according to claim 1 or 2, wherein the immunodeficient non-human mammal is an immunodeficient xenogeneic chimeric mammal.

8. An autoreactive T cell hybridoma detected by the method for detecting autoreactive T cells according to claim 1 or 2.

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