WO2021024742A1 - 細胞傷害アッセイ方法 - Google Patents

細胞傷害アッセイ方法 Download PDF

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WO2021024742A1
WO2021024742A1 PCT/JP2020/027655 JP2020027655W WO2021024742A1 WO 2021024742 A1 WO2021024742 A1 WO 2021024742A1 JP 2020027655 W JP2020027655 W JP 2020027655W WO 2021024742 A1 WO2021024742 A1 WO 2021024742A1
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cells
cytotoxic
cell
human animal
assay method
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PCT/JP2020/027655
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French (fr)
Japanese (ja)
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奈緒子 井川
明允 齊藤
裕輝 市村
美和子 正保
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国立大学法人筑波大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination

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  • the present invention relates to a cytotoxic assay method. More specifically, the present invention relates to a cytotoxic assay method, a method for screening a therapeutic agent for an immune-related disease, and a kit for screening a therapeutic agent for an immune-related disease.
  • the present application claims priority based on Japanese Patent Application No. 2019-144490 filed in Japan on August 6, 2019, the contents of which are incorporated herein by reference.
  • cytotoxic T cells As an in vitro experimental method for measuring the activity of cytotoxic T cells, a cytotoxic assay is known in which cytotoxic T cells and target cells are co-cultured to detect the number of damaged cells (for example, non-cytotoxic T cells). See Patent Document 1).
  • cytotoxic T cells it is necessary to activate the cytotoxic T cells in advance, and it is necessary to prepare cells that stimulate the cytotoxic T cells in addition to the cytotoxic T cells.
  • cells that stimulate cytotoxic T cells spleen cells of mice with different major histocompatibility complex (MHC) phenotypes and spleen cells of allogeneic mice immunized with antigen are used, although they differ depending on the experimental system. It is a very complicated experimental system.
  • MHC major histocompatibility complex
  • Non-Patent Document 2 a simple cytotoxic assay system that does not require stimulation of cytotoxic T cells.
  • an object of the present invention is to provide a cytotoxic assay method capable of preparing more cytotoxic T cells.
  • the present invention includes the following aspects.
  • T cell receptor gene introduction A step of co-culturing a cytotoxic T cell derived from a non-human animal and a target cell labeled with a labeling substance and recognized as the cytotoxic T cell in a buffer solution.
  • a step of measuring the labeling substance released from the target cell into the buffer solution the step of measuring the amount of the labeling substance indicating the cytotoxic activity of the cytotoxic T cell.
  • a cytotoxic assay method wherein the cytotoxic T cells are cells derived from the lymph nodes of the non-human animal.
  • the cytotoxic assay method according to [1] wherein the T cell receptor gene-introduced non-human animal has a gene mutation lacking T cells.
  • the target cells are EL-4 cell lines derived from mouse T-cell lymphoma, which presented a peptide-MHC class I complex consisting of the amino acid sequence of SIINFEKL (SEQ ID NO: 1), [1] to [4]. ] The cytotoxic assay method according to any one of.
  • the measured amount of the labeling substance is the labeling substance in the absence of the test substance.
  • a significant increase or decrease in comparison to the amount of the test substance comprises a step indicating that the test substance is a therapeutic agent for an immune-related disease, wherein the cytotoxic T cells are lymph nodes of the non-human animal.
  • a method for screening a therapeutic agent for an immune-related disease which is a cell derived from a node. [9] For screening of therapeutic agents for immune-related diseases, including cytotoxic T cells derived from lymph nodes of non-human animals into which a T cell receptor gene has been introduced and target cells recognized by the cytotoxic T cells. kit.
  • cytotoxic assay method capable of preparing more cytotoxic T cells.
  • the present invention buffers cytotoxic T cells derived from a non-human animal into which a T cell receptor gene has been introduced and target cells labeled with a labeling substance and recognized by the cytotoxic T cells.
  • a step of co-culturing in the cells and a step of measuring the labeling substance released from the target cells into the buffer, and the measured amount of the labeling substance determines the cytotoxic activity of the cytotoxic T cells.
  • a cytotoxic assay method comprising the steps shown, wherein the cytotoxic T cells are cells derived from the lymph nodes of the non-human animal.
  • Non-Patent Document 2 cytotoxic T cells were recovered from the spleen of a non-human animal.
  • the inventors have found that collecting cytotoxic T cells from the lymph nodes of non-human animals can prepare more cytotoxic T cells from the same number of non-human animals. Completed the invention.
  • the number of cells recovered from the spleen of a non-human animal and the number of cells recovered from the lymph nodes of a non-human animal are not so different.
  • lymph node-derived cytotoxic T cells were more proliferative than spleen-derived cytotoxic T cells, resulting in more. It was clarified that cytotoxic T cells of the above were obtained.
  • cytotoxic assay method of the present embodiment cells derived from non-human animals into which the T cell receptor gene has been introduced are used as cytotoxic T cells. This allows all cytotoxic T cells to have the introduced T cell receptor, simplifying the cytotoxic assay system.
  • non-human animals examples include mice, rats and the like.
  • the T cell receptor gene is preferably introduced so that it can be expressed in cytotoxic T cells.
  • the T cell receptor gene to be transferred into a non-human animal is not particularly limited, but it is preferable that the antigen peptide-major histocompatibility complex (Major Histocompatibility Complex, MHC) complex to be recognized has been clarified.
  • MHC Major Histocompatibility Complex
  • T cells into which such a T cell receptor gene has been introduced include a complex of a peptide consisting of the 258-265th amino acid sequence (SIINFEKL, SEQ ID NO: 1) of obalbumin and an MHC class I molecule. Recognizing OT-I cells, OT-II cells recognizing a complex of a peptide consisting of amino acid sequence 323-339 of obalbumin and MHC class II molecule, amino acid sequence 9-23 of insulin ⁇ chain Examples include NOD T cells that recognize a complex of MHC molecule and MHC molecule.
  • T cell receptor gene-introduced non-human animals include OT-I mice, OT-II mice, NOD mice and the like.
  • the T cell receptor gene-introduced non-human animal has a gene mutation lacking T cells.
  • a non-human animal having a T cell receptor gene transfer having such a mutation has no T cells other than the T cell having the gene-transfected T cell receptor. Therefore, the cytotoxic T cells prepared from this non-human animal will have only the surely introduced T cell receptor, and the cytotoxic assay system can be simplified.
  • T cell-deficient gene-abnormal mouse examples include a Rag1 gene-deficient mouse, a Rag2 gene-deficient mouse, and a SCID mouse.
  • the target cell recognized by the cytotoxic T cell is a cell that receives cytotoxicity by the above-mentioned cytotoxic T cell.
  • the target cell is a complex of a peptide consisting of the amino acid sequence of SIINFEKL (SEQ ID NO: 1) and an MHC class I molecule. Examples of cells presenting on the cell surface.
  • a peptide-MHC complex is brought to the cell surface by contacting an EL-4 cell line derived from mouse T cell lymphoma with a peptide consisting of the amino acid sequence of SIINFEKL (SEQ ID NO: 1). Examples thereof include the presented EL-4 cell line.
  • target cells are labeled with a labeling substance before co-culturing with effector cells (cytotoxic T cells).
  • cytotoxic T cells effector cells
  • the labeling substance examples include radioactive isotopes and fluorescent substances. It is preferable that the labeling substance is a fluorescent substance because the cytotoxicity assay can be performed more easily.
  • the radioactive isotope examples include 51 Cr.
  • the fluorescent substance examples include Fluorescein (CAS number: 1461-15-0), Carboxyfluorescein succinidiyl ester (CFSE, CAS number: 117548-22-8), and Carboxy fluorescein dialatete (CFDA, CAS number: 124387-). , Fluorescein diacenate (FDA, CAS number: 596-09-8), Tetramethylrhodamine substance (TMRM, CAS number: 115532-50-8) and the like.
  • Calcein-AM CAS number: 14854-34-1
  • CAS number: 14854-34-1 CAS number: 14854-34-1
  • AM acetoxymethyl esterified
  • Examples of the method of labeling the target cell with the labeling substance include adding the labeling substance to the medium of the target cell.
  • cytotoxic T cells derived from a non-human animal having a T cell receptor gene introduced and target cells labeled with a labeling substance are co-cultured in a buffer solution.
  • the buffer solution is not particularly limited and may be, for example, a medium.
  • serum may be added to the buffer solution to suppress damage to cells.
  • the labeling substance released from the target cells into the buffer solution is measured.
  • the labeling substance can be appropriately measured by a measuring method corresponding to the labeling substance.
  • a measuring method corresponding to the labeling substance For example, if the labeling substance is a radioisotope, a scintillation counter may be used.
  • the labeling substance is a fluorescent substance, a fluorescent plate reader or the like can be used.
  • the amount of labeling substance released from the target cells into the buffer solution corresponds to the number of target cells that have been cytotoxicized by cytotoxic T cells. Therefore, the cytotoxic activity of cytotoxic T cells can be evaluated by measuring the labeling substance released in the buffer solution.
  • cytotoxic T cells and target cells 2.5 ⁇ 10 6 or less cytotoxic T cells may be co-cultured with 7.5 ⁇ 10 4 or less target cells.
  • This cell number is about half the number of cells used in the cytotoxic assay described in Non-Patent Document 2.
  • the inventors have shown that a reliable cytotoxic assay can be performed even with the above cell numbers. If a cytotoxic assay can be performed with a smaller number of cells, more assays can be performed from the same number of non-human animals.
  • the T cell receptor gene-introduced non-human animal may have a mutation in a gene involved in cytotoxicity.
  • genes involved in cell injury include perforin gene, granzyme A gene, granzyme B gene, FasL gene, Tumor necrosis factor (TNF) gene, Interferon-gamma (IFN- ⁇ ) gene and the like.
  • the mutation in the gene involved in cytotoxicity may be a deletion mutation or a mutation that changes the activity of the gene product.
  • the present invention uses a T cell receptor gene-introduced non-human animal-derived cytotoxic T cell and a target cell labeled with a labeling substance and recognized by the cytotoxic T cell as a test substance.
  • the measured amount of the labeling substance is not the test substance.
  • the cytotoxic T cells include a step of indicating that the test substance is a therapeutic agent for an immune-related disease by significantly increasing or decreasing the amount of the labeling substance in the presence of the cell.
  • a method for screening a therapeutic agent for an immune-related disease which is a cell derived from a lymph node of the non-human animal.
  • a therapeutic agent for an immune-related disease can be screened.
  • Immune-related diseases include cancer, collagen disease, infectious diseases, graft-versus-host disease and the like.
  • Substances that improve the cytotoxicity of cytotoxic T cells can be applied, for example, to cancer immunotherapy and infectious disease treatment.
  • a substance that reduces the cytotoxicity of cytotoxic T cells can be used as a therapeutic agent for, for example, collagen disease, graft-versus-host disease, and the like.
  • test substance is not particularly limited, and examples thereof include a natural compound library, a synthetic compound library, an existing drug library, and a metabolite library.
  • the cytotoxic T cells, non-human animals, T cell receptor genes, target cells, target substances, co-cultured cytotoxic T cells, the number of target cells, and the like are described above. Similar to the one. Further, the T cell receptor gene-introduced non-human animal may have a mutation in a gene involved in cytotoxicity, and the mutation of a gene involved in cytotoxicity is the same as described above.
  • the present invention comprises an immune-related disorder comprising cytotoxic T cells derived from lymph nodes of a non-human animal into which a T cell receptor gene has been introduced and target cells recognized by the cytotoxic T cells.
  • an immune-related disorder comprising cytotoxic T cells derived from lymph nodes of a non-human animal into which a T cell receptor gene has been introduced and target cells recognized by the cytotoxic T cells.
  • the kit of the present embodiment can suitably screen the therapeutic agent for the above-mentioned immune-related diseases.
  • the kit of the present embodiment may further contain a labeling substance, a reagent for measuring the labeling substance, and the like.
  • the cytotoxic T cells, non-human animals, T cell receptor genes, target cells, target substances, etc. are the same as those described above.
  • the T cell receptor gene-introduced non-human animal may have a mutation in a gene involved in cytotoxicity, and the mutation of a gene involved in cytotoxicity is the same as described above.
  • the top part of the inner cylinder was rinsed with a small amount of 10% fetal bovine serum (FBS) -RPMI medium, and the cell suspension was collected. Subsequently, the collected cell suspension was passed through a 100 ⁇ m filter and placed in a 50 mL tube.
  • FBS fetal bovine serum
  • 10% FBS-RPMI medium was added to make up to 50 mL, mixed by inversion, and then centrifuged to discard the supernatant. Subsequently, 10% FBS-RPMI medium was added to loosen the cells, the cells were scalpel-up to 50 mL, and the cells were centrifuged again to discard the supernatant.
  • the cRPMI medium having the composition shown in Table 1 below was added and mixed. Subsequently, a 40 ⁇ m filter was placed in a new 50 mL tube and passed through the cell suspension. Subsequently, the number of cells was counted using a part of the cell suspension. Subsequently, cRPMI medium was added to adjust the cell density to 5 ⁇ 10 5 cells / mL. Approximately 9 ⁇ 10 6 cells were recovered from the spleen per mouse.
  • peptide (1 ⁇ g / ⁇ L) consisting of the amino acid sequence of SIINFEKL (SEQ ID NO: 1), 30 ⁇ L of mouse recombinant interleukin (IL) -2 (10 ⁇ g / mL), and mouse recombinant IL per 10 mL of cRPMI medium.
  • IL interleukin
  • -4 (10 ⁇ g / mL) was added in an amount of 30 ⁇ L and mixed.
  • the cell suspension was seeded on a 6-well plate at 5 mL / well and cultured in a CO 2 incubator at 37 ° C. for 2 days.
  • mouse recombinant IL-2 (10 ⁇ g / mL) and 30 ⁇ L of mouse recombinant IL-4 (10 ⁇ g / mL) were added and mixed per 10 mL of cRPMI medium.
  • the cell suspension was seeded on a 6-well plate at 5 mL / well and cultured in a CO 2 incubator at 37 ° C. for 2 days.
  • HBSSF a buffer
  • FBS FBS
  • HBSS HBSS
  • cytotoxic T cells approximately 7 ⁇ 10 6 cytotoxic T cells were obtained from the spleen per mouse. It is clear that the spleen-derived cytotoxic T cells have less cell proliferation and the number of finally obtained cytotoxic T cells is smaller than that of the lymph node-derived cytotoxic T cells described later. became.
  • cytotoxic T cells derived from lymph nodes On day 1, mesenteric lymph nodes, cervical lymph nodes, axillary lymph nodes, and inguinal lymph nodes were removed from OT-I +/+ Rag1 -/- GFP +/+ mice and 10 cm with a 100 ⁇ m filter. Grinded on a planet using the top of the inner cylinder of a 1 mL syringe.
  • the top part of the inner cylinder was rinsed with a small amount of 10% fetal bovine serum (FBS) -RPMI medium, and the cell suspension was collected. Subsequently, the collected cell suspension was passed through a 100 ⁇ m filter and placed in a 50 mL tube.
  • FBS fetal bovine serum
  • 10% FBS-RPMI medium was added to make up to 50 mL, mixed by inversion, and then centrifuged to discard the supernatant. Subsequently, 10% FBS-RPMI medium was added to loosen the cells, the cells were scalpel-up to 50 mL, and the cells were centrifuged again to discard the supernatant.
  • cRPMI medium was added and mixed. Subsequently, a 40 ⁇ m filter was placed in a new 50 mL tube and passed through the cell suspension. Subsequently, the number of cells was counted using a part of the cell suspension. Subsequently, cRPMI medium was added to adjust the cell density to 5 ⁇ 10 5 cells / mL. Approximately 6 ⁇ 10 5 cells were recovered from the lymph nodes per mouse.
  • lymph nodes per mouse one animal, about 1.5 ⁇ 10 7 cells cytotoxic T cells were obtained. Lymph node-derived cytotoxic T cells have more cell proliferation than the spleen-derived cytotoxic T cells described above, and the number of finally obtained cytotoxic T cells may be significantly higher. It became clear.
  • OT-I +/+ Rag1 ⁇ / ⁇ Gzmb ⁇ / ⁇ GFP +/ + mice, OT-I +/+ Rag1 ⁇ / ⁇ GFP +/+ mice and Granzyme A gene (Gzma) knockout mice The mesenteric lymph node, cervical lymph node, axillary lymph node and inguinal lymph node were excised from each of the obtained OT-I +/+ Rag1 ⁇ / ⁇ Gzma ⁇ / ⁇ GFP + / + mice and tested. Cytotoxic T cells were prepared in the same manner as in Example 1 up to the 6th day, and effector cells were obtained.
  • ⁇ Preparation of target cells The EL-4 cell line was adjusted to a cell density of 2.5 ⁇ 10 5 cells / mL and suspended in 10% FBS-DMEM medium. Subsequently, 10 ⁇ L of mouse recombinant interferon (IFN) - ⁇ (100 ng / mL) was added per 20 mL of the cell suspension and mixed. Subsequently, the cell suspension was placed in a T-flask and cultured in a CO 2 incubator at 37 ° C. for 2 days.
  • IFN mouse recombinant interferon
  • the cell suspension was transferred to a 50 mL tube and centrifuged. Subsequently, the supernatant was discarded, washed twice with 40 mL of HBSSF, and 1 mL of HBSSF was added to suspend the cells. Subsequently, the number of cells was counted using a part of the cell suspension. Subsequently, HBSSF was added to adjust the cell density to 3.0 ⁇ 10 6 cells / mL.
  • the cell suspension was centrifuged and the supernatant was discarded. At this point, the cells were colored yellow to yellow-green. Subsequently, it was washed 3 times with 40 mL of HBSSF. Subsequently, HBSSF was added to adjust the cell density to 7.5 ⁇ 10 4 cells / mL, and target cells were obtained.
  • ⁇ Mixing effector cells and target cells To a 96-well plate, 100 ⁇ L of 7.5 ⁇ 10 4 cells / mL target cells were added per well. Subsequently, the wells containing target cells, 2.5 ⁇ 10 6 cells /ML,7.5 ⁇ 10 5 pieces /ML,2.5 ⁇ 10 5 pieces /ML,7.5 ⁇ 10 4 cells / mL Diluted series of effector cells of 100 ⁇ L each was added. The experiment was performed in triplicate.
  • each cell was cultured at 37 ° C. in the absence of CO 2 for 3 hours. Subsequently, the 96-well plate was centrifuged, and 100 ⁇ L of the supernatant of each well was transferred to a new 96-well plate, being careful not to contaminate the cell pellet.
  • Dissolution rate (%) (sample value-negative control value) / (positive control value-negative control value) x 100 ... (1)
  • FIG. 1 is a graph showing the results of a cytotoxic assay.
  • E indicates an effector cell
  • T indicates a target cell.
  • wild type indicates that it is the result of using cytotoxic T cells derived from OT-I +/+ Rag1 -/- GFP +/+ mice, and "perforin-KO” is OT-I +/.
  • cytotoxic assay can be performed by co-culturing 2.5 ⁇ 10 6 or less cytotoxic T cells with 7.5 ⁇ 10 4 or less target cells. This cell number is about half that of conventional cytotoxic assay systems.
  • effector cells and target cells were prepared in the same manner as in Experimental Example 2.
  • As the effector cells cytotoxic T cells derived from OT-I +/+ Rag1 -/- GFP +/+ mice were used.
  • each cell was cultured at 37 ° C. in the absence of CO 2 for 3 hours. Subsequently, the 96-well plate was centrifuged, and 100 ⁇ L of the supernatant of each well was transferred to a new 96-well plate, being careful not to contaminate the cell pellet.
  • the fluorescence of Calcein was measured at an excitation wavelength of 485 nm and a fluorescence wavelength of 535 nm using a fluorescent plate reader (product name “Varioscan LUX”, Thermo Fisher Scientific Co., Ltd.). Subsequently, the dissolution rate (%) was calculated based on the above formula (1).
  • FIG. 2 is a graph showing the results of the cytotoxic assay.
  • control indicates that it is the result of a sample to which the recombinant Serpina3n protein was not added.
  • the cytotoxic activity of cytotoxic T cells decreases depending on the concentration of Serpina3n protein. This result is consistent with the conventional findings and further supports that the cytotoxic assay system of this experimental example is functioning effectively.
  • this result indicates that a drug that affects the cytotoxic activity of cytotoxic T cells can be screened by adding a test substance instead of Serpina3n and performing a cytotoxic assay.
  • cytotoxic assay method capable of preparing more cytotoxic T cells.

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PCT/JP2020/027655 2019-08-06 2020-07-16 細胞傷害アッセイ方法 WO2021024742A1 (ja)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005087215A (ja) * 1998-12-23 2005-04-07 Pfizer Inc Ｃｔｌａ−４に対するヒトモノクローナル抗体
JP2010248200A (ja) * 2001-03-14 2010-11-04 Dako Denmark As 新規なｍｈｃ分子構築物、ならびに診断および処置のためにこれらの構築物を用いる方法、ならびにｍｈｃ分子の使用
JP2012082213A (ja) * 2003-01-30 2012-04-26 Survac Aps スルビビン誘導ペプチドおよびその使用
JP2016535060A (ja) * 2013-08-27 2016-11-10 ドイチェス クレブスフォルシュングスツェントルムＤｅｕｔｓｃｈｅｓ Ｋｒｅｂｓｆｏｒｓｃｈｕｎｇｓｚｅｎｔｒｕｍ 細胞傷害性ｔ細胞応答の修飾物質

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005087215A (ja) * 1998-12-23 2005-04-07 Pfizer Inc Ｃｔｌａ−４に対するヒトモノクローナル抗体
JP2010248200A (ja) * 2001-03-14 2010-11-04 Dako Denmark As 新規なｍｈｃ分子構築物、ならびに診断および処置のためにこれらの構築物を用いる方法、ならびにｍｈｃ分子の使用
JP2012082213A (ja) * 2003-01-30 2012-04-26 Survac Aps スルビビン誘導ペプチドおよびその使用
JP2016535060A (ja) * 2013-08-27 2016-11-10 ドイチェス クレブスフォルシュングスツェントルムＤｅｕｔｓｃｈｅｓ Ｋｒｅｂｓｆｏｒｓｃｈｕｎｇｓｚｅｎｔｒｕｍ 細胞傷害性ｔ細胞応答の修飾物質

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SAITO AKIMASA, OKIYAMA NAOKO, KUBOTA NORIKO, NAKAMURA YOSHIYUKI, FUJISAWA YASUHIRO, WATANABE REI, ISHITSUKA YOSUKE, BLEACKLEY R. C: "Blockade of granzyme B remarkably improves mucocutaneous diseases with keratinocyte death in interface dermatitis", JOURNAL OF INVESTIGATIVE DERMATOLOGY, vol. 138, 2018, pages 2079 - 2083, XP055791309 *

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