WO2009145238A1 - Agent immunomodulateur et son utilisation - Google Patents

Agent immunomodulateur et son utilisation Download PDF

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WO2009145238A1
WO2009145238A1 PCT/JP2009/059728 JP2009059728W WO2009145238A1 WO 2009145238 A1 WO2009145238 A1 WO 2009145238A1 JP 2009059728 W JP2009059728 W JP 2009059728W WO 2009145238 A1 WO2009145238 A1 WO 2009145238A1
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positive
cells
cxcr3
cell
immune
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PCT/JP2009/059728
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鈴木　治彦
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国立大学法人名古屋大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464429—Molecules with a "CD" designation not provided for elsewhere
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/14—Decongestants or antiallergics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg

Definitions

  • the present invention relates to an immunomodulator and use thereof, and more particularly to a CD8-positive CXCR3-positive T cell and use thereof.
  • the immune system in animals such as humans is a biological defense mechanism that detects and eliminates foreign antigens such as pathogenic bacteria and mutated self-antigens such as cancer cells.
  • Such biological defense is due to the respective responses of cellular immunity by T cells and humoral immunity mainly consisting of B cells.
  • the immune system is established as a biological defense mechanism on a control system that maintains a balance between activation and suppression. For this reason, such a disruption of the control system may cause diseases such as allergic diseases, autoimmune diseases and carcinogenesis. Therefore, detecting and isolating cells that control the immune system is extremely useful for diagnosis, prevention and treatment of diseases.
  • Non-patent Documents 1 and 2 The basic control system of the immune system is thought to be borne by various T cells including CD4-positive cells and CD8-positive cells. CD25-positive CD4-positive cells suppress the progression of various autoimmune diseases and organ transplantation. It is known to be effective for later immune tolerance (Non-patent Documents 1 and 2). The present inventors have already separated CD8-positive CD122-positive T cells as CD8-positive regulatory T cells that control CD8-positive killer T cells and CD4-positive helper T cells (Patent Document 1, Non-Patent Document 3). And Non-Patent Document 4).
  • CD8-positive regulatory T cells were separated from other normal effector T cells by the expression of a marker molecule called CD122.
  • CD122 positive cells could not be found in CD8 positive T cells. Therefore, CD8 positive regulatory T cells have not yet been isolated in humans.
  • An object of the present invention is to provide a practical CD8-positive regulatory T cell and its use according to immune-related diseases in animals including humans.
  • the present inventor found several candidate genes, and among these, one of the chemokine receptors I got the knowledge that there is a CXCR3. Furthermore, by flow cytometry using a specific antibody, it was found that the CD122 positive cell population in the CD8 positive cell population is consistent with the CXCR3 positive cell population. The present inventor has further obtained the knowledge that a CXCR3-positive cell population exists in a CD122-positive cell population in humans and that the cell population functions as a CD8-positive regulatory T cell. The present invention has been made based on these findings. According to the present invention, the following means are provided.
  • an immunomodulator containing CD8-positive CXCR3-positive T cells is provided.
  • the CD8-positive CXCR3-positive T cells can have interleukin 10 production activity.
  • the CD8-positive CXCR3-positive T cells can be derived from primates.
  • the immunomodulator of the present invention can also be used as a prophylactic or therapeutic agent for immune-related disorders in which immunosuppression is effective for the prevention or treatment thereof.
  • the immune-related disorder may be an autoimmune disease or a rejection after organ transplantation.
  • a preventive or therapeutic agent for an immune-related disorder which comprises any of the above-mentioned immunomodulators and a pharmaceutically acceptable carrier.
  • the activation step is preferably a step of expressing interleukin 10 production activity of the CD8-positive CXCR3-positive T cells.
  • the activation step may be a step of culturing the CD8-positive CXCR3-positive T cells in the presence of a specific antigen to activate CD8-positive CXCR3-positive T cells having specificity for the antigen.
  • the antigen can be selected from the group consisting of a causative agent of autoimmune disease, a causative agent of allergic disease, and a donor-derived cell or a part thereof at the time of organ transplantation.
  • the method for producing activated CD8-positive CXCR3-positive T cells of the present invention may further comprise a step of selecting the activated CD8-positive CXCR3-positive T cells using IL10 production activity as an index. Furthermore, the method can further comprise a step of culturing and proliferating the activated CD8-positive CXCR3-positive T cells.
  • kits for detecting or separating CD8-positive regulatory T cells comprising a reagent for detecting or separating CD8-positive CXCR3-positive T cells.
  • a method for producing an agent for preventing or treating an immune-related disorder comprising the step of preparing CD8-positive CXCR3-positive T cells, and the CD8-positive CXCR3-positive T cells and a pharmaceutically acceptable carrier. And a step of mixing.
  • a screening method for a means for preventing or treating an immune-related disorder wherein one or more test conditions are applied to CD8-positive CXCR3-positive T cells to detect the immunosuppressive activity of the T cells.
  • a screening method comprising the steps of: In the screening method of the present invention, the one or more test conditions that enhance the immunosuppressive activity among the one or two or more test conditions are further used to prevent an immune-related disorder in which immunosuppression is effective or A step of selecting as a treatment means can also be provided. Furthermore, among the one or more test conditions, one or more test conditions that reduce the immunosuppressive activity are selected as a means for preventing or treating an immune-related disorder in which immune activation is effective. A process.
  • a method for preventing or treating an immune-related disorder comprising the step of administering CD8-positive CXCR3-positive T cells to an individual in need of prevention or treatment of the immune-related disorder.
  • a method for preventing or treating an immune-related disorder comprising the step of removing at least a part of CD8-positive CXCR3-positive T cells from an individual in need of prevention or treatment of the immune-related disorder. A method is provided.
  • FIG. 5 shows the expression profile of CXCR3 as a dot plot.
  • the percentage of cells showing IL10 production activity or not showing in all CD8 positive cells is shown in each panel. It is a graph which shows the average value of the number of IL10 producing cells in all the CD8 positive cells obtained from 3 times of experiments with the error bar of standard deviation. It is a graph which shows the IL10 density
  • the peak filled in gray represents CFSE fluorescence when cultured alone with CD8-positive CXCR3-negative T cells, and the thick open peak represents CFDSE fluorescence when cultured with CD8-positive CXCR3-positive T cells.
  • a dot plot developed with CD8 and CXCR3 and a dot plot developed with CD8 and CD122 are shown, respectively.
  • the percentage of CD8-positive CXCR3-positive T cells in each quadrant is shown inside each panel.
  • the left figure shows the expression profiles of CD4 and CD25 in human peripheral blood mononuclear cells as dot plots.
  • the right figure shows the expression profiles of CD25 and CXCR3 gated on the CD4 positive cell population. The percentage of cells in each quadrant is shown in the panel.
  • the gate position for obtaining CD8-positive CXCR3-negative T cells and CD8-positive CXCR3-positive T cells by a cell sorter is shown as a rectangular region.
  • the present invention relates to DC8-positive regulatory T cells and uses thereof, specifically, preventive or therapeutic agents for immune-related disorders containing the regulatory T cells and methods for producing the same, and immunosuppressive agents containing the regulatory T cells Further, the present invention relates to a method for screening a means for preventing or treating immune related disorders, and a method for preventing or treating immune related disorders.
  • the present invention is based on CD8-positive CXCR3-positive T cells isolated by the present inventors.
  • the present inventors have confirmed that CD8-positive CD122-positive cells isolated as CD8-positive control cells in mice correspond to CD8-positive CXCR3-positive cells in humans.
  • CD8-positive CXCR3-positive T cells can function as CD8-positive regulatory T cells in animals, preferably mammals, including at least mice that are non-primate mammals and humans that are primates.
  • CD8-positive CXCR3-positive T cells are considered to function as regulatory T cells when they have their own interleukin 10 (IL10) production activity. Therefore, it is possible to contribute to the control of the immune system based on the production activity and also based on suppression of IFN- ⁇ production of CD8 positive CXCR3 negative T cells as target cells. Furthermore, CD8 positive CXCR3 positive T cells suppress the production of interferon ⁇ (IFN- ⁇ ) from the target cells CD positive CD122 negative T cells or CD4 positive CD25 negative T cells and produce interleukin 2 (IL2) Suppression can contribute to the control of the immune system.
  • IFN- ⁇ interferon ⁇
  • such CD8-positive regulatory T cells and their progenitor cells could be isolated and identified.
  • immune-related disorders associated with excess or activation of regulatory T cells these can be removed, etc. It is possible to prevent or ameliorate the disorder, and to improve or prevent the disorder by supplementing or activating the immune-related disorder associated with the decrease or inactivation of regulatory T cells. be able to.
  • CD8 positive regulatory T cells could be isolated and identified, immunosuppression or immune activation can be achieved by finding test conditions that promote or suppress the immunoregulatory activity of the regulatory T cells. It becomes possible to screen effective preventive or therapeutic means for immune related disorders.
  • the immunomodulator of the present invention contains CD8-positive CXCR3-positive T cells.
  • the T cells have CD8-positive and CXCR3-positive phenotypes and belong to the CD8-positive CXCR3-positive cell population.
  • the T cell can function as a CD8-positive regulatory T cell when activated in vitro and in vivo.
  • the immunomodulator of the present invention can be used as an immunomodulator, particularly an immunosuppressant, based on the immunosuppressive ability of CD8 positive regulatory T cells.
  • regulatory T cells suppresses activation (proliferation, cytokine production, etc.) of target cells of the regulatory T cells when stimulated via a T cell receptor with an antigen or the like. It means a T cell having ability (immunosuppressive ability).
  • target cells targeted by CD8 positive CXCR3 positive T cells used in the present invention include all CD8 positive T cells and CD8 positive CXCR3 negative cells.
  • the CD8-positive CXCR3-positive T cells used in the present invention can self-produce interleukin 10 (IL10) as an effect (immunosuppressive ability) exerting factor when stimulated via a T cell receptor. Thereby, the production of IFN- ⁇ in the target cell can be suppressed.
  • the ability to produce IL10 can be detected and analyzed by, for example, flow cytometry or ELISA using an antibody specific for IL10. Further, the CD8-positive CXCR3-positive T cells used in the present invention can suppress the proliferation of target cells such as CD8-positive CXCR3-negative cells.
  • the phenotype of a cell when expressed by the presence / absence or strength of marker molecule (antigen) expression, unless otherwise specified, it is expressed by the presence / absence or strength of specific binding by an antibody to the marker molecule.
  • the determination of the phenotype of a cell based on the presence or absence of the marker molecule and its strength is usually performed by flow cytometry analysis using a specific antibody against the marker molecule.
  • “Positive” expression of a marker molecule means that the marker molecule is expressed on the cell surface (or in the cell) and specific binding by an antibody to the marker molecule can be confirmed.
  • the T cells used in the present invention are preferably derived from mammals.
  • the mammal is not particularly limited as long as the specific phenotypic cell population described above can be used as the CD8-positive regulatory T cell of the present invention, and examples thereof include rodents such as mice, rats, hamsters, guinea pigs, and rabbits. Examples include laboratory animals, domestic animals such as pigs, cows, goats, horses, sheep and minks, pets such as dogs and cats, and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans and chimpanzees.
  • the mammal is preferably a primate, more preferably a human.
  • T cells used in the present invention are CD8-positive CXCR3-positive T cells of any one of thymus, umbilical cord blood, and peripheral tissue. Therefore, CD8 positive regulatory T cells used in the present invention may be cells existing in peripheral tissues (tissues other than thymus and umbilical cord blood: for example, peripheral blood, spleen, lymph nodes, intestinal tract, liver, etc.). . Considering the ease of the preparation operation, cells in peripheral blood or spleen are preferably used.
  • CD8-positive CXCR3-positive T cells are preferably isolated and purified.
  • the T cells can be isolated and purified by known methods based on phenotypes and the like from various tissues such as mammals.
  • a mononuclear cell fraction is prepared from peripheral tissues (for example, peripheral blood).
  • the mononuclear cell fraction is prepared, for example, by density gradient centrifugation.
  • the mononuclear cell fraction is stained with a specific antibody against CD8 and / or CXCR3 labeled with a fluorescent dye or magnetic beads, and the desired fraction is isolated using a cell sorter or a magnetic column. Purify.
  • a cell sorter is preferably used.
  • CD8-positive CXCR3-positive T cells may be in an activated state in which IL10 is produced by applying an appropriate stimulus ex vivo. In this way, it can function as regulatory T cells in vivo immediately after administration. Such activated CD8-positive CXCR3-positive T cells can be detected and separated by the presence or absence of IL10 production. Hereinafter, production of activated CD8-positive CXCR3-positive T cells will be described.
  • the method for producing an activated CD8-positive CXCR3-positive T cell of the present invention comprises a step of preparing a CD8-positive CXCR3-positive T cell, and a substance capable of stimulating the T cell via a T-cell receptor. And culturing and activating. According to the production method of the present invention, activated CD8-positive CXCR3-positive T cells can be obtained, and a reliable and prompt effect is expected by administering the activated CD8-positive CXCR3-positive T cells.
  • the CD8-positive CXCR3-positive T cell preparation step can be performed by collecting CD8-positive CXCR3-positive T cells from an appropriate site of an appropriate source (for example, a human patient) as described above.
  • CD8 positive CXCR3 positive T cells are preferably isolated and purified as described above.
  • a T cell receptor stimulating substance capable of applying stimulation via the T cell receptor is used.
  • various antigens that can be used for activating CD8-positive CXCR3-positive T cells are used as such substances.
  • the activation step can be carried out by culturing CD8-positive CXCR3-positive T cells in the presence of such a T cell receptor stimulating substance.
  • the culture conditions in the activation step are not limited as long as CD8-positive CXCR3-positive T cells can be activated, and can be appropriately selected from conditions known to those skilled in the art for lymphocyte culture, or can be appropriately modified and used.
  • an antigen comprehensively means substances that can be recognized by an antigen receptor (eg, a T cell receptor) on cultured cells and can stimulate cells via the receptor.
  • antigens include not only antigen molecules such as peptides, proteins, lipids and glycolipids, but also immunological non-self cells, antigen receptor constituent molecules (CD3, TCR ⁇ , TCR ⁇ , etc.) and costimulatory molecules (CD28, etc.) May contain an antigen mimic such as a superantigen (eg, an anti-human CD3 antibody, OKT-3).
  • an antigen mimic such as a superantigen (eg, an anti-human CD3 antibody, OKT-3).
  • the activation step can be carried out in various modes depending on the specificity of the activated CD8-positive CXCR3-positive T cells to be obtained. For example, when maintaining the diversity of the antigen receptor repertoire possessed by the above-mentioned CD8-positive CXCR3-positive T cell population to be cultured, and producing a regulatory T cell population reflecting the diversity Include CD8-positive CXCR3-positive T cells that recognize antigen-receptor molecules (CD3, TCR ⁇ , TCR ⁇ , etc.) and other agonistic antibodies (eg, anti-human CD3 antibody OKT-3), costimulatory molecules (CD28 Antigen mimics such as superantigens (Blood, 104, p. 895-903, 2004, Blood, 104, p.
  • the antigen mimic can be used in combination of a plurality of types. For example, a combination of an agonistic antibody that recognizes CD3 and an agonistic antibody that recognizes CD28 can be used. By using the antigen mimic, a diverse population of regulatory T cells can be obtained.
  • CD8-positive CXCR3-positive T cells may be cultured in the presence of a specific antigen to obtain activated CD8-positive CXCR3-positive T cells having antigen specificity.
  • CD8-positive CXCR3-positive T cells specifically activated can be selected based on IL10 production activity. Thereby, an activated CD8-positive CXCR3-positive T cell population preferable for suppressing the enhancement of the immune system by a specific antigen can be obtained.
  • Antigen molecules include, for example, cell- and tissue-derived antigens such as histocompatibility antigens, causative antigens of allergic diseases, or causative antigens of autoimmune diseases (food-derived antigens, drugs or preparations expected to exhibit antigenicity) Substances, artificial organ-related substances, or modified products thereof (for example, heat-modified products)).
  • Histocompatibility antigens include major histocompatibility antigens (MHC antigens) and non-major histocompatibility antigens.
  • Substances that cause allergies include environmental / pollen antigens, fungal antigens, food antigens, artificial antigens, etc., for example, environmental / pollen antigens such as mites, house dust, cedar pollen and ragweed, and fungal antigens such as Candida, Arte Lunaria, Aspergillus, Cladosporium, Penicillium, etc., such as egg white, milk, soybean, flour, buckwheat, mackerel, sardine, horse mackerel, shrimp, crab, pork, beef, chicken meat, etc. And artificial organs.
  • Examples of the causative substance of the autoimmune disease include corresponding antigens of autoantibodies that cause the disease.
  • CD8-positive CXCR3-positive T cells Prior to collection of CD8-positive CXCR3-positive T cells from the collection source, a specific antigen is supplied to an individual to form CD8-positive CXCR3-positive T cells, and CD8-positive CXCR3-positive specific to the antigen is supplied. You may make it easy to collect T cells. In this way, activated CD8-positive CXCR3-positive T cells that can suppress the enhancement of the immune system due to antigens that may be exposed in the future can be collected.
  • immunological non-self such as organ transplantation
  • immunological non-self cells can be obtained by supplying the individual with specific immunological non-self cells or a part thereof that may be exposed to the immune system of the individual as the collection source.
  • CD8-positive CXCR3-positive T cells specific for the cells can be collected from the individual.
  • the immunological non-self cells are preferably inactivated by a known method such as irradiation with radiation (gamma rays or the like) or treatment with an anticancer agent (mitomycin C or the like).
  • the type of immunological non-self cells is not particularly limited, and cells derived from a desired tissue (for example, peripheral blood mononuclear cells (PBMC)) can be used.
  • PBMC peripheral blood mononuclear cells
  • donor-derived cells are selected if production of recipient regulatory T cells specific for the donor-derived cells is intended.
  • the donor-derived cell may be a cell derived from the same organ as the organ that can be transplanted or may be a cell derived from a different tissue.
  • antigen-presenting cells may be selected.
  • the antigen-presenting cell is not particularly limited as long as regulatory T cells can be produced by the method of the present invention.
  • antigen-presenting cells that are allogeneic with CD8-positive CXCR3-positive T cells for example, the T cells are derived from them).
  • Antigen-presenting cells obtained from an individual are used.
  • the type of antigen-presenting cell is not particularly limited as long as it has antigen-presenting ability and regulatory T cells can be produced by the method of the present invention.
  • PBMC, dendritic cells and the like are used.
  • Antigen-presenting cells are preferably inactivated by a method known per se, such as irradiation with radiation (gamma rays or the like) or treatment with an anticancer agent (mitomycin C or the like).
  • a step of selecting CD8-positive CXCR3-positive T cells activated by the activation step using IL10 production activity as an index can be provided. By doing so, a highly active CD8-positive CXCR3-positive T cell population that selectively contains activated CD8-positive CXCR3-positive T cells can be obtained.
  • an antigen-specific and highly active CD8-positive CXCR3-positive T cell population containing activated CD8-positive CXCR3-positive T cells that are selective for antigen specificity can be obtained. it can.
  • a cell sorter or the like is used. The selection process is performed as necessary.
  • a step of culturing and proliferating the activated CD8-positive CXCR3-positive T cells can be provided.
  • the proliferation step is set so that the activation state of CD8-positive CXCR3-positive T cells can be maintained, but is preferably performed in the absence of a T cell receptor stimulating substance such as an antigen and in the presence of IL2 or the like. . It is preferable that CD8-positive CXCR3-positive T cells activated by the proliferation step can be proliferated because a necessary amount of CD8-positive CXCR3-positive T cells can be obtained while suppressing the burden on the individual. In addition, a cell population with a higher immunomodulatory effect can be obtained by growing an antigen-specific and highly active CD8-positive CXCR3-positive T cell population.
  • a desired activated CD8-positive CXCR3-positive T cell population can be obtained by performing an activation step, and further, a selection step and / or a proliferation step as necessary.
  • the obtained CD8-positive CXCR3-positive T cell population has IL10 production activity, and can suppress the proliferation and IFN- ⁇ production activity of CD8-positive CXCR3-negative T cells as target cells.
  • the activated CD8-positive CXCR3-positive T cells are isolated and purified in the same manner as the CD8-positive CXCR3-positive T cells before activation.
  • the preventive or therapeutic agent for immune-related disorders of the present invention contains the immunomodulator of the present invention, that is, CD8-positive CXCR3-positive T cells (including activated cells, the same applies hereinafter) as an active ingredient.
  • the method for preventing or treating an immune-related disorder of the present invention can include a step of administering CD8-positive CXCR3-positive T cells to an individual who needs to prevent or treat an immune-related disorder.
  • the preventive or therapeutic agent of the present invention that is, CD8 positive CXCR3 positive T cells are administered to an individual who needs prevention or treatment of immune related disorders by increasing the amount or ratio of the T cells in the immune system.
  • a preventive or therapeutic effect based on the immunosuppressive effect of CD8 positive regulatory T cells can be obtained. That is, when the immune response is abnormally enhanced in vivo, when an undesirable immune response occurs in vivo, or when an undesirable immune response is predicted to occur in the future, etc.
  • CD8-positive CXCR3-positive cells By administering CD8-positive CXCR3-positive cells to the cells, abnormally enhanced immune responses can be suppressed, undesirable immune responses can be suppressed, or undesirable immune responses can be avoided.
  • the preventive or therapeutic agent and the preventive or therapeutic method of the present invention are effective for disorders in which immunosuppression is effective for the prevention or treatment.
  • immune-related disorders include autoimmune diseases (polymyositis, chronic rheumatism, systemic lupus erythematosis, systemic sclerosis, blistering, cutaneous lupus erythematosis, psoriasis, Crohn's disease, ulcerative colitis, autoimmune hepatitis, Multiple sclerosis, type 1 diabetes, aplastic anemia, etc., organ transplant rejection, allergic diseases (hay fever, food allergies, drug allergies, asthma, atopic dermatitis, eczema, food hypersensitivity, urticaria, (Allergic rhinitis, allergic conjunctivitis), graft-versus-host disease (GVHD), and infertility are useful for prevention and treatment.
  • autoimmune diseases polymyositis, chronic rheumatism, systemic lupus ery
  • administration of CD8-positive CXCR3-positive T cells as prevention or treatment of the above-mentioned various diseases can avoid or suppress an immune enhancement state that may occur due to the disease or accompanying it.
  • antigen-specific CD8-positive CXCR3-positive T cells activated by stimulation with antigens effective for the prevention or treatment from CD8-positive CXCR3-positive T cells derived from the patient are used. It may be administered. For example, prior to organ transplantation from an allogeneic donor, first, a donor-derived cell or a part thereof is administered to a recipient to obtain a CD8-positive CXCR3-positive T cell specific to the donor-derived cell in vivo. The T cells thus obtained are collected, activated and expanded as necessary, and then administered to the recipient to induce tolerance of the graft, avoid rejection of the graft, Can be promoted.
  • the CD8-positive CXCR3-positive T cells derived from the patient were specifically activated by culturing in the presence of the causative antigen of the autoimmune disease.
  • the autoimmune reaction can be suppressed.
  • CD8 positive CXCR3 positive T cells derived from the patient are cultured in the presence of allergic causative antigens to obtain CD8 positive CXCR3 positive T cells specific for the causative antigens, Allergic reaction can be suppressed by administering this T cell to a patient.
  • CD8-positive CXCR3-positive T cells derived from the patient are cultured in the presence of the partner-derived cells, so that CD8-positive specific to the partner-derived cells (or antigens) is obtained.
  • CD8-positive specific to the partner-derived cells or antigens
  • CXCR3-positive T cells By obtaining CXCR3-positive T cells and administering these T cells to a patient, induction of immune tolerance to a partner-derived cell (or antigen) or a fetus carrying the partner-derived antigen is avoided, and rejection to the fetus is avoided. Can promote the maintenance of pregnancy.
  • the preventive or therapeutic agent of the present invention is prepared by preparing an effective amount of CD8-positive CXCR3-positive T cells according to a technique well known to those skilled in the art, mixing the T cells and a pharmaceutically acceptable carrier, etc. It can be produced as an oral / parenteral formulation.
  • the immunomodulator of the present invention is usually produced as a parenteral preparation such as an injection, a suspension, and a drip.
  • Carriers that can be included in the parenteral preparations include, for example, aqueous solutions for injection such as physiological saline, isotonic solutions containing glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride). A liquid can be mentioned.
  • the immunomodulating agent of the present invention includes, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalkonium chloride, procaine, etc.), a stabilizer (eg, human serum). Albumin, polyethylene glycol, etc.), preservatives, antioxidants and the like. Since the preparation thus obtained is safe and has low toxicity, it can be administered to the above-mentioned mammals such as humans.
  • a buffer eg, phosphate buffer, sodium acetate buffer
  • a soothing agent eg, benzalkonium chloride, procaine, etc.
  • a stabilizer eg, human serum
  • Albumin polyethylene glycol, etc.
  • preservatives e.g, antioxidants and the like. Since the preparation thus obtained is safe and has low toxicity, it can be administered to the above-mentioned mammals such as humans.
  • the dose of the prophylactic or therapeutic agent of the present invention varies depending on the administration subject, target organ, symptom, administration method and the like.
  • An effective dose can be administered up to about 10 9 per day.
  • a step of removing at least a part of CD8-positive CXCR3-positive T cells from an individual in need of prevention or treatment of an immune-related disorder can be provided.
  • the immune system can be improved or activated.
  • the immune response against the tumor can be enhanced to prevent or treat the tumor.
  • an anti-CXCR3 antibody In order to remove CD8-positive CXCR3-positive T cells from an individual, it is possible to intravenously administer an anti-CXCR3 antibody. For example, for humans, it is effective to administer an anti-human CXCR3 antibody.
  • an antibody produced in a heterologous animal it is preferable to use a human-type antibody in which the Fc portion of the antibody is replaced with the Fc of a human antibody in order to suppress the immune response or suppress or avoid anaphylaxis. .
  • Such antibody production is well known to those skilled in the art.
  • the immune-related disorder in this aspect includes a disease in which immune activation is effective for prevention or treatment.
  • diseases include cancer in general.
  • the CXCR3 antibody can be used as an immune activator and a prophylactic or therapeutic agent for diseases effective for prevention or treatment with effective immune activation.
  • the kit of the present invention can contain a reagent for detecting or separating CD8-positive CXCR3-positive T cells.
  • CD8 positive regulatory T cells can be detected or separated from an appropriate collection source.
  • the kit of the present invention may contain labeled antibodies against CD8 and CXCR3 molecular markers in order to isolate or detect CD8 positive regulatory T cells.
  • a labeled antibody against IL10 for selecting activated CD8-positive CXCR3-positive T cells can be included.
  • various reagents (medium, IL2, etc.) for activating and culturing CD8-positive CXCR3-positive T cells can be included.
  • Such a detection or separation kit detects CD8-positive CXCR3-positive T cells such as in peripheral blood collected from an individual, and is used to evaluate the content of the cell and the antigen specificity of the cell. It can also be used as a kit for diagnosis of immune-related disorders such as the immune system status and autoimmune diseases. Such a detection or separation kit can also be used as a kit for using CD8-positive CXCR3-positive T cells as a preventive or therapeutic agent for immune-related disorders.
  • the screening method of the present invention can comprise a step of applying a plurality of test conditions to CD8-positive CXCR3-positive T cells and detecting the immunosuppressive activity of the T cells for each test condition.
  • test conditions that enhance or decrease the immunosuppressive activity of CD8-positive CXCR3-positive T cells can be selected.
  • the test condition may be, for example, contacting the test compound with a CD8-positive CXCR3-positive T cell, or may be gene transfer involving gene modification including knockout and / or expression of an appropriate gene, Environmental conditions such as temperature may be used.
  • the screening method may further include a step of selecting a test condition that enhances the immunosuppressive activity as a means for preventing or treating an immune-related disorder in which immunosuppression is effective.
  • a prophylactic or therapeutic means effective for an immune-related disorder of the immunosuppressive agent and the preventive or therapeutic agent of the present invention is provided.
  • the enhancement of immunosuppressive activity of CD8-positive CXCR3-positive T cells may be an increase in CD8-positive CXCR3-positive T cells, or may be an enhancement of the activity of CD8-positive CXCR3-positive T cells themselves.
  • the screening method may further include a step of selecting the test condition that reduces the immunosuppressive activity as a means for preventing or treating an immune-related disorder in which immune activation is effective.
  • an immune activator and a preventive or therapeutic means effective for various diseases including cancer in general in which immune activation is effective for prevention or treatment are provided.
  • the decrease in the immunosuppressive activity of CD8-positive CXCR3-positive T cells may be a decrease in CD8-positive CXCR3-positive T cells or a decrease in the activity of CD8-positive CXCR3-positive T cells themselves.
  • mice C57BL / 6 and BALB / c mice were purchased from SLC, Japan, and various weekly mice were prepared. CB-17 SCID mice were purchased from Clea Japan.
  • the human mononuclear cell fraction was collected by centrifuging peripheral blood collected from healthy volunteers with Ficoll-Paque Plus (Amersham Bioscience) and thoroughly washing with PBS. (antibody) All antibodies were obtained commercially.
  • Flow cytometry A FACS caliber flow cytometer (BD bioscience) was used for analysis, and a FACS vantage cell sorter (BD bioscience) was used for cell sorting.
  • CD8 positive cells were separated according to the protocol using an anti-CD8 antibody conjugated to magnetic microbeads and a column (Miltenyi Biotech).
  • Cell culture For each mouse and human derived cell, 2 ⁇ 10 5 cells in 200 ⁇ l complete medium (RPMI1640 + 10% FCS) containing 10 ng / .ml IL2 in one well with a flat bottom on a 96 well plate. And 50 ⁇ M 2-ME).
  • ELISA Regarding the IL10 concentration in the culture supernatant of mouse cells, an ELISA kit for mouse IL10 (R & D Systems) was used. SRL was requested for IL10 concentration in the culture supernatant of human cells.
  • CD8 positive CXCR3 positive T cells and CD8 positive CXCR3 negative T cells were collected from spleen cells of C57BL / 6 mice using an EFACS Vantage cell sorter. mRNA was extracted from the collected cells, and DNA microarray analysis was requested from Hokkaido System Science.
  • 10 or more genes showed more than twice the expression level in CD8 positive CD122 positive T cells than in CD8 positive CD122 negative T cells.
  • the CXCR3 gene was expressed more than 10 times in CD8 positive CD122 positive T cells than in CD8 positive CD122 negative T cells.
  • CXCR3-positive T cells As shown in FIGS. 1A and 1B, it was found that some of the CD8 positive T cells expressed CXCR3. It was also found that the proportion of CXCR3-positive T cells tends to depend on the individual age of the mouse. That is, newborn mice hardly had CD8 positive cells, but CD8 positive cells began to appear around one week old mice. Young mice contained a high percentage of CXCR3-positive cells, but a similar phenomenon was observed in CD122-positive cells. In addition, analysis of changes in the percentage of CXCR3-positive cells with the age of the mouse revealed that the pattern was similar to that in CD122-positive cells.
  • CXCR3 and CD122 in the CD8 positive cell group were analyzed. Specifically, the expression profiles of CD122 and CXCR3 were analyzed for cells that were gated to CD8 positive cells and the expression profiles of CD25 and CXCR3 were analyzed for cells that were gated to CD4 positive cells in 4-week-old mice by flow cytometry. The results are shown in FIG. 1C.
  • CD8 positive cells are divided into three cell populations based on the expression state of CD44 and CD62L (CD44 weak negative CD62L strong positive, CD44 strong positive CD62L weak positive, CD44 strong positive CD62L strong positive). And the expression profile of CXCR3 was confirmed. Specifically, CD8 positive cells collected from 6-week-old mice were stained with anti-CD44 antibody, anti-CD62L antibody, anti-CD122 antibody and anti-CXCR3 antibody using magnetic beads coated with anti-CD8 antibody.
  • the CD8 positive CD122 positive T cell population (CD44 strong positive CD62L strong positive T cell population) contains cells expressing both CD122 and CXCR3 at a high rate, and a naive T cell population (CD44 negative CD62L).
  • the strong positive T cell population was found to be CD8 positive CXCR3 negative T cells.
  • the effector memory cell population (CD44 strong positive CD62L weak positive T cell population) contains cells that express CXCR3 or CD122, but is clearly distinct from CD44 strong positive CD62L strong positive T cell population. It did not indicate a group.
  • FIG. 2A shows the results of analysis by flow cytometry after the bead treatment and before cell sorting and by flow cytometry after cell sorting.
  • the cells used in the experiment were collected from BALB / c mice by cell sorting, and their purity was confirmed.
  • the CD8-positive CXCR3-negative T cells prepared in this way were transplanted into CB-17CIDSCID mice with or without mixing with CD8-positive CD122-positive T cells.
  • mice that received these T cells were CD69 positive activated CD8 positive T cells and It becomes unhealthy with an increase in granulocytes in the spleen, and finally shows a tendency to form hematopoiesis predominantly granulocytes in which erythropoiesis is suppressed. Such a hematopoietic tendency can be a good marker for the lack of CD8 + CD122 + regulatory T cells. From the above, in this example, T cell activation was measured using CD69 expression of CD8 positive CXCR3 negative T cells and granulocyte increase in the spleen as indicators.
  • CXCR3 can be used as a marker for CD8-positive regulatory T cells in addition to CD122 in mice.
  • CD8-positive CXCR3-positive T cells and CD8-positive CXCR3-negative T cells were recovered from C57BL / 6 mice by cell sorting and cultured under stimulation with microbeads coated with anti-mouse CD3 antibody and anti-mouse CD28 antibody, respectively. did. After 48 hours, the cells were first stained with CD8 antibody, and then intracellular IL10 was detected using Cytofix / Cytopem (BD Bioscience).
  • IL10 concentration of each culture supernatant obtained by culturing in the same manner was measured by ELISA.
  • the results of flow cytometry for these cells are shown in FIGS. 3A and 3B.
  • the measurement result of IL10 by ELISA is shown in FIG. 3C.
  • IL10 is the most important effector produced by CD8 + CD122 + regulatory T cells.
  • FIG. 3C the same phenomenon was confirmed in the ELISA for the culture supernatant.
  • CD8-positive CXCR3-negative T cells collected from C57BL / 6 mice by cell sorting were first labeled with CFSE, and then cultured alone or with CD8-positive CXCR3-positive T cells. After culturing for 48 hours under stimulation with microbeads coated with anti-CD3 antibody and anti-CD28 antibody, the IFN- ⁇ production activity of CFSE cells (CD8-positive CXCR3-negative T cells) was examined by flow cytometry. The results are shown in FIGS. 3D and 3E. In addition, cell culture was performed in the same manner as described above, and cell proliferation was evaluated by a decrease in CFSE fluorescence. The result is shown in FIG. 3F.
  • CD8-positive CXCR3-negative T cells without CD8-positive CXCR3-positive T cells produced IFN- ⁇ .
  • FIG. 3E such IFN- ⁇ production is clearly suppressed when CD8-positive CXCR3-negative T cells are cultured together with CD8-positive CXCR3-positive T cells, and the regulatory activity of CD8-positive CXCR3-positive T cells.
  • FIG. 3F from the proliferation state of CD8 positive CXCR3 negative T cells measured by the decrease in CFSE fluorescence, CD8 positive when cocultured with CD8 positive CXCR3 positive T cells as compared with single culture.
  • CD8-positive CXCR3-positive T cells have a growth-suppressing ability with respect to CD8-positive CXCR3-negative T cells.
  • CD8 positive CXCR3 positive T cells and CD8 positive CXCR3 negative T cells is very similar to the relationship between CD8 positive CD122 positive T cells and CD8 positive CD122 negative T cells.
  • CD8-positive CD122-positive T cells could not be clearly confirmed in lymphocytes collected from human peripheral blood, but CD122-positive in low-intermediate level CD8-positive T cells (CD8 dim ). I found a cell. However, most of these CD8 dim CD122 positive cells were CD3 negative NK-like cells (data not shown). In contrast, as shown in FIGS. 4A and 4B, CXCR3-positive cells were clearly observed in the CD8 strong positive cell population. That is, the expression profile of CXCR3 was different from the CD122 expression profile in human peripheral blood lymphocytes. In addition, a positive correlation was observed in the expression of CD122 and CXCR3 in the mouse CD8 positive cell population (Example 1, FIG. 1C), whereas there was no expression of CD122 and CXCR3 in the human CD8 strong positive cell population. No correlation was found ( Figure 4B).
  • CD8-positive CXCR3-positive T cells are related to central memory cells and effector memory cells.
  • human cells were stained with anti-CD45RA antibody and anti-CCR7 antibody and subjected to flow cytometry. The results are shown in FIG. 4C.
  • CD8 positive CXCR3 positive T cells contain more CD45RA negative memory cells than CD8 positive CXCR3 negative T cells, but CD8 positive CXCR3 positive T cells and central memory cells (CD45RA negative CCR7 positive). There was no clear correlation with effector memory cells (CD45RA negative CCR7 negative).
  • the ratio of CXCR3 positive cells in all CD8 positive cell populations was analyzed by flow cytometry for the expression profiles of CD8 and CXCR3 on peripheral blood lymphocytes (mononuclear cells) collected from healthy volunteers of different ages. The result is shown in FIG. 4D.
  • CD4 and CD25 expression profiles of human peripheral blood lymphocytes were analyzed by flow cytometry, and CD25 and CXCR3 expression profiles of CD4-positive gated cell populations were analyzed by flow cytometry.
  • FIG. 4E The result is shown in FIG. 4E.
  • FIG. 4D it was suggested to some extent that relatively many CD8-positive CXCR3-positive T cells tend to exist in the young and old age groups.
  • the change in the ratio of CD8 positive CXCR3 positive T cells among all CD8 positive cells was not clearly observed as in mice (Example 1, FIG. 1A and FIG. 1B).
  • FIG. 4E in humans, as in the case of mice, there was no correlation between CD25 and CXCR3 in the CD4 positive cell population.
  • Example 3 In vitro functional analysis of human CD8-positive CXCR3-positive T cells as regulatory T cells. In Example 3, CD8 positive CXCR3 positive T cells functioned as regulatory T cells in mice. In this example, it was confirmed whether CD8-positive CXCR3-positive T cells function as regulatory T cells in humans.
  • the human peripheral blood mononuclear cell fraction was separated and subjected to cell sorting to collect CD8-positive CXCR3-positive T cells and CD8-positive CXCR3-negative T cells, respectively.
  • the result is shown in FIG. 5A.
  • CD8-positive CXCR3-positive T cells and CD8-positive CXCR3-negative T cells are cultured separately, and the culture supernatant
  • the IL10 concentration was measured by ELISA.
  • the cultured cells were first stained with an anti-CD8 antibody, and then intracellular IL10 was stained using Cytofix / Cytopem, and these cells were analyzed by flow cytometry. These results are shown in FIGS. 5B and 5C and 5D. As shown in FIGS. 5B to 5D, it was found that CD8-positive CXCR3-positive T cells produced more IL10 than CD8-positive CXCR3-positive T cells.
  • CD8-positive CXCR3-negative T cells CD8-positive CXCR3-negative T cells
  • CD8 positive CXCR3 negative T cells collected from human peripheral blood by cell sorting were first labeled with CFSE, and then cultured alone or with CD8 positive CXCR3 positive T cells. After culturing for 96 hours under stimulation with microbeads coated with anti-CD3 antibody and anti-CD28 antibody, staining with anti-CD8 antibody and intracellular staining with anti-human IFN- ⁇ antibody were performed to determine the IFN- ⁇ production activity It was evaluated by measurement. The results are shown in FIGS. 5E and 5F.
  • CD8-positive CXCR3-positive T cells function as regulatory T cells in a human in vitro experiment system. It was also found that CXCR3 can be used as a marker for human regulatory T cells instead of CD122 that is not expressed on human CD8-positive T cells. As already shown in Examples 2 and 3, it was shown that CD8-positive CXCR3-positive T cells are substantially equivalent as CD8-positive CD122-positive regulatory T cells in the mouse experimental system, but human CD8-positive CXCR3-positive T cells. The cells were found to be human counterparts of mouse CD8 + CD122 + regulatory T cells.
  • EAE mice Effect of administration of CD8 + CD122 + cells on human multiple sclerosis (MS) model mice (EAE mice)
  • MS multiple sclerosis
  • EAE mice mice
  • CD8 positive CD122 positive T cells were administered to EAE mice to evaluate improvement of symptoms.
  • EAE symptom evaluation was score 0: no symptom, score 1: tail or hindlimb weakness, score 2: tail and hindlimb weakness, score 3: incomplete paralysis of hind limbs, score 4: complete hind limbs Paralysis, score 5: Mice died.
  • CD8 + CD122 + cells or CD8 + CD122 ⁇ cells were separated and collected from mouse spleen cells using anti-CD8 antibody-bound magnetic beads (Milteny), and What was stained with the fluorescence-labeled anti-CD8 antibody and the fluorescence-labeled anti-CD122 antibody was recovered with a purity of 99% or more using a cell sorter (FACSVantage SE manufactured by BD), and the cells were injected from the tail vein of the mouse.
  • FIG. 6 shows the outline of the protocol and the results of immunizing B6 mice with MOG peptide to induce EAE, scoring symptoms, and following them.
  • CD8 + CD122 + regulatory T cells play an essential role in the recovery of EAE symptoms.
  • CD8 + CD122 + regulatory T cells correspond to human CD8 + CXCR3 + regulatory T cells, indicating that CD8 + CXCR3 + regulatory T cells can be used for the prevention or treatment of human MS It was.
  • IBD inflammatory bowel disease
  • CD4 + cells or CD8 + cells are separated and collected by magnetic beads (Milteny) bound with anti-CD4 or anti-CD8 antibody, and then fluorescently labeled anti-CD4, anti-CD45RB, anti-CD8 After staining with anti-CD122 antibody, the target cells were separated and recovered with high purity using a cell sorter (FACSVantage SE manufactured by BD).
  • FACSVantage SE manufactured by BD cell sorter
  • CD4 + CD45RB + cells come going to not include CD4 + regulatory T cells (CD4 + CD25 + cells). Symptoms (diarrhea, bloody stool, weight loss) will not occur if CD4 + CD45RB ⁇ cells (including CD4 + CD25 + regulatory T cells) are transferred at the same time. In addition, IBD symptoms did not occur when CD8 + CD122 + regulatory T cells collected from normal mice instead of CD4 + CD45RB ⁇ cells were transferred simultaneously with CD4 + CD45RB + cells.
  • CD8 + CD122 + regulatory T cells are effective in suppressing the onset of IBD
  • CD8 + CD122 + regulatory T cells CD8 + CXCR3 + regulatory T cells in humans
  • CD8 + CD122 + cells were purely collected using a cell sorter and stimulated with an anti-CD3 antibody (BDBiocoat, manufactured by BD) for 24 hours to be activated. Thereafter, it was found that the cells can be grown up to 100 times or more by maintaining and growing the cells in a medium containing only human recombinant IL-2 (50 ng / ml). This proliferated cell expresses IL-10, which is an essential molecule for CD8 + regulatory T cells to exert a suppressive action.
  • IL-10 an essential molecule for CD8 + regulatory T cells to exert a suppressive action.
  • IL-10 in the culture supernatant is expressed by ELISA (Quantikine mouse IL -10 measurement kit (manufactured by R & D) was confirmed, and it was found that mouse CD8 + regulatory T cells can be cultured and proliferated while maintaining the function as regulatory cells. . It is considered that human CD8 + CXCR3 + regulatory T cells can be cultured by the same method.

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Abstract

La présente invention concerne un lymphocyte T CD8-positif régulateur qui est plus pratique pour une maladie immunitaire chez un animal y compris un corps humain. Un lymphocyte T CD8-positif CXCR3-positif est utilisé en tant qu’agent immunomodulateur.
PCT/JP2009/059728 2008-05-27 2009-05-27 Agent immunomodulateur et son utilisation WO2009145238A1 (fr)

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