WO2008126940A1 - Nouveau lymphocyte t - Google Patents

Nouveau lymphocyte t Download PDF

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WO2008126940A1
WO2008126940A1 PCT/JP2008/057460 JP2008057460W WO2008126940A1 WO 2008126940 A1 WO2008126940 A1 WO 2008126940A1 JP 2008057460 W JP2008057460 W JP 2008057460W WO 2008126940 A1 WO2008126940 A1 WO 2008126940A1
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positive
cells
lag
cell
negative
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PCT/JP2008/057460
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Japanese (ja)
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Keishi Fujio
Tomohisa Okamura
Kazuhiko Yamamoto
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The University Of Tokyo
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    • 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
    • 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
    • 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
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • 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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • the present invention relates to providing a novel ⁇ cell population derived from a mammal. More specifically, the present invention relates to providing a novel regulatory rod cell population derived from mammals.
  • Organism's immune system is inherently tolerant to its constituent proteins, but under certain conditions an immune response to the self-protein may occur.
  • autoimmune diseases occur.
  • collagen diseases, rheumatoid arthritis, etc. are known as autoimmune diseases, but it is becoming clear that autoimmune responses are also involved in arteriosclerosis, cancer, and neurodegenerative diseases.
  • Autoimmune response is thought to be involved in the pathology of various diseases.
  • non-antigen-specific treatment methods such as administration of corticosteroids and administration of immunosuppressants are used.
  • these currently applied therapies also cause severe problems such as opportunistic infections because they cause systemic high immune suppression.
  • Such a situation also occurs when an immunosuppressant is administered after organ transplantation. Therefore, in the field of treatment of autoimmune diseases and organ transplantation, there is a strong demand for immunosuppressive treatments specific to autoantigens that cause autoimmune diseases and immunosuppressive treatments specific to transplanted antigens. Yes.
  • regulatory atory cells are important in the formation of autoimmune tolerance specific to self-antigens.
  • Mammalian T cells are a type of lymphocyte that refers to the progenitor cells produced in the bone marrow that have been differentiated and matured through selection in the thymus. It is known that 70-80% of lymphocytes in peripheral blood are T cells. This T cell is characterized by expressing CD4 or CD8 as a cell surface marker molecule. T4 cells that express CD4 function as helper T cells that induce the functional expression of other T cells, induce the differentiation and maturation of B cells, and induce antibody production by producing lymphoin, etc. . On the other hand, CD8 positive T The cell functions as a CTL (killer T cell) that destroys virus-infected cells.
  • CTL killer T cell
  • CD4 positive regulatory T cells have a function of suppressing the activity of other T cells.
  • Regulatory T cells known so far include regulatory T cells (Foxp3 Treg cells) that express CD4 and CD25 molecules on the cell surface and express the transcription factor Foxp3 (Non-patent Document 1). It was known to be the most common.
  • regulatory T cells that produce IL-10 T R 1 cells
  • regulatory T cells that produce TGF3 T H 3 cells
  • T R 1 cells regulatory T cells that produce TGF3
  • T H 3 cells regulatory T cells that produce TGF3
  • the expression profile of the cell surface antigen of these cells is unknown, and its substance has not been clarified.
  • the Egr-2 (Early Growth Response Gene-2) gene is 2862 bp (CDS), also called Krox20, NGF1-B, Zfp-25, Zfp-6, NGFl-) 3 (nerve growth factor inducible gene 1- / 3) Is a molecule that is cloned from mouse and human as a Zinc-finger type transcription factor (Non-patent document 5; Non-patent document 6).
  • This gene has been reported to be a transcription factor associated with T cell anagenesis (refractory) (Non-patent Document 7). However, none of the known regulatory T cells mentioned above was associated with this.
  • Non-Patent Document 1 Sakaguchi S., et al., J Immunol 1995; 155: 1151-1164
  • Non-Patent Document 2 Groux H. A, et al., Nature 1997; 389: 737-742
  • Non-Patent Document 3 Chen Y., et al., Science 1994; 265: 1237-1240
  • Non-Patent Document 4 Chen Z. et al., Pro atl. Acad. Sci. USA, 2005, 102, 14735-14740
  • Non-Patent Document 5 Chavrier P, et al., Mol. Cell. Biol. 8 1988, pp. 1319-1326
  • Non-Patent Document 6 Joseph LJ, et al., Proc. Nat l. Acad. Sci. USA 85 1988, 7164-716
  • Non-Patent Document 7 Saf ford M. et al., Nat. Immunol., 2005, 6, 472-480 Disclosure of the Invention
  • CD4 positive CD25 positive regulatory T cells which are conventionally known regulatory T cells, cannot sufficiently control autoimmune diseases. Therefore, CD4 can sufficiently control autoimmune diseases.
  • the objective is to search for regulatory T cells other than positive CD25 positive regulatory T cells.
  • the inventors of the present invention examined the relationship between the Egr-2 gene reported to be a transcription factor related to T cell anergy (refractory) and regulatory T cells, and thus developed novel T cells. It was shown that the above problems can be solved by isolating and clarifying the action.
  • Novel T cells with negative (CD25-), CD45R0 positive (CD45R0 +), and LAG-3 positive (LAG-3 +) properties are shown to have a function as regulatory T cells.
  • the function as a regulatory T cell is to suppress the activation reaction of other T cells, and its characteristics include anaphylaxis (refractory).
  • These cells are derived from any mammal (eg, human, primate, Inu, cat, etc.), and may be isolated from any mammal.
  • the present invention also provides from a mammalian lymphocyte population or T cell population that: (i) CD4 is positive; Collecting a certain cell (CD4 +), (ii) collecting a CD25 negative cell (CD25-), (iii) a CD45RB negative (low positive) cell (CD45RB- (low)) or CD45R0 By collecting the cells (CD45R0 +) positive for, and (iv) collecting the cells (LAG3 +) positive for LAG-3, by performing steps (i) to (iv) in any order.
  • a method for isolating mammalian T cells having the cell surface antigen characteristics of CD45R0 positive (CD45R0 +) and LAG-3 positive (LAG-3 +) can be provided.
  • the mammal may be any mammal (eg, human, primate, Inu, cat, etc.).
  • a mammalian lymphocyte population or T cell population may be collected from peripheral blood and then the steps (i) to (iv) may be performed as they are, or a mammalian lymphocyte population or T cell population. May be collected from peripheral blood and the lymphocyte population or T cell population may be expanded before the steps (i) to (iv) are performed.
  • the T cell population isolated by performing the steps (i) to (iv) may be used as it is, or the steps (i) to (iv) are performed.
  • a more isolated T cell population of the present invention may be used after being expanded.
  • a pharmaceutical composition comprising mammalian T cells having negative (CD25-), CD45R0 positive (CD45R0 +), and LAG-3 positive (LAG-3 +) cell surface antigen characteristics. It can. By administering this pharmaceutical composition, it is possible to induce functional suppression of pathogenic T cells that cause inflammation. Therefore, the pharmaceutical composition of the present invention is capable of eliminating an antigen by an antigen-antibody reaction. It can be used to treat various diseases caused.
  • various diseases caused by an immune reaction against an autoantigen or due to an antigen being eliminated by an antigen-antibody reaction include, for example, autoimmune diseases (eg, rheumatoid arthritis, collagen disease, inflammatory bowel disease) , Multiple sclerosis, type 1 diabetes, etc.) or transplant organ rejection.
  • CD4 positive CD4 +
  • CD25 CD25 negative
  • CD45RB negative low positive
  • LAG-3 positive LAG3 +
  • CD4 positive CD4 +
  • CD25 Screen for compounds to increase the number of mammalian T cells with negative (CD25-), CD45R0 positive (CD45R0 +), and LAG-3 positive (LAG-3 +) cell surface antigen properties
  • a method is also provided. The method for screening the target compound having the above-mentioned action is:
  • CD4 positive CD4 +
  • CD25 negative CD25-
  • CD45RB negative low positive
  • LAG-3 + LAG-3 positive
  • CD4 positive CD4 +
  • CD25 negative CD25-
  • CD45R0 CD45R0 +
  • LAG-3 positive LAG-3 +
  • FIG. 1 shows a schematic diagram of a retroviral vector pMX-Egr2-IRES-GFP and a control vector mock pMX-IRES-GFP vector used in the present invention.
  • FIG. 2 shows mouse CD4-positive splenocytes infected with Mock (pMX-IRES-GFP) or Egr2 (p X-Egr2-IRES-GFP) using a flow cytometer and GFP expression as an indicator. The process of separating and collecting in 4 groups (negative, low, mid, high) is shown.
  • Fig. 3 shows the results of real-time PCR analysis of cDNA synthesis from each of the 4 groups of cells collected and collected in Fig. 2, and the expression at the Egr2 mRNA and LAG-3 mRNA levels. Show.
  • Fig. 4 shows that co-transfer of OVA-specific T cell receptor (D01 1.10) significantly enhances its suppressive effect and enables antigen-specific immunosuppression. It is.
  • Fig. 5 shows the spleen mononuclear cells of mice co-transfected with Egr2 and D01 1.10 by flow cytometry. After lymphocyte and CD4 positive cell gate, D01 1.10 T cell receptor This is the process of developing each cell population separately using the KJ-1 antibody and GFP.
  • Figure 6 shows that Egr2 (pMX-Egr2-IRES-GFP) and DO 1 1.10 (pMX-DOTAE, pMX-DOTBE) co-transfected cells were treated with pMX-Egr2-IRES-GFP. Overexpression,
  • Fig. 7 shows that among mouse spleen mononuclear cells, LAG-3 surface antigen is CD4 positive among CD25 negative cells (Fig. 7a) and CD45RB negative to CD45RB weak positive cells (Fig. 7b). It is a figure which shows that expression is enhanced.
  • FIG. 8 is a graph showing that expression of Egr2 mRNA, IL-10 mRNA, and LAG-3 mRNA is enhanced in mouse LAG-3 expression cells that are CD45RB negative to CD45RB weak positive.
  • FIG. 9 shows cells gated in CD4 positive CD45RB ex low cell group, LAG-3,
  • FIG. 4 shows the results of separating and collecting three groups of cell populations, CD45RB ex low and CD4 + CD25 + CD45RB ex low.
  • FIG. 10 shows that Egr2 expression was strongest in CD4 + CD25-LAG3 + CD45RB ex low (Egr2-Treg), and the same expression tendency was not observed in Foxp3 expression.
  • Fig. 1 1 shows cells gated in CD4 positive CD25 negative cells group developed with LAG-3 and CD45RB, and each cell group was separated and collected by flow cytometry as shown in the figure. Show process It is a figure.
  • FIG. 12 shows that Egr2 expression is strongest in CD4 + CD25-LAG3 + CD45RB ex low, and Egr2-Treg different from the known Foxp3-Treg exists.
  • FIG. 13 shows that CD4 + CD25-LAG3 + CD45RBex low (Egr2-Treg) is still present after type II collagen immunization, and further enhancement of I-10 expression occurs in “Egr2-Treg” after immunization
  • FIG. 13 shows that CD4 + CD25-LAG3 + CD45RBex low (Egr2-Treg) is still present after type II collagen immunization, and further enhancement of I-10 expression occurs in “Egr2-Treg” after immunization
  • FIG. 14 shows a process in which CD4 + CD25- CD45RB ex low cell population (Thyl.2 +) is divided into high, low and nega '3 groups by LAG-3 expression as suppressor cells.
  • FIG. 15 shows that Egr-2Treg cells suppress cell division of a cytotoxic T cell subset (CD45RBhi cells), and the suppressive ability depends on LAG-3 expression.
  • Fig.16 shows the suppression of enteritis caused by CD4 + CD25- CD45RBM cells administration in CD4 + CD25-LAG3 + CD45RB ex low (Egr2-Treg) cells in RAG ⁇ /-mice. It is a figure which shows what can be done.
  • Fig. 17 shows CD4 + CD25-LAG3 + CD45RB ex low (Egr2-Treg) in the histology of the large intestine. ) It is a figure showing that the improvement was observed by co-transfer of cells.
  • Figure 18 shows that CD4 + CD25- LAG3 + CD45RB ex low (Egr2-Treg) cells are also present in the splenocytes of Scurfy mice lacking the functional Foxp3 gene as analyzed by flow cytometry.
  • FIG. 1 shows that CD4 + CD25- LAG3 + CD45RB ex low (Egr2-Treg) cells are also present in the splenocytes of Scurfy mice lacking the functional Foxp3 gene as analyzed by flow cytometry.
  • FIG. 19 is a view showing that Egr2-Treg cells of Scurfy mice have an in vitro suppressive ability, and that the suppressive ability is attenuated by LAG-3 neutralizing antibody.
  • Figure 20 shows the results of intraperitoneal administration of C57BL / 6 mouse spleen CD4 + CD25- CD45RBhi cells 1X10 5 cells to Rag ⁇ /-mice. It is a figure which shows suppressing the enteritis in.
  • FIG. 21 is a schematic diagram showing a technique for producing a shRNA-Egr2 bone marrow chimeric mouse in which Egr2 gene expression is suppressed.
  • FIG. 22 is a diagram showing that shRNA-Egr2 bone marrow chimeric mice develop marked thickening of the intestinal tract, which is characteristic of the onset of enteritis.
  • Fig. 23 shows that CD4 + CD25- LAG3 + CD45RB ex l ow (Egr2-Treg) cells were hardly observed in At MT mice lacking B cells, and Egr2- Treg cells were introduced by adoptive introduction of B cells. It is a figure which shows that this cell population is induced
  • Figure 24 shows flow cytometry of LAG-3 expression and CD45RB expression in thymus and spleen of OT-11 TCR transgenic mice with or without RIP-mOVA transgene.
  • Figure 25 shows a normal TEa offspring (left) gated with CD4 + CD25 "T cells
  • FIG. 4 shows flow cytometry of LAG-3 expression and CD45RB expression in spleen and Peyer's patch (PP) of 0T-I I (middle) and TEa TCR transgenic mice (right).
  • FIG. 26 shows the number of Egr-2 positive cells in the follicle.
  • FIG. 4 shows the results of immunohistochemical examination of cells expressing Egr-2 molecules in spleen tissue sections derived from TEa mice, 0T-I I mice and 7BL / 6 mice.
  • FIG. 27 shows that spontaneous development of colitis is inhibited in TEa TCR transgenic mice by adoptive transfer of CD4 + CD25 "LAG3 + T cells.
  • Fig. 28 shows that LAG-3 surface antigen is enhanced in CD25 negative cells (Fig. 28a) and CD45R0 positive cells (Fig. 28b) among human tonsil cells.
  • FIG. 28a shows that LAG-3 surface antigen is enhanced in CD25 negative cells (Fig. 28a) and CD45R0 positive cells (Fig. 28b) among human tonsil cells.
  • FIG. 29 shows the expression of Egr2 mRNA, CD45R0 positive human LAG-3 expressing cells
  • the inventors of the present invention first examined whether the Egr-2 gene, a gene reported to be a transcription factor associated with T cell anergy (refractory), is associated with the activity of regulatory T cells. Went. Specifically, the Egr-2 gene was introduced into CD4 + T cells, and the traits were introduced When T cells were transferred to mice, it was revealed that delayed hypersensitivity reaction to foreign antigens was remarkably suppressed. This indicates that the Egr-2 gene is associated with the activity of regulatory T cells. It was also shown that co-introduction of antigen-specific T cell receptor with Egr-2 gene enhances its suppressive ability and induces antigen-specific suppression.
  • T cells having such cell surface antigen characteristics have never been known, the present inventors have developed a new T cell population having the characteristics of CD positive CD25 negative CD45RB negative LAG-3 positive. A detailed analysis was performed, considering that it is a regulatory T cell that plays an important role in the immune tolerance induction mechanism of T cells and can sufficiently control autoimmune diseases.
  • IL-10 which is known as a cytokine with strong anti-inflammatory action, is highly expressed, and as a characteristic of the cell itself, in vitro, CD4 positive CD45RB highly expressed naive T cell division is observed.
  • enteritis model in which naive T cells with high expression of CD4 + CD45RB are transferred to RAG1-mouse mice, the onset of enteritis is induced by transfer of CD4 + CD25 negative CD45RB negative LAG-3 + T cells. It became clear to suppress.
  • T cells with cell surface antigen characteristics of CD positive CD4 +
  • CD25 negative CD25-
  • CD45RB negative low positive
  • LAG3 + LAG3 positive
  • CD4 positive CD25 negative CD45R0 positive LAG-3 positive T cells The mRNA expression level of 2 was found to be extremely high.
  • the CD45RB negative (low positive) memory T cell population in mice is the CD45R0 fluorescence intensity in humans.
  • the CD4 positive CD25 negative CD45R0 positive LAG-3 positive T cells like mouse CD4 positive CD25 negative CD45RB negative LAG-3 positive T cells, also show high IL-10 expression. It was done.
  • the inventors of the present invention named these cells “Egr-2 Treg cells” because these cells have the feature that the expression level of Egr-2 is extremely high.
  • the “Egr-2 Treg cells” obtained in the present invention were compared with the regulatory T cells “Foxp3 Treg cells” (CD4-positive CD25-positive Foxp3-positive T cells) known so far.
  • the regulatory T cells “Foxp3 Treg cells” CD4-positive CD25-positive Foxp3-positive T cells
  • high expression of the Foxp3 gene was not observed in the “Egr-2 Treg cell” of the present invention
  • the high Egr-2 gene was found in the conventionally known regulatory T cell “Foxp3 Treg cell”. It became clear that it showed a contrasting characteristic that no expression was seen. From these results, it was considered that the “Egr-2 Treg cells” of the present invention and the “Foxp3 Treg cells” of the prior art are independent T cell populations different from each other.
  • a mouse called scur fy which lacks the functional Foxp3 gene, is known to cause autoimmune-like disease with inflammatory cell infiltration into various organs and die within 4 weeks of birth.
  • T cells in the spleen of this scur fy mouse were examined, the same T4 subcells with the characteristics of CD4 positive CD25 negative CD45RB negative LAG-3 positive as the "Egr-2 Treg cells" of the present invention proliferated remarkably. It was shown that T cell subsets with this cell surface antigen profile were collected and examined for function in vitro.
  • T cell subsets with CD4-positive CD25-negative CD45RB-negative LAG-3 positive characteristics in scur fy mice are ⁇ Egr-2 "Treg cells”.
  • Egr-2 Treg cells are prominently present in scur fy mice even though scur fy mice lack the Foxp3 gene, “Egr-2 Treg cells” are It was confirmed that it has independent characteristics.
  • Egr-2 gene knockout mice are known to have the characteristics of embryonic lethality that develops symptoms such as impaired formation of the hindbrain and myelination of peripheral nerves and die during fetal period ( Genes Dev. 7 1993, pp. 207-2084). Therefore, the present inventors produced a bone marrow chimeric mouse in which a sh-RNA sequence that suppresses the expression of the Egr-2 gene was expressed in bone marrow cells using a retrovirus vector.
  • the mouse died approximately 4 weeks after bone marrow transplantation, and histological examination revealed that it developed enteritis.
  • the Egr-2 gene deficiency reduces the number of CD4-positive, CD25-negative, CD45RB-negative, LAG-3-positive cells that should be present in the body, resulting in the mechanism of regulatory T cells such as enteritis. It was shown to develop clinical symptoms associated with reduced performance.
  • CD4 positive CD25 negative CD45RB negative LAG-3 positive T cells or CD positive CD25 negative CD45R0 positive LAG-3 positive T cells are active in vitro or in vivo While the Tgr cell body and its transcription factor Egr-2 gene plays an important role in its differentiation, this regulatory T cell is shown to be independent of the functional control by the Foxp3 gene. It was done.
  • the present invention provides a novel regulatory T cell as a CD positive CD25 negative CD45RB negative LAG-3 positive T cell or CD4 positive CD25 negative CD45R0 positive LAG-3 positive T cell, "Egr-2 Treg cells” are provided. As described above, these cells are a cell population belonging to a T cell subset different from “Foxp3 Treg cells” which are regulatory T cells known so far.
  • Egr-2 refers to the protein having the amino acid sequence represented by GenBank Accession No. P 1 1 161 and the nucleotide sequence encoding it for human “Egr-2”.
  • GenBank Accession No. P 1 1 161 the protein having the amino acid sequence indicated by GenBank accession number NP_034248 and the nucleotide sequence encoding the protein, and for rat “Egr-2”, the amino acid indicated by GenBank accession number NP—446085.
  • a protein having a sequence and a nucleotide sequence encoding the protein are respectively referred to.
  • mRNA expressed from the Egr-2 gene or cDNA prepared therefrom is used as a saddle, and in the case of human gcaccagc tg tctgacaaca tctac (SEQ ID NO: 1) as a forward primer and agcaaagctg ctgggatatgg (SEQ ID NO: 2) as a reverse primer, for mice agccgt t tec ctgtcctctg (SEQ ID NO: 3) as a forward primer and gtccctcacc acctccactt (SEQ ID NO: 4) can be detected as a reverse primer by amplification according to a general PCR protocol.
  • LAG-3 for human “LAG-3”, the protein having the amino acid sequence represented by GenBank accession number P_002277 and the nucleotide sequence encoding it are as follows:
  • mouse “LAG-3” refer to the protein having the amino acid sequence indicated by GenBank accession number P_032505 and the nucleotide sequence encoding it.
  • GenBank accession number NP. refers to the protein having the amino acid sequence represented by 997678 and the nucleotide sequence encoding it, respectively.
  • mRNA expressed from the LAG-3 gene or cDNA prepared therefrom is used as a saddle type, and in humans atctgcagga acagcagctcaa (SEQ ID NO: 5) is forwarded.
  • Reverse primer as a primer and agggatccag gtgacccaaag (SEQ ID NO: 6) as reverse primer, for mice t tgct tctgg gactgctttg (SEQ ID NO: 7) as forward primer and gccactgtct ggttgatgtt g (SEQ ID NO: 8) As a primer, it can be detected by amplification according to a general PCR protocol.
  • the term “Foxp3” in the present invention refers to the protein having the amino acid sequence represented by GenBank accession number NP-054728 and the nucleotide sequence encoding the same for human “Foxp3”, and for mouse “Foxp3” It refers to the protein having the amino acid sequence represented by GenBank accession number NP-473380 and the nucleotide sequence encoding it.
  • mRNA expressed from the Foxp3 gene or cDNA prepared therefrom is used as a saddle type, and in the case of a mouse, cagctgccta cagtgccctag (SEQ ID NO: 9) is used as a forward primer and catttgccag cagtgggtag (SEQ ID NO: 10) as a reverse primer and a general PCR protocol Can be detected by amplification according to
  • Egr-2 Treg cells are CD4, CD25, CD45RB or CD45R0, and LAG-3 cell surface antigens, CD4 positive, CD25 negative, CD45RB negative ( (Low positive) or CD45R0 positive, and LAG-3 positive as an indicator, isolated from a leukocyte population or T cell population collected from a living body as a single cell population it can.
  • the “Egr-2 Treg cell” of the present invention can be isolated by sorting with CD5, CD25, CD45RB or CD45R0 and LAG-3 in a cell saw. Sorting based on the expression of CD4, CD25, CD45RB or CD45R0, and LAG-3 may be done in any order.
  • the lymphocyte population or T cell population obtained from the peripheral blood of the living body can be used as the T cell population that is the source of ⁇ 81 "-268 cells".
  • peripheral blood mononuclear cells are separated by extracorporeal circulation (pheresis) using the blood component separation device of peripheral blood, and four types of CD4, CD25, CD45RB or CD45R0, and LAG-3
  • the Egr-2 Treg cells can be isolated and collected using Celso overnight.
  • “Egr-2 Treg cells” can be separated and collected using a magnetic cell separation device such as Isolex or CliniMACS instead of Cell Soryu.
  • the above-mentioned lymphocyte population or T cell population may be subjected to cell sorter treatment for the above-mentioned CD4, CD25, CD45RB or CD45RO, and LAG-3 as it is to isolate “Egr-2 Treg cells”. If the number of cells is insufficient, the above-mentioned lymphocyte population or T-cell population is expanded to a desired level, and then subjected to cell sorter treatment for the above-mentioned CD4, CD25, CD45RB or CD45R0, and LAG-3. “Egr-2 Treg cells” may be isolated. In such cases, lymphocyte populations or T cell populations can be grown by culturing in the presence of CD3.CD28 costimulation, TGF-beta and IL-2.
  • CD81, CD25, CD45RB or CD45RO, and LAG-3 can be used as they are. If the number is insufficient, the isolated ⁇ 1-2 6 ⁇ cells can be further expanded by culturing in the presence of CD3 'CD28 co- stimulation , TGF- beta and IL-2. . In addition, for treatments that require strong organ migration, they were isolated using the above method.
  • the antigen-specific therapeutic effect can be enhanced.
  • cells that are positive for the cell surface antigen CD4 are sorted based on the fact that the cells are clearly one group in which the CD4 fluorescence intensity is enhanced more and more by Celso using an antibody against CD4. Can be isolated.
  • antibodies to CD4 Al 1 ophycocyan in (APC) labeled anti-CD antibody (available from BDbioscience, Beckman Coulter, AbD Serotec, etc.) for humans, FITC labeled anti-CD4 antibody (BD bioscience) for mice Available from Beckman Coulter, BioLegend, etc.).
  • cells that are negative for the cell surface antigen CD25 are sorted based on the fact that the cells are a population obtained by removing one population in which the CD25 fluorescence intensity is clearly enhanced by a cell sorter using an antibody against CD25. Can be isolated.
  • antibodies against CD25 allophycocyanin-Cyanin-7 (APC-Cy7) labeled anti-CD25 antibody (available from BD bioscience, BioLegend, etc.) for humans, and APC-labeled anti-CD25 antibody (BD bioscience) for mice. Available from BioLegend, etc.).
  • cells that are negative (low positive) for the cell surface antigen CD45RB are 70% of cells that are relatively strongly expressing CD45RB among CD4 positive cells according to CELSO overnight using an antibody against CD45RB.
  • the antibody against CD45RB can be isolated by using Phycoerythrin (PE) as an antibody against CD45RB (hereinafter, synonymous with “CD45RB ex low” in the main text).
  • PE Phycoerythrin
  • the cells positive for the cell surface antigen LAG-3 in the present invention are based on the fact that the cells are a group in which the LAG-3 fluorescence intensity is clearly enhanced by a cell saw using an antibody against LAG-3. It can be isolated by separating it.
  • ATT0 488-labeled anti-LAG-3 antibody available from Alex is Biochem ls
  • PE-labeled anti-LAG-3 antibody available from companies.
  • the present invention provides an isolated “Egr-2 Treg cell”.
  • ”A As a constituent component, for treating various diseases caused by an immune reaction against a self-antigen or due to an antigen being eliminated by an antigen-antibody reaction, such as various autoimmune diseases, transplanted organ rejection, etc.
  • a pharmaceutical composition can be provided.
  • the isolated “Egr-2 Treg cell” of the present invention When used in a pharmaceutical composition for treating various autoimmune diseases, transplant organ rejection, etc., 2 ⁇ 10 6 cells / kg to 4 This can be done by intravenous administration of 100 to 500 ml of a cell suspension of X 10 6 cels / kg.
  • the isolated “Egr-2 Treg cells” of the present invention administered in this manner are carried throughout the body in the bloodstream and can induce local immune tolerance.
  • variable diseases caused by antigens being eliminated by antigen-antibody reaction mainly refers to diseases such as rejection of transplanted organs, but is not limited thereto.
  • variant diseases caused by immune responses to self antigens refers to autoimmune diseases such as rheumatoid arthritis, collagen disease, inflammatory bowel disease, multiple sclerosis, type 1 diabetes.
  • the present invention also provides a method of screening for a compound capable of inducing the proliferation of the isolated ⁇ ⁇ 1 "-26 £ cell” of the present invention. Specifically, (a) ⁇ 1 of the present invention "-2 1 ⁇ a ⁇ cell", step culturing in the presence of a test compound; (b) cultured "Egr-2
  • Such compounds can also be used to grow "Egr-2 Treg cells” isolated in vitro or immunize by growing "Egr-2 Treg cells” in vivo. It can also be used to induce tolerance.
  • the purpose of this example is to clarify the mechanism by which the transcription factor Egr-2, which is expected to be related to T cell analogy, is related to the T cell suppressive ability.
  • the Egr-2 gene was introduced into T cells, and the effects of Egr-2 transformed T cells were examined.
  • Egr2 cDNA obtained from the cDNA library was introduced into the retroviral vector pMX (Onishi M, et al., 1996 Exp. Hematol. 24: 324-329) and pMX-Egr2-IRES-GFP A retroviral vector was prepared ( Figure 1).
  • pMX Foxp3, D0TAE, and D0TBE
  • OVA ovalbumin
  • Mock pMX-IRES-GFP
  • Egr2 pMX-Egr2-IRES-GFP
  • flow cytometry cells were divided into 4 groups each using GFP expression as an indicator.
  • Fig. 2 Separated and collected (negative, low, mid, high) (Fig. 2). Then, cDNA synthesis was performed from each cell of each of the 4 groups separated and collected in Fig. 2, and Egr2 mRNA and LAG-3 mRNA were synthesized. Expression at the level was examined by real-time PCR ( Figure 3). The mRNA expression is a relative expression level normalized with / 3 -actin. The real-time PCR method was analyzed using the same method. As a result, LAG-3 expression was enhanced along with Egr2 expression (Fig. 3).
  • OVA ovalbumin
  • isolated splenic mononuclear cells (Balb / c mice (6-8 weeks old)) were treated with pMX-Egr2- IRES-GFP, pMX-Foxp3-IRES-GFP, pMX-DOTAE, 72 hours after each infection with pMX-DOTBE retrovirus, CD8, -CDl lb, -CD19 positive cells were biotinylated using a MACS column (Milttenyi Biotec), and CD19 antibody and Strepton were biotinylated.
  • Egr2 suppressed delayed hypersensitivity reaction like Foxp3.
  • co-transfer of 0VA-specific T cell receptor significantly enhanced the suppressive effect, indicating that antigen-specific immunosuppression was possible (Fig. 4).
  • mice co-transfected with Egr2 p X-Egr2-IRES-GFP
  • DO 11.10 pMX-DOTAE, pMX-DOTBE
  • the cells were separated and analyzed by flow cytometry. Spleen mononuclear cells are lymphocytes and flow cytometry
  • CD positive cell gate After CD positive cell gate, it was developed with D011. 10T cell receptor specific antibody 26-26 antibody and GFP, and each cell population was separated and collected as shown in the figure.
  • the cells isolated and collected in Fig. 3 CDNA synthesis was performed, and expression at the Egr2 mRNA, LAG-3 mRNA, and Foxp3 mRNA levels was examined by real-time PCR.
  • the transferred cells overexpress Egr2 with PMX-Egr2-IRES-GFP, enhance the expression of LAG-3, and co-express the OVA-specific T cell receptor. Expression was further enhanced, indicating that an antigen-specific T cell receptor signal is important for the ability to suppress Egr-2 ( Figures 5 and 6).
  • Egr2 induces regulatory T cell-like activity in vivo.
  • Egr2-transformed sputum cells express LAG-3 significantly, and the expression level of Egr2 and LAG-3 are positively correlated.
  • LAG-3 expression is consistent with antigen-specific T cell reception. It became clear that it was strengthened more.
  • cell sorting was performed for the purpose of collecting T cells expressing the Egr-2 gene.
  • CD4 positive T cells were isolated and collected using flow cytometry according to the presence or absence of LAG-3 expression.
  • LAG-3 mRNA, Egr2 mRNA, Foxp3 mRNA, and IL were synthesized by real-time PCR.
  • -10 mRNA expression was examined, and enhanced expression of Egr 2 mRNA, IL-10 mRNA, and LAG-3 mRNA in LAG-3 expressing cells was observed (Fig. 8). Foxp3 expression did not show significant correlation with LAG expression.
  • CD4 positive CD45RB ex low cell group were expanded with LAG-3 and CD25, and then CD4 + CD25- LAG3 + CD45RB ex low (Egr2- Treg), CD4 + CD25- LAG3- CD45RB ex l ow,
  • Egr2 mRNA and Foxp3 mRNA were examined by the method. As a result, it was shown that Egr2 expression was strongest in CD4 + CD25- LAG-3 + CD45RB ex low, and there was Egr2-Treg different from the known Foxp3-Treg (Fig. 11, Fig. 12).
  • Egr2-Treg was examined using a type II collagen-induced arthritis onset model.
  • DBA-1J mice (6-8 weeks old) were injected intradermally with a mixture of sushi-derived collagen type II and complete Freund's adjuvant (complete Freund's adjuvant, CFA, Chondrex or BD Di fco) (dayO) ), And then boosted with type II collagen (day 21) to induce type II collagen-induced arthritis.
  • each cell group was separated and collected by the same method as in Fig. 11 and analyzed by real-time PCR.
  • CD4 + CD25-LAG3 + CD45RB ex low (Egr2-Treg) still existed after immunization with type II collagen, and further enhancement of IL-10 expression in “Egr2-Treg” was observed after immunization (FIG. 13).
  • Egr-2 remained highly expressed even after the onset of arthritis.
  • CD25-LAG-3 + CD45RB ex low (Egr2-Treg) was examined.
  • CFSE-labeled CD4 + CD25-CD45RB high cell population (Thyl. H) 5X10 4 cells and spleen cells (Thy 1.2+) lX10 5 cells irradiated with 1500 rad as antigen-presenting cells were used.
  • the CD4 + CD25- CD45RB ex low cell population (Thyl.2 +) was separated and collected into 3 groups of high, low and nega by LAG-3 expression (each group 5X10 4 cells) (Fig. 14) .
  • C57BL / 6 mouse spleen CD4 + CD25- CD45RBhi cells 1X10 5 cells were intraperitoneally administered to Rag ⁇ / ⁇ mice to produce an inflammatory enteritis model.
  • Enteritis was assessed by weight loss and histology. Body weight was 100% on the day of cell transfer (dayO). Each group consists of 3 groups of CD4 + CD25- CD45RBhi cells 1X10 5 cells only, CD4 + CD25- CD45RBhi cells 1X10 5 cells + CD4 + CD25-LAG3 + CD45RB ex low (Egr2-Treg) cells IX 10 5 cells and PBS only (control) Evaluated.
  • Egr2-Treg enteritis caused by C57BL / 6 mouse spleen CD4 + CD25- CD45RBhi cell administration could be suppressed by co-transfer of CD4 + CD25- LAG3 + CD45RB ex low (Egr2-Treg) cells (Fig.
  • CD4 + CD25- LAG3 + CD45RB ex low (Egr2-Treg) cells co-transferred with inflammatory cell infiltration and intestinal wall thickening, etc., observed with single administration of CD4 + CD25- CD45RBhi cells. (Fig. 17).
  • Foxp3 is currently thought to be a mass gene for suppressor T cells. Therefore, in this example, Scurfy mice lacking the Foxp3 functional gene were used.
  • spleen cells of Scurfy mice lacking the functional gene of Foxp3 were analyzed by flow cytometry using CD4-Cy7AP CD25-APC and CD45RB-FIT LAG-3-PE. As a result, CD4 + was also detected in spleen cells of Scurfy mice.
  • CD25- LAG3 + CD45RB ex low (Egr2- Treg) cells were shown to be present ( Figure 18).
  • the CD45RB high cell population (Thyl.) 5X10 4 cells was used as antigen-presenting cells, and whole splenocyte (Thyl.2 +) 1X10 5 cells of 1500 rad irradiated C57BL / 6 mice were used.
  • Suppressor cells include Scurfy mouse CD4 + CD25- CD45RB ex low cell population (Thyl.2 +)
  • C57BL / 6 mouse spleen CD4 + CD25- CD45RBh i cells 1X10 5 cells were intraperitoneally administered to Ragl ⁇ / ⁇ mice to prepare a model for the development of inflammatory bowel disease. Enteritis was assessed by weight loss and histology. Body weight was defined as 100% on the day of cell transfer (dayO).
  • Each group consists of C57BL / 6 mouse spleen CD4 + CD25- CD45RBhi cells 1X10 5 cells only, C57BL / 6 mouse spleen CD4 + CD25- CD45RBhi cells 1 X 10 5 cells + Scurfy mouse CD4 + CD25- LAG-3 + CD45RB ex low (Egr2 -Treg) Cells were evaluated in 3 groups: 1 x 10 5 eel Is and PBS only (control). As a result, it was shown that enteritis in the inflammatory enteritis onset model of Ragl-/-mice was also suppressed in vivo (Fig. 20).
  • Egr2- Treg cells are a novel inhibitory T cell population that exerts suppressive ability independent of Foxp3 function, and that the suppressive ability is mediated by LAG-3.
  • a shRNA-Egr2 retrovirus was prepared, the Egr2 gene was silencing, and mouse phenotype analysis was performed. Specifically, bone marrow cells of C57BL / 6 mice treated with 5FU were collected, infected with shRNA-Egr2 retrovirus, and then transferred to irradiated C57BL / 6 mice, so that shRNA-Egr2 bone marrow chimeric mice were Fabricated (Fig. 21). As a result, LAG-3 expression was suppressed by silencing Egr2 gene expression, and shRNA-Egr2 bone marrow chimeric mice showed marked thickening of the intestinal tract. . After bone marrow transplantation, he developed severe enteritis and died in about 4 weeks.
  • CD4 + T cells were collected from genase (Sigma-Aldrich, St. Louis, MO) and PE-anti-LAG-3 monoclonal antibody (LAG-3-PE) was used as described in Example 2 for these cells.
  • the analysis was performed on a flow cytometer using the FITC-anti-CD45RB monoclonal antibody (CD45RB-FITC).
  • both spleen-derived cells and Peyer's patch-derived cells have T cells with the same phenotype as the Egr2-Treg cells of the present invention (ie, LAG-3 + CD45RB ex low).
  • LAG-3 + CD45RB ex low the Egr2-Treg cells of the present invention
  • B cells may have some function in inducing differentiation of Egr2-Treg cells in vivo.
  • it is further a B cell-deficient mouse;
  • B cells collected from the spleen of C57BL / 6 mice were transplanted to examine whether the Egr2- Treg cells of the present invention were induced in the living mouse.
  • C57BL / 6 mouse sputum cells can be obtained from MACS using a single cell suspension of spleen from C57BL / 6 mice using the S cell isolation kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer's protocol. Purification was done by negative selection. The purity of B cells sorted by MACS was> 98%. 2X10 7 B cells purified in this way were injected intravenously into / MT mice. Control mice were irradiated with PBS. Nine weeks after cell transplantation, mice were sacrificed and spleen cells were analyzed by flow cytometry.
  • T cells having the same phenotype (LAG-3 + CD45RB ex low) as the Egr2-Treg cells of the present invention were significantly compared to ⁇ mice. (Fig. 23). Therefore, it has been clarified that the presence of B cells is indispensable for inducing differentiation of the Egr2-Treg cells of the present invention in vivo.
  • Foxp3 + Treg cells are required to have higher affinity agonist interactions with self-peptide / MHC expressed by thymic stromal cells to induce differentiation.
  • CD4 + CD25— LAG3 + T cells were induced to differentiate through the same thymic selection process as Foxp3 + Treg cells.
  • TCR Transgenic 0T-II mice (Taconic) and RIP-mOVA mice (Jackson Laborat ory) Transgenic TCRs that express membrane-bound OVA under the control of the rat insulin gene promoter in the islets and thymus while simultaneously recognizing OVA peptides in the context of IA b RIP-mOVA / OT-I I double-transgenic mice were also generated that also express V a 2 and V) 3 5.1 / 5.
  • 0T-II transgenic mice contained as many CD4 + CD25- LAG3 + T cells as normal mice in the spleen and Peyer's patches, but ⁇ - ⁇ ⁇ -chain-specific IA b -restricted transgenig TEa transgenic mice expressing TCR had few CD4 + CD25- LAG3 + T cells, especially in the spleen ( Figure 25).
  • TEa mice also contained very few 1 ⁇ 2r-2-expressing cells in splenic follicles in histological examination (FIG. 26).
  • each bar indicates the number of measurements from one follicle. This data was collected from 3 mice in each group. All error bars indicate standard deviation; an asterisk indicates ⁇ 0. 01.
  • Example 2 based on the mouse knowledge obtained in Example 2, cell sorting was performed for the purpose of collecting T cells expressing the Egr-2 gene from human tonsil-derived cells. .
  • CD4 positive T cells were isolated and collected using flow cytometry according to the presence or absence of LAG-3 expression.
  • LAG-3 mRNA, Egr2 mRNA, Foxp3 mRNA, and IL were synthesized by real-time PCR.
  • -10 When mRNA expression was examined, enhanced expression of Egr 2 mRNA, IL-10 mRNA, and LAG-3 mRNA in LAG-3 expressing cells was observed (Fig. 29). Foxp3 expression was not significantly correlated with LAG expression.

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Abstract

L'invention concerne un lymphocyte T régulateur positif du CD4, positif du CD5, qui est un lymphocyte T régulateur connu, qui ne peut pas réguler une maladie auto-immune par lui-même d'une manière satisfaisante. Ainsi, le but est de rechercher un lymphocyte T régulateur autre qu'un lymphocyte T régulateur positif du CD4, positif du CD25, qui peut réguler une maladie auto-immune d'une manière satisfaisante. Une étude a été effectuée sur la relation entre un lymphocyte T régulateur et le gène Egr-2 qui a été signalé comme étant un facteur de transcription impliqué dans l'anergie de lymphocyte T. En résultat, un nouveau lymphocyte T peut être isolé, et les activités du lymphocyte T peuvent être élucidées.
PCT/JP2008/057460 2007-04-10 2008-04-10 Nouveau lymphocyte t WO2008126940A1 (fr)

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JP2011518796A (ja) * 2008-04-28 2011-06-30 ティクセル 炎症性自己免疫病態を治療するための組成物
JP2013523886A (ja) * 2010-04-13 2013-06-17 イミュノバティブ セラピーズ， リミテッド 制御性ｔ細胞の阻害のための方法および組成物
WO2016084412A1 (fr) * 2014-11-28 2016-06-02 国立大学法人東京大学 Inhibiteur d'activation des lymphocytes b, et agent thérapeutique pour maladies auto-immunes
JP2019208501A (ja) * 2018-05-31 2019-12-12 公益財団法人ヒューマンサイエンス振興財団 インビトロでの制御性ｔ細胞の特異性評価方法

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011518796A (ja) * 2008-04-28 2011-06-30 ティクセル 炎症性自己免疫病態を治療するための組成物
JP2014167019A (ja) * 2008-04-28 2014-09-11 Txcell 炎症性自己免疫病態を治療するための組成物
JP2013523886A (ja) * 2010-04-13 2013-06-17 イミュノバティブ セラピーズ， リミテッド 制御性ｔ細胞の阻害のための方法および組成物
WO2016084412A1 (fr) * 2014-11-28 2016-06-02 国立大学法人東京大学 Inhibiteur d'activation des lymphocytes b, et agent thérapeutique pour maladies auto-immunes
JP2019208501A (ja) * 2018-05-31 2019-12-12 公益財団法人ヒューマンサイエンス振興財団 インビトロでの制御性ｔ細胞の特異性評価方法
JP7277255B2 (ja) 2018-05-31 2023-05-18 佳寛 大矢 インビトロでの制御性ｔ細胞の特異性評価方法

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