WO2009145238A1 - Immunomodulatory agent and use thereof - Google Patents
Immunomodulatory agent and use thereof Download PDFInfo
- Publication number
- 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
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- positive
- cells
- cxcr3
- cell
- immune
- Prior art date
Links
- 230000002519 immonomodulatory effect Effects 0.000 title abstract description 3
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 284
- 102100028990 C-X-C chemokine receptor type 3 Human genes 0.000 claims abstract description 255
- 101000916050 Homo sapiens C-X-C chemokine receptor type 3 Proteins 0.000 claims abstract description 254
- 210000003289 regulatory T cell Anatomy 0.000 claims abstract description 72
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 61
- 210000004027 cell Anatomy 0.000 claims description 223
- 239000000427 antigen Substances 0.000 claims description 58
- 102000036639 antigens Human genes 0.000 claims description 57
- 108091007433 antigens Proteins 0.000 claims description 57
- 208000035475 disorder Diseases 0.000 claims description 48
- 238000000034 method Methods 0.000 claims description 43
- 238000004519 manufacturing process Methods 0.000 claims description 34
- 230000000694 effects Effects 0.000 claims description 30
- 230000001506 immunosuppresive effect Effects 0.000 claims description 23
- 230000002265 prevention Effects 0.000 claims description 23
- 239000002955 immunomodulating agent Substances 0.000 claims description 19
- 230000004913 activation Effects 0.000 claims description 18
- 229940121354 immunomodulator Drugs 0.000 claims description 18
- 238000012360 testing method Methods 0.000 claims description 18
- 230000002584 immunomodulator Effects 0.000 claims description 17
- 230000031261 interleukin-10 production Effects 0.000 claims description 15
- 238000012216 screening Methods 0.000 claims description 15
- 238000012258 culturing Methods 0.000 claims description 14
- 239000003814 drug Substances 0.000 claims description 14
- 210000000056 organ Anatomy 0.000 claims description 14
- 239000000126 substance Substances 0.000 claims description 14
- 208000023275 Autoimmune disease Diseases 0.000 claims description 13
- 230000003449 preventive effect Effects 0.000 claims description 13
- 229940124597 therapeutic agent Drugs 0.000 claims description 13
- 108091008874 T cell receptors Proteins 0.000 claims description 10
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 claims description 10
- 238000002054 transplantation Methods 0.000 claims description 8
- 206010062016 Immunosuppression Diseases 0.000 claims description 7
- 230000005934 immune activation Effects 0.000 claims description 7
- 241000288906 Primates Species 0.000 claims description 6
- 208000026935 allergic disease Diseases 0.000 claims description 6
- 239000003937 drug carrier Substances 0.000 claims description 5
- 230000000069 prophylactic effect Effects 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 230000002062 proliferating effect Effects 0.000 claims description 3
- 201000010099 disease Diseases 0.000 abstract description 13
- 241001465754 Metazoa Species 0.000 abstract description 7
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 description 348
- 241000699670 Mus sp. Species 0.000 description 48
- 241000699666 Mus <mouse, genus> Species 0.000 description 31
- 238000000684 flow cytometry Methods 0.000 description 28
- 102000003814 Interleukin-10 Human genes 0.000 description 25
- 108090000174 Interleukin-10 Proteins 0.000 description 25
- 229940076144 interleukin-10 Drugs 0.000 description 23
- 102100037850 Interferon gamma Human genes 0.000 description 20
- 108010074328 Interferon-gamma Proteins 0.000 description 20
- 102100032912 CD44 antigen Human genes 0.000 description 15
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 15
- 101001018097 Homo sapiens L-selectin Proteins 0.000 description 15
- 102100033467 L-selectin Human genes 0.000 description 15
- 241000282412 Homo Species 0.000 description 13
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 13
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 13
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 description 13
- 210000000987 immune system Anatomy 0.000 description 13
- 208000024891 symptom Diseases 0.000 description 13
- 239000011324 bead Substances 0.000 description 12
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 11
- 239000003550 marker Substances 0.000 description 11
- 208000032116 Autoimmune Experimental Encephalomyelitis Diseases 0.000 description 10
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 10
- 208000012997 experimental autoimmune encephalomyelitis Diseases 0.000 description 10
- 210000005259 peripheral blood Anatomy 0.000 description 10
- 239000011886 peripheral blood Substances 0.000 description 10
- 230000035755 proliferation Effects 0.000 description 10
- 210000004989 spleen cell Anatomy 0.000 description 10
- 230000028993 immune response Effects 0.000 description 9
- 210000004698 lymphocyte Anatomy 0.000 description 9
- 210000005087 mononuclear cell Anatomy 0.000 description 9
- 238000002965 ELISA Methods 0.000 description 8
- 102000000588 Interleukin-2 Human genes 0.000 description 8
- 108010002350 Interleukin-2 Proteins 0.000 description 8
- 239000012228 culture supernatant Substances 0.000 description 8
- 230000006870 function Effects 0.000 description 8
- 210000000952 spleen Anatomy 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 238000012546 transfer Methods 0.000 description 8
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 7
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 7
- 210000003714 granulocyte Anatomy 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 230000000638 stimulation Effects 0.000 description 7
- 102100025137 Early activation antigen CD69 Human genes 0.000 description 6
- 101000934374 Homo sapiens Early activation antigen CD69 Proteins 0.000 description 6
- 241000124008 Mammalia Species 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 210000000612 antigen-presenting cell Anatomy 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 6
- 238000000018 DNA microarray Methods 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 5
- 238000011579 SCID mouse model Methods 0.000 description 5
- 230000003213 activating effect Effects 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 230000001900 immune effect Effects 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 230000004936 stimulating effect Effects 0.000 description 5
- 102000006306 Antigen Receptors Human genes 0.000 description 4
- 108010083359 Antigen Receptors Proteins 0.000 description 4
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 4
- 230000037396 body weight Effects 0.000 description 4
- 239000012636 effector Substances 0.000 description 4
- 238000011813 knockout mouse model Methods 0.000 description 4
- 210000003141 lower extremity Anatomy 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000011325 microbead Substances 0.000 description 4
- 230000002093 peripheral effect Effects 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 3
- 102100036301 C-C chemokine receptor type 7 Human genes 0.000 description 3
- 101000716065 Homo sapiens C-C chemokine receptor type 7 Proteins 0.000 description 3
- 230000001270 agonistic effect Effects 0.000 description 3
- 230000000735 allogeneic effect Effects 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 238000003501 co-culture Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 238000010230 functional analysis Methods 0.000 description 3
- 239000003018 immunosuppressive agent Substances 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 230000003278 mimic effect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000010172 mouse model Methods 0.000 description 3
- 201000006417 multiple sclerosis Diseases 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 230000001629 suppression Effects 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- 206010009900 Colitis ulcerative Diseases 0.000 description 2
- 208000011231 Crohn disease Diseases 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- 208000004262 Food Hypersensitivity Diseases 0.000 description 2
- 229930186217 Glycolipid Natural products 0.000 description 2
- 208000009329 Graft vs Host Disease Diseases 0.000 description 2
- 101001013648 Homo sapiens Methionine synthase Proteins 0.000 description 2
- 206010020751 Hypersensitivity Diseases 0.000 description 2
- 101100340718 Mus musculus Il10 gene Proteins 0.000 description 2
- 102100023302 Myelin-oligodendrocyte glycoprotein Human genes 0.000 description 2
- 206010033799 Paralysis Diseases 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 201000006704 Ulcerative Colitis Diseases 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 208000010668 atopic eczema Diseases 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 230000000139 costimulatory effect Effects 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 230000008260 defense mechanism Effects 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 231100000676 disease causative agent Toxicity 0.000 description 2
- 230000010437 erythropoiesis Effects 0.000 description 2
- 210000004700 fetal blood Anatomy 0.000 description 2
- 210000003754 fetus Anatomy 0.000 description 2
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 208000024908 graft versus host disease Diseases 0.000 description 2
- 230000003394 haemopoietic effect Effects 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 210000005104 human peripheral blood lymphocyte Anatomy 0.000 description 2
- 230000006058 immune tolerance Effects 0.000 description 2
- 229940125721 immunosuppressive agent Drugs 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 208000000509 infertility Diseases 0.000 description 2
- 230000036512 infertility Effects 0.000 description 2
- 231100000535 infertility Toxicity 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 229960004857 mitomycin Drugs 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 231100000617 superantigen Toxicity 0.000 description 2
- 210000001541 thymus gland Anatomy 0.000 description 2
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 241000238876 Acari Species 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 208000035285 Allergic Seasonal Rhinitis Diseases 0.000 description 1
- 244000036975 Ambrosia artemisiifolia Species 0.000 description 1
- 235000003129 Ambrosia artemisiifolia var elatior Nutrition 0.000 description 1
- 206010002198 Anaphylactic reaction Diseases 0.000 description 1
- 208000032467 Aplastic anaemia Diseases 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 206010003827 Autoimmune hepatitis Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108700031361 Brachyury Proteins 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 102000010910 CD28 Antigens Human genes 0.000 description 1
- 108010062433 CD28 Antigens Proteins 0.000 description 1
- 210000003359 CD4-positive helper T lymphocyte Anatomy 0.000 description 1
- 210000001239 CD8-positive, alpha-beta cytotoxic T lymphocyte Anatomy 0.000 description 1
- 101150004010 CXCR3 gene Proteins 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 241000218645 Cedrus Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 102000009410 Chemokine receptor Human genes 0.000 description 1
- 108050000299 Chemokine receptor Proteins 0.000 description 1
- 241000222290 Cladosporium Species 0.000 description 1
- 241000725101 Clea Species 0.000 description 1
- 206010010099 Combined immunodeficiency Diseases 0.000 description 1
- 206010010744 Conjunctivitis allergic Diseases 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- 206010012438 Dermatitis atopic Diseases 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 206010013700 Drug hypersensitivity Diseases 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 240000008620 Fagopyrum esculentum Species 0.000 description 1
- 235000009419 Fagopyrum esculentum Nutrition 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 206010016946 Food allergy Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 241001656403 Lunaria Species 0.000 description 1
- 241000282560 Macaca mulatta Species 0.000 description 1
- 101000686985 Mouse mammary tumor virus (strain C3H) Protein PR73 Proteins 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 241000282339 Mustela Species 0.000 description 1
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 108010081690 Pertussis Toxin Proteins 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 241000282405 Pongo abelii Species 0.000 description 1
- 201000004681 Psoriasis Diseases 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 206010039085 Rhinitis allergic Diseases 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 241001125046 Sardina pilchardus Species 0.000 description 1
- 241000269821 Scombridae Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 201000009594 Systemic Scleroderma Diseases 0.000 description 1
- 206010042953 Systemic sclerosis Diseases 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 241001504592 Trachurus trachurus Species 0.000 description 1
- 206010052779 Transplant rejections Diseases 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 208000024780 Urticaria Diseases 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 208000002205 allergic conjunctivitis Diseases 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 208000030961 allergic reaction Diseases 0.000 description 1
- 201000010105 allergic rhinitis Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 230000036783 anaphylactic response Effects 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 235000003484 annual ragweed Nutrition 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 208000024998 atopic conjunctivitis Diseases 0.000 description 1
- 201000008937 atopic dermatitis Diseases 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 229960000686 benzalkonium chloride Drugs 0.000 description 1
- CADWTSSKOVRVJC-UHFFFAOYSA-N benzyl(dimethyl)azanium;chloride Chemical compound [Cl-].C[NH+](C)CC1=CC=CC=C1 CADWTSSKOVRVJC-UHFFFAOYSA-N 0.000 description 1
- 208000002352 blister Diseases 0.000 description 1
- 208000027503 bloody stool Diseases 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 235000006263 bur ragweed Nutrition 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 235000003488 common ragweed Nutrition 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 210000003162 effector t lymphocyte Anatomy 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 210000002375 erythropoietic cell Anatomy 0.000 description 1
- -1 etc.) Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 239000012997 ficoll-paque Substances 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 235000020932 food allergy Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 208000035861 hematochezia Diseases 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 229960003130 interferon gamma Drugs 0.000 description 1
- 238000010212 intracellular staining Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000000644 isotonic solution Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 206010025135 lupus erythematosus Diseases 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 235000020640 mackerel Nutrition 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 241001515942 marmosets Species 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 1
- 229940021222 peritoneal dialysis isotonic solution Drugs 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 208000005987 polymyositis Diseases 0.000 description 1
- 235000015277 pork Nutrition 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- MFDFERRIHVXMIY-UHFFFAOYSA-N procaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 1
- 229960004919 procaine Drugs 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 235000009736 ragweed Nutrition 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 235000019512 sardine Nutrition 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229960002920 sorbitol Drugs 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
- 208000016261 weight loss Diseases 0.000 description 1
Images
Classifications
-
- 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
-
- 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
- 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
- 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
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.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Diabetes (AREA)
- Pulmonology (AREA)
- Hematology (AREA)
- Reproductive Health (AREA)
- Endocrinology (AREA)
- Zoology (AREA)
- Neurology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Dermatology (AREA)
- Biochemistry (AREA)
- Emergency Medicine (AREA)
- Gynecology & Obstetrics (AREA)
- Physical Education & Sports Medicine (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Gastroenterology & Hepatology (AREA)
- Neurosurgery (AREA)
- Transplantation (AREA)
Abstract
Disclosed is a CD8-positive regulatory T cell which is more practical for an immune-related disease in an animal including a human body.
A CD8-positive CXCR3-positive T cell is used as an immunomodulatory agent.
Description
本発明は、免疫調節剤及びその利用に関し、詳しくはCD8陽性CXCR3陽性T細胞及びその利用に関する。
The present invention relates to an immunomodulator and use thereof, and more particularly to a CD8-positive CXCR3-positive T cell and use thereof.
ヒトなどの動物における免疫系は、病原菌などの外来抗原やがん細胞などの変異性自己抗原を検知し駆除する生体防御機構である。かかる生体防御は、Ｔ細胞による細胞性免疫及びＢ細胞を主体とする体液性免疫のそれぞれの応答によるものである。
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.
免疫系は活性化と抑制とのバランスを保つ制御システムの上に生体防御機構として成立している。このため、こうした制御系の崩壊が、アレルギー性疾患、自己免疫疾患並びに発癌といった疾患を発症させる場合もありうる。したがって、免疫系を制御する細胞を検出し分離することは、疾患の診断並びに予防及び治療に極めて有用である。
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.
現在、免疫系を正常に機能させるための制御機構は必ずしも明らかになっているわけではない。免疫系の制御システムの基本は、CＤ4陽性細胞やＣＤ８陽性細胞を含めた各種Ｔ細胞が担っていると考えられており、CD25陽性CD4陽性細胞が種々の自己免疫疾患の進展の抑制や臓器移植後の免疫寛容に有効であることが知られている（非特許文献１、２）。また、本発明者らは、CD8陽性キラーT細胞やCD4陽性ヘルパーＴ細胞を制御するCD8陽性制御性Ｔ細胞としてCD8陽性CD122陽性T細胞を既に分離している(特許文献１、非特許文献３及び非特許文献４）。
Currently, the control mechanism for the normal functioning of the immune system is not always clear. 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).
特許文献２及び非特許文献２に開示されるように、マウスにおいては、CD8陽性制御性Ｔ細胞は、CD122というマーカー分子の発現によって他の通常のエフェクターT細胞等から分離された。しかしながら、ヒトにおいては、CD8陽性T細胞中に、CD122陽性細胞を見出すことができなかった。したがって、ヒトにおいては、CD8陽性制御性T細胞は未だ分離されていない。
As disclosed in Patent Document 2 and Non-Patent Document 2, in mice, CD8-positive regulatory T cells were separated from other normal effector T cells by the expression of a marker molecule called CD122. However, in humans, 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.
本発明は、ヒトを含む動物の免疫関連疾患により実用的なCD8陽性制御性T細胞及びその利用を提供することを一つの目的とする。
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.
本発明者は、マウスのCD8 陽性CD122陽性T細胞に特異的に発現している分子をＤＮＡマイクロアレイ法で検索した結果、いくつかの候補遺伝子を見出し、これらの中にケモカイン受容体の一つであるCXCR3があるという知見を得た。さらに、特異的抗体を用いたフローサイトメトリーにより、CD8陽性細胞集団中のCD122陽性細胞集団は、CXCR3陽性細胞集団と一致しているという知見を得た。本発明者は、さらに、ヒトにおいてCD122陽性細胞集団中にCXCR3陽性細胞集団が存在し、当該細胞集団がCD8陽性制御性T細胞として機能するという知見を得た。本発明はこれらの知見によってなされたものである。本発明によれば以下の手段が提供される。
As a result of searching for molecules specifically expressed in mouse CD8 + -positive CD122-positive T cells by the DNA microarray method, 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.
本発明によれば、CD8陽性CXCR3陽性T細胞を含有する、免疫調節剤が提供される。本発明の免疫調節剤において、前記CD8陽性CXCR3陽性T細胞はインターロイキン１０の産生活性を有することができる。また、前記CD8陽性CXCR3陽性T細胞は霊長類由来とすることもできる。
According to the present invention, an immunomodulator containing CD8-positive CXCR3-positive T cells is provided. In the immunomodulator of the present invention, the CD8-positive CXCR3-positive T cells can have interleukin 10 production activity. Further, the CD8-positive CXCR3-positive T cells can be derived from primates.
本発明の免疫調節剤は、免疫抑制がその予防又は治療に有効である免疫関連障害の予防又は治療剤とすることもできる。この態様において、前記免疫関連障害は自己免疫疾患であってもよいし、臓器移植後の拒絶反応であってもよい。
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. In this embodiment, the immune-related disorder may be an autoimmune disease or a rejection after organ transplantation.
本発明によれば、免疫関連障害の予防又は治療剤であって、上記いずれかの免疫調節剤と、薬学的に許容される担体と、を含有する、予防又は治療剤が提供される。
According to the present invention, there is provided a preventive or therapeutic agent for an immune-related disorder, which comprises any of the above-mentioned immunomodulators and a pharmaceutically acceptable carrier.
本発明によれば、活性化されたCD8陽性制御性T細胞の製造方法であって、CD8陽性CXCR3陽性Ｔ細胞を準備する工程と、前記CD8陽性CXCR3陽性T細胞を、当該細胞のT細胞受容体を介して刺激する物質の存在下で培養する活性化工程と、を備える、製造方法が提供される。本発明の製造方法においては、前記活性化工程は、前記CD8陽性CXCR3陽性T細胞のインターロイキン１０の産生活性を発現させる工程であることが好ましい。また、前記活性化工程は、前記CD8陽性CXCR3陽性T細胞を特定の抗原の存在下で培養して当該抗原についての特異性を有するCD8陽性CXCR3陽性T細胞を活性化する工程とすることができる。この態様において、前記抗原は、自己免疫疾患の原因物質、アレルギー疾患の原因物質及び臓器移植時のドナー由来細胞又はその一部からなる群から選択することができる。
According to the present invention, there is provided a method for producing an activated CD8-positive regulatory T cell, the step of preparing a CD8-positive CXCR3-positive T cell, and the CD8-positive CXCR3-positive T cell, And an activating step of culturing in the presence of a substance that stimulates through the body. In the production method of the present invention, 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. . In this embodiment, 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.
本発明の活性化されたCD8陽性CXCR3陽性T細胞の製造方法においては、さらに、前記活性化したCD8陽性CXCR3陽性T細胞をIL10産生活性を指標として選抜する工程を備えることもできる。また、さらに、活性化された前記CD8陽性CXCR3陽性T細胞を培養して増殖させる工程を備えることもできる。
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.
本発明によれば、CD8陽性制御性Ｔ細胞の検出又は分離用キットであって、CD8陽性CXCR3陽性T細胞を検出又は分離する試薬を含む、キットが提供される。
According to the present invention, there is provided a kit for detecting or separating CD8-positive regulatory T cells, comprising a reagent for detecting or separating CD8-positive CXCR3-positive T cells.
本発明によれば、免疫関連障害の予防又は治療剤の製造方法であって、CD8陽性CXCR3陽性T細胞を準備する工程と、前記CD8陽性CXCR3陽性T細胞と薬学的に許容される担体とを混合する工程と、を備える、製造方法が提供される。
According to the present invention, there is provided 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.
本発明によれば、免疫関連障害の予防又は治療手段のスクリーニング方法であって、CD8陽性CXCR3陽性T細胞に１種又は２種以上の被験条件を付与して前記T細胞の免疫抑制活性を検出する工程、を備える、スクリーニング方法が提供される。本発明のスクリーニング方法においては、さらに、前記１種又は２種以上の被験条件中、前記免疫抑制活性を増強する１種又は２種以上の被験条件を免疫抑制が有効な免疫関連障害の予防又は治療手段として選択する工程を備えることもできる。また、さらに、前記１種又は２種以上の被験条件中、前記免疫抑制活性を低下させる１種又は２種以上の被験条件を免疫活性化が有効な免疫関連障害の予防又は治療手段として選択する工程、を備えることもできる。
According to the present invention, there is provided 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.
本発明によれば、免疫関連障害の予防又は治療方法であって、前記免疫関連障害の予防又は治療を要する個体に、CD8陽性CXCR3陽性T細胞を投与する工程、を備える、方法が提供される。また、本発明によれば、免疫関連障害の予防又は治療方法であって、前記免疫関連障害の予防又は治療を要する個体から、CD8陽性CXCR3陽性T細胞の少なくとも一部を除去する工程、を備える、方法が提供される。
According to the present invention, there is provided 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. . Further, according to the present invention, there is provided 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.
本発明は、DC8陽性制御性T細胞とその利用に関し、具体的には、当該制御性T細胞を含む免疫関連障害の予防又は治療剤及びその製造方法、該制御性T細胞を含む免疫抑制剤、免疫関連障害の予防又は治療手段のスクリーニング方法並びに免疫関連障害の予防又は治療方法等に関する。
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.
本発明は、本発明者らが分離したCD8陽性CXCR3陽性T細胞に基づいている。本発明者らは、マウスにおいてCD8陽性制御細胞として分離したCD8陽性CD122陽性細胞が、ヒトにおいてはCD8陽性CXCR3陽性細胞に相当することを確認した。したがって、CD8陽性CXCR3陽性T細胞は、少なくとも非霊長哺乳類であるマウスと霊長類であるヒトを含む動物、好ましくは哺乳動物において、CD8陽性制御性T細胞として機能することができる。
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. Accordingly, 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.
例えば、CD8陽性CXCR3陽性T細胞は、自身のインターロイキン１０（IL10）の産生活性を有するとき制御性T細胞として機能すると考えられる。したがって、当該産生活性に基づいて、また、標的細胞であるCD8陽性CXCR3陰性T細胞のIFN-γの産生の抑制に基づいて、免疫系の制御に寄与することができる。さらに、CD8陽性CXCR3陽性T細胞は、標的細胞であるCD陽性CD122陰性T細胞又はCD4陽性CD25陰性T細胞のインターフェロンγ（IFN-γ）の産生を抑制するとともにインターロイキン２（IL2）の産生を抑制することにより、免疫系の制御に寄与することができる。
For example, 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-γ production of CD8 positive CXCR3 negative T cells as target cells. Furthermore, CD8 positive CXCR3 positive T cells suppress the production of interferon γ (IFN-γ) 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.
本発明によれば、かかるCD8陽性制御性T細胞及びその前駆細胞を分離同定できたことから、当該制御性Ｔ細胞の過剰又は活性化を伴う免疫関連障害については、これらを除去等することにより当該障害を予防又は改善することが可能であり、また、当該制御性T細胞の減少又は不活性化を伴う免疫関連障害については、これらを補充又は活性化することにより当該障害を改善又は予防することができる。
According to the present invention, such CD8-positive regulatory T cells and their progenitor cells could be isolated and identified. For 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.
さらに、本発明によれば、CD8陽性制御性T細胞を分離同定できたことから、当該制御性T細胞の免疫制御活性を促進又は抑制する被験条件を見出すことにより、免疫抑制又は免疫活性化が有効な免疫関連障害の予防又は治療手段をスクリーニングすることができるようになる。
Furthermore, according to the present invention, since 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.
以下、本発明の各種実施形態について詳細に説明する。
Hereinafter, various embodiments of the present invention will be described in detail.
（免疫調節剤）
（CD8陽性CXCR3陽性Ｔ細胞）
本発明の免疫調節剤は、CD8陽性CXCR3陽性T細胞を含有している。当該T細胞は、CD8陽性及びCXCR3陽性の表現型を有しており、CD8陽性CXCR3陽性細胞集団に属している。当該T細胞は、in vitro及びin vivoにて活性化されると、CD8陽性制御性T細胞として機能することができる。このため、本発明の免疫調節剤は、CD8陽性制御性T細胞の免疫抑制能に基づき、免疫調節剤、特に免疫抑制剤として利用できる。 (Immunomodulator)
(CD8 positive CXCR3 positive T cells)
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. For this reason, 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.
（CD8陽性CXCR3陽性Ｔ細胞）
本発明の免疫調節剤は、CD8陽性CXCR3陽性T細胞を含有している。当該T細胞は、CD8陽性及びCXCR3陽性の表現型を有しており、CD8陽性CXCR3陽性細胞集団に属している。当該T細胞は、in vitro及びin vivoにて活性化されると、CD8陽性制御性T細胞として機能することができる。このため、本発明の免疫調節剤は、CD8陽性制御性T細胞の免疫抑制能に基づき、免疫調節剤、特に免疫抑制剤として利用できる。 (Immunomodulator)
(CD8 positive CXCR3 positive T cells)
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. For this reason, 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.
本明細書において、「制御性T細胞」とは、抗原等によりT細胞受容体を介する刺激を受けたとき、当該制御性T細胞の標的細胞の活性化（増殖、サイトカイン産生等）を抑制する能力（免疫抑制能）を有するT細胞を意味している。
In the present specification, “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).
本発明で用いるCD8陽性CXCR3陽性T細胞が標的とする標的細胞は、例えば全CD8陽性T細胞、CD8陽性CXCR3陰性細胞が挙げられる。
Examples of 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.
本発明で用いるCD8陽性CXCR3陽性T細胞は、T細胞受容体を介する刺激を受けたとき、効果（免疫抑制能）発揮因子としてインターロイキン１０(IL10)を自己産生することができる。これにより、標的細胞のIFN-γの産生を抑制できる。IL10の生産能は、例えば、IL10に特異的な抗体を用いてフローサイトメトリーやELISA等により検出・解析することができる。また、本発明で用いるCD8陽性CXCR3陽性T細胞は、標的細胞であるCD8陽性CXCR3陰性細胞等の増殖を抑制することができる。
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-γ 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.
なお、本明細書において、細胞の表現型がマーカー分子（抗原）発現の有無や強弱で表される場合、特に断りのない限り、当該マーカー分子に対する抗体による特異的結合の有無や強弱で表記される。マーカー分子の発現の有無や強弱による細胞の表現型の決定は、通常、当該マーカー分子に対する特異的抗体等を用いたフローサイトメトリー解析等により行われる。マーカー分子の発現が「陽性」とは、該マーカー分子が細胞表面上（或いは細胞内）に発現しており、当該マーカー分子に対する抗体による特異的結合が確認できることをいう。
In this specification, when the phenotype of a cell is 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 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.
本発明で用いるT細胞は、哺乳動物由来であることが好ましい。哺乳動物としては、上記した特定の表現型細胞集団が本発明のCD8陽性制御性T細胞として利用できる限り特に限定されないが、例えば、マウス、ラット、ハムスター、モルモット等のげっ歯類やウサギ等の実験動物、ブタ、ウシ、ヤギ、ウマ、ヒツジ、ミンク等の家畜、イヌ、ネコ等のペット、ヒト、サル、アカゲザル、マーモセット、オラウータン、チンパンジーなどの霊長類を挙げることが出来る。哺乳動物は、好ましくは霊長類であり、より好ましくはヒトである。
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.
本発明で用いるT細胞は、胸腺、臍帯血及び抹消組織のいずれかのCD8陽性CXCR3陽性T細胞である。したがって、本発明で用いるCD8陽性制御性T細胞は、末梢組織（胸腺及び臍帯血以外の組織：例えば末梢血液、脾臓、リンパ節、腸管、肝臓等）に存在している細胞であってもよい。調製操作の容易性を考慮すると、末梢血液又は脾臓中の細胞が好ましく用いられる。
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陽性CXCR3陽性T細胞は、単離・精製されていることが好ましい。当該T細胞の単離・精製は、哺乳動物等の各種組織から表現型等に基づき、公知の方法により行うことができる。例えば、ヒトのCD8陽性CXCR3陽性T細胞を有する末梢T細胞を単離・精製するには、まず、末梢組織（例えば末梢血液等）より、単核球分画が調製される。単核球分画の調製は、例えば、密度勾配遠心等により行われる。次に、蛍光色素や磁気ビーズ等により標識された、CD8及び／又はCXCR3に対する特異的抗体により単核球分画を染色し、セルソーターや磁性カラム等を用いて目的とする分画を単離・精製する。高い精製度を達成するため、好ましくは、セルソーターが用いられる。
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. For example, in order to isolate and purify peripheral T cells having human CD8-positive CXCR3-positive T cells, first, 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. Next, 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. In order to achieve a high degree of purification, a cell sorter is preferably used.
CD8陽性CXCR3陽性T細胞は、ex vivoで適切な刺激を付与してIL10を産生する活性化した状態としてもよい。こうすることで、投与後、速やかにin vivoにおいて制御性T細胞として機能させることができる。このような活性化されたCD8陽性CXCR3陽性T細胞は、IL10の産生の有無にて検出し分離することができる。以下、活性化されたCD8陽性CXCR3陽性T細胞の製造について説明する。
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.
（活性化されたCD8陽性CXCR3陽性T細胞の製造方法）
本発明の活性化されたCD8陽性CXCR3陽性T細胞の製造方法は、CD8陽性CXCR3陽性T細胞を準備する工程と、前記T細胞をT細胞受容体を介した刺激が可能な物質等の存在下、培養して活性化する工程と、を備えることができる。本発明の製造方法によれば、活性化したCD8陽性CXCR3陽性T細胞を得ることができ、この活性化したCD8陽性CXCR3陽性T細胞を投与することで、確実で速やかな効果が期待される。 (Method for producing activated CD8-positive CXCR3-positive T cells)
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.
本発明の活性化されたCD8陽性CXCR3陽性T細胞の製造方法は、CD8陽性CXCR3陽性T細胞を準備する工程と、前記T細胞をT細胞受容体を介した刺激が可能な物質等の存在下、培養して活性化する工程と、を備えることができる。本発明の製造方法によれば、活性化したCD8陽性CXCR3陽性T細胞を得ることができ、この活性化したCD8陽性CXCR3陽性T細胞を投与することで、確実で速やかな効果が期待される。 (Method for producing activated CD8-positive CXCR3-positive T cells)
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.
CD8陽性CXCR3陽性T細胞準備工程は、CD8陽性CXCR3陽性T細胞を、既に説明したように、適切な由来の採取源（例えば、ヒト患者等）の適切な部位から採取することによって実施できる。CD8陽性CXCR3陽性T細胞は、既に説明したように、単離・精製されていることが好ましい。
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.
活性化工程は、T細胞受容体を介した刺激を付与可能なT細胞受容体刺激物質を用いる。例えば、かかる物質としてCD8陽性CXCR3陽性T細胞の活性化に利用できる各種の抗原の１種又は２種以上を用いる。こうしたT細胞受容体刺激物質の存在下、CD8陽性CXCR3陽性T細胞を培養することによって活性化工程を実施できる。活性化工程における培養条件は、CD8陽性CXCR3陽性T細胞を活性化できればよく、リンパ球の培養において当業者に公知の条件から適宜選択し、あるいはこれらを適宜改変択して用いることができる。
In the activation step, a T cell receptor stimulating substance capable of applying stimulation via the T cell receptor is used. For example, one or more of 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.
CD8陽性CXCR3陽性T細胞を活性化してIL10産生活性を付与するには、IL2の存在下CD8陽性CXCR3陽性T細胞にT細胞受容体を介した刺激を付与することが好ましい。ここで、「抗原」とは、培養細胞上の抗原受容体（例えばＴ細胞受容体）に認識され、該受容体を介して細胞を刺激し得る物質を包括的に意味する。抗原としては、例えばペプチド、タンパク質、脂質、糖脂質等の抗原分子のみならず、免疫学的非自己細胞、抗原受容体の構成分子（CD3、TCRβ、TCRα等）や補刺激分子（CD28等）を認識する作動性抗体（例えば抗ヒトCD3抗体であるOKT-3等）やスーパー抗原などの抗原ミミックを含んでいてもよい。
In order to activate CD8-positive CXCR3-positive T cells and impart IL10 production activity, it is preferable to impart stimulation via a T-cell receptor to CD8-positive CXCR3-positive T cells in the presence of IL2. Here, “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. Examples of antigens include not only antigen molecules such as peptides, proteins, lipids and glycolipids, but also immunological non-self cells, antigen receptor constituent molecules (CD3, TCRβ, TCRα, etc.) and costimulatory molecules (CD28, etc.) May contain an antigen mimic such as a superantigen (eg, an anti-human CD3 antibody, OKT-3).
活性化工程は、得ようとする活性化されたCD8陽性CXCR3陽性T細胞の抗原に対する特異性に応じて種々の態様で実施できる。例えば、培養に供される上記CD8陽性CXCR3陽性T細胞の集団が有している抗原受容体のレパートリーの多様性を保持し、当該多様性を反映した制御性Ｔ細胞集団を製造しようとする場合には、CD8陽性CXCR3陽性T細胞が、抗原受容体の構成分子（CD3、TCRβ、TCRα等）を認識する作動性抗体（例えば抗ヒトCD3抗体であるOKT-3等）、補刺激分子（CD28等）を認識する作動性抗体（例えば抗ヒトCD28抗体）、スーパー抗原などの抗原ミミックが選択される（Blood, 104, p. 895-903, 2004、Blood, 104, p. 453-61, 2004）。当該抗原ミミックは複数種類を組合せて用いることも可能であり、例えばCD3を認識する作動性抗体と、CD28を認識する作動性抗体との組み合わせ等が用いられ得る。当該抗原ミミックを使用することで、多様性を有する制御性Ｔ細胞集団が得られる。
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, TCRβ, TCRα, 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. 453-61, 2004) ). 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.
また、活性化工程は、CD8陽性CXCR3陽性T細胞を特定の抗原の存在下で培養して抗原特異性を有する活性化されたCD8陽性CXCR3陽性T細胞を取得するようにしてもよい。後述するように特異的に活性化されたCD8陽性CXCR3陽性T細胞はIL10産生活性に基づき選抜できる。これにより、特定抗原による免疫系の亢進を抑制するのに好ましい活性化されたCD8陽性CXCR3陽性T細胞集団を得ることができる。
In the activation step, 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. As described later, 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.
例えば、ある特定の抗原分子（ペプチド、タンパク質、脂質、糖脂質等）に特異的なCD8陽性CXCR3陽性T細胞を製造しようとする場合は、当該抗原分子が選択される。抗原分子としては、例えば、組織適合抗原など細胞及び組織由来抗原、アレルギー疾患の原因抗原又は自己免疫疾患の原因抗原（食品由来抗原、抗原性を示すことが予想される薬剤若しくは製剤中に共存する物質、又は人工臓器関連物質、又はそれらの変性物（例えば熱変性物等））などが挙げられる。組織適合抗原としては主要組織適合抗原（ＭＨＣ抗原）や非主要組織適合抗原が挙げられる。アレルギーの原因物質には環境・花粉抗原、真菌抗原、食物抗原、人工抗原等が含まれ、例えば、環境・花粉抗原としてはダニ、ハウスダスト、スギ花粉、ブタクサ等、真菌抗原としてはカンジダ、アルテルナリア、アスペルギルス、クラドスポリウム、ペニシリウム等、食物抗原としては卵白、牛乳、大豆、小麦粉、ソバ粉、サバ、イワシ、アジ、エビ、カニ、ブタ肉、牛肉、トリ肉等、人工抗原としては薬剤、人工臓器等が挙げられる。また、自己免疫疾患の原因物質としては、疾患の原因となる自己抗体の対応抗原等が挙げられる。
For example, when a CD8-positive CXCR3-positive T cell specific to a specific antigen molecule (peptide, protein, lipid, glycolipid, etc.) is to be produced, the antigen molecule is selected. 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.
なお、採取源からのCD8陽性CXCR3陽性T細胞の採取に先立って、個体にCD8陽性CXCR3陽性T細胞を形成させる程度に特定の抗原を供給しておき、当該抗原に特異的なCD8陽性CXCR3陽性T細胞を採取しやすい状態にしておいてもよい。こうすることで、将来的に暴露される可能性のある抗原による免疫系の亢進を抑制可能な活性化されたCD8陽性CXCR3陽性T細胞を採取できる。
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.
このような手法は、臓器移植の拒絶反応抑制に有用である。例えば、臓器移植など、採取源である個体の免疫系が暴露される可能性のある特定の免疫学的非自己細胞又はその一部を予め個体に供給しておくことで、免疫学的非自己細胞に特異的なCD8陽性CXCR3陽性T細胞を当該個体から採取することが可能である。免疫学的非自己細胞は、公知の方法、例えば放射線（ガンマ線等）照射や抗癌剤（マイトマイシンＣ等）処理等で不活化されていることが好ましい。また、免疫学的非自己細胞の種類は特に限定されず、所望の組織由来の細胞（例えば末梢血単核球細胞（ＰＢＭＣ）等）を使用することが可能である。例えば、同種異系のドナーからの移植に先立ち、当該ドナー由来細胞に特異的なレシピエントの制御性Ｔ細胞の製造が意図されている場合にはドナー由来の細胞が選択される。この場合、該ドナー由来の細胞は、移植されうる臓器と同一臓器由来の細胞であっても、異なる組織由来の細胞であってもよい。
Such a technique is useful for suppressing rejection of organ transplants. For example, immunological non-self, such as organ transplantation, 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. For example, prior to transplantation from allogeneic donors, donor-derived cells are selected if production of recipient regulatory T cells specific for the donor-derived cells is intended. In this case, 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.
抗原提示を確実に達成するには、抗原提示細胞が選択されてもよい。抗原提示細胞としては、本発明の方法により制御性Ｔ細胞を製造し得る限り特に限定されないが、通常は、CD8陽性CXCR3陽性T細胞と同種同系の抗原提示細胞（例えば、該T細胞が由来する個体から得られた抗原提示細胞）が用いられる。また、抗原提示細胞の種類は、抗原提示能を有し、本発明の方法により制御性Ｔ細胞を製造し得る限り特に限定されないが、例えばPBMC、樹状細胞等が用いられる。抗原提示細胞は、自体公知の方法、例えば放射線（ガンマ線等）照射や抗癌剤（マイトマイシンＣ等）処理等で不活化されていることが好ましい。
In order to reliably achieve antigen presentation, 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. Usually, 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. For example, 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).
なお、抗原としては、複数種類の抗原を組合せて用いることも可能であり、例えば免疫学的非自己細胞と抗原受容体の構成分子を認識する作動性抗体との組み合わせ（同種異系細胞と抗ＣＤ３抗体との組み合わせ等）を用いることが可能である。
In addition, it is also possible to use a combination of a plurality of types of antigens as an antigen. Combinations with CD3 antibodies, etc.) can be used.
活性化工程によって活性化したCD8陽性CXCR3陽性T細胞をIL10産生活性を指標として選抜する工程を備えることができる。こうすることで、活性化したCD8陽性CXCR3陽性T細胞を選択的に含む高活性なCD8陽性CXCR3陽性T細胞集団を得ることができる。また、特定抗原で活性化した場合には、抗原特異性につき選択的である活性化されたCD8陽性CXCR3陽性T細胞を含む抗原特異的かつ高活性なCD8陽性CXCR3陽性T細胞集団を得ることができる。IL10産生活性を指標としてCD8陽性CXCR3陽性T細胞を選択するには、セルソーター等などが用いられる。なお、選抜工程は、必要に応じて実施される。
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. In addition, when activated with a specific antigen, 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. To select CD8-positive CXCR3-positive T cells using IL10 production activity as an index, a cell sorter or the like is used. The selection process is performed as necessary.
活性化工程後又は選択工程後において、活性化後のCD8陽性CXCR3陽性T細胞を培養して増殖させる工程を備えることができる。増殖工程は、CD8陽性CXCR3陽性T細胞の活性化状態を維持できるよう条件設定されるが、抗原などT細胞受容体刺激物質の非存在下でありかつIL2等の存在下で実施することが好ましい。増殖工程によって活性化されたCD8陽性CXCR3陽性T細胞を増殖させることができれば、個体の負担を抑制して必要量のCD8陽性CXCR3陽性T細胞を得ることができるため好ましい。また、抗原特異的かつ高活性のCD8陽性CXCR3陽性T細胞集団を増殖させることでより高い免疫調節効果の高い細胞集団を得ることができる。
After the activation step or after the selection step, 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.
以上説明したように、活性化工程、さらには必要に応じて選択工程及び／又は増殖工程を実施することで、所望の活性化されたCD8陽性CXCR3陽性T細胞集団を得ることができる。得られたCD8陽性CXCR3陽性T細胞集団は、IL10産生活性を有し、標的細胞であるCD8陽性CXCR3陰性T細胞の増殖やIFN-γ産生活性を抑制できる。
As described above, 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-γ production activity of CD8-positive CXCR3-negative T cells as target cells.
活性化されたCD8陽性CXCR3陽性T細胞も、活性化前のCD8陽性CXCR3陽性T細胞と同様に単離・精製されていることが好ましい。
It is preferable that 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.
（免疫関連障害の予防又は治療剤及びその製造方法並びに免疫関連障害の予防又は治療方法）
本発明の免疫関連障害の予防又は治療剤は、本発明の免疫調節剤、すなわち、CD8陽性CXCR3陽性T細胞（活性化されたものを含む。以下同じ。）を有効成分として含有している。また、本発明の免疫関連障害の予防又は治療方法は、免疫関連障害の予防又は治療を要する個体に、CD8陽性CXCR3陽性T細胞を投与する工程を備えることができる。 (Prevention or treatment of immune-related disorders and method for producing the same, and prevention or treatment of immune-related disorders)
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. In addition, 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.
本発明の免疫関連障害の予防又は治療剤は、本発明の免疫調節剤、すなわち、CD8陽性CXCR3陽性T細胞（活性化されたものを含む。以下同じ。）を有効成分として含有している。また、本発明の免疫関連障害の予防又は治療方法は、免疫関連障害の予防又は治療を要する個体に、CD8陽性CXCR3陽性T細胞を投与する工程を備えることができる。 (Prevention or treatment of immune-related disorders and method for producing the same, and prevention or treatment of immune-related disorders)
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. In addition, 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.
本発明の予防又は治療剤、すなわち、CD8陽性CXCR3陽性T細胞を免疫関連当該T細胞を免疫関連障害の予防又は治療を要する個体に投与することで、免疫系における当該T細胞量又は比率を増大させて、CD8陽性制御性T細胞の免疫抑制効果等に基づく予防又は治療効果を得ることができる。すなわち、生体内で免疫反応が異常に亢進している場合、生体内で望ましくない免疫反応が起こっている場合、あるいは望ましくない免疫応答が起こることが将来的に予測される場合等に、当該個体にCD8陽性CXCR3陽性細胞を投与することなどにより、異常に亢進した免疫反応を抑制し、望ましくない免疫反応を抑制し、あるいは望ましくない免疫反応を回避することができる。
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. Thus, 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. 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.
本発明の予防又は治療剤並びに予防又は治療方法は、免疫抑制がその予防又は治療に有効である障害に有効である。こうした免疫関連障害としては、自己免疫疾患（多発性筋炎、慢性リュウマチ、全身性エリテマトーシス、全身性硬化症、水ほう症、皮膚エリテマトーシス、乾癬症、クローンズ病、潰瘍性大腸炎、自己免疫性肝炎、多発性硬化症、１型糖尿病、再生不良性貧血等）、臓器移植における拒絶反応、アレルギー疾患（花粉症、食品アレルギー、薬剤アレルギー、喘息、アトピー性皮膚炎、湿疹、食物過敏症、蕁麻疹、アレルギー性鼻炎、アレルギー性結膜炎）、移植片対宿主病（ＧＶＨＤ）、不妊症等の予防・治療に有用である。
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. These 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.
例えば、上記各種疾患の予防又は治療として、CD8陽性CXCR3陽性T細胞を投与しておくことで、上記疾患によりあるいはそれに伴って発生する可能性のある免疫亢進状態を回避又は抑制することができる。
For example, 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.
また、上記各種疾患の予防又は治療として、当該患者由来のCD8陽性CXCR3陽性T細胞からこれらの予防又は治療に有効な抗原を用いて刺激して活性化した抗原特異的CD8陽性CXCR3陽性T細胞を投与してもよい。例えば、同種異系のドナーからの臓器移植に先立ち、まず、レシピエントにドナー由来の細胞又はその一部を投与して当該ドナー由来細胞に特異的なCD8陽性CXCR3陽性T細胞を生体内において取得し、こうして得られたT細胞を採取し、必要に応じて活性化し増殖させた後レシピエントに投与することで、移植片に対する免疫寛容を誘導し、移植片に対する拒絶反応を回避し、移植片の生着を促進することができる。
In addition, for the prevention or treatment of the above-mentioned various diseases, 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.
また、例えば、自己免疫疾患の患者において、当該患者由来のCD8陽性CXCR3陽性T細胞を、当該自己免疫疾患の原因抗原の存在下で培養することで、当該原因抗原に特異的に活性化された制御性Ｔ細胞を取得し、このＴ細胞を患者に投与することで、自己免疫反応を抑制することができる。
In addition, for example, in a patient with an autoimmune disease, 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. By obtaining regulatory T cells and administering these T cells to a patient, the autoimmune reaction can be suppressed.
さらに、アレルギー疾患の患者において、当該患者由来のCD8陽性CXCR3陽性T細胞を、アレルギーの原因抗原の存在下で培養することで、当該原因抗原に特異的なCD8陽性CXCR3陽性T細胞を取得し、このＴ細胞を患者に投与することで、アレルギー反応を抑制することができる。
Furthermore, in patients with allergic diseases, 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.
さらに、例えば、不妊症の患者においては、当該患者由来のCD8陽性CXCR3陽性T細胞を、パートナー由来の細胞の存在下で培養することで、当該パートナー由来細胞（又は抗原）に特異的なCD8陽性CXCR3陽性T細胞を取得し、このＴ細胞を患者に投与することで、パートナー由来細胞（又は抗原）或いは、当該パートナー由来抗原を保持する胎児に対する免疫寛容を誘導し、胎児に対する拒絶反応を回避し、妊娠の維持を促進することができる。
Furthermore, for example, in patients with infertility, 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. 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.
なお、本発明の予防又は治療剤は、当業者に周知の手法に従って、有効量のCD8陽性CXCR3陽性T細胞を準備、当該T細胞と薬学的に許容される担体とを混合する等して、経口／非経口製剤として製造することが出来る。本発明の免疫調節剤は、通常は、注射剤、懸濁剤、点滴剤等の非経口製剤として製造される。当該非経口製剤に含まれ得る担体としては、例えば、生理食塩水、ブドウ糖やその他の補助薬を含む等張液（例えば、Ｄ－ソルビトール、Ｄ－マンニトール、塩化ナトリウムなど）などの注射用の水性液を挙げることが出来る。本発明の免疫調節剤は、例えば、緩衝剤（例えば、リン酸塩緩衝液、酢酸ナトリウム緩衝液）、無痛化剤（例えば、塩化ベンザルコニウム、塩酸プロカインなど）、安定剤（例えば、ヒト血清アルブミン、ポリエチレングリコールなど）、保存剤、酸化防止剤などと配合してもよい。このようにして得られる製剤は安全で低毒性であるので、例えば、ヒト等の上述の哺乳動物に対して投与することができる。
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.
本発明の予防又は治療剤の投与量は、投与対象、対象臓器、症状、投与方法などにより差異はあるが、例えば、ヒト成人の患者（体重６０Ｋｇとして）においては、例えば非経口投与の場合、約１０9個程度／日を上限として有効量を投与することができる。
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. For example, in a human adult patient (with a body weight of 60 kg), for example, in the case of parenteral administration, An effective dose can be administered up to about 10 9 per day.
本発明の免疫関連障害の予防又は治療方法の別の態様として、免疫関連障害の予防又は治療を要する個体から、CD8陽性CXCR3陽性T細胞の少なくとも一部を除去する工程を備えることができる。過剰にCD8陽性CXCR3陽性T細胞を保持したり過剰に活性化したCD8陽性CXCR3陽性T細胞を保持する個体から、こうしたCD8陽性CXCR3陽性T細胞の少なくとも一部を除去することにより、例えば、免疫系の機能を改善又は活性化することができる。例えば、腫瘍に対する免疫応答を強化して腫瘍の予防又は治療が可能となる。
As another aspect of the method for preventing or treating an immune-related disorder of the present invention, 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. By removing at least some of these CD8-positive CXCR3-positive T cells from an individual that holds excessive CD8-positive CXCR3-positive T cells or over-activated CD8-positive CXCR3-positive T cells, for example, the immune system Can be improved or activated. For example, the immune response against the tumor can be enhanced to prevent or treat the tumor.
個体から、CD8陽性CXCR3陽性T細胞を除去するには、抗CXCR3抗体を静脈投与等することによって可能である。例えば、ヒトに対しては、抗ヒトCXCR3抗体を投与するのが有効である。なお、異種動物で作製された抗体を使用する場合には、免疫応答を抑制又はアナフィラキシーを抑制又は回避するために、抗体のFc部分をヒト抗体のFcに置き換えたヒト型抗体を用いることが好ましい。こうした抗体作製は当業者において周知である。
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. When using 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.
この態様の免疫関連障害としては、免疫活性化が予防又は治療に有効である疾患が挙げられる。このような疾患としては、ガン全般挙げられる。以上のことから、CXCR3抗体は、免疫活性化剤及び免疫活性化が有効な予防又治療に有効な疾患の予防又は治療剤として用いることができる。
The immune-related disorder in this aspect includes a disease in which immune activation is effective for prevention or treatment. Such diseases include cancer in general. From the above, 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.
（CD8陽性制御性Ｔ細胞の検出又は分離用キット）
本発明のキットは、CD8陽性CXCR3陽性T細胞を検出又は分離する試薬を含むことができる。本発明のキットによれば、適当な採取源からCD8陽性制御性T細胞を検出又は分離することができる。本発明のキットは、CD8陽性制御性T細胞を分離又は検出するためにCD8及びCXCR3の分子マーカーに対する標識抗体を含むことができる。さらに、活性化したCD8陽性CXCR3陽性T細胞を選抜するためのIL10に対する標識抗体を含むことができる。さらに、CD8陽性CXCR3陽性T細胞の活性化及び培養のための各種試薬（培地、IL2など）を含むことができる。このような検出又は分離用キットは、個体から採取した末梢血中等のCD8陽性CXCR3陽性T細胞を検出し、当該細胞の含有量や当該細胞の抗原特異性を評価するのに用いることで、個体の免疫系の状態や自己免疫疾患などの免疫関連障害の診断用キットとしても用いることができる。また、このような検出又は分離用キットは、CD8陽性CXCR3陽性T細胞を免疫関連障害の予防又は治療剤として用いるためのキットとしても用いることができる。 (CD8 positive regulatory T cell detection or isolation kit)
The kit of the present invention can contain a reagent for detecting or separating CD8-positive CXCR3-positive T cells. According to the kit of the present invention, 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. Furthermore, a labeled antibody against IL10 for selecting activated CD8-positive CXCR3-positive T cells can be included. Furthermore, 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.
本発明のキットは、CD8陽性CXCR3陽性T細胞を検出又は分離する試薬を含むことができる。本発明のキットによれば、適当な採取源からCD8陽性制御性T細胞を検出又は分離することができる。本発明のキットは、CD8陽性制御性T細胞を分離又は検出するためにCD8及びCXCR3の分子マーカーに対する標識抗体を含むことができる。さらに、活性化したCD8陽性CXCR3陽性T細胞を選抜するためのIL10に対する標識抗体を含むことができる。さらに、CD8陽性CXCR3陽性T細胞の活性化及び培養のための各種試薬（培地、IL2など）を含むことができる。このような検出又は分離用キットは、個体から採取した末梢血中等のCD8陽性CXCR3陽性T細胞を検出し、当該細胞の含有量や当該細胞の抗原特異性を評価するのに用いることで、個体の免疫系の状態や自己免疫疾患などの免疫関連障害の診断用キットとしても用いることができる。また、このような検出又は分離用キットは、CD8陽性CXCR3陽性T細胞を免疫関連障害の予防又は治療剤として用いるためのキットとしても用いることができる。 (CD8 positive regulatory T cell detection or isolation kit)
The kit of the present invention can contain a reagent for detecting or separating CD8-positive CXCR3-positive T cells. According to the kit of the present invention, 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. Furthermore, a labeled antibody against IL10 for selecting activated CD8-positive CXCR3-positive T cells can be included. Furthermore, 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.
（免疫関連障害の予防又は治療手段のスクリーニング方法）
本発明のスクリーニング方法は、CD8陽性CXCR3陽性T細胞に複数の被験条件を付与して各被験条件につき前記T細胞の免疫抑制活性を検出する工程、を備えることができる。このスクリーニング方法によればCD8陽性CXCR3陽性T細胞の免疫抑制活性を増強又は低下する被験条件を選択することができる。被験条件は、例えば、被験化合物をCD8陽性CXCR3陽性T細胞に接触させることであってもよいし、適当な遺伝子のノックアウト及び／又は発現を含む遺伝子改変を伴う遺伝子導入であってもよいし、温度等の環境条件であってもよい。 (Screening method for prevention or treatment of 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. According to this screening method, 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.
本発明のスクリーニング方法は、CD8陽性CXCR3陽性T細胞に複数の被験条件を付与して各被験条件につき前記T細胞の免疫抑制活性を検出する工程、を備えることができる。このスクリーニング方法によればCD8陽性CXCR3陽性T細胞の免疫抑制活性を増強又は低下する被験条件を選択することができる。被験条件は、例えば、被験化合物をCD8陽性CXCR3陽性T細胞に接触させることであってもよいし、適当な遺伝子のノックアウト及び／又は発現を含む遺伝子改変を伴う遺伝子導入であってもよいし、温度等の環境条件であってもよい。 (Screening method for prevention or treatment of 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. According to this screening method, 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.
このスクリーニング方法において、さらに、前記免疫抑制活性を増強する被験条件を、免疫抑制が有効な免疫関連障害の予防又は治療手段として選択する工程を備えることができる。こうしたスクリーニング方法によれば、免疫抑制剤及び本発明の予防又は治療剤の免疫関連障害に有効な予防又は治療手段が提供される。なお、CD8陽性CXCR3陽性T細胞の免疫抑制活性の増強は、CD8陽性CXCR3陽性T細胞の増加であってもよいし、CD8陽性CXCR3陽性T細胞自体の活性増強であってもよい。
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. According to such a screening method, 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.
このスクリーニング方法において、さらに、前記免疫抑制活性が低下する被験条件を、免疫活性化が有効な免疫関連障害の予防又は治療手段として選択する工程、を備えることができる。こうしたスクリーニング方法によれば、免疫活性化剤及び免疫活性化が予防又は治療に有効であるガン全般を始めとする各種疾患に有効な予防又は治療手段が提供される。なお、CD8陽性CXCR3陽性T細胞の免疫抑制活性の低下は、CD8陽性CXCR3陽性T細胞の減少であってもよいし、CD8陽性CXCR3陽性T細胞自体の活性低下であってもよい。
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. According to such a screening method, 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.
以下、本発明を実施例を挙げて具体的に説明するが、本発明は以下の実施例に限定されるものではない。なお、以下の実施例において特に断りのない限り、以下に示す材料及び方法を用いた。
Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to the following examples. In the following examples, the materials and methods shown below were used unless otherwise specified.
（マウス）
C57BL/６及びBALB/cマウスは、SLC、ジャパンより購入し、各種週令マウスを準備した。CB-17 SCIDマウスは、Cleaジャパンより購入した。
（ヒト単核球画分の調製）
ヒト単核球画分は健常人ボランティアから採取した末梢血をFicoll-Paque Plus（アマーシャムバイオサイエンス）で遠心分離しＰＢＳにて十分に洗浄することにより回収した。
（抗体）
全ての抗体は、商業的に入手した。
（フローサイトメトリー）
分析用途にはFACSキャリバーフローサイトメーター（BDバイオサイエンス）を用い、セルソーティング用途にはFACSヴァンテージセルソーター（BDバイオサイエンス）を用いた。
（磁性ビーズによる細胞精製）
CD8陽性細胞は、磁性マイクロビーズに結合させた抗CD8抗体及びカラム（ミルテニイバイオテック）を用いてそのプロトコールに従い分離した。
（細胞培養）
マウス及びヒト由来のそれぞれの細胞につき、２×１０5個の細胞を９６ウェルプレート上の平坦な底部を有する１ウェル内で、１０ng／.mlのIL2を含有する２００μｌの完全培地（RPMI1640＋１０％FCS及び５０μM 2-ME）中で培養した。
（ELISA）
マウス細胞の培養上清中のIL10濃度については、マウスIL10用のELISAキット（R＆Dシステムズ）を用いた。ヒト細胞の培養上清中のIL10濃度については、SRLに依頼した。
（DNAマイクロアレイ）
それぞれ１×１０6個のCD8陽性CXCR3陽性T細胞及びCD8陽性CXCR3陰性T細胞は、C57BL/６マウスの脾臓細胞からEFACSヴァンテージセルソーターを用いて回収した。ｍRNAを回収した細胞から抽出し、DNAマイクロアレイ解析は北海道システムサイエンスに依頼した。 (mouse)
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.
(Preparation of human mononuclear cell fraction)
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.
(Cell purification using magnetic beads)
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 μl complete medium (RPMI1640 + 10% FCS) containing 10 ng / .ml IL2 in one well with a flat bottom on a 96 well plate. And 50 μM 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.
(DNA microarray)
1 × 10 6 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.
C57BL/６及びBALB/cマウスは、SLC、ジャパンより購入し、各種週令マウスを準備した。CB-17 SCIDマウスは、Cleaジャパンより購入した。
（ヒト単核球画分の調製）
ヒト単核球画分は健常人ボランティアから採取した末梢血をFicoll-Paque Plus（アマーシャムバイオサイエンス）で遠心分離しＰＢＳにて十分に洗浄することにより回収した。
（抗体）
全ての抗体は、商業的に入手した。
（フローサイトメトリー）
分析用途にはFACSキャリバーフローサイトメーター（BDバイオサイエンス）を用い、セルソーティング用途にはFACSヴァンテージセルソーター（BDバイオサイエンス）を用いた。
（磁性ビーズによる細胞精製）
CD8陽性細胞は、磁性マイクロビーズに結合させた抗CD8抗体及びカラム（ミルテニイバイオテック）を用いてそのプロトコールに従い分離した。
（細胞培養）
マウス及びヒト由来のそれぞれの細胞につき、２×１０5個の細胞を９６ウェルプレート上の平坦な底部を有する１ウェル内で、１０ng／.mlのIL2を含有する２００μｌの完全培地（RPMI1640＋１０％FCS及び５０μM 2-ME）中で培養した。
（ELISA）
マウス細胞の培養上清中のIL10濃度については、マウスIL10用のELISAキット（R＆Dシステムズ）を用いた。ヒト細胞の培養上清中のIL10濃度については、SRLに依頼した。
（DNAマイクロアレイ）
それぞれ１×１０6個のCD8陽性CXCR3陽性T細胞及びCD8陽性CXCR3陰性T細胞は、C57BL/６マウスの脾臓細胞からEFACSヴァンテージセルソーターを用いて回収した。ｍRNAを回収した細胞から抽出し、DNAマイクロアレイ解析は北海道システムサイエンスに依頼した。 (mouse)
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.
(Preparation of human mononuclear cell fraction)
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.
(Cell purification using magnetic beads)
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 μl complete medium (RPMI1640 + 10% FCS) containing 10 ng / .ml IL2 in one well with a flat bottom on a 96 well plate. And 50 μM 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.
(DNA microarray)
1 × 10 6 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.
（マウスにおけるCD8陽性CXCR3陽性細胞とCD8陽性CD122陽性細胞の発現の比較解析）
マウスにおけるCD8陽性CXCR3陽性T細胞とCD8陽性CD122陽性T細胞との発現プロファイルを比較するために、まず、DNAマイクロアレイを実施した。セルソーティングによりC57BL/6マウスの脾臓からCD8陽性CD122陽性T細胞及びCD8陽性CD122陰性T細胞のそれぞれをおおよそ100万個程度採取し、これらの細胞集団から抽出したmRNAにつきDNAマイクロアレイ解析を行った。 (Comparative analysis of the expression of CD8 positive CXCR3 positive cells and CD8 positive CD122 positive cells in mice)
In order to compare the expression profiles of CD8 positive CXCR3 positive T cells and CD8 positive CD122 positive T cells in mice, DNA microarray was first performed. Approximately 1 million each of CD8 positive CD122 positive T cells and CD8 positive CD122 negative T cells were collected from the spleen of C57BL / 6 mice by cell sorting, and DNA microarray analysis was performed on mRNA extracted from these cell populations.
マウスにおけるCD8陽性CXCR3陽性T細胞とCD8陽性CD122陽性T細胞との発現プロファイルを比較するために、まず、DNAマイクロアレイを実施した。セルソーティングによりC57BL/6マウスの脾臓からCD8陽性CD122陽性T細胞及びCD8陽性CD122陰性T細胞のそれぞれをおおよそ100万個程度採取し、これらの細胞集団から抽出したmRNAにつきDNAマイクロアレイ解析を行った。 (Comparative analysis of the expression of CD8 positive CXCR3 positive cells and CD8 positive CD122 positive cells in mice)
In order to compare the expression profiles of CD8 positive CXCR3 positive T cells and CD8 positive CD122 positive T cells in mice, DNA microarray was first performed. Approximately 1 million each of CD8 positive CD122 positive T cells and CD8 positive CD122 negative T cells were collected from the spleen of C57BL / 6 mice by cell sorting, and DNA microarray analysis was performed on mRNA extracted from these cell populations.
マイクロアレイの結果、１０個以上の遺伝子がCD8陽性CD122陰性T細胞におけるよりもCD8陽性CD122陽性T細胞において２倍以上の発現レベルを示した。なかでも、CXCR3遺伝子がCD8陽性CD122陰性T細胞よりもCD8陽性CD122陽性T細胞において１０倍以上発現していることがわかった。
As a result of the microarray, 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. In particular, it was found that the CXCR3 gene was expressed more than 10 times in CD8 positive CD122 positive T cells than in CD8 positive CD122 negative T cells.
次いで、マウス抗CXCR3抗体を用いてマウスの脾臓におけるCXCR3（CD183）遺伝子の発現レベルをフローサイトメトリーで分析した。具体的には、脾臓細胞を異なる週令のC57BL/6マウスから採取し、抗CD8抗体及び抗CXCR3抗体で染色し、その後、フローサイトメトリーにより分析した。結果を図１Ａ及び図１Ｂに示す。
Subsequently, the expression level of the CXCR3 (CD183) gene in the spleen of the mouse was analyzed by flow cytometry using a mouse anti-CXCR3 antibody. Specifically, spleen cells were collected from C57BL / 6 mice of different ages, stained with anti-CD8 antibody and anti-CXCR3 antibody, and then analyzed by flow cytometry. The results are shown in FIGS. 1A and 1B.
図１Ａ及び図１Ｂに示すように、CD8陽性T細胞のうちの一部がCXCR3を発現していることがわかった。また、CXCR3陽性T細胞の割合は、マウスの個体年齢に依存する傾向があることがわかった。すなわち、新生仔マウスはほとんどCD8陽性細胞を有していなかったが、１週令マウスあたりからCD8陽性細胞が現れはじめた。幼若マウスでは、高い割合でCXCR3陽性細胞を含んでいたが、同様の現象は、CD122陽性細胞にも観察されていた。また、マウスの週令によるCXCR3陽性細胞の割合の変動を解析してみると、CD122陽性細胞におけるのと類似のパターン示すこともわかった。
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.
同時に、CD8陽性細胞群におけるCXCR3及びCD122の発現を解析した。具体的には、４週令マウスについて、CD8陽性細胞にゲートされた細胞についてCD122及びCXCR3の発現プロファイル及びCD4陽性細胞にゲートされた細胞についてCD25及びCXCR3の発現プロファイルをフローサイトメトリーにより分析した。結果を図１Ｃに示す。
Simultaneously, the expression of CXCR3 and CD122 in the CD8 positive cell group was 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.
図１Ｃに示すように、CD122及びCXCR3の双方を発現する明瞭な細胞集団があることがわかった。また、図１Ｃに示すように、CD25及びCXCR3の双方を発現する細胞集団を見出すことはできなかった。以上の結果から、マウスのCD8陽性細胞集団において、CD122及びCXCR3の発現には強い相関関係があることがわかった。一方、マウスのCD4陽性細胞において、CD25及びCXCR3の発現にはなんら相関関係がないことがわかった。
As shown in FIG. 1C, it was found that there was a clear cell population expressing both CD122 and CXCR3. In addition, as shown in FIG. 1C, a cell population expressing both CD25 and CXCR3 could not be found. From the above results, it was found that the expression of CD122 and CXCR3 has a strong correlation in the mouse CD8 positive cell population. On the other hand, it was found that there is no correlation between the expression of CD25 and CXCR3 in mouse CD4-positive cells.
また、CD8陽性細胞をCD44及びCD62L（CD44弱陰性CD62L強陽性、CD44強陽性CD62L弱陽性、CD44強陽性CD62L強陽性）の発現状態に基づき３つの細胞集団に分けて、これらの各集団のCD122及びCXCR3の発現プロファイルを確認した。具体的には、抗CD8抗体でコートしたマグネティックビーズを用いて６週令マウスから回収したCD8陽性細胞を抗CD44抗体、抗CD62L抗体及び抗CD122抗体並びに抗CXCR3抗体で染色した。これらの細胞をフローサイトメトリーで分析して、CD44及びCD62Lの発現パターンで決定される３つの細胞集団（#1:CD44弱陽性CD62L強陽性、#2：CD44強陽性CD62L強陽性、#3:CD44強陽性CD62L弱陽性）におけるCD122及びCXCR3の発現プロファイルを確認した。結果を図１Ｄに示す。
In addition, 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. These cells were analyzed by flow cytometry, and three cell populations determined by the expression pattern of CD44 and CD62L (# 1: CD44 weak positive CD62L strong positive, # 2: CD44 strong positive CD62L strong positive, # 3: CD122 and CXCR3 expression profiles in CD44 strong positive CD62L weak positive) were confirmed. The results are shown in FIG. 1D.
図１Ｄに示すように、CD8陽性CD122陽性T細胞集団（CD44強陽性CD62L強陽性T細胞集団）は、CD122及びCXCR3の双方を発現する細胞を高率で含み、ナイーブT細胞集団（CD44陰性CD62L強陽性T細胞集団）がCD8陽性CXCR3陰性T細胞であることがわかった。さらに、エフェクターメモリー細胞集団（CD44強陽性CD62L弱陽性T細胞集団）は、CXCR3又はCD122を発現する細胞を含むものの、CD44強陽性CD62L強陽性T細胞集団と比べて明確なCD8陽性CXCR3陽性T細胞集団を示すものではなかった。
As shown in FIG. 1D, 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. In addition, 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.
（マウスCD8陽性CXCR3陽性T細胞の制御性T細胞としてin vivoにおける機能解析）
本実施例では、リンパ球欠損重症複合型免疫不全（SCID）マウスにCD8陽性CXCR3陽性T細胞を伴って又は伴わずにCD8陽性CXCR3陰性T細胞の移植実験を行った。最初に、CD8陽性CXCR3陽性T細胞及びCD8陽性CXCR3陰性T細胞をBALB/cマウスから抗CD8抗体マグネティックビーズ及びセルソーターを用いて回収した。ビーズ処理後であってセルソーティング前のフローサイトメトリー及びセルソーティング後のフローサイトメトリーによる分析結果を図２Ａに示す。 (Functional analysis in vivo as regulatory T cells of mouse CD8 positive CXCR3 positive T cells)
In this example, lymphocyte-deficient severe combined immunodeficiency (SCID) mice were transplanted with CD8-positive CXCR3-negative T cells with or without CD8-positive CXCR3-positive T cells. First, CD8-positive CXCR3-positive T cells and CD8-positive CXCR3-negative T cells were collected from BALB / c mice using anti-CD8 antibody magnetic beads and a cell sorter. 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.
本実施例では、リンパ球欠損重症複合型免疫不全（SCID）マウスにCD8陽性CXCR3陽性T細胞を伴って又は伴わずにCD8陽性CXCR3陰性T細胞の移植実験を行った。最初に、CD8陽性CXCR3陽性T細胞及びCD8陽性CXCR3陰性T細胞をBALB/cマウスから抗CD8抗体マグネティックビーズ及びセルソーターを用いて回収した。ビーズ処理後であってセルソーティング前のフローサイトメトリー及びセルソーティング後のフローサイトメトリーによる分析結果を図２Ａに示す。 (Functional analysis in vivo as regulatory T cells of mouse CD8 positive CXCR3 positive T cells)
In this example, lymphocyte-deficient severe combined immunodeficiency (SCID) mice were transplanted with CD8-positive CXCR3-negative T cells with or without CD8-positive CXCR3-positive T cells. First, CD8-positive CXCR3-positive T cells and CD8-positive CXCR3-negative T cells were collected from BALB / c mice using anti-CD8 antibody magnetic beads and a cell sorter. 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.
図２Ａに示すように、実験に用いた細胞は、BALB/ｃマウスからセルソーティングにより採取し、純度が確認された。こうして調製されたCD8陽性CXCR3陰性T細胞をCD8陽性CD122陽性T細胞と混合した状態又は混合しない状態で、CB-17 SCIDマウスに移植した。
As shown in FIG. 2A, 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.
なお、リンパ球欠損SCIDマウス又はRAG－/－マウス（データ示さず）にCD8陽性CXCR3陰性T細胞を移植する場合には、このT細胞を受容したマウスは、CD69陽性活性化CD8陽性T細胞及び脾臓における顆粒球の増加を伴って不健康になり、最終的には赤血球生成が抑制される顆粒球優位の造血傾向を示すようになる。このような造血傾向は、CD8陽性CD122陽性制御性T細胞の不足の良好なマーカーになりうる。以上のことから、本実施例では、CD8陽性CXCR3陰性T細胞のCD69発現と脾臓における顆粒球の増大を指標としてT細胞の活性化を測定することとした。
When transplanting CD8 positive CXCR3-negative T cells into lymphocyte-deficient SCID mice or RAG − / − mice (data not shown), the 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.
具体的には、３×１０5個のCD8陽性CXCR3陰性T細胞のみ、又は１０5個のCD8陽性CXCR3陽性T細胞及び３×１０5個のCD8陽性CXCR3陰性T細胞との混合物を静脈経由でCB-17 SCIDマウスに移植した。１０週間経過後、脾臓細胞をフローサイトメーターにより分析した。結果を図２Ｂ～図２Dに示す。
Specifically, only 3 × 10 5 CD8-positive CXCR3-negative T cells, or 10 5 through the vein of a mixture of CD8-positive CXCR3 positive T cells and 3 × 10 5 CD8-positive CXCR3-negative T cells Transplanted into CB-17 SCID mice. After 10 weeks, spleen cells were analyzed with a flow cytometer. The results are shown in FIGS. 2B to 2D.
図２Ｂに示すように、CD8陽性CXCR3陰性T細胞のみを受容したSCIDマウスは、CD69陽性活性化CD8陽性T細胞の増加が認められたが、CD8陽性CD122陰性T細胞とCD8陽性CD122陽性T細胞とを受容したマウスでは、このような活性化されたT細胞の増加は認められなかった。図２Ｃに示すように、CD8陽性CXCR3陰性T細胞のみを受容したマウスの脾臓におけるGr-I陽性顆粒球の増加は、双方のT細胞を受容したマウスにおいては観察されなかった。さらに、図２Ｄに示すように、CD8陽性CXCR3陰性T細胞のみを受容したマウスの骨髄においては顆粒球優位でありかつ赤血球生成が抑制された造血状態が認められたが、双方のT細胞を受容したマウスの骨髄においてはこうした状態は改善されていた。
As shown in FIG. 2B, SCID mice receiving only CD8-positive CXCR3-negative T cells showed an increase in CD69-positive activated CD8-positive T cells, but CD8-positive CD122-negative T cells and CD8-positive CD122-positive T cells. In mice that received and, such an increase in activated T cells was not observed. As shown in FIG. 2C, an increase in Gr-I positive granulocytes in the spleen of mice that received only CD8 positive CXCR3 negative T cells was not observed in mice that received both T cells. Furthermore, as shown in FIG. 2D, in the bone marrow of mice that received only CD8-positive CXCR3-negative T cells, a hematopoietic state in which granulocyte was dominant and erythropoiesis was suppressed was observed, but both T cells were received. This condition was improved in the bone marrow of the isolated mice.
以上の結果から、マウスにおいてはCD122に加えてCXCR3もCD8陽性制御性T細胞のマーカーとして利用できることがわかった。
From the above results, it was found that CXCR3 can be used as a marker for CD8-positive regulatory T cells in addition to CD122 in mice.
（マウスCD8陽性CXCR3陽性T細胞の制御性T細胞としてのin vitroでの機能解析）
本実施例では、試験管内におけるマウスのCD8陽性CXCR3陽性T細胞の制御性T細胞の作用を調べた。マウスの脾臓細胞をセルソーティングにかけて、CD8陽性CXCR3陽性T細胞及びCD8陽性CXCR3陰性T細胞をそれぞれ回収した。 (In vitro functional analysis of mouse CD8-positive CXCR3-positive T cells as regulatory T cells)
In this example, the effect of regulatory T cells on mouse CD8-positive CXCR3-positive T cells in vitro was examined. Mouse spleen cells were subjected to cell sorting to collect CD8-positive CXCR3-positive T cells and CD8-positive CXCR3-negative T cells, respectively.
本実施例では、試験管内におけるマウスのCD8陽性CXCR3陽性T細胞の制御性T細胞の作用を調べた。マウスの脾臓細胞をセルソーティングにかけて、CD8陽性CXCR3陽性T細胞及びCD8陽性CXCR3陰性T細胞をそれぞれ回収した。 (In vitro functional analysis of mouse CD8-positive CXCR3-positive T cells as regulatory T cells)
In this example, the effect of regulatory T cells on mouse CD8-positive CXCR3-positive T cells in vitro was examined. Mouse spleen cells were subjected to cell sorting to collect CD8-positive CXCR3-positive T cells and CD8-positive CXCR3-negative T cells, respectively.
それぞれの細胞を、抗マウスCD3抗体+抗マウスCD28抗体コーティングビーズ（Dynabeads MouseCD3/CD28 T Cell Expander）で刺激して培養した。具体的には、CD8陽性CXCR3陽性T細胞及びCD8陽性CXCR3陰性T細胞をC57BL/6マウスからセルソーティングにより回収し、それぞれ抗マウスCD3抗体及び抗マウスCD28抗体でコーティングしたマイクロビーズによる刺激下で培養した。４８時間経過後、まずCD8抗体で染色し、その後、Cytofix/Cytopem（BDバイオサイエンス）を用いて細胞内IL10を検出した。また、同様にして培養して得られた各培養上清のIL10濃度をELISAにより測定した。これらの細胞についてのフローサイトメトリーの結果を図３Ａ及び図３Ｂに示す。また、ELISAによるIL10の測定結果を図３Ｃに示す。
Each cell was stimulated with anti-mouse CD3 antibody + anti-mouse CD28 antibody-coated beads (DynabeadsadMouseCD3 / CD28 T Cell Expander) and cultured. Specifically, 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). Further, the 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. Moreover, the measurement result of IL10 by ELISA is shown in FIG. 3C.
図３Ａ及び図３Ｂに示すように、CD8陽性CXCR3陰性T細胞に比べてより多くのCD8陽性CXCR3陽性T細胞がIL10を産生するようになった。IL10は、CD8陽性CD122陽性制御性T細胞によって産生される最も重要な作用伝達因子である。図３Cに示すように、培養上清についてのELISAにおいても、同様の現象が確認された。
As shown in FIGS. 3A and 3B, more CD8-positive CXCR3-positive T cells produced IL10 than CD8-positive CXCR3-negative T cells. IL10 is the most important effector produced by CD8 + CD122 + regulatory T cells. As shown in FIG. 3C, the same phenomenon was confirmed in the ELISA for the culture supernatant.
また、先と同様の抗体コーティングビーズを用いて刺激したCD8陽性CXCR3陰性T細胞培養におけるIFN-γ量を測定した。具体的には、セルソーティングによりC57BL/6マウスから採取したCD8陽性CXCR3陰性T細胞をまずCFSEによりラベルし、その後、単独又はCD8陽性CXCR3陽性T細胞と共に培養した。抗CD3抗体及び抗CD28抗体をコートしたマイクロビーズによる刺激下で４８時間培養した後、CFSE細胞（CD8陽性CXCR3陰性T細胞）のIFN-γの産生活性をフローサイトメトリーにより調べた。結果を図３Ｄ及び図３Ｅに示す。また、上記と同様にして細胞培養を行い、CFSE蛍光の減少によって細胞増殖を評価した。結果を図３Ｆに示す。
In addition, the amount of IFN-γ in CD8 positive CXCR3 negative T cell culture stimulated with the same antibody-coated beads as above was measured. Specifically, 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-γ 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.
図３Ｄに示すように、CD8陽性CXCR3陽性T細胞を伴わないCD8陽性CXCR3陰性T細胞は、IFN-γを産生した。一方、図３Ｅに示すように、このようなIFN-γ産生は、CD8陽性CXCR3陰性T細胞がCD8陽性CXCR3陽性T細胞とともに培養されたときには明らかに抑制され、CD8陽性CXCR3陽性T細胞の制御活性を呈していた。また、図３Ｆに示すように、CFSE蛍光の減少によって計測されたCD8陽性CXCR3陰性T細胞の増殖状態からは、単独培養時に比較して、CD8陽性CXCR3陽性T細胞と共培養したとき、CD8陽性CXCR3陰性T細胞の増殖が抑制されていることがわかった。すなわち、CD8陽性CXCR3陽性T細胞がCD8陽性CXCR3陰性T細胞に対して増殖抑制能を有していることがわかった。
As shown in FIG. 3D, CD8-positive CXCR3-negative T cells without CD8-positive CXCR3-positive T cells produced IFN-γ. On the other hand, as shown in FIG. 3E, such IFN-γ 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. Was presenting. In addition, as shown in 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. It was found that the proliferation of CXCR3-negative T cells was suppressed. That is, it was found that CD8-positive CXCR3-positive T cells have a growth-suppressing ability with respect to CD8-positive CXCR3-negative T cells.
以上の結果から、CD8陽性CXCR3陽性T細胞及びCD8陽性CXCR3陰性T細胞の関係は、CD8陽性CD122陽性T細胞及びCD8陽性CD122陰性T細胞の関係に極めて類似していることがわかった。
From the above results, it was found that the relationship between 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.
（ヒトCD8陽性CXCR3陽性T細胞の発現解析）
Ficoll重層遠心分離によってヒト末梢血から単核球を分離し、抗ヒトCD8抗体、抗ヒトCXCR3抗体及び抗ヒトCD122抗体で染色し、フローサイトメトリーにより分析した。結果を図４Ａ及び図４Ｂに示す。 (Expression analysis of human CD8-positive CXCR3-positive T cells)
Mononuclear cells were separated from human peripheral blood by Ficoll multilayer centrifugation, stained with anti-human CD8 antibody, anti-human CXCR3 antibody and anti-human CD122 antibody, and analyzed by flow cytometry. The results are shown in FIGS. 4A and 4B.
Ficoll重層遠心分離によってヒト末梢血から単核球を分離し、抗ヒトCD8抗体、抗ヒトCXCR3抗体及び抗ヒトCD122抗体で染色し、フローサイトメトリーにより分析した。結果を図４Ａ及び図４Ｂに示す。 (Expression analysis of human CD8-positive CXCR3-positive T cells)
Mononuclear cells were separated from human peripheral blood by Ficoll multilayer centrifugation, stained with anti-human CD8 antibody, anti-human CXCR3 antibody and anti-human CD122 antibody, and analyzed by flow cytometry. The results are shown in FIGS. 4A and 4B.
図４Ａに示すように、ヒト末梢血から採取したリンパ球には、明確にCD8陽性CD122陽性T細胞を確認できなかったが、Low-intermediate レベルのCD8陽性T細胞（CD8dim）中にCD122陽性細胞を見出した。しかしながら、これらのCD8dimCD122陽性細胞のほとんどがCD3陰性NK様細胞であった（データ示さず）。これに対して、図４Ａ及び図４Ｂに示すように、CD8強陽性細胞集団中に明らかにCXCR3陽性細胞が観察された。すなわち、CXCR3の発現プロファイルは、ヒト末梢血リンパ球におけるCD122発現プロファイルとは異なっていた。また、マウスCD8陽性細胞集団におけるCD122及びCXCR3の発現に関しては正の相関関係が認められた（実施例１、図１Ｃ）のに対し、ヒトCD8強陽性細胞集団におけるCD122及びCXCR3の発現には何ら相関関係は見出されなかった（図４Ｂ）。
As shown in FIG. 4A, 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).
さらに、CD8陽性CXCR3陽性T細胞がセントラルメモリー細胞やエフェクターメモリ細胞と関連があるかとうかを調べるために、ヒト細胞を抗CD45RA抗体及び抗CCR7抗体で染色し、フローサイトメトリーを行った。結果を図４Ｃに示す。
Furthermore, in order to examine whether 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.
図４Ｃに示すように、CD8陽性CXCR3陽性T細胞はCD8陽性CXCR3陰性T細胞よりは多くのCD45RA陰性メモリー細胞を含んでいるものの、CD8陽性CXCR3陽性T細胞とセントラルメモリー細胞（CD45RA陰性CCR7陽性）やエフェクターメモリー細胞（CD45RA陰性CCR7陰性）とは明らかな相関関係は認めらなかった。
As 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).
全てのCD8陽性細胞集団におけるCXCR3陽性細胞の比率を、異なる年齢の健常人ボランティアから採取した末梢血リンパ球（単核球）について、CD8及びCXCR3の発現プロファイルをフローサイトメトリーにより分析した。結果を図４Ｄに示す。また、ヒト末梢血リンパ球についての、CD4及びCD25の発現プロファイルをフローサイトメトリーにより分析するとともに、CD4陽性にゲートされた細胞集団についてのCD25及びCXCR3の発現プロファイルをフローサイトメトリーで分析した。結果を図４Ｅに示す。
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. In addition, 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. The result is shown in FIG. 4E.
図４Ｄに示すように、相対的に多くのCD8陽性CXCR3陽性T細胞が若年層と老年層に存在する傾向がある程度示唆された。しかしながら、全CD8陽性細胞におけるCD8陽性CXCR3陽性T細胞の比率の変化は、マウス（実施例１、図１Ａ及び図１Ｂ）におけるほど明確に観察されなかった。また、図４Ｅに示すように、ヒトにおいては、マウスと同様、CD4陽性細胞集団においてCD25及びCXCR3の相関関係は認められなかった。
As shown in 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. However, 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). Further, as shown in FIG. 4E, in humans, as in the case of mice, there was no correlation between CD25 and CXCR3 in the CD4 positive cell population.
（ヒトCD8陽性CXCR3陽性T細胞の制御性Ｔ細胞としてのin vitroでの機能解析）
実施例３においては、CD8陽性CXCR3陽性T細胞は、マウスにおいて制御性T細胞として機能した。本実施例では、CD8陽性CXCR3陽性T細胞がヒトにおいても制御性T細胞として機能するかどうかを確認した。 (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.
実施例３においては、CD8陽性CXCR3陽性T細胞は、マウスにおいて制御性T細胞として機能した。本実施例では、CD8陽性CXCR3陽性T細胞がヒトにおいても制御性T細胞として機能するかどうかを確認した。 (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.
まず、ヒト末梢血単核球画分を分離し、セルソーティングにかけて、CD8陽性CXCR3陽性T細胞及びCD8陽性CXCR3陰性T細胞をそれぞれ回収した。結果を図５Ａに示す。次に、抗ヒトCD3+抗ヒトCD28抗体コーティングビーズ（Dynabeads MouseCD3/CD28 T Cell Expander）の刺激下で、CD8陽性CXCR3陽性T細胞とCD8陽性CXCR3陰性T細胞とをそれぞれ別々に培養し、培養上清のIL10濃度をELISAで測定した。また、培養細胞をまず抗CD8抗体で染色し、その後、細胞内IL10をCytofix/Cytopemを用いて染色し、これらの細胞をフローサイトメトリーで分析した。これらの結果を図５Ｂ並びに図５Ｃ及び図５Ｄに示す。図５Ｂ～図５Ｄに示すように、CD8陽性CXCR3陽性T細胞はCD8陽性CXCR3陽性T細胞に比較してより多くのIL10を産生していることがわかった。
First, 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. Next, under the stimulation of anti-human CD3 + anti-human CD28 antibody-coated beads (Dynabeads MouseCD3 / CD28 T Cell Expander), 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. In addition, 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.
また、CD8陽性CXCR3陰性T細胞の単独培養及びCD8陽性CXCR3陽性T細胞との共培養につき、CFSE標識細胞（CD8陽性CXCR3陰性T細胞）のIFN-γ産生活性を調べた。具体的には、セルソーティングによりヒト末梢血から採取したCD8陽性CXCR3陰性T細胞をまずCFSEによりラベルし、その後、単独又はCD8陽性CXCR3陽性T細胞と共に培養した。抗CD3抗体及び抗CD28抗体をコートしたマイクロビーズによる刺激下で９６時間培養した後、抗CD8抗体による染色及び抗ヒトIFN-γ抗体による細胞内染色を行い、IFN-γ産生活性をフローサイトメトリーにより評価した。結果を図５Ｅ及び図５Ｆに示す。
In addition, the IFN-γ production activity of CFSE-labeled cells (CD8-positive CXCR3-negative T cells) was examined for single culture of CD8-positive CXCR3-negative T cells and co-culture with CD8-positive CXCR3-positive T cells. Specifically, 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-γ antibody were performed to determine the IFN-γ production activity It was evaluated by measurement. The results are shown in FIGS. 5E and 5F.
図５Ｅ及び図５Ｆに示すように、CD8陽性CXCR3陰性T細胞のみを培養したときにはIFN-γを産生したが、CD8陽性CXCR3陽性T細胞と共培養したときには、明らかにより少量のIFN-γしか産生しなかった。
As shown in FIGS. 5E and 5F, when only CD8-positive CXCR3-negative T cells were cultured, IFN-γ was produced, but when co-cultured with CD8-positive CXCR3-positive T cells, apparently a smaller amount of IFN-γ was produced. I did not.
さらに、CFSE蛍光の減少に基づく細胞増殖についても調べた。結果は、図５Ｇに示すように、単独培養時に比較して、CD8陽性CXCR3陽性T細胞と共培養したとき、CD8陽性CXCR3陰性T細胞の増殖が抑制されていた。すなわち、CD8陽性CXCR3陽性T細胞は、CD8陽性CXCR3陰性T細胞の増殖を抑制する能力があることがわかった。
Furthermore, cell proliferation based on the decrease in CFSE fluorescence was also examined. As a result, as shown in FIG. 5G, when co-cultured with CD8-positive CXCR3-positive T cells, proliferation of CD8-positive CXCR3-negative T cells was suppressed as compared with the case of single culture. That is, it was found that CD8-positive CXCR3-positive T cells have the ability to suppress the proliferation of CD8-positive CXCR3-negative T cells.
以上の結果から、ヒトのin vitro実験系において、CD8陽性CXCR3陽性T細胞は制御性T細胞として機能することがわかった。また、ヒトCD8陽性T細胞では発現されないCD122に代わり、CXCR3は、ヒト制御性T細胞のマーカーとして利用できることがわかった。既に、実施例２及び３において示したように、マウス実験系において、CD8陽性CXCR3陽性T細胞がCD8陽性CD122陽性制御性T細胞として実質的に等しいことを示したが、ヒトCD8陽性CXCR3陽性T細胞がマウスCD8陽性CD122陽性制御性T細胞のヒトにおける対応細胞であることがわかった。
From the above results, it was found that 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.
（ヒト多発性硬化症（ＭＳ）モデルマウス（EAEマウス）に対するCD8陽性CD122陽性細胞投与の効果）
ヒトMSの動物モデルとしてマウスEAE（実験的自己免疫性脳脊髄炎）がある。本実施例では、以下のプロトコールに従い、ＥＡＥマウスに対してCD8陽性CD122陽性T細胞を投与してその症状の改善について評価した。 (Effect of administration of CD8 + CD122 + cells on human multiple sclerosis (MS) model mice (EAE mice))
There is mouse EAE (experimental autoimmune encephalomyelitis) as an animal model of human MS. In this example, according to the following protocol, CD8 positive CD122 positive T cells were administered to EAE mice to evaluate improvement of symptoms.
ヒトMSの動物モデルとしてマウスEAE（実験的自己免疫性脳脊髄炎）がある。本実施例では、以下のプロトコールに従い、ＥＡＥマウスに対してCD8陽性CD122陽性T細胞を投与してその症状の改善について評価した。 (Effect of administration of CD8 + CD122 + cells on human multiple sclerosis (MS) model mice (EAE mice))
There is mouse EAE (experimental autoimmune encephalomyelitis) as an animal model of human MS. In this example, according to the following protocol, CD8 positive CD122 positive T cells were administered to EAE mice to evaluate improvement of symptoms.
（プロトコール）
（１）抗マウスCD122抗体（クローンTM-b1）は、ハイブリドーマ細胞をヌードマウスの腹腔に植え付けて増殖させた後に、腹水を回収し、硫安沈殿をして精製したものを使用した。マウス１匹あたり25mgの抗体を尾静脈より注射した。
（２）EAEの誘導には、メスのB6マウスを使用し、200mgのMOG35-55ペプチドを4mg/mlの結核菌死菌を含んだ完全フロイントアジュバントとよく混合し、エマルジョン化したものを尾の付け根の皮内に注射（この時をday0とする）し、同日と２日後の２回にわたりPertussis Toxin 200ngを腹腔内に注射することで行った。
（３）EAE症状の評価は、スコア0：症状なし、スコア1：尾または片側の後肢の脱力、スコア２：尾と後肢の脱力、スコア３：後肢の不完全麻痺、スコア４：後肢の完全麻痺、スコア５：マウス死亡、として行った。
（４）CD8+CD122+細胞もしくはCD8+CD122-細胞の移入には、マウス脾細胞からCD8+細胞を抗CD8抗体の結合した磁気ビーズ（ミルテニー社製）を用いて分離回収し、さらにそれを蛍光標識抗CD8抗体と蛍光標識抗CD122抗体で染色したものをセルソーター（BD社製FACSVantage SE）を用いて99％以上の純度で回収し、その細胞をマウスの尾静脈から注射した。プロトコールの概要とB6マウスにMOGペプチドを免疫してEAEを誘導し、症状をスコア化して追跡した結果を図６に示す。 (Protocol)
(1) As anti-mouse CD122 antibody (clone TM-b1), hybridoma cells were planted in the abdominal cavity of nude mice and proliferated, and ascites was collected and purified by ammonium sulfate precipitation. 25 mg antibody per mouse was injected from the tail vein.
(2) For induction of EAE, female B6 mice were used, and 200 mg of MOG 35-55 peptide was mixed well with complete Freund's adjuvant containing 4 mg / ml of Mycobacterium tuberculosis killed and emulsified. This was performed by intraperitoneally injecting 200 ng Pertussis Toxin twice a day and two days later.
(3) 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.
(4) For the transfer of CD8 + CD122 + cells or CD8 + CD122 − cells, CD8 + 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.
（１）抗マウスCD122抗体（クローンTM-b1）は、ハイブリドーマ細胞をヌードマウスの腹腔に植え付けて増殖させた後に、腹水を回収し、硫安沈殿をして精製したものを使用した。マウス１匹あたり25mgの抗体を尾静脈より注射した。
（２）EAEの誘導には、メスのB6マウスを使用し、200mgのMOG35-55ペプチドを4mg/mlの結核菌死菌を含んだ完全フロイントアジュバントとよく混合し、エマルジョン化したものを尾の付け根の皮内に注射（この時をday0とする）し、同日と２日後の２回にわたりPertussis Toxin 200ngを腹腔内に注射することで行った。
（３）EAE症状の評価は、スコア0：症状なし、スコア1：尾または片側の後肢の脱力、スコア２：尾と後肢の脱力、スコア３：後肢の不完全麻痺、スコア４：後肢の完全麻痺、スコア５：マウス死亡、として行った。
（４）CD8+CD122+細胞もしくはCD8+CD122-細胞の移入には、マウス脾細胞からCD8+細胞を抗CD8抗体の結合した磁気ビーズ（ミルテニー社製）を用いて分離回収し、さらにそれを蛍光標識抗CD8抗体と蛍光標識抗CD122抗体で染色したものをセルソーター（BD社製FACSVantage SE）を用いて99％以上の純度で回収し、その細胞をマウスの尾静脈から注射した。プロトコールの概要とB6マウスにMOGペプチドを免疫してEAEを誘導し、症状をスコア化して追跡した結果を図６に示す。 (Protocol)
(1) As anti-mouse CD122 antibody (clone TM-b1), hybridoma cells were planted in the abdominal cavity of nude mice and proliferated, and ascites was collected and purified by ammonium sulfate precipitation. 25 mg antibody per mouse was injected from the tail vein.
(2) For induction of EAE, female B6 mice were used, and 200 mg of MOG 35-55 peptide was mixed well with complete Freund's adjuvant containing 4 mg / ml of Mycobacterium tuberculosis killed and emulsified. This was performed by intraperitoneally injecting 200 ng Pertussis Toxin twice a day and two days later.
(3) 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.
(4) For the transfer of CD8 + CD122 + cells or CD8 + CD122 − cells, CD8 + 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.
図６に示すように、抗CD122抗体を注射してCD8+CD122+細胞を除去したマウスにＥＡＥを導入すると、ピークに達した症状が回復せずに重症のまま経過した。これに対して、こうしたマウスに、他の同系マウスから採取したCD8+CD122+制御性T細胞を移入すると速やかな症状の快復が認められた。CD8+CD122－制御性T細胞を移入しても症状は回復しなかった。以上のことから、CD8+CD122+制御性T細胞がEAEの症状快復に必須の役割をしていることがわかった。CD8+CD122+制御性T細胞は、ヒトのCD8+CXCR3+制御性T細胞に相当することから、CD8+CXCR3+制御性T細胞は、ヒトのＭＳの予防又は治療に用いることができることがわかった。
As shown in FIG. 6, when EAE was introduced into a mouse from which CD8 + CD122 + cells were removed by injection of an anti-CD122 antibody, the symptoms that reached the peak did not recover and remained severe. In contrast, when these mice were transfected with CD8 + CD122 + regulatory T cells collected from other syngeneic mice, rapid recovery of symptoms was observed. Symptoms did not recover after transfer of CD8 + CD122 − regulatory T cells. Based on the above, it was found that 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.
（炎症性腸疾患モデルマウスへに対するCD8陽性CD122陽性T細胞投与の効果）
潰瘍性大腸炎とクローン病を総称して炎症性腸疾患（IBD）というが、これらも免疫応答が関わった自己免疫性疾患の一つである。IBDモデルマウスにもいくつかの種類があるが、よく使われるものとしてリンパ球を欠損するRAGノックアウトマウスにCD4+CD45RB+細胞を養子移入するという系がある。本実施例では、RAGノックアウトマウスに対してCD8+CD122+制御性T細胞を投与したときのIBDの症状の変化を観察した。なお、IBDの症状が体重変化に如実に反映されることは、これまでの多くの論文によって証明されている。 (Effect of CD8 + CD122 + T cell administration to inflammatory bowel disease model mice)
Ulcerative colitis and Crohn's disease are collectively called inflammatory bowel disease (IBD), but these are also autoimmune diseases that involve immune responses. There are several types of IBD model mice, but the most commonly used system is adoptive transfer of CD4 + CD45RB + cells to RAG knockout mice lacking lymphocytes. In this example, changes in IBD symptoms were observed when CD8 + CD122 + regulatory T cells were administered to RAG knockout mice. The fact that IBD symptoms are reflected in changes in body weight has been proven by many previous papers.
潰瘍性大腸炎とクローン病を総称して炎症性腸疾患（IBD）というが、これらも免疫応答が関わった自己免疫性疾患の一つである。IBDモデルマウスにもいくつかの種類があるが、よく使われるものとしてリンパ球を欠損するRAGノックアウトマウスにCD4+CD45RB+細胞を養子移入するという系がある。本実施例では、RAGノックアウトマウスに対してCD8+CD122+制御性T細胞を投与したときのIBDの症状の変化を観察した。なお、IBDの症状が体重変化に如実に反映されることは、これまでの多くの論文によって証明されている。 (Effect of CD8 + CD122 + T cell administration to inflammatory bowel disease model mice)
Ulcerative colitis and Crohn's disease are collectively called inflammatory bowel disease (IBD), but these are also autoimmune diseases that involve immune responses. There are several types of IBD model mice, but the most commonly used system is adoptive transfer of CD4 + CD45RB + cells to RAG knockout mice lacking lymphocytes. In this example, changes in IBD symptoms were observed when CD8 + CD122 + regulatory T cells were administered to RAG knockout mice. The fact that IBD symptoms are reflected in changes in body weight has been proven by many previous papers.
（プロトコール）
（１）リンパ球を持たないRAGノックアウトマウス（遺伝的背景はB6）に各種の細胞を移入した後、マウスの体重を暫時計測し、細胞移入前の体重に対する増減を百分率で記録した。
（２）CD4+CD45RB+細胞、CD4+CD45RB-細胞、CD8+CD122+細胞、CD8+CD122-細胞はすべてB6マウスの脾細胞から調製した。
（３）第一段階として、抗CD4もしくは抗CD8抗体を結合させた磁気ビーズ（ミルテニー社製）によってCD4+細胞もしくはCD8+細胞を分離回収し、その後蛍光標識した抗CD4、抗CD45RB、抗CD8、抗CD122抗体で染色して、目的の細胞をセルソーター（BD社製FACSVantage SE）を用いて高純度で分離回収した。
（４）CD4+CD45RB+細胞はいずれの組み合わせの細胞移入においても4x105個を用い、そこにCD8+CD122+細胞、CD8+CD122-細胞、CD4+CD45RB-細胞はそれぞれ2x105個を加える形で混ぜ合わせて経静脈的に移入した。
結果を図７に示す。 (Protocol)
(1) After transferring various cells into RAG knockout mice (genetic background is B6) without lymphocytes, the body weight of the mice was measured for a while, and the increase / decrease relative to the body weight before cell transfer was recorded as a percentage.
(2) CD4 + CD45RB + cells, CD4 + CD45RB − cells, CD8 + CD122 + cells, and CD8 + CD122 − cells were all prepared from spleen cells of B6 mice.
(3) As a first step, 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).
(4) Use 4x10 5 CD4 + CD45RB + cells for any combination of cell transfer, and add 2x10 5 each for CD8 + CD122 + cells, CD8 + CD122 - cells, and CD4 + CD45RB - cells Mixed and transferred intravenously.
The results are shown in FIG.
（１）リンパ球を持たないRAGノックアウトマウス（遺伝的背景はB6）に各種の細胞を移入した後、マウスの体重を暫時計測し、細胞移入前の体重に対する増減を百分率で記録した。
（２）CD4+CD45RB+細胞、CD4+CD45RB-細胞、CD8+CD122+細胞、CD8+CD122-細胞はすべてB6マウスの脾細胞から調製した。
（３）第一段階として、抗CD4もしくは抗CD8抗体を結合させた磁気ビーズ（ミルテニー社製）によってCD4+細胞もしくはCD8+細胞を分離回収し、その後蛍光標識した抗CD4、抗CD45RB、抗CD8、抗CD122抗体で染色して、目的の細胞をセルソーター（BD社製FACSVantage SE）を用いて高純度で分離回収した。
（４）CD4+CD45RB+細胞はいずれの組み合わせの細胞移入においても4x105個を用い、そこにCD8+CD122+細胞、CD8+CD122-細胞、CD4+CD45RB-細胞はそれぞれ2x105個を加える形で混ぜ合わせて経静脈的に移入した。
結果を図７に示す。 (Protocol)
(1) After transferring various cells into RAG knockout mice (genetic background is B6) without lymphocytes, the body weight of the mice was measured for a while, and the increase / decrease relative to the body weight before cell transfer was recorded as a percentage.
(2) CD4 + CD45RB + cells, CD4 + CD45RB − cells, CD8 + CD122 + cells, and CD8 + CD122 − cells were all prepared from spleen cells of B6 mice.
(3) As a first step, 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).
(4) Use 4x10 5 CD4 + CD45RB + cells for any combination of cell transfer, and add 2x10 5 each for CD8 + CD122 + cells, CD8 + CD122 - cells, and CD4 + CD45RB - cells Mixed and transferred intravenously.
The results are shown in FIG.
RAGノックアウトマウスに見られるIBDは、移入したCD4+CD45RB+細胞がCD4+の制御性T細胞（CD4+CD25+細胞）を含まないために起こってくるものである。CD4+CD45RB-細胞（CD4+CD25+制御性T細胞を含む）を同時に移入すれば症状（下痢、血便、体重減少）は起こってこない。また、CD4+CD45RB-細胞の代わりに正常マウスから採取したCD8+CD122+制御性T細胞をCD4+CD45RB+細胞と同時に移入してもIBD症状は起こってこなかった。この実験結果は、CD8+CD122+制御性T細胞がIBDの発症抑制に効果的に働くことを示しており、CD8+CD122+制御性T細胞（ヒトではCD8+CXCR3+制御性T細胞）がIBDの予防／治療に使えることがわかった。
IBD seen in RAG knockout mice, in which populated the 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. The results of this experiment show that CD8 + CD122 + regulatory T cells are effective in suppressing the onset of IBD, and CD8 + CD122 + regulatory T cells (CD8 + CXCR3 + regulatory T cells in humans) It was found that it can be used for prevention / treatment of IBD.
（CD8+CD122+制御性T細胞（ヒトCD8+CXCR3+制御性T細胞）のin vitroでの培養）
ヒト疾患に対する制御性T細胞の有用性を示すには、その制御性T細胞をin vitroで培養増殖させることが必要である。具体的方法としては、末梢血から採取した制御性T細胞を抗CD3抗体などで刺激して活性化状態にし、IL-2添加培地中で培養して増殖させる技術を確立する必要がある。本実施例では、培養増殖させることが簡単なマウスT細胞を用いて実験を行った。 (In vitro culture of CD8 + CD122 + regulatory T cells (human CD8 + CXCR3 + regulatory T cells))
In order to show the usefulness of regulatory T cells for human diseases, it is necessary to culture and propagate the regulatory T cells in vitro. As a specific method, it is necessary to establish a technique for stimulating regulatory T cells collected from peripheral blood with an anti-CD3 antibody to be activated and cultivating them in a medium supplemented with IL-2. In this example, experiments were conducted using mouse T cells that were easy to grow and grow.
ヒト疾患に対する制御性T細胞の有用性を示すには、その制御性T細胞をin vitroで培養増殖させることが必要である。具体的方法としては、末梢血から採取した制御性T細胞を抗CD3抗体などで刺激して活性化状態にし、IL-2添加培地中で培養して増殖させる技術を確立する必要がある。本実施例では、培養増殖させることが簡単なマウスT細胞を用いて実験を行った。 (In vitro culture of CD8 + CD122 + regulatory T cells (human CD8 + CXCR3 + regulatory T cells))
In order to show the usefulness of regulatory T cells for human diseases, it is necessary to culture and propagate the regulatory T cells in vitro. As a specific method, it is necessary to establish a technique for stimulating regulatory T cells collected from peripheral blood with an anti-CD3 antibody to be activated and cultivating them in a medium supplemented with IL-2. In this example, experiments were conducted using mouse T cells that were easy to grow and grow.
CD8+CD122+細胞を、セルソーターを用いて純粋に採取し、抗CD3抗体（BDBiocoat、BD社製）で24時間刺激し活性化状態にした。以後は、細胞の維持・増殖にはヒトレコンビナントIL-2（50ng/ml）のみを加えた培地で行い、細胞を100倍以上まで増殖させ得ることがわかった。この増殖させた細胞には、CD8+制御性T細胞が抑制作用を発揮するための必須分子であるIL-10を発現することが、培養上清中のIL-10をELISA法（QuantikineマウスIL-10測定キット、R＆D社製を使用）で測定した結果確かめられており、制御性細胞としての機能を保ったままマウスCD8+制御性T細胞を培養増殖させることが可能であることがわかった。同様の方法により、ヒトCD8+CXCR3+制御性T細胞を培養できると考えられる。
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 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.
Claims (20)
- CD8陽性CXCR3陽性T細胞を含有する、免疫調節剤。 An immunomodulator containing CD8-positive CXCR3-positive T cells.
- 前記CD8陽性CXCR3陽性T細胞はインターロイキン１０の産生活性を有する、請求項１に記載の免疫調節剤。 The immunomodulator according to claim 1, wherein the CD8-positive CXCR3-positive T cells have interleukin 10 production activity.
- 前記CD8陽性CXCR3陽性T細胞は霊長類由来である、請求項１又は２に記載の免疫調節剤。 The immunomodulator according to claim 1 or 2, wherein the CD8-positive CXCR3-positive T cells are derived from primates.
- 免疫抑制がその予防又は治療に有効である免疫関連障害の予防又は治療用である、請求項１～３のいずれかに記載の免疫調節剤。 The immunomodulator according to any one of claims 1 to 3, which is used for prevention or treatment of an immune-related disorder in which immunosuppression is effective for the prevention or treatment.
- 前記免疫関連障害は自己免疫疾患である、請求項４記載の免疫調節剤。 The immunomodulator according to claim 4, wherein the immune-related disorder is an autoimmune disease.
- 前記免疫関連障害は臓器移植後の拒絶反応である、請求項４に記載の免疫調節剤。 The immunomodulator according to claim 4, wherein the immune-related disorder is rejection after organ transplantation.
- 免疫関連障害の予防又は治療剤であって、
請求項１～６のいずれかに記載の免疫調節剤と、
薬学的に許容される担体と、
を含有する、予防又は治療剤。 A preventive or therapeutic agent for immune related disorders,
An immunomodulator according to any one of claims 1 to 6;
A pharmaceutically acceptable carrier;
A prophylactic or therapeutic agent comprising: - 活性化されたCD8陽性制御性T細胞の製造方法であって、
CD8陽性CXCR3陽性Ｔ細胞を準備する工程と、
前記CD8陽性CXCR3陽性T細胞を、当該細胞のT細胞受容体を介して刺激する物質の存在下培養する活性化工程と、
を備える、製造方法。 A method for producing activated CD8-positive regulatory T cells, comprising:
Preparing a CD8 positive CXCR3 positive T cell;
An activation step of culturing the CD8-positive CXCR3-positive T cell in the presence of a substance that stimulates the cell via the T cell receptor;
A manufacturing method comprising: - 前記活性化工程は、前記CD8陽性CXCR3陽性T細胞のインターロイキン１０の産生活性を発現させる工程である、請求項８に記載の製造方法。 The production method according to claim 8, wherein the activation step is a step of expressing interleukin 10 production activity of the CD8-positive CXCR3-positive T cells.
- 前記活性化工程は、前記CD8陽性CXCR3陽性T細胞を特定の抗原の存在下で培養して当該抗原についての特異性を有するCD8陽性CXCR3陽性T細胞を活性化する工程である、請求項８又は９に記載の製造方法。 The activation step is a step of culturing the CD8-positive CXCR3-positive T cells in the presence of a specific antigen to activate the CD8-positive CXCR3-positive T cells having specificity for the antigen. 9. The production method according to 9.
- 前記抗原は、自己免疫疾患の原因物質、アレルギー疾患の原因物質及び臓器移植時のドナー由来細胞又はその一部から選択される、請求項１０に記載の製造方法。 The production method according to claim 10, wherein the antigen is selected from a causative substance of an autoimmune disease, a causative substance of an allergic disease, and a donor-derived cell or a part thereof at the time of organ transplantation.
- さらに、前記活性化したCD8陽性CXCR3陽性T細胞をIL10産生活性を指標として選抜する工程を備える、請求項８～１１のいずれかに記載の製造方法。 The production method according to any one of claims 8 to 11, further comprising a step of selecting the activated CD8-positive CXCR3-positive T cells using IL10 production activity as an index.
- さらに、活性化された前記CD8陽性CXCR3陽性T細胞を培養して増殖させる工程を備える、請求項８～１２のいずれかに記載の製造方法。 The production method according to any one of claims 8 to 12, further comprising a step of culturing and proliferating the activated CD8-positive CXCR3-positive T cells.
- CD8陽性制御性Ｔ細胞の検出又は分離用キットであって、
CD8陽性CXCR3陽性T細胞を検出又は分離する試薬を含む、キット。 A kit for detecting or separating CD8 positive regulatory T cells,
A kit comprising a reagent for detecting or separating CD8-positive CXCR3-positive T cells. - 免疫関連障害の予防又は治療用剤の製造方法であって、
CD8陽性CXCR3陽性T細胞を準備する工程と、
前記CD8陽性CXCR3陽性T細胞と薬学的に許容される担体とを混合する工程と、
を備える、製造方法。 A method for producing an agent for preventing or treating an immune related disorder, comprising:
Preparing CD8-positive CXCR3-positive T cells;
Mixing the CD8-positive CXCR3-positive T cells with a pharmaceutically acceptable carrier;
A manufacturing method comprising: - 免疫関連障害の予防又は治療手段のスクリーニング方法であって、
CD8陽性CXCR3陽性T細胞に１種又は２種以上の被験条件を付与して前記T細胞の免疫抑制活性を検出する工程、
を備える、スクリーニング方法。 A screening method for a means for preventing or treating an immune related disorder, comprising:
A step of applying one or more test conditions to CD8-positive CXCR3-positive T cells to detect immunosuppressive activity of the T cells;
A screening method comprising: - さらに、前記１種又は２種以上の被験条件中、前記免疫抑制活性を増強する１種又は２種以上の被験条件を免疫抑制が有効な免疫関連障害の予防又は治療手段として選択する工程を備える、請求項１６に記載のスクリーニング方法。 Further, the method includes a step of selecting one or two or more test conditions that enhance the immunosuppressive activity among the one or more test conditions as a means for preventing or treating an immune-related disorder in which immunosuppression is effective. The screening method according to claim 16.
- さらに、前記１種又は２種以上の被験条件中、前記免疫抑制活性を低下させる１種又は２種以上の被験条件を免疫活性化が有効な免疫関連障害の予防又は治療手段として選択する工程、を備える、請求項１６に記載のスクリーニング方法。 A step of selecting one or more test conditions that reduce the immunosuppressive activity among the one or more test conditions as a means for preventing or treating immune-related disorders in which immune activation is effective; The screening method according to claim 16, comprising:
- 免疫関連障害の予防又は治療方法であって、
前記免疫関連障害の予防又は治療を要する個体に、CD8陽性CXCR3陽性T細胞を投与する工程、
を備える、方法。 A method for preventing or treating an immune related disorder comprising:
A step of administering a CD8-positive CXCR3-positive T cell to an individual in need of prevention or treatment of the immune-related disorder;
A method comprising: - 免疫関連障害の予防又は治療方法であって、
前記免疫関連障害の予防又は治療を要する個体から、CD8陽性CXCR3陽性T細胞の少なくとも一部を除去する工程、
を備える、方法。 A method for preventing or treating an immune related disorder comprising:
Removing at least a portion of CD8-positive CXCR3-positive T cells from an individual in need of prevention or treatment of the immune-related disorder,
A method comprising:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2010514521A JP5549014B2 (en) | 2008-05-27 | 2009-05-27 | Immunomodulators and uses thereof |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2008137622 | 2008-05-27 | ||
| JP2008-137622 | 2008-05-27 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2009145238A1 true WO2009145238A1 (en) | 2009-12-03 |
Family
ID=41377109
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/JP2009/059728 WO2009145238A1 (en) | 2008-05-27 | 2009-05-27 | Immunomodulatory agent and use thereof |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JP5549014B2 (en) |
| WO (1) | WO2009145238A1 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2704715A2 (en) * | 2011-04-29 | 2014-03-12 | Selecta Biosciences, Inc. | Tolerogenic synthetic nanocarriers for generating cd8+regulatory t cells |
| US10046064B2 (en) | 2014-09-07 | 2018-08-14 | Selecta Biosciences, Inc. | Methods and compositions for attenuating exon skipping anti-viral transfer vector immune responses |
| US10335395B2 (en) | 2013-05-03 | 2019-07-02 | Selecta Biosciences, Inc. | Methods of administering immunosuppressants having a specified pharmacodynamic effective life and therapeutic macromolecules for the induction of immune tolerance |
| JP2020523018A (en) * | 2017-06-09 | 2020-08-06 | プロビデンス ヘルス アンド サービシーズ−オレゴン | Use of CD39 and CD103 for identification of tumor-reactive human T cells for treatment of cancer |
| US11426451B2 (en) | 2017-03-11 | 2022-08-30 | Selecta Biosciences, Inc. | Methods and compositions related to combined treatment with antiinflammatories and synthetic nanocarriers comprising an immunosuppressant |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2312820T3 (en) * | 2002-11-29 | 2009-03-01 | Maria Grazia Roncarolo | PARAMYCIN AND IL-10 FOR THE TREATMENT OF AUTOIMMUNE DISEASES. |
| AU2006232374B2 (en) * | 2005-04-01 | 2011-08-25 | University Of Southern California | Preventing rejection of transplanted tissue using regulatory T cells |
| CN101217875A (en) * | 2005-07-07 | 2008-07-09 | 建新公司 | Prevention of chronic rejection in solid organ transplantation |
-
2009
- 2009-05-27 JP JP2010514521A patent/JP5549014B2/en not_active Expired - Fee Related
- 2009-05-27 WO PCT/JP2009/059728 patent/WO2009145238A1/en active Application Filing
Non-Patent Citations (10)
| Title |
|---|
| COSMI L. ET AL.: "Human CD8+CD25+ thymocytes share phenotypic and functional features with CD4+CD25+ regulatorythy mocytes", BLOOD, vol. 102, no. 12, 2003, pages 4107 - 4114 * |
| FILACI G. ET AL.: "CD8+CD28- T Regulatory Lymphocytes Inhibiting T Cell Proliferative and Cytotoxic Functions Infiltrate Human Cancers", THE JOURNAL OF IMMUNOLOGY, vol. 179, no. 7, 2007, pages 4323 - 4334 * |
| GILLIET M. ET AL.: "Generation of Human CD8 T Regulatory Cells by CD40 Ligand activated Plasmacytoid Dendritic Cells", JOURNAL OF EXPERIMENTAL MEDICINE, vol. 195, no. 6, 2002, pages 695 - 704 * |
| SUZUKI H. ET AL.: "Are CD8+CD122+ cells regulatory T cells or memory T cells?", HUMAN IMMUNOLOGY, vol. 69, no. 11, November 2008 (2008-11-01), pages 751 - 754 * |
| SUZUKI H. ET AL.: "CD8+CD122+ Seigyosei T-saibo ni yoru Men'eki Seigyo", IGAKU NO AYUMI, vol. 220, no. 6, 2007, pages 525 - 526 * |
| SUZUKI H.: "CD8+ Seigyosei T-saibo no Rinsho Oyo eno Tenkai", JAPANESE SOCIETY FOR APOPTOSIS PROGRAM YOSHISHU, vol. 17TH, August 2008 (2008-08-01), pages 27 * |
| SUZUKI H.: "CD8+CD122+ Seigyosei T-saibo o Riyo shita Gan Men'eki Ryoho no Kaihatsu", GRANT-IN-AIDS FOR SCIENTIFIC RESEARCH ON PRIORITY AREAS CANCER HEISEI 19 NENDO, 18 August 2008 (2008-08-18), pages 381 * |
| SUZUKI H.: "CD8+CD122+ Seigyosei T-saibo", EXPERIMENTAL MEDICINE, vol. 24, no. 20, 2006, pages 3192 - 3199 * |
| SUZUKI M. ET AL.: "Inhibitory CD8+ T cells in autoimmune disease", HUMAN IMMUNOLOGY, vol. 69, no. 11, November 2008 (2008-11-01), pages 781 - 789 * |
| WINTER D ET AL.: "Down-modulation of CXCR3 surface expression and function in CD8+ T cells from cutaneous T cell lymphoma patients", THE JOURNAL OF IMMUNOLOGY, vol. 179, no. 6, 2007, pages 4272 - 4282 * |
Cited By (28)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11235057B2 (en) | 2011-04-29 | 2022-02-01 | Selecta Biosciences, Inc. | Methods for providing polymeric synthetic nanocarriers for generating antigen-specific tolerance immune responses |
| US9987354B2 (en) | 2011-04-29 | 2018-06-05 | Selecta Biosciences, Inc. | Tolerogenic synthetic nanocarriers for antigen-specific deletion of T effector cells |
| EP2704715A4 (en) * | 2011-04-29 | 2015-03-04 | Selecta Biosciences Inc | Tolerogenic synthetic nanocarriers for generating cd8+regulatory t cells |
| US9265815B2 (en) | 2011-04-29 | 2016-02-23 | Selecta Biosciences, Inc. | Tolerogenic synthetic nanocarriers |
| US9289477B2 (en) | 2011-04-29 | 2016-03-22 | Selecta Biosciences, Inc. | Tolerogenic synthetic nanocarriers to reduce cytotoxic T lymphocyte responses |
| US9289476B2 (en) | 2011-04-29 | 2016-03-22 | Selecta Biosciences, Inc. | Tolerogenic synthetic nanocarriers for allergy therapy |
| US9295718B2 (en) | 2011-04-29 | 2016-03-29 | Selecta Biosciences, Inc. | Tolerogenic synthetic nanocarriers to reduce immune responses to therapeutic proteins |
| US10420835B2 (en) | 2011-04-29 | 2019-09-24 | Selecta Biosciences, Inc. | Tolerogenic synthetic nanocarriers for antigen-specific deletion of T effector cells |
| US9993548B2 (en) | 2011-04-29 | 2018-06-12 | Selecta Biosciences, Inc. | Tolerogenic synthetic nanocarriers for inducing regulatory B cells |
| US10004802B2 (en) | 2011-04-29 | 2018-06-26 | Selecta Biosciences, Inc. | Tolerogenic synthetic nanocarriers for generating CD8+ regulatory T cells |
| US10039822B2 (en) | 2011-04-29 | 2018-08-07 | Selecta Biosciences, Inc. | Method for providing polymeric synthetic nanocarriers for generating antigen-specific tolerance immune responses |
| US11717569B2 (en) | 2011-04-29 | 2023-08-08 | Selecta Biosciences, Inc. | Tolerogenic synthetic nanocarriers |
| US11779641B2 (en) | 2011-04-29 | 2023-10-10 | Selecta Biosciences, Inc. | Tolerogenic synthetic nanocarriers for allergy therapy |
| EP2704715A2 (en) * | 2011-04-29 | 2014-03-12 | Selecta Biosciences, Inc. | Tolerogenic synthetic nanocarriers for generating cd8+regulatory t cells |
| US10441651B2 (en) | 2011-04-29 | 2019-10-15 | Selecta Biosciences, Inc. | Tolerogenic synthetic nanocarriers for generating CD8+ regulatory T cells |
| JP2014514334A (en) * | 2011-04-29 | 2014-06-19 | セレクタ バイオサイエンシーズ インコーポレーテッド | Tolerogenic synthetic nanocarriers for generating CD8 + regulatory T cells |
| US10668053B2 (en) | 2013-05-03 | 2020-06-02 | Selecta Biosciences, Inc. | Tolerogenic synthetic nanocarriers to reduce or prevent anaphylaxis in response to a non-allergenic antigen |
| US10434088B2 (en) | 2013-05-03 | 2019-10-08 | Selecta Biosciences, Inc. | Methods related to administering immunosuppressants and therapeutic macromolecules at a reduced pharmacodynamically effective dose |
| US10357482B2 (en) | 2013-05-03 | 2019-07-23 | Selecta Biosciences, Inc. | Methods providing a therapeutic macromolecule and synthetic nanocarriers comprising immunosuppressant locally and concomitantly to reduce both type I and type IV hypersensitivity |
| US10357483B2 (en) | 2013-05-03 | 2019-07-23 | Selecta Biosciences, Inc. | Methods comprising dosing combinations for reducing undesired humoral immune responses |
| US11298342B2 (en) | 2013-05-03 | 2022-04-12 | Selecta Biosciences, Inc. | Methods providing a therapeutic macromolecule and synthetic nanocarriers comprising immunosuppressant locally and concomitantly to reduce both type I and type IV hypersensitivity |
| US10335395B2 (en) | 2013-05-03 | 2019-07-02 | Selecta Biosciences, Inc. | Methods of administering immunosuppressants having a specified pharmacodynamic effective life and therapeutic macromolecules for the induction of immune tolerance |
| US11633422B2 (en) | 2014-09-07 | 2023-04-25 | Selecta Biosciences, Inc. | Methods and compositions for attenuating anti-viral transfer vector immune responses |
| US10046064B2 (en) | 2014-09-07 | 2018-08-14 | Selecta Biosciences, Inc. | Methods and compositions for attenuating exon skipping anti-viral transfer vector immune responses |
| US10071114B2 (en) | 2014-09-07 | 2018-09-11 | Selecta Biosciences, Inc. | Methods and compositions for attenuating gene expression modulating anti-viral transfer vector immune responses |
| US11426451B2 (en) | 2017-03-11 | 2022-08-30 | Selecta Biosciences, Inc. | Methods and compositions related to combined treatment with antiinflammatories and synthetic nanocarriers comprising an immunosuppressant |
| JP2020523018A (en) * | 2017-06-09 | 2020-08-06 | プロビデンス ヘルス アンド サービシーズ−オレゴン | Use of CD39 and CD103 for identification of tumor-reactive human T cells for treatment of cancer |
| US11572541B2 (en) | 2017-06-09 | 2023-02-07 | Providence Health & Services—Oregon | Utilization of CD39 and CD103 for identification of human tumor reactive T cells for treatment of cancer |
Also Published As
| Publication number | Publication date |
|---|---|
| JP5549014B2 (en) | 2014-07-16 |
| JPWO2009145238A1 (en) | 2011-10-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7001575B2 (en) | How to increase and evaluate B cells and how to use increased B cells for disease treatment | |
| JP7242761B2 (en) | T cells to express chimeric antigen receptors and other receptors | |
| KR102073901B1 (en) | Anti third party central memory t cells, methods of producing same and use of same in transplantation and disease treatment | |
| US10577586B2 (en) | Compositions and methods for modulating an immune response | |
| JP2018516586A (en) | Method for producing TCRγδ + T cells | |
| JP2002507881A (en) | Method of in vitro expansion of dendritic cell precursors and its use in immunogen production | |
| TW202112806A (en) | Methods for manufacturing t cells by direct sorting and compositions therof | |
| JP2007538000A (en) | Regulatory T cells suppress autoimmunity | |
| JP6543375B1 (en) | Population of CD3 negative cells expressing chemokine receptor and cell adhesion molecule and use thereof | |
| JP2013537187A (en) | Use of anti-third party central memory T cells for anti-leukemia / lymphoma treatment | |
| JP2006280307A (en) | Method for producing controllable t cell | |
| US10149863B2 (en) | NKT-like cell subpopulation and method of using the same in the treatment of tumor | |
| WO2019195657A1 (en) | Compositions for establishing mixed chimerism and methods of manufacture thereof | |
| JP5549014B2 (en) | Immunomodulators and uses thereof | |
| EP2689009A1 (en) | Method for using regulatory t cells in therapy | |
| CA2653848A1 (en) | Methods of using anti-thymocyte globulin and related agents | |
| US20240075063A1 (en) | Use of mait cells for controlling graft versus host disease | |
| KR20130093083A (en) | Il-13 producing tr1-like cells and use thereof | |
| US20070128670A1 (en) | Methods for the identification and preparation of regulator/suppressor t lymphocytes, compositions and use thereof | |
| AU2013354909A1 (en) | A method of generating multilineage potential cells | |
| JP2003529363A (en) | Production of TcR gamma delta T cells | |
| JP7277255B2 (en) | Method for evaluating specificity of regulatory T cells in vitro | |
| Rogers | Identification and Isolation of Five Developmental Stages in GM-CSF-Stimulated Murine Bone Marrow Culture | |
| JP2003033175A (en) | Peripheral blood dendritic cell subset inducting selective immune response suppression |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 09754746 Country of ref document: EP Kind code of ref document: A1 |
|
| WWE | Wipo information: entry into national phase |
Ref document number: 2010514521 Country of ref document: JP |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 09754746 Country of ref document: EP Kind code of ref document: A1 |