CN104428411A - 用于cd4+t细胞群之抗原特异性扩增的标准化离体平台 - Google Patents
用于cd4+t细胞群之抗原特异性扩增的标准化离体平台 Download PDFInfo
- Publication number
- CN104428411A CN104428411A CN201380033983.2A CN201380033983A CN104428411A CN 104428411 A CN104428411 A CN 104428411A CN 201380033983 A CN201380033983 A CN 201380033983A CN 104428411 A CN104428411 A CN 104428411A
- Authority
- CN
- China
- Prior art keywords
- cell
- peptide
- hesc
- hla
- antigen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000001744 T-lymphocyte Anatomy 0.000 title claims abstract description 128
- 239000000427 antigen Substances 0.000 title claims abstract description 50
- 108091007433 antigens Proteins 0.000 title claims abstract description 50
- 102000036639 antigens Human genes 0.000 title claims abstract description 50
- 210000004027 cell Anatomy 0.000 claims abstract description 201
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 77
- 238000000034 method Methods 0.000 claims abstract description 75
- 241000282414 Homo sapiens Species 0.000 claims abstract description 16
- 210000000130 stem cell Anatomy 0.000 claims abstract description 15
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 6
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 6
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 6
- 210000004443 dendritic cell Anatomy 0.000 claims description 152
- 230000004044 response Effects 0.000 claims description 41
- 230000004927 fusion Effects 0.000 claims description 33
- 210000004369 blood Anatomy 0.000 claims description 30
- 239000008280 blood Substances 0.000 claims description 30
- 239000000203 mixture Substances 0.000 claims description 29
- 208000024827 Alzheimer disease Diseases 0.000 claims description 20
- 229960005486 vaccine Drugs 0.000 claims description 19
- 230000003321 amplification Effects 0.000 claims description 17
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 17
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 16
- 239000012634 fragment Substances 0.000 claims description 14
- 210000003643 myeloid progenitor cell Anatomy 0.000 claims description 14
- 201000010099 disease Diseases 0.000 claims description 13
- 108020001507 fusion proteins Proteins 0.000 claims description 10
- 102000037865 fusion proteins Human genes 0.000 claims description 10
- 206010036590 Premature baby Diseases 0.000 claims description 6
- 210000001161 mammalian embryo Anatomy 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 230000000890 antigenic effect Effects 0.000 claims description 3
- 229940124873 Influenza virus vaccine Drugs 0.000 claims description 2
- 230000024245 cell differentiation Effects 0.000 claims description 2
- 108020003175 receptors Proteins 0.000 claims description 2
- 102000005962 receptors Human genes 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 6
- 108010064539 amyloid beta-protein (1-42) Proteins 0.000 claims 3
- 230000022131 cell cycle Effects 0.000 claims 1
- 210000000612 antigen-presenting cell Anatomy 0.000 abstract description 20
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 13
- 230000001225 therapeutic effect Effects 0.000 abstract description 3
- 230000030741 antigen processing and presentation Effects 0.000 abstract description 2
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 87
- 102100040505 HLA class II histocompatibility antigen, DR alpha chain Human genes 0.000 description 63
- 108010067802 HLA-DR alpha-Chains Proteins 0.000 description 63
- 238000012258 culturing Methods 0.000 description 56
- 150000001413 amino acids Chemical group 0.000 description 40
- 239000000243 solution Substances 0.000 description 26
- 230000009977 dual effect Effects 0.000 description 23
- 238000012360 testing method Methods 0.000 description 23
- 235000001014 amino acid Nutrition 0.000 description 22
- 238000000684 flow cytometry Methods 0.000 description 20
- 230000004069 differentiation Effects 0.000 description 19
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 description 16
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 description 16
- 238000012744 immunostaining Methods 0.000 description 16
- 230000008569 process Effects 0.000 description 16
- 239000006228 supernatant Substances 0.000 description 16
- 238000012546 transfer Methods 0.000 description 16
- 206010022000 influenza Diseases 0.000 description 15
- 239000001963 growth medium Substances 0.000 description 14
- 108010076504 Protein Sorting Signals Proteins 0.000 description 13
- 230000005867 T cell response Effects 0.000 description 13
- 210000003995 blood forming stem cell Anatomy 0.000 description 13
- 230000035800 maturation Effects 0.000 description 13
- 241000700605 Viruses Species 0.000 description 12
- 239000006285 cell suspension Substances 0.000 description 12
- 238000005516 engineering process Methods 0.000 description 12
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 11
- 239000013612 plasmid Substances 0.000 description 11
- 238000005406 washing Methods 0.000 description 11
- 108010058597 HLA-DR Antigens Proteins 0.000 description 10
- 102000006354 HLA-DR Antigens Human genes 0.000 description 10
- 108010002350 Interleukin-2 Proteins 0.000 description 10
- 239000002158 endotoxin Substances 0.000 description 10
- 230000012447 hatching Effects 0.000 description 10
- 238000011081 inoculation Methods 0.000 description 10
- 229920006008 lipopolysaccharide Polymers 0.000 description 10
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 10
- 238000007747 plating Methods 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 9
- 239000004677 Nylon Substances 0.000 description 9
- 229920001778 nylon Polymers 0.000 description 9
- 238000001556 precipitation Methods 0.000 description 9
- 208000023275 Autoimmune disease Diseases 0.000 description 8
- 230000004913 activation Effects 0.000 description 8
- 238000010586 diagram Methods 0.000 description 8
- 239000000975 dye Substances 0.000 description 8
- 238000004043 dyeing Methods 0.000 description 8
- 210000000066 myeloid cell Anatomy 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 7
- 229940032046 DTaP vaccine Drugs 0.000 description 7
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 7
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 7
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 7
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 7
- 239000011324 bead Substances 0.000 description 7
- 238000003745 diagnosis Methods 0.000 description 7
- 208000011580 syndromic disease Diseases 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 239000013598 vector Substances 0.000 description 7
- 206010015150 Erythema Diseases 0.000 description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 description 6
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 238000005520 cutting process Methods 0.000 description 6
- 239000007758 minimum essential medium Substances 0.000 description 6
- 239000011049 pearl Substances 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 230000009182 swimming Effects 0.000 description 6
- 102000029816 Collagenase Human genes 0.000 description 5
- 108060005980 Collagenase Proteins 0.000 description 5
- 229940032070 DTaP-IPV/Hib vaccine Drugs 0.000 description 5
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 5
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 5
- 229940124908 Pediarix Drugs 0.000 description 5
- 229940124910 Pentacel Drugs 0.000 description 5
- 229960002424 collagenase Drugs 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000010410 layer Substances 0.000 description 5
- 239000002502 liposome Substances 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 108010082117 matrigel Proteins 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000011435 rock Substances 0.000 description 5
- 108010090849 Amyloid beta-Peptides Proteins 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 4
- 229940124919 Kinrix Drugs 0.000 description 4
- 229940124876 ProQuad Drugs 0.000 description 4
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 4
- 206010047115 Vasculitis Diseases 0.000 description 4
- 210000003719 b-lymphocyte Anatomy 0.000 description 4
- 230000004992 fission Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 239000004816 latex Substances 0.000 description 4
- 229920000126 latex Polymers 0.000 description 4
- -1 length is 3 Chemical class 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 230000001717 pathogenic effect Effects 0.000 description 4
- 230000007170 pathology Effects 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000002741 site-directed mutagenesis Methods 0.000 description 4
- 239000013589 supplement Substances 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 3
- 208000030507 AIDS Diseases 0.000 description 3
- 206010001052 Acute respiratory distress syndrome Diseases 0.000 description 3
- 102000013455 Amyloid beta-Peptides Human genes 0.000 description 3
- 102100022704 Amyloid-beta precursor protein Human genes 0.000 description 3
- 108010060215 Apolipoprotein E3 Proteins 0.000 description 3
- 102000008128 Apolipoprotein E3 Human genes 0.000 description 3
- 229940124900 Boostrix Drugs 0.000 description 3
- 101100028791 Caenorhabditis elegans pbs-5 gene Proteins 0.000 description 3
- 229940124902 Daptacel Drugs 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 206010014596 Encephalitis Japanese B Diseases 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
- HVLSXIKZNLPZJJ-TXZCQADKSA-N HA peptide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HVLSXIKZNLPZJJ-TXZCQADKSA-N 0.000 description 3
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 3
- 229940124915 Infanrix Drugs 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- 201000005807 Japanese encephalitis Diseases 0.000 description 3
- 241000710842 Japanese encephalitis virus Species 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 208000013616 Respiratory Distress Syndrome Diseases 0.000 description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 3
- 208000021386 Sjogren Syndrome Diseases 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 3
- 229940032047 Tdap vaccine Drugs 0.000 description 3
- 229940124923 Tripedia Drugs 0.000 description 3
- 208000037386 Typhoid Diseases 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 229940102614 adacel Drugs 0.000 description 3
- 230000001363 autoimmune Effects 0.000 description 3
- 230000005784 autoimmunity Effects 0.000 description 3
- 230000006399 behavior Effects 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- 150000007942 carboxylates Chemical class 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000005138 cryopreservation Methods 0.000 description 3
- 230000005016 dendritic process Effects 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 210000001671 embryonic stem cell Anatomy 0.000 description 3
- 208000024908 graft versus host disease Diseases 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000008595 infiltration Effects 0.000 description 3
- 238000001764 infiltration Methods 0.000 description 3
- 229960003971 influenza vaccine Drugs 0.000 description 3
- 201000006417 multiple sclerosis Diseases 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 206010039073 rheumatoid arthritis Diseases 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 210000002536 stromal cell Anatomy 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 201000008297 typhoid fever Diseases 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 101710137189 Amyloid-beta A4 protein Proteins 0.000 description 2
- 101710151993 Amyloid-beta precursor protein Proteins 0.000 description 2
- 101001026137 Cavia porcellus Glutathione S-transferase A Proteins 0.000 description 2
- 102100026735 Coagulation factor VIII Human genes 0.000 description 2
- 208000015943 Coeliac disease Diseases 0.000 description 2
- 206010010099 Combined immunodeficiency Diseases 0.000 description 2
- 229940124901 Comvax Drugs 0.000 description 2
- 208000011231 Crohn disease Diseases 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 201000004624 Dermatitis Diseases 0.000 description 2
- 239000012983 Dulbecco’s minimal essential medium Substances 0.000 description 2
- 108700024394 Exon Proteins 0.000 description 2
- 101001026109 Gallus gallus Glutathione S-transferase Proteins 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 208000024869 Goodpasture syndrome Diseases 0.000 description 2
- 208000015023 Graves' disease Diseases 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 208000030836 Hashimoto thyroiditis Diseases 0.000 description 2
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 2
- 101000608935 Homo sapiens Leukosialin Proteins 0.000 description 2
- 241000701806 Human papillomavirus Species 0.000 description 2
- 206010021245 Idiopathic thrombocytopenic purpura Diseases 0.000 description 2
- 208000010159 IgA glomerulonephritis Diseases 0.000 description 2
- 206010021263 IgA nephropathy Diseases 0.000 description 2
- 102000004388 Interleukin-4 Human genes 0.000 description 2
- 108090000978 Interleukin-4 Proteins 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- 102100039564 Leukosialin Human genes 0.000 description 2
- 201000009906 Meningitis Diseases 0.000 description 2
- 206010029240 Neuritis Diseases 0.000 description 2
- 206010034277 Pemphigoid Diseases 0.000 description 2
- 208000003670 Pure Red-Cell Aplasia Diseases 0.000 description 2
- 206010037742 Rabies Diseases 0.000 description 2
- 206010041047 Slow virus infection Diseases 0.000 description 2
- 206010042033 Stevens-Johnson syndrome Diseases 0.000 description 2
- 201000009594 Systemic Scleroderma Diseases 0.000 description 2
- 206010042953 Systemic sclerosis Diseases 0.000 description 2
- 208000031981 Thrombocytopenic Idiopathic Purpura Diseases 0.000 description 2
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 2
- 229940124922 Twinrix Drugs 0.000 description 2
- 229940124924 Varivax Drugs 0.000 description 2
- 229940124925 Zostavax Drugs 0.000 description 2
- 208000011341 adult acute respiratory distress syndrome Diseases 0.000 description 2
- 201000000028 adult respiratory distress syndrome Diseases 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- DZHSAHHDTRWUTF-SIQRNXPUSA-N amyloid-beta polypeptide 42 Chemical compound C([C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O)[C@@H](C)CC)C(C)C)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C(C)C)C1=CC=CC=C1 DZHSAHHDTRWUTF-SIQRNXPUSA-N 0.000 description 2
- 206010002026 amyotrophic lateral sclerosis Diseases 0.000 description 2
- 208000010668 atopic eczema Diseases 0.000 description 2
- 201000003710 autoimmune thrombocytopenic purpura Diseases 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000001149 cognitive effect Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000003750 conditioning effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 206010014599 encephalitis Diseases 0.000 description 2
- 210000001163 endosome Anatomy 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000005484 gravity Effects 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000003394 haemopoietic effect Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 208000003532 hypothyroidism Diseases 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 206010025135 lupus erythematosus Diseases 0.000 description 2
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 206010028417 myasthenia gravis Diseases 0.000 description 2
- 201000004071 non-specific interstitial pneumonia Diseases 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- CWEFIMQKSZFZNY-UHFFFAOYSA-N pentyl 2-[4-[[4-[4-[[4-[[4-(pentoxycarbonylamino)phenyl]methyl]phenyl]carbamoyloxy]butoxycarbonylamino]phenyl]methyl]phenyl]acetate Chemical compound C1=CC(CC(=O)OCCCCC)=CC=C1CC(C=C1)=CC=C1NC(=O)OCCCCOC(=O)NC(C=C1)=CC=C1CC1=CC=C(NC(=O)OCCCCC)C=C1 CWEFIMQKSZFZNY-UHFFFAOYSA-N 0.000 description 2
- 108010094020 polyglycine Proteins 0.000 description 2
- 229920000232 polyglycine polymer Polymers 0.000 description 2
- 208000005987 polymyositis Diseases 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000001185 psoriatic effect Effects 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 208000002491 severe combined immunodeficiency Diseases 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 201000008827 tuberculosis Diseases 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- 229940031767 13-valent pneumococcal conjugate vaccine Drugs 0.000 description 1
- XGWFJBFNAQHLEF-UHFFFAOYSA-N 9-anthroic acid Chemical compound C1=CC=C2C(C(=O)O)=C(C=CC=C3)C3=CC2=C1 XGWFJBFNAQHLEF-UHFFFAOYSA-N 0.000 description 1
- 229940124965 ACAM2000 Drugs 0.000 description 1
- 229940124962 ActHIB Drugs 0.000 description 1
- 208000026872 Addison Disease Diseases 0.000 description 1
- 229940124963 Afluria Drugs 0.000 description 1
- 201000010000 Agranulocytosis Diseases 0.000 description 1
- 229940124838 Agriflu Drugs 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 206010002556 Ankylosing Spondylitis Diseases 0.000 description 1
- 208000003343 Antiphospholipid Syndrome Diseases 0.000 description 1
- 208000032467 Aplastic anaemia Diseases 0.000 description 1
- 108010060159 Apolipoprotein E4 Proteins 0.000 description 1
- 101000993093 Arabidopsis thaliana Heat stress transcription factor B-2a Proteins 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 206010003805 Autism Diseases 0.000 description 1
- 208000020706 Autistic disease Diseases 0.000 description 1
- 208000032116 Autoimmune Experimental Encephalomyelitis Diseases 0.000 description 1
- 206010003827 Autoimmune hepatitis Diseases 0.000 description 1
- 206010064539 Autoimmune myocarditis Diseases 0.000 description 1
- 208000031212 Autoimmune polyendocrinopathy Diseases 0.000 description 1
- 235000000832 Ayote Nutrition 0.000 description 1
- 241000193738 Bacillus anthracis Species 0.000 description 1
- 201000001178 Bacterial Pneumonia Diseases 0.000 description 1
- 208000023328 Basedow disease Diseases 0.000 description 1
- 208000009137 Behcet syndrome Diseases 0.000 description 1
- 208000008439 Biliary Liver Cirrhosis Diseases 0.000 description 1
- 208000033222 Biliary cirrhosis primary Diseases 0.000 description 1
- 229940124899 Biothrax Drugs 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 235000002687 Caesalpinia echinata Nutrition 0.000 description 1
- 208000020119 Caplan syndrome Diseases 0.000 description 1
- 229940124957 Cervarix Drugs 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 206010009900 Colitis ulcerative Diseases 0.000 description 1
- 235000009854 Cucurbita moschata Nutrition 0.000 description 1
- 240000001980 Cucurbita pepo Species 0.000 description 1
- 235000009804 Cucurbita pepo subsp pepo Nutrition 0.000 description 1
- 229940124888 DECAVAC Drugs 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 101150034979 DRB3 gene Proteins 0.000 description 1
- 101150082328 DRB5 gene Proteins 0.000 description 1
- 206010011968 Decreased immune responsiveness Diseases 0.000 description 1
- 241001115402 Ebolavirus Species 0.000 description 1
- 229940124884 Engerix-B Drugs 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 206010016077 Factor IX deficiency Diseases 0.000 description 1
- 201000003542 Factor VIII deficiency Diseases 0.000 description 1
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 1
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 229940124895 FluMist Drugs 0.000 description 1
- 229940124896 Fluarix Drugs 0.000 description 1
- YCKRFDGAMUMZLT-UHFFFAOYSA-N Fluorine atom Chemical compound [F] YCKRFDGAMUMZLT-UHFFFAOYSA-N 0.000 description 1
- 229940124893 Fluvirin Drugs 0.000 description 1
- 229940124894 Fluzone Drugs 0.000 description 1
- 229940124897 Gardasil Drugs 0.000 description 1
- 208000007465 Giant cell arteritis Diseases 0.000 description 1
- 206010018364 Glomerulonephritis Diseases 0.000 description 1
- 206010018498 Goitre Diseases 0.000 description 1
- 208000009329 Graft vs Host Disease Diseases 0.000 description 1
- 208000003807 Graves Disease Diseases 0.000 description 1
- 102100028972 HLA class I histocompatibility antigen, A alpha chain Human genes 0.000 description 1
- 108010075704 HLA-A Antigens Proteins 0.000 description 1
- 241000606768 Haemophilus influenzae Species 0.000 description 1
- 108010050195 Haemophilus influenzae-type b polysaccharide-Neisseria meningitidis outer membrane protein conjugate vaccine Proteins 0.000 description 1
- 208000001204 Hashimoto Disease Diseases 0.000 description 1
- 229940124914 Havrix Drugs 0.000 description 1
- 102100032606 Heat shock factor protein 1 Human genes 0.000 description 1
- 101710190344 Heat shock factor protein 1 Proteins 0.000 description 1
- 208000035186 Hemolytic Autoimmune Anemia Diseases 0.000 description 1
- 208000009292 Hemophilia A Diseases 0.000 description 1
- 208000007514 Herpes zoster Diseases 0.000 description 1
- 101150094793 Hes3 gene Proteins 0.000 description 1
- 101150029234 Hes5 gene Proteins 0.000 description 1
- 229940124885 Hiberix Drugs 0.000 description 1
- 108700020122 Hiberix Proteins 0.000 description 1
- 101100005713 Homo sapiens CD4 gene Proteins 0.000 description 1
- 101001046686 Homo sapiens Integrin alpha-M Proteins 0.000 description 1
- 101001002657 Homo sapiens Interleukin-2 Proteins 0.000 description 1
- 101001002709 Homo sapiens Interleukin-4 Proteins 0.000 description 1
- 101001100327 Homo sapiens RNA-binding protein 45 Proteins 0.000 description 1
- 101000843556 Homo sapiens Transcription factor HES-1 Proteins 0.000 description 1
- 101000611183 Homo sapiens Tumor necrosis factor Proteins 0.000 description 1
- WOBHKFSMXKNTIM-UHFFFAOYSA-N Hydroxyethyl methacrylate Chemical compound CC(=C)C(=O)OCCO WOBHKFSMXKNTIM-UHFFFAOYSA-N 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010021067 Hypopituitarism Diseases 0.000 description 1
- 229940124913 IPOL Drugs 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 1
- 102100022338 Integrin alpha-M Human genes 0.000 description 1
- 102100022297 Integrin alpha-X Human genes 0.000 description 1
- 208000029523 Interstitial Lung disease Diseases 0.000 description 1
- 229940124956 Ixiaro Drugs 0.000 description 1
- 229940124918 JE-Vax Drugs 0.000 description 1
- 208000003456 Juvenile Arthritis Diseases 0.000 description 1
- 206010059176 Juvenile idiopathic arthritis Diseases 0.000 description 1
- 208000011200 Kawasaki disease Diseases 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 201000001779 Leukocyte adhesion deficiency Diseases 0.000 description 1
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 1
- 201000005505 Measles Diseases 0.000 description 1
- 229940124904 Menactra Drugs 0.000 description 1
- 206010027336 Menstruation delayed Diseases 0.000 description 1
- 229940124951 Menveo Drugs 0.000 description 1
- 240000000249 Morus alba Species 0.000 description 1
- 235000008708 Morus alba Nutrition 0.000 description 1
- 208000005647 Mumps Diseases 0.000 description 1
- 206010028424 Myasthenic syndrome Diseases 0.000 description 1
- 244000223072 Narcissus jonquilla Species 0.000 description 1
- 235000013862 Narcissus jonquilla Nutrition 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 206010029888 Obliterative bronchiolitis Diseases 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 101100278514 Oryza sativa subsp. japonica DRB2 gene Proteins 0.000 description 1
- 101100117565 Oryza sativa subsp. japonica DRB4 gene Proteins 0.000 description 1
- 101100117569 Oryza sativa subsp. japonica DRB6 gene Proteins 0.000 description 1
- 208000012868 Overgrowth Diseases 0.000 description 1
- 206010033661 Pancytopenia Diseases 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 241000127464 Paubrasilia echinata Species 0.000 description 1
- 229940124909 PedvaxHIB Drugs 0.000 description 1
- 241000721454 Pemphigus Species 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 208000031845 Pernicious anaemia Diseases 0.000 description 1
- 208000000474 Poliomyelitis Diseases 0.000 description 1
- 208000007048 Polymyalgia Rheumatica Diseases 0.000 description 1
- 206010036105 Polyneuropathy Diseases 0.000 description 1
- 206010036297 Postpartum hypopituitarism Diseases 0.000 description 1
- 208000012654 Primary biliary cholangitis Diseases 0.000 description 1
- 206010036697 Primary hypothyroidism Diseases 0.000 description 1
- 201000001263 Psoriatic Arthritis Diseases 0.000 description 1
- 208000036824 Psoriatic arthropathy Diseases 0.000 description 1
- 102100038823 RNA-binding protein 45 Human genes 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 229940124875 RabAvert Drugs 0.000 description 1
- 101100016889 Rattus norvegicus Hes2 gene Proteins 0.000 description 1
- 208000003782 Raynaud disease Diseases 0.000 description 1
- 208000012322 Raynaud phenomenon Diseases 0.000 description 1
- 208000033464 Reiter syndrome Diseases 0.000 description 1
- 229940124878 RotaTeq Drugs 0.000 description 1
- 229940124941 Rotarix Drugs 0.000 description 1
- 241000702670 Rotavirus Species 0.000 description 1
- 206010039361 Sacroiliitis Diseases 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 201000009895 Sheehan syndrome Diseases 0.000 description 1
- 231100000168 Stevens-Johnson syndrome Toxicity 0.000 description 1
- 206010072148 Stiff-Person syndrome Diseases 0.000 description 1
- 229940124929 TYPHIM Vi Drugs 0.000 description 1
- 206010043561 Thrombocytopenic purpura Diseases 0.000 description 1
- 201000007023 Thrombotic Thrombocytopenic Purpura Diseases 0.000 description 1
- 206010043781 Thyroiditis chronic Diseases 0.000 description 1
- 206010043784 Thyroiditis subacute Diseases 0.000 description 1
- 206010044223 Toxic epidermal necrolysis Diseases 0.000 description 1
- 231100000087 Toxic epidermal necrolysis Toxicity 0.000 description 1
- 102100030798 Transcription factor HES-1 Human genes 0.000 description 1
- 206010052779 Transplant rejections Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 206010053613 Type IV hypersensitivity reaction Diseases 0.000 description 1
- 201000006704 Ulcerative Colitis Diseases 0.000 description 1
- 206010046851 Uveitis Diseases 0.000 description 1
- 206010046865 Vaccinia virus infection Diseases 0.000 description 1
- 229940124937 Vaqta Drugs 0.000 description 1
- 241000700647 Variola virus Species 0.000 description 1
- 206010047642 Vitiligo Diseases 0.000 description 1
- 229940124928 YF-Vax Drugs 0.000 description 1
- 208000003152 Yellow Fever Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000004721 adaptive immunity Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 150000003862 amino acid derivatives Chemical class 0.000 description 1
- 230000002052 anaphylactic effect Effects 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 206010003230 arteritis Diseases 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 201000000448 autoimmune hemolytic anemia Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 201000003848 bronchiolitis obliterans Diseases 0.000 description 1
- 208000023367 bronchiolitis obliterans with obstructive pulmonary disease Diseases 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000011748 cell maturation Effects 0.000 description 1
- 229940030156 cell vaccine Drugs 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 208000019069 chronic childhood arthritis Diseases 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 208000037893 chronic inflammatory disorder Diseases 0.000 description 1
- 230000012085 chronic inflammatory response Effects 0.000 description 1
- 208000025302 chronic primary adrenal insufficiency Diseases 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 206010009887 colitis Diseases 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 229940000425 combination drug Drugs 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 201000003278 cryoglobulinemia Diseases 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 210000005220 cytoplasmic tail Anatomy 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000000280 densification Methods 0.000 description 1
- 201000001981 dermatomyositis Diseases 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000001152 differential interference contrast microscopy Methods 0.000 description 1
- 206010013023 diphtheria Diseases 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000012953 feeding on blood of other organism Effects 0.000 description 1
- 239000012997 ficoll-paque Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000000799 fusogenic effect Effects 0.000 description 1
- 238000001879 gelation Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 208000007475 hemolytic anemia Diseases 0.000 description 1
- 208000009429 hemophilia B Diseases 0.000 description 1
- 208000005252 hepatitis A Diseases 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 231100000652 hormesis Toxicity 0.000 description 1
- 102000055229 human IL4 Human genes 0.000 description 1
- 102000057041 human TNF Human genes 0.000 description 1
- 230000002989 hypothyroidism Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 210000000428 immunological synapse Anatomy 0.000 description 1
- 229940026063 imovax Drugs 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 201000002215 juvenile rheumatoid arthritis Diseases 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 208000013104 leukocyte disease Diseases 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000011325 microbead Substances 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 230000001617 migratory effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 208000005871 monkeypox Diseases 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 208000001725 mucocutaneous lymph node syndrome Diseases 0.000 description 1
- 208000037890 multiple organ injury Diseases 0.000 description 1
- 208000010805 mumps infectious disease Diseases 0.000 description 1
- 201000006938 muscular dystrophy Diseases 0.000 description 1
- 201000003631 narcolepsy Diseases 0.000 description 1
- 201000008383 nephritis Diseases 0.000 description 1
- 201000001119 neuropathy Diseases 0.000 description 1
- 230000007823 neuropathy Effects 0.000 description 1
- 210000004493 neutrocyte Anatomy 0.000 description 1
- 238000012758 nuclear staining Methods 0.000 description 1
- 238000000399 optical microscopy Methods 0.000 description 1
- 201000005737 orchitis Diseases 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 208000033808 peripheral neuropathy Diseases 0.000 description 1
- 238000002135 phase contrast microscopy Methods 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229940033515 pneumovax 23 Drugs 0.000 description 1
- 201000006292 polyarteritis nodosa Diseases 0.000 description 1
- 230000007824 polyneuropathy Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 210000004986 primary T-cell Anatomy 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000009696 proliferative response Effects 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 208000008128 pulmonary tuberculosis Diseases 0.000 description 1
- 235000015136 pumpkin Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 201000008158 rapidly progressive glomerulonephritis Diseases 0.000 description 1
- 208000002574 reactive arthritis Diseases 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 210000003289 regulatory T cell Anatomy 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 230000005070 ripening Effects 0.000 description 1
- 201000005404 rubella Diseases 0.000 description 1
- 201000000306 sarcoidosis Diseases 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 238000004092 self-diagnosis Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000011146 sterile filtration Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000013517 stratification Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 201000007497 subacute thyroiditis Diseases 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 206010043207 temporal arteritis Diseases 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000003582 thrombocytopenic effect Effects 0.000 description 1
- 230000005313 thymus development Effects 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 229940111100 tice bcg Drugs 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 208000009174 transverse myelitis Diseases 0.000 description 1
- 229960001814 trypan blue Drugs 0.000 description 1
- 230000005951 type IV hypersensitivity Effects 0.000 description 1
- 208000027930 type IV hypersensitivity disease Diseases 0.000 description 1
- 230000034512 ubiquitination Effects 0.000 description 1
- 238000010798 ubiquitination Methods 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 208000007089 vaccinia Diseases 0.000 description 1
- 210000003934 vacuole Anatomy 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 229940104152 vivotif Drugs 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
Classifications
-
- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
- D21H17/00—Non-fibrous material added to the pulp, characterised by its constitution; Paper-impregnating material characterised by its constitution
- D21H17/63—Inorganic compounds
-
- 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
- A61K39/0005—Vertebrate antigens
- A61K39/0007—Nervous system antigens; Prions
-
- 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
- A61K39/4615—Dendritic cells
-
- 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
- A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
- A61K39/4622—Antigen presenting cells
-
- 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
-
- 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
-
- 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
- C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M10/00—Physical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. ultrasonic, corona discharge, irradiation, electric currents, or magnetic fields; Physical treatment combined with treatment with chemical compounds or elements
- D06M10/02—Physical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. ultrasonic, corona discharge, irradiation, electric currents, or magnetic fields; Physical treatment combined with treatment with chemical compounds or elements ultrasonic or sonic; Corona discharge
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M10/00—Physical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. ultrasonic, corona discharge, irradiation, electric currents, or magnetic fields; Physical treatment combined with treatment with chemical compounds or elements
- D06M10/04—Physical treatment combined with treatment with chemical compounds or elements
- D06M10/06—Inorganic compounds or elements
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M10/00—Physical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. ultrasonic, corona discharge, irradiation, electric currents, or magnetic fields; Physical treatment combined with treatment with chemical compounds or elements
- D06M10/04—Physical treatment combined with treatment with chemical compounds or elements
- D06M10/08—Organic compounds
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M11/00—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising
- D06M11/32—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising with oxygen, ozone, ozonides, oxides, hydroxides or percompounds; Salts derived from anions with an amphoteric element-oxygen bond
- D06M11/36—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising with oxygen, ozone, ozonides, oxides, hydroxides or percompounds; Salts derived from anions with an amphoteric element-oxygen bond with oxides, hydroxides or mixed oxides; with salts derived from anions with an amphoteric element-oxygen bond
- D06M11/38—Oxides or hydroxides of elements of Groups 1 or 11 of the Periodic Table
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M11/00—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising
- D06M11/32—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising with oxygen, ozone, ozonides, oxides, hydroxides or percompounds; Salts derived from anions with an amphoteric element-oxygen bond
- D06M11/36—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising with oxygen, ozone, ozonides, oxides, hydroxides or percompounds; Salts derived from anions with an amphoteric element-oxygen bond with oxides, hydroxides or mixed oxides; with salts derived from anions with an amphoteric element-oxygen bond
- D06M11/44—Oxides or hydroxides of elements of Groups 2 or 12 of the Periodic Table; Zincates; Cadmates
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M11/00—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising
- D06M11/32—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising with oxygen, ozone, ozonides, oxides, hydroxides or percompounds; Salts derived from anions with an amphoteric element-oxygen bond
- D06M11/36—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising with oxygen, ozone, ozonides, oxides, hydroxides or percompounds; Salts derived from anions with an amphoteric element-oxygen bond with oxides, hydroxides or mixed oxides; with salts derived from anions with an amphoteric element-oxygen bond
- D06M11/45—Oxides or hydroxides of elements of Groups 3 or 13 of the Periodic Table; Aluminates
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M11/00—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising
- D06M11/32—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising with oxygen, ozone, ozonides, oxides, hydroxides or percompounds; Salts derived from anions with an amphoteric element-oxygen bond
- D06M11/36—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising with oxygen, ozone, ozonides, oxides, hydroxides or percompounds; Salts derived from anions with an amphoteric element-oxygen bond with oxides, hydroxides or mixed oxides; with salts derived from anions with an amphoteric element-oxygen bond
- D06M11/46—Oxides or hydroxides of elements of Groups 4 or 14 of the Periodic Table; Titanates; Zirconates; Stannates; Plumbates
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M11/00—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising
- D06M11/83—Treating fibres, threads, yarns, fabrics or fibrous goods made from such materials, with inorganic substances or complexes thereof; Such treatment combined with mechanical treatment, e.g. mercerising with metals; with metal-generating compounds, e.g. metal carbonyls; Reduction of metal compounds on textiles
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M23/00—Treatment of fibres, threads, yarns, fabrics or fibrous goods made from such materials, characterised by the process
- D06M23/08—Processes in which the treating agent is applied in powder or granular form
-
- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M23/00—Treatment of fibres, threads, yarns, fabrics or fibrous goods made from such materials, characterised by the process
- D06M23/12—Processes in which the treating agent is incorporated in microcapsules
-
- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21H—PULP COMPOSITIONS; PREPARATION THEREOF NOT COVERED BY SUBCLASSES D21C OR D21D; IMPREGNATING OR COATING OF PAPER; TREATMENT OF FINISHED PAPER NOT COVERED BY CLASS B31 OR SUBCLASS D21G; PAPER NOT OTHERWISE PROVIDED FOR
- D21H21/00—Non-fibrous material added to the pulp, characterised by its function, form or properties; Paper-impregnating or coating material, characterised by its function, form or properties
- D21H21/14—Non-fibrous material added to the pulp, characterised by its function, form or properties; Paper-impregnating or coating material, characterised by its function, form or properties characterised by function or properties in or on the paper
- D21H21/36—Biocidal agents, e.g. fungicidal, bactericidal, insecticidal agents
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
-
- 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
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/44—Thiols, e.g. mercaptoethanol
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/05—Adjuvants
- C12N2501/052—Lipopolysaccharides [LPS]
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2304—Interleukin-4 (IL-4)
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/25—Tumour necrosing factors [TNF]
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/50—Cell markers; Cell surface determinants
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
- C12N2502/1121—Dendritic cells
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
- C12N2502/1394—Bone marrow stromal cells; whole marrow
-
- 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
- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
-
- 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
- C12N2510/00—Genetically modified cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Textile Engineering (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Hematology (AREA)
- Inorganic Chemistry (AREA)
- Neurosurgery (AREA)
- Neurology (AREA)
- Plasma & Fusion (AREA)
- General Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pest Control & Pesticides (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明涉及出于免疫诊断或治疗目的,用于以抗原特异性的方式分离和扩增人T细胞群的方法、肽、核酸和细胞。本发明还涉及衍生自人多能干细胞的专职抗原呈递细胞,以及通过所述抗原呈递细胞实现的可定制抗原呈递。
Description
相关申请的交叉引用
本申请要求2012年5月8日提交的序列号为61/644265的美国申请的优先权,并且其公开内容通过引用包括在本文中。
技术领域
本发明涉及出于免疫诊断或治疗目的,用于以抗原特异性的方式分离和扩增人T细胞群的方法、肽、核酸和细胞。本发明还涉及衍生自人多能干细胞的专职抗原呈递细胞(professional antigen presenting cell),以及通过抗原呈递细胞实现的可定制(customizable)抗原呈递。
背景技术
CD4+T细胞在适应性免疫中作为炎症、耐受性的介导者和作为B细胞成熟的促进者发挥重要作用(Pao等,1996;Klein等,2010)。人CD4+T细胞库包含以百万计的不同的细胞,这些细胞表达不同的T细胞受体(TCR)。的确,许多人类病症涉及CD4+T细胞的离散亚群,其响应于高特异性抗原而增殖,并且分离可能与特定疾病相关的小比例的CD4+细胞是一项艰巨的任务。因此,在临床或治疗条件(setting)中尚未能实现具有应答特定肽之TCR的CD4+T细胞的常规分离或表征。
由T细胞群所示出的巨大TCR库是在胸腺发育期间存在于胸腺的未成熟T细胞的阿尔法(α)和贝塔(β),或者伽马(γ)和德尔塔(δ)TCR基因体细胞重组事件的结果(Haas等,1993;Hayday等,1995)。该重组过程产生了具有以百万计的不同TCR组合的T细胞库,其集体地与每个可能的氨基酸序列结合(Haas等,1993;Hayday等,1995)。该库在静息状态通过血管和淋巴系统循环,直到T细胞遇到具有组织相容性复合物(HLA II类)的专职APC(例如树突细胞(DC)),所述专职APC呈递以适当的亲和力与其TCR结合的肽(Wen等,1998;Sakaguchi等,2000;Huang等,2012)。细胞和CD4+T细胞之间形成的免疫突触刺激增殖,从而产生数十到数千个克隆。新产生的T细胞离开次级淋巴器官,借助循环通过身体并且在呈递抗原的部位积聚。活化的CD4+T细胞释放细胞因子,所述细胞因子作用于邻近的细胞并协调适应性免疫应答。数天后,在抗原的来源被减弱后,大多数克隆通过激活诱导的细胞死亡而消除,少数作为记忆细胞(准备好只要抗原(病原体)再次出现即触发快速应答)保留的克隆除外(Wen等,1998;Villadangos等,2005)。
从新鲜全血中分离的许多CD4+T细胞将分裂一次或两次,但为了临床应用目的的实现需要额外的增殖周期。例如,在测定期间相对稳健地分裂(例如,超过5次)的CD4+T细胞的比例可用于指示对未知肽的反应性。包含单一的蛋白质或肽的不同片段的组可以用来创建与响应于该抗原的CD4+T细胞有关的增殖谱。然后从这些结果推导的信息可用于确定多种人类病症的状态和可能的作用进程。然而,CD4+T细胞的临床表征受到成功扩增抗原特异性CD4+T细胞群所需的适当的APC之制备的可用性和可变性的限制。
使分离或表征抗原特异性CD4+T细胞群的诊断和治疗方法的开发进一步复杂化的是人群中的人HLA-DR受体的异质性。简言之,II类HLA复合物包含HLA-DRB和HLA-DRA受体的异源二聚体,其形成含有肽的结合口袋(binding pocket)。人群中包含数十种,也许数百种不同的HLA-DRB等位基因。因此,在人群中,大量的HLA II类等位基因与以百万计的可能的TCR组合相组合,使得技术上难以从病源T细胞驱动的自身免疫病症中分离抗原特异性CD4+T细胞(Villdangos等,2005;Huang等,2012),例如I型糖尿病(Haskins等,2011;Goianovich等,2012;Delong等,2012)、类风湿性关节炎(Nikken等,2011;Albani等,2011)和多发性硬化(McFarland等,2007;Codarri等,2012;Sallusto等,2012)。简而言之,在一个人中与给定肽结合的TCR在另一人中可能不与相同的肽结合,这是因为个体表达不同的HLA-DRB等位基因,从而致使通用探针的开发复杂化而且在某种程度上难以再现。然而,HLA-DRA等位基因只有两个,其中一个见于98%的人群中。因此,利用广泛表达的HLA-DRA等位基因评估特异性CD4+T细胞应答的方法将适用于绝大部分人群。
因此,鉴于上述讨论,本文中描述的组合物和方法用于提供对CD4+T细胞应答进行标准化分析的平台。这些方法和组合物包括一个分化方案,其由人胚胎干细胞(hESC)有效地产生髓系树突细胞(DC),其可用于刺激从全血中分离的抗原特异性CD4+T细胞。总之,虽然CD4+T细胞的临床表征受到原代树突细胞(DC)制备的可变性的限制,但是使用干细胞衍生的DC,通过提供一种充分表征的并且功能上可修改的APC群来分析T细胞对任何给定抗原的应答避免了这个问题。
发明内容
本发明提供了利用人胚胎干细胞(hESC)衍生的树突细胞以抗原特异性的方式离体刺激CD4+T细胞群增殖的组合物和方法。本发明的树突细胞是可定制的,因为任何可想到的驱动抗原特异性CD4+T细胞应答的肽抗原均可被呈递到树突细胞的表面,并用于表征特异性CD4+T细胞应答。因此,本发明包括任何用于对在多个对象个体中对特定抗原的免疫应答进行比较或者在同一对象中在不同时间对特定抗原的免疫应答进行比较的标准化方法。类似地,本发明提供了为了多种其他目的扩增抗原特异性CD4+T细胞群的方法,例如用作细胞疫苗或作为可以被重新引入到患者的活化的CD4+T细胞群以治疗可通过增强对特定肽靶标之免疫应答缓解的疾病。例如,在某一抗原特异性CD4+T细胞群被认为有利但数目不足的情况下,可以体外扩增那些细胞并将其返回至对象。此外,在其中高增殖性CD4+T细胞被认为有害(例如,自身免疫)的情况下,可体外扩增那些细胞并将其重编程为调节性T细胞(Treg),其在返回到对象时将抑制那些特异性T细胞应答。
本发明的优点包括但不限于,使用相对少量的血液从任何人中鉴别和分离人CD4+T细胞亚群的能力。本发明的用途可包括但不限于,表征对疫苗的应答,以及诊断和/或治疗涉及CD4+T细胞应答和/或CD4+T细胞库变化的人疾病。例如,本发明提供了用于诊断和治疗以下疾病的方法和组合物:阿尔茨海默病、多发性硬化、狼疮、银屑病、获得性免疫缺陷综合征(AIDS)、重症综合性免疫缺陷综合征(SCID)、重症肌无力、类风湿性关节炎、I型糖尿病、格雷夫斯病、桥本甲状腺炎、桥本脑炎、多肌炎、舍格伦综合征、克罗恩病、横贯性脊髓炎、血管炎、韦格纳肉芽肿病、帕金森病、肌营养不良、白癜风、自身免疫性心脏病、腹腔疾病、吉兰-巴雷综合征、发作性睡病、病因不明的自身免疫性疾病、自闭症和过敏。
附图说明
图1举例说明了hESC向共同髓系祖细胞的分化,其包括:
(A)示出光滑边缘的多能hESC(H9)集落的显微照片;(B)表明H9细胞多能性的SSEA4免疫染色的流式细胞术直方图;(C)平板接种(plating)后不久30至50个hESC细胞的小团块附着到OP9细胞上;(D)hESC/OP9共培养4天后分化的集落;(E)共培养8天后示出大的分化集落;(F)流式细胞术直方图示出共培养8天后,分离自集落的CD34+细胞(红色),同种型对照是黑色;(G)在平板接种到pHEMA包被的培养瓶1天后,单细胞悬液重新形成聚集体;(H)是(G)的插图,示出具有南瓜形无颗粒形态的髓系细胞的大聚集体,且OP9细胞没有附着在经包被的培养瓶上并经历细胞凋亡;(I)在含GM-CSF的培养基中培养3天后,大多数髓系细胞开始从聚集体分离,培养物中出现单个的CMP;(J-L)流式细胞术点状图示出CD34和CD45的相对表达,包括(J)CD34lo/CD45hi群由第一天的2-4%增加至(K)第三天的20-30%,以及(L)第12天的60-70%;(M)J-L的同种型对照;(N)12天后CD45+染色的直方图;和(O)CMP分化和扩增12天后CD43+免疫染色的直方图。标尺=10微米。
图2举例说明了由图1中举例说明的干细胞标志物(HSC)向共同髓系祖细胞分化的十二天期间内,HSC的表达减少而髓系细胞标志物的表达增加。在直方图中,抗原和同种型对照分别用红色和黑色表示。
图3举例说明了hESC衍生的树突细胞(DC)的形态和功能活性,包括:(A)用GM-CSF和IL-4培养10天后,显著比例的细胞分化为未成熟(i)DC,其(箭头)比未分化的CMP(楔形)更大更亮;(B)显微照片显示被iDC吞噬羧化物修饰的红色荧光乳胶珠(2微米大小),明场图像的荧光叠加示出iDC中的荧光珠(箭头);(C)前侧散射和侧向散射(FSC/SSC)的点状图示出具有内化珠的iDC;(D)流式细胞术的直方图显示由iDC内化的红色荧光珠(FL4通道);(E)用TNF-α和LPS刺激了72个小时的iDC的图像;(F)是B框住的细胞的插图,箭表示长突起(processes)以及成熟DC的形态;(G)使用霍夫曼光学显示的成熟DC的DIC图像;(H)成熟DC的苏木精染色示出长树枝状突起(箭)和暗紫色的核;(I)流式细胞术显示用TNF-α和LPS诱导成熟iDC的DC标志(DCsign)免疫染色(红色)和未用TNF-α和LPS诱导成熟iDC的DC标志免疫染色(蓝色);(J)流式细胞术示出DC成熟之后CD80+免疫染色增加;(K)TNF-α和LPS诱导成熟后CD86+免疫染色;和(L)在成熟DC上HLA-DR的表面表达。
图4举例说明了流式细胞术结果,示出在CMP向DC分化期间的第8、12、和14天,DC标志物的表达。在第12天时给出了DC成熟的因素。在该直方图中,抗原和同种型对照分别用红色和黑色表示。
图5举例说明了hESC衍生的DC需要两个群以形成激活岛(islandsof activation,IOA),包括:(A)用于处理DC以与T细胞共培养的方案的示意图;(B)iDC的流式细胞术点状图,示出FSC/SSC点状图上选通的群;(C)在B中选通之群的DC标志/FSC图,Q1-UR(42.8%)是DC标志+细胞。(D)在图B中选通之群的CD14+/FSC点状图,Q5-UR(51.3%);(E)DC标志的点状图:CD14示出CD14lo/DC标志hi与CD14hi/DC标志lo的比例为1∶1.5,定义为比例均衡的DC;(F)DC与成熟因子混合的显微照片,示出激活岛(IOA);(G)IOA的高倍率明场图像;(H)IOA的相差图像,注意薄的边缘;(I)用抗-CD14的磁性微珠纯化的DC不包括DC标志hi/CD14lo群,(J)不形成IOA;(K)用抗-DC标志MACS微珠纯化的DC不包括DC标志lo/CD14hi群,和(L)不形成IOA。
图6举例说明了CD4+T细胞的纯化和用成熟DC刺激,其包括:(A)概述从全血中纯化T细胞的示意图;(B)流式细胞术的点状图(FSC/SSC)显示选通的T细胞群,和(C)直方图显示选通之群的抗CD4免疫染色;(D)经纯化T细胞的低倍率显微照片显示出均一的形态。(E)直方图示出第0天CD4+T细胞的CFSE免疫染色及(F)与DC和IL-2共培养十天后,其示出随着每次细胞分裂,子代细胞的荧光减半,如在从1至7次每次的细胞分裂由红色数字所指示;(G)由Dil标记的T细胞和DC组成的激活岛的照片;(H)使用DAPI对细胞进行核染色,将假着色的红色作为对照,示出中间的大DC核和周界附近的小T细胞核;(I)是(G)和(H)的合并图像;(J)用IL-2和来自标准制备物的DC孵育的CD4+T细胞中CFSE荧光的直方图,和(K)DC标志纯化的制备物,和(L)CD14纯化的制备物。
图7举例说明了DC-诱导的T细胞增殖的分析,包括:(A)三个直方图显示在接种疫苗之前从人对象(对象1至3)中分离的并且已与hESC衍生的DC共培养之CD4+T细胞的CFSE荧光,所述hESC衍生的DC已被预先加载(pre-loaded)了流感肽(INF,蓝色)或无肽(对照,红色);(B)三个直方图显示接种流感疫苗1周后从同一对象中分离的并且已与已被预先加载流感肽(INF,蓝色)或无肽(对照,红色)的DC共培养之CD4+T细胞的CFSE荧光;(C)与对照DC(对照)共培养的疫苗接种后CD4+T细胞CFSE荧光的三个直方图,其中虚线表示从含IL-2的同伴培养物(companion culture)计算的第五次细胞分裂;(D)与已被预先加载流感肽(INF)的DC共培养的疫苗接种后CD4+T细胞的CFSE荧光;(E)柱状图示出在接种流感疫苗之前和之后三个人对象的CD4+T细胞的相对增殖(分裂5至7次)。白色柱表示在含预先加载流感肽(INF)之DC的培养物中CD4+T细胞的增殖;黑色柱表示含有未加载肽(对照)之DC培养物中CD4+T细胞的增殖;(F)柱状图显示在三个对照对象(c1至3)和六个诊断推定为患有阿尔茨海默病的对象(AD1至6)中CD4+T细胞的增殖(5到7次分裂)。白色柱表示在含有预先加载人Aβ1-42肽之DC的培养物中CD4+T细胞的增殖(5至7次分裂);黑色柱表示含有未预先加载肽之DC的培养物中CD4+T细胞的增殖(5至7次分裂)。
图8示出pCR8/GW-TOPO-HLADRA+接头载体的质粒图谱。
图9示出HLA-DRA的全长DNA和氨基酸序列。
图10示出[Genbank登录号CAI18477.1](人淀粉样蛋白β)的全长DNA序列和氨基酸序列。
图11示出HLA-DRA/Aβ1-13融合蛋白的氨基酸序列,其包含信号肽(No.1)、信号肽切割位点(No.2)、Aβ1-13(No.3)、接头(No.4)和HLA-DRA(No.5)。
图12示出HLA-DRA/Aβ7-13融合蛋白的氨基酸序列,其包含信号肽(No.1)、信号肽切割位点(No.2)、Aβ7-13(No.3)、接头(No.4)和HLA-DRA(No.5)。
图13示出HLA-DRA/Aβ16-30融合蛋白的氨基酸序列,其包含信号肽(No.1)、信号肽切割位点(No.2)、Aβ16-30(No.3)、接头(No.4)和HLA-DRA(No.5)。
图14示出HLA-DRA/Aβ24-35融合蛋白的氨基酸序列,其包含信号肽(No.1)、信号肽切割位点(No.2)、Aβ24-35(No.3)、接头(No.4)和HLA-DRA(No.5)。
图15示出HLA-DRA/Aβ31-42融合蛋白的氨基酸序列,其包含信号肽(No.1)、信号肽切割位点(No.2)、Aβ31-42(No.3)、接头(No.4)和HLA-DRA(No.5)。该Aβ31-42包含T411替换。
图16示出HLA-DRA/Aβ7-23融合蛋白的氨基酸序列,其包含信号肽(No.1)、信号肽切割位点(No.2)、Aβ7-23(No.3)、接头(No.4)和HLA-DRA(No.5)。
图17示出HLA-DRA/Aβ1-13融合构建体的DNA和氨基酸序列。
图18示出HLA-DRA/Aβ7-13融合构建体的DNA和氨基酸序列。
图19示出HLA-DRA/Aβ16-30融合构建体的DNA和氨基酸序列。
图20示出HLA-DRA/Aβ24-35融合构建体的DNA和氨基酸序列。
图21示出HLA-DRA/Aβ31-42融合构建体的DNA和氨基酸序列。
图22示出HLA-DRA/Aβ7-23融合构建体的DNA和氨基酸序列。
图23示出感染了改造为表达任一绿色荧光蛋白(GFP)(其充当载体对照,泳道1)和下面的各HLA-DRA/Aβ融合蛋白之慢病毒的293细胞之细胞裂解物的Western印迹,分别是:HLA-DRA/Aβ1-13(泳道2);HLA-DRA/Aβ7-13(泳道3);HLA-DRA/Aβ16-30(泳道4);HLA-DRA/Aβ24-35(泳道5),和HLA-DRA/Aβ31-42(泳道6)。
图24示出在人树突细胞系KG-1中表达的重组HLA-DRA构建体的表面表达。左上方显示具有所示选通的KG-1细胞的FDC/SSC散点图。上部中图显示,在无刺激的条件下,KG-1细胞的抗HLA-DRA免疫染色。右上图显示,LPS刺激后KG-1细胞的抗HLA-DRA免疫染色。与感染了表达HLA-DRA/Aβ7-13的慢病毒的细胞(蓝色)相比,对照细胞(红色)示出较少的HLA-DRA染色。同种型对照染色在所有图中用黑色显示。下方四个图示出感染含有上述HLA-DRA/Aβ7-13、HLA-DRA/Aβ16-30、HLA-DRA/Aβ24-35和HLA-DRA/Aβ31-42之慢病毒载体的KG-1细胞的HLA-DRA免疫染色类似的增加。
图25示出:(A)FSC/SSC散点图表示在那些细胞的混合物中检测到hESC-衍生的髓系树突细胞(DC)和从一个或更多个新鲜血液样品分离的T细胞。在流式细胞仪分析之前,用CFSE预先标记T细胞,然后与DC混合,接着混合物中的所有细胞用与荧光缀合的抗CD4染色。在这一步中fsc/ssc用来将潜在的T细胞与其它选通出来的群(包括DC和非DC)分离。我们未证明它们是DC。荧光标记的CD4+细胞从一个较小的亚群中选通。并不是所有在DC群中的检测事件都是实际上的DC;(B)仅(A)所示的CD4+T细胞群的T细胞选通的FSC/SSC散点图;(B)中描述了CD4+T细胞群的散点图(x轴为CFSE荧光,y轴为抗CD4)显示已分裂(即,增殖)的T细胞中包含较少的CFSE,从而由图左侧表示细胞群,并且在实验开始时没有可测量的增殖,因此在左边没有细胞;(D)示出(C)中所示的散点图,只是T细胞和DC的混合物在进行分析之前在对照条件下(T细胞与不表达HLA-DRA(Ab)构建体的DC一起培养)或试验条件下(T细胞与表达HLA-DRA(Ab)构建体的DC一起培养)一起培养十天。对照的细胞群,其被表示为绿色的事件,具有其CD4+T细胞总群的2.6%(图左侧描绘的),即,图的这一侧示出的是分裂的细胞。实验的细胞群,其被表示为红色的事件,具有其CD4+T细胞总群的2.6%(图左侧描绘的)。3.8和2.6之间的差异表示Aβ特异性T细胞群。图25(E)示出已感染了被改造以表达HLA-DRA构建体(分别含有全长Aβ、A1(Aβ1-13)、B2(Aβ7-13)、C3(Aβ16-30)、D4(Aβ24-35)或E5(Aβ1-42))之慢病毒的CD4+T细胞群的FSC/SSC散点图(x轴为CFSE荧光,y轴为抗CD4)。
图26显示从具有ApoE3/E3基因型,并且没有痴呆迹象的47岁男性之血液中分离的CD4+T细胞的细胞增殖响应的柱状图。该CD4+T细胞与hESC衍生的已感染了被改造以表达HLA-DRA构建体之慢病毒的DC共培养,所述HLA-DRA构建体分别包含仅HLA-DRA(对照)、A1(Aβ1-13)、B2(Aβ7-13)、C3(Aβ16-30)、D4(Aβ24-35)或E5(Aβ1-42)。柱代表经历5次或更多次细胞分裂的细胞的比例,即,指示一个持续和特定的增殖响应。
图27示出与图26所示相同类型的数据,只是供血者是一位具有ApoE3/E3基因型的、没有阿尔茨海默病迹象的88岁男性。
图28示出与图26所示相同类型的数据,只是供血者是一位具有ApoE4/E4基因型的、已被诊断出患有阿尔茨海默病14年的86岁女性。
图29示出与图26所示相同类型的数据,只是供血者是一位具有ApoE3/E4基因型的58岁的女性,并且是一位患有晚期阿尔茨海默病的母亲的女儿。该供血者是图30和图31中供血者的姐姐/妹妹。
图30示出与图26所示相同类型的数据,该供血者是图29图和图31中供血者56岁的妹妹,和她姐姐一样,具有APOE3/E4基因型。
图32示出与图26所示相同类型的数据,该供血者是图30和图31中供血者的61岁的姐姐。和她妹妹一样,具有APOE3/E4基因型。
具体实施方式
本文所公开的本发明的方法和组合物涉及到评估人对象对多种肽抗原的免疫应答。换言之,本发明涉及一种用于对多个对象个体中对特定抗原免疫应答进行比较或者在同一对象中在不同的时间对特定抗原的免疫应答进行比较的标准化方法。本发明包括在人II类白细胞(HLA)的环境下对通过抗原呈递细胞(APC)呈递之任何肽抗原的免疫应答的分析。本发明还特别涉及评估免疫应答的CD4+T细胞组分。
在多个实施方案中,本发明的方法将人胚胎干细胞(hESC)分化为成熟的专职APC。例如,本发明的方法可以诱导hESC分化为共同的髓系祖细胞,其进而可以分化为未成熟树突细胞,并且然后进一步分化为成熟树突细胞。在多个实施方案中,本发明使H9hESC分化;然而,本领域技术人员可以选择使用本领域中已知的任何人多能干细胞。例如,胚胎干细胞可以选自胚胎干细胞系或者可以直接获自原代胚胎组织。已经建立了多种胚胎干细胞系,包括但不限于:H1、H7、H9、H13和H14(Thompson等);hESBGN-01、hESBGN-02、hESBGN-03(BresaGen公司,Athens,Ga.);HES-1、HES-2、HES-3、HES-4、HES-5、HES-6(ES Cell International公司,Singapore);HSF-1、HSF-6(旧金山加利福尼亚大学);13、14、16(Technion-Israel Institute of Technology,Haifa,Israel);UCSF-1和UCSF-2(Genbacev等,Fertil.Steril.83(5):1517-29,2005);HUES 1-17系(Cowan等,NEJM 350(13):1353-56,2004);和ACT-14系(Klimanskaya等,Lancet,365(9471):1636-41,2005)。
如上所述,本发明的hESC衍生的成熟树突细胞在HLA II类分子的情况中主要呈递单个肽抗原的片段。在本发明的APC上呈递的肽可以是由本领域技术人员选择的为了评估针对包含该肽之抗原的CD4+T细胞应答特异性的任意肽。在多个实施方案中,所述抗原包含的肽是疫苗组合物的组分。例如,在多个实施方案中,抗原性肽包含流感HA的氨基酸126至138(即,NH2-HNTNGVTAACSHE-OH),并且可在本发明的方法中用以评估已施用(一种由GlaxoSmithKline,PLC销售的流感病毒疫苗)之对象中的CD4+T细胞应答。
然而,由于本发明的方法涉及可用于对任何可引起CD4+T细胞应答的肽的免疫应答进行评估的标准平台,本领域技术人员可以使用本发明的方法来评估CD4+T细胞对任何可在对象中引起这样的应答的疫苗的应答。因此,本发明可包括本领域中已知的疫苗的实例,还包括用于炭疽病的AVA(BioThrax);用于水痘的VAR(Varivax)和MMRV(ProQuad);用于白喉的DTaP(Daptacel、Infanrix、Tripedia)、Td(Decavaca、generic)、DT(-generic-)、Tdap(Boostrix、Adacel)、DTaP-IPV(Kinrix)、DTaP-HepB-IPV(Pediarix)、DTaP-IPV/Hib(Pentacel)和DTaP/Hib(TriHIBit);用于甲型肝炎的HepA(Havrix、Vaqta)和HepA-HepB(Twinrix);用于乙型肝炎的HepB(Engerix-B、RecombiVaxHB)、Hib-HepB(Comvax)、DTaP-HepB-IPV(Pediarix)和HepA-HepB(Twinrix);用于B型流感嗜血杆菌的Hib(ActHIB、PedvaxHIB、Hiberix)、Hib-HepB(Comvax)、DTaP/Hib(TriHIBit)和DTaP-IPV/Hib(Pentacel);用于人乳头瘤病毒(HPV)的HPV4(Gardasil)和HPV2(Cervarix);用于流行性感冒的TIV(Afluria,Agriflu、Fluarix、Fluvirin、Fluzone)和LAIV(FluMist);用于日本脑炎(JE)的JE(Ixiaro和JE-Vax);用于麻疹的MMR(M-M-RII)和MMRV(ProQuad);用于脑膜炎的MCV4(Menactra)、MPSV4(Menomune)和MODC(Menveo);用于流行性腮腺炎的MMR(M-M-RII)和MMRV(ProQuad);用于百日咳的DTaP(Daptacel、Infanrix、Tripedia)、Tdap(Adacel、Boostrix)、DTaP-IPV(Kinrix)、DTaP-HepB-IPV(Pediarix)、DTaP-IPV/Hib(Pentacel)和DTaP/Hib(TriHIBit);用于细菌性肺炎的PCV7(Prevnar)、PCV13(Prevuar13)和PPSV23(Pneumovax 23);用于脊髓灰质炎的Polio(Ipol)、DTaP-IPV(Kinrix)、DTaP-HepB-IPV(Pediarix)和DTaP-IPV/Hib(Pentacel);狂犬病(Imovax Rabies和RabAvert);用于轮状病毒的RV1(Rotarix)和RV5(RotaTeq);用于风疹的MMR(M-M-RII)和MMRV(ProQuad);用于带状疱疹的ZOS(Zostavax);用于天花和猴痘的Vaccinia(ACAM2000、Dryvax);用于破伤风的DTaP(Daptacel、Infanrix、Tripedia)、Td(Decavac、generic)、DT(-generic-)、TT(-generic-)、Tdap(Boostrix、Adacel)、DTaP-IPV(Kinrix)、DTaP-HepB-IPV(Pediarix)、DTaP-IPV/Hib(Pentacel)和DTaP/Hib(TriHIBit);用于肺结核(TB)的BCG(TICE BCG、Mycobax);用于伤寒的Typhoid Oral(Vivotif)和Typhoid Polysaccharide(Typhim Vi)和用于黄热病的YF(YF-Vax)。
除了涉及疫苗的肽之外,本发明的方法也基于CD4+T细胞对与指定的自身免疫疾病状态相关联的特定肽的反应来对多种自身免疫疾病的治疗进展进行诊断和追踪。如本文中所理解的,“自身免疫疾病”是由个体自身组织产生并且针对个体自身组织的疾病或病症。自身免疫性疾病或病症的实例包括,但不限于:关节炎(类风湿关节炎、幼年型类风湿关节炎、骨关节炎、银屑病关节炎)、卡普兰综合征、费尔蒂氏综合征、银屑病、皮炎、舍格伦综合征、施蒂林病、多肌炎/皮肌炎、中毒性表皮坏死松解症、全身性硬皮病和硬化、与炎性肠病有关的响应(responses associatedwith inflammatory bowel disease)、克罗恩病、溃疡性结肠炎、呼吸窘迫综合征、成人呼吸窘迫综合征(ARDS)、脑膜炎、脑炎、葡萄膜炎、结肠炎、肾小球肾炎、过敏性疾病、湿疹、哮喘、涉及T细胞的浸润和慢性炎症响应的病症(conditions involving infiltration of T cells and chronicinflammatory responses)、动脉粥样硬化、自身免疫性心肌炎、白细胞粘附缺陷、系统性红斑狼疮(SLE)、幼年型糖尿病、多发性硬化、过敏性脑脊髓炎、与由细胞因子和T-淋巴细胞介导的急性和迟发型超敏反应相关的免疫应答、结核病、结节病、包括韦格纳肉芽肿病的肉芽肿病、粒细胞缺乏症、血管炎(包括ANCA)、再生障碍性贫血、戴-布贫血、包括自身免疫性溶血性贫血(MBA)的免疫性溶血性贫血、恶性贫血、纯红细胞再生障碍(PRCA)、因子VIII缺乏症、血友病A、自身免疫性中性白细胞减少、全血细胞减少、白细胞减少、涉及白细胞渗出的疾病、中枢神经系统(CNS)炎性疾病、多器官损伤综合征、重症肌无力、抗原-抗体复合物介导的疾病、抗肾小球基底膜病、抗磷脂抗体综合征、变应性神经炎、白塞氏病(Bechet disease)、卡斯尔曼综合征、古德帕斯丘综合征、兰伯特-伊顿肌无力综合征、雷诺氏综合征(Reynaud′s syndrome)、舍格伦综合征、史-约综合征、实体器官移植排斥、移植物抗宿主病(GVHD)、类天疱疮(pemphigoid bullous)、天疱疮、自身免疫性多内分泌腺病、莱特尔氏病、僵人综合征、巨细胞动脉炎、免疫复合物肾炎、IgA肾病、IgM多神经病或IgM介导的神经病、特发性血小板减少性紫癜(ITP)、血栓性血小板减少性紫癜(TTP)、自身免疫性血小板减少、睾丸和卵巢的自身免疫性疾病(包括自身免疫性睾丸炎和卵巢炎)、原发性甲状腺功能减退症;自身免疫性内分泌疾病,包括自身免疫性甲状腺炎、慢性甲状腺炎(桥本氏甲状腺炎)、亚急性甲状腺炎、特发性甲状腺功能减退症、阿狄森氏病、格雷夫斯氏病、自身免疫性多腺体综合征(或者多腺体内分泌病综合征)、I型糖尿病也被称为胰岛素依赖性糖尿病(IDDM)和席汉氏综合征;自身免疫性肝炎、淋巴细胞间质性肺炎(HIV)、闭塞性细支气管炎(非移植)和非特异性间质肺炎(NSIP)、吉-巴综合征、大血管血管炎(包括风湿性多肌痛和巨细胞(高安)动脉炎)、中血管血管炎(包括川崎氏病和结节性多动脉炎)、强直性脊柱炎、贝尔热病(IgA肾病)、急进性肾小球肾炎、原发性胆汁性肝硬化、口炎性腹泻(麸质肠病)、冷球蛋白血症、肌萎缩性侧索硬化症(ALS)、冠状动脉疾病等。
在多个实施方案中,本发明的组合物和方法可用于评估CD4+T细胞库对阿尔茨海默肽淀粉样蛋白-β(Aβ)的应答。简言之,Aβ是具有来源于蛋白水解处理淀粉样前体蛋白(APP)得到的42个氨基酸的多肽。如本文中所理解的,Aβ蛋白质还包括突变体和其通过氨基酸交换而衍生的等位基因变体。在多个实施方案中,本发明的方法使用可包含Aβ1-13、Aβ7-13、Aβ16-30、Aβ24-35、Aβ31-42和Aβ7-23的肽来替代全长Aβ。
在多个实施方案中,对所有Aβ肽广泛和强烈的应答表明正在进行对Aβ和Aβ所有片段的免疫应答。本领域技术人员能解释这样的应答是对积累的Aβ病理持续响应的指示,最终变得像无反应性开始一样变得较低强健,最终导致在阿尔茨海默病后期阶段较低强健的CD4+应答。也就是说,对Aβ或者某些Aβ片段具有相对强健CD4+响应的个体即便可能没有发现认知上或行为上的改变,也将被认为患前阿尔茨海默病。
通常,在本发明的方法中可使用任何可使APC优选地呈递特定单一肽片段的方法。例如,在多个实施方案中,本发明的方法通过直接向APC培养物中添加足量的肽以以使APC的HLA II类分子呈递该肽或其片段来加载APC。在本发明的多种另外的方法中,肽抗原在其中已引入编码该肽之核酸的APC表面呈递。例如,可以在病毒或质粒载体中编码该肽。
在本发明的多种实施方案中,由APC呈递的肽是作为融合蛋白的肽组分来被呈递的,所述融合蛋白还包括至少一部分HLA-II蛋白。例如,本发明的融合蛋白可包含来源于人白细胞抗原(HLA)II类-DRA(HLAII类α链的旁系同源)的核酸和氨基酸序列。在多种另外的实施方案中,本发明的融合蛋白包括包含Aβ片段的HLA-DRA融合构建体。例如,作为全长Aβ的替代,融合蛋白可包含Aβ1-13、Aβ7-13、Aβ16-30、Aβ24-35、Aβ31-42和Aβ7-23。
HLA-DRAα链约33-35kDa并且它的基因含有5个外显子。外显子1编码前导肽,外显子2和外显子3编码两个胞外结构域,和外显子4编码跨膜结构域和胞质尾区。HLA-DRA在该肽结合部分不具有多态性,并且作为用于β链DRB1、DRB3、DRB4和DRB5的唯一的α链。HLA-DRA序列是公开可得的。例如,核苷酸和氨基酸序列可见于:GenBank登录号CAI18477.1(氨基酸序列)、M60334.1(DNA序列)、NP_061984.2(氨基酸序列)、AAA36275.1(氨基酸序列)、AAA59785.1(氨基酸序列)、AAA36302.1(氨基酸序列)、AAAH71659.1、CAI18476.1(氨基酸序列)和G13122(基因序列);以及在EMBL登录号AL935032.13(DNA序列)、CAG33294.1(氨基酸序列)、CAQ08811.1(DNA序列)和EAX03629.1(DNA序列)。本领域技术人员将理解,HLA-DRA核酸和蛋白质分子可不同于这些可公开获得的,例如导致一个或更多个替换、缺失、插入或其组合同时仍保持了HLA-DRA之生物活性的多态性。因此,在本发明的多个实施方案中,本发明融合蛋白的HLA-DRA组分的氨基酸序列可以与图2中示例的HLA-DRA序列或其片段有约90%、约91%、约92%、约93%、约94%、约95%、约96%、约97%、约98%、约99%的同一性。
在多个实施方案中,本发明的融合蛋白包含接头。通常接头是位于所述HLA-DRA和融合蛋白所需的融合肽组分之间的柔性肽接头元件。接头元件优选具有25个氨基酸或更小的长度。在某些实施方案中,接头元件的长度为3至30个氨基酸,特别是长度为3、4、5、6、7、8、9、10、11、12至13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28或30个氨基酸。在一个实施方案中,接头元件的长度是5至25个氨基酸、8至20个氨基酸或10至20个氨基酸。更优选地,接头的长度为9至15个氨基酸。通常本文所用的接头元件可以由任何已知的氨基酸或人工氨基酸衍生物组成。在某些实施方案中,接头元件由小的并且亲水的非带电氨基酸构成。通常,根据本发明的接头元件可以包含选自G、S、A和T的氨基酸。接头元件优选为甘氨酸/丝氨酸接头,即,基本上由氨基酸甘氨酸和丝氨酸组成的肽接头,如聚甘氨酸。在本发明的某些实施方案中,接头序列是IGGGSGGGGSGGGGS。
如上所述,本发明还涉及扩增肽抗原限定性CD4+T细胞群的方法。在多个实施方案中,如上所示,本发明的方法涉及CD4+T细胞群的抗原特异性扩增以对例如疫苗、自身免疫疾病、以及与Aβ沉积相关的认知疾病产生免疫应答。
通常,本发明的方法可从使用标准的放血方法分离的全血中获得人CD4+T细胞。初始的纯化去除了大多数髓系细胞和B细胞,富集T细胞群。然后用羧基荧光素二乙酸琥珀酰亚胺酯(CFSE)将T细胞染色,然后与表达一组HLA-Aβ融合蛋白的APC混合并共培养7至10天。在这个孵育期后,细胞混合物用CD4-特异性荧光抗体染色并通过流式细胞仪,例如荧光激活细胞扫描仪或分选仪。用CD4-阳性(CD4+)荧光来鉴别CD4+T细胞,并使用CFSE的强度来确定自试验开始经历了多少次增殖。
如上所述,本发明的方法在APC中表达HLA-DRA融合蛋白。因此,本发明的方法和组合物包括能够在APC中表达本发明的融合构建体的DNA或RNA载体。通常载体包含启动子、待表达核苷酸序列的下游终止子。在本发明的多个实施方案中,所述载体编码重组病毒。例如,在本发明的某些实施方案中,所述载体包含来自慢病毒基因组的序列,例如FUW慢病毒载体(描述于Science.2002年2月1日.295(5556):868-72.Epub2002年1月10日)。
在一些实施方案中,本发明的组合物和方法可以用作直接免疫方案的一部分。例如,病毒载体、脂质体或其它递送方法可用来将HLA-DRA融合蛋白构建体直接递送到淋巴结内。这种方法将允许在内源性APC的HLA类II复合物中快速呈递未知肽。因此,本发明提供了传统疫苗方法的理想替代方案,所述传统方法引入肽作为辅助复合物,其必须由APC细胞进行处理然后搬运至淋巴结,这个过程可能需要进行三到七天。未知结合HLA-DRA融合蛋白的直接递送和及时表达在产生对迅速致死的病原体(如埃博拉病毒)的免疫反应中尤其有用。
实施例
实施例1.使用加载了流感抗原的hESC-衍生的DC来评估CD4+T细胞应答的方法。
hESC的造血干细胞(HSC)分化。在具有5×补充剂的mTeSR培养基(Stem Cell Technologies)中培养H9(NSCB代码WA09,第23代)hESC细胞系,该培养基补充有额外的bFGF(4微克/毫升,LifeTechnologies)。使用标准的方法在基质胶(matrigel)中培养多能性H9hESC的集落(图1A),并且每5代用SSEA4染色检测多能性,通过显微镜和流式细胞术对其进行评估(图1B)。轻柔地将集落刮为30至60个细胞的团块,并将其收集到15毫升试管中,然后以300×g离心5分钟,并重悬于HSC分化培养基中(HDM:α-MEM,10%FBS,100μM的单硫代甘油)。
在收获H9细胞进行分化之前,清洗大约70%汇合的OP9小鼠基质细胞(ATCC)的培养瓶并用HDM预孵育。OP9细胞在经凝胶化的(G1393,Sigma)T75培养瓶中于OP9生长培养基(OP9M:α-MEM(LifeTechnologies),含有20%FBS(HyClone))中培养。将含有hESC细胞团块的HDM直接加入到培养瓶中。在第4天将所有的HDM更换为新鲜的HDM,并在第6天和第8天,将一半HDM培养基更换为新鲜的HDM。
在HDM中共培养2天后,H9集落的含HSC团块的初始造血分化附着到OP9饲养层并开始(bega)(图1C)。共培养4天后,集落形态类似于hESC集落,但细胞大小更易变(图1D)。8天后,含有HSC之集落的形态是高度可变的,并且明显不同于光滑边缘,甚至出现了多能性hESC集落(图1E)。CD34的免疫染色示出了共培养8天后从集落分离的细胞的表达分析(图1F)。
共同髓系祖细胞的分化。在pHEMA包被的(Sigma)的T25培养瓶内维持髓系细胞。为了诱导HSC分化为共同髓系祖细胞(CMP),将细胞用胶原酶IV(1毫克/毫升)在KnockOut DMEM/F-12(LifeTechnologies)中于37℃处理20分钟,接着用0.05%胰蛋白酶-EDTA在37℃处理15分钟。用10%的FBS淬灭胰蛋白酶活性,沉淀(300×g),重悬于髓系分化培养基(Myeloid differentiation medium,MDM)中,并平板接种于pHEMA包被的培养瓶中。髓系扩增进行10至12天。MDM包含α-MEM、10%FBS、100纳克/毫升GM-CSF、100μM的单硫代甘油。MDM也可用来扩增髓系细胞数目。在重新平板接种的一天之内,分化的CMP前体重新形成具有南瓜形核的团块(图1G,H);3天后,团块开始分解然后单细胞出现(图11)。包括CD34在内的HSC标志物的表面表达降低的同时CMP标志物表达增加,所述CMP标志物包括CD14、CD11b(SI2)、CD45(图1J-N)和CD43(图10;图2)。
未成熟树突细胞(iDC)的产生。为了在髓系扩增后产生髓系DC,将细胞通过70微米的过滤器过滤并用25%分级以去除死细胞和细胞团块(图5A)。用含5%FBS的PBS清洗细胞,并重新平板接种于DC分化培养基(DDM)中8至10天。DDM是StemSFEM培养基(Stem Cell Technologies),补充有生长补充剂(Millipore)、100纳克/毫升的GM-CSF、100纳克/毫升的IL-4(Endogen)。每四天用新鲜的DDM更换iDC培养物中一半体积的DDM。为了扩大iDC的数目,使它们在相同的条件下分裂和维持。在补充有TNF-α(100纳克/毫升)和LPS(250纳克/毫升)的DDM中诱导DC成熟,保持3天。在DC分化条件下8天后,iDC具有皱的形态(图3A),并示出DC标志、CD80和CD86的表面表达(图3I-K)。iDC的特征行为是病原体和细胞碎片的吞噬作用,这是使用人免疫球蛋白包被的荧光胶乳珠测定的(Dagenault等,2010)。更具体地说,将平均直径为2微米的羧化物修饰的红色荧光胶乳珠(L3030,Sigma)在具有PBS的50%人AB血清中在37℃调理30分钟。在孵育后,将这些经调理的乳胶珠添加到DC培养物中并且在37℃轻柔摇动30分钟进行孵育。相同的对照在4℃孵育。30分钟后,通过加入2毫升的冰冷的PBS终止吞噬作用,随后用冰冷的PBS洗涤细胞两次。使DC重悬于1毫升冷的PBS中,且通过加入等体积的4%多聚甲醛来固定,保持在4℃的暗处直到成像。iDC内化了这些珠子(图3B)且流式细胞术显示大部分iDC具有吞噬的珠子(图3C,D)。
树突细胞的成熟。在用补充有100纳克/毫升的TNF-α(PeproTech)和250纳克/毫升的LPS(Sigma)的DDM触发成熟之前,未成熟DC被预先加载了目的肽。用相差显微术(图3E,F)、苏木精和伊红(H&E)染色的光学显微术(图3G)以及DIC显微术(图3H)观察到成熟DC具有分叉的树枝状突起。使用由细胞离心涂片器(Cytospin)铺展到玻片上的经固定细胞(2%多聚甲醛)进行H&E染色。用H&E溶液覆盖具有iDC和成熟DC的载玻片并孵育15分钟,随后用酸和蓝化溶液(bluingsolutions)(Vector Laboratories)处理。用水清洗载玻片,经过醇系列(50%、70%、95%、100%乙醇)并用Permount(Fisher)封装盖玻片。H&E染色的图像用安装在显微镜上的彩色CCD摄像头捕捉。
流式细胞术证实iDC和成熟DC两者都表达DC标志(图3I)。更具体地,iDC培养物用Percoll分离进行纯化(图5A),含有表达高水平DC标志(图5B,C)或CD14(图5D)的群。一个群是DC标志hi/CD14lo,另一个群是DC标志lo/CD14hi(图5E)。这些群通常分别是约1∶1.5的比例。为了比例平衡的研究,用抗-CD14或抗-DC标志的磁珠纯化iDC。简言之,将DC用70微米过滤器过滤以去除聚集体并用髓系树突细胞的试剂盒纯化,用以分离表达CD14的细胞,随后通过MACS柱(Miltenyi)纯化。将分离的细胞在DDM中培养4周。为了富集分离的DC标志hi细胞,我们使用人DC标志试剂盒(Miltenyi)来纯化细胞并且它们也在DDM中扩增4周。
当将成熟DC与原代T细胞进行混合时,培养物在3到5天内形成了细胞聚集体,称为“激活岛”(Islands of activation,IOA)(图5F-H)。IOA通过第一荧光标记的DC成像,将它们在预热的含有Dil(2μM,LifeTechnologies)的无血清培养基中在37℃孵育20分钟,且经分离的T细胞用DiO(1μM)以相同的方式标记。孵育后,加入10倍体积的T细胞培养基并在300×g离心5分钟沉淀细胞。然后用含有0.1%BSA的PBS洗涤细胞两次。将荧光标记的DC(红色)和T细胞(绿色)混合,并在一个pHEMA包被的培养瓶中过夜(16小时)培养,然后通过加入等体积的4%多聚甲醛固定并静置30分钟。通过直接将DAPI添加到定影溶液(20μM的终浓度)中来用DAPI标记细胞核。不对细胞进行洗涤,因为离心和重悬可能会破坏IOA,所以用显微镜挑取IOA并放置于盖玻璃底下的室来成像。IOA的图像由C1共聚焦显微镜捕捉。
iDC的纯化,用抗DC标志的磁珠富集DC标志hi/CD14lo群并去除大部分DC标志lo/CD14hi群(图5I),但防止IOA的形成(图5J)。互补性研究中,用抗CD14磁珠富集DC标志lo/CD14hi细胞,降低了DC标志hi/CD14lo群(图5K),并且还防止了IOA的形成(图5L)。
流式细胞术也证实,iDC和成熟DC两者都表达CD80(图3J)、CD86(图3K)以及CD11c(图3)。HLA II类复合物在iDC的内体(endosome)中隔离,并随着DC成熟被运输到表面(图3L)。
为了确定hESC-衍生的DC是否可以刺激T细胞增殖,从全血中分离人T细胞(图6A),用CFSE标记,并与加载抗原的成熟DC共培养7至10天。之后,CD4+T细胞用抗CD4进行免疫染色并通过CD4+群的CFSE荧光评估其增殖(图6B,C)。使用与DC和IL-2(Life Technologies)一起孵育的T细胞(图6D)作为阳性对照,以确定连续细胞分裂的CFSE荧光(图6E,F)。为了检测IOA,我们在将T细胞与成熟DC混合前,用具有亲脂性的DiI荧光标记T细胞。第二天可以看到具有T细胞围绕较大DC的IOA,(图6G-I)。与成熟DC和IL-2混合的T细胞的增殖多达7代(图6J),用抗DC标志(图6K)或抗CD14(图6L)磁珠纯化的DC仅发生了5代增殖。
hESC-衍生的DC刺激流感HA肽特异性CD4+T细胞的增殖。T细胞是从通过肌肉内注射接受市售流感疫苗由GlaxoSmithKline分布)之前和一周后的人对象中抽取的20毫升全血样品收集的。从对象抽取的全血在含EDTA的血管(#366643,BDBiosciences)。在无菌条件下用PBS将血样稀释至1∶1,并且40毫升的稀释样品在30毫升的的顶部分层,并以400×g旋转30分钟。收集外周血淋巴细胞(PBL)层(图6A)并用PBS稀释(1∶7),随后以300×g离心10分钟。细胞用在通过将沉淀重悬在PBS中再清洗细胞两次。存活的细胞用台盼蓝排除法(exclusion)计数。除去贴壁细胞以使用尼龙羊毛柱富集T细胞。简言之,用T细胞培养基(RPMI1640,具有10%FBS)洗涤尼龙羊毛柱并在37℃孵育一小时。然后,将107个细胞/毫升的PBL添加到预热的柱中并在37℃孵育1小时,在这之后通过打开柱的旋塞来收集非贴壁细胞。该柱用5毫升T细胞培养基洗涤两次以收集总流出物。T细胞富集的级分用CFSE溶液(4μM,在PBS中)在37℃水浴中孵育8分钟。孵育后,将溶液用10倍体积的T细胞培养基稀释,通过离心(300×g,10分钟)沉淀,并用含有0.1%BSA的PBS洗涤两次。对CFSE标记的细胞进行重悬、计数,并且一部分被用来通过流式细胞术BD Biosciences)评估CFSE荧光。在流式细胞术之前,细胞用647缀合的抗CD4抗体(BD Biosciences)进行免疫染色以允许分析CD4+群中的CFSE荧光。另外流式细胞仪的数据和直方图的分析已由FCS express 4流式细胞仪软件(全新的软件(De Novo Software))完成。
将CFSE标记的T细胞与成熟DC共培养,所述成熟DC已被预先加载了流感HA的免疫原性肽(氨基酸126至138,H-HNTNGVTAACSHE-OH,Anaspec)。(Le等,2010;Schmidt等,2012)。DC的肽加载包含在水中重悬冻干的流感HA肽和十分之一体积的10×PBS以中和pH值。在培养基中用终浓度为每毫升10微克的流感肽加载未成熟DC,在湿润的含5%CO2的培养箱中培养4天。然后用如前所述的TNF-α和LPS使未成熟DC成熟。
对CD4+T细胞增殖的分析显示在疫苗前样品中对对照DC几乎没有应答(图7A),但疫苗接种后样品示出CD4+T细胞增殖的显著增加(图7B,C),可能是由于疫苗中佐剂的非特异性效应(Fox等,2012;Tetsutani等,2012;Caproni等,2012)。被预先加载了流感肽的成熟DC也对疫苗接种前的CD4+T细胞几乎没有刺激作用(图7A),但疫苗接种后的T细胞以增加的增殖来应答,该应答远高于对对照DC的应答(7B-E)。在疫苗接种后测定中使用对照DC和流感-DC之间的唯一区别是流感肽的预先加载,提示应答增加是由于流感特异性CD4+T细胞的增殖。
简言之,涉及人对象的所有程序都是在健康科学西部大学(Pomona,CA)、箭头地区医疗中心(Colton,CA)和Casa科利纳(Pomona,CA)伦理审查委员会的批准下进行的。向所有参与者解释了知情同意书,并由所有对象和/或法定监护人签字。
实施例2.阿尔茨海默病患者的Aβ-特异性CD4+T细胞应答的分析
为了评价hESC-衍生的DC是否可以检测慢性病症中的CD4+T细胞应答,我们招募了六个推定诊断患有阿尔茨海默病(AD)的对象和三个年龄匹配的对照者。根据与实施例1中所述相同的方法采集全血、处理并分离T细胞。通过对根据上述用流感HA肽加载DC的相同方法预先加载有10毫克/毫升的Aβ1-42肽(BioMer Technology)的成熟DC的应答来评估CD4+T细胞增殖。
这种肽的不溶性沉积物积聚成神经炎脑斑(neuritic brain plaque),是阿尔茨海默病的病理的明确标志(Braak和Braak,1997)。Aβ特异性的CD4+T细胞减弱了疾病的小鼠模型中阿尔茨海默病的病理和行为(Ethell等,2006;Cao等,2009)。尝试用Aβ1-42疫苗接种作为阿尔茨海默病的治疗方案,虽然有前景,但是由于对象子集(6%)中的脑膜炎症,该试验被叫停(Orgogozo等,2003)。至今仍不清楚,在AD病因中是否出现了Aβ特异性的T细胞应答,以及他们是否可以提供随着病情的发展而淹没的早期有益影响(Ethell等,2002;Ethell和Buhler,2003;Cao等,2009年)。按照上述实施例1来制备T细胞,并将其与预先加载Aβ1-42的成熟DC共培养。6个AD对象中的5个示出响应呈递Aβ之DC的CD4+T细胞增殖比对照DC高三倍,然而对照对象均未表现出高于2倍对照的响应(图7F)。对加载Aβ之DC没有增殖反应的一个AD对象(AD4),可能是由于不正确诊断,因为AD的确诊只能通过死后的大脑病理做出(Alzheimer,1907;Fischer,1907)。
实施例3.用转染了改造为表达HLA-DRA-Aβ融合蛋白之慢病毒构建体之hESC衍生的DC来评估CD4+T细胞应答的方法。
本发明的组合物和方法已被用来在36个人对象中评估CD4+T细胞库对阿尔茨海默病的肽、淀粉样蛋白(Aβ)的应答。CD4+T细胞是从使用标准放血的方法得到的全血中获得的。初始的基于尼龙羊毛的纯化方法除去大部分的髓系细胞和B细胞,从而富集了T细胞群。然后用羧化物荧光二乙酸琥珀酰亚胺酯(CFSE)将T细胞染色,然后与表达HLA-DRA-Aβ融合蛋白的APC组混合并共培养7至10天。融合蛋白的组包括全长Aβ、Aβ1-13、Aβ7-13、Aβ16-30、Aβ24-35、Aβ31-42、和Aβ7-23。在孵育期后,将细胞混合物用CD4特异性荧光抗体进行染色,并通过流式细胞仪。利用CD4特异性荧光来鉴别CD4+T细胞,并利用CFSE强度来确定那些CD4+T细胞都经历了多少次增殖,因为T细胞最初是用CFSE染色的。以下实施例举例说明了本发明的多个步骤。
HLA-DRA/淀粉样蛋白β融合构建体。人HLA II类α链(HLA-DRA,提供了基因库入口)是按照如下方法克隆的:采用标准方法从1毫升全血的人血外周血单核细胞(PBMC)中分离RNA,然后使用标准的反转录方法来建立HLA-DRA cDNA模板。接着通过向含校对DNA聚合酶“Phusion”(New England Biolabs)的标准聚合酶链反应(PCR)中添加)HLA-DRA cDNA模板来扩增HLA-DRA核苷酸序列(引物是由发明人Doug Ethell设计的HLA-DRAF(正向):5′-TTATTCTTGTCTGTTCTGCCTC;HLA-DRAR(反向):5′-CTTCTCTCTAAGAAACACCATCACCTC。PCR的产物溶解在琼脂糖凝胶上,并且从凝胶中分离出正确大小(约2kb)的单一条带来,然后按照制造商的说明连接到8/GWTA载体上(Invitrogen,LifeTechnologies,Carisbad,CA)。选择一个包含有正确序列并且无突变之HLA-DRA克隆的TA质粒。用定点诱变(SDM)在HLA-DRA序列的5′末端引入编码多聚甘氨酸/丝氨酸接头的核苷酸序列。从SDM得到的后续克隆的标准DNA测序来鉴定具有正确的序列的克隆。图9示出所得到的8/GWHLA-DRA+接头质粒的质粒图谱。SDM被用来与该质粒一起用于生成在这些研究中使用的各个淀粉样蛋白β(Aβ)的融合构建体。具体地说,制造的Aβ+接头+HLA-DRA融合构建体包含以下Aβ片段:Aβ1-13(图11)、Aβ7-13(图12)、Aβ16-30(图13)、Aβ24-35(图14)、Aβ31-42(图15)和Aβ7-23(图16)。
使用标准的分子生物学克隆方法在FUW载体的多克隆位点EcoRI位点处将各个上述Aβ+接头+HLA-DRA融合构建体从它们各自的8/GW载体亚克隆到FUW慢病毒载体上(在Science.2002年2月1日.295(5556):868-72.Epub 2002 Jan 10中描述的)。在插入到FUW载体之前,对每个Aβ+接头+HLA-DRA融合限制性片段进行凝胶纯化。Aβ+接头+HLA-DRA插入的方向由限制性酶切消化来验证。具体地,使用核酸酶XbaI和PstI来确认插入片段的方向。
Aβ+接头+HLA-DRA融合蛋白的蛋白表达由存在于FUW质粒上的泛素化启动子驱动。Western印迹分析表明当构建体被引入到293细胞中时,FUW构建体表达Aβ+接头+HLA-DRA融合蛋白。参见图15。细胞裂解物的Western印迹分析是通过使用HLA-DRA特异性抗体(Abnova目录#MAB7134,克隆L243)探测印迹进行的。
人类胚胎干细胞(hESC)衍生的树突细胞。通过在37℃水浴中持续地轻轻摇晃冻存管直到只有小冷冻块(pellet)残留使冷冻小瓶的hESC之H9系(WiCell/国家干细胞库)迅速解冻。然后从水浴中取出冻存管并擦干,用异丙醇擦拭。使用1毫升移液枪头(pepitte tip)将冻存管中的内容物转移到15毫升锥形管中。将9毫升预热的干细胞培养基(完全mTeSR,来自Stem Cell Technologies,Vancouver,Canada)逐滴加入到该管中,随着培养基的加入轻柔地混合。然后将细胞在室温(25℃),300×g离心5分钟。将培养基吸出,让细胞沉淀保持完整。使用1毫升移液枪头,将细胞沉淀轻轻地重悬,同时小心维持细胞为聚集体。确保团块均匀分布,将1毫升细胞聚集体的细胞转移到包被有基质胶(Becton-Dickinson,根据制造商的说明制备的)的T25培养瓶中。然后将培养瓶快速地从一边到另一边晃动,随后前后晃动直到细胞聚集体均匀地分布在培养基中。在湿润的培养箱(37℃,5%CO2)中培养细胞。每隔一天向每个T25培养瓶(4毫升)的hESC培养物供给,用新鲜mTeSR培养基100%更换。在解冻后约5至7天,检查hESC培养物中存在准备好传代的hESC集落(即致密居中具有光滑的边缘)。
未分化的hESC的传代。用显微镜检查如上所述准备传代的hESC的集落以鉴定未分化的集落区域,其用移液枪头刮基质胶表面来移除。在此步骤中,每个培养瓶的表面面积的至多20%被刮下。脱落的细胞被转移至基质胶包被的含有4毫升的mTeSR培养基的T25培养瓶中。在未分化的细胞被转移到一个新的T25培养瓶中之后,存在的任何相对较大的集落均通过用1毫升移液枪头轻轻上下吹吸集落而被打散(breakup)。未分化的细胞通过摇动T25培养瓶轻柔地混合,然后将培养瓶放置在培养箱中培养。未分化的hESC集落可通过不存在粗糙的集落边缘来识别,由每隔一天供给培养物4毫升的mTeSR培养基来维持。未分化的hESC在传代后5至6天后被用于实验中。
OP9细胞的培养。使用OP9骨髓基质细胞(ATCC目录号CRL-2749)来刺激hESC的造血干细胞(HSC)系的分化,尽管其它的骨髓基质细胞在此过程中可以取代。OP9细胞保持在OP9培养基中:1×的α-MEM(LifeTechnologie)培养基中补充有20%FBS(未加热灭活)、L-谷氨酰胺。OP9培养物维持在T75培养瓶中,所述T75培养瓶在湿润的培养箱中37℃和5%CO2,如在ATCC方案中所描述的。简言之,OP9的培养物每2天供给一次,每4天传代一次。为了使OP9培养物传代,将培养基从培养瓶中吸出,并用8毫升PBS清洗OP9单层细胞。为了使OP9细胞从培养瓶表面脱落,将TrypLE表达(Life Technologies)溶液(4毫升)加入到该培养瓶中,并将培养瓶置于37℃培养箱中5分钟。通过加入4毫升含0.5%BSA的PBS终止TRYPLE处理。将经处理的细胞和溶液转移到15毫升的试管中,将试管以300×g离心5分钟。离心后,吸出上清液,并将细胞重悬于1毫升OP9培养基中。细胞溶液重悬于30毫升OP9培养基中并将10毫升的重悬细胞分别加入到3个新的T75培养瓶中。在约4天后,当OP9细胞生长至85%汇合度时,按照上述方法再次对它们进行分离。
hESC通过与OP9基质细胞共培养进行造血分化。为了准备hESC分化,T75培养瓶中的OP9细胞过度生长(即,分离后4至5天)。将培养基吸出并将10毫升的hESC-HSC分化培养基(1×α-MEM中补充有10%FBS(未加热灭活的)和100μM的单硫代甘油(MTG,Sigma目录#M6145))加入到培养瓶中,并将培养物孵育20分钟。当用hESC-HSC分化培养基孵育OP9细胞时,将mTeSR培养基从hESC集落(分离后5-6天)的T25培养瓶中除去,并用5毫升的PBS清洗hESC培养物。用3毫升补充有5%FBS的PBS覆盖细胞,然后用细胞刮(BD Falcon目录#353085)使hESC集落从T25培养瓶的表面脱落并被打散。接着用移液管将脱落的hESC集落转移到15毫升锥形管中。然后用另外3毫升的PBS/5%FBS清洗培养瓶,将其添加到含有刮下来的集落同一个15毫升试管中。将含有分散的hESC集落的15毫升试管以300×g离心5分钟。吸出上清液,将沉淀轻轻悬浮于1毫升的hESC分化培养基中,通过轻敲该培养瓶并用1毫升移液管轻柔地磨碎,并小心不要打碎细胞团块。细胞悬液的体积为1.5毫升至2毫升,并且通常含有约106个细胞/毫升。将另外的9毫升的hESC-HSC分化培养基轻柔地加入到hESC细胞悬液中,并小心保持hESC细胞团块的完好。然后将10毫升含hESC的hESC-HSC培养基均匀地分布到如上文所述已用10毫升hESC-HSC分化培养基孵育的OP9培养瓶中。然后将培养瓶放置在一个温润的培养箱(37℃和5%CO2)中。孵育1天后,所有的培养基都吸走。将20毫升新鲜hESC-HSC分化培养基加入到培养瓶中,将培养瓶放回培养箱中。合并的hESC/OP9培养物自起始平板接种起的第4天和第6天供给,每次供给用新鲜的hESC-HSC分化培养基更换50%的培养基(10毫升)。在第4天开始时,在显微镜下检查共培养物,以观察hESC集落的分化。好的HSC分化的特点是与未分化的hESC集落形成对比的畸形集落并且培养基不含有成脂肪的(含大液泡)OP9细胞。在初始平板接种后7至8天收获HSC集落。
在GM-CSF批量培养物中髓系祖细胞的扩增。用pHEMA包被塑料表面。通过在50毫升试管中向10毫升含有10mM NaOH的95%乙醇中加入4克的pHEMA制备聚(2-羟乙基甲基丙烯酸酯)/pHEMA(Sigma目录#P3932)包被溶液。并且在37℃培养箱中使其旋转过夜。为了包被T25培养瓶,使用2毫升的pHEMA包被溶液,并且旋转倾斜该培养瓶,直到整个底部表面被覆盖。将培养瓶侧立放置,过量的pHEMA包被溶液被排入一个角落,并用吸管除去。培养瓶立即置于水平位置且将盖子取下,并使其在组织培养罩中干燥过夜(无UV)。第二天将盖子盖在培养瓶上,并在室温下储存直到使用。第8天的hESC/OP9共培养物的单细胞悬液是通过如下的用胶原酶-胰蛋白酶溶液连续的酶处理而制备的。首先,将hESC/OP9共培养的培养基吸出并用10毫升PBS清洗。然后每个T75培养瓶中加入5毫升的胶原酶溶液(在DMEM/F12(1毫克/毫升)中无菌过滤的IV型胶原酶,Life Technologies 17104-019号),并将培养瓶在37℃孵育20分钟。将胶原酶溶液从培养瓶中移出,并转移到一个50毫升锥形管中。然后将5毫升的胰蛋白酶-EDTA溶液(在0.5mM EDTA溶液中含0.05%胰蛋白酶,Life Technologies#25300-054)加入到相同的hESC/OP9共培养培养瓶中,将培养瓶在37℃下孵育20分钟。将5毫升的PBS-5%FBS加入到含有胰蛋白酶-EDTA溶液的培养瓶中,并用10毫升的血清移液管(serological pipet)通过吹吸将脱落的细胞重悬。然后将细胞转移到含有所收集的胶原酶溶液的同样的50毫升收集管中,通过封闭收集管并使其颠倒来使细胞混合。将细胞在同一管中以400×g离心10分钟。除去上清液,将细胞用PBS-5%FBS洗涤,并再次以400×g离心10分钟。再重复这个洗涤步骤两次,并在最后一次洗涤后,将上清液除去。将细胞重悬于10毫升的髓系细胞扩增培养基(MCEM。MCEM含补充有10%非加热灭活的Hyclone FBS(Hyclone)、1×MTG(100μM)、和100纳克/毫升的GM-CSF(R&D系统目录215-GM)的α-MEM。GM-CSF是在临用前加入的。在MCEM中重悬细胞后,将细胞悬液通过安装到一个50毫升试管上的70微米的尼龙过滤器来过滤。过滤步骤后,用血细胞计数器和台盼蓝来计算细胞的总细胞数。细胞最终以2至3×106个细胞/毫升的浓度悬浮。将包括hESC衍生的共同髓系祖细胞的悬浮细胞分配到pHEMA包被的T25培养瓶中(5毫升/T25培养瓶),并在37℃和5%CO2下孵育。在平板接种后第4、8和12天,通过用新鲜的MCEM更换每个培养瓶50%的培养基来供给培养物新的MCEM。在每次供给时,取出2.5毫升的培养基,并以300×g离心10分钟。将上清液完全除去,将细胞沉淀重悬于新鲜的MCEM中,并返回到培养瓶中。在建立共培养后11天或12天后收集细胞。
髓系在细胞分化成DC。在直接从T25培养瓶中收集以上步骤中描述的髓系祖细胞,然后通过70微米尼龙筛过滤,转移到15毫升锥形管中,并以400×g离心10分钟。使用移液管来除去上清液,然后将细胞在5毫升的PBS中重悬。将细胞悬液用5毫升25%的Percoll(Sigma目录.No.P1644)悬起,然后以400×g离心15分钟。将细胞用PBS-5%FBS洗涤两次并以450×g离心10分钟,小心保持细胞沉淀完好。在最后一次洗涤后,将细胞重悬于8毫升的树突细胞分化培养基(DCDM)中。DCDM包括由StemSpan无血清扩增培养基(SFEM)(Stem Cell Tech目录#09650)构成,并补充有1/500稀释的EX-CYTE生长增强补充剂(Millipore,目录#81-129-N)、100纳克/毫升的重组人GM-CSF(Sigma)和100纳克/毫升的重组人IL-4(R&D Biosystems)。向两个T25培养瓶的每一个中加入一半的含有细胞的溶液。每个培养瓶中细胞悬液的最终浓度为每毫升DCDM2×105至1×106个细胞。在平板接种后4天和8天通过更换50%的DCDM供给细胞。从每个培养瓶中取出2毫升培养基并以400×g离心10分钟。除去上清液并将细胞重悬于2毫升的新鲜DCDM中,并将该溶液加回到培养瓶中,将培养瓶放回培养箱中。
树突细胞(DC)的成熟(一般的成熟过程)。使用标准的胰蛋白酶溶液细胞分离的方法,将10至12天的树突细胞培养物收集到15毫升锥形管中,并以400×g离心10分钟。通过移液管吸除去上清液,将沉淀重悬,并与4毫升新鲜制备的DC成熟培养基(补充有EX-CYTE生长增强补充剂(Millipore,目录#81-129-N)的SFEM)轻柔地混合。将重悬的DC转移到pHEMA包被的T25培养瓶中,并在37℃和5%CO2下孵育。DC从平板接种细胞2至3天后开始形成树枝状突起。
树突细胞的感染与成熟。慢病毒的制备。慢病毒是通过在HEK293细胞中转染质粒产生的。在湿润的培养箱37℃,5%CO2下,于293培养基(杜尔贝科最低必需培养基(Dulbecco Minimal Essential Medium,DMEM),补充有10%FBS和L-谷氨酰胺)中培养人胚胎肾293细胞/HEK293(以下简称为293)细胞。在细胞80%汇合时,吸除培养基,用PBS(直径10厘米的培养皿用5毫升)清洗细胞,并将胰蛋白酶-EDTA放在细胞上3至4分钟(直径10厘米的培养皿用5毫升)以将细胞分开。经胰蛋白酶-EDTA处理后,将5毫升的293培养基加入到培养皿中,通过上下吹吸溶液轻柔地移动细胞。将重悬的293细胞和培养基转移到15毫升试管中,并以500×g离心5分钟。吸除上清液,将细胞团块重悬于1毫升的PBS中,并用血细胞计数器对细胞计数。在293培养基中稀释细胞并且在6孔板的每个孔中平板接种2毫升的5×105个细胞。将该板放置在培养箱中过夜,并在第二天用质粒转染细胞。用脂质体2000(LifeTechnologies)将质粒转染到293细胞中。使用以下方式分别在不同孔中制成所有表达融合蛋白的慢病毒。将50微升的OptiMEM(LifeTechnologies)放到两个1.5毫升的微量离心管中,并使其在组织培养罩中升温至室温10分钟。在一个试管加入下面的质粒:2微克表达Aβ-HLA-DRA融合蛋白构建体的慢病毒、2微克的VSVG、2微克的Δ8.9。所有质粒都是用使用Endo-Free Maxi试剂盒(Qiagen)的氨苄青霉素抗性的大肠杆菌培养物制备的。在第二试管中加入10毫升的脂质体2000。将两个管中的内容物加到一起,混合并留在组织培养罩中20分钟。在这段时间后,按照制造商的脂质体方案(Life Technologies)将溶液逐滴加入到293细胞的孔中,。在将含有脂质体的溶液加入到各孔之后,将平板返回培养箱中。第二天,从293细胞除去培养基,并用1毫升的DCDM更换。将细胞再孵育2天,此时含有病毒的培养基被除去,并准备好感染未成熟DC。在DCDM中10天后,收集树突细胞,并在15毫升锥形管中以400×g进行沉淀。用移液管除去上清液,并用1毫升的DCDM重悬含DC的沉淀物,并用血细胞计数器对细胞计数。通常,每个Aβ融合的慢病毒感染和实验对照(即,IL-2,Aβ肽,未处理的对照)使用5×105个细胞。24孔板中总共有10个孔被准备成pHEMA包被用于感染和对照。按照如上所述完成pHEMA包被,只是每孔中使用500微升的pHEMA包被溶液。将来自于转染有融合蛋白载体之293的含有慢病毒的培养基(600微升)加入到7个孔中。将来自于已转染有空的FUGW慢病毒载体或含Aβ融合蛋白DNA序列(Aβ1-13、Aβ7-13、Aβ16-30、Aβ24-35和Aβ31-42)的FUGW载体的293细胞的条件培养基分别加入到6个孔中。剩下的三个孔做如下处理:1)用含10微克/毫升Aβ1-42多肽的DC的分化培养基(Biomer Technology,Pleasanton CA)处理;2)用含IL-2的DC分化培养基处理;和3)仅用DC分化培养基处理。然后向每个孔加入5×105个分化的DC。在加入分化的DC后,将平板来回晃动,然后在37℃和5%CO2孵育2天。在开始感染后的第2天,从各孔移除培养基,并用1毫升DC成熟培养基(DCMM)更换。DCMM由补充有100微克/毫升的重组人TNF-α和250纳克/毫升的脂多糖(LPS)的DCDM构成。用DCMM另外孵育感染的DC3天。
T细胞的分离。使用标准的放血技术从志愿者采集全血。从每个对象采集约20毫升的全血置于紫色顶EDTA包被的放血管中(Becton-Dickinson)。将血液用PBS(Gibco目录#14040)1∶1稀释,并且在50毫升锥形管中,使24毫升的稀释血液在18毫升的Ficoll-PaquePlus(比例1∶1.4,GE Healthcare Biosciences,Upsala,SE,目录#17-1440-02)上成层。包含经稀释的血液和Ficoll-Paque Plus的试管在室温下以400×g离心30分钟。将上清液(即黄色血浆成分)吸走并弃去。小心地移出血浆成分和Ficoll层之间的界面层并转移到新试管中。此界面层包含有外周血淋巴细胞(PBL)和单核细胞。在含PBL的试管中填充25毫升1×PBS,混合然后以1500×g离心10分钟。通过抽吸除去上清液,并用10毫升PBS将沉淀物重悬。再重复两次1500×g离心10分钟和洗涤步骤。在最后清洗后,将细胞重悬于1毫升的PBS中,并用血细胞计数器计数。然后将细胞重悬于RPMI中达到20-50×106个细胞/毫升的细胞浓度。
通过PBL传代并在无菌尼龙羊毛柱(Polysciences公司,目录号21759)中孵育它们而从贴壁细胞(例如B细胞和单核细胞)纯化T细胞。简言之,通过将5毫升的RPMI培养基加入到柱的顶部使10毫升的尼龙羊毛柱变湿,并通过打开柱底部的旋塞使培养基通过该柱。还用5毫升的RPMI培养基完成第二次洗涤,然后通过在顶部加入培养基同时关闭旋塞,将5毫升的RPMI培养基留在柱中。然后用注射器柱塞密封该柱的顶部,随后将含有RPMI培养基的柱于37℃孵育1小时。在该孵育后,将该柱夹持在竖直位置并打开旋塞,以允许RPMI培养基穿过该柱。然后用另外5毫升的RPMI清洗该柱并使打开旋塞。将4毫升的细胞悬液(2-5×106个细胞/毫升)加入到柱的顶部(总共4毫升的PBL),并使溶液进入该柱,关闭旋塞。然后使1毫升的RPMI在柱的顶部分层,并使其进入尼龙羊毛柱。关闭旋塞,将柱的顶部用注射器柱塞密封,并且使该柱在37℃孵育1小时。在孵育1小时后,将柱竖直地安装在组织培养罩中,移走柱塞。打开旋塞,靠重力收集流出柱的流出物。此流出物由T细胞富集的级分组成。用10毫升的RPMI培养基再洗涤该柱一次,并且靠重力流动来收集流出物。将两种流出物合并在50毫升锥形管中,并以1500×g离心10分钟。吸出上清液,并将T细胞富集的制备物重悬于1毫升PBS中。用血细胞计数器对细胞计数。在细胞悬液中仅相对比较大的细胞作为目的T细胞进行计数。
T细胞的羧基荧光素二乙酸琥珀酰亚胺酯(CFSE)染色。在对从尼龙柱收集的T细胞悬液进行了计数之后,将该悬液以1500×g离心10分钟,并重悬于4毫升含有0.1%BSA的PBS(预先温热至37℃)中。然后将4毫升含8μM CFSE(CellTraceTM CFSE细胞增殖试剂盒,Invitrogen)的PBS/BSA加入到4毫升细胞悬液中,并将该混合物在37℃水浴中孵育8分钟。孵育后立即用10毫升含10%hyclone胎牛血清(FBS)的RPMI稀释细胞溶液。然后将细胞悬液在25℃以1500×g离心10分钟。用10毫升含有0.1%BSA的PBS(牛血清白蛋白级分V,Calbiochem目录#2930)洗涤CFSE染色的T细胞两次,在25℃以1500×g离心5分钟。在最后一次洗涤后,将T细胞重悬于1毫升的RPMI中并计数。通过流式细胞术(Accuri/BD)分析CFSE染色的重悬T细胞的等分试样来确认CFSE染色。通过流式细胞术(FL1通道)进行分析以确认CFSE染色的均匀强度。在被感染的3天前从成熟DC除去成熟分化培养基。
将成熟的呈递HLA-DRA-Aβ的树突细胞和来自诊断患有AD之对象的具有CFSE标记的T细胞共培养。然后将一百万(1×106)个CFSE染色的T细胞加入到已被表达HLA-DRA-Aβ融合蛋白之慢病毒感染的成熟树突细胞的各孔中,预先加载有Aβ1-42肽(10微克/毫升),或两个未处理的孔。通过从一边到另一边晃动、随后前后晃动24孔板来混合细胞,以使T细胞均匀地分布在孔中。将平板放回培养箱中。在第二天,每个孔中加入1微升的人IL-2(GIBCO,目录号PHC0026)。IL-2的终浓度为200纳克/毫升。通过使平板经受从一侧到另一侧的温柔晃动将IL-2分散开来,然后将细胞在37℃孵育9天。在第10天,如下面所述,通过ACCURITM流式细胞仪测定CD4阳性T细胞群。
用抗CD4免疫染色。通过对每个孔中用新的1毫升移液枪头将每孔中的培养基转移到一个单独的15毫升锥形管中,收集10天的T细胞和改造的DC的共培养物。用1毫升PBS洗涤平孔板的表面,并且将洗涤液与对应孔中先前的洗涤液合并。一旦所有孔中的细胞被收集,将该试管在450×g离心10分钟。弃去上清液,将细胞用5毫升PBS洗涤一次,然后以450×g再离心10分钟。弃去上清液,收集沉淀物。将细胞用荧光缀合的抗CD4抗体进行免疫染色(活)并通过流式细胞术测定。孵育后,将50微升的每种细胞悬液(即,1×106个细胞)转移到新的流式细胞管中。每个管中加入1微升氟Alexa647缀合的抗CD4抗体(Alexa647小鼠抗人CD4抗体,BD Biosciences,目录号557707)。将剩余的50微升NMS-封闭的T细胞用作抗体的同种型对照。将1微升的同种型对照抗体(Alexa647小鼠IgG1k同种型对照,BD Biosciences目录号557714)加到用于每个实验条件下的同种型对照的细胞中。抗CD4标记的细胞和同种型对照的细胞在4℃于黑暗中孵育1小时。孵育后,在每个试管中加入1毫升冷的含0.5%BSA的PBS(Calbiochem目录号2930),并将试管以200×g离心5分钟。弃去上清液,并且重复两次洗涤步骤。在最后一次的洗涤液中仅包含PBS而无0.5%BSA。用200微升冰冷的1×PBS重悬各管中的细胞。
通过ACCURITM流式细胞仪测量CD4+T细胞增殖。用Accuri流式细胞仪测定细胞。使用前向散射(FSC)和侧向散射(SSC)的点状图来创建围绕含T细胞的群的选通,其是在初步研究中所确定。该选通内的细胞事件绘制在直方图FL2上,其示出Alexa647荧光(红色)。由抗CD4抗体标记的细胞在直方图中具有显著更多的荧光并且选通被用于分离那个群。FL1(绿色)荧光直方图显示CD4+细胞中的CFSE标记。最强烈的标记见于(最右边)没有分裂的细胞中。进行细胞分裂之细胞的CFSE荧光逐步减少。IL-2处理条件CFSE图被用来确定每个步骤(即分裂)的荧光程度。这些标志物被转移到每个实验条件的CFSE图来确定在实验过程中已分裂5次以上的CD4+T细胞。在每种条件下的T细胞增殖谱的比较被用来确定各血液样品中Aβ-特异性CD4+T细胞的相对丰度和响应性。
参考文献:
Agrawal,A.,and Gupta S.(2011).Impact of aging on dendritic cell functions in humans.Ageing Res.Rev.10,336-345.
Albani S,et al.(2011).Induction of immune tolerance in the treatment of rheumatoid arthritis.Nat.Rev.Rheumatol.7,272-281.
Braak,H.,and Braak,E.(1997).Diagnostic criteria for neuropathologic assessment of Alzheimer′sdisease.Neurobiol.Aging 18,S85-S88.
Cao,C.,et al.(2009).Aβ-specific T cells are sufficient for beneficial effects in the APP+PS1 mouse modelfor Alzheimer′s disease.Neurobiol.Dis.34,63-70.
Choi,K.D.,et al.(2009).Generation of mature human myelomonocytic cells through expansion anddifferentiation of pluripotent stem cell-derived lin-CD34+CD43+CD45+ progenitors.J.Clin.Invest.119,2818-2829.
Chow,A.,et al.(2002).Dendritic cell maturation triggers retrograde MHC class II transport fromlysosomes to the plasma membrane Nature 418,988-994.
Codarri,L.,et al.(2012).Communication between pathogenic T cells and myeloid cells inneuroinflammatory disease.Trends Immunol.Oct 29.pii:S1471-4906(12)00174-3.doi:10.1016/j.it.2012.09.007.[Epub ahead of print]
Daigneault,M.,et al.(2010).The identification of markers of macrophage differentiation in PMA-stimulated THP-1 cells and monocyte-derived macrophages.PLoS One 5,e8668.
Delong,T.,et al.(2012).Novel autoantigens for diabetogenic CD4T cells in autoimmune diabetes.Immunol.Res.Sep 13.[Epub ahead of print].
Ethell,D.W.,et al.(2002).Metalloproteinase shedding of Fas ligand regulates 3-amyloid neurotoxicity.Curr.Biol.12,1595-1600.
Ethell,D.W.,and Buhler,L.A.(2003).Fas ligand-mediated apoptosis in degenerative disorders of thebrain.J.Clin.lmmunol.23,363-370.
Ethell,D.W.et al.(2006).Aβ-specific T cells reverse cognitive decline and synaptic loss in Alzheimer′smice.Neurobiol.Dis.23,351-361.
Fox,C.B.,et al.(2012).Adjuvanted pandemic influenza vaccine:variation of emulsion componentsaffects stability,antigen structure,and vaccine efficacy.Influenza Other Respi Viruses.Nov 5.doi:10.1111/irv.12031.
Frasca,D.,and Blomberg,B.B.(2011).Aging impairs murine B cell differentiation and function in primaryand secondary lymphoid tissues.Aging Dis.2,361-373.
Gojanovich,G.S.,and Hess P.R.(2012).Making the most of major histocompatibility complex moleculemultimers:applications in type 1diabetes.Clin.Dev.Immunol.,380289.doi:10.1155/2012/380289.Epub 2012 May 28.
Haas,W.,et al.(1993).Gamma/delta T cells.Ann.Rev.Immunol.11,637-685.
Haskins,K.,and Cooke,A.(2011).CD4T cells and their antigens in the pathogenesis of autoimmunediabetes.Curr.Opin.Immunol.23,739-745.
Hayday,A.C.(1995).γδT cell specificity and function:how much like who?In T Cell Receptors,J.I.Bell,M.J.Owen,and E.Simpson,eds.(Oxford,U.K:Oxford University Press)1995,pp.70.
Hosken,N.A.,et al.(1995).The effect of antigen dose on CD41 T helper cell phenotype development in aT cell receptor-αβ-transgenic model.J.Exp.Med.182,1579-1584.
Huang,J.,et al.(2012).T cell antigen recognition at the cell membrane Mol.Immunol.52,155-164.
Klein,L.,et al.(2010).Autophagy-mediated antigen processing in CD4(+)T cell tolerance and immunity.FEBS Lett.584,1405-1410.
LeC.,et al.(2010).Effects of human respiratory syncytial virus,metapneumovirus,parainfluenza virus 3 and influenza virus on CD4+ T cell activation by dendritic cells.PLoS One 5,e15017
McFarland,H.F.,and Martin,R.(2007).Multiple sclerosis:a complicated picture of autoimmunity.Nat.Immunol.8,913-919.
Nakken,B.,et al.(2011).B-cells and their targeting in rheumatoid arthritis-current concepts and futureperspectives.Autoimmun.Rev.11,28-34.
Orgogozo,J.M.,et al.(2003).Subacute meningoencephalitis in a subset of patients with AD afterAbeta42 immunization.Neurology 61,46-54.
Pao,W.,et al.(1996).γδT cell help of B cells is induced by repeated parasitic infection in the absence ofother T cells.Curr.Biol.6,1317-1325.
Pereira,L.F.,et al.(2011).Impaired in vivo CD4+ T cell expansion and differentiation in aged mice is notsolely due to T cell defects:decreased stimulation by aged dendritic cells.Mech.Ageing Dev.132,187-194.
Philpott,K.L.,et al.(1992).Lymphoid development in mice congenitally lacking T cell receptorαβ-expressing cells.Science 256,1448-1452.
Sallusto,F.,et al.(2012).T-cell trafficking in the central nervous system.Immunol.Rev.248,216-227.
S.Sakaguchi.(2000).Regulatory T cells:key controllers of immunologic self-tolerance.Cell 101,455-458.
Schmidt,T.,et al.(2012).CD4+ T-cell immunity after pandemic influenza vaccination cross-reacts withseasonal antigens and functionally differs from active influenza infection.Eur.J.Immunol.42,1755-1766.
Small,E.J.,et al.(2000).lmmunotherapy of hormone-refractory prostate cancer with antigen-loadeddendritic cells.J.Clin.Oncol.18,3894-3903.
Tetsutani,K.,and Ishii,K.J.(2012).Adjuvants in influenza vaccines.Vaccine 30,7658-7661.
J.A.Villadangos,P.Schnorrer,N.S.Wilson.(2005).Control of MHC class II antigen presentation indendritic cells:a balance between destructive forces.Immunol.Rev.207,191-205.
Vodyanik,M.A.,et al.(2005).Human embryonic stem cell-derived CD34+cells:efficient production inthe coculture with OP9stromal cells and analysis of lymphohematopoietic potential.Blood 105,617-626.
Vodynik,M.A.,and Sluvkin,I.I.(2007).Directed differentiation of human embryonic stem cells todendritic cells.Methods Mol.Biol.,407,275-293.
Wen,L.,Barber,D.F.,Pao,W.,Wong,F.S.,Owen,M.J.,and Hayday,A.(1998).Primary gamma delta cellclones can be defined phenotypically and functionally as Th1/Th2 cells and illustrate theassociation of CD4 with Th2 differentiation.J.Immunol.160,1965-1974.
Claims (11)
1.将人胚胎干细胞(hESC)分化为成熟的专职抗原呈递细胞(APC)的方法,其包括:
诱导所述hESC分化为共同髓系祖细胞;
诱导所述髓系祖细胞分化为未成熟树突细胞;和
诱导所述未成熟树突细胞分化为成熟树突细胞,其中所述成熟树突细胞是成熟的专职抗原呈递细胞。
2.根据权利要求1所述的将hESC分化为成熟的专职APC的方法,其中所述成熟树突细胞在其HLA II类分子的环境下主要呈递单一肽抗原的片段。
3.扩增肽抗原限定性CD4+T细胞群的方法,其包括以下步骤:
从对象的全血分离CD4+T细胞群;
用权利要求1所述的成熟树突细胞培养所述CD4+T细胞足够长的时间以允许CD4+T细胞经历至少3个细胞分裂周期,其中由所述成熟树突细胞呈递的单一肽抗原特异性地与至少一种CD4+T细胞的T细胞受体相互作用。
4.根据权利要求2所述的扩增肽抗原限定性CD4+T细胞群的方法,其中所述肽抗原包含疫苗组合物之肽的抗原性氨基酸序列,并且其中所述CD4+T细胞群扩增至少三个周期表明对象对所述疫苗组合物的肽产生了免疫应答。
5.根据权利要求3所述的扩增肽抗原限定性CD4+T细胞群的方法,其中所述疫苗组合物是流感病毒疫苗组合物。
6.根据权利要求2所述的扩增肽抗原限定性CD4+T细胞群的方法,其中所述肽抗原包含所述对象已对其产生免疫应答的肽的抗原性氨基酸序列,并且其中所述免疫应答是疾病或病症发生或发展的指示。
7.根据权利要求5所述的扩增肽抗原限定性CD4+T细胞群的方法,其中所述疾病或病症是阿尔茨海默病。
8.根据权利要求5所述的扩增肽抗原限定性CD4+T细胞群的方法,其中,所述肽是淀粉样蛋白-β(1-42)或其片段。
9.根据权利要求1所述的将hESC分化为成熟的专职APC的方法,其中所述成熟树突细胞包含编码融合蛋白的重组核酸,所述融合蛋白包含肽抗原的氨基酸序列、肽接头和与图9中所示氨基酸序列有至少90%同一性的氨基酸序列。
10.根据权利要求8所述的将hESC分化为成熟的专职APC的方法,其中所述肽抗原包含淀粉样蛋白-β(1-42)或其片段的氨基酸序列。
11.根据权利要求9所述的将hESC分化为成熟的专职APC的方法,其中所述淀粉样蛋白-β(1-42)的片段选自Aβ1-13、Aβ7-13、Aβ16-30、Aβ24-35、Aβ31-42、Aβ7-23,或其任何组合。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201261644265P | 2012-05-08 | 2012-05-08 | |
US61/644,265 | 2012-05-08 | ||
PCT/US2013/040172 WO2013169922A1 (en) | 2012-05-08 | 2013-05-08 | Standardized ex vivo platforms for the antigen-specific expansion of cd4+ t cell populations |
Publications (2)
Publication Number | Publication Date |
---|---|
CN104428411A true CN104428411A (zh) | 2015-03-18 |
CN104428411B CN104428411B (zh) | 2018-11-23 |
Family
ID=49551254
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201380033983.2A Expired - Fee Related CN104428411B (zh) | 2012-05-08 | 2013-05-08 | 用于cd4+t细胞群之抗原特异性扩增的标准化离体平台 |
Country Status (8)
Country | Link |
---|---|
US (2) | US9943578B2 (zh) |
EP (1) | EP2847322B1 (zh) |
JP (1) | JP6445427B2 (zh) |
CN (1) | CN104428411B (zh) |
AU (1) | AU2013259598B2 (zh) |
CA (1) | CA2872656A1 (zh) |
HK (2) | HK1207395A1 (zh) |
WO (1) | WO2013169922A1 (zh) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2791322A1 (en) | 2011-12-12 | 2014-10-22 | Cell Medica Limited | Process of expanding t cells |
GB201121308D0 (en) | 2011-12-12 | 2012-01-25 | Cell Medica Ltd | Process |
US20150010519A1 (en) | 2012-02-09 | 2015-01-08 | Baylor College Of Medicine | Pepmixes to generate multiviral ctls with broad specificity |
WO2017049291A1 (en) | 2015-09-18 | 2017-03-23 | Baylor College Of Medicine | Immunogenic antigen identification from a pathogen and correlation to clinical efficacy |
EP3419656A4 (en) * | 2016-02-22 | 2019-10-30 | Oceanside Biotechnology | NEOANTIGEN COMPOSITIONS AND THEIR METHODS OF USE IN IMMUNO-ONCOTHERAPY |
Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20020034513A1 (en) * | 1996-01-31 | 2002-03-21 | Sunol Molecular Corporation | MHC complexes and uses thereof |
US20020137108A1 (en) * | 2000-11-08 | 2002-09-26 | Michael Mullan | CD45 isoform alteration in CD4+ T cells as a potential diagnostic marker in alzheimer's disease |
CN1468109A (zh) * | 2000-10-18 | 2004-01-14 | Ӣ����������ҽҩ�о���չ�ɷݹ�˾ | 疫苗组合物 |
US20050003431A1 (en) * | 1996-08-16 | 2005-01-06 | Wucherpfennig Kai W. | Monovalent, multivalent, and multimeric MHC binding domain fusion proteins and conjugates, and uses therefor |
WO2009015841A1 (en) * | 2007-07-27 | 2009-02-05 | Immatics Biotechnologies Gmbh | Composition of tumour-associated peptides and related anti-cancer vaccine |
CN102016007A (zh) * | 2008-03-27 | 2011-04-13 | 杰龙公司 | 使灵长类多能干细胞分化成为造血谱系细胞 |
US20110086035A1 (en) * | 2008-03-11 | 2011-04-14 | Cell Medica Limited | Immunotherapeutic methods and molecules |
CN102091327A (zh) * | 2010-12-27 | 2011-06-15 | 蔡建辉 | 一种负载自体肿瘤相关全抗原的树突状细胞疫苗的制备方法 |
CN102428101A (zh) * | 2009-03-18 | 2012-04-25 | Ac免疫有限公司 | 用于治疗用途的方法 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006077152A2 (en) * | 2005-01-21 | 2006-07-27 | Utku Nalan | Hla fusion molecules and uses thereof |
US7910565B2 (en) * | 2006-09-01 | 2011-03-22 | Wisconsin Alumni Research Foundation | Prostate cancer vaccine |
US20120015040A1 (en) | 2008-03-03 | 2012-01-19 | University Of Toledo | Dendritic Cell Precursor Populations, Dendritic Cell Populations Derived Therefrom and Uses Thereof |
-
2013
- 2013-05-08 EP EP13788471.4A patent/EP2847322B1/en active Active
- 2013-05-08 US US14/398,496 patent/US9943578B2/en active Active - Reinstated
- 2013-05-08 CA CA2872656A patent/CA2872656A1/en not_active Abandoned
- 2013-05-08 WO PCT/US2013/040172 patent/WO2013169922A1/en active Application Filing
- 2013-05-08 CN CN201380033983.2A patent/CN104428411B/zh not_active Expired - Fee Related
- 2013-05-08 AU AU2013259598A patent/AU2013259598B2/en not_active Ceased
- 2013-05-08 JP JP2015511666A patent/JP6445427B2/ja not_active Expired - Fee Related
-
2015
- 2015-08-21 HK HK15108110.1A patent/HK1207395A1/zh not_active IP Right Cessation
- 2015-08-26 HK HK15108298.5A patent/HK1207667A1/zh unknown
-
2018
- 2018-02-13 US US15/895,734 patent/US20180202104A1/en not_active Abandoned
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20020034513A1 (en) * | 1996-01-31 | 2002-03-21 | Sunol Molecular Corporation | MHC complexes and uses thereof |
US20050003431A1 (en) * | 1996-08-16 | 2005-01-06 | Wucherpfennig Kai W. | Monovalent, multivalent, and multimeric MHC binding domain fusion proteins and conjugates, and uses therefor |
CN1468109A (zh) * | 2000-10-18 | 2004-01-14 | Ӣ����������ҽҩ�о���չ�ɷݹ�˾ | 疫苗组合物 |
US20020137108A1 (en) * | 2000-11-08 | 2002-09-26 | Michael Mullan | CD45 isoform alteration in CD4+ T cells as a potential diagnostic marker in alzheimer's disease |
WO2009015841A1 (en) * | 2007-07-27 | 2009-02-05 | Immatics Biotechnologies Gmbh | Composition of tumour-associated peptides and related anti-cancer vaccine |
US20110086035A1 (en) * | 2008-03-11 | 2011-04-14 | Cell Medica Limited | Immunotherapeutic methods and molecules |
CN102016007A (zh) * | 2008-03-27 | 2011-04-13 | 杰龙公司 | 使灵长类多能干细胞分化成为造血谱系细胞 |
CN102428101A (zh) * | 2009-03-18 | 2012-04-25 | Ac免疫有限公司 | 用于治疗用途的方法 |
CN102091327A (zh) * | 2010-12-27 | 2011-06-15 | 蔡建辉 | 一种负载自体肿瘤相关全抗原的树突状细胞疫苗的制备方法 |
Non-Patent Citations (1)
Title |
---|
ROMAN SPORRI1等: "Inflammatory mediators are insufficient for full dendritic cell activation and promote expansion of CD4+ T cell populations lacking helper function", 《NATURE IMMUNOLOGY》 * |
Also Published As
Publication number | Publication date |
---|---|
CN104428411B (zh) | 2018-11-23 |
US9943578B2 (en) | 2018-04-17 |
US20150337262A1 (en) | 2015-11-26 |
JP6445427B2 (ja) | 2018-12-26 |
WO2013169922A1 (en) | 2013-11-14 |
US20180202104A1 (en) | 2018-07-19 |
CA2872656A1 (en) | 2013-11-14 |
AU2013259598B2 (en) | 2018-10-04 |
HK1207395A1 (zh) | 2016-01-29 |
EP2847322A1 (en) | 2015-03-18 |
EP2847322B1 (en) | 2019-06-26 |
HK1207667A1 (zh) | 2016-02-05 |
JP2015516164A (ja) | 2015-06-11 |
EP2847322A4 (en) | 2016-05-18 |
AU2013259598A1 (en) | 2014-11-20 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20180202104A1 (en) | Standardized ex vivo platforms for the antigen-specific expansion of cd4+ t cell populations | |
JP6092357B2 (ja) | 生体組織から単離できる多能性幹細胞 | |
Tay et al. | CD40L expression allows CD8+ T cells to promote their own expansion and differentiation through dendritic cells | |
US10034900B2 (en) | Method of producing myeloid blood cells | |
TW202344686A (zh) | 從幹細胞產生t細胞之方法及使用該t細胞之免疫療法 | |
WO2014153346A1 (en) | Engineering a heterogeneous tissue pluripotent stem cells | |
US20190099540A1 (en) | A closed system for labelling and selecting live cells | |
CN113383070A (zh) | 用于分化天然杀伤细胞的培养基和方法 | |
JP2021176323A (ja) | ヒト褐色脂肪由来幹細胞およびその使用 | |
CN111954684A (zh) | Car-t细胞与自身免疫性疾病 | |
JPWO2020045368A1 (ja) | 不死化単球細胞および誘導細胞を用いた抗感染症薬,ワクチンなどの評価方法 | |
US20170247657A1 (en) | Method for generation of a cell composition of mesencephalic dopaminergic progenitor cells | |
WO2012137538A1 (ja) | 細胞傷害性t細胞誘導用組成物 | |
JP2009514509A (ja) | 肺幹細胞および関連の方法およびキット | |
US20170304430A1 (en) | Virus vector, cell, and construct | |
US20210332324A1 (en) | Method for producing a corneal epithelial cell population | |
JP5888852B2 (ja) | 免疫不全動物を用いた細胞の製法 | |
US20200362014A1 (en) | Applications of soluble protein baff in b cell in-vitro culture and proliferation | |
JP2019170393A (ja) | iPS細胞の作製方法 | |
CN117761308A (zh) | 筛选目标抗原特异性t细胞的标志物组合及其用途 | |
CN102183640A (zh) | 筛选和鉴定乙肝病毒特异性细胞毒性t淋巴细胞表位方法 | |
Hua et al. | Isolation and Culture of Bone Marrow Mesenchymal Stem Cells (MSCs) of Tibetan miniature pig, and Lentivirus-mediated EGFP Gene Transfer into MSCs | |
EP3427052A1 (en) | A closed system for labelling and selecting live cells |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 1207395 Country of ref document: HK |
|
GR01 | Patent grant | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20181123 Termination date: 20210508 |