WO2012018930A1 - Methods of isolating and expanding human t regulatory cells and uses thereof for cellular therapy - Google Patents
Methods of isolating and expanding human t regulatory cells and uses thereof for cellular therapy Download PDFInfo
- Publication number
- WO2012018930A1 WO2012018930A1 PCT/US2011/046447 US2011046447W WO2012018930A1 WO 2012018930 A1 WO2012018930 A1 WO 2012018930A1 US 2011046447 W US2011046447 W US 2011046447W WO 2012018930 A1 WO2012018930 A1 WO 2012018930A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- bright
- foxp3
- expanded
- ntreg
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 80
- 210000003289 regulatory T cell Anatomy 0.000 title abstract description 54
- 238000002659 cell therapy Methods 0.000 title abstract description 17
- 210000004027 cell Anatomy 0.000 claims abstract description 332
- 210000002501 natural regulatory T cell Anatomy 0.000 claims abstract description 173
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 claims abstract description 157
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 claims abstract description 156
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 claims abstract description 156
- 102000004127 Cytokines Human genes 0.000 claims abstract description 82
- 108090000695 Cytokines Proteins 0.000 claims abstract description 82
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 60
- 210000000056 organ Anatomy 0.000 claims abstract description 25
- 238000002054 transplantation Methods 0.000 claims abstract description 23
- 230000001506 immunosuppresive effect Effects 0.000 claims abstract description 11
- 230000006698 induction Effects 0.000 claims abstract description 11
- 208000024908 graft versus host disease Diseases 0.000 claims abstract description 8
- 206010062016 Immunosuppression Diseases 0.000 claims abstract description 7
- 239000007787 solid Substances 0.000 claims abstract description 7
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 claims description 224
- ZAHRKKWIAAJSAO-UHFFFAOYSA-N rapamycin Natural products COCC(O)C(=C/C(C)C(=O)CC(OC(=O)C1CCCCN1C(=O)C(=O)C2(O)OC(CC(OC)C(=CC=CC=CC(C)CC(C)C(=O)C)C)CCC2C)C(C)CC3CCC(O)C(C3)OC)C ZAHRKKWIAAJSAO-UHFFFAOYSA-N 0.000 claims description 45
- 229960002930 sirolimus Drugs 0.000 claims description 45
- QFJCIRLUMZQUOT-HPLJOQBZSA-N sirolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 QFJCIRLUMZQUOT-HPLJOQBZSA-N 0.000 claims description 45
- 239000000203 mixture Substances 0.000 claims description 29
- 210000004369 blood Anatomy 0.000 claims description 25
- 239000008280 blood Substances 0.000 claims description 25
- 238000003556 assay Methods 0.000 claims description 20
- 101000946843 Homo sapiens T-cell surface glycoprotein CD8 alpha chain Proteins 0.000 claims description 17
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 claims description 17
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 17
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 claims description 16
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 claims description 16
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 claims description 16
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 claims description 16
- 239000003795 chemical substances by application Substances 0.000 claims description 16
- 238000002955 isolation Methods 0.000 claims description 16
- 201000010099 disease Diseases 0.000 claims description 15
- -1 TCRγδ Proteins 0.000 claims description 11
- 230000028993 immune response Effects 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 9
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 claims description 7
- 210000005259 peripheral blood Anatomy 0.000 claims description 7
- 239000011886 peripheral blood Substances 0.000 claims description 7
- 102000049320 CD36 Human genes 0.000 claims description 5
- 108010045374 CD36 Antigens Proteins 0.000 claims description 5
- 102100025470 Carcinoembryonic antigen-related cell adhesion molecule 8 Human genes 0.000 claims description 5
- 102100035716 Glycophorin-A Human genes 0.000 claims description 5
- 108091005250 Glycophorins Proteins 0.000 claims description 5
- 101000914320 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 8 Proteins 0.000 claims description 5
- 101000917858 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-A Proteins 0.000 claims description 5
- 101000917839 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor III-B Proteins 0.000 claims description 5
- 206010020751 Hypersensitivity Diseases 0.000 claims description 5
- 102100029185 Low affinity immunoglobulin gamma Fc region receptor III-B Human genes 0.000 claims description 5
- 230000007815 allergy Effects 0.000 claims description 5
- 230000005784 autoimmunity Effects 0.000 claims description 5
- 230000001105 regulatory effect Effects 0.000 claims description 5
- 206010052779 Transplant rejections Diseases 0.000 claims description 4
- 206010012601 diabetes mellitus Diseases 0.000 claims description 4
- 239000003937 drug carrier Substances 0.000 claims description 4
- 206010025327 Lymphopenia Diseases 0.000 claims description 3
- 229940112129 campath Drugs 0.000 claims description 3
- 230000003247 decreasing effect Effects 0.000 claims description 3
- 231100001023 lymphopenia Toxicity 0.000 claims description 3
- 206010061218 Inflammation Diseases 0.000 claims description 2
- 208000022559 Inflammatory bowel disease Diseases 0.000 claims description 2
- 230000004054 inflammatory process Effects 0.000 claims description 2
- 230000003612 virological effect Effects 0.000 claims description 2
- 239000012636 effector Substances 0.000 abstract description 18
- 230000000638 stimulation Effects 0.000 abstract description 17
- 238000000338 in vitro Methods 0.000 abstract description 15
- PGHMRUGBZOYCAA-UHFFFAOYSA-N ionomycin Natural products O1C(CC(O)C(C)C(O)C(C)C=CCC(C)CC(C)C(O)=CC(=O)C(C)CC(C)CC(CCC(O)=O)C)CCC1(C)C1OC(C)(C(C)O)CC1 PGHMRUGBZOYCAA-UHFFFAOYSA-N 0.000 abstract description 14
- PGHMRUGBZOYCAA-ADZNBVRBSA-N ionomycin Chemical compound O1[C@H](C[C@H](O)[C@H](C)[C@H](O)[C@H](C)/C=C/C[C@@H](C)C[C@@H](C)C(/O)=C/C(=O)[C@@H](C)C[C@@H](C)C[C@@H](CCC(O)=O)C)CC[C@@]1(C)[C@@H]1O[C@](C)([C@@H](C)O)CC1 PGHMRUGBZOYCAA-ADZNBVRBSA-N 0.000 abstract description 14
- 230000000770 proinflammatory effect Effects 0.000 abstract description 10
- 238000004519 manufacturing process Methods 0.000 abstract description 9
- ZOOGRGPOEVQQDX-UUOKFMHZSA-N 3',5'-cyclic GMP Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=C(NC2=O)N)=C2N=C1 ZOOGRGPOEVQQDX-UUOKFMHZSA-N 0.000 abstract description 6
- 208000023275 Autoimmune disease Diseases 0.000 abstract description 6
- 238000006243 chemical reaction Methods 0.000 abstract description 5
- 101000716102 Homo sapiens T-cell surface glycoprotein CD4 Proteins 0.000 description 207
- 238000004458 analytical method Methods 0.000 description 42
- 230000014509 gene expression Effects 0.000 description 27
- 108010002350 Interleukin-2 Proteins 0.000 description 26
- 102000000588 Interleukin-2 Human genes 0.000 description 26
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 21
- 108050005493 CD3 protein, epsilon/gamma/delta subunit Proteins 0.000 description 21
- 238000002474 experimental method Methods 0.000 description 20
- 230000001629 suppression Effects 0.000 description 17
- 101001043809 Homo sapiens Interleukin-7 receptor subunit alpha Proteins 0.000 description 16
- 102100021593 Interleukin-7 receptor subunit alpha Human genes 0.000 description 16
- 210000001519 tissue Anatomy 0.000 description 16
- 238000000684 flow cytometry Methods 0.000 description 15
- PHEDXBVPIONUQT-RGYGYFBISA-N phorbol 13-acetate 12-myristate Chemical compound C([C@]1(O)C(=O)C(C)=C[C@H]1[C@@]1(O)[C@H](C)[C@H]2OC(=O)CCCCCCCCCCCCC)C(CO)=C[C@H]1[C@H]1[C@]2(OC(C)=O)C1(C)C PHEDXBVPIONUQT-RGYGYFBISA-N 0.000 description 15
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 13
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 13
- 238000011109 contamination Methods 0.000 description 13
- 102000003814 Interleukin-10 Human genes 0.000 description 12
- 108090000174 Interleukin-10 Proteins 0.000 description 12
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 12
- 239000011324 bead Substances 0.000 description 12
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 description 11
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 description 11
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 11
- 239000000975 dye Substances 0.000 description 11
- 239000001963 growth medium Substances 0.000 description 11
- 230000003834 intracellular effect Effects 0.000 description 11
- 101000998146 Homo sapiens Interleukin-17A Proteins 0.000 description 10
- 102100033461 Interleukin-17A Human genes 0.000 description 10
- 238000005516 engineering process Methods 0.000 description 10
- 230000003389 potentiating effect Effects 0.000 description 10
- 230000035755 proliferation Effects 0.000 description 10
- 210000000130 stem cell Anatomy 0.000 description 10
- 230000006870 function Effects 0.000 description 9
- 101001023379 Homo sapiens Lysosome-associated membrane glycoprotein 1 Proteins 0.000 description 8
- 102100035133 Lysosome-associated membrane glycoprotein 1 Human genes 0.000 description 8
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 8
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 8
- 230000003248 secreting effect Effects 0.000 description 8
- 230000028327 secretion Effects 0.000 description 8
- 230000001225 therapeutic effect Effects 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- 230000000735 allogeneic effect Effects 0.000 description 6
- 239000012228 culture supernatant Substances 0.000 description 6
- 239000012091 fetal bovine serum Substances 0.000 description 6
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 6
- 238000001802 infusion Methods 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 210000001616 monocyte Anatomy 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 230000035899 viability Effects 0.000 description 6
- 102100027207 CD27 antigen Human genes 0.000 description 5
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 description 5
- 102000014150 Interferons Human genes 0.000 description 5
- 108010050904 Interferons Proteins 0.000 description 5
- 108050003558 Interleukin-17 Proteins 0.000 description 5
- 102000013691 Interleukin-17 Human genes 0.000 description 5
- 239000004698 Polyethylene Substances 0.000 description 5
- 210000003719 b-lymphocyte Anatomy 0.000 description 5
- 230000001413 cellular effect Effects 0.000 description 5
- 238000012512 characterization method Methods 0.000 description 5
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 5
- 238000010212 intracellular staining Methods 0.000 description 5
- 210000000822 natural killer cell Anatomy 0.000 description 5
- 239000002953 phosphate buffered saline Substances 0.000 description 5
- 101100005713 Homo sapiens CD4 gene Proteins 0.000 description 4
- 241000124008 Mammalia Species 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 4
- 210000004700 fetal blood Anatomy 0.000 description 4
- 230000001900 immune effect Effects 0.000 description 4
- 210000002602 induced regulatory T cell Anatomy 0.000 description 4
- 230000001939 inductive effect Effects 0.000 description 4
- 229940079322 interferon Drugs 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 4
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 4
- 239000012103 Alexa Fluor 488 Substances 0.000 description 3
- 206010002199 Anaphylactic shock Diseases 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 102100040247 Tumor necrosis factor Human genes 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 208000026935 allergic disease Diseases 0.000 description 3
- 208000003455 anaphylaxis Diseases 0.000 description 3
- 208000006673 asthma Diseases 0.000 description 3
- 230000001363 autoimmune Effects 0.000 description 3
- 230000032823 cell division Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 210000003162 effector t lymphocyte Anatomy 0.000 description 3
- 230000036512 infertility Effects 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 239000011325 microbead Substances 0.000 description 3
- 230000002093 peripheral effect Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 230000000284 resting effect Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 239000012117 Alexa Fluor 700 Substances 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 101000611183 Homo sapiens Tumor necrosis factor Proteins 0.000 description 2
- 101800001155 Latency-associated peptide Proteins 0.000 description 2
- 102400000401 Latency-associated peptide Human genes 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 238000002716 delivery method Methods 0.000 description 2
- 210000004443 dendritic cell Anatomy 0.000 description 2
- 238000000432 density-gradient centrifugation Methods 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
- 229940079593 drug Drugs 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000013632 homeostatic process Effects 0.000 description 2
- 230000002519 immonomodulatory effect Effects 0.000 description 2
- 229960003444 immunosuppressant agent Drugs 0.000 description 2
- 239000003018 immunosuppressive agent Substances 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 239000002122 magnetic nanoparticle Substances 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 230000002062 proliferating effect Effects 0.000 description 2
- 230000000069 prophylactic effect Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 230000010473 stable expression Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 210000001541 thymus gland Anatomy 0.000 description 2
- 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 1
- 108010082808 4-1BB Ligand Proteins 0.000 description 1
- VDABVNMGKGUPEY-UHFFFAOYSA-N 6-carboxyfluorescein succinimidyl ester Chemical compound C=1C(O)=CC=C2C=1OC1=CC(O)=CC=C1C2(C1=C2)OC(=O)C1=CC=C2C(=O)ON1C(=O)CCC1=O VDABVNMGKGUPEY-UHFFFAOYSA-N 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 239000012114 Alexa Fluor 647 Substances 0.000 description 1
- 206010003827 Autoimmune hepatitis Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000015943 Coeliac disease Diseases 0.000 description 1
- 206010009900 Colitis ulcerative Diseases 0.000 description 1
- 208000011231 Crohn disease Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 102100021260 Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1 Human genes 0.000 description 1
- 208000009329 Graft vs Host Disease Diseases 0.000 description 1
- 208000035186 Hemolytic Autoimmune Anemia Diseases 0.000 description 1
- 101000894906 Homo sapiens Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 1 Proteins 0.000 description 1
- 101001076422 Homo sapiens Interleukin-1 receptor type 2 Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000019223 Interleukin-1 receptor Human genes 0.000 description 1
- 108050006617 Interleukin-1 receptor Proteins 0.000 description 1
- 102100026017 Interleukin-1 receptor type 2 Human genes 0.000 description 1
- 108010065805 Interleukin-12 Proteins 0.000 description 1
- 108090000176 Interleukin-13 Proteins 0.000 description 1
- 108090000172 Interleukin-15 Proteins 0.000 description 1
- 102000049772 Interleukin-16 Human genes 0.000 description 1
- 108010038453 Interleukin-2 Receptors Proteins 0.000 description 1
- 102000010789 Interleukin-2 Receptors Human genes 0.000 description 1
- 102000013264 Interleukin-23 Human genes 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 108010002616 Interleukin-5 Proteins 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 108090001007 Interleukin-8 Proteins 0.000 description 1
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 1
- 229930191564 Monensin Natural products 0.000 description 1
- GAOZTHIDHYLHMS-UHFFFAOYSA-N Monensin A Natural products O1C(CC)(C2C(CC(O2)C2C(CC(C)C(O)(CO)O2)C)C)CCC1C(O1)(C)CCC21CC(O)C(C)C(C(C)C(OC)C(C)C(O)=O)O2 GAOZTHIDHYLHMS-UHFFFAOYSA-N 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- 108010042215 OX40 Ligand Proteins 0.000 description 1
- 206010035148 Plague Diseases 0.000 description 1
- 201000004681 Psoriasis Diseases 0.000 description 1
- 230000017274 T cell anergy Effects 0.000 description 1
- 230000033540 T cell apoptotic process Effects 0.000 description 1
- 102000013530 TOR Serine-Threonine Kinases Human genes 0.000 description 1
- 108010065917 TOR Serine-Threonine Kinases Proteins 0.000 description 1
- 102100026890 Tumor necrosis factor ligand superfamily member 4 Human genes 0.000 description 1
- 102100032101 Tumor necrosis factor ligand superfamily member 9 Human genes 0.000 description 1
- 201000006704 Ulcerative Colitis Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 229960004308 acetylcysteine Drugs 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 201000004984 autoimmune cardiomyopathy Diseases 0.000 description 1
- 201000000448 autoimmune hemolytic anemia Diseases 0.000 description 1
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 210000001772 blood platelet Anatomy 0.000 description 1
- KQNZDYYTLMIZCT-KQPMLPITSA-N brefeldin A Chemical compound O[C@@H]1\C=C\C(=O)O[C@@H](C)CCC\C=C\[C@@H]2C[C@H](O)C[C@H]21 KQNZDYYTLMIZCT-KQPMLPITSA-N 0.000 description 1
- JUMGSHROWPPKFX-UHFFFAOYSA-N brefeldin-A Natural products CC1CCCC=CC2(C)CC(O)CC2(C)C(O)C=CC(=O)O1 JUMGSHROWPPKFX-UHFFFAOYSA-N 0.000 description 1
- 210000004970 cd4 cell Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000025084 cell cycle arrest Effects 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000011260 co-administration Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000004940 costimulation Effects 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 239000012997 ficoll-paque Substances 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 230000003832 immune regulation Effects 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 210000004153 islets of langerhan Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 206010025135 lupus erythematosus Diseases 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000007799 mixed lymphocyte reaction assay Methods 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229960005358 monensin Drugs 0.000 description 1
- GAOZTHIDHYLHMS-KEOBGNEYSA-N monensin A Chemical compound C([C@@](O1)(C)[C@H]2CC[C@@](O2)(CC)[C@H]2[C@H](C[C@@H](O2)[C@@H]2[C@H](C[C@@H](C)[C@](O)(CO)O2)C)C)C[C@@]21C[C@H](O)[C@@H](C)[C@@H]([C@@H](C)[C@@H](OC)[C@H](C)C(O)=O)O2 GAOZTHIDHYLHMS-KEOBGNEYSA-N 0.000 description 1
- 206010028417 myasthenia gravis Diseases 0.000 description 1
- 210000000581 natural killer T-cell Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- VYNDHICBIRRPFP-UHFFFAOYSA-N pacific blue Chemical compound FC1=C(O)C(F)=C2OC(=O)C(C(=O)O)=CC2=C1 VYNDHICBIRRPFP-UHFFFAOYSA-N 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 229940056360 penicillin g Drugs 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 230000008823 permeabilization Effects 0.000 description 1
- 238000009520 phase I clinical trial Methods 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000000985 reactive dye Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000012121 regulation of immune response Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 238000012882 sequential analysis Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 102000055501 telomere Human genes 0.000 description 1
- 108091035539 telomere Proteins 0.000 description 1
- 210000003411 telomere Anatomy 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000002992 thymic effect Effects 0.000 description 1
- 230000003614 tolerogenic effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 238000003211 trypan blue cell staining Methods 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
- C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
- A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/46434—Antigens related to induction of tolerance to non-self
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/04—Immunosuppressors, e.g. cyclosporin, tacrolimus
-
- 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
- C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere
Definitions
- the invention relates generally to the fields of immunology, molecular biology, cell biology, and medicines. More particularly, the invention relates to methods of isolating and expanding (enriching) human T regulatory cells and uses thereof. BACKGROUND
- Tregs T regulatory cells
- the CD4 derived major Treg populations include natural T regulatory cells (nTreg) which originate in the thymus, induced Tregs (iTreg) derived from naive CD4 + T cells in the periphery, Tr1 cells which secrete predominantly IL-10 and TH3 cells which secrete predominantly TGF .
- Other cell populations such as NKT cells and CD8 T suppressor cells can also mediate immune regulation.
- Natural T regulatory cells play a key role in inducing and maintaining immunological tolerance and immune homeostasis (Sakaguchi, S., 2004 Annu Rev Immunol 22, 531-62; Sakaguchi, S.
- T cells This specialized subpopulation of T cells is critical for maintaining unresponsiveness to self- antigens.
- These cells are of considerable interest from the viewpoint of cellular therapy in the therapeutic management of autoimmune disorders such as diabetes type 1, for short term immunosuppression in graft versus host disease, and for induction of tolerance in solid organ transplantation to prevent graft rejection.
- autoimmune disorders such as diabetes type 1
- Tregs are under way, the procedures for deriving sufficient quantities of cells which have desirable characteristics of nTregs have yet to be optimized.
- the expanded CD4 + T cell population should ideally have the following four characteristic of nTregs: 1) a stable phenotype of CD25 ++bright Foxp3 + expression; 2) functional ability to suppress immune reactive T cells by a mechanism that does not involve secretion of IL-10 or TGF- ; 3) no evidence of conversion to effector T cells or TH17 cells and 4) no secretion of pro-inflammatory cytokines upon in-vitro stimulation.
- nTregs To accomplish the goal of expanding nTregs ex vivo for therapeutic purposes, it is critical to begin with the appropriate starting cell population and to use culture conditions that selectively favor the expansion of Tregs with properties that best characterize nTregs.
- Cell- based therapy using purified nTregs is under consideration for several conditions, but procedures employed to date have resulted in cell populations that are contaminated with cytokine secreting effector cells.
- the expanded nTregs had the following properties: (1) stable expression of CD4 + CD25 ++brightFoxp3+ with N97–98% purity; (2) potent functional ability to suppress CD4 + CD25 negative T cells without secretion of IL-10 or TGF- ⁇ ; (3) no conversion into effector T cells or TH17 cells and (4) no production of pro-inflammatory cytokine upon in vitro stimulation with PMA/Ionomycin.
- Two critical determinants in the quality and quantity of expanded nTregs were first, the successful isolation of purified nTregs from peripheral blood, and second, the use of rapamycin in the expansion protocol.
- the isolated and expanded Tregs can be used in any therapeutic strategy to restore self-tolerance in autoimmune disorders (e.g., type 1 diabetes, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, celiac disease, Crohn’s disease, lupus erythematosus, myasthenia gravis, psoriasis, rheumatoid arthritis, ulcerative colitis), for immunosuppression in host vs. graft disease, and for induction of tolerance in solid organ, tissue or cellular transplantation to prevent graft rejection.
- autoimmune disorders e.g., type 1 diabetes, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, celiac disease, Crohn’s disease, lupus erythematosus, myasthenia gravis, psoriasis, rheumatoid arthritis, ulcerative colitis
- nTreg natural T regulatory cells
- obtaining blood from an individual obtaining at least one Buffy coat from the blood; isolating CD4 + CD25 ++bright Foxp3 + nTreg cells from the at least one Buffy coat; and expanding the isolated CD4 + CD25 ++bright Foxp3 + nTreg cells by culturing the CD4 + CD25 ++bright Foxp3 + nTreg cells in the presence of rapamycin.
- the blood may be peripheral blood and the individual is typically a healthy donor.
- the isolated and expanded CD4 + CD25 ++bright Foxp3 + nTreg cells are capable of suppressing CD4 + CD25 negative T cells as measured by at least one T cell assay and do not express cytokines as measured by at least one cytokine assay.
- Isolating CD4 + CD25 ++bright Foxp3 + nTreg cells from the Buffy coat can include enrichment of CD4 + T cells by negative selection using agents which specifically bind cell markers including: CD8, CD16, CD19, CD36, CD56, CD66b, TCR , glycophorin A and P9, and isolation of CD25 ++bright cells by positive selection from the enriched CD4 + T cells using at least one agent which specifically binds to CD25.
- the step of positive selection from the enriched CD4 + T cells using at least one agent which specifically binds to CD25 is performed in an immunomagnetic cell separator, and the at least one agent which specifically binds to CD25 can be a monoclonal antibody to CD25.
- the method includes: providing CD4 + CD25 ++bright Foxp3 + nTreg cells isolated and expanded by a method including obtaining blood from an individual, obtaining at least one Buffy coat from the blood, isolating CD4 + CD25 ++bright Foxp3 + nTreg cells from the at least one Buffy coat, and expanding the isolated CD4 + CD25 ++bright Foxp3 + nTreg cells by culturing the CD4 + CD25 ++bright Foxp3 + nTreg cells in the presence of rapamycin; and administering the cells to the subject in a therapeutically effective amount for decreasing the immune response in the subject.
- Diseases or conditions associated with immune responses include: autoimmunity, allergies, diabetes, inflammation, graft versus host reactions, organ transplantation, inflammatory bowel disease and viral diseases.
- Administering the cells to the subject results in at least one of: alleviation of type 1 diabetes, immunosuppression in host vs. graft disease, and induction of tolerance in solid organ transplantation to prevent graft rejection.
- compositions including a pharmaceutically acceptable carrier and a therapeutically effective amount of CD4 + CD25 ++bright Foxp3 + nTreg cells isolated and expanded by a method including obtaining blood from an individual, obtaining at least one Buffy coat from the blood, isolating CD4 + CD25 ++bright Foxp3 + nTreg cells from the at least one Buffy coat, and expanding the isolated CD4 + CD25 ++bright Foxp3 + nTreg cells by culturing the CD4 + CD25 ++bright Foxp3 + nTreg cells in the presence of rapamycin.
- the isolated CD4 + CD25 ++bright Foxp3 + nTreg cells are capable of suppressing CD4 + CD25 negative T cells as measured by at least one T cell assay.
- the isolated CD4 + CD25 ++bright Foxp3+ nTreg cells typically do not express cytokines as measured by at least one cytokine assay.
- tissue or organ transplantation to a subject (e.g., human).
- the method includes: (a) obtaining the tissue or organ to be transplanted from a donor; (b) transplanting said tissue or organ to the subject; and (c) delivering a composition including a pharmaceutically acceptable carrier and a therapeutically effective amount of CD4 + CD25 ++bright Foxp3 + nTreg cells isolated and expanded by a method including obtaining blood from an individual, obtaining at least one Buffy coat from the blood, isolating CD4 + CD25 ++bright Foxp3 + nTreg cells from the at least one Buffy coat, and expanding the isolated CD4 + CD25 ++bright Foxp3 + nTreg cells by culturing the CD4 + CD25 ++bright Foxp3 + nTreg cells in the presence of rapamycin, to the subject prior to and/or subsequent to transplantation of the tissue or organ to the subject.
- compositions may be delivered to the subject after the transplantation at the time of maximum lymphopenia (e.g., 6 to 11 days after transplantation) in the subject with an immunosuppressive induction using a T cell depletion protocol such as Thymoglobuliun or Campath 1H treatment of the subject.
- T cell depletion protocol such as Thymoglobuliun or Campath 1H treatment of the subject.
- nTreg “nTregs” and“nTreg cells” is meant“natural T regulatory cells”, a subset of T regulatory cells which plays a key role in inducing and maintaining immunological tolerance and immune homeostasis.
- the methods described herein provide for the isolation of highly purified nTregs that maintain the nTreg phenotype throughout the expansion protocol, avoiding contamination with pro-inflammatory cytokine markers that plague other Treg expansion protocols.
- the phrases“expanded nTreg cells,”“expanded human nTreg cells,” and“expanded CD4 + CD25 ++ Foxp 3+ cells” mean natural T regulatory cells isolated and expanded from CD4 + T cells with a bright expression of CD25 (CD25 bright or CD25 ++ ) which remains stable through the expansion protocol.
- the expanded cells contain none or minimal IFN IL2, IL17, TNF and CD107a expressing (positive) cells.
- treatment and“treating” are intended to refer to inhibiting, eliminating, ameliorating, diminishing and/or reducing cellular damage and/or symptoms associated with a disease or condition, e.g. allograft rejection.
- Treating includes, but is not limited to, inducing tolerance to a cell, tissue or organ transplant, restoring self-tolerance in autoimmunity, restoring self-tolerance in conditions such as asthma, allergy and anaphylactic shock, etc.
- Compositions as described herein may also or alternatively be a prophylactic, i.e., used to partially or completely prevent a disease or condition or symptom thereof.
- patient and “subject” are used interchangeably in the present specification, they include animals.
- the patient is a mammal, and in a preferred embodiment, the patient is human.
- an effective amount” or“a therapeutically effective” amount is intended to refer to the total amount of the expanded Treg cells or a composition including expanded Treg cells, or of the method that is sufficient to show a meaningful patient benefit. This term is also intended to refer to an amount that returns to normal, either partially or completely, physiological or biochemical parameters associated with immunological intolerance.
- isolated or biologically pure refer to material (e.g., nucleic acids) which is substantially or essentially free from components which normally accompany it as found in its native state.
- the term“buffy coat” means the fraction of an anticoagulated blood sample after density gradient centrifugation that contains most of the white blood cells and platelets.
- FIG. 1 shows the purity of freshly isolated CD4 + CD25 ++bright nTregs showing minimal contamination with other cell populations.
- CD4 + CD25 ++bright cells isolated by the two step technique of negative selection of CD4 + T cells followed by positive selection of CD4 + CD25 ++bright cells using the Robosep® as per protocol were stained and analyzed by flow cytometry.
- Upper panel shows sequential analysis of frequencies of CD3, CD4, CD25 and Foxp3 expressing cells.
- Lower panel shows frequencies of individual CD19, CD14, CD56 and CD8 expressing cells on (A) open gate (B) cells gated on singlets. (Representative of one of the two experiments)
- FIG. 2 shows a gating strategy and phenotype analysis of ex vivo expanded CD4 + CD25 ++bright nTregs.
- CD4 + CD25 ++bright cells were expanded ex vivo as per protocol and were harvested on day 19.
- B Cells gated on singlets and analyzed in a similar manner. Figure is representative of 4 experiments.
- FIG. 3 shows ex vivo expanded nTregs exhibit potent suppressive function.
- Autologous CD4 + CD25 negative responder cells labeled with CFSE dye were analyzed for proliferation and cell division on day 4 following culture under the following conditions: (A) in medium, without stimulation, (B) after stimulation with anti-CD3/anti-CD28 coated micro beads (positive control), (C) with anti-CD3/anti-CD28 stimulation and addition of expanded CD4 + CD25 ++bright cells to responder cells in 1:1 and 1:10 ratios and (D) with anti-CD3/anti-CD28 stimulation and addition of expanded CD4 + CD25 dim cells to responder cells in 1:1 and 1:10 ratios.
- Figure is representative of 2 experiments showing potent suppression by the CD4 + CD25 ++bright cells at 1:1 ratio. The figure is representative of 3 experiments showing potent suppression by the CD4 + CD25 ++bright cells at 1:1 ratio.
- E Summary data from 3 donors showing the effect of
- FIG. 4 shows ex vivo expanded nTregs exhibit minimal cytokine expression following in-vitro stimulation.
- Robosep® isolated CD4 + CD25 ++bright , CD4 + CD25 dim and CD4 + CD25 negative cells were cultured as per the expansion protocol in X-vivo complete medium containing CD3/CD28 expander beads and rhIL2 (300 IU ml -1 ) and rapamycin (100 ng ml -1 ). Each cell population was harvested on day 19, stimulated with PMA/Ionomycin for 5 hrs and stained for cytokines IL17, IL2, IFN ⁇ , TNF ⁇ and for CD107a.
- Each cell population was analyzed by FLOWJO using (A) an open gate (B) cells gated on singlets and analyzed in a similar manner. Plots show frequencies of cells positive for each cytokine and for CD107a in CD4 + CD25 ++bright , CD4 + CD25 dim and CD4 + CD25 negative cells, demonstrating the very low frequencies for all measures in CD4 + CD25 ++bright cells compared to the other two populations.
- FIG. 5 shows Robosep® isolated nTregs exhibit low frequencies of cytokine expressing cells.
- CD4 + CD25 ++bright nTregs and CD4 + CD25 negative cells were isolated as described by Robosep® and stimulated with PMA/Ionomycin for 5 hrs and stained for IL17, IL2, IFN and TNF ⁇ .
- A Analysis of CD4 + CD25 ++bright cell fraction shown in open gate that includes doublets, triplets and other cells, After exclusion of dead cells with ViViD, gated CD4 cells were analyzed for IL17, IL2, IFN ⁇ and TNF ⁇ .
- FIG. 6 shows rapamycin curtails expansion of all populations of freshly isolated CD4 + cells. Isolated CD4 + CD25 ++bright , CD4 + CD25 dim and CD4 + CD25 negative cell fractions were expanded in X-Vivo complete medium containing CD3/CD28 expander beads + rhIL2 + rapamycin. Fold expansion data are shown at day 19 time point.
- FIG. 7 shows a phenotype analysis of expanded nTregs shows minimal contamination with other populations.
- Expanded, cryopreserved and thawed CD4 + CD25 ++bright cells were stained and analyzed on (A) open gate (B) on singlets for ViViD (Live/Dead cells), for frequencies of live cells expressing CD25, Foxp3, CD14, CD19, CD56 and CD8 (C) Mean and SD values of frequencies of CD14, CD19, CD56 and CD8 expressing cell populations in expanded CD4 + CD25 ++bright cell fractions from 4 different buffy coats.
- FIG. 8 shows that the addition of rapamycin curtails expansion of cytokine producing cells.
- Freshly isolated CD4 + CD25 ++bright nTregs were expanded in X-vivo complete medium containing CD3/CD28 expander beads and IL2 with and without the addition of rapamycin for 19 days.
- Cells were stimulated with PMA/Ionomycin for 5 hrs and stained for cytokines IL17, IL2, IFN ⁇ and TNF ⁇ to determine frequencies of cells expressing individual cytokines.
- Upper panels show the experiment with no rapamycin added in the culture and lower panel shows cells with addition of rapamycin to the culture, demonstrating the marked reduction in frequencies of cytokine expressing cells in the latter.
- Described herein are methods of isolating and expanding human nTregs ex vivo in cGMP conditions for use in cellular therapies (e.g., for treating autoimmune disorders, immunosuppression in graft vs. host disease, induction of tolerance in solid organ transplantation, etc.) and properties and uses of these expanded Treg cells. Described in the Examples section below is the successful expansion of a unique population of human natural Tregs in vitro.
- rapamycin has been shown to induce apoptosis of CD4 + CD8 + thymocytes and result in the expansion of peripheral regulatory CD4 + CD25 + T cells, and is described, e.g., in Tian et al., 2004, Transplantation 77: 183-89. These references are herein incorporated by reference.
- the methods result in the production of CD4 + CD25 ++ Foxp 3+ cells that may be used in cellular therapies.
- a typical method of isolating and expanding nTreg cells includes obtaining Treg cells from a donor and purifying them. The purified Treg cells are then cultured in the presence of rapamycin or other Treg promoting (Treg“permissive”) immunosuppressant agent.
- the method includes: obtaining blood from an individual; obtaining at least one Buffy coat from the blood; isolating CD4 + CD25 ++bright Foxp3 + nTreg cells from the at least one Buffy coat; and expanding the isolated CD4 + CD25 ++bright Foxp3 + nTreg cells by culturing the CD4 + CD25 ++bright Foxp3 + nTreg cells in the presence of rapamycin.
- the isolated and expanded CD4 + CD25 ++bright Foxp3 + nTreg cells are capable of suppressing CD4 + CD25 negative T cells as measured by at least one T cell assay, and do not express cytokines as measured by at least one cytokine assay.
- Isolating CD4 + CD25 ++bright Foxp3 + nTreg cells from the Buffy coat typically includes enrichment of CD4+ T cells by negative selection using agents which specifically bind cell markers comprising: CD8, CD16, CD19, CD36, CD56, CD66b, TCR , glycophorin A and P9, and isolation of CD25 ++bright cells by positive selection from the enriched CD4 + T cells using at least one agent which specifically binds to CD25.
- the step of positive selection from the enriched CD4 + T cells using at least one agent which specifically binds to CD25 is typically performed in an immunomagnetic cell separator, and the at least one agent which specifically binds to CD25 is typically a monoclonal antibody to CD25.
- Any suitable culture medium can be used for culturing the freshly isolated nTreg cells.
- An example of a suitable culture medium for freshly isolated nTreg cells is one that includes serum-free X-Vivo with CD3/CD28 T-cell expander Dyna beads, IL2 and rapamycin.
- any suitable culture medium can be used for culturing the nTreg cells during the expansion phase.
- a suitable culture medium for freshly isolated nTreg cells includes serum-free X-Vivo with CD3/CD28 T-cell expander Dyna beads, IL2 and rapamycin.
- AB serum is added (final concentration 10%).
- rapamycin or other suitable immunosuppressant is added. Rapamycin has a potent immunosuppressive activity and serves to prevent IL-2-mediated signaling and cell cycle arrest at the G1-S boundary, thereby leading to T-cell anergy and/or apoptosis and induction of operational tolerance.
- a phenotype analysis of expanded Treg cells is performed prior to using the Treg cells in a cellular therapy. Any suitable analysis of expanded Treg cells can be used. As described in the experiments described herein, a phenotype analysis of Treg cells can be performed by surface staining of cells with CD3, CD4, CD25, CD127, CD27, CD45RO and by using a‘dump’ channel for cells stained with ViViD dye, CD14, CD19, CD56, followed by intracellular staining for FoxP3 in a single tube.
- Flow data can be collected on any suitable FACS cytometer (e.g., FACS LSRII cytometer) and analyzed with appropriate software (e.g., FlowJo software, Mac version 8.6.8, Tree Star).
- FACS cytometer e.g., FACS LSRII cytometer
- appropriate software e.g., FlowJo software, Mac version 8.6.8, Tree Star
- intracellular and culture supernatant cytokine assays can be performed on freshly isolated and expanded nTregs (i.e., CD4 + CD25 ++ Foxp 3+ cells) as described in the Examples below for determining if the cells are producing pro-inflammatory cytokines (e.g., upon in vitro stimulation) prior to administering the nTregs to a recipient (e.g., patient).
- the CD4 + CD25 ++ Foxp 3+ cells can also be characterized functionally for their ability to suppress autologous CD4+CD25negative T cells.
- any negative selection cocktail of antibodies containing enrichment of agents which specifically bind cell markers comprising: CD8, CD16, CD19, CD36, CD56, CD66b, TCR ⁇ , glycophorin A and P9, could be used to isolate CD4 + T cells by negative selection, followed by any suitable method for isolation of CD25 ++bright cells by positive selection from the enriched CD4 + T cells using at least one agent which specifically binds to CD25.
- the step of positive selection from the enriched CD4 + T cells using at least one agent which specifically binds to CD25 is typically performed in an immunomagnetic cell separator, and the at least one agent which specifically binds to CD25 is typically a monoclonal antibody to CD25.
- nTreg cells can be obtained from any suitable source.
- nTreg cells were obtained from buffy coats obtained from healthy adult donors (e.g., peripheral blood from a healthy donor).
- nTreg cells can be obtained from other sources. Examples of other nTreg sources include any other suitable hematopoietic cell source such as umbilical cord blood.
- a subject to be treated receives cells obtained and expanded from that subject (i.e., autologous transplant).
- a subject may receive an infusion of nTreg cells obtained and expanded from another individual (e.g., allogeneic transplant).
- An allogeneic transplant of cells may find particular use, for example, in situations in which there might be a defect of nTregs from the subject to be treated because the subject has a specific disease condition associated with a defect in nTregs and would therefore require an infusion of nTregs obtained from a donor that does not have such a defect.
- compositions for modulating (e.g., decreasing) an immune response in a subject are described herein.
- Such compositions typically include a pharmaceutically acceptable carrier and a therapeutically effective amount of CD4 + CD25 ++bright Foxp3 + nTreg cells isolated and expanded by the methods described herein.
- the isolated and expanded CD4 + CD25 ++bright Foxp3 + nTreg cells are capable of suppressing CD4 + CD25 negative T cells as measured by at least one T cell assay and typically do not express cytokines as measured by at least one cytokine assay.
- Methods of tissue or organ transplantation to a subject are described herein. Such methods typically include: obtaining the tissue or organ to be transplanted from a donor; transplanting said tissue or organ to the subject; and delivering a composition of expanded nTregs to the subject prior to and/or subsequent to transplantation of the tissue or organ to the subject.
- the cells are delivered to the transplant recipient after the transplant at the time of maximum recipient lymphopenia (e.g. 6 to 11 days after transplantation) with an immunosuppressive induction using a T cell depletion protocol, such as Thymoglobuliun or Campath 1H treatment of the recipient.
- KITS For treatment in an autoimmune condition that may not require a transplant (e.g., type 1 diabetes at the onset, before the beta cells are destroyed), it may be preferable to just treat the patient as soon as possible at onset or even before onset if you identify patients at risk to prevent onset of disease (it is generally easier to restore self tolerance at the very first moments of detection of an immune imbalance or trend, rather that reversing an overt autoimmune process at clinical onset).
- kits for preventing allograft rejection, and/or restoring self- tolerance in autoimmunity e.g., in diseases such as type 1 diabetes
- a typical kit includes a therapeutically effective amount of a composition including expanded CD4 + CD25 ++bright Foxp3 + nTreg cells with instructions for administering the cells to the subject.
- the cells can be packaged by any suitable means for transporting and storing cells; such methods are well known in the art.
- the instructions generally include one or more of: a description of the cells; dosage schedule and administration for prevention of allograft rejection and/or restoring self-tolerance in autoimmunity, and restoring self-tolerance in conditions such as asthma, allergy and anaphylactic shock; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references.
- the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
- a kit as described herein also includes packaging.
- the kit includes a sterile container which contains a therapeutic or prophylactic composition; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding cells or medicaments. ADMINISTRATION OF COMPOSITIONS AND CELLS
- Ex vivo delivery of human Treg cells isolated and expanded according to the methods described herein i.e., CD4 + CD25 ++bright Foxp3 + nTreg cells
- compositions including the expanded human Treg cells is provided for within the invention.
- Ex vivo cellular therapy may be used to transplant expanded Treg cells isolated from a healthy host donor into a recipient in need thereof (e.g., a human patient). Any suitable delivery method may be used for delivering expanded Treg cells isolated from a host donor to a recipient in need thereof.
- Several suitable modes of delivery of cells into the recipient are encompassed, including intravenous injection, intraperitoneal injection, or in situ injection into target tissue.
- nTregs nTregs
- MSCs mesenchymal stem cells
- MDSC Marrow Derived Suppressor Cells
- Dendritic Cells tolerogenic Dendritic Cells as other immunomodulatory cell sources that could enhance nTreg engraftment and function.
- MSCs can be added to the nTreg infusion as MSCs have been already successfully used to provide temporary immunomodulation/tolerance in clinical protocols for treatment of GVHD or for enhancement of organ transplant survival and function.
- the therapeutic methods described herein in general include administration of a therapeutically effective amount of the compositions or cells described herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human.
- a subject e.g., animal, human
- Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects "at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider.
- the methods and compositions herein may be also used in the treatment of any disorders in which a lack of self-tolerance may be implicated.
- Suitable patients include human or other animals in need of treatment with expanded Tregs.
- patients suffering from or at risk for an autoimmune disease such as Type I diabetes, or patients receiving foreign graft transplants (i.e., allograft patients and xenograft patients), are examples of recipients in need of Treg expansion in accordance with the invention.
- compositions and cells described herein are preferably administered to a mammal (e.g., human) in an effective amount, that is, an amount capable of producing a desirable result in a treated mammal (e.g., preventing allograft rejection).
- a mammal e.g., human
- an effective amount that is, an amount capable of producing a desirable result in a treated mammal (e.g., preventing allograft rejection).
- a therapeutically effective amount can be determined according to standard methods.
- Toxicity and therapeutic efficacy of the compositions utilized in methods of the invention can be determined by standard pharmaceutical procedures.
- dosage for any one subject depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, time and route of administration, general health, and other drugs being administered concurrently.
- Example 1 Isolation and expansion of human natural T regulatory cells for cellular therapy.
- Natural T regulatory cells nTregs
- Cell based therapy using purified nTregs is under consideration for several conditions, but procedures employed to date have resulted in cell populations that are contaminated with cytokine secreting effector cells. Described herein is the establishment of a method for isolation and ex vivo expansion of human nTregs from healthy blood donors for cellular therapy aimed at preventing allograft rejection in organ transplants.
- the Robosep® instrument was used for initial nTreg isolation and rapamycin was included in the expansion phase of cell cultures.
- the resulting cell population exhibited a stable CD4 + CD25 ++bright Foxp3 + phenotype, had potent functional ability to suppress CD4 + CD25 negative T cells without evidence of conversion to effector T cells including TH17 cells, and manifested little to no production of pro- inflammatory cytokines upon in-vitro stimulation.
- Boolean gating analysis of cytokine- expressing cells by flow cytometry for 32 possible profile end points revealed that 96% of expanded nTregs did not express any cytokine. From a single buffy coat, approximately 80 million pure nTregs were harvested after expansion under cGMP conditions; these cell numbers are adequate for infusion of approximately one million cells Kg -1 for cell therapy in clinical trials.
- Human buffy coats Human Buffy coats containing approximately 3-4X10 10 cells were obtained from healthy adult donors from Community Blood Center of South Florida, Miami, FL, USA. Informed consent was obtained in accordance with standard policies and procedures. Samples were processed within 24 hours of collection and were required to have a lymphocyte viability of >90% in order to be processed.
- nTregs Natural T regulatory cell
- the buffy coat was diluted 1:2 with Ca 2+ Mg 2+ free phosphate buffered saline (PBS) (Stem Cell Technologies, Vancouver, BC, Canada) containing 2% fetal bovine serum (FBS) (Hyclone, South Logan, Utah, USA) and dispensed in 20ml volume each in 50ml conical tubes (Corning Life Sciences, Lowell, MA, USA).
- PBS Ca 2+ Mg 2+ free phosphate buffered saline
- FBS fetal bovine serum
- RosetteSep human CD4 + T Cell Enrichment Cocktail (Stem Cell Technologies, Vancouver, BC, Canada) which is a mixture of mAbs to CD8, CD16, CD19, CD36, CD56, CD66b, TCR , Glycophorin A and P9 was added to each tube at a concentration of 50 l ml -1 and tubes were incubated at room temperature for 20 minutes. Thereafter the cell suspension was diluted 1:1 with PBS, and the CD4 + enriched cells were harvested by Ficoll Paque Plus (GE Healthcare, Pittsburgh, PA , USA) density gradient centrifugation at 1200Xg for 20 minutes at 23 0 C.
- Ficoll Paque Plus GE Healthcare, Pittsburgh, PA , USA
- Isolated CD4 + cells were washed twice with PBS containing 10% FBS and re-suspended at a concentration of 5x10 7 cells ml -1 and dispersed in 4 ml aliquots in sterile 15ml round-bottom polyethylene tubes as required for processing on the Robosep® instrument.
- the Robosep® instrument was primed as per the manufacturer’s protocol by loading EasySep human CD25 Positive Selection Cocktail and EasySep Magnetic Nanoparticles (both from Stem Cell Technologies, Vancouver, BC, Canada) using the volume and concentration specified for the selection of CD4 + CD25 ++bright cells.
- the instrument was programmed for automatic separation of CD4 + CD25 ++bright cells followed by further separation of the remaining cells into CD4 + CD25 dim and CD4 + CD25 negative cells.
- the cells were cultured for a total of 19 ⁇ 1 days.
- cells were exposed to the Dyna cell magnetic particle separator (Invitrogen, Carlsbad, CA, USA) for 10 minutes to remove CD3/CD28 T-Cell expander Dyna beads.
- Cells were washed twice and were analyzed for cell count, viability, purity, potency of suppression and cryopreserved in 10% DMSO using an automated temperature controlled freezer (T.S. Scientific 17 Kryo 10 Series, Perkasie, PA, USA) in liquid nitrogen.
- T.S. Scientific 17 Kryo 10 Series, Perkasie, PA, USA automated temperature controlled freezer
- Monoclonal antibody reagents Flow cytometry panels for 10-12 color polychromatic flow cytometry analyses were utilized to analyze T cell phenotype and functions. Antibodies to IFN- -PECy7, IL2-PE, CD3-Amcyan, CD4-PercpCy5-5, TNF- - Alexafluor700, CD107a-PECy5, CD25-APCCy7, Foxp3-Alexafluor488 or Alexafluor647 (clone 259D/C7), CD127-PE, CD27-APC, CD45RO-FITC, CD14-Alexafluor700 or pacific blue, CD19-PECy7, CD8-APCCy7 were obtained from BD Pharmingen, San Jose, CA.
- IL17-Alexafluor488, CD56- Pacific blue were obtained from e-Bioscience, San Diego, CA, USA and CD56-Alexafluor488 was obtained from Biolegend, San Diego, CA, USA.
- CD25-PE was obtained from Stem cell technologies, Vancouver, BC, Canada.
- CD19-Pacific blue and a violet fluorescent reactive dye used as a viability marker to exclude dead cells from analysis were obtained from Invitrogen, Carlsbad, CA, USA.
- Flow cytometry analysis Polychromatic flow cytometry for surface and intracellular staining of freshly isolated and expanded nTregs was performed on a BD LSR II Flow Cytometer System (BD Biosciences, San Jose, CA, USA) as described (Perfetto, S., et al., 2004, Nat Rev Immunol 4, 648-55; Lamoreaux, L., et al., 2006, Nat Protoc 1, 1507-16; Darrah, P., et al., 2007, Nat Med 13, 843-50).
- Phenotypic analysis Phenotypic analysis of freshly isolated nTregs was performed by surface staining of 0.5 X 10 6 cells with CD3, CD4, CD25, CD8, CD14, CD19, CD56 followed by intracellular staining for Foxp3 according to manufacturing instructions. Surface staining for CD25 was done using CD25-PE from Stem cell Technologies, Vancouver, BC, Canada.
- Phenotype analysis of expanded nTregs was performed by surface staining of 1X10 6 cells with CD3, CD4, CD25, CD127, CD27, CD45RO and by using a‘dump’ channel for cells stained with ViViD dye, CD14, CD19, CD56, followed by intracellular staining for FoxP3 according to manufacturer’s instructions in a single tube. Cells were also stained in another tube with ViViD dye and for CD4, CD8, CD25, CD14, CD19, CD56 and FoxP3 to trace contamination with monocytes, B cells, NK cells and cytotoxic T cells in the live cells. Flow data was collected on a FACS LSRII cytometer and analyzed with FlowJo software (Mac version 8.6.8, Tree Star).
- Intracellular Cytokine analysis was performed in freshly isolated and expanded nTregs. nTreg populations at a concentration of 1X10 6 cells were cultured with phorbol myristate acetate (PMA) (Sigma-Aldrich, St. Louis, MO) 50ngml -1 , Ionomycin 1 ⁇ g ml -1 , Monensin 0.7 ⁇ l ml -1 (golgistop , BD Biosciences, San Jose, CA, USA) and Brefeldin-A 10 ⁇ g ml -1 (Sigma-Aldrich, St.
- PMA phorbol myristate acetate
- Cytokine assay in culture supernatants Freshly isolated nTregs and expanded nTregs were stimulated with PMA 50 ng ml -1 and Ionomycin 1 ⁇ g ml -1 for 5 hours at 37 o C.
- Cells were centrifuged at 850g for 10 minutes and supernatants were collected and were analyzed in a multiplex format using Quansys multiplex Elisa kit (Quansys Biosciences, West Logan, Utah, USA ) for cytokines interferon (IFN)- , IL-2, IL-4, IL5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-1a, IL-16, IL-17, IL-23, TNF- ⁇ , LTa, TGF , TGF ⁇ -RII, TGF ⁇ -RIII.
- IFN interferon
- In vitro suppression assay Frozen autologous CD4 + CD25 negative cells were thawed, washed twice and cell count and viability was assessed by trypan blue dye exclusion. Viable 2 x10 6 CD4 + CD25 negative (responder) cells were labeled with Carboxyfluorescein succinimidyl ester (CFSE, Invitrogen, Carlsbad, CA, USA) at a concentration of 4 ⁇ M/5 x10 6 cells for 10 minutes at 37 o C. Labeling was terminated by addition of an equal volume of 100% FBS.
- CFSE Carboxyfluorescein succinimidyl ester
- Isolation and characterization of freshly isolated n Tregulatory cells Isolation of nTregs was made from buffy coats of healthy donors in two steps. In the first step CD4 + cells were enriched by negative selection with a cocktail of nine monoclonal antibodies. From eight different buffy coat samples, recovery of CD4 + cells ranged from 135-240 X 10 6 cells.
- CD4 + CD25 ++bright cells were isolated from the CD4 + cells using positive selection with the Robosep® instrument.
- the yield of CD4 + CD25 ++bright cells ranged from 0.8 - 1.4 X 10 6 cells, which is approximately 0.5 - 0.71% of the starting population of CD4 + cells.
- the CD4 + CD25 dim and CD4 + CD25 negative cell fractions were also collected. In the flow cytometry open gate data for CD4 + CD25 ++bright cell fraction (Fig.
- CD4 + cells 99.8% were CD25 ++bright with 97.4% cells expressing Foxp3 (84.5% bright Foxp3 + and 12.9% dim Foxp3 + ) and had minimal contamination with other cellular subsets.
- the Robosep® isolated CD4 + CD25 ++bright cell fraction was designated as nTregs.
- the Robosep®-isolated nTregs were characterized functionally for ability to suppress autologous CD4 + CD25 negative T cells and for their capacity to produce cytokines upon in vitro stimulation.
- the nTregs mediated modest suppression of proliferation of autologous CD4 + CD25 negative T cells at 1:1 ratio as assessed by CFSE dye dilution with a decrease in CFSE low cells from 91.7% to 70.6%.
- the cytokine expression profile of the Robosep® isolated CD4 + CD25 ++bright and CD25 negative cell populations was examined following in vitro stimulation with PMA (phorbol myristate acetate) plus Ionomycin for 5 hours (Fig. 5).
- Fig.5A Very low frequencies of cytokine positive cells were identified in the total cell population, consisting of IL-17 (0.27%), IL-2 (3.9%), IFN ⁇ (0.79%) and TNF ⁇ (2.9%). A majority of the cytokine expressing cells were CD4 + T cells, showing expression of IL-17 (0.16%), IL-2 (2.7%), IFN ⁇ (0.59%), and TNF ⁇ (2.63%). Analysis performed on gated singlets (Fig.
- CD4 + CD25 ++bright and CD4 + CD25 negative cells did not alter the cytokine production. Based on these results, it is contended that the methodology described herein can lead to purification of a CD4 + CD25 ++bright cell fraction which is strongly Foxp3 + and exhibits preferred attributes of nTregs and constitutes an appropriate source of cells for ex vivo expansion of nTregs.
- Rapamycin is known to selectively block expansion of CD4 + CD25 negative T effector cells, while allowing the growth of CD4 + CD25 ++bright Tregs with maintenance of high Foxp3 protein expression and suppressor function (Powell et al., 1999 J. Immunol. 162, 2775; Kahan and Camardo, 2001, Transplantation 72, 1181; Battaglia et al., 2006, J. Immunol. 177, 8338). Cells cultured without rapamycin exhibited tremendous expansion (Fig 6B) and showed marked expansion in all 3 cell populations when rapamycin was omitted from the cultures. Isolated CD4 + CD25 ++bright CD4 + CD25 dim and CD4 + CD25 negative cells expanded up to an average of 1200-fold, 64000-fold and 66490-fold respectively without the addition of rapamycin.
- FIG. 7A demonstrates extremely low frequencies of contaminating monocytes (CD14, 0.057%), B cells (CD19, 0.16%), NK cells (CD56, 0.09%) and cytotoxic T cells (CD8, 0.099%). Similar results were obtained when cells were gated on singlets (Fig. 7B). Means and SD of frequencies of non-Treg contamination observed in expanded CD4 + CD25 ++bright cells from four different buffy coats consisted of monocytes (CD14 +, 0.16 ⁇ 0.09%), B cells, (CD19 + , 0.19 ⁇ 0.07%), NK cells (CD56 + , 0.19 ⁇ 0.08%) and CD8 T cells (0.09 ⁇ 0.03%) (Fig. 7C).
- nTregs exhibited potent suppressor activity in the classical suppression assay (Brusko et al., 2007 Immunol. Invest. 36, 607).
- Autologous CD4 + CD25 negative responder cells were labeled with CFSE dye, stimulated with CD3/CD28 dynal beads and expanded nTregs were mixed with responder cells in 1:1 and 1:10 ratio for 4 days. Proliferation and cell division were analyzed on day 4 (Fig. 3).
- Intracellular cytokine expression in stimulated expanded nTreg cells Assays for intracellular cytokines were performed on frozen and thawed expanded cells as described in the method section. Expanded CD4 + CD25 ++bright , CD4 + CD25 dim and CD4 + CD25 negative cells were stimulated with PMA plus Ionomycin for 5 hours, stained for surface markers and intracellular cytokines were analyzed by multicolor flow cytometry (Perfetto et al., 2004 Nat Rev. Immunol. 4, 648; Lamoreaux et al., Nat Protoc. 1, 1507; Darrah et al., 2007, Nat Med. 13, 843).
- Fig. 4A shows that expanded CD4 + CD25 ++bright nTregs do not convert to TH17 cells (0.13% IL-17 expression) and have an extremely reduced frequency of cytokine expressing cells for IFN ⁇ (0. 51%), TNF ⁇ (1.01%), IL- 2 (1.53%) and a low frequency of cells with the degranulation marker CD107a (0.06%).
- CD4 + CD25 dim and CD4 + CD25 negative cells expressed high frequencies of cytokine positive cells.
- Fig. 4B The intracellular cytokine analysis using singlet populations in CD4 + CD25 bright cells also revealed minimal cytokine expressing cells (Fig. 4B). Data for the percent cytokine expressing cells in four different experiments is presented (Fig.4C) and shows the minimal cytokine expression in CD4 + CD25 ++bright cells in comparison to CD4 + CD25 dim and CD4 + CD25 negative cells. Boolean gating analysis for all the cytokine combinations was determined and 32 possible profile end points were scored and frequencies of more than 0.1% were highlighted (Table 1). Only one triple combination clone of IL-2 + IFN ⁇ + TNF ⁇ + cells with frequency of 0.16% was observed. The same clone was observed in three additional experiments.
- cytokine profile of expanded CD4 + CD25 ++bright cells cultured without rapamycin manifests an outgrowth of cytokine expressing cells despite the fact that they were present at very low frequencies in freshly isolated cells. Addition of rapamycin inhibited the growth and expansion of cytokine- secreting cells in this population during the 19 day culture period.
- Analysis of culture supernatants of three different samples of freshly isolated nTregs and nTregs expanded according to the expansion protocol results in very low or undetectable levels of IL-10, TGF ⁇ , or pro- inflammatory cytokines in both fresh and expanded nTreg cell populations (Table 3).
- CD25 represents the chain of the IL-2 receptor that is essential for the generation and maintenance of nTregs and high expression of CD25 is commonly used in protocols for isolating peripheral Tregs.
- CD25 is also upregulated upon cellular activation, thus recently activated effector CD4 + T cells may be confused with nTregs and iTregs.
- CD4 + CD25 ++bright nTregs Despite there are differences between CD4 + CD25 ++bright nTregs and activated T cells with respect to the characteristics of CD25 expression.
- Human and mouse CD4 + cells with potent regulatory properties express high and sustained levels of CD25, whereas recently activated T cells express transient and low levels of CD25 (Kuniyasu et al., 2000, Int. Immunol. 12, 1145; Baecher-Allan et al., 2001, J. Immunol. 167, 1245).
- a stable and high expression of CD25 is an essential characteristic of nTregs.
- the expanded nTreg population derived from the CD4 + CD25 ++bright cell fraction maintained a stable CD25 bright expression.
- markers such as latency-associated peptide (LAP) and IL-1 receptor type I & II (CD121 a/CD121b) have also been used for Treg characterization. These markers are not expressed on resting or expanded Foxp3 + Tregs, but are rapidly induced and expressed on Foxp3 + Tregs for a short time period after TCR-mediated activation (Tran et al., 2009, Blood 113, 5125). Thus these markers can only isolate TCR activated Foxp3 + Tregs but not resting or expanded Foxp3 + Tregs.
- LAP latency-associated peptide
- CD121 a/CD121b IL-1 receptor type I & II
- a feature that is increasingly used for isolating Tregs from blood is the absence of CD127, the IL-7R ⁇ , which is abundantly expressed on na ⁇ ve cells. It is contended that CD127 negativity should not be used to select the initial starting population for nTreg expansion for several reasons. First, by doing so, it may also eliminate the thymic derived resting precursors of nTregs which may express CD127. Recent data of Treg expansion using umbilical cord blood which is enriched in na ⁇ ve cells supports this contention.
- CD127 negativity may be more useful for characterizing functional expanded nTregs and less so for initial selection of the population to be expanded.
- CD127 negativity as a biomarker cannot discriminate between Tregs and T effector cells.
- CD127 Upon cellular activation CD127 is downregulated in CD4 + cells including CD4 + CD25 ++bright nTregs.
- CD127 low/- expression when CD127 low/- expression is used in combination with CD25 ++bright expression for isolating Tregs, it can concentrate a heterogeneous subpopulation of cells consisting of nTregs, iTregs and activated CD4 + CD25 ++ non Tregs which can be transiently positive for Foxp3.
- the CD4 + CD25 ++ CD127 low/- population may have a greater potential for differentiating into cytokine secreting effector cells.
- Treg function is by cell-to-cell interaction and not via secretion of cytokines IL-10 and TGF- ⁇ which are rarely found in the supernatants of in vitro nTreg assays and that the use of anti-IL-10 or anti-TGF- ⁇ antibodies fails to abrogate suppression.
- Another distinct subset of regulatory T cells (Tr-1) suppresses immune responses via cell-to-cell interactions and/or the production of IL-10 and TGF- ⁇ for a variety of antigens.
- IL-10 is also secreted by other cells like Th-2 cells, macrophages, monocytes and dendritic cells. Thus it is important to demonstrate the suppressor function of nTregs without evidence of cytokine secretion.
- Rapamycin was used throughout the culture for ex vivo selective expansion of stable nTregs.
- CD28 costimulation, IL-2 and rapamycin were required to consistently expand nTregs that had suppressor activity in vitro, in the absence of contaminating cytokine secreting effector cells.
- Cells expanded in rapamycin have been shown to prevent xenogeneic GVHD.
- Rapamycin selectively blocks expansion of CD4 + CD25 negative T effector cells, whereas it allows CD4 + CD25 ++bright Treg growth. Inhibition of the mTOR pathway in presence of IL-2 allows Tregs to be constantly activated through the STAT-5 pathways and promotes their preferential expansion and Foxp3 expression.
- nTregs to suppress proliferation of responder T cells is mediated through a cell-cell contact-dependent mechanism and they do not secrete either IL-10 or TGF ⁇ , whereas stimulation of other regulatory cells results in secretion of IL10 and TGF .
- analysis of culture supernatant of expanded nTregs did not show detectable levels of TGF ⁇ and had extremely low levels of IL10.
- the procedure has the potential for further optimization by incorporating the recently described cell-based artificial antigen presenting cells (aAPCs) preloaded with anti CD3/CD28 mAbs to achieve higher level of Treg expansion (Godfrey et al., 2005Blood 105, 750).
- aAPCs cell-based artificial antigen presenting cells
- Modification of aAPCs to co-express OX40L or 4-1BBL was shown to achieve more than 1250-fold expansion of umbilical cord blood Tregs (Godfrey et al., 2005Blood 105, 750).
- Table 1 - Expanded nTregs Boolean gating analysis of cytokine expressing cells
- nTregs, expanded as per protocol contain none/minimal single or multiple cytokine or CD107a expressing cells: Boolean gating analysis of a representative experiment of expanded nTregs stimulated with PMA/Ionomycin for 5 hrs and stained for intracellular cytokines IL17, IL2, IFN ⁇ , TNF ⁇ , and CD107a and analyzed on FLOWJO. Frequencies of cells positive for 1,2,3,4 and 5 measures are depicted. * Frequencies > 0.1%.
- Table 2 - Freshly isolated nTregs Boolean gating analysis of cytokine expressing cells No of cytokines Cytokine expressing cells Total Frequency
- Freshly isolated nTregs as per protocol contain minimal single or multiple cytokine expressing cells.
- Boolean gating analysis of a representative experiment of freshly isolated CD4 + CD25 ++bright nTreg cells stimulated with PMA/Ionomycin for 5 hrs and stained for intracellular cytokines IL17, IL2, IFN ⁇ and TNF ⁇ were analyzed on FLOWJO. Frequencies of cells expressing 1, 2, 3 and 4 cytokines are depicted. *Frequencies >0.1% Table 3 - Analysis of cytokines in culture supernatants of freshly isolated and expanded nTregs Freshly isolated Expanded nTregs
- Freshly isolated and expanded nTregs secrete none/minimal cytokines in culture supernatants.
- Freshly isolated and expanded nTregs were cultured at a cell concentration of 1 X 10 6 each and stimulated with PMA/Ionomycin for 5 hrs.
- Supernatants were collected and analyzed for cytokines as described. Values of individual cytokines in pg/ml are shown. Data represents mean of 3 experiments. ** TGF ⁇ values were undetectable less than 470 pg ml -1 .
- OTHER EMBODIMENTS [0073] Any improvement may be made in part or all of the compositions, cells, kits, and method steps.
Abstract
Described herein are methods of isolating and expanding human nTregs ex vivo in cGMP conditions for use in cellular therapies (e.g., for treating autoimmune disorders, immunosuppression in graft vs. host disease, induction of tolerance in solid organ transplantation, etc.) and properties and uses of these expanded Treg cells. A unique population of human natural Tregs were successfully expanded in vitro. Properties of these expanded CD3+CD4+ T cells were close to optimal for nTregs which are as follows: phenotypic stable characteristics (CD4+CD25++Foxp3+); no evidence of conversion to effector CD4 subset of TH-17 cells upon in-vitro stimulation; functional ability to suppress CD4+CD25negative T cells; and lack of production of pro-inflammatory cytokines upon stimulation with PMA + Ionomycin.
Description
METHODS OF ISOLATING AND EXPANDING HUMAN T REGULATORY CELLS AND USES THEREOF FOR CELLULAR THERAPY CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of Provisional Application Serial No. 61/370,306 filed August 3, 2010, which is herein incorporated by reference in its entirety. FIELD OF THE INVENTION
[0002] The invention relates generally to the fields of immunology, molecular biology, cell biology, and medicines. More particularly, the invention relates to methods of isolating and expanding (enriching) human T regulatory cells and uses thereof. BACKGROUND
[0003] Several subsets of T regulatory cells (Tregs) have been described in humans. The CD4 derived major Treg populations include natural T regulatory cells (nTreg) which originate in the thymus, induced Tregs (iTreg) derived from naive CD4+ T cells in the periphery, Tr1 cells which secrete predominantly IL-10 and TH3 cells which secrete predominantly TGF . Other cell populations such as NKT cells and CD8 T suppressor cells can also mediate immune regulation. Natural T regulatory cells play a key role in inducing and maintaining immunological tolerance and immune homeostasis (Sakaguchi, S., 2004 Annu Rev Immunol 22, 531-62; Sakaguchi, S. et al., 2001, Immunol Rev 182, 18-32; Nagahama, K., et al. 2007, Methods Mol Biol 380, 431-42). This specialized subpopulation of T cells is critical for maintaining unresponsiveness to self- antigens. These cells are of considerable interest from the viewpoint of cellular therapy in the therapeutic management of autoimmune disorders such as diabetes type 1, for short term immunosuppression in graft versus host disease, and for induction of tolerance in solid organ transplantation to prevent graft rejection. Although clinical trials using Tregs are under way, the procedures for deriving sufficient quantities of cells which have desirable characteristics of nTregs have yet to be optimized. The expanded CD4+ T cell population should ideally have the following four characteristic of nTregs: 1) a stable phenotype of CD25++brightFoxp3+ expression; 2) functional ability to suppress immune reactive T cells by a mechanism that does not involve
secretion of IL-10 or TGF- ; 3) no evidence of conversion to effector T cells or TH17 cells and 4) no secretion of pro-inflammatory cytokines upon in-vitro stimulation.
[0004] To accomplish the goal of expanding nTregs ex vivo for therapeutic purposes, it is critical to begin with the appropriate starting cell population and to use culture conditions that selectively favor the expansion of Tregs with properties that best characterize nTregs. Cell- based therapy using purified nTregs is under consideration for several conditions, but procedures employed to date have resulted in cell populations that are contaminated with cytokine secreting effector cells.
SUMMARY
[0005] Methods of isolating and expanding human nTregs ex vivo in cGMP conditions for use in cellular therapies (e.g., for treating autoimmune disorders, immunosuppression in graft vs. host disease, induction of tolerance in solid organ transplantation, etc.) and properties and uses of these expanded Treg cells is described herein. Natural Tregs constitute a minor population in peripheral blood with a frequency of 1–2% of total circulating CD4+ cells (Baecher-Allan et al., 2001, J Immunol 167, 1245-53). Thus expansion (enrichment) of therapeutic quantities of nTregs, particularly under cGMP compliant conditions, is a challenge (Riley et al., 2009, Immunity 30, 656-65). Described herein is the methodology for isolation and expansion (enrichment) of a population of nTregs from peripheral blood in humans. Based on their phenotype and functional characteristics, the expanded Treg population met the criteria that most closely define thymus derived nTregs, thus making them ideally suited for cell therapy in clinical trials. The expanded nTregs had the following properties: (1) stable expression of CD4+CD25++brightFoxp3+ with N97–98% purity; (2) potent functional ability to suppress CD4+CD25negative T cells without secretion of IL-10 or TGF-β; (3) no conversion into effector T cells or TH17 cells and (4) no production of pro-inflammatory cytokine upon in vitro stimulation with PMA/Ionomycin. Two critical determinants in the quality and quantity of expanded nTregs were first, the successful isolation of purified nTregs from peripheral blood, and second, the use of rapamycin in the expansion protocol. The isolated and expanded Tregs can be used in any therapeutic strategy to restore self-tolerance in autoimmune disorders (e.g., type 1 diabetes, autoimmune cardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis, celiac disease, Crohn’s disease, lupus erythematosus, myasthenia gravis, psoriasis, rheumatoid arthritis,
ulcerative colitis), for immunosuppression in host vs. graft disease, and for induction of tolerance in solid organ, tissue or cellular transplantation to prevent graft rejection.
[0006] Accordingly, described herein is a method of isolating and expanding natural T regulatory (nTreg) cells including: obtaining blood from an individual; obtaining at least one Buffy coat from the blood; isolating CD4+CD25++brightFoxp3+ nTreg cells from the at least one Buffy coat; and expanding the isolated CD4+CD25++brightFoxp3+ nTreg cells by culturing the CD4+CD25++brightFoxp3+ nTreg cells in the presence of rapamycin. The blood may be peripheral blood and the individual is typically a healthy donor. The isolated and expanded CD4+CD25++brightFoxp3+ nTreg cells are capable of suppressing CD4+CD25negative T cells as measured by at least one T cell assay and do not express cytokines as measured by at least one cytokine assay. Isolating CD4+CD25++brightFoxp3+ nTreg cells from the Buffy coat can include enrichment of CD4+ T cells by negative selection using agents which specifically bind cell markers including: CD8, CD16, CD19, CD36, CD56, CD66b, TCR , glycophorin A and P9, and isolation of CD25++bright cells by positive selection from the enriched CD4+ T cells using at least one agent which specifically binds to CD25. The step of positive selection from the enriched CD4+ T cells using at least one agent which specifically binds to CD25 is performed in an immunomagnetic cell separator, and the at least one agent which specifically binds to CD25 can be a monoclonal antibody to CD25.
[0007] Also described herein is a method of preventing or treating a disease or condition associated with an immune response in a subject. The method includes: providing CD4+CD25++brightFoxp3+ nTreg cells isolated and expanded by a method including obtaining blood from an individual, obtaining at least one Buffy coat from the blood, isolating CD4+CD25++brightFoxp3+ nTreg cells from the at least one Buffy coat, and expanding the isolated CD4+CD25++brightFoxp3+ nTreg cells by culturing the CD4+CD25++brightFoxp3+ nTreg cells in the presence of rapamycin; and administering the cells to the subject in a therapeutically effective amount for decreasing the immune response in the subject. Diseases or conditions associated with immune responses include: autoimmunity, allergies, diabetes, inflammation, graft versus host reactions, organ transplantation, inflammatory bowel disease and viral diseases. Administering the cells to the subject results in at least one of: alleviation of type 1 diabetes, immunosuppression in host vs. graft disease, and induction of tolerance in solid organ transplantation to prevent graft rejection.
[0008] Further described herein is a composition including a pharmaceutically acceptable carrier and a therapeutically effective amount of CD4+CD25++brightFoxp3+ nTreg cells isolated and expanded by a method including obtaining blood from an individual, obtaining at least one Buffy coat from the blood, isolating CD4+CD25++brightFoxp3+ nTreg cells from the at least one Buffy coat, and expanding the isolated CD4+CD25++brightFoxp3+ nTreg cells by culturing the CD4+CD25++brightFoxp3+ nTreg cells in the presence of rapamycin. The isolated CD4+CD25++brightFoxp3+ nTreg cells are capable of suppressing CD4+CD25negative T cells as measured by at least one T cell assay. The isolated CD4+CD25++brightFoxp3+ nTreg cells typically do not express cytokines as measured by at least one cytokine assay.
[0009] Still further described herein is a method of tissue or organ transplantation to a subject (e.g., human). The method includes: (a) obtaining the tissue or organ to be transplanted from a donor; (b) transplanting said tissue or organ to the subject; and (c) delivering a composition including a pharmaceutically acceptable carrier and a therapeutically effective amount of CD4+CD25++brightFoxp3+ nTreg cells isolated and expanded by a method including obtaining blood from an individual, obtaining at least one Buffy coat from the blood, isolating CD4+CD25++brightFoxp3+ nTreg cells from the at least one Buffy coat, and expanding the isolated CD4+CD25++brightFoxp3+ nTreg cells by culturing the CD4+CD25++brightFoxp3+ nTreg cells in the presence of rapamycin, to the subject prior to and/or subsequent to transplantation of the tissue or organ to the subject. The composition (cells) may be delivered to the subject after the transplantation at the time of maximum lymphopenia (e.g., 6 to 11 days after transplantation) in the subject with an immunosuppressive induction using a T cell depletion protocol such as Thymoglobuliun or Campath 1H treatment of the subject.
[0010] By the terms“nTreg,”“nTregs” and“nTreg cells” is meant“natural T regulatory cells”, a subset of T regulatory cells which plays a key role in inducing and maintaining immunological tolerance and immune homeostasis. The methods described herein provide for the isolation of highly purified nTregs that maintain the nTreg phenotype throughout the expansion protocol, avoiding contamination with pro-inflammatory cytokine markers that plague other Treg expansion protocols.
[0011] When referring to isolated and expanded nTreg cells using the methods described herein, the phrases“expanded nTreg cells,”“expanded human nTreg cells,” and“expanded CD4+CD25++Foxp3+ cells” mean natural T regulatory cells isolated and expanded from CD4+ T
cells with a bright expression of CD25 (CD25bright or CD25++) which remains stable through the expansion protocol. The expanded cells contain none or minimal IFN IL2, IL17, TNF and CD107a expressing (positive) cells.
[0012] As used herein, "treatment" and“treating” are intended to refer to inhibiting, eliminating, ameliorating, diminishing and/or reducing cellular damage and/or symptoms associated with a disease or condition, e.g. allograft rejection. "Treating" includes, but is not limited to, inducing tolerance to a cell, tissue or organ transplant, restoring self-tolerance in autoimmunity, restoring self-tolerance in conditions such as asthma, allergy and anaphylactic shock, etc. Compositions as described herein may also or alternatively be a prophylactic, i.e., used to partially or completely prevent a disease or condition or symptom thereof.
[0013] Where the terms "patient" and "subject" are used interchangeably in the present specification, they include animals. In one embodiment, the patient is a mammal, and in a preferred embodiment, the patient is human.
[0014] As used herein,“an effective amount” or“a therapeutically effective” amount is intended to refer to the total amount of the expanded Treg cells or a composition including expanded Treg cells, or of the method that is sufficient to show a meaningful patient benefit. This term is also intended to refer to an amount that returns to normal, either partially or completely, physiological or biochemical parameters associated with immunological intolerance.
[0015] The phrases "isolated" or biologically pure" refer to material (e.g., nucleic acids) which is substantially or essentially free from components which normally accompany it as found in its native state.
[0016] As used herein, the term“buffy coat” means the fraction of an anticoagulated blood sample after density gradient centrifugation that contains most of the white blood cells and platelets.
[0017] Although compositions and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable compositions and methods are described below. All publications, patent applications, and patents mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. The particular embodiments discussed below are illustrative only and not intended to be limiting.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 shows the purity of freshly isolated CD4+CD25++bright nTregs showing minimal contamination with other cell populations. CD4+CD25++bright cells isolated by the two step technique of negative selection of CD4+ T cells followed by positive selection of CD4+CD25++bright cells using the Robosep® as per protocol were stained and analyzed by flow cytometry. Upper panel shows sequential analysis of frequencies of CD3, CD4, CD25 and Foxp3 expressing cells. Lower panel shows frequencies of individual CD19, CD14, CD56 and CD8 expressing cells on (A) open gate (B) cells gated on singlets. (Representative of one of the two experiments)
[0019] FIG. 2 shows a gating strategy and phenotype analysis of ex vivo expanded CD4+CD25++bright nTregs. CD4+CD25++bright cells were expanded ex vivo as per protocol and were harvested on day 19. (A) Expanded cells were stained and analyzed on open gate for viability using ViViD dye and for CD3, CD4, CD25 Foxp3, CD27, CD45RO and CD127. The CD3+CD4+ cells were gated for CD25 and Foxp3. FoxP3+ cells were analyzed for expression of CD127 and CD45RO+CD27+ phenotype demonstrating that they were CD127 negative memory cells. (B) Cells gated on singlets and analyzed in a similar manner. Figure is representative of 4 experiments.
[0020] FIG. 3 shows ex vivo expanded nTregs exhibit potent suppressive function. Autologous CD4+CD25negative responder cells labeled with CFSE dye were analyzed for proliferation and cell division on day 4 following culture under the following conditions: (A) in medium, without stimulation, (B) after stimulation with anti-CD3/anti-CD28 coated micro beads (positive control), (C) with anti-CD3/anti-CD28 stimulation and addition of expanded CD4+CD25++bright cells to responder cells in 1:1 and 1:10 ratios and (D) with anti-CD3/anti-CD28 stimulation and addition of expanded CD4+CD25dim cells to responder cells in 1:1 and 1:10 ratios. Figure is representative of 2 experiments showing potent suppression by the CD4+CD25++bright cells at 1:1 ratio. The figure is representative of 3 experiments showing potent suppression by the CD4+CD25++bright cells at 1:1 ratio. (E) Summary data from 3 donors showing the effect of
adding CD4+CD25++bright and CD4+CD25dim cells to autologous CD4+CD25negative cells stimulated with anti-CD3/anti-CD28 coated micro beads. Box plots represent mean and 95th
percentiles of proliferation response (%CFSE low cells). Asterisks indicate statistical significance (**pb0.01, ***pb0.001).
[0021] FIG. 4 shows ex vivo expanded nTregs exhibit minimal cytokine expression following in-vitro stimulation. Robosep® isolated CD4+CD25++bright, CD4+CD25dim and CD4+CD25negative cells were cultured as per the expansion protocol in X-vivo complete medium containing CD3/CD28 expander beads and rhIL2 (300 IU ml-1) and rapamycin (100 ng ml-1). Each cell population was harvested on day 19, stimulated with PMA/Ionomycin for 5 hrs and stained for cytokines IL17, IL2, IFNγ, TNFα and for CD107a. Each cell population was analyzed by FLOWJO using (A) an open gate (B) cells gated on singlets and analyzed in a similar manner. Plots show frequencies of cells positive for each cytokine and for CD107a in CD4+CD25++bright, CD4+CD25dim and CD4+CD25negative cells, demonstrating the very low frequencies for all measures in CD4+CD25++bright cells compared to the other two populations. (C) Summary data from 4 donors showing frequencies of IL17, IFN- , IL2, TNF and CD107a expressing cells in CD4+CD25++bright cells (blue) in comparison with CD4+CD25dim (red) and CD4+CD25negative (green) cell populations. Box plots represent mean and 95th percentiles. Asterisks indicate statistical significance p<0.05 (ANOVA, Tukey post test).
[0022] FIG. 5 shows Robosep® isolated nTregs exhibit low frequencies of cytokine expressing cells. CD4+CD25++bright nTregs and CD4+CD25negative cells were isolated as described by Robosep® and stimulated with PMA/Ionomycin for 5 hrs and stained for IL17, IL2, IFN and TNFα. (A) Analysis of CD4+CD25++bright cell fraction shown in open gate that includes doublets, triplets and other cells, After exclusion of dead cells with ViViD, gated CD4 cells were analyzed for IL17, IL2, IFNγ and TNFα. (B) cells gated on singlets on CD4+CD25bright cells (C) Analysis of CD4+CD25negative cell fraction shown in open gate performed in a similar manner to A. (D) Analysis of CD4+CD25negative cell fraction shown in singlet gate performed in a similar manner to B.
[0023] FIG. 6 shows rapamycin curtails expansion of all populations of freshly isolated CD4+ cells. Isolated CD4+CD25++bright, CD4+CD25dim and CD4+CD25negative cell fractions were expanded in X-Vivo complete medium containing CD3/CD28 expander beads + rhIL2 + rapamycin. Fold expansion data are shown at day 19 time point. (A) Fold expansion from starting cell numbers of CD4+CD25++bright (10 samples), CD4+CD25dim (8 samples) and CD4+CD25negative (3 samples) cells. (B) Comparison of CD4+CD25++bright (2 samples),
CD4+CD25dim (3 samples) and CD4+CD25negative (2 samples) cells expanded with rapamycin ( ) and without rapamycin ( ) at day 19 time point. Identical samples are connected in the figure with lines.
[0024] FIG. 7 shows a phenotype analysis of expanded nTregs shows minimal contamination with other populations. Expanded, cryopreserved and thawed CD4+CD25++bright cells were stained and analyzed on (A) open gate (B) on singlets for ViViD (Live/Dead cells), for frequencies of live cells expressing CD25, Foxp3, CD14, CD19, CD56 and CD8 (C) Mean and SD values of frequencies of CD14, CD19, CD56 and CD8 expressing cell populations in expanded CD4+CD25++bright cell fractions from 4 different buffy coats.
[0025] FIG. 8 shows that the addition of rapamycin curtails expansion of cytokine producing cells. Freshly isolated CD4+CD25++bright nTregs were expanded in X-vivo complete medium containing CD3/CD28 expander beads and IL2 with and without the addition of rapamycin for 19 days. Cells were stimulated with PMA/Ionomycin for 5 hrs and stained for cytokines IL17, IL2, IFNγ and TNFα to determine frequencies of cells expressing individual cytokines. Upper panels show the experiment with no rapamycin added in the culture and lower panel shows cells with addition of rapamycin to the culture, demonstrating the marked reduction in frequencies of cytokine expressing cells in the latter. DETAILED DESCRIPTION
[0026] Described herein are methods of isolating and expanding human nTregs ex vivo in cGMP conditions for use in cellular therapies (e.g., for treating autoimmune disorders, immunosuppression in graft vs. host disease, induction of tolerance in solid organ transplantation, etc.) and properties and uses of these expanded Treg cells. Described in the Examples section below is the successful expansion of a unique population of human natural Tregs in vitro. Properties of these expanded CD3+CD4+ T cells were close to optimal for nTregs which are as follows: phenotypic stable characteristics (CD4+CD25++Foxp3+); no evidence of conversion to effector CD4 subset of TH-17 cells upon in-vitro stimulation; functional ability to suppress CD4+CD25negative T cells; and lack of production of pro-inflammatory cytokines upon stimulation with PMA + Ionomycin. In addition, a Boolean gating analysis of cytokine- expressing cells by flow cytometry for 32 possible profile end points revealed that 96% of expanded nTregs did not express any cytokine. From a single buffy coat, approximately 80
million pure nTregs were harvested after expansion under cGMP conditions. Starting from 0.8 to 1.4 million purified CD4+CD25++bright cells from a single buffy coat, approximately 50 to 80 million cells can be harvested at the end of the expansion protocol and this number would yield approximately 1 million cells kg-1 for cellular therapy in an average adult. These expanded cells conform with the FDA requirement which mandates that the identity, purity, potency and sterility of a cell product should be demonstrated before they can be administered.
[0027] The below described preferred embodiments illustrate adaptations of these compositions and methods. Nonetheless, from the description of these embodiments, other aspects of the invention can be made and/or practiced based on the description provided below.
BIOLOGICAL METHODS
[0028] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manua1,3rd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Methods for culturing T regulatory cells are well known to those skilled in the art. See, e.g., Regulatory T Cells: Methods and Protocols (Methods in Molecular Biology) George Kassiotis (editor) and Adrian Liston (editor), Humana Press (New York, NY); 1st ed. (2011). Methods of suppressing immune responses are known in the art and are described, e.g., in Suppression and Regulation of Immune Responses: Methods and Protocols (Methods in Molecular Biology), Maria Cristina Cuturi (editor) and Ignacio Anegon (editor), Humana Press (New York, NY); 1st ed. (2010). Use of rapamycin has been shown to induce apoptosis of CD4+CD8+ thymocytes and result in the expansion of peripheral regulatory CD4+CD25+ T cells, and is described, e.g., in Tian et al., 2004, Transplantation 77: 183-89. These references are herein incorporated by reference.
METHOD OF ISOLATING AND EXPANDING NTREG CELLS
[0029] Described herein are methods of isolating and expanding nTreg cells for use in cellular therapies. The methods result in the production of CD4+CD25++Foxp3+ cells that may be used in cellular therapies. A typical method of isolating and expanding nTreg cells includes obtaining Treg cells from a donor and purifying them. The purified Treg cells are then cultured in the presence of rapamycin or other Treg promoting (Treg“permissive”) immunosuppressant
agent. In one example of a method of isolating and expanding nTreg cells, the method includes: obtaining blood from an individual; obtaining at least one Buffy coat from the blood; isolating CD4+CD25++brightFoxp3+ nTreg cells from the at least one Buffy coat; and expanding the isolated CD4+CD25++brightFoxp3+ nTreg cells by culturing the CD4+CD25++brightFoxp3+ nTreg cells in the presence of rapamycin. The isolated and expanded CD4+CD25++brightFoxp3+ nTreg cells are capable of suppressing CD4+CD25negative T cells as measured by at least one T cell assay, and do not express cytokines as measured by at least one cytokine assay. Isolating CD4+CD25++brightFoxp3+ nTreg cells from the Buffy coat typically includes enrichment of CD4+ T cells by negative selection using agents which specifically bind cell markers comprising: CD8, CD16, CD19, CD36, CD56, CD66b, TCR , glycophorin A and P9, and isolation of CD25++bright cells by positive selection from the enriched CD4+ T cells using at least one agent which specifically binds to CD25. The step of positive selection from the enriched CD4+ T cells using at least one agent which specifically binds to CD25 is typically performed in an immunomagnetic cell separator, and the at least one agent which specifically binds to CD25 is typically a monoclonal antibody to CD25.
[0030] Any suitable culture medium can be used for culturing the freshly isolated nTreg cells. An example of a suitable culture medium for freshly isolated nTreg cells is one that includes serum-free X-Vivo with CD3/CD28 T-cell expander Dyna beads, IL2 and rapamycin. Similarly, any suitable culture medium can be used for culturing the nTreg cells during the expansion phase. A suitable culture medium for freshly isolated nTreg cells includes serum-free X-Vivo with CD3/CD28 T-cell expander Dyna beads, IL2 and rapamycin. One day after culture initiation, AB serum is added (final concentration 10%). To the expansion phase culture medium, rapamycin or other suitable immunosuppressant is added. Rapamycin has a potent immunosuppressive activity and serves to prevent IL-2-mediated signaling and cell cycle arrest at the G1-S boundary, thereby leading to T-cell anergy and/or apoptosis and induction of operational tolerance.
[0031] In some embodiments, a phenotype analysis of expanded Treg cells is performed prior to using the Treg cells in a cellular therapy. Any suitable analysis of expanded Treg cells can be used. As described in the experiments described herein, a phenotype analysis of Treg cells can be performed by surface staining of cells with CD3, CD4, CD25, CD127, CD27, CD45RO and by using a‘dump’ channel for cells stained with ViViD dye, CD14, CD19, CD56,
followed by intracellular staining for FoxP3 in a single tube. In this analysis, cells are also stained in another tube with ViViD dye and for CD4, CD8, CD25, CD14, CD19, CD56 and FoxP3 to trace contamination with monocytes, B cells, NK cells, and cytotoxic T cells in the live cells. Flow data can be collected on any suitable FACS cytometer (e.g., FACS LSRII cytometer) and analyzed with appropriate software (e.g., FlowJo software, Mac version 8.6.8, Tree Star). In addition, intracellular and culture supernatant cytokine assays can be performed on freshly isolated and expanded nTregs (i.e., CD4+CD25++Foxp3+ cells) as described in the Examples below for determining if the cells are producing pro-inflammatory cytokines (e.g., upon in vitro stimulation) prior to administering the nTregs to a recipient (e.g., patient). The CD4+CD25++Foxp3+ cells can also be characterized functionally for their ability to suppress autologous CD4+CD25negative T cells.
[0032] In the experiments described herein, a Robosep® instrument (Stem Cell Technologies, Vancouver, BC, Canada) was used for isolating nTreg cells from buffy coats. However, any suitable immunomagnetic cell separator could be used to isolate nTreg cells from human buffy coats or other sources. Similarly, in addition to the RosetteSep human CD4+ T Cell Enrichment Cocktail (Stem Cell Technologies, Vancouver, BC, Canada) used in the experiments described herein, any other products that contain the selected combination/concentration of antibodies described herein for enriching CD4+ T cells could be used.
[0033] In addition to the EasySep human CD25 Positive Selection Cocktail and EasySep Magnetic Nanoparticles (both from Stem Cell Technologies, Vancouver, BC, Canada) used in the experiments described herein, any negative selection cocktail of antibodies containing enrichment of agents which specifically bind cell markers comprising: CD8, CD16, CD19, CD36, CD56, CD66b, TCRγδ, glycophorin A and P9, could be used to isolate CD4+ T cells by negative selection, followed by any suitable method for isolation of CD25++bright cells by positive selection from the enriched CD4+ T cells using at least one agent which specifically binds to CD25. The step of positive selection from the enriched CD4+ T cells using at least one agent which specifically binds to CD25 is typically performed in an immunomagnetic cell separator, and the at least one agent which specifically binds to CD25 is typically a monoclonal antibody to CD25.
[0034] In these methods, nTreg cells can be obtained from any suitable source. In the experiments described herein, nTreg cells were obtained from buffy coats obtained from healthy
adult donors (e.g., peripheral blood from a healthy donor). However, nTreg cells can be obtained from other sources. Examples of other nTreg sources include any other suitable hematopoietic cell source such as umbilical cord blood.
[0035] Typically, a subject to be treated receives cells obtained and expanded from that subject (i.e., autologous transplant). However, in some embodiments, a subject may receive an infusion of nTreg cells obtained and expanded from another individual (e.g., allogeneic transplant). An allogeneic transplant of cells may find particular use, for example, in situations in which there might be a defect of nTregs from the subject to be treated because the subject has a specific disease condition associated with a defect in nTregs and would therefore require an infusion of nTregs obtained from a donor that does not have such a defect. Therapeutic Compositions for Modulating an Immune Response In a Subject
[0036] Therapeutic compositions for modulating (e.g., decreasing) an immune response in a subject are described herein. Such compositions typically include a pharmaceutically acceptable carrier and a therapeutically effective amount of CD4+CD25++brightFoxp3+ nTreg cells isolated and expanded by the methods described herein. The isolated and expanded CD4+CD25++brightFoxp3+ nTreg cells are capable of suppressing CD4+CD25negative T cells as measured by at least one T cell assay and typically do not express cytokines as measured by at least one cytokine assay. METHODS OF TISSUE AND ORGAN TRANSPLANTATION TO A SUBJECT
[0037] Methods of tissue or organ transplantation to a subject are described herein. Such methods typically include: obtaining the tissue or organ to be transplanted from a donor; transplanting said tissue or organ to the subject; and delivering a composition of expanded nTregs to the subject prior to and/or subsequent to transplantation of the tissue or organ to the subject. In general transplant protocols, the cells are delivered to the transplant recipient after the transplant at the time of maximum recipient lymphopenia (e.g. 6 to 11 days after transplantation) with an immunosuppressive induction using a T cell depletion protocol, such as Thymoglobuliun or Campath 1H treatment of the recipient. This is to take advantage of the minimum level of recipient effector cells possible before infusing the expanded nTregs to give them the best chance of a biologic effect.
[0038] For treatment of an autoimmune condition that requires also a transplant (e.g., islet cells to replace the destroyed insulin producing cells), it may be preferable to treat the patient with expanded nTregs prior to the transplant, to restore self tolerance, and after the transplant to induce tolerance to the transplanted cells. For treatment in an autoimmune condition that may not require a transplant (e.g., type 1 diabetes at the onset, before the beta cells are destroyed), it may be preferable to just treat the patient as soon as possible at onset or even before onset if you identify patients at risk to prevent onset of disease (it is generally easier to restore self tolerance at the very first moments of detection of an immune imbalance or trend, rather that reversing an overt autoimmune process at clinical onset). KITS
[0039] Described herein are kits for preventing allograft rejection, and/or restoring self- tolerance in autoimmunity (e.g., in diseases such as type 1 diabetes), and restoring self-tolerance in conditions such as asthma, allergy and anaphylactic shock in a mammalian subject. A typical kit includes a therapeutically effective amount of a composition including expanded CD4+CD25++brightFoxp3+ nTreg cells with instructions for administering the cells to the subject. The cells can be packaged by any suitable means for transporting and storing cells; such methods are well known in the art. The instructions generally include one or more of: a description of the cells; dosage schedule and administration for prevention of allograft rejection and/or restoring self-tolerance in autoimmunity, and restoring self-tolerance in conditions such as asthma, allergy and anaphylactic shock; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. Generally, a kit as described herein also includes packaging. In some embodiments, the kit includes a sterile container which contains a therapeutic or prophylactic composition; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding cells or medicaments.
ADMINISTRATION OF COMPOSITIONS AND CELLS
[0040] Ex vivo delivery of human Treg cells isolated and expanded according to the methods described herein (i.e., CD4+CD25++brightFoxp3+ nTreg cells), and compositions including the expanded human Treg cells, is provided for within the invention. Ex vivo cellular therapy may be used to transplant expanded Treg cells isolated from a healthy host donor into a recipient in need thereof (e.g., a human patient). Any suitable delivery method may be used for delivering expanded Treg cells isolated from a host donor to a recipient in need thereof. Several suitable modes of delivery of cells into the recipient are encompassed, including including intravenous injection, intraperitoneal injection, or in situ injection into target tissue. One example of a delivery method involves co-administration of expanded nTregs with MSCs (mesenchymal stem cells) and/or Marrow Derived Suppressor Cells (MDSC) and/or tolerogenic Dendritic Cells as other immunomodulatory cell sources that could enhance nTreg engraftment and function. For example, MSCs can be added to the nTreg infusion as MSCs have been already successfully used to provide temporary immunomodulation/tolerance in clinical protocols for treatment of GVHD or for enhancement of organ transplant survival and function.
[0041] The therapeutic methods described herein in general include administration of a therapeutically effective amount of the compositions or cells described herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider. The methods and compositions herein may be also used in the treatment of any disorders in which a lack of self-tolerance may be implicated.
[0042] Suitable patients include human or other animals in need of treatment with expanded Tregs. For example, patients suffering from or at risk for an autoimmune disease, such as Type I diabetes, or patients receiving foreign graft transplants (i.e., allograft patients and xenograft patients), are examples of recipients in need of Treg expansion in accordance with the invention.
EFFECTIVE DOSES
[0043] The compositions and cells described herein are preferably administered to a mammal (e.g., human) in an effective amount, that is, an amount capable of producing a desirable result in a treated mammal (e.g., preventing allograft rejection). Such a therapeutically effective amount can be determined according to standard methods. Toxicity and therapeutic efficacy of the compositions utilized in methods of the invention can be determined by standard pharmaceutical procedures. As is well known in the medical and veterinary arts, dosage for any one subject depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, time and route of administration, general health, and other drugs being administered concurrently. EXAMPLES
[0044] The present invention is further illustrated by the following specific examples. The examples are provided for illustration only and should not be construed as limiting the scope of the invention in any way. [0045] Example 1 - Isolation and expansion of human natural T regulatory cells for cellular therapy. Natural T regulatory cells (nTregs) play a key role in inducing and maintaining immunological tolerance. Cell based therapy using purified nTregs is under consideration for several conditions, but procedures employed to date have resulted in cell populations that are contaminated with cytokine secreting effector cells. Described herein is the establishment of a method for isolation and ex vivo expansion of human nTregs from healthy blood donors for cellular therapy aimed at preventing allograft rejection in organ transplants. The Robosep® instrument was used for initial nTreg isolation and rapamycin was included in the expansion phase of cell cultures. The resulting cell population exhibited a stable CD4+CD25++brightFoxp3+ phenotype, had potent functional ability to suppress CD4+CD25negative T cells without evidence of conversion to effector T cells including TH17 cells, and manifested little to no production of pro- inflammatory cytokines upon in-vitro stimulation. Boolean gating analysis of cytokine- expressing cells by flow cytometry for 32 possible profile end points revealed that 96% of expanded nTregs did not express any cytokine. From a single buffy coat, approximately 80 million pure nTregs were harvested after expansion under cGMP conditions; these cell numbers
are adequate for infusion of approximately one million cells Kg-1 for cell therapy in clinical trials.
MATERIAL AND METHODS
[0046] Human buffy coats: Human Buffy coats containing approximately 3-4X1010 cells were obtained from healthy adult donors from Community Blood Center of South Florida, Miami, FL, USA. Informed consent was obtained in accordance with standard policies and procedures. Samples were processed within 24 hours of collection and were required to have a lymphocyte viability of >90% in order to be processed.
[0047] Isolation and culture of Natural T regulatory cell (nTregs): To obtain a purified population of nTregs for subsequent expansion, human CD4+CD25++bright cells were isolated from buffy coats in two steps. In the first step CD4+ T cells were enriched by negative selection using a cocktail of nine monoclonal antibodies. In the second step CD25++bright cells were isolated by positive selection from purified CD4+ cells using anti CD25 antibody in a Robosep® instrument (Stem cell Technologies, Vancouver, BC, Canada). For the first step, the buffy coat was diluted 1:2 with Ca2+ Mg2+ free phosphate buffered saline (PBS) (Stem Cell Technologies, Vancouver, BC, Canada) containing 2% fetal bovine serum (FBS) (Hyclone, South Logan, Utah, USA) and dispensed in 20ml volume each in 50ml conical tubes (Corning Life Sciences, Lowell, MA, USA). RosetteSep human CD4+ T Cell Enrichment Cocktail (Stem Cell Technologies, Vancouver, BC, Canada) which is a mixture of mAbs to CD8, CD16, CD19, CD36, CD56, CD66b, TCR , Glycophorin A and P9 was added to each tube at a concentration of 50 l ml-1 and tubes were incubated at room temperature for 20 minutes. Thereafter the cell suspension was diluted 1:1 with PBS, and the CD4+ enriched cells were harvested by Ficoll Paque Plus (GE Healthcare, Pittsburgh, PA , USA) density gradient centrifugation at 1200Xg for 20 minutes at 23 0C. Isolated CD4+ cells were washed twice with PBS containing 10% FBS and re-suspended at a concentration of 5x107 cells ml-1 and dispersed in 4 ml aliquots in sterile 15ml round-bottom polyethylene tubes as required for processing on the Robosep® instrument. For the second step, the Robosep® instrument was primed as per the manufacturer’s protocol by loading EasySep human CD25 Positive Selection Cocktail and EasySep Magnetic Nanoparticles (both from Stem Cell Technologies, Vancouver, BC, Canada) using the volume and concentration specified for the selection of CD4+CD25++bright cells. The instrument was programmed for automatic separation of CD4+CD25++bright cells followed by further separation of the remaining cells into
CD4+CD25dim and CD4+CD25negative cells. To prepare the isolated cells for the expansion phase, they were washed twice and re-suspended in 1ml of culture medium consisting of X-Vivo 15 (Lonza, Muenchensteinerstrasse, CH, Switzerland), 1% N-Acetylcysteine (American Reagent, Shirley, NY, USA) and 1% Pen-Step (Invitrogen, Carlsbad, CA, USA).
[0048] Ex vivo expansion of Robosep® isolated CD4+ cell populations: The Robosep®- isolated population of CD4+CD25++bright cells suspended in serum free X-Vivo culture medium were plated in a final volume of 300 μl in flat bottom 48-well microtiter plates (Corning Life Sciences, Lowell, MA, USA) at a concentration of 0.2x106 cells well-1 with CD3/CD28 T-cell expander Dyna beads at 3:1 ratio (Invitrogen, Carlsbad, CA, USA) and 1000 U ml-1 IL-2 (R&D systems, Minneapolis, MN, USA), in presence of 100 ng ml-1 of rapamycin (Wyeth, Philadelphia, PA, USA ), as has been previously described (Godfrey, W., et al., 2005, Blood 105, 750-8; Hippen, K., et al., 2008, Blood 112, 2847-57; Putnam, A., et al., 2009, Diabetes 58, 652- 62). Cells were cultured at 37 oC with 5% CO2 and 100% humidity. One day after culture initiation, 30 μl human AB serum (Valley Biomedical, Winchester, VA, USA) was added to the wells at a final concentration of 10%. On the second day, 700 μl X-Vivo culture media with 10% human AB serum with rapamycin 100 ng ml-1 was added to make the final volume to 1ml in each culture well. On day 5, cells from different wells were pooled, sampled for viability and cultured at a concentration of 0.3x106 cells in sterile T-25 tissue culture flasks (Corning Life Sciences, Lowell, MA, USA) in complete X-Vivo culture medium supplemented with 10% human AB serum, 300U ml-1 of IL-2 and rapamycin 100 ng ml-1. On day 8, of culture, cells from the T-25 tissue culture flasks were pooled, sampled for viable cells and re-distributed into sterile T-75 or T-175 tissue culture flasks at a concentration of 0.3x106 cells ml-1 in the culture medium of complete X-Vivo culture medium supplemented with 10% human AB serum, 300U ml-1 of IL-2 and rapamycin 100 ng ml-1. They were again pooled on day 12 and re-distributed into sterile T-75 or T-175 tissue culture flasks at a concentration of 0.3x106 cells ml-1 in the same media as above, and the procedure was repeated on day 15 in a similar manner. For the expansion phase, the cells were cultured for a total of 19 ± 1 days. At the end of the culture period, cells were exposed to the Dyna cell magnetic particle separator (Invitrogen, Carlsbad, CA, USA) for 10 minutes to remove CD3/CD28 T-Cell expander Dyna beads. Cells were washed twice and were analyzed for cell count, viability, purity, potency of suppression and cryopreserved in 10% DMSO using an automated temperature controlled freezer (T.S. Scientific 17
Kryo 10 Series, Perkasie, PA, USA) in liquid nitrogen. To evaluate properties of all 3 Robosep® isolated CD4+ cell populations, identical cultures were set up with the other two Robosep® isolated cell populations of CD4+CD25dim and CD4+CD25negative cells. In addition, to evaluate the effect of rapamycin, cultures were also set up without the addition of rapamycin.
[0049] Monoclonal antibody reagents: Flow cytometry panels for 10-12 color polychromatic flow cytometry analyses were utilized to analyze T cell phenotype and functions. Antibodies to IFN- -PECy7, IL2-PE, CD3-Amcyan, CD4-PercpCy5-5, TNF- - Alexafluor700, CD107a-PECy5, CD25-APCCy7, Foxp3-Alexafluor488 or Alexafluor647 (clone 259D/C7), CD127-PE, CD27-APC, CD45RO-FITC, CD14-Alexafluor700 or pacific blue, CD19-PECy7, CD8-APCCy7 were obtained from BD Pharmingen, San Jose, CA. IL17-Alexafluor488, CD56- Pacific blue were obtained from e-Bioscience, San Diego, CA, USA and CD56-Alexafluor488 was obtained from Biolegend, San Diego, CA, USA. CD25-PE was obtained from Stem cell technologies, Vancouver, BC, Canada. CD19-Pacific blue and a violet fluorescent reactive dye used as a viability marker to exclude dead cells from analysis (LIVE/DEAD® Fixable Dead Cell Stain Kit, ViViD) were obtained from Invitrogen, Carlsbad, CA, USA.
[0050] Flow cytometry analysis: Polychromatic flow cytometry for surface and intracellular staining of freshly isolated and expanded nTregs was performed on a BD LSR II Flow Cytometer System (BD Biosciences, San Jose, CA, USA) as described (Perfetto, S., et al., 2004, Nat Rev Immunol 4, 648-55; Lamoreaux, L., et al., 2006, Nat Protoc 1, 1507-16; Darrah, P., et al., 2007, Nat Med 13, 843-50). The procedure and method for 10-12 color flow cytometry was optimized in key steps which included appropriate concentrations of monoclonal antibodies, use of a dead-cell discriminator and‘dump’ channel, selection of a cytokine secretion inhibitor, selection of fixation and permeabilization reagents and inclusion of compensation controls as described (Lamoreaux, L., et al., 2006, Nat Protoc 1, 1507-16). This assay was used to detect four/five separate functions (production of three/four cytokines and degranulation) and simultaneous identification of surface markers on the subpopulation of cells. The same method was used for FoxP3 staining and characterization of nTregs. Cells were analyzed by flow cytometry using gated singlets as is usually recommended, as well as open gate to encompass all the cells to ensure that there were no contaminating cells being excluded in the analysis, because they would all be included in the expansion phase. It was suggested by FDA that an open gate analysis should be done on the expanded cell population because all the expanded cells are
planned to be infused when used in clinical trials. Open gates encompassing all cells (including doublets, triplets and other cells) were therefore used in addition to cell specific gates to conduct the phenotypic characterization and intracellular cytokine analysis.
[0051] Phenotypic analysis: Phenotypic analysis of freshly isolated nTregs was performed by surface staining of 0.5 X 106 cells with CD3, CD4, CD25, CD8, CD14, CD19, CD56 followed by intracellular staining for Foxp3 according to manufacturing instructions. Surface staining for CD25 was done using CD25-PE from Stem cell Technologies, Vancouver, BC, Canada.
[0052] Phenotype analysis of expanded nTregs was performed by surface staining of 1X106 cells with CD3, CD4, CD25, CD127, CD27, CD45RO and by using a‘dump’ channel for cells stained with ViViD dye, CD14, CD19, CD56, followed by intracellular staining for FoxP3 according to manufacturer’s instructions in a single tube. Cells were also stained in another tube with ViViD dye and for CD4, CD8, CD25, CD14, CD19, CD56 and FoxP3 to trace contamination with monocytes, B cells, NK cells and cytotoxic T cells in the live cells. Flow data was collected on a FACS LSRII cytometer and analyzed with FlowJo software (Mac version 8.6.8, Tree Star).
[0053] Intracellular Cytokine analysis: Intracellular cytokine analysis was performed in freshly isolated and expanded nTregs. nTreg populations at a concentration of 1X106 cells were cultured with phorbol myristate acetate (PMA) (Sigma-Aldrich, St. Louis, MO) 50ngml-1, Ionomycin 1 μg ml-1, Monensin 0.7 μl ml-1 (golgistop , BD Biosciences, San Jose, CA, USA) and Brefeldin-A 10 μg ml-1 (Sigma-Aldrich, St. Louis, MO) in complete media ( RPMI 1640 supplemented with 10% heat inactivated FBS, 100U/ml penicillin G, 100 g ml-1 streptomycin) for 5 hours at 37 oC in 5% CO2 incubator at 100% humidity. Cells were washed twice with RPMI 1640 and intracellular staining for cytokines IL-2, IL-17, IFN - , TNF- and CD107a was performed in a single tube with appropriate controls as described13. Flow data was collected on a FACS LSRII and analyzed with FlowJo software. Frequency of cytokine expression on per cell basis was analyzed by Boolean gating using FlowJo software.
[0054] Cytokine assay in culture supernatants: Freshly isolated nTregs and expanded nTregs were stimulated with PMA 50 ng ml-1 and Ionomycin 1 μg ml-1 for 5 hours at 37 oC. Cells were centrifuged at 850g for 10 minutes and supernatants were collected and were analyzed in a multiplex format using Quansys multiplex Elisa kit (Quansys Biosciences, West
Logan, Utah, USA ) for cytokines interferon (IFN)- , IL-2, IL-4, IL5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-1a, IL-16, IL-17, IL-23, TNF-α, LTa, TGF , TGFβ-RII, TGFβ-RIII.
[0055] In vitro suppression assay: Frozen autologous CD4+CD25negative cells were thawed, washed twice and cell count and viability was assessed by trypan blue dye exclusion. Viable 2 x106 CD4+CD25negative (responder) cells were labeled with Carboxyfluorescein succinimidyl ester (CFSE, Invitrogen, Carlsbad, CA, USA) at a concentration of 4 μM/5 x106 cells for 10 minutes at 37 oC. Labeling was terminated by addition of an equal volume of 100% FBS. After 4 washes in 10% FBS in complete media, cells were cultured alone and with unlabeled nTregs at 1:1 and 1:10 (responder: Treg) ratio and stimulated with 50 μl (7.8ul of beads suspended in 1 ml PBS and 4ml of complete media) of anti CD3/CD28 coated micro beads for 4 days at 37 oC in 5% CO2 incubator. CD4+CD25negative cells in medium alone were also cultured as control. On day 4, cells were washed twice and cell division was analyzed in all culture conditions. Cells undergoing division were identified by the decrease in CFSE, resulting from dilution of dye with each division. The medium-alone culture consisted of non-proliferating cells (CFSE bright) with less than 3.3% CFSE dim (proliferating) cells.
[0056] Sterility of Monoclonal antibodies: A custom batch of the RosetteSep human CD4+ cell enrichment cocktail and EasySep human CD25+ positive selection cocktail was produced by the manufacturer (Stem Cell Technologies, Vancouver, BC, Canada), in quantities sufficient to cover 60 patients for phase I clinical trials. All the products were tested for sterility and were free of murine retrovirus, adventitious virus (tested with highly sensitive assay, Wuxi Apptec Inc) mycoplasma, bacteria, fungi and endotoxin.
[0057] Statistical analysis: All group results are expressed as mean plus or minus S.D. if not stated otherwise. One way ANOVA-Tukey post test was used for the comparison of group values and discriminatory parameters, where appropriate P values less than 0.05 were considered significant.
RESULTS
[0058] As the procedure described here is aimed at developing nTreg cell therapies for infusion into patients, the flow cytometry analysis throughout the study utilized an open gate analysis that included the entire cell population without exclusion of doublets, triplets and other potentially contaminating non Treg cells. In addition, conventional analysis of gated singlets was performed.
[0059] Isolation and characterization of freshly isolated n Tregulatory cells: Isolation of nTregs was made from buffy coats of healthy donors in two steps. In the first step CD4+ cells were enriched by negative selection with a cocktail of nine monoclonal antibodies. From eight different buffy coat samples, recovery of CD4+ cells ranged from 135-240 X 106 cells. In the second step, CD4+CD25++bright cells were isolated from the CD4+ cells using positive selection with the Robosep® instrument. The yield of CD4+CD25++bright cells ranged from 0.8 - 1.4 X 106 cells, which is approximately 0.5 - 0.71% of the starting population of CD4+ cells. The CD4+CD25dim and CD4+CD25negative cell fractions were also collected. In the flow cytometry open gate data for CD4+CD25++bright cell fraction (Fig. 1A), 90% cells were CD3+ cells and were composed almost entirely of CD4+CD25++bright cells, with 97.4% cells expressing Foxp3, (86.7% bright Foxp3+ and 11.3% dim Foxp3) and had insignificant contamination with monocytes (CD14+, 0.2%), B cells (CD19+, 0.63%), NK cells (CD56+, 0.72%) and cytotoxic T cells (CD8+, 0.39%). Analysis of the same cell fraction gated on singlets demonstrated similar cell frequencies (Fig. 1B). Among CD4+ cells, 99.8% were CD25++bright with 97.4% cells expressing Foxp3 (84.5% bright Foxp3+ and 12.9% dim Foxp3+) and had minimal contamination with other cellular subsets. The Robosep® isolated CD4+CD25++bright cell fraction was designated as nTregs.
[0060] The Robosep®-isolated nTregs were characterized functionally for ability to suppress autologous CD4+CD25negative T cells and for their capacity to produce cytokines upon in vitro stimulation. The nTregs mediated modest suppression of proliferation of autologous CD4+CD25negative T cells at 1:1 ratio as assessed by CFSE dye dilution with a decrease in CFSE low cells from 91.7% to 70.6%. The cytokine expression profile of the Robosep® isolated CD4+CD25++bright and CD25negative cell populations was examined following in vitro stimulation with PMA (phorbol myristate acetate) plus Ionomycin for 5 hours (Fig. 5). To rule out contamination with non-nTreg cells that can be induced to secrete cytokines, an open gate was again used to encompass all cells in the Robosep®-isolated CD4+CD25++bright cell fraction including doublets, triplets and other possible contaminating cells (Fig.5A). Very low frequencies of cytokine positive cells were identified in the total cell population, consisting of IL-17 (0.27%), IL-2 (3.9%), IFNγ (0.79%) and TNFα (2.9%). A majority of the cytokine expressing cells were CD4+ T cells, showing expression of IL-17 (0.16%), IL-2 (2.7%), IFNγ (0.59%), and TNFα (2.63%). Analysis performed on gated singlets (Fig. 5B) showed slightly
lower cytokine expressing cell frequencies of IL17 (0.12%), IL2 (2.4%), IFNγ (0.63%) and TNFα (2.49%). In contrast, the CD4+CD25negative cells expressed very high percentages of all cytokines in both types of analyses (Figs. 5C, D). Boolean gating analysis (Table 2) of cytokine expressing cells in open gate for 16 possible profile end points revealed that 94.8% of cells in the CD4+CD25++bright cell fraction did not express any cytokine. A single triple positive IFNγ+IL2+IL17+ TNFα- clone with frequency of 0.18% was observed consistently in two experiments. Cryopreservation and thawing of CD4+CD25++bright and CD4+CD25negative cells did not alter the cytokine production. Based on these results, it is contended that the methodology described herein can lead to purification of a CD4+CD25++bright cell fraction which is strongly Foxp3+ and exhibits preferred attributes of nTregs and constitutes an appropriate source of cells for ex vivo expansion of nTregs.
[0061] Ex vivo expansion of nTregs: Freshly isolated CD4+CD25++bright cells were expanded in X-Vivo complete medium containing CD3/CD28 expander beads and rhIL-2, with and without addition of rapamycin for 19 days and compared with similarly expanded CD4+CD25dim and CD4+CD25negative cells (Fig. 6). Fold expansion of the cellular fractions in presence of rapamycin (Fig 6A), shows a mean of 60 fold expansion in CD4+CD25++bright cells (10 subjects) whereas the CD4+CD25dim (8 subjects) and CD4+CD25negative (3 subjects) cells expanded to an average of 216- and 432-fold respectively. Rapamycin is known to selectively block expansion of CD4+CD25negative T effector cells, while allowing the growth of CD4+CD25++bright Tregs with maintenance of high Foxp3 protein expression and suppressor function (Powell et al., 1999 J. Immunol. 162, 2775; Kahan and Camardo, 2001, Transplantation 72, 1181; Battaglia et al., 2006, J. Immunol. 177, 8338). Cells cultured without rapamycin exhibited tremendous expansion (Fig 6B) and showed marked expansion in all 3 cell populations when rapamycin was omitted from the cultures. Isolated CD4+CD25++bright CD4+CD25dim and CD4+CD25negative cells expanded up to an average of 1200-fold, 64000-fold and 66490-fold respectively without the addition of rapamycin.
[0062] Identity, Purity and Potency of expanded natural T regulatory cells: Phenotypic characterization of expanded nTregs was performed by multicolor flow cytometry analysis on cryopreserved cells (Perfetto et al., 2004 Nat Rev. Immunol. 4, 648; Lamoreaux et al., Nat Protoc. 1, 1507; Darrah et al., 2007, Nat Med. 13, 843). Because the expansion protocol has been developed for infusion of nTregs into patients, it was suggested by FDA that open gate
analysis be performed on the entire population to include doublets, triplets and other potentially contaminating cells. Using an open gate analysis (Fig. 2A), cell viability was 96%; CD4 purity was 99.2%, with 97.6% CD25++ bright cells of which 98.3% cells were Foxp3+. The CD3+ CD4+CD25++bright Foxp3+ cells were found to be mostly negative for expression of CD127, and 94.3% of cells were CD45RO+CD27+ indicating that they were of memory phenotype. These cells were CD57 negative implying that telomeres were preserved in these cells. Similar properties were observed by conventional flow cytometry analysis of singlets (Fig. 2B). The expanded nTregs showed minimal contamination with other cell populations (Fig. 7). A representative open gate analysis (Fig. 7A), demonstrates extremely low frequencies of contaminating monocytes (CD14, 0.057%), B cells (CD19, 0.16%), NK cells (CD56, 0.09%) and cytotoxic T cells (CD8, 0.099%). Similar results were obtained when cells were gated on singlets (Fig. 7B). Means and SD of frequencies of non-Treg contamination observed in expanded CD4+CD25++bright cells from four different buffy coats consisted of monocytes (CD14+, 0.16±0.09%), B cells, (CD19+, 0.19±0.07%), NK cells (CD56+, 0.19±0.08%) and CD8 T cells (0.09±0.03%) (Fig. 7C).
[0063] The expanded nTregs exhibited potent suppressor activity in the classical suppression assay (Brusko et al., 2007 Immunol. Invest. 36, 607). Autologous CD4+CD25negative responder cells were labeled with CFSE dye, stimulated with CD3/CD28 dynal beads and expanded nTregs were mixed with responder cells in 1:1 and 1:10 ratio for 4 days. Proliferation and cell division were analyzed on day 4 (Fig. 3). At a 1:1 ratio of CD4+CD25++bright cells with CD4+CD25negative responder cells, proliferation (as assessed by CFSE low cells) was reduced from 63.4% to 5.27% (91.6% suppression) whereas addition of CD+CD25dim to CD4+CD25negative responder cells at 1:1 ratio only reduced proliferation from 63.4% to 41.4% (34.7% suppression). Suppression was minimal at a 1:10 ratio regardless of the population used. Summary data from three donors (Fig. 3E) showing significant inhibition of proliferation function at 1:1ratio of CD4+CD25++Bright cells with CD4+CD25Negative responder cells which resulted in a decrease of CFSE low cells from 50.9±8.48% to 4.425±1.126% (92.07±0.98% suppression). Suppression/Inhibition of proliferation was statistically insignificant with addition of CD4+CD25Dim cells to autologous CD4+CD25Negative responder cells.
[0064] Intracellular cytokine expression in stimulated expanded nTreg cells: Assays for intracellular cytokines were performed on frozen and thawed expanded cells as described in the
method section. Expanded CD4+CD25++bright, CD4+CD25dim and CD4+CD25negative cells were stimulated with PMA plus Ionomycin for 5 hours, stained for surface markers and intracellular cytokines were analyzed by multicolor flow cytometry (Perfetto et al., 2004 Nat Rev. Immunol. 4, 648; Lamoreaux et al., Nat Protoc. 1, 1507; Darrah et al., 2007, Nat Med. 13, 843). In every experiment, extreme care was taken to use the ViViD dye in the staining protocol for live/dead cell discrimination. In all the experiments, gating was done on live cells where >95% cells were negative for ViViD dye. The analysis on open gate (Fig. 4A) shows that expanded CD4+CD25++bright nTregs do not convert to TH17 cells (0.13% IL-17 expression) and have an extremely reduced frequency of cytokine expressing cells for IFNγ (0. 51%), TNFα (1.01%), IL- 2 (1.53%) and a low frequency of cells with the degranulation marker CD107a (0.06%). In contrast, CD4+CD25dim and CD4+CD25negative cells expressed high frequencies of cytokine positive cells. The intracellular cytokine analysis using singlet populations in CD4+CD25bright cells also revealed minimal cytokine expressing cells (Fig. 4B). Data for the percent cytokine expressing cells in four different experiments is presented (Fig.4C) and shows the minimal cytokine expression in CD4+CD25++bright cells in comparison to CD4+CD25dim and CD4+CD25negative cells. Boolean gating analysis for all the cytokine combinations was determined and 32 possible profile end points were scored and frequencies of more than 0.1% were highlighted (Table 1). Only one triple combination clone of IL-2+ IFNγ+ TNFα+ cells with frequency of 0.16% was observed. The same clone was observed in three additional experiments. Approximately 96% of the cells did not express any cytokines. Interestingly, the cytokine profile of expanded CD4+CD25++bright cells cultured without rapamycin (Fig. 8) manifests an outgrowth of cytokine expressing cells despite the fact that they were present at very low frequencies in freshly isolated cells. Addition of rapamycin inhibited the growth and expansion of cytokine- secreting cells in this population during the 19 day culture period. Analysis of culture supernatants of three different samples of freshly isolated nTregs and nTregs expanded according to the expansion protocol results in very low or undetectable levels of IL-10, TGFβ, or pro- inflammatory cytokines in both fresh and expanded nTreg cell populations (Table 3).
DISCUSSION
[0065] For the initial isolation of nTregs, peripheral CD4+ cells with bright expression of CD25 were used as the main marker and the Robosep® magnetic cell isolation method was used. The choice of markers on which to isolate human nTregs for expansion has been controversial.
As Foxp3 is an intracellular protein it cannot be used to isolate viable cells. CD25 represents the chain of the IL-2 receptor that is essential for the generation and maintenance of nTregs and high expression of CD25 is commonly used in protocols for isolating peripheral Tregs. However CD25 is also upregulated upon cellular activation, thus recently activated effector CD4+T cells may be confused with nTregs and iTregs. Nevertheless there are differences between CD4+CD25++bright nTregs and activated T cells with respect to the characteristics of CD25 expression. Human and mouse CD4+ cells with potent regulatory properties express high and sustained levels of CD25, whereas recently activated T cells express transient and low levels of CD25 (Kuniyasu et al., 2000, Int. Immunol. 12, 1145; Baecher-Allan et al., 2001, J. Immunol. 167, 1245). Thus a stable and high expression of CD25 is an essential characteristic of nTregs. In the present report, the expanded nTreg population derived from the CD4+CD25++bright cell fraction maintained a stable CD25 bright expression. Other markers, such as latency-associated peptide (LAP) and IL-1 receptor type I & II (CD121 a/CD121b) have also been used for Treg characterization. These markers are not expressed on resting or expanded Foxp3+ Tregs, but are rapidly induced and expressed on Foxp3+ Tregs for a short time period after TCR-mediated activation (Tran et al., 2009, Blood 113, 5125). Thus these markers can only isolate TCR activated Foxp3+ Tregs but not resting or expanded Foxp3+Tregs.
[0066] A feature that is increasingly used for isolating Tregs from blood is the absence of CD127, the IL-7Rα, which is abundantly expressed on naïve cells. It is contended that CD127 negativity should not be used to select the initial starting population for nTreg expansion for several reasons. First, by doing so, it may also eliminate the thymic derived resting precursors of nTregs which may express CD127. Recent data of Treg expansion using umbilical cord blood which is enriched in naïve cells supports this contention. Umbilical cord blood T regulatory cells isolated by positive selection using either AutoMACS or CliniMACS based on CD4+CD25+ expression, not on absence of CD127expression, that were cultured with anti CD3/CD28 mAb coated Dynabeads with IL2 and rapamycin (Godfrey et al., 2005, Blood 105, 750; Hippen et al., 2008, Blood 112, 2847) showed approximately 100 fold to 199 fold expansion. Foxp3 expression was 72.6% in one report (Hippen et al., 2008, Blood 112, 2847) and they exhibited potent suppressor activity of >95% (Tran et al., 2009, Blood 113, 5125) and 58±11% (Hippen et al., 2008, Blood 112, 2847), respectively in allogeneic mixed lymphocyte reaction. In the freshly isolated CD4+CD25++bright population that was isolated on Robosep® in the experiments
described herein, the expression of CD127 was approximately 1%, and the final expanded population was negative for CD127. Thus CD127 negativity may be more useful for characterizing functional expanded nTregs and less so for initial selection of the population to be expanded. Another reason against using absence of CD127 expression for selecting the initial population is that CD127 negativity as a biomarker cannot discriminate between Tregs and T effector cells. Upon cellular activation CD127 is downregulated in CD4+ cells including CD4+ CD25++bright nTregs. Thus when CD127low/- expression is used in combination with CD25++bright expression for isolating Tregs, it can concentrate a heterogeneous subpopulation of cells consisting of nTregs, iTregs and activated CD4+CD25++ non Tregs which can be transiently positive for Foxp3. The CD4+CD25++CD127low/- population may have a greater potential for differentiating into cytokine secreting effector cells. A previous study that has used FACS sorting for isolating CD4+ Tregs based on CD25 expression and CD127 negativity and subsequent expansion resulted in contamination with effector cells based on their cytokine profile (Putnam e al., 2009 Diabetes 58, 652). In that report, the expanded cells, despite showing Foxp3 expression of >95%, manifested substantial cytokine producing cells. None of the previous culture systems described in the literature that have had different results of cytokine secreting effector cell- contamination have examined the multiple pro-inflammatory cytokine secreting potential of expanded Tregs on a single cell basis by multi color flow cytometry with proper compensation. The rigorous examination of cytokine expression of the expanded nTreg population described herein has ruled out the presence of contaminating effector cells to a major extent.
[0067] This is an important criterion because the mechanism of natural Treg function is by cell-to-cell interaction and not via secretion of cytokines IL-10 and TGF-β which are rarely found in the supernatants of in vitro nTreg assays and that the use of anti-IL-10 or anti-TGF-β antibodies fails to abrogate suppression. Another distinct subset of regulatory T cells (Tr-1) suppresses immune responses via cell-to-cell interactions and/or the production of IL-10 and TGF-β for a variety of antigens. IL-10 is also secreted by other cells like Th-2 cells, macrophages, monocytes and dendritic cells. Thus it is important to demonstrate the suppressor function of nTregs without evidence of cytokine secretion.
[0068] Rapamycin was used throughout the culture for ex vivo selective expansion of stable nTregs. CD28 costimulation, IL-2 and rapamycin were required to consistently expand nTregs
that had suppressor activity in vitro, in the absence of contaminating cytokine secreting effector cells. Cells expanded in rapamycin have been shown to prevent xenogeneic GVHD. Rapamycin selectively blocks expansion of CD4+CD25negative T effector cells, whereas it allows CD4+CD25++bright Treg growth. Inhibition of the mTOR pathway in presence of IL-2 allows Tregs to be constantly activated through the STAT-5 pathways and promotes their preferential expansion and Foxp3 expression. Limited use of rapamycin in the expansion phase is not effective in curtailing the expansion of T effector cells. Thus, in studies where rapamycin was only added early in the culture for 5 days or 7 days, FACS purified CD4+CD127lowCD25high cells expanded up to 1500 fold after 14 days in one study and MACS bead sorted CD4+CD127lowCD25high cells expanded up to 800 fold after 21 days in the other study. Surprisingly, CD4+CD25highCD49-CD127- cells used in another study showed very low ex vivo expansion of only 12-fold even in the absence of rapamycin after 33 days of culture. In all these instances, there was evidence of contamination by cytokine secreting effector cells. These studies also demonstrate less impressive results of suppression than was observed with cells derived in the expansion protocol described herein. As examples, Foxp3 was 76.4% and exhibited suppression of 60% of allogeneic CD8+ proliferation, or Foxp3 was 95.5% and still exhibited only 47% suppression of allogeneic CD4+CD25negative cells, or Foxp3 was 81.4% in bead purified expanded cells and showed 70 to 80 % suppressive activity on allogeneic CD4+ effector cells. In the experiments described herein, expanded nTregs were 98.3% Foxp3+ and had suppressor activity of 91.6%. The ability of nTregs to suppress proliferation of responder T cells is mediated through a cell-cell contact-dependent mechanism and they do not secrete either IL-10 or TGFβ, whereas stimulation of other regulatory cells results in secretion of IL10 and TGF . In the method described herein, analysis of culture supernatant of expanded nTregs did not show detectable levels of TGFβ and had extremely low levels of IL10.
[0069] In conclusion, the data described herein demonstrate that using an appropriate concentration of nine monoclonal antibodies to isolate CD4+ cells from buffy coat followed by isolation of CD25++bright cells from CD4+ cells on automatic Robosep® instrument in a custom protocol we can isolate a population of highly purified nTregs from human peripheral blood. These cells have been successfully expanded ex vivo by 60-fold resulting in an ideal population of human nTregs in sufficient quantities for possible cell therapy. Properties of expanded cells are close to optimal for nTregs, characterized by phenotypically stable expression of
CD4+CD25++brightFoxp3+ population, potent suppression of CD4+CD25negative T cells without secretion of IL-10 or TGF-β and no propensity to convert into effector TH17 cells or production of pro-inflammatory cytokine upon in-vitro stimulation with PMA/Ionomycin. Based on Boolean gating analysis of all the cytokine combination and frequency analysis, 96% of the cells did not express any cytokine. It is proposed that these cells are currently the most suitable for evaluating in clinical trials of Tregs. The procedure has the potential for further optimization by incorporating the recently described cell-based artificial antigen presenting cells (aAPCs) preloaded with anti CD3/CD28 mAbs to achieve higher level of Treg expansion (Godfrey et al., 2005Blood 105, 750). Modification of aAPCs to co-express OX40L or 4-1BBL was shown to achieve more than 1250-fold expansion of umbilical cord blood Tregs (Godfrey et al., 2005Blood 105, 750). Table 1 - Expanded nTregs: Boolean gating analysis of cytokine expressing cells
[0070] nTregs, expanded as per protocol contain none/minimal single or multiple cytokine or CD107a expressing cells: Boolean gating analysis of a representative experiment of expanded nTregs stimulated with PMA/Ionomycin for 5 hrs and stained for intracellular cytokines IL17, IL2, IFNγ, TNFα, and CD107a and analyzed on FLOWJO. Frequencies of cells positive for 1,2,3,4 and 5 measures are depicted. * Frequencies > 0.1%. Table 2 - Freshly isolated nTregs: Boolean gating analysis of cytokine expressing cells No of cytokines Cytokine expressing cells Total Frequency
[0071] Freshly isolated nTregs as per protocol contain minimal single or multiple cytokine expressing cells. Boolean gating analysis of a representative experiment of freshly isolated CD4+CD25++bright nTreg cells stimulated with PMA/Ionomycin for 5 hrs and stained for intracellular cytokines IL17, IL2, IFNγ and TNFα were analyzed on FLOWJO. Frequencies of cells expressing 1, 2, 3 and 4 cytokines are depicted. *Frequencies >0.1% Table 3 - Analysis of cytokines in culture supernatants of freshly isolated and expanded nTregs Freshly isolated Expanded nTregs
[0072] Freshly isolated and expanded nTregs secrete none/minimal cytokines in culture supernatants. Freshly isolated and expanded nTregs were cultured at a cell concentration of 1 X 106 each and stimulated with PMA/Ionomycin for 5 hrs. Supernatants were collected and analyzed for cytokines as described. Values of individual cytokines in pg/ml are shown. Data represents mean of 3 experiments. ** TGFβ values were undetectable less than 470 pg ml-1. OTHER EMBODIMENTS [0073] Any improvement may be made in part or all of the compositions, cells, kits, and method steps. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended to illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. Any statement herein as to the nature or benefits of the invention or of the preferred embodiments is not intended to be limiting, and the appended claims should not be deemed to be limited by such statements. More generally, no language in the specification should be construed as indicating any non-claimed element as being essential to the practice of the invention. This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contraindicated by context.
Claims
What is claimed is: 1. A method of isolating and expanding natural T regulatory (nTreg) cells comprising: obtaining blood from an individual;
obtaining at least one Buffy coat from the blood;
isolating CD4+CD25++brightFoxp3+ nTreg cells from the at least one Buffy coat; and expanding the isolated CD4+CD25++brightFoxp3+ nTreg cells by culturing the
CD4+CD25++brightFoxp3+ nTreg cells in the presence of rapamycin.
2. The method of claim 1, wherein the blood is peripheral blood and the individual is a healthy donor.
3. The method of claim 1, wherein the isolated and expanded CD4+CD25++brightFoxp3+ nTreg cells are capable of suppressing CD4+CD25negative T cells as measured by at least one T cell assay.
4. The method of claim 3, wherein the isolated and expanded CD4+CD25++brightFoxp3+ nTreg cells do not express cytokines as measured by at least one cytokine assay.
5. The method of claim 1, wherein isolating CD4+CD25++brightFoxp3+ nTreg cells from the Buffy coat comprises enrichment of CD4+ T cells by negative selection using agents which specifically bind cell markers comprising: CD8, CD16, CD19, CD36, CD56, CD66b, TCRγδ, glycophorin A and P9, and isolation of CD25++bright cells by positive selection from the enriched CD4+ T cells using at least one agent which specifically binds to CD25.
6. The method of claim 5, wherein the step of positive selection from the enriched CD4+ T cells using at least one agent which specifically binds to CD25 is performed in an
immunomagnetic cell separator, and the at least one agent which specifically binds to CD25 is a monoclonal antibody to CD25.
7. A method of preventing or treating a disease or condition associated with an immune response in a subject comprising: providing CD4+CD25++brightFoxp3+ nTreg cells isolated and expanded by the method of claim 1; and administering the cells to the subject in a therapeutically effective amount for decreasing the immune response in the subject.
8. The method of claim 7, wherein diseases or conditions associated with immune responses comprise: autoimmunity, allergies, diabetes, inflammation, graft versus host reactions, organ transplantation, inflammatory bowel disease and viral diseases.
9. The method of claim 7, wherein administering the cells to the subject results in at least one selected from the group consisting of: alleviation of type 1 diabetes, immunosuppression in host vs. graft disease, and induction of tolerance in solid organ transplantation to prevent graft rejection.
10. A composition consisting of a pharmaceutically acceptable carrier and a therapeutically effective amount of CD4+CD25++brightFoxp3+ nTreg cells isolated and expanded by the method of claim 1.
11. The composition of claim 10, wherein the isolated CD4+CD25++brightFoxp3+ nTreg cells are capable of suppressing CD4+CD25negative T cells as measured by at least one T cell assay.
12. The composition of claim 10, wherein the isolated CD4+CD25++brightFoxp3+ nTreg cells do not express cytokines as measured by at least one cytokine assay.
13. A method of tissue or organ transplantation to a subject comprising: (a) obtaining the tissue or organ to be transplanted from a donor; (b) transplanting said tissue or organ to the subject; (c) delivering the composition of claim 10 to the subject prior to and/or subsequent to transplantation of the tissue or organ to the subject.
14. The method of claim 13, wherein the composition of claim 10 is delivered to the subject after the transplantation at the time of maximum lymphopenia in the subject with an
immunosuppressive induction using a T cell depletion protocol.
15. The method of claim 14, wherein the composition of claim 10 is delivered to the subject 6 to 11 days after transplantation, and the T cell depletion protocol is Thymoglobuliun or Campath 1H treatment of the subject.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US37030610P | 2010-08-03 | 2010-08-03 | |
US61/370,306 | 2010-08-03 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2012018930A1 true WO2012018930A1 (en) | 2012-02-09 |
Family
ID=45559811
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2011/046447 WO2012018930A1 (en) | 2010-08-03 | 2011-08-03 | Methods of isolating and expanding human t regulatory cells and uses thereof for cellular therapy |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2012018930A1 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018080541A1 (en) * | 2016-10-31 | 2018-05-03 | Seattle Children's Hospital (dba Seattle Children's Research Institute) | Method for treating autoimmune disease using cd4 t-cells with engineered stabilization of expression of endogennous foxp3 gene |
CN114008192A (en) * | 2019-04-30 | 2022-02-01 | 加利福尼亚大学董事会 | Bead-free ex vivo expansion of human regulatory T cells |
US11384336B2 (en) | 2016-12-07 | 2022-07-12 | East Carolina University | Compositions and methods for in vitro cultivation and/or expansion of regulatory T cells |
US11713459B2 (en) | 2018-04-27 | 2023-08-01 | Seattle Children's Hospital | Expression of FOXP3 in edited CD34+ cells |
Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20020034501A1 (en) * | 2000-05-18 | 2002-03-21 | Robert Pawliuk | Methods and compositions for promoting angiogenesis using monocytes |
US20030092078A1 (en) * | 1999-05-28 | 2003-05-15 | Stemcell Technologies Inc. | Method for separating cells using immunorosettes |
US20070207161A1 (en) * | 2003-10-16 | 2007-09-06 | Ralph Stephen J | Immunomodulating Compositions and uses Therefor |
US20080279826A1 (en) * | 2005-02-25 | 2008-11-13 | Fondazione Centro San Rafaele Del Monte Tabor | Method for Expanding Cd4+ Cd25+ T Regulator Cells |
US20080287358A1 (en) * | 2005-04-25 | 2008-11-20 | Noelle Randolph J | Regulatory T Cell Mediator Proteins and Uses Thereof |
US20090208471A1 (en) * | 2006-04-07 | 2009-08-20 | Yun Theodore J | Isolation and Use of Human Regulatory T Cells |
US20100003271A1 (en) * | 2006-12-15 | 2010-01-07 | Board Of Regents Of The University Of Texas System | Nitric oxide increases switching of t cells into t regulatory cells |
US20100034782A1 (en) * | 2006-05-31 | 2010-02-11 | Nader Najafian | Methods of Using Anti-Thymocyte Globulin and Related Agents |
US20100150893A1 (en) * | 2002-06-27 | 2010-06-17 | The General Hospital Corporation | Methods of organ regeneration |
-
2011
- 2011-08-03 WO PCT/US2011/046447 patent/WO2012018930A1/en active Application Filing
Patent Citations (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030092078A1 (en) * | 1999-05-28 | 2003-05-15 | Stemcell Technologies Inc. | Method for separating cells using immunorosettes |
US20020034501A1 (en) * | 2000-05-18 | 2002-03-21 | Robert Pawliuk | Methods and compositions for promoting angiogenesis using monocytes |
US20100150893A1 (en) * | 2002-06-27 | 2010-06-17 | The General Hospital Corporation | Methods of organ regeneration |
US20070207161A1 (en) * | 2003-10-16 | 2007-09-06 | Ralph Stephen J | Immunomodulating Compositions and uses Therefor |
US20080279826A1 (en) * | 2005-02-25 | 2008-11-13 | Fondazione Centro San Rafaele Del Monte Tabor | Method for Expanding Cd4+ Cd25+ T Regulator Cells |
US20080287358A1 (en) * | 2005-04-25 | 2008-11-20 | Noelle Randolph J | Regulatory T Cell Mediator Proteins and Uses Thereof |
US20090208471A1 (en) * | 2006-04-07 | 2009-08-20 | Yun Theodore J | Isolation and Use of Human Regulatory T Cells |
US20100034782A1 (en) * | 2006-05-31 | 2010-02-11 | Nader Najafian | Methods of Using Anti-Thymocyte Globulin and Related Agents |
US20100003271A1 (en) * | 2006-12-15 | 2010-01-07 | Board Of Regents Of The University Of Texas System | Nitric oxide increases switching of t cells into t regulatory cells |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018080541A1 (en) * | 2016-10-31 | 2018-05-03 | Seattle Children's Hospital (dba Seattle Children's Research Institute) | Method for treating autoimmune disease using cd4 t-cells with engineered stabilization of expression of endogennous foxp3 gene |
CN110139675A (en) * | 2016-10-31 | 2019-08-16 | 西雅图儿童医院(Dba西雅图儿童研究所) | With the method with the CD4 T cell for being engineered stable endogenous FOXP3 gene expression treatment autoimmune disease |
US11712454B2 (en) | 2016-10-31 | 2023-08-01 | Seattle Children's Hospital | Method for treating autoimmune disease using CD4 T-cells with engineered stabilization of expression of endogenous FOXP3 gene |
CN110139675B (en) * | 2016-10-31 | 2023-09-29 | 西雅图儿童医院(Dba西雅图儿童研究所) | Method of treating autoimmune diseases with CD4T cells having engineered stable endogenous FOXP3 gene expression |
US11384336B2 (en) | 2016-12-07 | 2022-07-12 | East Carolina University | Compositions and methods for in vitro cultivation and/or expansion of regulatory T cells |
US11713459B2 (en) | 2018-04-27 | 2023-08-01 | Seattle Children's Hospital | Expression of FOXP3 in edited CD34+ cells |
CN114008192A (en) * | 2019-04-30 | 2022-02-01 | 加利福尼亚大学董事会 | Bead-free ex vivo expansion of human regulatory T cells |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Marín Morales et al. | Automated clinical grade expansion of regulatory T cells in a fully closed system | |
JP6422344B2 (en) | Methods for increasing allogeneic antigen-reactive regulatory T cells | |
Pahwa et al. | Isolation and expansion of human natural T regulatory cells for cellular therapy | |
Torelli et al. | A good manufacturing practice method to ex vivo expand natural killer cells for clinical use | |
JP2015513403A5 (en) | ||
US10857183B2 (en) | Method of hematopoietic stem cell transplants | |
Gregori et al. | Isolation, expansion, and characterization of human natural and adaptive regulatory T cells | |
KR20180054663A (en) | Novel populations of CD8 + CD45RClow TREGS and their uses | |
US20140044687A1 (en) | Method for using regulatory t cells in therapy | |
Ukena et al. | Human regulatory T cells of G-CSF mobilized allogeneic stem cell donors qualify for clinical application | |
KR20130065657A (en) | New methods for isolating tr1 cells | |
WO2012018930A1 (en) | Methods of isolating and expanding human t regulatory cells and uses thereof for cellular therapy | |
RU2391401C2 (en) | METHOD OF OBTAINING POPULATION OF HUMAN CD4+CD25+Foxp3+ T-LYMPHOCYTES ex vivo, DISEASE TREATMENT METHOD | |
Gołąb et al. | Impact of culture medium on CD4+ CD25highCD127lo/neg Treg expansion for the purpose of clinical application | |
Bresatz et al. | Isolation, propagation and characterization of cord blood derived CD4+ CD25+ regulatory T cells | |
KR20130093083A (en) | Il-13 producing tr1-like cells and use thereof | |
WO2011013568A1 (en) | Immunosuppressive γδt cell | |
EP2597463A1 (en) | Methods of enrichment and isolation of regulatory T-cells and use of the same | |
US20220072040A1 (en) | Treatment method for graft-versus-host disease | |
US20220333072A1 (en) | The process for manufacturing of antigen-specific t lymphocytes | |
Rubio‐Azpeitia et al. | Clinical grade production of IL‐15 stimulated NK cells for early infusion in adult AML patients undergoing haploidentical stem cell transplantation with post‐transplant cyclophosphamide | |
RU2437933C1 (en) | METHOD OF HUMAN REGULATORY CD4+CD25+FOXP3+T-CELL ENRICHMENT ex vivo | |
EP4101925A1 (en) | Method for isolating autologous tumor antigen-reactive cd8 t cell by using cd71, and application thereof | |
Sitnicka et al. | Functional Resolution of Long-Term-and Short-Term-Hematopoietic Stem Cells | |
RU2766691C9 (en) | NEW SUBPOPULATION OF TREG CD8+CD45RClow CELLS AND ITS APPLICATIONS |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 11815270 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 11815270 Country of ref document: EP Kind code of ref document: A1 |