CN102168066A - Method for in vitro induction of specific cytotoxic T lymphocytes of hepatitis B virus (HBV) - Google Patents
Method for in vitro induction of specific cytotoxic T lymphocytes of hepatitis B virus (HBV) Download PDFInfo
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- CN102168066A CN102168066A CN2011100333275A CN201110033327A CN102168066A CN 102168066 A CN102168066 A CN 102168066A CN 2011100333275 A CN2011100333275 A CN 2011100333275A CN 201110033327 A CN201110033327 A CN 201110033327A CN 102168066 A CN102168066 A CN 102168066A
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- 238000000034 method Methods 0.000 title claims abstract description 16
- 241000700721 Hepatitis B virus Species 0.000 title claims abstract description 7
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 title abstract description 6
- 238000000338 in vitro Methods 0.000 title abstract 3
- 230000006698 induction Effects 0.000 title abstract 3
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 37
- 239000000427 antigen Substances 0.000 claims abstract description 28
- 102000036639 antigens Human genes 0.000 claims abstract description 28
- 108091007433 antigens Proteins 0.000 claims abstract description 28
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 16
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 15
- 229920001184 polypeptide Polymers 0.000 claims abstract description 13
- 210000000612 antigen-presenting cell Anatomy 0.000 claims abstract description 10
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 8
- 108010090804 Streptavidin Proteins 0.000 claims abstract description 4
- 229960002685 biotin Drugs 0.000 claims abstract description 4
- 235000020958 biotin Nutrition 0.000 claims abstract description 4
- 239000011616 biotin Substances 0.000 claims abstract description 4
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000011324 bead Substances 0.000 claims description 15
- 230000000763 evoking effect Effects 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 6
- 238000005516 engineering process Methods 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 230000007541 cellular toxicity Effects 0.000 claims description 4
- 108010088729 HLA-A*02:01 antigen Proteins 0.000 claims description 3
- 108010002350 Interleukin-2 Proteins 0.000 claims description 3
- 238000005194 fractionation Methods 0.000 claims description 3
- 238000002826 magnetic-activated cell sorting Methods 0.000 claims description 3
- 210000005259 peripheral blood Anatomy 0.000 claims description 3
- 239000011886 peripheral blood Substances 0.000 claims description 3
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 238000001179 sorption measurement Methods 0.000 claims description 3
- 238000010186 staining Methods 0.000 claims description 3
- 230000003321 amplification Effects 0.000 abstract 1
- 150000001875 compounds Chemical class 0.000 abstract 1
- 238000005034 decoration Methods 0.000 abstract 1
- 238000000684 flow cytometry Methods 0.000 abstract 1
- 238000003199 nucleic acid amplification method Methods 0.000 abstract 1
- 208000002672 hepatitis B Diseases 0.000 description 5
- 208000000419 Chronic Hepatitis B Diseases 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000000890 antigenic effect Effects 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 206010059193 Acute hepatitis B Diseases 0.000 description 2
- 208000037628 acute hepatitis B virus infection Diseases 0.000 description 2
- 230000000840 anti-viral effect Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 210000005229 liver cell Anatomy 0.000 description 2
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 108091036055 CccDNA Proteins 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- OSBADCBXAMSPQD-YESZJQIVSA-N Phe-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CC=CC=C2)N OSBADCBXAMSPQD-YESZJQIVSA-N 0.000 description 1
- NJJBATPLUQHRBM-IHRRRGAJSA-N Phe-Pro-Ser Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)N)C(=O)N[C@@H](CO)C(=O)O NJJBATPLUQHRBM-IHRRRGAJSA-N 0.000 description 1
- OLIJLNWFEQEFDM-SRVKXCTJSA-N Ser-Asp-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 OLIJLNWFEQEFDM-SRVKXCTJSA-N 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000007849 functional defect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
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Abstract
The invention discloses a method for in vitro induction of specific cytotoxic T lymphocytes of hepatitis B virus (HBV). The method comprises the following steps of: 1, preparing artificial antigen presenting cells (aAPC) capable of loading any polypeptides; 2, loading aAPC on epitope peptides; and 3, loading aAPC in vitro induction HBV antigen specific CTL of antigen peptides; measuring the number of obtained antigen specific CTL by a Tetramer decoration method, namely combining streptavidin marked by fluorescein and four MHC-peptide compounds marked by biotin to form the Tetramer, namely MHC-peptide tetramer; and after the MHC-peptide tetramer is combined with TCR on the antigen specific CTL, measuring by a flow cytometry. The method can be applied to the amplification of T lymphocytes resisting the HBV.
Description
Technical field
The invention belongs to fields such as the cytobiology of biotechnology and immunology, relate to HBV cytotoxic T lymphocyte (Cytotoxic T Lymphocyte, CTL) the epitope peptide bag is by artificial antigen presenting cells (artificial antigen-presentingcells, aAPC), be used to kill and wound the antigenic cell of expression HBV at external evoked HBV specific CTL.
Background technology
The treatment of chronic HBV infection remains the great difficult problem that medical circle faces, and the medicine of present anti-HBV treatment can't thoroughly be removed virus, and how effectively removing HBV still is the focus for the treatment of hepatitis B research.
(1) (Cytotoxic T Lymphocytes CTLs) is the important factor that body is removed HBV to HBV specific cytotoxic T lymphocyte.The preparation method of research polyclone, polyspecific, the specific CTL of replying by force, significant to the treatment of chronic hepatitis B.
There are some researches show that the HBV specific CTL is the important factor that body is removed HBV: the acute hepatitis B patient removes HBV mainly by CD8
+The CTL mediation, CTL can be by causing the HBV (especially HBV cccDNA) in two approach removings of hepatolysis and/or non-dissolving liver cell, performance antiviral effect.The intravital CTL of acute hepatitis B patient is often for polyclone, polyspecific, reply and high IFN-γ excretory by force.The HBV persistent infection of opposite chronic hepatitis B patient is not enough relevant with host T cellullar immunologic response.Discover, chronic hepatitis B patient body endoantigen presenting cells (Antigen Presenting Cells, APC) angtigen presentation functional defect, the information of virus antigen can not be passed to the immunity system of body, so the intravital specific CTL of chronic hepatitis B patient often shows as few specificity, weak response.This shows that the polyclone of importing q.s of adopting, polyspecific, the specific CTL of replying are by force removed virus, can replenish mutually with present antiviral therapy, separate the problem that must not thoroughly remove the liver cell inner virus.
(2) aAPC is with artificial method simulation APC) function---T lymphocyte activation signal, can be at external activation specific T lymphocyte.Method commonly used at present is at crosslinked anti-CD28 monoclonal antibody of magnetic bead surfaces (mAb) and MHC I-Ig: antigenic peptide complexes, this magnetic bead can be induced the CTL of active antigen peptide specific.Since aAPC have higher can be handling, can artificially control the MHC antigenic peptide complexes, thereby realize CD8
+The regulation and control of T lymphocyte activation, and activation condition is arranged than homogeneous, system easy to control the quality, advantages such as T cell activation weak point proliferating cycle.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of method of external evoked HBV specificity cell toxicity T lymphocyte, adopts this method can be used to increase T lymphocyte of anti-HBV.
In order to solve the problems of the technologies described above, the invention provides a kind of method of external evoked hepatitis b virus specificity cell toxicity T lymphocyte, it is characterized in that may further comprise the steps:
1) but, the artificial antigen presenting cells aAPC of any polypeptide of preparation load:
Adopt MHC I-Ig and anti-CD28mAb bag by magnetic bead: with the direct coated method MHC I-Ig and anti-CD28 mAb to be coated on the magnetic bead, but to be built into the artificial antigen presenting cells aAPC of any polypeptide of load;
2), epitope peptide load aAPC:
The aAPC of MHC I-Ig and anti-CD28 mAb bag quilt washs through PBS, and with PBS epitope peptide being mixed with concentration respectively is 20~40 μ g/ml polypeptide solutions, and the aAPC after will washing again is with 4~8 * 10
8/ ml concentration is resuspended in the described polypeptide solution, gets the aAPC of load antigen peptide;
The sequence of the epitope peptide of load be following any one:
①FLPSD
FFPSV;
②ILCWG
ELMTL;
③QLLWF
HISCL;
④TLATW
VGVNL;
⑤YLVSFGVWIR;
⑥SWLSLLVPFV;
⑦FLLSLGIHLN;
⑧FLLTRILTIP;
⑨ILSTL
PETTV;
⑩NLEDP
ASRDL;
The external evoked HBV antigen-specific of the aAPC of 3), load antigen peptide CTL:
Separate HLA-A*0201 normal people's peripheral blood PBMC cell, obtain CD8 with the sorting of MACS technology
+The T cell; With CD8
+The aAPC of T cell and load antigen peptide in substratum 37 ℃, 5%CO2 cultivates to be stimulated for 3~4 weeks, and described substratum is OpTmizer
TMT-Cell Expansion SFM serum free, 2%heated inactivated humanserum, 100-200IU IL-2; Again with magnet fractionation by adsorption antigen-specific CTL;
Adopt the Tetramer staining to measure its quantity to the antigen-specific CTL that is obtained:
Fluorescein-labeled streptavidin combines with 4 biotin labeled MHC-peptide complex and forms the tetramer (Tetramer), gets the MHC-peptide tetramer; The MHC-peptide tetramer detects by flow cytometer with after TCR on the antigen-specific CTL combines.
Adopt method of the present invention can be used to the to increase T lymphocyte of anti-HBV.
Embodiment
1) but the artificial antigen presenting cells aAPC of preparation load any polypeptide:
Adopt MHC I-Ig and anti-CD28mAb bag by magnetic bead: with the direct coated method MHC I-Ig and anti-CD28 mAb to be coated on the magnetic bead (Dynabeads M-450 is available from invitrogen), but to be built into the artificial antigen presenting cells aAPC of any polypeptide of load;
Concrete grammar: the 0.1M borate buffer solution of sterilization washing magnetic bead twice, make magnetic bead and 1: 1 blended HLA-A2-Ig and anti-CD28 mAb9.3 (monoclonal antibody of anti-CD28, purchase) on turner 4 ℃ hatch 24h.With washing the magnetic bead buffer solution washed twice, hatch 24h in the magnetic bead buffer solution containing to wash again, it goes washing lotion then, adds fresh damping fluid.Last each magnetic bead of the aAPC of preparation contains 0.9 * 10
5The HLA-A2-Ig of molecule and 1.9 * 10
5Anti-CD28 mAb 9.3 molecules.The magnetic bead for preparing can be preserved 3 months for 4 ℃.Magnetic bead behind the bag quilt is aAPC.
2), epitope peptide load aAPC:
The aAPC of MHC I-Ig and anti-CD28 mAb bag quilt washs through PBS, and with PBS epitope peptide being mixed with concentration respectively is 20~40 μ g/ml polypeptide solutions, and the aAPC after will washing again is with 4~8 * 10
8/ ml concentration is resuspended in the described polypeptide solution, gets the aAPC of load antigen peptide;
Following any one of the optional usefulness of the sequence of the epitope peptide of load:
1. FLPSD
FFPSV, that is: Phe Leu Pro Ser Asp Phe Phe Pro Ser Val (SEQ ID NO:1);
②ILCWG
ELMTL;
③QLLWF
HISCL;
④TLATW
VGVNL;
⑤YLVSFGVWIR;
⑥SWLSLLVPFV;
⑦FLLSLGIHLN;
⑧FLLTRILTIP;
⑨ILSTL
PETTV;
⑩NLEDP
ASRDL。
What select for use in the present embodiment is the 1. bar.
The external evoked HBV antigen-specific of the aAPC of 3), load antigen peptide CTL:
Separate HLA-A*0201 normal people's peripheral blood PBMC cell, obtain CD8 with the sorting of MACS technology
+T cell (German milteny company's product " CD8+T Cell Isolation Kit " is pressed the operation of test kit specification sheets); With CD8
+The aAPC of T cell and load antigen peptide in substratum 37 ℃, 5%CO2 cultivates to be stimulated for 3~4 weeks, and described substratum is OpTmizer
TMT-Cell Expansion SFM serum free (Gibco), 2%heated inactivated humanserum (Gibco), 100-200IU IL-2; Again with magnet (DynaMag-2, Dynabeads) fractionation by adsorption antigen-specific CTL (pressing the working instructions operation).
Adopt the Tetramer staining to measure its quantity to the antigen-specific CTL that is obtained:
Fluorescein-labeled streptavidin (purchase) combines with 4 biotin labeled MHC-peptide complex (entrusting commercial company to synthesize) and forms the tetramer (Tetramer), gets the MHC-peptide tetramer; The MHC-peptide tetramer detects by flow cytometer with after TCR on the antigen-specific CTL combines.Antigen-specific CTL ratio improves more than 30% more than 10% than control group.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
SEQ?ID?NO:1
Phe?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val
1 5 10
Claims (1)
1. the method for external evoked hepatitis b virus specificity cell toxicity T lymphocyte is characterized in that may further comprise the steps:
1) but, the artificial antigen presenting cells aAPC of any polypeptide of preparation load:
Adopt MHC I-Ig and anti-CD28mAb bag by magnetic bead: with the direct coated method MHC I-Ig and anti-CD28mAb to be coated on the magnetic bead, but to be built into the artificial antigen presenting cells aAPC of any polypeptide of load;
2), epitope peptide load aAPC:
The aAPC of MHC I-Ig and anti-CD28mAb bag quilt washs through PBS, and with PBS epitope peptide being mixed with concentration respectively is 20~40 μ g/ml polypeptide solutions, and the aAPC after will washing again is with 4~8 * 10
8/ ml concentration is resuspended in the described polypeptide solution, gets the aAPC of load antigen peptide;
The sequence of the epitope peptide of load be following any one:
①FLPSD
FFPSV;
②ILCWG
ELMTL;
③QLLWF
HISCL;
④TLATW
VGVNL;
⑤YLVSFGVWIR;
⑥SWLSLLVPFV;
⑦FLLSLGIHLN;
⑧FLLTRILTIP;
⑨ILSTL
PETTV;
⑩NLEDP
ASRDL;
The external evoked HBV antigen-specific of the aAPC of 3), load antigen peptide CTL:
Separate HLA-A*0201 normal people's peripheral blood PBMC cell, obtain CD8 with the sorting of MACS technology
+The T cell; With CD8
+The aAPC of T cell and load antigen peptide in substratum 37 ℃, 5%CO2 cultivates to be stimulated for 3~4 weeks, and described substratum is OpTmizer
TMT-Cell Expansion SFM serum free, 2%heated inactivated humanserum, 100-200IU IL-2; Again with magnet fractionation by adsorption antigen-specific CTL;
Adopt the Tetramer staining to measure its quantity to the antigen-specific CTL that is obtained:
Fluorescein-labeled streptavidin combines with 4 biotin labeled MHC-peptide complex and forms the tetramer (Tetramer), gets the MHC-peptide tetramer; The MHC-peptide tetramer detects by flow cytometer with after TCR on the antigen-specific CTL combines.
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103667189A (en) * | 2013-09-24 | 2014-03-26 | 上海宇研生物技术有限公司 | CD8 cytotoxic T-lymphocyte for treating lung cancer and preparation method thereof |
CN106795492A (en) * | 2015-06-17 | 2017-05-31 | 深圳市达科为生物工程有限公司 | A kind of preparation method of tumor-specific CTL |
WO2018188235A1 (en) * | 2017-04-14 | 2018-10-18 | 黄月华 | Antigen-presenting signal group of hepatitis b virus antigen peptide combined with liver cancer cell antigen information and application thereof |
CN109475620A (en) * | 2016-03-16 | 2019-03-15 | 耐克西缪恩有限公司 | The production of T cells with antigenic specificity |
CN109913414A (en) * | 2019-03-21 | 2019-06-21 | 吉林省银丰生物工程技术有限公司 | The artificial antigen presenting cell induction agent box of liver cancer AFP specificity |
US10481158B2 (en) | 2015-06-01 | 2019-11-19 | California Institute Of Technology | Compositions and methods for screening T cells with antigens for specific populations |
CN111944018A (en) * | 2020-08-28 | 2020-11-17 | 深圳市乐土生物医药有限公司 | Antigen polypeptide for inducing liver cancer specific cytotoxic T lymphocyte and application thereof |
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Cited By (8)
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---|---|---|---|---|
CN103667189A (en) * | 2013-09-24 | 2014-03-26 | 上海宇研生物技术有限公司 | CD8 cytotoxic T-lymphocyte for treating lung cancer and preparation method thereof |
CN103667189B (en) * | 2013-09-24 | 2015-10-28 | 上海宇研生物技术有限公司 | CD8 toxic T lymphocyte being used for the treatment of lung cancer and preparation method thereof |
US10481158B2 (en) | 2015-06-01 | 2019-11-19 | California Institute Of Technology | Compositions and methods for screening T cells with antigens for specific populations |
CN106795492A (en) * | 2015-06-17 | 2017-05-31 | 深圳市达科为生物工程有限公司 | A kind of preparation method of tumor-specific CTL |
CN109475620A (en) * | 2016-03-16 | 2019-03-15 | 耐克西缪恩有限公司 | The production of T cells with antigenic specificity |
WO2018188235A1 (en) * | 2017-04-14 | 2018-10-18 | 黄月华 | Antigen-presenting signal group of hepatitis b virus antigen peptide combined with liver cancer cell antigen information and application thereof |
CN109913414A (en) * | 2019-03-21 | 2019-06-21 | 吉林省银丰生物工程技术有限公司 | The artificial antigen presenting cell induction agent box of liver cancer AFP specificity |
CN111944018A (en) * | 2020-08-28 | 2020-11-17 | 深圳市乐土生物医药有限公司 | Antigen polypeptide for inducing liver cancer specific cytotoxic T lymphocyte and application thereof |
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