CN102168066A - Method for in vitro induction of specific cytotoxic T lymphocytes of hepatitis B virus (HBV) - Google Patents

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

The method of external evoked hepatitis b virus specificity cell toxicity T lymphocyte

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.