CN106190975A - Melanoma-associated antigen specific CTL and preparation method thereof - Google Patents
Melanoma-associated antigen specific CTL and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of melanoma-associated antigen specific CTL and preparation method thereof, wherein, the preparation method of described melanoma-associated antigen specific CTL includes: obtain peripheral blood lymphocytes；The ripe DC cell of preparation load melanoma-associated antigen polypeptide；CD8 is obtained from described peripheral blood lymphocytes+T lymphocyte；With CD8 described in the ripe DC cytositimulation of described load melanoma-associated antigen polypeptide+T lymphocyte；Use the post-stimulatory described CD8 of Tetramer technical mark+T lymphocyte, and isolate Tetramer+CD8+T lymphocyte；Stimulate described Tetramer+CD8+T lymphocyte, and prepare melanoma-associated antigen specific CTL.The preparation time of the preparation method of melanoma-associated antigen specific CTL of the present invention is short, amplification times is high, CTL high purity 98%, and kills the effective of melanoma cell.
Description
Technical field
The present invention relates to immunocyte technical field, particularly relate to a kind of melanoma-associated antigen specific CTL and preparation thereof
Method.
Background technology
Cytotoxic T lymphocyte (cytotoxic lymphocyte, CTL) be by immunotherapy techniques obtain thin
Born of the same parents, because it has special, the ability of direct killing tumor target cell, therefore become the focus of research.The mechanism of action of CTL is such as
Under:
There is huge T B lymphocyte repertoire in body, the t lymphocyte receptor (TCR) different by its surface expression is special
Identify different extracellular antigens molecules (antibacterial, virus and tumor-antigen peptide), be activated as having the CTL killing target cell,
Bacteria removal, the infection of virus and tumor cell, and can produce immunological memory CTL, prevent antibacterial, the invasion again of virus and
The recurrence of tumor.CTL immunne response is roughly divided into four-stage:
1, antigen recognition: APC (professional antigen presenting cell, such as DC cell) is by surface receptor identification and captures antigen
(antibacterial, virus, tumor-antigen peptide)；
2, antigen processing and submission: antigen digests through APC, is cracked into peptide molecule, the MHC-that the latter is abundant with APC intracellular
I class or II quasi-molecule combine and form MHC-I-polypeptide or MHC-II-polypeptide complex respectively, and are transported to APC surface；
3, T lymphocyte activator (dual signal theory): APC with T lymphocyte meets, and T lymphocyte is known by self TCR
The MHC-I/II-polypeptide complex of other APC submission, wherein CD8+T lymphocyte identification MHC-I-polypeptide complex, CD4+T lymph
Cell recognition MHC-II-polypeptide complex, T lymphocyte receives the stimulus signal (the first signal) of antigen plus immunity thorn altogether
Energizing signal (secondary signal) starts activation, has cytotoxic effect, i.e. CTL；
4, target cell is killed: activation CTL is recycled to position, antigen place, by the TCR specific recognition antigen table on CTL surface
Kill after reaching cell and remove.
However, it has been discovered that there is a large amount of immunosuppressive factor in tumor patient body, affect effective activation and the propagation of CTL,
Quantity is the fewest, is not enough to contend with the tumor bred rapidly.If a large amount of Peptide-specific CTL can be prepared in vitro and feeds back to suffering from
Person, can play therapeutical effect by killing tumor cell direct, quick, and the remaining tumor cells cannot removed conventional therapy kills
Go out, recurrence and the transfer of tumor will be possible to prevent.But, the melanoma-associated antigen not providing external preparation in prior art is special
Opposite sex CTL, and then cannot directly, quickly kill human melanoma cells by feeding back patient.
Summary of the invention
Present invention is primarily targeted at the preparation method that a kind of melanoma-associated antigen specific CTL is provided, it is intended to obtain
Melanoma-associated antigen specific CTL, and directly, quickly kill human melanoma cells.
For achieving the above object, the present invention provides the preparation method of a kind of melanoma-associated antigen specific CTL, including step
As follows:
Obtain peripheral blood lymphocytes；
The ripe DC cell of preparation load melanoma-associated antigen polypeptide；
CD8 is obtained from described peripheral blood lymphocytes+T lymphocyte；
With CD8 described in the ripe DC cytositimulation of described load melanoma-associated antigen polypeptide+T lymphocyte；
Use the post-stimulatory described CD8 of Tetramer technical mark+T lymphocyte, and isolate Tetramer+CD8+T drenches
Bar cell；
Stimulate described Tetramer+CD8+T lymphocyte, and prepare melanoma-associated antigen specific CTL.
Preferably, during the ripe DC cell of described preparation load melanoma-associated antigen polypeptide, comprise the following steps that
Synthesis of melanin tumor antigen polypeptide；
Immature DC cell is obtained from described peripheral blood lymphocytes；
It is ripe DC cell by described immature DC cell induction；
Co-culture described ripe DC cell and described melanoma-associated antigen polypeptide, and prepare load melanoma-associated antigen polypeptide
Ripe DC cell.
Preferably, described described immature DC cell induction is ripe DC cell during, comprise the following steps that
The RPMI-1640 containing people's AB serum, GM-CSF, IL-4 and TGF-β 1 is added complete in described immature DC cell
Culture medium；
After 4～10 days, add β and PGE Han GM-CSF, IL-4, TNF, IL-6, IL-12Fresh culture；
After 24～72h, add the Serum-free complete medium containing people's B2M, after stimulating 1～4h, it is thus achieved that ripe
DC cell.
Preferably, in described RPMI-1640 complete medium, the percent by volume of people's AB serum is 5～20%, GM-CSF
Concentration be 500～1000U/ml, the concentration of IL-4 is 200～800U/ml and the concentration of TGF-β 1 is 2～10ng/ml；Described
In fresh culture, the concentration of GM-CSF is 500～1000U/ml, and the concentration of IL-4 is 200～800U/ml, and the concentration of TNF is
The concentration of 5～20ng/ml, IL-6 is 500～2000U/ml, and the concentration of IL-1 β is 5～20ng/ml, and the concentration of PGE2 is 0.5
～5 μ g/ml；In described Serum-free complete medium, the concentration of people's B2M is 2～20 μ g/ml.
Preferably, described immature DC cell and the CD8 obtained from described peripheral blood lymphocytes+T lymphocyte
Quantity is than for 1:(6～10).
Preferably, the described Tetramer of described stimulation+CD8+During T lymphocyte, comprise the following steps that
The bone-marrow-derived lymphocyte that peripheral blood lymphocytes described in radiation treatment part and people's Epstein-Barr virus convert；
The B lymph that described peripheral blood lymphocytes after lymphocyte activation stimulant and irradiation, people's Epstein-Barr virus are converted
Tetramer described in cytositimulation+CD8+T lymphocyte.
Preferably, described melanoma-associated antigen polypeptide is HLA-A2 restricted melanoma-associated antigen polypeptide.
Preferably, described melanoma-associated antigen polypeptide is HLA-A2 restricted Melan-A antigen polypeptide.
Preferably, described melanoma-associated antigen polypeptide is HLA-A2 restricted gp100 antigen polypeptide.
Additionally, for achieving the above object, the present invention also provides for a kind of melanoma-associated antigen specific CTL as above
The melanoma-associated antigen specific CTL that preparation method is obtained.
The preparation method preparation time of melanoma-associated antigen specific CTL of the present invention is short, amplification times is high, CTL purity high
Reach 98% and to kill the fragmentation effect of human melanoma cell good, in target CTL to the specific killing activity of target cell in effect
Target ratio is during for 10:1, up to 39%.
Detailed description of the invention
Technical scheme in the embodiment of the present invention will be clearly and completely described below, it is clear that described enforcement
Example is only a part of embodiment of the present invention rather than whole embodiments.Based on the embodiment in the present invention, this area is general
The every other embodiment that logical technical staff is obtained under not making creative work premise, broadly falls into present invention protection
Scope.
The present invention provides the preparation method of a kind of melanoma-associated antigen specific CTL, comprises the following steps that
S1, acquisition peripheral blood lymphocytes；
S2, the ripe DC cell of preparation load melanoma-associated antigen polypeptide；
S3, from peripheral blood lymphocytes obtain CD8+T lymphocyte；
S4, the ripe DC cytositimulation CD8 of use load melanoma-associated antigen polypeptide+T lymphocyte；
S5, the employing post-stimulatory CD8 of Tetramer technical mark+T lymphocyte, and isolate Tetramer+CD8+T drenches
Bar cell；
S6, stimulation Tetramer+CD8+T lymphocyte, and prepare melanoma-associated antigen specific CTL.
The preparation method of melanoma-associated antigen specific CTL of the present invention is by loading the ripe DC of melanoma-associated antigen polypeptide
Cytositimulation CTL precursor (CD8+T lymphocyte), be not only supplied T lymphocyte activation the first signal (melanoma resist
Former), and DC cell supply T lymphocyte activation necessary secondary signal (co-stimulators CD40, CD80, CD83
Deng)；Then, Tetramer is isolated+CD8+T lymphocyte, and reject the T cell of non-specific amplification, to reduce it to target
The impact of CTL propagation；Finally, through acquisition target CTL that stimulates proliferation；Preparation method preparation time is short, amplification times is high for this, CTL
The fragmentation effect of high purity 98% and killing human melanoma cell is good, lives the specific killing of target cell in target CTL
Property at effect target ratio during for 10:1, up to 39%.
It should be noted that this peripheral blood lymphocytes (is signed from the patient gathered by automatic mononuclear cell Acquisition Instrument
Administration's Informed Consent Form) peripheral blood in isolated, it is thus achieved that peripheral blood lymphocytes be at least divided into three parts: part is used for making
Standby DC cell, part is used for isolating CTL precursor, and part is for radiation treatment.This melanoma-associated antigen polypeptide can be
Multiple, owing to all can be identified by T lymphocyte, and then T lymphocyte all can be stimulated to produce the first signal, the most also
Being realized the load of antigen polypeptide by DC cell institute's antigen presentation, therefore, this melanoma-associated antigen polypeptide can be in table 1 below
Any one melanoma-associated antigen:
Table 1, the human melanoma antigen of T lymphocyte identification
Wherein, Melan-A/MART-1 (melanoma antigen recognized by T cell 1) is people's black
4th self differentiation antigen in element tumor, by containing 5 exons, the gene code that is about 18.5kb.Gp100 is also known as melanin
Cell/melanoma-specific protein Pmel17, Immunoelectron Microscopic Identification gp100 be predominantly located at I, II phase melanosome membranaceous,
In thread substrate, its wide expression is in Humanmachine tumour (50%).Further, the epitope of this melanoma-associated antigen polypeptide
Can be HLA-A3, HLA-A68, HLA-A2 or HLA-A24, and then all can be with the antigen receptor of corresponding T lymphocytic cell surface
In conjunction with, thus activated T lymphocytes, cause immunne response；Wherein HLA-A2 is modal HLA genotype, about 50% white
Planting people, Aisan and Spaniard and the African of 33% and American has this phenotype, China is the positive of HLA-A2
Area, the people of about about 50% expresses HLA-A2 antigen, and HLA-A2 genotype is closely related with melanoma in addition, so HLA-
A2 restricted CTL epitope more specific aim, the most more has realistic meaning.
Further, to specifically include step as follows for step S2:
S20, synthesis of melanin tumor antigen polypeptide；
S21, from peripheral blood lymphocytes, obtain immature DC cell；
S22, it is ripe DC cell by immature DC cell induction；
S23, co-culture ripe DC cell and melanoma-associated antigen polypeptide, and prepare the one-tenth of load melanoma-associated antigen polypeptide
Ripe DC cell.
By co-culturing of ripe DC cell and melanoma-associated antigen polypeptide, DC cell recognition also captures antigen polypeptide, resists
Former polypeptide through DC cell dissociation, be cracked into peptide molecule, the latter's abundant MHC-I class intracellular with DC or II quasi-molecule are combined point
Not Xing Cheng MHC-I-polypeptide or MHC-II-polypeptide complex, and be transported to DC cell surface, and realize the anti-of ripe DC cell
Former load, and then T lymphocyte activation can be stimulated；And, ripe DC cell prepare simple and direct, the time is short, efficiency is high.
Further, to specifically include step as follows for step S22:
S220, in immature DC cell add containing people's AB serum, GM-CSF (granulocyte-macrophage colony stimulate because of
Son), IL-4 and the RPMI-1640 complete medium of TGF-β 1 (transforming growth factor-beta 1)；
S221, after 4～10 days, add containing GM-CSF, IL-4, TNF (tumor necrosis factor), IL-6, IL-1 β and PGE2
(prostaglandin E2) fresh culture；
After S222,24～72h, add the Serum-free complete medium containing people's B2M (micro-globulin),
After stimulating 1～4h, it is thus achieved that ripe DC cell.
By the stimulation of different culture media, accelerate the maturation of DC cell, simultaneously facilitate the propagation of DC cell, and then greatly
Shorten preparation time, improve preparation efficiency.
Further, in RPMI-1640 complete medium, the percent by volume of people's AB serum is 5～20%, GM-CSF's
Concentration is 500～1000U/ml, and the concentration of IL-4 is 200～800U/ml and the concentration of TGF-β 1 is 2～10ng/ml.Fresh training
Supporting in base, the concentration of GM-CSF is 500～1000U/ml, and the concentration of IL-4 is 200～800U/ml, the concentration of TNF be 5～
The concentration of 20ng/ml, IL-6 is 500～2000U/ml, and the concentration of IL-1 β is 5～20ng/ml, and the concentration of PGE2 is 0.5～5 μ
g/ml.In Serum-free complete medium, the concentration of people's B2M is 2～20 μ g/ml.
Cell growth factor in above-mentioned amount ranges can be that the maturation of DC cell provides enough stimulation, and favorably
Fast-ripenin and propagation in DC cell.
Further, immature DC cell and the CD8 obtained from peripheral blood lymphocytes+The quantity ratio of T lymphocyte
For 1:(6～10).
Within the ratio range of this cell quantity, by the ripe DC of the Antigen polypeptide of immature DC cell transformation
Cell can be CD8+T lymphocyte provides enough stimulations, and then promotes CD8+T lymphocyte produces target CTL.
Further, step S5 is particularly as follows: use Tetramer labeling method and Flow cytometry purification again
Target CTL, i.e. selects Tetramer+CD8+T lymphocyte, rejects the T lymphocyte of non-specific amplification, to reduce it to mesh
The impact of mark CTL propagation.
Further, in step s 6, Tetramer is stimulated+CD8+It is as follows that the process of T lymphocyte specifically includes step:
The bone-marrow-derived lymphocyte that S60, radiation treatment portion perimeter blood monocyte and people's Epstein-Barr virus convert；
S61, the B lymph that the peripheral blood lymphocytes after lymphocyte activation stimulant and irradiation, people's Epstein-Barr virus are converted
Cytositimulation Tetramer+CD8+T lymphocyte.
The B lymph converted by the peripheral blood lymphocytes after lymphocyte activation stimulant and irradiation, people's Epstein-Barr virus is thin
The stimulation of born of the same parents, promote T lymphocyte propagation, accelerate cell growth, and a large amount of costimulatory molecules of PBMC surface expression and divide
Secreting cytokine profiles provides CTL good growing environment, and irradiated after will not breed and disturb the growth of CTL, thus protect
The efficient amplification of card CTL.
The present invention also provides for the melanoma prepared by preparation method of a kind of above-mentioned melanoma-associated antigen specific CTL and resists
Former specific CTL, it kills the effective of human melanoma cell, exists the specific killing activity of target cell in target CTL
Effect target ratio is during for 10:1, up to 39%.
Now by embodiment, melanoma-associated antigen specific CTL of the present invention and preparation method thereof is further explained, with
Describe its technical scheme and the technique effect brought in detail.
Embodiment 1
The preparation of Melan-A Peptide-specific CTL
One, the acquisition of peripheral blood lymphocytes (peripheral blood mononuclear cells, PBMCs)
The collection of PBMCs and purification: with singly adopting the peripheral blood lymphocytes of instrument collection patient, through Ficoll density gradient from
Purification PBMCs after heart method.PBMCs part after purification is for DC cell induction, and part is for CTL precursor (CD8+T lymph
Cell) enrichment and purification, part through gamma-rays irradiate and frozen in-80 DEG C.
Two, the preparation of the ripe DC cell of load melanoma-associated antigen polypeptide
1, the synthesis of HLA-A2 restricted Melan-A antigen polypeptide
HLA-A2 restricted Melan-A antigen polypeptide, site is 26～35, and sequence is the (letter below of ELAGIGILTV10 peptide
Claim Melan-A26～35Polypeptide), chemosynthesis (Tai Ye bio tech ltd, Nanjing), fully dissolve with aseptic double-distilled water,
Melan-A26～35Peptide concentration is 5mg/ml, and subpackage is stored in-80 DEG C；Wherein, Melan-A26～35Polypeptide fragment is Melan-
A26～35peptide(ELAGIGILTV)；
2, the preparation of immature DC cell
Prepared by the adherent method of DC cell: part PBMCs be after purification suspended in RPMI1640 culture medium, cell density
It is 3 × 106/ ml, is inoculated in 75cm2In culture bottle, and in 5%CO2, hatch in 37 DEG C of incubators 90 minutes, with pre-temperature physiology salt
Water washs 2～3 times gently, forms attached cell, i.e. immature DC cell；
3, the induction of immature DC cell
Adding the DC inducing culture of 30ml in the culture bottle fill attached cell, this DC inducing culture is containing 10%
(V/V) TGF-β 1 of people's AB serum, the GM-CSF of concentration 800U/ml, the IL-4 of concentration 500U/ml and concentration 5ng/ml
RPMI-1640 complete medium；6th day, add containing the GM-CSF of concentration 800U/ml, the IL-4 of concentration 500U/ml, concentration
The TNF of 10ng/ml, the IL-6 of concentration 1000U/ml, the IL-1 β of concentration 10ng/ml and the PGE of concentration 1 μ g/ml2Fresh training
Support base, after continuing to cultivate 48h, add the Serum-free complete medium of the people's B2M containing concentration 10 μ g/ml, at 37 DEG C
Under the conditions of stimulate 2h, it is thus achieved that ripe DC cell；
4, the antigen load of ripe DC cell
Ripe DC cell loading Melan-A26～35Polypeptide: by the ripe DC cell suspension of results in RPMI-1640 culture medium
In, cell density is 1 × 106/ ml, and be inoculated in cell and cultivate in 6 orifice plates, every hole 3ml, in hole, add Melan-A26～35Many
Peptide, to final concentration of 30 μ g/ml, is positioned over 37 DEG C, 5%CO2Results load Melan-A after educating altogether in incubator 2 hours26～35Many
The ripe DC cell of peptide；The cell of results RPMI-1640 culture medium is washed 2 times, more resuspended by RPMI-1640 culture medium, carefully
Born of the same parents' density is 1 × 106/ ml, for the stimulation of CTL precursor.
Three, CTL precursor (CD8+T lymphocyte) isolation and purification
In load Melan-A26～35The ripe DC cell harvesting same day of polypeptide, recovery part PBMCs after purification of thawing,
And wash PBMCs 2 times by the PBS solution of the mixing normal human AB serum containing 2% inactivation of pre-cooling, and resuspended PBMC cell, adjust
Whole PBMC cell density is 2 × 107/ ml, adds clinical grade CD4 in PBMCs cell+CD19+CD16/56+T sorts magnetic bead
(Miltenyi CliniMACS), crosses post, collects the CD8 flowed out after educating altogether 30 minutes+T lymphocyte；With the RPMI-of pre-cooling
1640 culture medium are washed 2 times, by CD8+T lymphocyte is resuspended in the RPMI-of the mixing normal human AB serum containing 10% inactivation
In 1640 complete mediums, CD8+The density of T lymphocyte is 1 × 106/ ml, and obtain CD8 after purification+T lymphocyte.Its
In, after purification for the CTL precursor of amplification and the quantity of immature DC cell ratio for 8:1.
Four, the amplification (the ripe DC cytositimulation CTL precursor of Antigen polypeptide) of CTL precursor
By CD8 after purification+T lymphocyte is inoculated in cell and cultivates in 96 orifice plates, adds load Melan-A26～35Polypeptide
Ripe DC cell, stimulate weekly once, gently mixing after put into 5%CO2, cultivate in 37 DEG C of cell culture incubators；Every day observes
The growth conditions of cell, supplements weekly the complete medium containing the T B cell growth factor (TCGF) of 3% twice；Cell is close
Carrying out when spending big expanding bottle, cell proliferation can be moved to 75cm after a certain amount of2Culture bottle continues amplification cultivate；Wherein, TCGF
Complete medium includes: the recombined human cytokine of 10ng/ml, IL-4,5ng/ml of GM-CSF, 500U/ml of 800U/ml
TGF-β 1, the interferon-γ of TNF, 2000U/ml of IL-6,10ng/ml of IL-1 β, 1000U/ml of 10ng/ml
(IFN-γ) and the PGE of 1 μ g/ml2。
Five, the sorting purification of the CTL precursor after amplification
Load Melan-A26～35The DC cell of polypeptide and CD8+After T lymphocyte educates 12～14 days altogether, harvesting, with pre-
The PBS solution washed cell of the cold mixing normal human AB serum containing 2% inactivation 2 times, and re-suspended cell, adjusting cell density is
1×107/ ml, adds the HLA-A2 of PE labelling in cell suspension+Melan-A26～35Tetramer and FITC labelling
CD8mAb, gently after mixing, educates 20 minutes in 4 DEG C of refrigerators altogether；With the mixing normal human AB serum's containing 2% inactivation of pre-cooling
PBS solution washed cell 2 times, re-suspended cell, adjusting cell density is 1 × 107/ ml, carries out airflow classification by cell, results
CD8+Tetramer+T lymphocyte, abandons supernatant after being centrifuged, with the RPMI-1640 of the mixing normal human AB serum containing 10% inactivation
The resuspended CD8 of complete medium+Tetramer+T lymphocyte, cell density is 2 × 106/ml。
Six, CD8+Tetramer+The amplification of T lymphocyte
Thin with bone-marrow-derived lymphocyte, the allogeneic PBMCs of irradiation and the lymph that allochthonous people's Epstein-Barr virus of irradiation converts
Born of the same parents activate stimulant PHA-L (1mg/ml) repetitive stimulation CD8+Tetramer+T lymphocyte, and expand；Wherein, irradiation
Allogeneic PBMCs is prepared by following method:
Leave and take the PBMCs obtained in part steps one, in preservation after the gamma-ray irradiation of 30～50GY (average 40GY)
Liquid preserves；Wherein, storage temperature is-80 DEG C of Excised Embryos, and preserving liquid containing 20% (V/V) DMSO and 20% (V/V) is just
The RPMI1640 culture fluid of ordinary person's AB serum.
Seven, the results of target CTL and Phenotypic examination
CD8+Tetramer+T Lymphocyte expansion about 10 days, gathers in the crops target CTL, then, with brine 2 times,
Being resuspended in 100ml normal saline, cell density is 107About/ml, for immunization therapy.
Eight, the killing ability of target CTL
By preparation target CTL respectively with load Melan-A26～35The HLA-A2 of polypeptide+T2 cell strain, HLA-A2+T2 is empty
Carry cell strain and three kinds of target cells of K562 (NK sensitive cells strain) according to imitating the target ratio mixing than 3:1,10:1 and 30:1, MTT
Method detection CTL killing activity.Result shows, the specific killing activity of target cell is being imitated by target CTL of the present embodiment 1 preparation
Target ratio, during for 10:1, reaches 35.5%.
In the embodiment of the present invention 1, after each step operation, cell quantity and phenotypic characteristic are shown in such as table 2 below:
Table 2
Embodiment 2
The preparation of gp100 Peptide-specific CTL
One, the acquisition of peripheral blood lymphocytes (peripheral blood mononuclear cells, PBMCs)
The collection of PBMCs and purification: with singly adopting the peripheral blood lymphocytes of instrument collection patient, through Ficoll density gradient from
Purification PBMCs after heart method.PBMCs part after purification is for DC cell induction, and part is for CTL precursor (CD8+T lymph
Cell) enrichment and purification, part through gamma-rays irradiate and frozen in-80 DEG C.
Two, the preparation of the ripe DC cell of load melanoma-associated antigen polypeptide
1, the synthesis of HLA-A2 restricted gp100 antigen polypeptide
HLA-A2 restricted gp100 antigen polypeptide, site is 209～217, sequence be ITDQVPFSV 9 peptide (hereinafter referred to as
gp100209～217Polypeptide), chemosynthesis (Tai Ye bio tech ltd, Nanjing), fully dissolve with aseptic double-distilled water,
gp100209～217Peptide concentration is 5mg/ml, and subpackage is stored in-80 DEG C.Wherein, the restricted anti-gp100 antigen polypeptide sheet of HLA-A2
Section is gp100209～217(ITDQVPFSV)。
2, the preparation of immature DC cell
Prepared by the adherent method of DC cell: part PBMCs be after purification suspended in RPMI1640 culture medium, cell density
It is 3 × 106/ ml, is inoculated in 75cm2In culture bottle, and in 5%CO2, hatch in 37 DEG C of incubators 90 minutes, with pre-temperature physiology salt
Water washs 2～3 times gently, forms attached cell, i.e. immature DC cell；
3, the induction of immature DC cell
Adding the DC inducing culture of 30ml in the culture bottle fill attached cell, this DC inducing culture is containing 10%
(V/V) TGF-β 1 of people's AB serum, the GM-CSF of concentration 800U/ml, the IL-4 of concentration 500U/ml and concentration 5ng/ml
RPMI-1640 complete medium；6th day, add containing the GM-CSF of concentration 800U/ml, the IL-4 of concentration 500U/ml, concentration
The TNF of 10ng/ml, the IL-6 of concentration 1000U/ml, the IL-1 β of concentration 10ng/ml and the PGE of concentration 1 μ g/ml2Fresh training
Support base, after continuing to cultivate 48h, add the Serum-free complete medium of the people's B2M containing concentration 10 μ g/ml, at 37 DEG C
Under the conditions of stimulate 2h, it is thus achieved that ripe DC cell；
4, the antigen load of ripe DC cell
Ripe DC cell loading gp100209～217Polypeptide: by the ripe DC cell suspension of results in RPMI-1640 culture medium
In, cell density is 1 × 106/ ml, and be inoculated in cell and cultivate in 6 orifice plates, every hole 3ml, in hole, add gp100209～217Many
Peptide, to final concentration of 30 μ g/ml, is positioned over 37 DEG C, 5%CO2Results load gp100 after educating altogether in incubator 2 hours209～217Many
The ripe DC cell of peptide；The cell of results RPMI-1640 culture medium is washed 2 times, more resuspended by RPMI-1640 culture medium, carefully
Born of the same parents' density is 1 × 106/ ml, for the stimulation of CTL precursor.
Three, CTL precursor (CD8+T lymphocyte) isolation and purification
In load gp100209～217The ripe DC cell harvesting same day of polypeptide, recovery part PBMCs after purification of thawing, and
PBMCs is washed 2 times by the PBS solution of the mixing normal human AB serum containing 2% inactivation of pre-cooling, and resuspended PBMC cell, adjust
PBMC cell density is 2 × 107/ ml, adds clinical grade CD4 in PBMCs cell+CD19+CD16/56+T sorts magnetic bead
(Miltenyi CliniMACS), crosses post, collects the CD8 flowed out after educating altogether 30 minutes+T lymphocyte；With the RPMI-of pre-cooling
1640 culture medium are washed 2 times, by CD8+T lymphocyte is resuspended in the RPMI-of the mixing normal human AB serum containing 10% inactivation
In 1640 complete mediums, CD8+The density of T lymphocyte is 1 × 106/ ml, and obtain CD8 after purification+T lymphocyte.Its
In, after purification for the CTL precursor of amplification and the quantity of immature DC cell ratio for 8:1.
Four, the amplification (the ripe DC cytositimulation CTL precursor of Antigen polypeptide) of CTL precursor
By CD8 after purification+T lymphocyte is inoculated in cell and cultivates in 96 orifice plates, adds load gp100209～217Polypeptide
Ripe DC cell, stimulate weekly once, gently mixing after put into 5%CO2, cultivate in 37 DEG C of cell culture incubators；Every day observes
The growth conditions of cell, supplements weekly the complete medium containing the T B cell growth factor (TCGF) of 3% twice；Cell is close
Carrying out when spending big expanding bottle, cell proliferation can be moved to 75cm after a certain amount of2Culture bottle continues amplification cultivate；Wherein, TCGF
Complete medium includes: the recombined human cytokine of 10ng/ml, IL-4,5ng/ml of GM-CSF, 500U/ml of 800U/ml
TGF-β 1, the interferon-γ of TNF, 2000U/ml of IL-6,10ng/ml of IL-1 β, 1000U/ml of 10ng/ml
(IFN-γ) and the PGE of 1 μ g/ml2。
Five, the sorting purification of the CTL precursor after amplification
Load gp100209～217The DC cell of polypeptide and CD8+After T lymphocyte educates 12～14 days altogether, harvesting, with pre-
The PBS solution washed cell of the cold mixing normal human AB serum containing 2% inactivation 2 times, and re-suspended cell, adjusting cell density is
1×107/ ml, adds the HLA-A2 of PE labelling in cell suspension+gp100209～217The CD8mAb of Tetramer and FITC labelling,
Gently after mixing, educate altogether in 4 DEG C of refrigerators 20 minutes；PBS solution with the mixing normal human AB serum containing 2% inactivation of pre-cooling
Washed cell 2 times, re-suspended cell, adjusting cell density is 1 × 107/ ml, carries out airflow classification by cell, gathers in the crops CD8+
Tetramer+T lymphocyte, abandons supernatant after being centrifuged, and the RPMI-1640 with the mixing normal human AB serum containing 10% inactivation is complete
The resuspended CD8 of culture medium+Tetramer+T lymphocyte, cell density is 2 × 106/ml。
Six, CD8+Tetramer+The amplification of T lymphocyte
Thin with bone-marrow-derived lymphocyte, the allogeneic PBMCs of irradiation and the lymph that allochthonous people's Epstein-Barr virus of irradiation converts
Born of the same parents activate stimulant PHA-L (1mg/ml) repetitive stimulation CD8+Tetramer+T lymphocyte, and expand；Wherein, irradiation
Allogeneic PBMCs is prepared by following method:
Leave and take the PBMCs obtained in part steps one, in preservation after the gamma-ray irradiation of 30～50GY (average 40GY)
Liquid preserves；Wherein, storage temperature is-80 DEG C of Excised Embryos, and preserving liquid containing 20% (V/V) DMSO and 20% (V/V) is just
The RPMI1640 culture fluid of ordinary person's AB serum.
Seven, the results of target CTL and Phenotypic examination
CD8+Tetramer+T Lymphocyte expansion about 10 days, gathers in the crops target CTL, then, with brine 2 times,
Being resuspended in 100ml normal saline, cell density is 107About/ml, for immunization therapy.
Eight, the killing ability of target CTL
By preparation target CTL respectively with load gp100209～217The HLA-A2 of polypeptide+T2 cell strain, HLA-A2+T2 is unloaded
Cell strain and three kinds of target cells of K562 (NK sensitive cells strain) are according to imitating the target ratio mixing than 3:1,10:1 and 30:1, mtt assay
Detection CTL killing activity.Result shows, the specific killing activity of target cell is being imitated target by target CTL of the present embodiment 1 preparation
Than during for 10:1, reach 39.1%.
In the embodiment of the present invention 2, after each step operation, cell quantity and phenotypic characteristic are shown in such as table 3 below:
Table 3
The foregoing is only the preferred embodiments of the present invention, not thereby limit the scope of the claims of the present invention, every at this
Under the inventive concept of invention, utilize the equivalent transformation that description of the invention is made, or directly/indirectly it is used in other relevant skills
Art field is included in the scope of patent protection of the present invention.
Claims (10)
1. the preparation method of a melanoma-associated antigen specific CTL, it is characterised in that comprise the following steps that
Obtain peripheral blood lymphocytes；
The ripe DC cell of preparation load melanoma-associated antigen polypeptide；
CD8 is obtained from described peripheral blood lymphocytes+T lymphocyte；
With CD8 described in the ripe DC cytositimulation of described load melanoma-associated antigen polypeptide+T lymphocyte；
Use the post-stimulatory described CD8 of Tetramer technical mark+T lymphocyte, and isolate Tetramer+CD8+T lymph is thin
Born of the same parents；
Stimulate described Tetramer+CD8+T lymphocyte, and prepare melanoma-associated antigen specific CTL.
2. the preparation method of melanoma-associated antigen specific CTL as claimed in claim 1, it is characterised in that described preparation is born
During carrying the ripe DC cell of melanoma-associated antigen polypeptide, comprise the following steps that
Synthesis of melanin tumor antigen polypeptide；
Immature DC cell is obtained from described peripheral blood lymphocytes；
It is ripe DC cell by described immature DC cell induction；
Co-culture described ripe DC cell and described melanoma-associated antigen polypeptide, and prepare the one-tenth of load melanoma-associated antigen polypeptide
Ripe DC cell.
3. the preparation method of melanoma-associated antigen specific CTL as claimed in claim 2, it is characterised in that described by described
During immature DC cell induction is ripe DC cell, comprise the following steps that
In described immature DC cell, add the RPMI-1640 containing people's AB serum, GM-CSF, IL-4 and TGF-β 1 cultivate completely
Base；
After 4～10 days, add β and PGE Han GM-CSF, IL-4, TNF, IL-6, IL-12Fresh culture；
After 24～72h, add the Serum-free complete medium containing people's B2M, after stimulating 1～4h, it is thus achieved that ripe DC is thin
Born of the same parents.
4. the preparation method of melanoma-associated antigen specific CTL as claimed in claim 3, it is characterised in that
In described RPMI-1640 complete medium, the percent by volume of people's AB serum is 5～20%, and the concentration of GM-CSF is 500
～the concentration of 1000U/ml, IL-4 is 200～800U/ml and the concentration of TGF-β 1 is 2～10ng/ml；
In described fresh culture, the concentration of GM-CSF is 500～1000U/ml, and the concentration of IL-4 is 200～800U/ml, TNF
Concentration be 5～20ng/ml, the concentration of IL-6 is 500～2000U/ml, and the concentration of IL-1 β is 5～20ng/ml, and PGE2's is dense
Degree is 0.5～5 μ g/ml；
In described Serum-free complete medium, the concentration of people's B2M is 2～20 μ g/ml.
5. the preparation method of melanoma-associated antigen specific CTL as claimed in claim 2, it is characterised in that described immaturity
DC cell and the CD8 obtained from described peripheral blood lymphocytes+The quantity of T lymphocyte is than for 1:(6～10).
6. the preparation method of melanoma-associated antigen specific CTL as claimed in claim 1, it is characterised in that described stimulation institute
State Tetramer+CD8+During T lymphocyte, comprise the following steps that
The bone-marrow-derived lymphocyte that peripheral blood lymphocytes described in radiation treatment part and people's Epstein-Barr virus convert；
The bone-marrow-derived lymphocyte that described peripheral blood lymphocytes after lymphocyte activation stimulant and irradiation, people's Epstein-Barr virus are converted
Stimulate described Tetramer+CD8+T lymphocyte.
7. the preparation method of the melanoma-associated antigen specific CTL as described in claim 1 to 6 any one, it is characterised in that
Described melanoma-associated antigen polypeptide is HLA-A2 restricted melanoma-associated antigen polypeptide.
8. the preparation method of melanoma-associated antigen specific CTL as claimed in claim 7, it is characterised in that described melanin
Tumor antigen polypeptide is HLA-A2 restricted Melan-A antigen polypeptide.
9. the preparation method of melanoma-associated antigen specific CTL as claimed in claim 7, it is characterised in that described melanin
Tumor antigen polypeptide is HLA-A2 restricted gp100 antigen polypeptide.
10. the preparation method of the melanoma-associated antigen specific CTL as described in any one of claim 1 to 9 is obtained
Melanoma-associated antigen specific CTL.
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