CN108220239B - A kind of stimulation induction mononuclearcell amplification is composition and its application of gamma delta T cells - Google Patents
A kind of stimulation induction mononuclearcell amplification is composition and its application of gamma delta T cells Download PDFInfo
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Abstract
The present invention relates to the compositions that a kind of stimulation induction mononuclearcell amplification is gamma delta T cells, the composition includes azoles Lai phosphoric acid, anti-human CD3Ab, anti-human CD28Ab, IL-15, IL-21 and IL-2, the present invention also provides a kind of methods using the external massive amplification gamma delta T cells of the composition, it is specially a kind of to induce massive amplification for the method for gamma delta T cells in vitro with peripheral blood mononuclear cells (PBMCs), the present invention is by being added zoledronic acid induced activation, anti-human CD3Ab is added, anti-human CD28Ab, IL-15, IL-21, induced activation proliferation is carried out with IL-2, it is lured using a variety of amplification factors combination Co stituation Amplification gamma delta T cells are led, the gamma delta T cells of acquisition have the characteristics that big quantity, purity is high, cytotoxicity are strong, have extraordinary clinical value.
Description
Technical field
The present invention relates to one kind, and mononuclearcell can be stimulated to make the composition of its induced amplification gamma delta T cells, and using should
Method of the composition to mononuclearcell (PBMCs) massive amplification gamma delta T cell in vitro.
Background technique
Tumour is one of the major disease to threaten at present to human health, and no matter the death rate and disease incidence occupy height
Position.Tumour immunotherapy is the 4th kind of tumor therapeuticing method after operation, radiotherapy, chemotherapy, and at present it is of greatest concern and
The best therapy of therapeutic effect.Mucous membrane (epidermis) tissue as physical barriers contain extensive cell type include non-Lymphatic System and
Lymphatic System immunocyte, especially T cell.
Verified T cell, the especially T cell of expression γ δ receptor are maintaining mucosal tissue balance and are preventing disease for research
Key effect is played in epidermal tissues' pressure such as pathogenic microorganism infection and pernicious canceration.Gamma delta T cells mainly develop into thymus gland
It is ripe, gamma delta T cells receptor (TCR) is generated by V (D) J genetic recombination.By the gene rearrangement of specificity, from a common lymphoid
Cell precursors (common lymphoidprecursor, CLP) is divided into the T cell system of express alpha beta receptor and γ δ receptor.γδT
Cell conducts TCR signal by association CD3 compound, and in the peripheral blood of normal adults, gamma delta T cells only account for circulation T cell
1-10%；α β T cell accounts for 90% or more of circulation T cell, and directly conducts intracellular signal by being associated with CD3 compound.
Different from α β TCR, gamma delta T CR can bind directly antigen and not depend on the numerator mediated antigen presentation of MHC, therefore thin in gamma delta T
Cellular surface does not express CD4 and CD8 molecule.Widely studies have shown that gamma delta T cells can be divided into three according to the expression of δ chain
The gamma delta T cells of major class, expression 1 chain of V δ are mainly distributed on the endodermis of mucomembranous surface, and seldom a part is distributed in peripheral blood.V
1 type gamma delta T cells of δ play a significant role in resisting damage, infection and vicious transformation epidermis intact.Gamma delta T cells are another kind of
Main subgroup is the gamma delta T cells for expressing 2 chain of V δ, and 2 chain of V δ is almost only matched with 9 chain of V γ, is main in normal adults
The circulation gamma delta T cells wanted, can achieve the 50-90% of Peripheral Circulation gamma delta T cells.It is interesting that be once activated, V δ 2T
Cell obtains the characteristic that professional antigen is in delivery cell (APCs), including expression costimulatory molecules, adhesion molecule and antigen presentation point
Sub (for example, CD80, CD86, CD11b, CD18, CD54 and MHC II).3 chain of third class gamma delta T cells Expression of Subsets V δ, accounts for about
Recycle the 0.2% of T cell, including CD4+, CD8+ and CD4-CD8- subgroup.The unstable expression CD56 of 3 gamma delta T cells of V δ,
CD161, HLA-DR and NKG2D.
Different from α β T cell, the influence of missing is not processed vulnerable to antigen and presented to gamma delta T cells, therefore gamma delta T cells have
Very high clinical tumor immunization therapy potential using value.Gamma delta T cells are sent out in cancer immunosurveillance and anti tumor immune response
Wave important function.Girardi's et al. research shows that the gamma delta T cells of epidermis positioning play weight in prevention mouse skin is formed
It acts on, the mouse of gamma delta T cells missing is easy to get cutaneum carcinoma (Science.2001；294(5542):605-609.).Liu et al.
People's studies have shown that, by the gamma delta T of tail vein injection homogenic type, can be significantly reduced prostate compared with control group mice
The disease incidence of tumour tumor-bearing mice tumour and life cycle (the J Immunol.2008 for significantly extending tumor-bearing mice；180(9):
6044-6053.).Zhao's et al. studies have shown that in dysgerminoma and seminoma, the relevant granulation of tumor tissues
A large amount of gamma delta T infiltrating T lymphocytes are accumulated in swollen property inflammation, the gamma delta T cells of these infiltrations have killing autologous tumor thin
The activity of born of the same parents can pass through the killing activity of the monoclonal antibodies block gamma delta T cell of identification V δ；Gamma delta T cells secretion is a large amount of
Proinflammatory cytokines, such as IFN-γ and TNF-α (Immunol Invest.1995；24(4):607-618.).Todaro et al.
Studies have shown that gamma delta T cells can kill colorectal cancer tumor stem cell, tumor stem cell originates in tumour, growth, transfer,
Traditional tumour treatment means are resisted to (the J Immunol.2009 that plays a significant role in recurrence；182(11):7287-
7296.).It has been separated at present from kinds of tumors (including colorectal cancer, breast cancer, prostate cancer, oophoroma and kidney etc.)
Infiltrating T lymphocytes (TILs) identify gamma delta T cells (J Immunol.2005；174(3):1338-1347.； J
Immunol.2005；175(8):5481-5488.；Mol Immunol.2007； 44(4):302-310.).
Since content of the gamma delta T cells in peripheral blood is extremely low, greatly limits gamma delta T cells and exist as adoptive immunity cell
Application clinically.Gamma delta T cells are expanded from peripheral blood mononuclear cells at present, amplification times are low, and cell purity is not high.Therefore,
Invent it is a kind of it is practical and efficient, at low cost, technically simple, can massive amplification high-purity, high cytotoxic activity gamma delta T cells become
Problem in the urgent need to address at present.
Summary of the invention
It in view of the above problems, is gamma delta T cells the invention proposes a kind of stimulation induction mononuclearcell amplification
Composition and its application combine Co stituation induced amplification using a variety of amplification factors by the way that zoledronic acid induced activation is added
Gamma delta T cells, the gamma delta T cells of acquisition have the characteristics that big quantity, purity is high, cytotoxicity are strong, there is extraordinary clinic to answer
With value.
In order to achieve the above purpose, the following technical solution is employed by the present invention:
It is a kind of stimulation induction mononuclearcell amplification be gamma delta T cells composition, the composition include zoledronic acid,
Anti-human CD3Ab, anti-human CD28Ab, IL-15, IL-21 and IL-2.
Preferably, the concentration of zoledronic acid be 1-100 μM, the concentration of anti-human CD3Ab be 10~500ng/mL,
The concentration of anti-human CD28Ab is 10~500ng/mL, the concentration of IL-15 is 10~200ng/mL, the concentration of IL-21
Concentration for 10~200ng/mL, IL-2 is 500~3000U/mL.
Preferably, zoledronic acid concentration is 5 μM, anti-human CD3Ab concentration is 50 ng/mL, anti-human
It is 50ng/mL, IL-21 concentration be 50ng/mL, IL-2 concentration is 1000U/ that CD28Ab concentration, which is 50ng/mL, IL-15 concentration,
mL。
Preferably, mononuclearcell derives from peripheral blood, Cord blood, marrow or inductive pluripotent stem cells (iPSC).
Preferably, a kind of to stimulate induction mononuclearcell amplification in vitro for the side of gamma delta T cells using combinations of the above object
Method, comprising the following steps:
1) preparation of mononuclearcell: separation mononuclearcell contains 10vt% with the 50%-100% containing percent by volume
Cell is resuspended in the mixed culture medium of the RMPI 1640 and 0%-50%X-vivo15 of FBS or autoserum, be seeded in culture bottle or
It is cultivated in culture bag；
2) zoledronic acid and 500~3000U/mL IL-2 that are 1-100 μM containing concentration the induction of gamma delta T cells: is added
It is added after mixed culture medium stimulated in vitro 4 days and contains 10~500 ng/mL anti-human CD3Ab, 10~500ng/mL
Anti-human CD28Ab, 10~200ng/mL IL-15,10~200ng/mL IL-21 and 500~3000U/mL IL-2
Mixed culture medium carry out half amount and change liquid, be placed in 37 DEG C, cultivate in 5%CO2 incubator；
3) every 2~3 days according to count results fluid infusion, 14~21 days harvest gamma delta T cells.
Preferably, the zoledronic acid being added in step 2) is 5 μM final concentration of, anti-human CD3Ab is final concentration of
50ng/mL, anti-human CD28Ab final concentration of 50ng/mL, IL-15 final concentration of 50ng/mL, IL-21 are final concentration of
The final concentration of 1000U/mL of 50ng/mL and IL-2.
Preferably, the concentration of the peripheral blood mononuclear cells (PBMCs) described in a case study on implementation is 1 × 106/
mL。
Other phosphoantigens also can be used in one embodiment instead of zoledronic acid, including isopentenyl pyrophosphate
(IPP), (E) -4- hydroxy-3-methyl-but-2-ene base pyrophosphate (HMB-PP), ethyl pyrophosphate (EPP), conversion
Ester (FPP), dimethyl-allyl phosphate (DMAP), dimethylallylpyrophosphate ester (DMAPP), ethyl-adenosine triphosphoric acid
Ester (EPPPA), geranylpyrophosphate ester (GPP), yak geranylpyrophosphate ester (GGPP), isopentenyl-adenosine pyrophosphate
(IPPPA), etherophosphoric acid (MEP), the biphosphonate containing nitrogen such as one ethyl ester of pyrophosphoric acid (MEPP).
Preferably, RMPI 1640 in a case study on implementation containing 10vt%FBS or autoserum in mixed culture medium
Ratio is 50vt%, and the ratio of X-vivo15 culture medium is 50vt%.
The raw materials used in the present invention and reagent are commercially available in addition to having specified otherwise.
Due to use above-mentioned technical solution, the beneficial effects of the present invention are: the present invention provides one kind by cell factor,
The composition of zoledronic acid and antibody composition, and using the method for the composition stimulation external massive amplification of gamma delta T cells, it should
Method can obtain a large amount of, high proliferation ability, high-purity and high cytotoxic activity gamma delta T cells, can be used for the cell of tumour
Immunization therapy.
Detailed description of the invention
Fig. 1 is the conventional culture methods and cell factor, zoledronic acid and anti-of cell factor and zoledronic acid combined induction
Body combines the flow analysis chart of the gamma delta T cells of modified cultural method amplification cultivation；
Fig. 2 is modified cultural method amplification cultivation cell growth curve；
Fig. 3 is the amplification times of the 7th, 14 and 21 day cell of modified cultural method amplification cultivation；
Fig. 4 is modified cultural method amplification cultivation the 0th day and the flow analysis chart of the 21st day gamma delta T cells；
Fig. 5 is real-time n cell functional analysis instrument detection curve；
Fig. 6 is that the gamma delta T cells of modified cultural method amplification cultivation and breast cancer cell line SKBR-3 cell are incubated for 4 altogether
The cell index and different effect targets of hour is than gamma delta T cells to the killing percentage of SKBR-3.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention,
Technical scheme in the embodiment of the invention is clearly and completely described.Based on the embodiment of the present invention, the common skill in this field
Art personnel every other embodiment obtained without creative efforts belongs to the model that the present invention protects
It encloses.
" mixed culture medium " described in the embodiment of the present invention refers both to the 50vt% prepared according to existing method containing 10vt%
The mixed culture medium of the RMPI1640+50vt%X-vivo15 of autoserum.
Embodiment 1: the induction of gamma delta T cells
Mononuclearcell (PBMCs) is separated from peripheral blood and expands gamma delta T cells:
1. using first 30 minutes unlatching Biohazard Safety Equipments；
2. taking out D-PBS from refrigerator using preceding, it is placed at room temperature for 30 minutes；
3. 30ml peripheral blood sample (anticoagulant heparin) is transferred to two sterile 50ml centrifuge tubes, every pipe 15ml, then
The sterile D-PBS of 22.5ml is added to every pipe, overturns centrifuge tube repeatedly, mixes well；
4. separately taking two 50ml sterile centrifugation tubes, it is separately added into 15ml Ficoll-Paque Plus solution, is then distinguished
The blood (by drawing in two sterile tubes in step 3) after diluting in 24ml step 3 is slowly added, is formed and is layered, 20 DEG C,
400 × g is centrifuged 30 minutes；
5. two 50ml centrifuge tubes in step 4 are put into Biohazard Safety Equipment, 15ml blood then is sopped up using 10ml suction pipe
Clear layer is fitted into a new sterile 50ml centrifuge tube, 56 DEG C of inactivations, 30 minutes stand-by (for preparing mixed culture medium)；
6. drawing the leukocytic cream (PBMCs) in each 50ml centrifuge tube, it is transferred to a new 50ml sterile centrifugation tube；
7. to step 6 equipped with being added the sterile PBS of its 3 times of volumes in the centrifuge tube of PBMCs cell suspension, and with sterile shifting
Liquid pipe mixes, and 20 DEG C, 400 × g, is centrifuged 10 minutes；
8. abandoning supernatant, the sterile PBS of 50ml is added, PBMC is slowly resuspended；
9. 20 DEG C, 400 × g, being centrifuged 10 minutes；
10. the mixed culture of the RMPI1640+50vt% X-vivo15 of 5ml 50vt% autoserum containing step 5 is added
Base mixes, and takes 10 μ l for counting；
By taking the peripheral blood mononuclear cells (PBMCs) of half amount in step 10, addition certain volume contains azoles and carrys out phosphine
The mixed culture medium of acid adjusts cell concentration to 1 × 106cells/ml；The PBMCs addition of the other half amount contains zoledronic acid
1640 culture medium of RPMI, adjustment cell concentration to 1 × 106cells/ml；
Use T-175 culture bottle culture cell；
Tissue Culture Flask is placed in 5%CO2, carries out cell culture in 37 DEG C of cell incubators.
Embodiment 2: culture medium changes liquid
1. mixed culture medium is put into warm bath in 37 DEG C of water-baths, or equilibrium at room temperature 1 hour is placed on to room temperature；
2. Tissue Culture Flask is taken in Biohazard Safety Equipment, cell is resuspended, is then blown with 25ml pipettor even, takes 10 μ l
Cell suspension is counted；
3. when on day 4, all cells are transferred in 50ml sterile centrifugation tube, and replace amplification culture medium, it is specific to replace
It operates as follows:
4. adjustment final cell concentration is 1 × 10 according to cell total amount6cells/ml.Wherein, improved method are as follows: take one
It partly measures the new improved method that contains and expands required amplification factor (anti-human CD3Ab, anti-human CD28Ab, IL-
15, IL-21 and IL-2) mixed culture medium of composition is added in empty Tissue Culture Flask；Conventional culture methods are as follows: take half
The new RPMI 1640 containing amplification factor (IL-15, IL-21 and IL-2) composition needed for conventional culture methods of amount is cultivated
Base is added in empty Tissue Culture Flask；
5. taking the cell of step 3,20 DEG C, 400 × g, it is centrifuged 5 minutes；
6. the cell culture medium supernatant after step 5 centrifugation is transferred in 50ml sterile centrifugation tube, half amount cell is taken to train
It supports base supernatant and cell is resuspended, the cell after resuspension is put into continues to cultivate in the Tissue Culture Flask of step 4 respectively；
7. Tissue Culture Flask is put into cell incubator, 5%CO2,37 DEG C continue to cultivate；
8. concrete operations were as follows in the 6th, 8,10,12,14,16,18 and 20 day replacement amplification culture medium:
9. mixed culture medium or 1640 culture medium of RPMI are put into warm bath in 37 DEG C of water-baths, or to be placed on equilibrium at room temperature 1 small
Up to room temperature；
10. Tissue Culture Flask is taken in Biohazard Safety Equipment, cell is resuspended, is then blown using 25ml pipettor even, takes 10 μ
L cell suspension is counted；
According to cell total amount, take mixed containing amplification factor needed for improved method or conventional method amplification cultivation in right amount
It closes culture medium or RMPI1640 is added in Tissue Culture Flask, adjustment final cell concentration is 1 × 106cells/ml；
Cell is put into cell incubator, 5%CO2,37 DEG C continue to cultivate.
By the phenotype for the gamma delta T cells that flow cytomery conventional amplification method and modified amplification method are turned out,
The cell mass of the CD3 positive gamma delta T CR positive is gamma delta T cells, as shown in the region Q2 in Fig. 1.Figure 1A is using conventional method culture
14 days cell phenotype testing results, gamma delta T cells purity as shown in the figure are that 35.5%, Figure 1B is using improved method culture
14 days cell phenotype testing results illustrate that modified cultural method can expand as shown, gamma delta T cells purity is 95.5%
Increase the gamma delta T cells for turning out higher purity.
Culture the 0th, 4,6,7,8,10,12,14,16,18,20 and 21 day statistics inoculating cell number and amplification cultivation it is thin
Born of the same parents' number makes cell growth curve, as shown in Fig. 2, 3,002,000,000 cells of inoculation, expand 21 days, total number of cells can be of about 500
Hundred million, fully meet clinical application needs.It counts the 0th day, the 7th day, the 14th day and the 21st day, calculates gamma delta T cells expanded,
It can be seen that a large amount of gamma delta T cells can be obtained using modified amplification method, as a result referring to Fig. 3.
Embodiment 3: the gamma delta T cells Phenotypic examination of modified cultural method amplification cultivation
1. taking the cell of culture the 21st day, it is put into the EP pipe of 2 1.5mL, every pipe 1 × 106A cell, 400 × g from
The heart 5 minutes, remove supernatant；
2. being separately added into 1mLPBS to wash one time, 400 × g is centrifuged 5 minutes, removes supernatant；
3. the PBS of 100 μ L is added, it is then respectively adding control antibodies Mouse IgG1-PerCP, Mouse IgG1-FITC
(Ctrl in Fig. 4) and detection antibody mouse anti-human CD3-PerCP, anti-human gamma delta T CR-FITC fluorescence antibody
(CD3/ γ δ-TCR in Fig. 4), 4 DEG C are placed 30 minutes；
4. PBS is washed twice, supernatant is abandoned, cell finally is resuspended using 200 μ LPBS.
It is detected using the gamma delta T cells that AECANovocyte flow cytometer obtains culture, as shown in figure 4, culture
21 days cells, the purity of gamma delta T cells is up to 97.8%.
Embodiment 4: the gamma delta T cells killing activity detection that modified cultural method expands
Different effect targets are detected than gamma delta T cells to breast cancer using the real-time n cell functional analysis instrument of Essen Biology
Cell line SKBR-3 cell killing activity.
1. digesting SKBR-3 cell, concentration of cell suspension is prepared to 2.6667 × 105/mL；
2. spreading cell on real-time 8 plate of n cell functional analysis instrument E-Plate, 300 μ L, 37 DEG C of trainings are added in every hole
It supports 18-24 hours；
3. by the gamma delta T cells suspension of 100 μ L or isometric culture medium (blank control) with different effect target ratio 5:1；
10:1；20:1 is added in culture plate；
4. 8 detection plate of E-Plate is placed in real-time monitoring on real-time n cell functional analysis instrument monitor station, observe
Influence (Fig. 5) of the gamma delta T cells to SKBR-3 cell.
Experimental result is analyzed, at the time point that gamma delta T cells are added, uniforms cell index value, homogenization cell is obtained and refers to
Number (NCI), the NCI value after statistics addition gamma delta T cells 4 hours, it is thin to calculate different proportion (20:1,10:1,5:1 and 0:1) gamma delta T
Born of the same parents are to the killing activity of breast cancer cell line SKBR-3, as shown in fig. 6, the killing activity of gamma delta T cells is with gamma delta T cells ratio
Increase and increases.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although with reference to the foregoing embodiments
Invention is explained in detail, those skilled in the art should understand that: it still can be to aforementioned each implementation
Technical solution documented by example is modified or equivalent replacement of some of the technical features；And these modification or
Replacement, the spirit and scope for technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution.
Claims (7)
1. the composition that a kind of stimulation induction mononuclearcell amplification is gamma delta T cells, which is characterized in that the composition includes azoles
Lai phosphoric acid, anti-human CD3 Ab, anti-human CD28 Ab, IL-15, IL-21 and IL-2；The concentration of azoles Lai phosphoric acid be 1-100 μM, it is anti-human
The concentration of CD3 Ab is 10~500ng/mL, the concentration of anti-human CD28 Ab is 10~500ng/mL, the concentration of IL-15 be 10~
The concentration of 200ng/mL, IL-21 are 10~200ng/mL, the concentration of IL-2 is 500~3000U/mL.
2. the composition that stimulation induction mononuclearcell amplification according to claim 1 is gamma delta T cells, it is characterised in that:
Azoles Lai phosphoric acid concentration is 5 μM, anti-human CD3 Ab concentration is 50ng/mL, anti-human CD28Ab concentration is that 50ng/mL, IL-15 concentration are
50ng/mL, IL-21 concentration are that 50ng/mL, IL-2 concentration are 1000U/mL.
3. the composition that stimulation induction mononuclearcell amplification according to claim 1 is gamma delta T cells, it is characterised in that:
The mononuclearcell derives from peripheral blood, Cord blood, marrow or inductive pluripotent stem cells.
4. a kind of thin for gamma delta T using the described in any item composition stimulation induction mononuclearcell amplification in vitros of claim 1-3
The method of born of the same parents, comprising the following steps:
1) preparation of mononuclearcell: separation mononuclearcell, with percent by volume 50%-100% containing 10vt%FBS or certainly
Cell is resuspended in the mixed culture medium of the RMPI 1640 and 0%-50%X-vivo15 of body serum, is seeded in culture bottle or culture bag
Middle culture；
2) mixing of the azoles Lai phosphoric acid and 500~3000U/mL IL-2 that are 1-100 μM containing concentration the induction of gamma delta T cells: is added
After medium body external stimulus 4 days be added containing anti-human CD3 Ab of 10~500ng/mL, the anti-human CD28Ab of 10~500ng/mL, 10~
The mixed culture medium progress half of 200ng/mL IL-15,10~200ng/mL IL-21 and 500~3000U/mL IL-2, which are measured, changes
Liquid is placed in 37 DEG C, cultivates in 5%CO2 incubator；
3) every 2~3 days according to count results fluid infusion, 14~21 days harvest gamma delta T cells.
5. composition stimulation induction mononuclearcell amplification in vitro according to claim 4 is the method for gamma delta T cells,
Be characterized in that: the azoles Lai phosphoric acid being added in step 2) is 5 μM final concentration of, the final concentration of 50ng/mL of anti-human CD3 Ab, anti-human CD28
Ab final concentration of 50ng/mL, IL-15 final concentration of 50ng/mL, IL-21 final concentration of 50ng/mL and IL-2 is final concentration of
1000U/mL。
6. composition stimulation induction mononuclearcell amplification in vitro according to claim 4 is the method for gamma delta T cells,
Be characterized in that: the concentration of the peripheral blood mononuclear cells (PBMCs) is 1 × 106/mL。
7. composition stimulation induction mononuclearcell amplification in vitro according to claim 4 is the method for gamma delta T cells,
Be characterized in that: the ratio of the RMPI 1640 containing 10vt%FBS or autoserum in mixed culture medium is 50vt%, X-vivo15
The ratio of culture medium is 50vt%.
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| CN114591907A (en) * | 2020-12-03 | 2022-06-07 | 复旦大学 | Preparation and amplification method and application of gamma delta T cells |
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