CN108220239A - A kind of composition for stimulating induction mononuclearcell amplification as gamma delta T cells and its application - Google Patents
A kind of composition for stimulating induction mononuclearcell amplification as gamma delta T cells and its application Download PDFInfo
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Abstract
The present invention relates to a kind of compositions for stimulating induction mononuclearcell amplification as 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 for largely expanding gamma delta T cells in vitro using the composition,It is specially a kind of to induce methods of a large amount of amplifications for gamma delta T cells in vitro with peripheral blood mononuclear cells (PBMCs),The present invention is by adding in zoledronic acid induced activation,Add in anti human CD3Ab,anti‑human CD28Ab,IL‑15,IL‑21,Induced activation proliferation is carried out with IL 2,Co stituation induced amplification gamma delta T cells are combined using a variety of amplification factors,The gamma delta T cells of acquisition are big with quantity,Purity is high,The features such as cytotoxicity is strong,With extraordinary clinical value.
Description
Technical field
Mononuclearcell is stimulated to make its induced amplification should for the composition of gamma delta T cells and use the present invention relates to a kind of
The method that composition largely expands mononuclearcell (PBMCs) in gamma delta T cell in vitro.
Background technology
Tumour is one of major disease to threaten at present to human health, and no matter the death rate and incidence occupy height
Position.Tumour immunotherapy be the 4th kind of tumor therapeuticing method after operation, radiotherapy, chemotherapy and it is of greatest concern at present 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, particularly T cell.
Verified T cell, the T cell for particularly expressing γ δ receptors 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 receptors.γδT
Cell conducts TCR signals by being associated with CD3 compounds, and in the peripheral blood of normal adults, gamma delta T cells only account for cycle T cell
1-10%；α β T cells account for more than 90% cycle T cell, and directly conduct intracellular signal by being associated with CD3 compounds.
Different from α β TCR, gamma delta T CR directly in conjunction with antigen and can not depend on the numerator mediated antigen presentations of MHC, therefore thin in gamma delta T
Cellular surface does not express CD4 and CD8 molecules.Widely research shows that, according to the expression of δ chains, gamma delta T cells can be divided into three
Major class, the gamma delta T cells of 1 chains of expression V δ are mainly distributed on the endodermis of mucomembranous surface, and a seldom part is distributed in peripheral blood.V
1 type gamma delta T cells of δ play a significant role in damage, infection and vicious transformation epidermis intact is resisted.Gamma delta T cells are another kind of
Main subgroup is to express the gamma delta T cells of 2 chains of V δ, and 2 chains of V δ are almost only matched with 9 chains of V γ, is main in normal adults
The cycle gamma delta T cells wanted can reach 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 chains of third class gamma delta T cells Expression of Subsets V δ, account for about
The 0.2% of T cell is recycled, including CD4+, CD8+ and CD4-CD8- subgroups.The unstable expression CD56 of 3 gamma delta T cells of V δ,
CD161, HLA-DR and NKG2D.
Different from α β T cells, gamma delta T cells are not easy to be processed and presented to lack by antigen to be influenced, 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 mouse skin is prevented to be 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 research shows that, compared with control group mice, by the gamma delta T of tail vein injection homogenic type, prostate can be significantly reduced
The 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. research shows 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 are thin with killing autologous tumor
The activity of born of the same parents can pass through the killing activity for the monoclonal antibodies block gamma delta T cell for identifying 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.
Research shows that, gamma delta T cells can kill colorectal cancer tumor stem cell, tumor stem cell is originated in tumour, growth, transfer,
Traditional tumour treatment means are resisted so as to (the J Immunol.2009 that play a significant role in recurring；182(11):7287-
7296.).It has been detached 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 a kind of practicality and high efficiency, it is at low cost, technically simple, can largely expand high-purity, the gamma delta T cells of high cytotoxic activity become
The problem of in the urgent need to address at present.
Invention content
In view of the above problems, the present invention proposes a kind of stimulate and induces mononuclearcell amplification as gamma delta T cells
By adding in zoledronic acid induced activation, Co stituation induced amplification is combined using a variety of amplification factors for composition and its application
Gamma delta T cells, the gamma delta T cells of acquisition have the characteristics that quantity is big, purity is high, cytotoxicity is strong, have extraordinary clinic should
With value.
In order to realize above-mentioned purpose, the present invention uses following technical solution：
It is a kind of stimulate induction mononuclearcell amplification for gamma delta T cells composition, the composition include zoledronic acid,
Anti-human CD3Ab, anti-human CD28Ab, IL-15, IL-21 and IL-2.
Preferably, a concentration of 1-100 μM of zoledronic acid, a concentration of 10~500ng/mL of anti-human CD3Ab,
A concentration of 10~500ng/mL of anti-human CD28Ab, a concentration of 10~200ng/mL of IL-15, IL-21 concentration
For 10~200ng/mL, a concentration of 500~3000U/mL of IL-2.
Preferably, zoledronic acid is 5 μM a concentration of, anti-human CD3Ab a concentration of 50 ng/mL, anti-human
A concentration of 1000U/ of CD28Ab a concentration of 50ng/mL, IL-15 a concentration of 50ng/mL, IL-21 a concentration of 50ng/mL, IL-2
mL。
Preferably, mononuclearcell derives from peripheral blood, Cord blood, marrow or inductive pluripotent stem cells (iPSC).
Preferably, it is a kind of that the side that induction mononuclearcell amplification in vitro is gamma delta T cells is stimulated using combinations of the above object
Method includes the following steps：
1) preparation of mononuclearcell：Mononuclearcell is detached, 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) induction of gamma delta T cells：It adds in containing a concentration of 1-100 μM of zoledronic acid and 500~3000U/mL IL-2
Mixed culture medium stimulated in vitro adds in after 4 days 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 incubators；
3) every 2~3 days according to count results fluid infusion, 14~21 days harvest gamma delta T cells.
Preferably, the middle zoledronic acid added in of step 2) is 5 μM final concentration of, anti-human CD3Ab are 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, a concentration of 1 × 10 of the peripheral blood mononuclear cells (PBMCs) described in a case study on implementation6/
mL。
Other phosphoantigens can also be used to replace zoledronic acid in one embodiment, including isopentenyl pyrophosphate
(IPP), (E) -4- hydroxy-3-methyls-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), the biphosphonate containing nitrogen such as etherophosphoric acid (MEP), 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 mediums is 50vt%.
The raw materials used in the present invention and reagent are commercially available in addition to having specified otherwise.
Due to using above-mentioned technical solution, the beneficial effects of the invention are as follows：The present invention provides one kind by cell factor,
The composition and the method largely expanded in vitro using the composition stimulation gamma delta T cells that zoledronic acid and antibody form, should
Method can obtain the gamma delta T cells of a large amount of, high proliferation ability, high-purity and high cytotoxic activity, the cell available for tumour
Immunization therapy.
Description of the drawings
Fig. 1 is the conventional culture methods of cell factor and zoledronic acid combined induction and cell factor, zoledronic acid and anti-
The flow cytometer showed figure of the gamma delta T cells of body joint 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 cytometer showed figure 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 cells are incubated 4 altogether
Hour cell index and it is different effect target than gamma delta T cells to the killing percentage of SKBR-3.
Specific embodiment
Purpose, technical scheme and advantage to make the embodiment of the present invention are clearer, below in conjunction with the embodiment of the present invention,
Technical solution in the embodiment of the present invention is clearly and completely described.Based on the embodiment of the present invention, the common skill in this field
Art personnel all other embodiments obtained without creative efforts belong to the model that the present invention protects
It encloses.
" mixed culture medium " described in the embodiment of the present invention refers both to contain 10vt% according to the 50vt% that existing method is prepared
The mixed culture medium of the RMPI1640+50vt%X-vivo15 of autoserum.
Embodiment 1：The induction of gamma delta T cells
Mononuclearcell (PBMCs) is detached from peripheral blood and expands gamma delta T cells：
1. opened Biohazard Safety Equipment using first 30 minutes；
2. taking out D-PBS from refrigerator using preceding, it is placed at room temperature for 30 minutes；
3. 30ml peripheral bloods sample (anticoagulant heparin) is transferred to two sterile 50ml centrifuge tubes, often pipe 15ml, then
The sterile D-PBS of 22.5ml are added in every pipe, overturn centrifuge tube, abundant mixing repeatedly；
4. separately taking two 50ml sterile centrifugation tubes, 15ml Ficoll-Paque Plus solution is separately added into, is then distinguished
The blood (by being drawn in two sterile tubes in step 3) after diluting slowly is added in 24ml steps 3, 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, then 15ml blood is sopped up using 10ml suction pipes
Clear layer is fitted into a new sterile 50ml centrifuge tube, 56 DEG C of inactivations 30 minutes for use (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. the sterile PBS of its 3 times of volumes is added in into centrifuge tube of the step 6 equipped with PBMCs cell suspensions, and with sterile shifting
Liquid pipe mixing, centrifuges 10 minutes by 20 DEG C, 400 × g；
8. abandoning supernatant, the sterile PBS of 50ml are added in, PBMC is slowly resuspended；
9. it 20 DEG C, 400 × g, centrifuges 10 minutes；
10. add in the mixed culture of the RMPI1640+50vt% X-vivo15 of 5ml 50vt% autoserums containing step 5
Base, mixing take 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 additions of the other half amount contain zoledronic acid
1640 culture mediums of RPMI, adjustment cell concentration to 1 × 106cells/ml；
Use T-175 culture bottle culture cells；
Tissue Culture Flask is positioned in 5%CO2,37 DEG C of cell incubators and carries out cell culture.
Embodiment 2：Culture medium changes liquid
1. mixed culture medium is put into 37 DEG C of water-baths warm bath or is placed on equilibrium at room temperature 1 hour to room temperature；
2. Tissue Culture Flask is taken in Biohazard Safety Equipment, cell is resuspended, is then blown with 25ml pipettors even, takes 10 μ l
Cell suspension is counted；
3. when on day 4, all cells are transferred in 50ml sterile centrifugation tubes, and replace amplification culture medium, it is specific to replace
Operation is as follows：
4. according to cell total amount, final cell a concentration of 1 × 10 is adjusted6cells/ml.Wherein, improved method is：Take one
What partly amount was new contains amplification factor (anti-human CD3Ab, anti-human CD28Ab, IL- needed for improved method amplification
15, IL-21 and IL-2) mixed culture medium of composition added in empty Tissue Culture Flask；Conventional culture methods are：Take half
Amount is new to cultivate containing the RPMI 1640 of amplification factor (IL-15, IL-21 and IL-2) composition needed for conventional culture methods
Base is added in empty Tissue Culture Flask；
5. taking the cell of step 3,20 DEG C, 400 × g, centrifuge 5 minutes；
6. the cell culture medium supernatant after step 5 is centrifuged is transferred in 50ml sterile centrifugation tubes, half amount cell is taken to train
It supports base supernatant and cell is resuspended, the cell after resuspension is put into the Tissue Culture Flask of step 4 continues to cultivate respectively；
7. Tissue Culture Flask is put into cell incubator, 5%CO2,37 DEG C continue to cultivate；
8. replacing amplification culture medium at the 6th, 8,10,12,14,16,18 and 20 day, concrete operations are as follows：
9. mixed culture medium or 1640 culture mediums of RPMI are put into 37 DEG C of water-baths warm bath 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 pipettors even, takes 10 μ
L cell suspensions are counted；
According to cell total amount, the mixing in right amount containing amplification factor needed for improved method or conventional method amplification cultivation is taken
Culture medium or RMPI1640 are added in Tissue Culture Flask, adjust final cell a concentration of 1 × 106cells/ml；
Cell is put into cell incubator, 5%CO2,37 DEG C continue to cultivate.
The phenotype of gamma delta T cells turned out by flow cytomery conventional amplification method and modified amplification method,
The cell mass of the CD3 positive gamma delta T CR positives is gamma delta T cells, as shown in Q2 regions in Fig. 1.Figure 1A is using conventional method culture
The cell phenotype testing result of 14 days, gamma delta T cells purity is using improved method culture for 35.5%, Figure 1B as shown in the figure
The cell phenotype testing result of 14 days as shown in the figure, gamma delta T cells purity is 95.5%, illustrates that modified cultural method can expand
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 practice 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 pipes of 2 1.5mL, often pipe 1 × 106A cell, 400 × g from
The heart 5 minutes, removes supernatant；
2. being separately added into 1mLPBS to wash one time, 400 × g is centrifuged 5 minutes, removes supernatant；
3. adding in the PBS of 100 μ L, 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 antibodies
(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.
The gamma delta T cells obtained using AECANovocyte flow cytometers to culture are detected, as shown in figure 4, culture
The cell of 21 days, 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
Using the different effect targets of real-time n cell functional analysis instrument detection of Essen Biology than gamma delta T cells to breast cancer
Cell line SKBR-3 cell killing activities.
1. digesting SKBR-3 cells, concentration of cell suspension is prepared to 2.6667 × 105/mL；
2. spreading cell on real-time 8 plates of n cell functional analysis instrument E-Plate, adding in 300 μ L per hole, 37 DEG C are trained
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 targets than 5:1；
10:1；20:1 adds in culture plate；
It monitors, observes in real time 4. 8 detection plates of E-Plate are placed on real-time n cell functional analysis instrument monitor station
Influence (Fig. 5) of the gamma delta T cells to SKBR-3 cells.
Experimental result is analyzed, at the time point for adding in gamma delta T cells, uniforms cell index value, homogenization cell is obtained and refers to
Number (NCI), statistics add in NCI value of the gamma delta T cells after 4 hours, calculate different proportion (20:1,10:1,5:1 and 0:1) gamma delta T is thin
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 increase.
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
The present invention is described in detail, it will be understood by those of ordinary skill in the art that：It still can be to aforementioned each implementation
Technical solution recorded in example modifies or carries out equivalent replacement to which part technical characteristic；And these modification or
It replaces, the spirit and scope for various embodiments of the present invention technical solution that it does not separate the essence of the corresponding technical solution.
Claims (8)
1. a kind of composition for stimulating induction mononuclearcell amplification as 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.
2. composition of the stimulation induction mononuclearcell amplification according to claim 1 for gamma delta T cells, it is characterised in that：
A concentration of 1-100 μM of azoles Lai phosphoric acid, a concentration of 10~500ng/mL of anti-human CD3 Ab, anti-human CD28
A concentration of 10~500ng/mL of Ab, a concentration of 10~200ng/mL of IL-15, IL-21 a concentration of 10~200ng/mL,
A concentration of 500~3000U/mL of IL-2.
3. composition of the stimulation induction mononuclearcell amplification according to claim 2 for gamma delta T cells, it is characterised in that：
Azoles Lai phosphoric acid concentrations are 5 μM, a concentration of 50ng/mL of anti-human CD3 Ab, anti-human CD28 Ab are a concentration of
A concentration of 1000U/mL of 50ng/mL, IL-15 a concentration of 50ng/mL, IL-21 a concentration of 50ng/mL, IL-2.
4. composition of the stimulation induction mononuclearcell amplification according to claim 1 for gamma delta T cells, it is characterised in that：
The mononuclearcell derives from peripheral blood, Cord blood, marrow or inductive pluripotent stem cells.
It is 5. a kind of thin for gamma delta T using claim 1-4 any one of them composition stimulation induction mononuclearcell amplification in vitro
The method of born of the same parents, includes the following steps：
1) preparation of mononuclearcell：Mononuclearcell is detached, 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) induction of gamma delta T cells：Add in the mixing containing a concentration of 1-100 μM of azoles Lai phosphoric acid and 500~3000U/mL IL-2
Medium body external stimulus adds in after 4 days contains 10~500ng/mL anti-human CD3 Ab, 10~500ng/mL anti-
The mixing of human CD28 Ab, 10~200ng/mL IL-15,10~200ng/mL IL-21 and 500~3000U/mL IL-2
Culture medium carries out half amount and changes liquid, is placed in 37 DEG C, cultivates in 5%CO2 incubators；
3) every 2~3 days according to count results fluid infusion, 14~21 days harvest gamma delta T cells.
6. composition stimulation induction mononuclearcell amplification in vitro according to claim 5 is the method for gamma delta T cells,
It is characterized in that：The azoles Lai phosphoric acid that adds in step 2) is 5 μM final concentration of, the final concentration of 50ng/mL of anti-human CD3 Ab,
The final concentration of 50ng/mL of anti-human CD28 Ab final concentration of 50ng/mL, IL-15 final concentration of 50ng/mL, IL-21,
With the final concentration of 1000U/mL of IL-2.
7. composition stimulation induction mononuclearcell amplification in vitro according to claim 5 is the method for gamma delta T cells,
It is characterized in that：A concentration of the 1 × 10 of the peripheral blood mononuclear cells (PBMCs)6/mL。
8. composition stimulation induction mononuclearcell amplification in vitro according to claim 5 is the method for gamma delta T cells,
It is characterized in that：The ratio of 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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