CN102443567A - In vitro amplification method of tumor specific gamma-delta T lymphocytes - Google Patents
In vitro amplification method of tumor specific gamma-delta T lymphocytes Download PDFInfo
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Abstract
The invention discloses an amplification method of tumor specific gamma-delta T lymphocytes. In the Method, inactivated autologous leukemia cells, recombinant human IL2 (rhIL-2), phytohemagglutinin (PHA) and autologous mononuclear cell components (CD4-CD8-PBMCs) are jointly used so as to corporately culture and amplify the tumor specific gamma-delta T lymphocytes. The tumor specific gamma-delta T lymphocytes can be used for anti-leukaemia immunologic reaction and can be used as an effective leukaemia treatment scheme.
Description
Technical field
The invention belongs to medical field, specifically, relate to a kind of amplification in vitro method of tumour-specific gamma delta T lymphocytes.
Background technology
The T cell surface has can discern antigenic structure specifically, is called T cell antigen receptor (TCR).Dissimilar according to TCR, the T cell can be divided into α β T cell and gamma delta T cells.
α β T cell is mainly participated in the acquired immunity process, through the restricted tumor cell of MHC.Yet tumour cell can suddenly change, weaken antigen presentation, induce mechanism such as the immune evasion that produces the inhibition immunocyte, blocking-up antigen recognition process through MHC.
Gamma delta T cells is considered to specifc immunity cell type (the ChemImmunol Allergy.2005 between acquired and the natural immunity; 86:151-183.); Account for 1%～5% of CD3+T cell, most of gamma delta T cells is not expressed CD4, CD8 molecule, the specific recognition antigen function is arranged and does not have MHC restriction; And body anti-infective with autoimmune disease and process such as antitumor in play an important role (Nat Rev Immune.2002,2:336-345).The activatory gamma delta T cells has the various kinds of cell function, and it both can directly kill special cells, showed the Tc activity; But secrete cytokines is coordinated immunoreation again, shows the Th activity.Because gamma delta T cells is cell function and unique antigen recognition spectrum flexibly, it plays a part indispensable in the immunne response of body and immunosurveillance.
Because low, the limited amount of gamma delta T cells ratio in human body; In view of its powerful anti-tumor activity; Seek a kind of easy, excite and the method for the gamma delta T cells that increases specifically; To help undoubtedly the biological characteristics of this group cell and the research of function, and the utilization of the possibility in the tumour adoptive immunotherapy.And the antigen specific immune cell therapy malignant tumour of using external evoked amplification has become a kind of effective clinical immunotherapy method (TRENDS in Immunology.2001,22 (9): 516-523.).The gamma delta T cells of amplification in vitro also has been applied to clinical examination and has controlled (Blood.2003,102:200-206; Immunol Res.2009,45 (1): 85-95; ExpMol Pathol.2004,76 (2): 117-121; Cancer Res.2010,70:10024-10027).
The report that many vitro culture gamma delta T cells methods are arranged at present; Comprise with anti-cd 3 antibodies, IL-2 and IL-4 (Leukemia.2011; Doi:10.1038), anti-TCR γ δ and IL-2 (Chinese Journal of Immunology .2011; 739-743), non-peptide phosphoric acid antigen and IL-2 (Blood.2003,102:200-206) etc.Though cell cultures and amplification method constantly improve and are perfect; But the research to the gamma delta T cells adoptive immunotherapy is research object with the healthy subjects gamma delta T cells mostly both at home and abroad; So the gamma delta T cells specificity that amplification is come out is not high; Reduced its tumor-killing effect, causing with the gamma delta T cells is that basic immunotherapy has received certain restriction.Therefore, seek new efficiently from body gamma delta T cells amplification method to improve the efficient that it is used for adoptive immunotherapy, seem particularly important.
Summary of the invention
The present inventor utilizes the leukaemic from the monocyte component CD4-CD8-PBMCs of body to be: " seed cell "; Through with deactivation from body leukemia cell, recombination human interleukin 2 (rhIL-2) and phytohemagglutinin (PHA) co-cultivation; Stimulate and induce gamma delta T lymphocytes to be able to selective amplification with real tumor-killing effect, thus the tumour-specific gamma delta T lymphocytes that acquisition has individuation effect characteristic.
The amplification in vitro method that the purpose of this invention is to provide a kind of tumour-specific gamma delta T lymphocytes.
The amplification in vitro method of tumour-specific gamma delta T lymphocytes of the present invention may further comprise the steps:
(1) obtains leukemia cell and deactivation by separating in the blood patient peripheral blood that just turns white;
(2) obtain monocyte component CD4-CD8-PBMCs by separating in the leukaemic of alleviating the peripheral blood;
(3) in the presence of recombination human interleukin 2 (rhIL-2) and phytohemagglutinin (PHA), with the CD4-CD8-PBMCs co-cultivation that obtains in the leukemia cell of the deactivation that obtains in the step (1) and the step (2), amplification tumour-specific gamma delta T lymphocytes.
According to a preferred embodiment of the invention, said step (1) comprises the step through the deactivation of radionuclide cobalt irradiation with the leukemia cell.
According to a preferred embodiment of the invention, culture condition is 37 ℃ and 5%CO in the said step (3)
2
The present invention has following beneficial effect:
(1) directly with deactivation from the body leukemia cell cultivation that contacts with gamma delta T lymphocytes, the screening step that can remove loaded down with trivial details LAA from is a kind of succinct effective means.
(2) can produce tumour-specific gamma delta T lymphocytes, can be used as a kind of effective leukemia treating scheme with individuation effect characteristic.
Among the present invention; The tumour antigen from the body leukemia cell through deactivation is mutually crosslinked with the corresponding acceptor of gamma delta T lymphocytes; And under the stimulation of the growth factor of recombination human interleukin 2 and phytohemagglutinin, make that being selected property of the gamma delta T lymphocytes propagation to specific for tumour antigen activates.The present invention increases the gamma delta T lymphocytes that obtains for from body leukemia cell and heterology leukemia cell good inhibition effect being arranged, in leukemic adoptive immunotherapy, has wide practical use.
Description of drawings
Fig. 1 is the growth of colony clone property for the gamma delta T lymphocytes of microscopic examination amplification.
Fig. 2 is different biotic factor or its combination influence to gamma delta T lymphocytes propagation.
Fig. 3 is the phenotypic alternation before and after the amplification of CD4-CD8-PBMCs cellular component.Wherein, A is for before increasing, and B is for after increasing.
Fig. 4 is the cytokine secretion spectrum of amplification property gamma delta T lymphocytes.
Fig. 5 is the CDCC of amplification property gamma delta T lymphocytes to the leukemia cell.Wherein, A is to the killing activity from the body leukemia cell, and B is the killing activity to the heterology leukemia cell line.
Fig. 6 is the white blood disease effect in vivo of amplification property gamma delta T lymphocytes.Wherein, A is anti-heterology white blood disease effect in vivo, and B is in vivo anti-from the effect of body white blood disease.
Embodiment
Below in conjunction with specific embodiment, the present invention is further specified.Should be understood that following examples only are used to the present invention is described but not are used to limit scope of the present invention.
" from body " among the present invention is meant and derives from same individual patient." just send out " and be meant that the patient is diagnosed as white blood disease for the first time, and before not through any chemotherapeutics treatment." alleviation " is meant the patient that leukemic sings and symptoms disappears, its blood picture Hb>=100g/L (man) or 90g/L (women and children), neutrophil leucocyte absolute value>=1.5 * 10
9/ L, thrombocyte>=100 * 10
9/ L, do not have the leukemia cell, bone marrow smear in the PBL classification: myeloblast+promyelocyte (former list+promonocyte or former lymph+prolymphocyte)≤5%, red corpuscle and megalokaryocyte series are normal.
1.1, leukemia cell's separation and preservation
Collect four examples becoming impatient property white blood disease (AML) patient (the high leukemia cell's type of peripheral blood:＞10 * 10 just
9/ L) fresh peripheral blood is used anticoagulant heparin, uses equal-volume PBS dilute blood then.Get the equal-volume lymphocyte separation medium and add at the bottom of the centrifuge tube pipe, and be warmed up to room temperature.Draw the blood sample after diluting with suction pipe, slowly be taped against above the lymphocyte separation medium, do not upset the liquid layer interface along tube wall.At ambient temperature, centrifugal 20 minutes of 700g level is drawn lymphocyte separation medium interface oyster white mononuclearcell layer.Add 10ml saline water and dilute isolating lymphocyte, at ambient temperature, centrifugal 10 minutes of 250g abandons supernatant.With saline water repeated washing 1～2 time, remove thrombocyte and anticoagulant substances, use the whole serum frozen storing liquid that the leukemia cell is frozen at last, it is subsequent use to insert liquid nitrogen cryopreservation.
1.2, leukemia cell's deactivation
Get leukemia cell frozen described in 1.1, place 37 ℃ of water-baths to thawing fully.Transitional cell dropwise adds 1ml reagent I (the RPMI1640 substratum that contains 20%FBS) in the 15ml centrifuge tube, 37 ℃ of water-baths 10 minutes.At ambient temperature, 400g is centrifugal 10 minutes.Abandon supernatant, add 5ml reagent I, room temperature was placed 1 hour.At ambient temperature, centrifugal 10 minutes of 400g, collecting precipitation is used cell.It is resuspended to add the 10mlRPMI1640 substratum, 37 ℃ of (5%CO
2) hatched 24 hours.Behind the washed cell, with radionuclide cobalt irradiation (35Gy) deactivation leukemia cell, 37 ℃ of (5%CO then
2) continue to cultivate 24 hours subsequent use.Leukemia cell through deactivation can not continue division growth, but still keeps the antigenic activity to tumour, is used for follow-up amplification tumour-specific gamma delta T lymphocytes.
In the present embodiment; From leukaemic's autologous peripheral blood, obtain the monocyte component and be used to cultivate the tumour-specific gamma delta T lymphocytes; This monocyte component is called CD4-CD8-PBMCs, comprises gamma delta T lymphocytes, BMDC, NK cell, NKT cell, B cell, granulocyte etc.The isolating concrete operations of CD4-CD8-PBMCs are following in the peripheral blood:
Four routine acute leukemias are in complete paracmastic patient's peripheral blood 10ml, anticoagulant heparin among the aseptic collection embodiment 1.Add each 500 μ l of people CD4 Depletion Cocktail and people CD8 Depletion Cocktail reagent (available from U.S. StemCell Technologies company), mixing, room temperature left standstill 20 minutes, added the PBS doubling dilution that equal-volume contains 2%FBS.Get the equal-volume lymphocyte separation medium and add at the bottom of the centrifuge tube pipe, and be warmed up to room temperature.Draw the blood sample after diluting with suction pipe, slowly be taped against above the lymphocyte separation medium, do not upset the liquid layer interface along tube wall.At ambient temperature, centrifugal 20 minutes of 700g level is drawn lymphocyte separation medium interface oyster white mononuclearcell layer.Add 10ml saline water and dilute isolating lymphocyte, at ambient temperature, centrifugal 10 minutes of 250g abandons supernatant.Repeated washing 1～2 time is removed thrombocyte and anticoagulant substances.With perfect medium (the RPMI1640 substratum that contains 10%FBS) adjustment cell concn to 10
6Cell/ml is subsequent use.
Four routine acute leukemia reduction of patient peripheral bloods by obtaining among the embodiment 2 separate the CD4-CD8-PBMCs that obtains; With being added with recombination human interleukin 2 (rhIL-2; 50U/ml) and the perfect medium (the RPMI1640 substratum that contains 10%FBS) of phytohemagglutinin (PHA, 0.5 μ g/ml) be adjusted to 5 * 10
5/ ml places 24 well culture plates, and adds 1.5 * 10
5The four routine deactivations that obtain among the individual embodiment 1 from the body leukemia cell, 37 ℃ of (5%CO
2) cultivate under the condition.Every half liquid that changed at a distance from 3 days whenever added stimulating again from the body leukemia cell of deactivation at a distance from 7 days, can see that four routine amplifying cells all are the growth of colony clone property, and representational microscope imaging figure is as shown in Figure 1.The gamma delta T lymphocytes that last centrifugal collection amplification obtains.
Embodiment4, different biotic factor or its combination to the detection of the influence of gamma delta T lymphocytes propagation (
3The H-TdR infiltration method)
With the amplification property gamma delta T lymphocytes (1 * 10 that obtains among the embodiment 3
5Cells/well) places 24 well culture plates, 37 ℃ of (5%CO
2) in tranquillization 24 hours, then respectively with deactivation from the ratio of body leukemia cell, or/and rhIL-2 (100U/ml), PHA (1 μ g/ml) carry out common cultivation 3 days in 3: 1.Simultaneously, be control group with the gamma delta T lymphocytes that does not add any other component.Then, every hole adds
3H-TdR 1 μ Ci continues to cultivate 12 hours.Adopt liquid scintillation instrument to measure its radioactivity (cpm), and calculate its amplification times.Three multiple holes are all established in all experiments.Amplification times is calculated and is used formula: amplification times=[test holes (cpm)-blank well (cpm)]/[control wells (cpm)-blank well (cpm)].As shown in Figure 2; That uses separately deactivation can the obvious stimulation gamma delta T lymphocytes from the body leukemia cell, makes it breed about 3 times and rhIL-2 or the PHA coupling can make gamma delta T lymphocytes increase about 7 times and 4 times respectively; And three's coupling expanding effect is the most remarkable, and amplification is about 8 times.Experimental result show that rhIL-2 and PHA can synergistic inactivations from the multiplication capacity of body leukemia cell to gamma delta T lymphocytes.
EmbodimentThe FCM of 5, amplification property gamma delta T lymphocytes phenotype analyzes
The CD4-CD8-PBMCs cell that obtains among the embodiment 2 is handling with fluorescently-labeled CD3, CD4, CD8, V δ 1, V δ 2, TCR γ δ, TCR monoclonal antibody respectively before body leukemia cell, rhIL-2 and the activation of PHA mixture stimulate and after the activation stimulation through deactivation in 3 weeks.Carry out flow cytometry (FCM) analysis after cultivating for 3 weeks, the immunophenotype that detects CD4-CD8-PBMCs cell amplification front and back changes.Visible like Fig. 3, before amplification cultivation, about 35%～48% is CD3+TCR γ δ+T cell among the CD4-CD8-PBMCs, and wherein V δ 2+ gamma delta T cells ratio accounts for 78%～89% (Fig. 3 A).Amplification cultivation was analyzed the proportional range that shows CD3+TCR γ δ+T cell in the expanded cells and is expanded to 73%～87% after 3 weeks, be main with V δ 1+ gamma delta T cells wherein, and V δ 2+ gamma delta T cells ratio was less than 15% (Fig. 3 B) of gamma delta T cells.Because V δ 1+ gamma delta T cells has better anti-tumor activity than V δ 2+ gamma delta T cells, explain that the gamma delta T lymphocytes that the present invention obtains has good tumour-specific.
EmbodimentThe detection of cytokine levels in the culture supernatant of 6, amplification property gamma delta T lymphocytes
In this enforcement, the level of cytokine in the culture supernatant of use ELISA test kit (available from R&D systems company) detection amplification property gamma delta T lymphocytes, concrete grammar is following:
Collect the culture supernatant of the amplification property gamma delta T lymphocytes that obtains among the embodiment 3; Analyze the concentration of IFN-γ, TNF-α, IL-3, IL-4, IL-10, IL-10 and IL-17 in the culture supernatant liquid with the ELISA test kit; Collect simultaneously from stimulate deactivation from body leukemia cell's culture supernatant liquid as control group, amplification property gamma delta T lymphocytes is carried out the cytokine spectrum analysis.As shown in Figure 4, amplification property gamma delta T lymphocytes is mainly secreted a large amount of IL-6, IL-12, TNF-α and IFN-γ, and IL-3, IL-4, IL-10 and IL-17 secretion are not obvious.Wherein, tumour necrosis factor (TNF-α) can kill and wound and suppress tumour cell, and amplification property gamma delta T lymphocytes of the present invention is TNF secretion-α in a large number, explains that it has good tumour-specific.
EmbodimentThe CDCC of 7, amplification property gamma delta T lymphocytes
In the present embodiment; Use the amplification property gamma delta T lymphocytes that obtains among apoptosis test kit (comprising annexin V-PE and 7 '-AAD dyestuff) the detection embodiment 3 to CDCC from body leukemia cell and heterology leukemia cell line available from BDPharmigen company.That uses in the present embodiment derives from first the becoming impatient property leukaemic of four examples among the embodiment 1 from the body leukemia cell.The heterology leukemia cell line that uses in the present embodiment is provided by Shanghai City hematology institute, comprises following cell strain: U973, HL-60, Kasumi-1 and K562.Concrete grammar is following:
With the four example amplification property gamma delta T lymphocytes that obtain among the embodiment 3 with the CFSE mark of 5 μ M action effect cell after 10 minutes; Then with the target cell of mark according to imitate target than (effector: target, E: T) be respectively 0: 1 (as the control group), 0.5: 1,1: 1,2.5: 1,5: 1 at 37 ℃ of (5%CO
2) cultivated altogether 4 hours, use annexinV-PE and 7 '-AAD dyestuff room temperature to dye then 15 minutes.With FCM check and analysis immediately, establish door with the CFSE negative cells, with annexin V or with 7 '-AAD positive cell as apoptotic cell.Calculation formula is: kill rate (%)=experimental group (apoptosis rate %)-control group (apoptosis rate %).As shown in Figure 5; Under the condition of 5: 1 effect target ratios; Amplification property gamma delta T cells system is 20%～30% (Fig. 5 A) to the kill rate from the body leukemia cell, is 10%～25% (Fig. 5 B) to the kill rate of heterology leukemia cell line, and along with the quantity of gamma delta T lymphocytes increases; Its lethality is strong more, and the kill capability that shows gamma delta T lymphocytes of the present invention is that quantity relies on.
Embodiment 8, the leukemia resisting action of amplification property gamma delta T cells in the SCID mouse
8.1, amplification property gamma delta T lymphocytes is in vivo to the leukemia resisting action of heterology leukemia cell line
Adopt the SCID female mice in 6 ages in week, be divided into two groups at random: control group and treatment group, 3 every group.Get well-grown AML clone Kasumi-1 cell (institute provides by the Shanghai City hematology),, be resuspended in serum-free RPMI-1640 substratum, 4 * 10 with perfect medium (the RPMI1640 substratum that contains 10%FBS) washing 2 times
6Injection cell is to every mouse peritoneal.Treatment group mouse is simultaneously with an example amplification property gamma delta T lymphocytes 2 * 10 that obtains among the good embodiment 3 of position injection growth conditions
7Individual cell is injected the gamma delta T lymphocytes of equal amts once more after 4 days, control group is used the RPMI-1640 substratum injection with volume.In above-mentioned inoculation, only at same position injection rhIL-2 2000IU/.Gamma delta T lymphocytes kills and wounds the effect of heterology leukemia cell line in vivo and estimates through the mouse survival rate.Experimental result shown in Fig. 6 A, all mouse, if do not inoculate gamma delta T lymphocytes, then swelling progressively appears in mouse peritoneal, after 50 days, begin gradually dead, all dead after 70 days.On the contrary, the survival time of mice of inoculating with gamma delta T lymphocytes obviously prolongs, and after 80 days, just begins death to occur, and mouse survival (P＜0.01) was still arranged after 100 days.
8.2, amplification property gamma delta T lymphocytes is in vivo to the leukemia resisting action from the body leukemia cell
Adopt the NOD-SCID female mice in 6 ages in week, be divided into two groups at random: control group and treatment group, 7 every group.Radioactive rays (150cGy) with sublethal dose are handled mouse, and bone marrow transplantation is just sent out the leukemia cell in leukemia patient source subsequently.The leukemia cell who just sends out is injected into mouse shin bone chamber, every injected in mice 1 * 10
7Individual cell, the enchylema scale of construction of final every injected in mice is 50 μ l.Then, in the treatment group, an example amplification property gamma delta T lymphocytes 2 * 10 that obtained among the tail vein injection embodiment 3 at the 1st day and the 4th day respectively
7Individual cell and 2000IU rhIL-2 are to the mouse body.Simultaneously, control group is through RPMI-1640 substratum and the 2000IU rhIL-2 of mouse tail vein injection with volume.After 8 weeks, gather the mouse peripheral blood, detect the percentage ratio of people CD45 positive cell.Gamma delta T lymphocytes in vivo kills and wounds from body leukemia cell, the effect of organizing the leukemia cell to implant and assesses through the mouse survival rate.Experimental result in the mouse of gamma delta T lymphocytes inoculation of no use, has 5 mouse people CD45 positive cell (2%～11%) to occur at peripheral blood shown in Fig. 6 B, after 60 days, begins to occur dead.On the contrary, the mouse that inoculated of useful gamma delta T lymphocytes obviously prolong (P＜0.05) lifetime in the observation period.
Claims (3)
1. the amplification in vitro method of a tumour-specific gamma delta T lymphocytes is characterized in that, said method comprising the steps of:
(1) obtains leukemia cell and deactivation by separating in the blood patient peripheral blood that just turns white;
(2) obtain monocyte component CD4-CD8-PBMCs by separating in the leukaemic of alleviating the peripheral blood;
(3) in the presence of recombination human interleukin 2 (rhIL-2) and phytohemagglutinin (PHA), with the CD4-CD8-PBMCs co-cultivation that obtains in the leukemia cell of the deactivation that obtains in the step (1) and the step (2), amplification tumour-specific gamma delta T lymphocytes.
2. the method for claim 1 is characterized in that, said step (1) comprises the step through the deactivation of radionuclide cobalt irradiation with the leukemia cell.
3. the method for claim 1 is characterized in that, culture condition is 37 ℃ and 5%CO in the said step (3)
2
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102994448A (en) * | 2012-12-13 | 2013-03-27 | 上海柯莱逊生物技术有限公司 | Method for in-vitro amplification of gamma-delta-T cells |
| CN104388386A (en) * | 2014-09-30 | 2015-03-04 | 浙江大学 | In-vitro activation amplification method for V delta 1 gamma delta T cell coming from peripheral blood |
| CN108070554A (en) * | 2016-11-15 | 2018-05-25 | 首都医科大学附属北京天坛医院 | A kind of pharmaceutical composition and its amplification method for amplification in vitro cytotoxicity gamma delta T lymphocytes |
| CN109517793A (en) * | 2018-11-30 | 2019-03-26 | 广州长峰生物技术有限公司 | A kind of method for building up of NK cell and gamma delta T cells co-cultivation |
| CN112410297A (en) * | 2020-11-30 | 2021-02-26 | 北京荟科柘生物科技有限公司 | Gamma delta T-like alpha beta T cell and preparation method and application thereof |
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2011
- 2011-12-29 CN CN2011104527513A patent/CN102443567A/en active Pending
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| CHARLES R. MACKAY ET AL: "Gamma delta T cells express a unique surface molecule appearing late during thymic development", 《EUR.J.IMMUNOL》 * |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994448A (en) * | 2012-12-13 | 2013-03-27 | 上海柯莱逊生物技术有限公司 | Method for in-vitro amplification of gamma-delta-T cells |
| CN104388386A (en) * | 2014-09-30 | 2015-03-04 | 浙江大学 | In-vitro activation amplification method for V delta 1 gamma delta T cell coming from peripheral blood |
| CN104388386B (en) * | 2014-09-30 | 2017-02-22 | 浙江大学 | In-vitro activation amplification method for V delta 1 gamma delta T cell coming from peripheral blood |
| CN108070554A (en) * | 2016-11-15 | 2018-05-25 | 首都医科大学附属北京天坛医院 | A kind of pharmaceutical composition and its amplification method for amplification in vitro cytotoxicity gamma delta T lymphocytes |
| CN109517793A (en) * | 2018-11-30 | 2019-03-26 | 广州长峰生物技术有限公司 | A kind of method for building up of NK cell and gamma delta T cells co-cultivation |
| CN109517793B (en) * | 2018-11-30 | 2022-05-10 | 广州长峰生物技术有限公司 | Establishment method for NK cell and gamma delta T cell co-culture |
| CN112410297A (en) * | 2020-11-30 | 2021-02-26 | 北京荟科柘生物科技有限公司 | Gamma delta T-like alpha beta T cell and preparation method and application thereof |
| CN112410297B (en) * | 2020-11-30 | 2022-05-24 | 北京荟科柘生物科技有限公司 | Gamma delta T-like alpha beta T cell and preparation method and application thereof |
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Application publication date: 20120509 |