CN107904203B - Method for directionally inducing and amplifying Thymus-derived iNKT cells - Google Patents

Method for directionally inducing and amplifying Thymus-derived iNKT cells Download PDF

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CN107904203B
CN107904203B CN201711221227.9A CN201711221227A CN107904203B CN 107904203 B CN107904203 B CN 107904203B CN 201711221227 A CN201711221227 A CN 201711221227A CN 107904203 B CN107904203 B CN 107904203B
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孟明
王园园
刘慧芳
陈冬志
张金库
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Abstract

The invention provides a method for directionally inducing and amplifying iNKT cells from thymus, which comprises two stages, namely stimulating iNKT cells to proliferate in vivo in a first stage, stimulating iNKT cells to proliferate in a mouse body by using a specific stimulator α -Galcer, and directionally inducing iNKT cells to amplify in vitro in a second stage, preparing single cell suspension by taking α -Galcer-stimulated mouse thymus, and adding α -Galcer and an inducer to assist the iNKT cells to amplify and guide the iNKT cells to differentiate.

Description

Method for directionally inducing and amplifying Thymus-derived iNKT cells
Technical Field
The invention relates to the field of immunology, in particular to a method for directionally inducing and amplifying thymic-derived iNKT cells.
Background
iNKT cells are a group of special immune cells that combine NK cell functions with T cell characteristics, and are the bridge for communicating adaptive and innate immunity. iNKT cells differ from conventional T cells in that they lack diversity in surface TCR, have a narrow antigen recognition spectrum, recognize common lipid and glycolipid antigens presented by CD1 molecules on the surface of different target cells (a-Galcer), and are not MHC-restricted. iNKT cells are innate immune cells whose main biological functions are cytotoxicity and immunoregulatory effects. iNKT cells can act as a center for immune regulation to interact with other immune cells and play a crucial role in the development of infection, tumors and autoimmune diseases.
iNKT cells are a heterogeneous population of cells, which are divided into three subgroups iNKT1, iNKT2, and iNKT17, depending on the expression of their transcription factors and the levels of the major cytokines produced. The iNKT cells can control and alleviate the progress of rejection of diabetes, tumors and organ transplantation, and can expand and promote the occurrence and development of lupus erythematosus, allergic asthma and the like, and the contradictory experimental results are presumed to be related to the function of iNKT cells of different subgroups. iNKT cells are known as the "swiss military knife" of the immune system in view of the diversity of functions and uses.
Cellular immunotherapy is a new biological treatment method in recent years, and autologous cells induced in vitro by infusion can effectively regulate and control the dysfunction of the immune system of the body, and plays an important role in the treatment of various diseases such as tumors, infections, autoimmune diseases and the like. Therefore, the research of obtaining specific immune cells with regulation effect for cellular immunotherapy is of great significance, while iNKT cells serve as the center of immune regulation, which is of no help for cellular immunotherapy, and it is understood that cellular immunotherapy by adoptive infusion of iNKT cells can become a new strategy for the treatment of tumors, infections and many immune diseases. However, because the number of iNKT cells in vivo is small, the frequency is only 0.3% of the total number of mononuclear cells in peripheral blood, and the potential of the iNKT cells in clinical application is severely limited, so that the acquisition of a large number of directionally differentiated iNKT cells for cellular immunotherapy becomes a hot spot of the current research.
The methods for inducing expansion of iNKT cells reported in the literature include: 1. mature NKT nuclei are transferred to embryonic stem cells (ES), thereby expanding NKT cells in large numbers. 2. Adult hematopoietic stem cells (HSPCs) of bone marrow origin are induced in large numbers into iNKT cells. 3. Mouse spleen iNKT cells were isolated and purified for extensive expansion.
1. Mature iNKT cell nuclei are transferred to embryonic stem cells (ES), and iNKT cells are cloned in a large quantity, so that the method is high in technical difficulty, difficult in obtaining embryonic stem cells and low in cloning efficiency; 2. the massive induction of adult hematopoietic stem cells (HSPCs) of bone marrow origin into iNKT cells has proved to be very difficult by this method, which has a great limitation to the basic research before clinical. 3. Purification and amplification of mouse spleen-derived iNKT cells are complex and time-consuming to operate, and the spleen iNKT cells are mature iNKTs, have poor differentiation capability and cannot directionally induce the differentiation direction. The common disadvantages of the above three methods are: the high cost, complex process, long cycle, small quantity of iNKT cells obtained and difficult directional acquisition of iNKT cells with specific functions create great limitations for cellular immunotherapy and related research of adoptive infusion of iNKT cells for tumors, infections and many autoimmune diseases.
Disclosure of Invention
The invention aims to provide a thymus-derived iNKT cell directional induction amplification method, which aims to solve the problems that the existing method is complex in process, long in period, small in quantity of obtained iNKT cells and difficult to directionally obtain iNKT cells with specific functions.
The purpose of the invention is realized by the following technical scheme: a method for the directed induction of expansion of thymic-derived iNKT cells, comprising two stages:
the first stage is in vivo stimulation of iNKT cell proliferation, and specific stimulator α -Galcer is used for stimulating iNKT cell proliferation in mice;
the second stage is in vitro directional induction of iNKT cell expansion, mouse thymus stimulated by α -Galcer is taken to prepare single cell suspension, α -Galcer and an inducer are added to assist iNKT cell expansion and guide iNKT cell differentiation.
The two phases mainly comprise the following steps:
a. a single subcutaneous injection of a stimulant α -Galcer at the root of the tail of a 6-week-old DBA/1 male mouse;
b. after 8 days, the mice are killed after neck removal, the thymus is taken to prepare single cell suspension, and the cells are counted;
c. adjusting the cell density to 2 × 10 with a medium containing α -Galcer, an inducer6Per ml, and placed in 5vol% CO at 37 deg.C2Culturing in an incubator; changing the cell liquid and carrying out passage every 4-5 days, and harvesting the cells on the 14 th day.
In step a, the α -Galcer is used in an amount of 2 mu g/mouse.
In step c, the concentration of α -Galcer is 100 ng/ml.
In the step C, the inducer comprises IL-2, IL-4 and vitamin C, wherein the concentration of the IL-2 is 100IU/ml, the concentration of the IL-4 is 25IU/ml, and the concentration of the vitamin C is 1 mu mol/ml.
In step c, penicillin, streptomycin and 15vol% FBS are added into KBM-551 serving as the culture medium.
In step c, β -mercaptoethanol was added to the KBM-551 medium to a final concentration of β -mercaptoethanol of 0.05 mmol/ml.
The invention adopts a method combining in-vivo proliferation stimulation and in-vitro amplification induction to directionally induce the iNKT cells, and realizes that a large number of iNKT cells with specific functions are directionally differentiated in a short time (14 days) (the frequency is up to 40.07 percent, and the number of the cells is improved by 6 ten thousand times) by means of specific antigen stimulation time, stimulation intensity, stimulation frequency, specific mouse week age, sex, organs (thymus), different in-vitro stimulation culture conditions and the like.
Compared with the activation of in vitro experiments, the in vivo stimulation can enable the iNKT cells to be more fully activated, is beneficial to the amplification of the iNKT cells when in vitro cell culture, and lays a foundation for in vitro induced amplification. The combination of in vivo stimulation and in vitro experiments can effectively reduce the complicated and fussy steps during the in vitro amplification culture of cells, shorten the time of cell culture, and reduce the problems of cell morphological function variation, pollution and the like generated during the in vitro cell induction, which cause headache to people. In addition, the invention also has the advantages of strong repeatability and relatively low cost.
Drawings
FIG. 1 is a graph of iNKT cell frequency and the detection of each subpopulation of cells.
Detailed Description
The invention is illustrated in detail below by way of examples, the reagents used in the examples of the invention:
α galactose sphingosine (α -Galcer) is a product of ENZO Life Sciences;
FITC Hamster Anti-Mouse TCR β Chain (553170), PerCP-CyTM5.5 Mouse Anti-T-beta (561316), Alexa Fluor 647 Mouse Anti-PLZF (563490) purchased from BD Co;
PE-T-selected-CD1d tetramer was purchased from MBL International, Woburn, MA, Japan;
foxp 3/transformation Factor stabilizing Buffer was purchased from eBiosdence;
KBM551 Medium was purchased from kangning;
fetal bovine serum, purchased from Gibco;
penicillin and streptomycin were purchased from Invitrogen; 2-Hydrophobic ethanol was purchased from Amresco;
human IL-2 was purchased from Mihenyi Biotec, Inc.;
murine IL-4 was purchased from Peprotech;
vitamin C (VC), available from Sigama.
Example 1: directionally induced expansion of iNKT cells
(1) A single subcutaneous injection of stimulator α -Galcer (2. mu.g/mouse) at the tail root of 6-week-old DBA/1 male mice;
(2) after 8 days, the eyeballs of the mice are taken out of the necks and killed;
(3) completely soaking the mouse in 75% alcohol for 1min, and fixing the limbs of the mouse on an anatomical plate to obtain thymus;
(4) placing the thymus in a culture dish containing PBS, and washing for 2-3 times by using PBS to remove blood stains;
(5) then, the thymus is placed on a 200-mesh screen (the screen is placed on a culture dish containing PBS), the thymus is cut into blocks with the size of 2-3 mm by using an ophthalmic scissors, then the thymus is ground, the thymus is fully ground, tissue fluid is transferred into a 10ml centrifuge tube by using a fluid transfer gun, the tissue fluid is centrifuged for 5min at 1000r/min, the tissue fluid is discarded, and then the thymus is washed twice (1000 r/min, 5 min) by using PBS (the whole process is operated on ice);
the iNKT frequency of the obtained cells can reach 7.95% through FACS detection, and the iNKT purity can reach 70-80% through MACS magnetic bead separation;
(6) after counting the cells obtained in the step (5), using a reagent containing α -Galcer (100 ng/ml),The cell density of KBM-551 (specially used for culturing NK cells, and having higher protein content) complete medium containing IL-2 (100 IU/ml), IL-4 (25 IU/ml), vitamin C (1. mu. mol/ml), β -mercaptoethanol (0.05 mmol/ml), penicillin (100U/ml), streptomycin (0.1 mg/ml) and 15vol% FBS was adjusted to 2 × 106One/ml, then transferred to a six-well plate and 5vol% CO at 37 ℃2Culturing in an incubator;
(7) and (5) changing the cell liquid and carrying out passage every 4-5 days, observing the cell growth condition, and harvesting the cells on the 14 th day.
Frequency and subpopulation of iNKT cells cultured cells were harvested, washed twice with PBS, and placed in flow tubes (1 × 10)6Tube), 5. mu.l of α -Galcer-loaded PE-T-selected-CD1d tetramer and 2. mu.l of FITCHAster Anti-Mouse TCR β Chain were added, incubated at room temperature for 30min in the absence of light, washed twice with PBS, permeabilized and fixed according to the specific protocol of Foxp3/Transcription Factor stabilizing Buffer, followed by 5. mu.l of PerCP-CyTM5.5 Mouse Anti-T-beta and 5 microliter Alexa Fluor 647 Mouse Anti-PLZF, incubated at room temperature in dark place for at least 30min, PBS washed twice, 500 microliter PBS resuspended cells, FACS detection, the result is shown in FIG. 1, the frequency of iNKT can reach 40.07%, wherein iNKT1 accounts for 92.28%.
Example 2: assay for iNKT cell killing Activity
iNKT cytotoxicity detection51Cr release test. K562 as target cell and Na51CrO4Incubating at 37 deg.C for 1h, washing with PBS, and adjusting the density of target cells to 5 × 105And (3) adding effector cells and target cells into a 96-well U-shaped culture plate according to a ratio of 20:1, and arranging a plurality of multiple wells. Placing at 37 ℃ and 5vol% CO2Incubate in incubator for 4 h, remove, aspirate 0.1ml of supernatant from each well with micropipette, add to small plastic tubes (without aspirating cells), measure supernatant primer using gamma counter (Beckman LS 6500)51And calculating the cytotoxicity activity, namely the cell killing rate = (experimental pore release amount-natural release amount)/(maximum release amount-natural release amount) × 100%.
By the optimized combination of in vivo stimulation proliferation and in vitro induction amplification, the invention can realize that a large amount of iNKT cells with directional differentiation and specific functions (the frequency is as high as 40.07%) can be obtained in a short time (14 days), thereby providing guarantee for the cell immunotherapy of adoptive infusion of iNKT cells of tumors, infections and a plurality of autoimmune diseases and related research.

Claims (1)

1. A method for directionally inducing and amplifying Thymus-derived iNKT cells is characterized by comprising the following two stages:
the first stage is in vivo stimulation of iNKT cell proliferation, and specific stimulator α -Galcer is used for stimulating iNKT cell proliferation in mice;
the second stage is in vitro directional induction of iNKT cell expansion, the mouse thymus stimulated by α -Galcer is taken to prepare single cell suspension, α -Galcer and an inducer are added to assist iNKT cell expansion and guide iNKT cell differentiation;
the two phases comprise the following steps:
a. a single subcutaneous injection of a stimulant α -Galcer into the tail root of a DBA/1 male mouse at the age of 6 weeks, wherein the dosage of α -Galcer is 2 mug/mouse;
b. after 8 days, the mice are killed after neck removal, the thymus is taken to prepare single cell suspension, and the cells are counted;
c. cell density was adjusted to 2 × 10 using KBM-551 complete medium containing α -Galcer, IL-2, IL-4, ascorbic acid, β -mercaptoethanol, penicillin, streptomycin, and 15vol% FBS6Per ml, and placed in 5vol% CO at 37 deg.C2Culturing in an incubator, changing and passaging cells every 4-5 days, and harvesting cells on the 14 th day, wherein the concentration of α -Galcer in the culture medium is 100ng/ml, the concentration of IL-2 is 100IU/ml, the concentration of IL-4 is 25IU/ml, the concentration of vitamin C is 1 mu mol/ml, the concentration of β -mercaptoethanol is 0.05mmol/ml, the concentration of penicillin is 100U/ml, and the concentration of streptomycin is 0.1 mg/ml.
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