CN113462646A

CN113462646A - Simple and effective method for induced amplification of iNKT cells and application

Info

Publication number: CN113462646A
Application number: CN202110827989.3A
Authority: CN
Inventors: 李慧忠; 郑骏年; 王刚; 刘宜林; 张连军
Original assignee: Xuzhou Medical University
Current assignee: Xuzhou Medical University
Priority date: 2021-06-30
Filing date: 2021-07-22
Publication date: 2021-10-01
Anticipated expiration: 2041-07-22
Also published as: CN113549600A; CN113549600B; CN113462646B

Abstract

The invention provides a method for inducing and amplifying iNKT cells, which has small initial demand (10 to 10)⁶Order of magnitude), the proportion of the obtained iNKT cells is high, the proportion of the iNKT cells is improved to 20-80% from 0.01-1% after 7-9 days, the efficiency can be obviously improved, and finally the iNKT cells are expanded by more than 2000 times; and the prepared cell can secrete Th1 type cytokines, highly express CD62L, has long survival time in vivo and can meet the requirement of clinical treatment.

Description

Simple and effective method for induced amplification of iNKT cells and application

Technical Field

The invention belongs to the field of cellular immunotherapy, and relates to a simple and effective method for induced amplification of iNKT cells and application thereof.

Background

iNKT cells are so named for their expression of a unique invariant TCR, which primarily recognizes the glycolipid antigen presented by the CD1d molecule, by secreting large amounts of cytokines, mediating both innate and adaptive immunity. iNKT cells are currently considered to be a rare but powerful immunoregulatory and effector group of T cells that play important roles in antitumor immunity.

Several studies have shown that patients with tumors have low levels of iNKT cells or impaired function (reduced ability to secrete IFN- γ). The number of iNKT cells infiltrating into the tumor is closely related to the clinical prognosis of neuroblastoma, colon cancer and the like; a study with median follow-up times up to 8.7 years reports that patients with head and neck squamous cell carcinoma who are severely deficient iN circulating iN KT cells have poor clinical prognosis; reconstitution of iNK T cells in peripheral blood after hematopoietic stem cell transplantation is useful for long-term remission of childhood leukemia and the like. These findings suggest that iNKT cells have potent antitumor activity, and iNKT immunocytotherapy has attracted much attention in recent years.

It is currently believed that iNKT cells exert an anti-tumor effect mainly through the following mechanisms:

1. CD1 d-dependent direct cytotoxic killing activity: mainly mediated by Perforin, FasL, granB, TNF-alpha and the like;

2. CD1 d-independent cytotoxic killing activity: once activated, the activation of both innate and adaptive immunity in the body is accompanied by secretion of large amounts of Th1/Th2 type cytokines, where the iNKT cell-DC cell "cross-talk" mediated by CD40-CD40L or CD1d lipid antigen/iTCR and activation of CD8 by secretion of IFN- γ⁺T cells are particularly important and thus increaseStrong anti-tumor effect;

3. the immune regulation mechanism is as follows: recent studies report that iNKT cells mediate anti-tumor activity by killing tumor-associated macrophages (TAMs) in primary neuroblastoma; furthermore, iNKT cells were found to be able to reduce immunosuppressive activity of myeloid-derived suppressor cells (MDS C) in models of human and mouse influenza a virus infection. These findings suggest that iNKT cells have a novel mechanism of action, i.e., modulating the tumor microenvironment by killing immunosuppressive cells, thereby enhancing the anti-tumor immune effect.

In recent years, clinical trials for treating tumors using iNKT cells have been carried out in succession. Clinical data show that the iNKT cells subjected to amplification culture have no obvious side effect in adoptive immunotherapy of tumors, and have good clinical application prospects. However, because the iNKT cell content in human peripheral blood is low (about 0.01-1% of lymphocytes), the existing amplification method has the technical problems of large initial cell demand, long amplification period, low purity of cell preparations, weak activity and the like, and finally results in low clinical overall remission rate and unsatisfactory anti-tumor effect.

Disclosure of Invention

Optimized lymphocyte culture medium

The invention provides an optimized lymphocyte culture medium, which is prepared by adding cell agonist and cytokine into a basic lymphocyte culture medium.

Preferably, the cell agonist is alpha galactosyl ceramide (alpha GalCer).

Preferably, the cytokine includes GM-CSF (granulocyte macrophage colony stimulating factor), sICAM-1 (human soluble intercellular adhesion molecule 1), IL-1 (interleukin 1), IL-1alpha receptor (interleukin 1alpha receptor), IL 1alpha (interleukin 1 alpha), IL-3 (interleukin 3), IL-2 (interleukin 2), IL-21 (interleukin 21), IL-10 (interleukin 10), IL-16 (interleukin 16), IL-13 (interleukin 13), IL-17 (interleukin 17), IP-10 (interferon inducible protein 10), SCYA2 (small inducible cytokine A2), MIG (interferon gamma inducible monokine), MIP-1alpha (macrophage inflammatory protein l alpha), TGF-beta (transforming growth factor beta), IL-4 (interleukin 4), TRAF6(TNF receptor-related factor 6), FGF (fibroblast growth factor), IGF (insulin-like growth factor), PDGF (platelet-derived growth factor), LIF (leukemia inhibitory factor), mTOR (rapamycin target protein), LPS (intracellular endotoxin), TLRl (Toll-like receptor 1), IL-12 (interleukin 12), IL-23 (interleukin 23), NGF (nerve growth factor), TNF alpha (interferon-alpha), IL-1beta (interleukin 1 beta), or a functional fragment of any of the foregoing.

Preferably, the cytokines include a combination of one or more of: IL-2 or a functional fragment thereof, IL-21 or a functional fragment thereof, IL-4 or a functional fragment thereof, GM-CSF or a functional fragment thereof.

Preferably, the cytokine is a combination of commercially available IL-2, IL-21, IL-4 and GM-CSF.

Preferably, the final concentration (working concentration) of the alpha-Galcer is 50-1000 ng/mL.

Preferably, the final concentration of α -Galcer is 50-500 ng/mL.

Preferably, the final concentration of α -Galcer is 100 ng/mL.

Preferably, the final concentration of the IL-2 is 10-200U/mL.

Preferably, the final concentration of the IL-2 is 10-100U/mL.

Preferably, the final concentration of IL-2 is 50U/mL.

Preferably, the final concentration of IL-21 is 5-100 ng/mL.

Preferably, the final concentration of IL-21 is 5-50 ng/mL.

Preferably, the final concentration of IL-21 is 10 ng/mL.

Preferably, the final concentration of the GM-CSF is 100-1000U/mL.

Preferably, the final concentration of GM-CSF is 300-700U/mL.

Preferably, the concentration of GM-CSF is 500U/mL.

Preferably, the final concentration of the IL-4 is 100-1000U/mL.

Preferably, the final concentration of the IL-4 is 300-700U/mL.

Preferably, the final concentration of IL-4 is 500U/mL.

Preferably, the basal lymphocyte culture medium is X-VIVO 15 medium containing FCS.

Preferably, the basal lymphocyte culture medium is X-VIVO 15 medium containing 5% FCS.

Antibody fluids to activate NKT cells

The invention provides an antibody fluid for activating NKT cells, which contains a CD3 monoclonal antibody and a CD28 monoclonal antibody.

Preferably, the NKT cells are iNKT cells.

Preferably, the working concentration of the CD3 monoclonal antibody is 1-10 mug/mL.

Preferably, the working concentration of the CD3 monoclonal antibody is 1 μ g/mL.

Preferably, the working concentration of the CD28 monoclonal antibody is 1-10 mug/mL.

Preferably, the working concentration of the CD28 monoclonal antibody is 1 μ g/mL.

Preferably, the antibody coated well plates can be used to activate NKT cells.

Preferably, the well plate is a 24 well plate.

Lymphocyte culture medium containing IL-7 and IL-15

The invention provides a lymphocyte culture medium containing IL-7 and IL-15, which is prepared by adding IL-7 and IL-15 into a basic lymphocyte culture medium.

Preferably, the working concentration of the IL-7 is 5-50 ng/mL.

Preferably, the working concentration of IL-7 is 10 ng/mL.

Preferably, the working concentration of the IL-15 is 1-50 ng/mL.

Preferably, the working concentration of IL-15 is 5 ng/mL.

Methods of inducing NKT cells

The present invention provides a method for inducing NKT cells comprising the step of culturing a sample using the aforementioned optimized lymphocyte culture medium.

Preferably, the NKT cells are iNKT cells.

Preferably, the initial concentration of cells in the culture is 1X 10⁶/mL～50×10⁶/mL。

Preferably, the initial concentration of cells in the culture is 1X 10⁶/mL～10×10⁶/mL。

Preferably, the initial concentration of cells in the culture is 2X 10⁶/mL。

Preferably, the culture conditions are 37 ℃ and 5% CO₂。

Preferably, the culture is changed every other day.

Preferably, the liquid change is a half-amount liquid change.

Preferably, the sample contains iNKT cells or does not contain iNKT cells.

Preferably, the sources of the samples include bone marrow, umbilical cord blood and placental blood, peripheral blood.

Preferably, the sample is derived from peripheral blood.

Preferably, the sample is Peripheral Blood Mononuclear Cells (PBMC) isolated from Peripheral blood.

Preferably, the peripheral blood mononuclear cells isolated from the peripheral blood are well known in the art, including but not limited to, Ficoll apheresis (Ficoll density gradient centrifugation) or Percoll apheresis.

Preferably, the PBMC obtained by peripheral blood separation is subjected to Ficoll density gradient centrifugation.

Method for expanding NKT cells

The invention provides a method for expanding NKT cells, which comprises the steps of culturing cell fluid containing NKT cells by using the lymphocyte culture medium containing IL-7 and IL-15, and then inoculating the cells to the anti-body fluid coated pore plate for culturing;

preferably, the NKT cells are iNKT cells.

Preferably, the cell fluid containing NKT cells is sorted by magnetic beads.

Preferably, the cell fluid containing NKT cells is obtained by magnetic bead sorting of cells prepared by the aforementioned method for inducing NKT cells.

Preferably, Anti-iNKT MicroBeads is selected as the magnetic beads in the magnetic bead sorting.

Preferably, the buffer used in any of the resuspension, washing and elution in the magnetic bead sorting is a MACS buffer.

Preferably, an LS sorting column is used in the magnetic bead sorting.

Preferably, the culture conditions are 37 ℃ and 5% CO₂。

Preferably, the concentration of cells in the culture is maintained at 1X 10⁶about/mL.

Preferably, the culture is changed every other day.

Preferably, the liquid change is a half-amount liquid change.

Reagent kit

The invention provides a kit for preparing NKT cells; the kit comprises the optimized lymphocyte culture medium, the antibody liquid for activating NKT cells, the lymphocyte culture medium containing IL-7 and IL-15, a reagent for preparing the optimized lymphocyte culture medium, a reagent for preparing the antibody liquid for activating NKT cells, and a reagent for preparing the lymphocyte culture medium containing IL-7 and IL-15;

preferably, the NKT cells are iNKT cells.

Cells

In another aspect, the present invention provides an NKT cell capable of killing tumor cells (cancer cells), which NKT cell is prepared by the aforementioned method for inducing NKT cells or the method for expanding NKT cells.

Preferably, the tumor cell is a kidney cancer cell.

Preferably, the NKT cells are iNKT cells.

Method

The invention provides a method for killing tumor cells, which comprises the step of contacting the NKT cells with the tumor cells.

Preferably, said killing of tumor cells occurs in vitro.

Preferably, the tumor cell is a kidney cancer cell.

Preferably, the tumor cell is a human kidney cancer cell line.

Pharmaceutical composition

In another aspect, the present invention provides a pharmaceutical composition for treating cancer, comprising NKT cells prepared by the aforementioned method for inducing NKT cells and/or method for expanding NKT cells.

Preferably, the cancer is renal cancer.

Preferably, the pharmaceutical composition further comprises other ingredients useful for treating cancer or for health benefits.

Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients and/or additives.

Preferably, the excipients include, but are not limited to, buffer systems, thickeners, stabilizers, neutralizing agents, humectants.

Preferably, the additives include, but are not limited to, fillers, binders, moisturizers, glidants, stabilizers, preservatives, emulsifiers.

Preferably, the NKT cells are iNKT cells.

Applications of

In another aspect, the invention provides the use of any of the aforementioned optimized lymphocyte culture medium, the aforementioned antibody fluid for activating NKT cells, the aforementioned lymphocyte culture medium comprising IL-7 and IL-15, and the aforementioned kit for inducing (preparing) NKT cells.

In another aspect, the invention provides the use of any of α -GalCer, IL-2, IL-21, IL-4 and GM-CSF for inducing NKT cells.

In another aspect the invention provides the use of IL-7 or IL-15 for inducing NKT cells.

In another aspect, the invention provides the use of a CD3 monoclonal antibody or a CD28 monoclonal antibody for inducing NKT cells.

In another aspect, the present invention also provides the use of the aforementioned NKT cells or pharmaceutical composition for the preparation of a medicament for the treatment of cancer.

Preferably, the cancer is renal cancer.

Preferably, the NKT cells are iNKT cells.

Drawings

FIG. 1 is a graph showing the results of flow cytometry analysis of iNKT cell phenotypes; FIG. 1A shows PBMCs cells prior to induction, FIG. 1B shows cells at day 9 after induction by conventional methods, FIG. 1B' shows cells at day 9 after induction by the method of the present invention, and FIG. 1C shows cells after magnetic bead sorting.

FIG. 2 is a graph showing the results of RTCA assay for in vitro killing activity of iNKT cells; FIG. 2A is the normalized cell index when 786-O cells were killed, FIG. 2B is the normalized cell index when OSRC-2 cells were killed, FIG. 2C is the cytotoxic activity of iNKT cells against 786-O cells, and FIG. 2D is the cytotoxic activity of iNKT cells against OSRC-2 cells.

FIG. 3 is a graph showing the results of detection of the levels of secreted cytokines in coculture of iNKT cells with renal cancer cells 786-O, OSRC-2 by ELISA; IFN-gamma concentration, B.IL-2 concentration, C.TNF-alpha concentration.

Detailed Description

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. As used herein, the term "lymphocyte culture medium" refers to a solution containing nutrients that allow lymphocytes to grow, and that can maintain the viability of the lymphocytes. The lymphocyte culture medium is well known to those skilled in the art and typically provides standard inorganic salts such as zinc, iron, magnesium, calcium and potassium, as well as vitamins, glucose, buffer systems and essential amino acids.

Culture media (including lymphocyte culture media) that can be used in the present application include, but are not limited to, TeSR-E8, mTESR1, E8, Essential 8^TMMedium, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), eagle's minimal Medium (BME), F-10, F-12, alpha-minimal essential Medium (alpha-MEM), G-minimal essential Medium (G-MEM), IMPM (IMDM), AmnioMa x, novel dibasic amniotic fluid Medium (Amino Max II complex Medium), Chang's Medium, Mesemcult-XF Medium, RPMI 1640, Ham's F12, DMEM/F12, Ham's F-12K Medium m, HepatozYME-SFM, William's EMedium, Waymouth's Medium, Heapatocycle Medium.

The lymphocyte culture medium can be a commercial culture medium, and can also be prepared according to a formula; other substances can be added according to different requirements.

As used herein, the term Peripheral Blood Mononuclear Cells (PBMCs) refers to the generic term for cells in peripheral blood having a single nucleus, including but not limited to lymphocytes (T cells, B cells, NK cells, NKT cells), monocytes or dendritic cells.

The invention will be described in detail below with reference to the accompanying drawings and examples. It should be noted that the drawings and their embodiments of the present invention are for illustrative purposes and are not to be construed as limiting the invention. The embodiments and features of the embodiments in the present application may be combined with each other without contradiction.

The reagents used in the examples of the invention were as follows:

TABLE 1 reagents used in the examples of the invention

Reagent	Company(s)
		X-VIVO 15 culture medium	Lonza Corp
Lymphocyte separation liquid	Axis-shield company
		CD3 monoclonal antibody, CD28 monoclonal antibody	Biolegend Corp
rhIL-2、rhIL-7、rhIL-15、rhIL-21、rhIL-4、rhGM-CSF	PrimeGene Inc
		α-Galcer	Funakoshi Co Ltd
Anti-iNKT microbeads	Miltenyi Biotec Inc
		PerCP-Cy5.5 CD3、PE invariant NKT	BD Biosciences

Example 1 preparation of iNKT cells

1) Isolation of PBMCs: collecting peripheral blood of a donor, diluting the whole blood with equal amount of physiological saline, adding lymphocyte separation liquid and the diluted blood into a centrifugal tube according to the proportion of 1:2, centrifuging at 2000rpm/min for 20 minutes, collecting leucocyte layer cells, cleaning twice with the physiological saline, and centrifuging at 1500rpm/min for 8 minutes to obtain the PBMCs.

2) Induction of iNKT cells: suspending PBMCs in lymphocyte culture medium, adjusting cell concentration to 2 × 10⁶Add 100ng/mL α -Galcer, 50U/mL IL-2, 10ng/mL IL-21, 500U/mL IL-4 and 500U/mL GM-CSF to/mL and inoculate cells in 24-well plates in 5% CO2 incubators at 37 ℃. Cell status was observed daily, fluid was changed half day after day, amplification effect was monitored by cell count, and iNKT cell phenotype was analyzed by flow cytometry.

3) Magnetic bead sorting iNKT cells: collecting the induced cells on the 10 th day, re-suspending the cells by using 500 mu L of MACS buffer, adding Anti-iNKT MicroBeads according to the dosage of the instruction, uniformly mixing, incubating at 4 ℃ for 30 minutes, adding 5mL of MACS buffer for washing, centrifuging at 400 Xg for 5 minutes, and discarding the supernatant; resuspending with 500 μ LMACS buffer, loading onto LS sorting column, and washing with MACS buffer 3 times (3 mL each time); finally, the sorting column was placed in a collection tube and 500. mu.L of MACS buffer was added for elution to obtain iNKT positive cells. Figure 1C shows iNKT cell purity after flow cytometry analysis sorting.

4) Activation expanded iNKT cells: on day 9 of incubation, 1. mu.g/mL of CD3 and CD28 monoclonal antibody working solutions were prepared, a new 24-well plate was prepared, 0.5mL of antibody diluent (antibody working solution) was added to each well, the wells were covered with the antibody diluent by gentle shaking, and the plate was sealed with a sealing film and placed in a refrigerator at 4 ℃ overnight. The cells obtained were further purified by resuspension on day 10 in lymphocyte cultures containing 10ng/mL IL-7 and 5ng/mL IL-15, seeded on CD3Ab and CD28Ab pre-coated plates and subjected to extensive expansion in a 5% CO2 incubator at 37 ℃.

The experimental results are as follows:

the cells were examined at different stages using a flow cytometer and the results are shown in FIG. 1: FIG. 1A is PBMCs cells prior to induction, FIG. 1B is cells at day 9 after induction by conventional methods, FIG. 1B' is cells at day 9 after induction by the methods of the invention, and FIG. 1C is cells after magnetic bead sorting;

table 2 shows the comparison of the number of cells in the experimental process between the method of the present invention and the conventional method, and the statistical result of the number of cells shows that the initial demand of the present invention is small (10. about.⁶Order of magnitude), a high proportion of iNKT cells is obtained, 7 ℃ toAfter 9 days, the proportion of the iNKT cells is increased from 0.01-1% to 20-80%, and finally the iNKT cells are expanded by 2595 times.

Table 2: comparison of the amplification effects of the present invention with those of the conventional methods

The experimental results prove that the method can obviously improve the efficiency of preparing the iNTK cells.

Example 2 in vitro killing of tumor cells by iNKT cells

Effector cells: iNKT cells obtained in example 1

Target cell: 786-O cells and OSRC-2 cells (in this example, human renal carcinoma cells are used as an example)

Firstly, adding 50 mu L of tumor cell complete culture medium into an E-Plate detection Plate of an xCELLigence cell function analyzer, and measuring a background impedance value; collecting target cells in logarithmic phase, adjusting the concentration of cell suspension to 1 × 105/mL, adding 100 μ L of cell suspension into an E-Plate detection Plate, standing at room temperature for 30 min, and placing on a detection table; dynamically observing the proliferation of the target cells in a plateau stage in real time, respectively adding 50 mu L of effector cells according to the effective target ratio of 20:1, 10:1 and 5:1, and only adding a T cell culture medium to a control group; real-time observation of iNKT cell-mediated cell killing response curves was continued.

FIG. 2A shows that the iNKT cells obtained in example 1 can kill 786-O cells when the effective target ratio is 20:1, 10:1, or 5:1, and the killing effect is better when the ratio of the effector cells is larger;

FIG. 2B demonstrates that the iNKT cells obtained in example 1 can kill OSRC-2 cells when the ratio of effective target is 20:1, 10:1, 5:1, the larger the ratio of effective cells is, the better the killing effect is;

FIG. 2C demonstrates that the cytotoxic killing activity of iNKT cells obtained in example 1 on renal cancer cells 786-O was higher than 4 hours for 24 hours at the same effective target ratio;

FIG. 2D demonstrates that the cytotoxic activity of iNKT cells obtained in example 1 on renal carcinoma cells OSRC-2 was higher than 4 hours at 24 hours under the same effective target ratio.

Example 3 ability of iNKT cells to secrete cytokines

Digesting tumor cells, cleaning, counting, and adjusting density to 1 × 10⁵Laying 24-well plates, and adhering 2mL of plates to the wall per well overnight; collecting the amplified iNKT cells, adding the iNKT cells into tumor cells according to the proportion of 1:1 for co-culture, collecting supernatant after 24h, and detecting IFN-gamma, IL-2 and TNF-alpha by an ELISA method.

FIG. 3 shows the results: the iNKT cells and the kidney cancer cells are incubated together and can secrete cytokines A, IFN-gamma, B, IL-2 and C, TNF-alpha.

The above results demonstrate that iNKT cells obtained in example 1 have the ability to secrete cytokines.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Claims

1. An optimized lymphocyte culture medium, which is prepared by adding cell agonist and cytokine into a basic lymphocyte culture medium;

preferably, the cell agonist is α -Galcer;

preferably, the cytokines include a combination of one or more of: a combination of IL-2 or a functional fragment thereof, IL-21 or a functional fragment thereof, IL-4 or a functional fragment thereof, GM-CSF or a functional fragment thereof;

preferably, the final concentration of the alpha-Galcer is 100-1000 ng/mL;

preferably, the final concentration of α -Galcer is 100 ng/mL;

preferably, the final concentration of the IL-2 is 10-200U/mL;

preferably, the final concentration of IL-2 is 50U/mL;

preferably, the final concentration of the IL-21 is 10-100 ng/mL;

preferably, the final concentration of IL-21 is 10 ng/mL;

preferably, the final concentration of the GM-CSF is 500-1000U/mL;

preferably, the concentration of GM-CSF is 500U/mL;

preferably, the final concentration of the IL-4 is 500-1000U/mL;

preferably, the final concentration of IL-4 is 500U/mL;

2. An antibody solution for activating NKT cells, said antibody solution comprising a CD3 monoclonal antibody and a CD28 monoclonal antibody.

Preferably, the NKT cells are iNKT cells;

preferably, the antibody fluid comprises a CD3 monoclonal antibody and a CD28 monoclonal antibody;

preferably, the working concentration of the CD3 monoclonal antibody is 1 μ g/mL;

3. A lymphocyte culture medium containing IL-7 and IL-15, wherein the lymphocyte culture medium containing IL-7 and IL-15 is prepared by adding IL-7 and IL-15 to a basic lymphocyte culture medium;

preferably, the final concentration of the IL-7 is 10-50 ng/mL;

preferably, the final concentration of IL-7 is 10 ng/mL;

preferably, the final concentration of the IL-15 is 5-50 ng/mL;

preferably, the final concentration of IL-15 is 5 ng/mL;

4. A method of inducing NKT cells comprising the step of culturing a sample using the optimized lymphocyte culture medium of claim 1;

preferably, the NKT cells are iNKT cells;

preferably, the initial concentration of cells in the culture is 1X 10⁶/mL～10×10⁶/mL；

Preferably, the initial concentration of cells in the culture is 2X 10⁶/mL；

Preferably, the source of the sample comprises bone marrow, umbilical cord blood, placental blood, peripheral blood;

preferably, the sample is derived from peripheral blood;

preferably, the sample is PBMCs isolated from peripheral blood;

preferably, the PBMC obtained by separating from the peripheral blood is obtained by Ficoll density gradient centrifugation.

5. A method for expanding NKT cells, comprising culturing a cell culture fluid containing NKT cells using the lymphocyte culture medium containing IL-7 and IL-15 according to claim 3, and then inoculating the cells onto the antibody-coated well plate according to claim 2 for culture;

preferably, the NKT cells are iNKT cells.

6. The method of claim 5, wherein the cell fluid comprising NKT cells is sorted from magnetic beads;

preferably, the cell fluid containing NKT cells is derived from the cell fluid obtained by the method for inducing NKT cells according to claim 4.

7. A kit for preparing NKT cells comprising one or more of the optimized lymphocyte culture medium of claim 1, the antibody fluid for activating NKT cells of claim 2, the lymphocyte culture medium of claim 3 comprising IL-7 and IL-15, reagents for formulating the optimized lymphocyte culture medium of claim 1, reagents for formulating the antibody fluid for activating NKT cells of claim 2, reagents for formulating the lymphocyte culture medium of claim 3 comprising IL-7 and IL-15;

preferably, the NKT cells are iNKT cells.

8. An NKT cell capable of killing a tumor cell, which NKT cell is produced by the method for inducing NKT cell according to claim 4 and/or the method for expanding NKT cell according to claim 5;

preferably, the NKT cells are iNKT cells;

preferably, the killing occurs in vitro;

preferably, the tumor cell is a kidney cancer cell.

9. A pharmaceutical composition for treating cancer, comprising NKT cells produced by the method for inducing NKT cells according to claim 4 and/or the method for expanding NKT cells according to claim 5;

preferably, the cancer is renal cancer;

preferably, the NKT cells are iNKT cells.

10. An application, characterized in that the application comprises any one of the following:

1) use of any of the optimized lymphocyte culture medium of claim 1, the antibody fluid for activating NKT cells of claim 2, the lymphocyte culture medium comprising IL-7 and IL-15 of claim 3, the kit of claim 7 for the preparation of NKT cells;

2) use of any one of α -GalCer, IL-2, IL-21, IL-4 and GM-CSF for the preparation of iNKT cells;

3) use of a CD3 monoclonal antibody or a CD28 monoclonal antibody in the preparation of iNKT cells;

4) the use of IL-7 and IL-15 in the preparation of NKT cells;

5) use of the NKT cell of claim 8 or the pharmaceutical composition of claim 9 in the manufacture of a medicament for the treatment of cancer;

preferably, the cancer is renal cancer;

preferably, the NKT cells are iNKT cells.