Method for selectively amplifying natural killer T cells in vitro, natural killer T cells and application thereof

Abstract

The invention provides a method for selectively amplifying natural killer T cells in vitro. The method comprises the following steps: (1) obtaining peripheral blood mononuclear cells from peripheral blood; (2) preparing peripheral blood mononuclear cells into a first cell suspension by using a cell culture medium, treating by adopting an adherence method, and collecting a non-adherent cell suspension after the treatment is completed; and (3) centrifugally collecting non-adherent cells from the non-adherent cell suspension, preparing the non-adherent cells into a second cell suspension by using a cell culture medium, and carrying out I-type natural killer T cell proliferation. The invention also provides the natural killer T cell obtained by the method and application of the natural killer T cell. The method disclosed by the invention is simple to operate, and the I-type natural killer T cells with high purity and high effector capability can be obtained by efficiently amplifying a small amount of peripheral blood mononuclear cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell biology. Specifically, the present invention relates to a method for selectively amplifying natural killer T cells in vitro, natural killer T cells obtained by this method, and the use of natural killer T cells. Background Art

[0002] Natural killer T cells (NKT cells) are a type of αβ-T cells that restrictively recognize lipid antigens presented by CD1d molecules. Because they simultaneously express T cell receptors (TCRs) and natural killer cell (NK cell) receptors, NKT cells have the response characteristics of innate immunity and adaptive immunity. Specifically, like T cells, NKT cells recognize antigens through TCRs, specifically recognize lipid antigens presented by the MHC-like molecule CD1d, and like NK cells, they can rapidly respond, kill target cells, and simultaneously produce a series of cytokines and chemokines, thus playing an important immune regulatory role and participating in immune responses against tumors, infections, and autoimmune diseases. Among them, αβ-T cells with fixed transcription TCR Vα24Jα18 are also known as type I natural killer cells and are one of the most intensively studied NKT cells. Vα24 preferentially combines with Vβ11 to form a complete TCR, and α-galactosylceramide (α-GalCer or KRN7000) is its most effective stimulator. After NKT cells recognize antigens, they can rapidly respond and directly kill target cells by secreting perforin and granzyme. In addition, NKT cells secrete a variety of cytokines, including IFN-γ and IL-2, etc., to assist the activation and proliferation of CD8+ T cells, NK cells, and DC cells, etc., and promote the development of adaptive immune responses towards Th1-type immune responses. Therefore, NKT cells play an important role in the body's anti-tumor and anti-infection immune responses.

[0003] Numerous preclinical and clinical studies have shown that NKT cells have important functions such as inhibiting tumor growth, enhancing the body's anti-tumor immune response, and enhancing the adaptability of organ transplantation. Therefore, NKT cells are important candidate cells for cell therapy and have received attention in the fields of anti-tumor, anti-infection, and treatment of autoimmune diseases. High-quality in vitro amplified products of cells are an important guarantee for the application of this cell therapy strategy in clinical practice. However, because the proportion of NKT cells in peripheral blood mononuclear cells (PBMCs) is insufficient, only 0.01%-0.5%, realizing the specific proliferation of this small cell subset is the main problem to be solved in the in vitro culture of NKT cells.

[0004] At present, the in vitro culture protocols for NKT cells in the field are mainly divided into two categories. One type of protocol starts from PBMC, and by adding NKT cell-specific stimulants (α-GalCer) and appropriate cytokines (such as IL-2, IL-7, IL-15, etc.), the specific in vitro expansion of this cell subset is achieved, thereby increasing the proportion of target cells in the culture product. However, due to the cell mixture in PBMC, the purity of NKT cells in the cell product of this culture protocol is often affected. Another type of protocol first enriches NKT cells through cell sorting, and then adds NKT cell-specific stimulants (α-GalCer) and appropriate cytokines (such as IL-2, IL-7, IL-15, etc.) to achieve the specific in vitro expansion of this cell subset. Such a culture protocol can obtain in vitro expanded products of iNKT cells with a relatively high purity. However, due to the extremely low proportion of NKT cells in peripheral blood, a relatively large initial cell amount is required for this type of method.

[0005] Therefore, providing a method for the in vitro selective expansion of natural killer T cells to obtain cell products with high cell purity and high effector function with a relatively small initial cell amount is a technical problem that urgently needs to be solved in the current industry. Summary of the Invention

[0006] Therefore, the object of the present invention is to provide, in view of the deficiencies of the prior art, a method for the in vitro selective expansion of natural killer T cells, the natural killer T cells obtained by this method, and the uses of the natural killer T cells.

[0007] The object of the present invention is achieved by the following technical solutions:

[0008] On the one hand, the present invention provides a method for the in vitro selective expansion of natural killer T cells, which comprises the following steps:

[0009] (1) Obtaining peripheral blood mononuclear cells from peripheral blood;

[0010] (2) Using a cell culture medium to prepare the peripheral blood mononuclear cells into a first cell suspension, and performing treatment by the adherent method. After the treatment is completed, the non-adherent cell suspension is collected;

[0011] (3) Centrifuging the non-adherent cell suspension to collect non-adherent cells, using a cell culture medium to prepare the non-adherent cells into a second cell suspension, and performing the proliferation of type I natural killer T cells.

[0012] Preferably, the cell concentration of the first cell suspension is 1×10 6 cells / mL to 2×10 6 cells / mL; the cell concentration of the second cell suspension is 3×10 5 to 1×106 cells / mL.

[0013] Preferably, in step (2), the treatment by the adherent method is carried out by a method comprising the following steps:

[0014] Place the first cell suspension in a glass or plastic container and incubate statically for 45 minutes to 2 hours;

[0015] Transfer the obtained supernatant and non-adherent cells to a centrifuge tube, wash the cell surface of the container with cell culture medium, and transfer the washing solution to the centrifuge tube to obtain the non-adherent cell suspension.

[0016] Preferably, in step (3), the proliferation of type I natural killer T cells is carried out by a method comprising the following steps:

[0017] Add a specific stimulant to the second cell suspension for culturing type I natural killer T cells; preferably, the specific stimulant is α-GalCer;

[0018] On the 7th day of culture, add CD1d-expressing cells loaded with a specific stimulant to the culture system, and add cytokines IL-2 and IL-7;

[0019] On the 14th day of culture, add CD1d-expressing cells loaded with a specific stimulant to the culture system, and add cytokines IL-15 and IL-12;

[0020] Collect cells on the 21st to 28th day of culture.

[0021] Preferably, the CD1d-expressing cells loaded with a specific stimulant are DC cells loaded with α-GalCer obtained by inducing and differentiating adherent cells treated by the adherent method in step (2); preferably, the preparation method of the DC cells loaded with α-GalCer includes:

[0022] Resuspend the adherent cells with DC cell culture medium; preferably, the DC cell culture medium is RPMI1640 medium containing 10% FBS or autologous serum;

[0023] Add cytokines GM-CSF and IL-4 to the adherent cell suspension so that the concentration of GM-CSF in the adherent cell suspension is 500-1000 U / ml and the concentration of IL-4 is 50-200 ng / ml, and incubate statically;

[0024] On the 6th day of culture, add α-GalCer to the adherent cell suspension so that the concentration of α-GalCer in the adherent cell suspension is 50-500 ng / ml;

[0025] On the 7th day of culture, DC cells loaded with α-GalCer were collected.

[0026] Preferably, the working concentration of the specific stimulant added to the second cell suspension is 50 ng / mL to 500 ng / mL; the working concentration of IL-2 is 10 U / mL to 100 U / mL; the working concentration of IL-7 is 20 ng / mL to 200 ng / mL; the working concentration of IL-15 is 10 ng / mL to 100 ng / mL; the working concentration of IL-12 is 10 ng / mL to 100 ng / mL.

[0027] Preferably, the proliferation of type I natural killer T cells further includes the following step: supplementing the specific stimulant and / or cytokine to the culture system every 2 to 3 days to maintain their working concentrations.

[0028] Preferably, the cell culture medium is X-VIVO-15 serum-free medium or RPMI1640 medium containing 10% FBS or autologous serum.

[0029] On the other hand, the present invention provides a selectively amplified natural killer T cell prepared by the method according to the present invention.

[0030] On another aspect, the present invention provides the use of the selectively amplified natural killer T cell prepared by the method according to the present invention in the preparation of a drug for preventing and / or treating tumors, autoimmune diseases, and / or inflammatory diseases.

[0031] As can be seen from the above technical solutions, the method for in vitro selective amplification of natural killer T cells, the natural killer T cells, and their uses of the present invention at least have the following beneficial effects:

[0032] The method of the present invention reduces activated non-target T cells in peripheral blood mononuclear cells, weakens the interference with the proliferation of type I NKT cells existing in the culture system, and improves the in vitro amplification efficiency of type I NKT cells by using the adherent method, thereby being able to reduce the dosage of initial cells and simultaneously achieving efficient amplification of type I NKT cells.

[0033] The method of the present invention can improve the purity of target cells, the level of secreted effector cytokines, and the killing ability in the culture product.

[0034] The method of the present invention is simple to operate and is beneficial to the large-scale production and clinical application of type I NKT cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Hereinafter, the embodiments of the present invention will be described in detail with reference to the drawings, wherein:

[0036] Figure 1It is a process flow diagram schematically illustrating a method for in vitro selectively amplifying natural killer T cells of the present invention according to a preferred embodiment of the present invention.

[0037] Figure 2 It is a flow cytometry plot showing the change in the proportion of NKT cells in different experimental groups over the culture time in Experimental Example 1.

[0038] Figure 3 It is a statistical chart showing the change in the proportion of NKT cells in different experimental groups over the culture time in Experimental Example 1. Among them, (A) is a plot showing the data of each single cell in each group, and (B) is a statistical chart of the data of two groups. Among them, A i represents the i-th cell in Group A, and B i corresponds to the i-th cell in Group B.

[0039] Figure 4 It shows the secretion of effector cytokines by the NKT cell culture products in different experimental groups in Experimental Example 2. Among them, (A) shows the secretion level of IFN-γ, and (B) shows the ratio of IFN-γ to IL-4.

[0040] Figure 5 It shows the killing ability of NKT cells in different experimental groups in Experimental Example 3.

[0041] Figure 6 It shows the cell composition before and after the pre-culture treatment steps in different experimental groups in Experimental Example 4. Among them, (A) is a flow cytometry plot, and (B) is a statistical chart. Among them, 001, 002, and 003 correspond to 3 replicates of each experimental group.

[0042] Figure 7 It shows the purity of NKT cells in the cell products of different experimental groups in Experimental Example 4. Among them, (A) is a flow cytometry plot, and (B) is a statistical chart. Among them, 001, 002, and 003 correspond to 3 replicates of each experimental group.

[0043] Figure 8 It shows the purity of NKT cells in the cell products of different experimental groups in Experimental Example 5. Among them, (A) is a schematic diagram of the CD3+CD18 high gating during flow sorting operation, and (B) is a statistical chart of the purity of the NKT cell product.

[0044] Figure 9 It shows the purity of NKT cells in the culture products of different experimental groups in Experimental Example 6. Among them, (A) is a schematic diagram of the CD3+CD18 high gating during flow sorting operation, and (B) is a statistical chart of the purity of the NKT cell product. Detailed implementation mode

[0045] The present invention will be further described below in conjunction with the accompanying drawings and through the description of specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of the present invention, but as long as they do not depart from the basic idea of the present invention, such modifications or improvements are within the scope of the present invention.

[0046] In a first aspect, the present invention provides a method for in vitro selectively amplifying natural killer T cells. This method can specifically selectively amplify type I natural killer T cells in peripheral blood mononuclear cells. In the present invention, "type I natural killer T cells", "type I NKT cells", "iNKT cells", and "NKT cells" have the same meaning and can be used interchangeably.

[0047] The following is combined with Figure 1 , and a preferred embodiment of the method for in vitro selectively amplifying natural killer T cells of the present invention will be described in detail.

[0048] (1) Obtain peripheral blood mononuclear cells from peripheral blood.

[0049] The specific method for obtaining peripheral blood mononuclear cells (PBMC) from peripheral blood can refer to relevant protocols in the prior art. In the following examples, the separation of PBMC uses the Ficoll density gradient centrifugation method, but this is only exemplary. In practical applications, those skilled in the art can select a suitable PBMC separation method according to needs, and the present invention will not elaborate.

[0050] (2) Use a cell culture medium to prepare the peripheral blood mononuclear cells into a first cell suspension, and perform treatment by the adherent method. After the treatment is completed, collect the non-adherent cell suspension.

[0051] Before treating the PBMC separated in step (1), first use a cell culture medium to prepare it into a cell suspension, that is, the first cell suspension. The concentration of PBMC in this cell suspension is preferably 1×10 6 cells / mL to 2×10 6 cells / mL. The cell culture medium can be X-VIVO-15 serum-free medium or RPMI1640 medium containing 10% FBS or autologous serum.

[0052] Then, treat the first cell suspension by the adherent method to separate the adherent cells and non-adherent cells in the first cell suspension. Specifically, the treatment of the first cell suspension by the adherent method includes the following steps:

[0053] ① Place the first cell suspension in a glass or plastic container so that the cells in the first cell suspension are evenly distributed on the surface of the container, and perform static incubation. Preferably, perform static incubation at 37°C for 45 minutes to 2 hours.

[0054] ②After the static incubation is completed, transfer the obtained supernatant and non-adherent cells to a centrifuge tube. Wash the cell surface of the container with cell culture medium and transfer the washing solution to the centrifuge tube to obtain a non-adherent cell suspension.

[0055] In the present invention, by using the adherent method to treat the first cell suspension, the proliferation ability of non-target T cells in the culture system can be weakened, so as to achieve the specific proliferation of iNKT cells in the subsequent culture process, thereby improving the purity of target cells in the product.

[0056] It is generally considered that the adherent method, like the magnetic bead sorting method, is a common method for separating monocytes from PBMC. However, the inventors of the present invention unexpectedly found through research that in the method of the present invention, using the adherent method to treat the first cell suspension and formulating the obtained non-adherent cells into a cell suspension (i.e., the second cell suspension in step (3)) for the in vitro proliferation of iNKT cells can significantly improve the in vitro proliferation efficiency of iNKT cells. And when delving into its mechanism, it was unexpectedly found that it was not due to the adherent operation separating and removing monocytes, but due to the adherent operation separating and removing CD3-positive cells (CD3+) and cells highly expressing CD18 (CD18 high ).

[0057] (3) Centrifuge the non-adherent cell suspension to collect non-adherent cells, and formulate the non-adherent cells into a second cell suspension with cell culture medium for the specific proliferation of type I natural killer T cells.

[0058] Before the specific proliferation of iNKT cells, first centrifuge the non-adherent cell suspension collected in step (2) to collect non-adherent cells, and formulate the non-adherent cells into a cell suspension with cell culture medium, that is, the second cell suspension. The concentration of non-adherent cells in this cell suspension is preferably 3×10 5 to 1×10 6 cells / mL. The cell culture medium can be X-VIVO-15 serum-free medium or RPMI1640 medium containing 10% FBS or autologous serum.

[0059] Then, perform the specific proliferation of iNKT cells. Specifically, the specific proliferation of iNKT cells includes the following steps:

[0060] ①Add a specific stimulant to the second cell suspension to form a culture system for iNKT cell culture. Preferably, the specific stimulant is α-GalCer. Preferably, the working concentration of the specific stimulant in the culture system is 50 ng / mL to 500 ng / mL.

[0061] ②On the 7th day of culture, CD1d-expressing cells loaded with the specific stimulant α-GalCer, cytokines IL-2 and IL-7 are added to the culture system. Preferably, the working concentration of cytokine IL-2 in the culture system is 10 U / mL to 100 U / mL. Preferably, the working concentration of cytokine IL-7 in the culture system is 20 ng / mL to 200 ng / mL.

[0062] ③On the 14th day of culture, CD1d-expressing cells loaded with the specific stimulant α-GalCer, cytokines IL-15 and IL-12 are added to the culture system. Preferably, the working concentration of cytokine IL-15 in the culture system is 10 ng / mL to 100 ng / mL. Preferably, the working concentration of cytokine IL-12 in the culture system is 10 ng / mL to 100 ng / mL.

[0063] ④On the 21st - 28th day of culture, the cells are collected to obtain a selectively amplified NKT cell product.

[0064] During the process of specific proliferation of iNKT cells, the working concentrations of the specific stimulant and various cytokines should be maintained constant. Therefore, preferably, according to the consumption of materials in the culture system, the specific stimulant and / or cytokines are supplemented to the culture system every 2 to 3 days. For example, on the 3rd day of culture, cell culture medium is supplemented to the culture system, and α-GalCer is supplemented to make the concentration of α-GalCer in the culture system reach its working concentration. For example, on the 10th day of culture, α-GalCer, IL-2 and IL-7 are supplemented to the system to make the concentrations of α-GalCer, IL-2 and IL-7 in the culture system reach their respective working concentrations.

[0065] Preferably, in step ② above, the CD1d-expressing cells loaded with the specific stimulant added to the culture system are DC cells (dendritic cells) loaded with α-GalCer. The DC cells loaded with α-GalCer are prepared by inducing the differentiation of adherent cells obtained by treating the first cell suspension by the adherent method in step (2). Specifically, the preparation method of the DC cells loaded with α-GalCer includes the following steps:

[0066] ①Resuspend the adherent cells with DC cell medium. Preferably, the DC cell medium is RPMI1640 medium containing 10% FBS or autologous serum.

[0067] ② Add cytokines GM-CSF and IL-4 to the obtained adherent cell suspension so that the concentration of GM-CSF in the adherent cell suspension is 500 - 1000 U / ml and the concentration of IL-4 is 50 - 200 ng / ml. Place the adherent cell suspension in a 37°C 5% CO 2 cell incubator for static culture. Optionally, according to the consumption of materials in the adherent cell suspension, on the 4th day of culture, an appropriate amount of DC cell medium containing the aforementioned concentrations of GM-CSF and IL-4 can be supplemented to the adherent cell suspension.

[0068] ③ On the 6th day of culture, add α-GalCer to the adherent cell suspension so that the concentration of α-GalCer in the adherent cell suspension is 50 - 500 ng / ml.

[0069] ④ On the 7th day of culture, collect the DC cells loaded with α-GalCer.

[0070] In the specific proliferation process of iNKT cells in the present invention, CD1d-expressing cells loaded with a specific stimulant, such as DC cells loaded with α-GalCer, are used for stimulation, and cytokines are added simultaneously to assist the growth of NKT cells. Among them, through the combined stimulation of the specific stimulant and cytokines, NKT cells are selectively amplified. In addition, CD1d-expressing cells are supplemented to improve the effective stimulation of NKT cells.

[0071] In particular, existing in vitro expansion methods for NKT cells require additional use of PBMC to induce the expression cells loaded with stimulants, so a large number of starting cells are needed, and the purity of iNKT cells in the culture product is low. In the method of the present invention, adherent cells are used to culture DC cells loaded with α-GalCer as cells for stimulating the proliferation of iNKT cells, while non-adherent cells are used to culture iNKT cells, without the need to additionally use PBMC to induce the expression cells loaded with stimulants, thus greatly reducing the initial cell dosage and being beneficial to the large-scale production and clinical application of NKT cells.

[0072] By using the method for in vitro selective expansion of natural killer T cells of the present invention, a selectively expanded NKT cell product (selectively expanded NKT cells, seNKT) can be obtained. Therefore, in the second aspect, the present invention provides a selectively expanded NKT cell product (seNKT). The purity of NKT cells, the level of secreted effector cytokines, and the killing ability of this cell product are all improved.

[0073] The selectively expanded NKT cell product of the present invention has the advantages of high NKT cell purity and high effector function, so this cell product has a wide range of uses in the field of cell therapy. Therefore, in the third aspect, the present invention provides the use of the selectively expanded NKT cell product in the preparation of a medicament for preventing and / or treating tumors, autoimmune diseases, and / or inflammatory diseases.

[0074] Example

[0075] The following examples are only used to illustrate the present invention, but not to limit the scope of the present invention.

[0076] The experimental methods used in the following examples are all conventional experimental methods in the art unless otherwise specified. The experimental materials used in the following examples are all purchased from biochemical reagent sales companies unless otherwise specified.

[0077] Example 1: Isolation of peripheral blood PBMC

[0078] Add 30 mL to 50 mL of heparinized human peripheral blood into centrifuge tube 1, and dilute the anticoagulated peripheral blood with physiological saline. The volume ratio of physiological saline to anticoagulated peripheral blood is 1:1, and mix evenly to obtain a blood dilution.

[0079] Take another new 50 mL centrifuge tube 2, add 15 mL of lymphocyte separation solution (ficoll) into it, and then slowly add the blood dilution along the tube wall to the upper layer of the lymphocyte separation solution according to the volume ratio of ficoll:blood dilution of 1:2, so that the two form a clear layer. Centrifuge tube 2 is centrifuged at 3000 rpm for 30 minutes, and the acceleration is selected as 3 up and 4 down (that is, the acceleration of increasing speed is 3 and the acceleration of decreasing speed is 4).

[0080] After centrifugation, aspirate the mononuclear cell layer and transfer it to a new 50 ml centrifuge tube 3. Add 30 mL of X-VIVO-15 medium to centrifuge tube 3 and wash once. Centrifuge tube 3 is centrifuged at 800 g for 5 minutes. After centrifugation, discard the supernatant in centrifuge tube 3.

[0081] Add 20 mL of X-VIVO-15 medium to centrifuge tube 3 and pipette to mix evenly. At room temperature, centrifuge tube 3 is centrifuged at 200 g for 10 minutes. After centrifugation, discard the supernatant in centrifuge tube 3 to obtain the isolated PBMC.

[0082] Example 2: Adhesion treatment

[0083] Count the cells of the PBMC isolated in Example 1. Take 2×10 7 PBMCs, and adjust the PBMC concentration to 1.5×10 6cells / mL to obtain a PBMC suspension. The PBMC suspension was seeded into a T25 cell culture flask 1. The T25 cell culture flask 1 was placed in a cell culture incubator and statically cultured for 1 hour under the conditions of 37°C and 5% CO 2 Then, the T25 cell culture flask 1 was taken out, and the supernatant and non-adherent cells therein were transferred into a 50 mL centrifuge tube 4. The cell surface of the T25 cell culture flask 1 was washed 2 times with X-VIVO-15 cell culture medium, and the washing solution was transferred into the 50 mL centrifuge tube 4.

[0084] The non-adherent cells were in the centrifuge tube 4, and the cell surface of the T25 cell culture flask 1 was adherent cells.

[0085] Example 3: Inductive differentiation of DC cells loaded with α-GalCer

[0086] The RPMI1640 medium containing 10% FBS was used as the DC cell culture medium. 12 mL of the DC cell culture medium was added to the T25 cell culture flask 1 obtained in Example 2, and GM-CSF and IL-4 were added to the T25 cell culture flask 1 so that the concentration of GM-CSF in the culture system of the T25 cell culture flask 1 was 500 U / mL and the concentration of IL-4 was 50 ng / mL. It was placed in a 37°C 5% CO 2 cell culture incubator and statically cultured.

[0087] On the 4th day of culture, 3 mL of the DC cell culture medium containing 500 U / mL GM-CSF and 50 ng / mL IL-4 was supplemented into the culture system of the T25 cell culture flask 1.

[0088] On the 6th day of culture, α-Galcer was added to the culture system of the T25 cell culture flask 1 so that the concentration of α-Galcer in the culture system was 100 ng / mL.

[0089] On the 7th day of culture, the cells were collected to obtain DC cells loaded with α-Galcer for standby use.

[0090] Example 4: Proliferation of type I natural killer T cells

[0091] The centrifuge tube 4 obtained in Example 2 was centrifuged at 600 g for 10 minutes at room temperature. After centrifugation, the supernatant in the centrifuge tube 4 was discarded. The cells in the centrifuge tube 4 were resuspended with 8 ml of X-VIVO-15 cell culture medium, and the obtained cell suspension was transferred into a T25 cell culture flask 2. The concentration of adherent cells in the cell suspension was 1×10 6cells / mL. Add α-Galcer to T25 cell culture flask 2 so that the concentration of α-Galcer in the culture system of T25 cell culture flask 2 is 100 ng / mL. After shaking and mixing evenly, place T25 cell culture flask 2 in the cell culture incubator for static culture to specifically stimulate NKT cells.

[0092] On the 3rd day of culture, supplement 3 mL of X-VIVO-15 cell culture medium to the culture system of T25 cell culture flask 2, and add α-GalCer so that the concentration of α-Galcer in the culture system of T25 cell culture flask 2 is 100 ng / mL.

[0093] On the 7th day of culture, add the α-GalCer-loaded DC cells prepared in Example 3 to the culture system of T25 cell culture flask 2. At the same time, supplement the stimulatory factor α-GalCer to the culture system of T25 cell culture flask 2 so that the concentration of α-GalCer in the culture system is 100 ng / mL. And add cytokines IL-2 and IL-7 to the culture system of T25 cell culture flask 2 so that the concentration of IL-2 in the culture system is 100 U / mL and the concentration of IL-7 is 20 ng / mL.

[0094] At the same time, resuscitate a vial of PBMCs for inducing the differentiation of α-GalCer-loaded DC cells to re-stimulate NKT cells. The specific method is the same as in Example 3. The obtained α-GalCer-loaded DC cells are used to be added to the culture system on the 14th day.

[0095] On the 10th day of culture, supplement X-VIVO-15 cell culture medium to the culture system of T25 cell culture flask 2 so that the cell concentration is 1×10 6 / mL, and add α-GalCer, IL-2 and IL-7 so that the concentration of α-GalCer in the culture system is 100 ng / mL, the concentration of IL-2 is 100 U / mL, and the concentration of IL-7 is 20 ng / mL.

[0096] On the 14th day of culture, add the α-GalCer-loaded DC cells to the culture system of T25 cell culture flask 2 again. At the same time, supplement the stimulatory factors α-GalCer, IL-2 and IL-7 to the culture system of T25 cell culture flask 2 so that the concentration of α-GalCer in the culture system is 100 ng / mL, the concentration of IL-2 is 100 U / mL, and the concentration of IL-7 is 20 ng / mL. And add IL-15 and IL-12 to the culture system of T25 cell culture flask 2 so that the concentration of IL-15 in the culture system is 20 ng / mL and the concentration of IL-12 is 20 ng / mL.

[0097] On the 21st day of culture, the cells were collected.

[0098] Comparative Example 1:

[0099] (1) Isolation of peripheral blood PBMCs: The same as in Example 1.

[0100] (2) Inducing and differentiating autologous DC cells for stimulating NKT cell proliferation:

[0101] The PBMCs isolated in step (1) were prepared into a PBMC suspension with a concentration of 1×10 6 cells / mL using RPMI1640 medium. The PBMC suspension was seeded into T25 cell culture flasks 3. The T25 cell culture flasks 3 were placed in a cell culture incubator and statically cultured for 1 hour under the conditions of 37°C and 5% CO 2 .

[0102] The T25 cell culture flasks 3 were taken out, and the supernatant and non-adherent cells were removed. The RPMI1640 medium containing 10% FBS was used as the DC cell medium. The cell surface of the T25 cell culture flasks 3 was washed 2 times with the DC cell medium, and then 5 mL of the DC cell medium was added to the T25 cell culture flasks 3. Moreover, cytokines GM-CSF and IL-4 were added to the T25 cell culture flasks 3 such that the concentration of GM-CSF was 500 U / mL and the concentration of IL-4 was 50 ng / mL.

[0103] On the 4th day of culture, 3 mL of the DC cell medium containing 500 U / mL GM-CSF and 50 ng / mL IL-4 was supplemented into the culture system of the T25 cell culture flasks 3.

[0104] On the 6th day of culture, α-Galce was added to the culture system of the T25 cell culture flasks 3 such that the concentration of α-Galce was 100 ng / mL.

[0105] On the 7th day of culture, the cells were collected to obtain DC cells loaded with α-Galcer.

[0106] (3) In vitro expansion of NKT cells:

[0107] Without performing adherent treatment, directly add α-Galcer to the PBMC separated in step (1) to make the concentration of α-Galcer 100 ng / mL, and then culture. The remaining operation steps are the same as those in Example 4. Specifically: On the 7th day, add DC cells loaded with α-GalCer to the NKT cell culture system, and add IL-2 and IL-7 to their respective working concentrations (so that the concentration of α-GalCer in the culture system is 100 ng / mL, the concentration of IL-2 is 100 U / mL, and the concentration of IL-7 is 20 ng / mL); On the 14th day of culture, add DC cells loaded with α-GalCer again, and add IL-2, IL-7, and IL-15 to their respective working concentrations; On the 21st day of culture, collect the cells.

[0108] Experimental Example 1: Influence of the pre-treatment step of culture by adherent method on the purity of NKT cell amplification products

[0109] (1) Experimental grouping:

[0110] PBMC: PBMC separated in Example 1

[0111] Group A: Cells cultured in Example 4

[0112] Group B: Cells cultured in Comparative Example 1

[0113] The following Experimental Examples 2 to 4 adopt the same grouping method as this Experimental Example.

[0114] (2) Detection method

[0115] Respectively take the NKT cells cultured in Example 4 (Group A) and Comparative Example 1 (Group B) under the same culture conditions (37 °C, 5% CO 2 concentration) for effect testing. Specifically, on the 10th, 14th, and 25th days of culture, respectively take the cultured cells of the two experimental groups, and detect the proportion of iNKT cells in them by flow cytometry.

[0116] The flow cytometry detection method is as follows:

[0117] Take 5×10 5Place the cells in an Ep tube. Centrifuge the Ep tube at 600 g for 5 minutes at room temperature. After centrifugation, discard the supernatant in the Ep tube. Resuspend the cells in the Ep tube with 500 μL of staining buffer (PBS buffer containing 2% FBS by volume), and mix well to obtain a cell suspension. Centrifuge the Ep tube at 600 g for 5 minutes at room temperature. After centrifugation, discard the supernatant in the Ep tube. Resuspend the cells in the Ep tube with 30 μl of staining buffer, and add the following fluorescent antibodies to the obtained cell suspension: anti-TCR Vα24-PE (Beckman Coulter, clone#C15), anti-TCR Vβ11-FITC (Beckman Coulter, clone#C21), anti-CD3-PB (BD Pharmingen, clone#SP34-2), and then incubate at 4 °C for 30 minutes. After incubation, add 500 μL of staining buffer to the system in the Ep tube and mix well. Centrifuge the Ep tube at 600 g for 5 minutes at room temperature. After centrifugation, discard the supernatant in the Ep tube. Resuspend the cells in the Ep tube with 200 μL of staining buffer, mix well, transfer the cell suspension into a flow tube, and use a flow cytometer to detect the proportion of iNKT cells (CD3+TCR Vα24+Vβ11+).

[0118] The detection results of this experimental example are as Figure 2 and Figure 3 shown.

[0119] It can be seen from Figure 2 and Figure 3 that the proportion of NKT cells in the group (group A) treated with the adherent method for PBMC before culture is significantly higher than that in the group (group B) not treated with the adherent method. Figure 2 and Figure 3 The results of

[0120] show that treating PBMC with the adherent method before culture and then amplifying NKT cells using non-adherent cells can significantly improve the purity of NKT cells in the culture product.

[0121] Existing research results show that the ability of NKT cells to secrete Th1-type cytokines is positively correlated with their anti-tumor ability. Therefore, in this experimental example, the ratio of IFN-γ to IL-4 content in the supernatant of the cell product will be detected as one of the indicators to evaluate the effector function of the product.

[0122] Specifically, in this experimental example, the ratio of IFN-γ:IL-4 in the supernatant of the cell culture products in Example 4 (Group A) and Comparative Example 1 (Group B) was detected by using the ELISA method (Dakewei, Human IFN-γ Precoated ELISA Kit, 1110002; Dakewei, Human IL-4 Precoated ELISA Kit, 1110402), so as to evaluate the ability of the NKT cell culture products to secrete effector cytokines. The results are as Figure 4 shown.

[0123] It can be Figure 4 seen that treating PBMC by the adherent method before culturing NKT cells can significantly increase the secretion level of IFN-γ in the product supernatant and the ratio of IFN-γ:IL-4. This indicates that the ability of the NKT cell culture products to secrete effector cytokines has been improved.

[0124] Experimental Example 3: Influence of the pre-culture treatment step by the adherent method on the killing ability of the culture products

[0125] Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme. When cells lyse, LDH will be released into the extracellular space and catalyze its substrate tetrazolium salt (INT) to produce a red product. Therefore, the amount of the red product generated is proportional to the amount of cell lysis. In this experimental example, the killing ability of the amplified NKT cell products against target cells was evaluated by detecting the amount of INT. In this experimental example, an LDH detection kit (product number CK12, Dongren Chemical) was used for detection, and the operation was carried out according to the kit instructions as follows:

[0126] The target cells K562 cells were centrifuged, and the density of the target cells was adjusted to 1×10 5 cells / mL. The cell products cultured in Example 4 (Group A) and Comparative Example 1 (Group B) were centrifuged and collected as effector cells. The number ratios of effector cells to target cells were set to 5:1, 10:1, and 20:1 respectively. Three replicate wells were set for both Group A and Group B. The two experimental groups were incubated in an incubator at 37°C and 5% CO 2 for 4 hours respectively. After the cells in the two experimental groups were fully lysed and precipitated, the absorbance was detected with an enzyme-linked immunosorbent detector, and the killing rate was calculated. The calculation formula for the killing rate is: Killing rate (%) = (OD490 实验孔 -OD490 阴性孔 ) / (OD490 阳性孔 -OD490 阴性孔 ))×100%.

[0127] The comparison results of the killing ability are as Figure 5 shown.

[0128] It can beFigure 5 It can be seen that treating PBMC by the adherent method before NKT cell culture can significantly improve the killing ability of the culture product.

[0129] Experimental Example 4: Influence of the pre-culture treatment step using the adherent method and the pre-culture treatment step using magnetic bead sorting to reduce monocytes on the purity of NKT cell products

[0130] Since the adherent method is a common method for separating monocytes from PBMC, in order to clarify the influence of removing monocytes on the purity of NKT cell culture products, this experimental example uses the magnetic bead sorting method to separate and remove monocytes in PBMC, and compares the influence of the two pre-treatment steps on the purity of NKT cell products.

[0131] Experimental grouping:

[0132] Group D: No pre-culture treatment, directly starting with PBMC for in vitro expansion of iNKT cells;

[0133] Group E: Pre-culture treatment using the adherent method;

[0134] Group F: Pre-culture treatment using magnetic bead sorting to reduce CD14+ monocytes in PBMC.

[0135] Experimental method:

[0136] (1) Isolation of peripheral blood PBMC: The same as in Example 1.

[0137] (2) Pre-culture treatment:

[0138] Group D does not perform pre-culture treatment.

[0139] The pre-culture treatment step of the adherent method in Group E is the same as that in Example 2.

[0140] For the magnetic bead sorting method in Group F, Miltenyi Biotec CD14+ monocyte positive selection kit (Miltenyi Biotec, CD14 microbeads human, 130 - 050 - 201) is used and operated according to the kit instructions. Specifically: Mix the CD14 microbeads evenly, then mix them with the PBMC separated in step (1) to obtain a mixture, and incubate the obtained mixture at room temperature for 15 minutes; then pass the incubation product through the column, wash it twice with the washing buffer, and collect the cells not bound to and bound to the column respectively.

[0141] For the culture systems before and after the pre-culture treatment steps of Group E and Group F, flow cytometry is used to detect the composition ratio of monocytes in the culture systems respectively, and the results are as Figure 6 shown. From Figure 6It can be seen that the adherent method will reduce the proportion of monocytes in the culture system, and almost all monocytes in the culture system are removed after positive selection of CD14+ monocytes.

[0142] (3) The step of massive expansion of NKT cells in each experimental group is the same as that in Example 4.

[0143] On the 14th day of culture, flow cytometry was used to detect the purity of NKT cells in the culture products of each experimental group, and the detection method was the same as that in Experimental Example 1. The detection results are as Figure 7 shown.

[0144] As Figure 7 can be seen, treating PBMC with the adherent method before culturing NKT cells can significantly improve the purity of NKT cells in the product. However, removing CD14+ monocytes by magnetic bead sorting before culturing NKT cells cannot achieve the same effect.

[0145] From the experimental results of Experimental Example 4, it can be seen that although the adherent method is a classic method for separating monocytes from PBMC, in the present invention, the adherent method pretreatment does not play a role in enhancing the in vitro proliferation ability of iNKT cells by removing monocytes, but there is an unexpected mechanism of action.

[0146] Experimental Example 5: Effect of removing CD3+CD18 high cell population in PBMC by flow sorting on the purity of NKT cell products

[0147] CD18 is an important molecule mediating the adherent characteristics of T cells. To further confirm the effect of T cells with adherent characteristics on the purity of NKT cell in vitro culture products, in this experimental example, the CD3+CD18 high cell population in PBMC was removed by flow sorting to verify the adverse effect of adherent T cells on the purity of NKT cell products.

[0148] Experimental grouping:

[0149] Group D: Without pretreatment before culture, directly start in vitro expansion of iNKT cells with PBMC;

[0150] Group E: The pretreatment group using the adherent method;

[0151] Group G: The pretreatment group using the BD Aria II flow sorter to remove the CD3+CD18 high cell population in PBMC.

[0152] Experimental method:

[0153] (1) Isolation of peripheral blood PBMC: The same as in Example 1.

[0154] (2) Pretreatment before culture:

[0155] Group D does not undergo pretreatment before culture.

[0156] The pretreatment steps before adherent culture in Group E are the same as those in Example 2.

[0157] In Group G, the PBMC isolated in step (1) is stained with flow antibodies (BV421-anti-CD3: 562426, BD; PE-anti-CD18: 373408, Biolegend). Then, the BD Aria II flow cytometer is used to sort and remove the CD3+CD18 high cell population in PBMC, and the remaining cells are used for NKT cell culture.

[0158] (3) The steps for large-scale expansion of NKT cells in each experimental group are the same as those in Example 4.

[0159] On the 14th day of culture, flow cytometry is used to detect the purity of NKT cells in the culture products of each experimental group. The detection method is the same as that in Experimental Example 1. The detection results are as Figure 8 shown.

[0160] It can be seen from Figure 8 that the pretreatment operation before culturing to remove the CD3+CD18 high cell population in PBMC can significantly improve the purity of NKT cells in the culture products, and there is no significant difference from the purity of NKT cell products obtained by the adherent method. This indicates that treating PBMC by the adherent method before NKT cell culture can reduce the CD3+CD18 high cell population in the culture system, thereby weakening the interference with the proliferation of NKT cells and finally obtaining a selectively amplified NKT cell product.

[0161] Experimental Example 6: Effect of adding anti-CD18 blocking antibody to the culture system on the purity of NKT cell products

[0162] To further confirm the effect of T cells with adherent characteristics on the purity of in vitro cultured NKT cell products, in this experimental example, an anti-CD18 blocking antibody is added to the culture conditions of Comparative Example 1 to verify the adverse effect of adherent T cells on the purity of NKT cell products.

[0163] Experimental grouping:

[0164] Group D: Without pretreatment before culture, directly start in vitro expansion of iNKT cells with PBMC;

[0165] Group E: The pretreatment group using the adherent method;

[0166] Group H: Without pre - culture treatment, directly start with PBMC for in vitro expansion of iNKT cells, and add CD18 blocking antibody to the culture system.

[0167] Experimental method:

[0168] (1) Isolation of peripheral blood PBMC: The same as in Example 1.

[0169] (2) Pre - culture treatment:

[0170] Group D does not perform pre - culture treatment, which is the same as Comparative Example 1.

[0171] The adherent method pre - culture treatment steps of Group E are the same as in Example 2.

[0172] Group H does not perform pre - culture treatment. Add CD18 blocking antibody to step (1) of Comparative Example 1, and its concentration is 2 μg / ml. The remaining culture reagents, operations and steps are the same as in Comparative Example 1.

[0173] On the 14th day of culture, flow cytometry was used to detect the purity of NKT cells in the culture products of each experimental group. The detection method is the same as in Experimental Example 1. The detection results are as Figure 9 shown.

[0174] It can be seen from Figure 9 that adding CD18 blocking antibody can significantly improve the purity of iNKT cells in the culture products. This indicates that inhibiting the cells expressing CD18 can achieve an effect similar to that of the adherent method in improving the purity of iNKT cell products. Further suggesting that the pre - culture operation of the adherent method is through weakening the CD3 + CD18 high cell population in PBMC, and finally obtaining a selectively amplified NKT cell product.

[0175] The above are only several exemplary embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention is disclosed as above with preferred embodiments, it is not intended to limit the present invention. Any equivalent or equivalent embodiments obtained by making some changes or modifications by those skilled in the art within the scope of the technical solution of the present invention using the disclosed technical content all belong to the scope of the present invention.

Claims

1. A method for in vitro selectively amplifying natural killer T cells, characterized in that, the method comprises the following steps: (1) Obtaining peripheral blood mononuclear cells from peripheral blood; (2) Using a cell culture medium to prepare the peripheral blood mononuclear cells into a first cell suspension, and performing treatment by the adherent method. After the treatment is completed, collecting the non-adherent cell suspension; (3) Centrifuging the non-adherent cell suspension to collect non-adherent cells, using a cell culture medium to prepare the non-adherent cells into a second cell suspension, and performing type I natural killer T cell proliferation.

2. The method according to claim 1, characterized in that, The cell concentration of the first cell suspension is 1×10 6 cells / mL to 2×10 6 cells / mL; the cell concentration of the second cell suspension is 3×10 5 to 1×10 6 cells / mL.

3. The method according to claim 1, characterized in that, In step (2), the treatment by the adherent method is carried out by a method comprising the following steps: Placing the first cell suspension in a glass or plastic container, and statically incubating for 45 minutes to 2 hours; Transferring the obtained supernatant and non-adherent cells to a centrifuge tube, washing the cell surface of the container with a cell culture medium, and transferring the washing solution to the centrifuge tube to obtain the non-adherent cell suspension.

4. The method according to claim 1, characterized in that, In step (3), the type I natural killer T cell proliferation is carried out by a method comprising the following steps: Adding a specific stimulant to the second cell suspension to perform type I natural killer T cell culture; preferably, the specific stimulant is α-GalCer; On the 7th day of culture, adding CD1d-expressing cells loaded with the specific stimulant to the culture system, and adding cytokines IL-2 and IL-7; On the 14th day of culture, adding CD1d-expressing cells loaded with the specific stimulant to the culture system, and adding cytokines IL-15 and IL-12; On the 21st to 28th day of culture, collecting cells.

5. The method according to claim 4, characterized in that, the CD1d-expressing cells loaded with the specific stimulant are DC cells loaded with α-GalCer obtained by inducing and differentiating the adherent cells obtained by the adherent method treatment in step (2); preferably, the preparation method of the DC cells loaded with α-GalCer comprises: Resuspending the adherent cells with a DC cell culture medium to obtain an adherent cell suspension; preferably, the DC cell culture medium is RPMI1640 medium containing 10% FBS or autologous serum; Adding cytokines GM-CSF and IL-4 to the adherent cell suspension so that the concentration of GM-CSF in the adherent cell suspension is 500-1000 U / ml and the concentration of IL-4 is 50-200 ng / ml, and performing static culture; On the 6th day of culture, adding α-GalCer to the adherent cell suspension so that the concentration of α-GalCer in the adherent cell suspension is 50-500 ng / ml; On the 7th day of culture, collecting the DC cells loaded with α-GalCer.

6. The method according to claim 4, characterized in that, The working concentration of the specific stimulant added to the second cell suspension is 50 ng / mL to 500 ng / mL; the working concentration of IL-2 is 10 U / mL to 100 U / mL; the working concentration of IL-7 is 20 ng / mL to 200 ng / mL; the working concentration of IL-15 is 10 ng / mL to 100 ng / mL; the working concentration of IL-12 is 10 ng / mL to 100 ng / mL.

7. According to the method described in claim 4, wherein, the proliferation of the type I natural killer T cells further comprises the following step: supplementing the specific stimulant and / or cytokine to the culture system every 2 to 3 days to maintain their working concentrations.

8. According to the method described in claim 1, wherein, the cell culture medium is X-VIVO-15 serum-free medium or RPMI1640 medium containing 10% FBS or autologous serum.

9. A selectively amplified natural killer T cell prepared by the method according to any one of claims 1 to 8.

10. Use of the selectively amplified natural killer T cell prepared by the method according to any one of claims 1 to 8 in the preparation of a drug for preventing and / or treating tumors, autoimmune diseases and / or inflammatory diseases.

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