Gamma delta t cell as well as culture method and application thereof

Abstract

The invention provides a gamma delta t cell as well as a culture method and application thereof, and the method comprises the following steps: separating human peripheral blood mononuclear cells from malignant pleuroperitoneal fluid, adding normal saline into the human peripheral blood mononuclear cells, and centrifuging; separating gamma delta t cells from the cell precipitate by adopting an immunomagnetic negative selection method, and resuspending the gamma delta t cells by adopting a first culture medium; adding the gamma delta t cell suspension into a T-175 culture flask coated with a PD-1 monoclonal antibody, adding a second culture medium into the T-175 culture flask, and then adding 0.05-0.2 mg / mL TLR8 agonist which accounts for 1-3% of the cell volume; and culturing the T-175 culture bottle for 2-4 days to obtain the gamma delta t cell which is jointly cultured by coating the PD-1 monoclonal antibody with the TLR8 agonist. The killing efficiency of the gamma delta t cells obtained through culture in the embodiment of the invention on K562 target cells can be larger than or equal to 82.12%.

Description

Technical Field

[0001] The present invention relates to the technical field of γδt cell preparation, and particularly to a γδt cell, a culture method thereof and an application thereof. Background Art

[0002] γδt cells are a unique type of innate-like immune cells that can infiltrate into various human solid tumors and are key coordinators of monitoring infections, immune defense and anti-malignant tumor immune responses. Its T cell receptor is composed of a γ chain and a δ chain, which is different from traditional αβ T cells. γδT cells directly recognize antigens without antigen processing and presentation and are not restricted by the major histocompatibility complex (MHC molecules). Instead, γδT cells carry a large number of natural killer cell receptors (NKRs) on their surface. These unique characteristics enable γδT cells to quickly respond to infected or transformed cells, thus becoming the first line of defense before the antigen-specific αβ T cell response. More and more clinical studies have shown that γδT cells infiltrate into various types of human tumors including CRC and are widely involved in anti-tumor immune responses.

[0003] However, the immune prospects and functional status of tumor-infiltrating γδt cells in the tumor microenvironment are still unclear, and the immunosuppressive function of γδt cells has not been solved. A large number of research results have shown that most tumor-derived γδt cells have a strong immunosuppressive function, significantly inhibiting the proliferation of naive / effector T cells and the secretion of IL-2, and inhibiting the maturation and function of DCs. Immunosuppression leads to cell exhaustion. Tumor-infiltrating γδt cells show an exhausted phenotype in the tumor microenvironment and show more severe functional exhaustion than tumor-infiltrating CD8+ T cells or NK cells. And single-cell RNA sequencing (scRNA-seq) analysis shows that the population of γδt cells infiltrating tumors is heterogeneous, and exhausted cells are the dominant subpopulation.

[0004] Therefore, how to reverse the immunosuppressive function of γδt cells and restore the anti-tumor immune response will help to optimize γδt cell-based cancer immunotherapy. Summary of the Invention

[0005] The purpose of the present invention is to provide a γδt cell, a culture method thereof and an application thereof, aiming to reverse the immunosuppressive effect of γδt cells by combining in vitro PD-1 monoclonal antibody coating and TLR8 agonist, activate and amplify γδt cells in vitro, and transfuse them into the body to play an anti-tumor role.

[0006] In the first aspect, the present invention provides a culture method of γδt cells, characterized in that the method includes:

[0007] Step 1: Collect malignant pleural effusion and ascites from tumor patients, isolate human peripheral blood mononuclear cells from the malignant pleural effusion and ascites, add physiological saline to the isolated human peripheral blood mononuclear cells, and centrifuge to obtain cell pellets.

[0008] Step 2: Isolate γδt cells from the cell pellets by immunomagnetic negative selection method, and resuspend the γδt cells with a pre-prepared first culture medium to obtain a γδt cell suspension.

[0009] Step 3: Add the γδt cell suspension to a T-175 culture flask coated with anti-PD-1 monoclonal antibody, add the pre-prepared second culture medium to the T-175 culture flask, and then add a TLR8 agonist with a concentration of 0.05 - 0.2 mg / mL at 1% - 3% of the cell volume.

[0010] Step 4: Place the T-175 culture flask in an environment of 3% - 7% CO 2 , 25 - 50 °C and culture for 2 - 4 days to obtain γδt cells co-cultured with anti-PD-1 monoclonal antibody and TLR8 agonist.

[0011] Further, Step 1 includes:

[0012] Perform a first centrifugation on the malignant pleural effusion and ascites, discard the supernatant to obtain a first pellet, resuspend the first pellet with 15 - 25 ml of physiological saline to obtain a first resuspension, filter the first resuspension and collect the filtrate.

[0013] Perform a second centrifugation on the filtrate, discard the supernatant to obtain a second pellet, and resuspend the second pellet with physiological saline to obtain a second resuspension.

[0014] Add 10 - 30 ml of human lymphocyte separation medium to a 50 ml centrifuge tube, slowly aspirate 10 - 30 ml of the second resuspension and add it to the upper layer of the human lymphocyte separation medium, then perform centrifugation, and use a Pasteur pipette to aspirate the PBMC cells in the white film layer into a new 50 ml centrifuge tube, and add physiological saline to obtain a third resuspension.

[0015] Perform a third centrifugation on the third resuspension, discard the supernatant to obtain cell pellets.

[0016] Further, Step 2 includes:

[0017] Resuspend the cell pellets with 2 - 10 ml of γδt cell sorting buffer to obtain a fourth resuspension, and use a Pasteur pipette to aspirate the fourth cell suspension and add it to a centrifuge tube containing magnetic bead particles.

[0018] Incubate the centrifuge tube in the refrigerator for 20 - 40 min, gently mix and shake it every 2 - 8 min. After the incubation, place the centrifuge tube in a magnetic rack and let it stand for 1 - 3 min.

[0019] Insert a Pasteur pipette vertically along the side wall without magnetic beads, aspirate the supernatant completely, and perform a fourth centrifugation on the supernatant. Discard the supernatant to obtain the third precipitate.

[0020] Resuspend the third precipitate with the first culture medium to obtain a γδt cell suspension.

[0021] Further, step 3 includes:

[0022] In a T-175 culture flask, add PBS and PD-1 monoclonal antibody solution at a volume ratio of 3-5:1, shake gently to allow the solution to spread on the bottom of the culture flask and cover the bottom, and let it stand overnight at 4°C.

[0023] Wash the bottom of the T-175 culture flask with PBS, and then add the γδt cell suspension to the T-175 culture flask coated with PD-1 monoclonal antibody.

[0024] Further, the first culture medium is 581 complete medium containing 100 IU / ml IL-2 and 3% human AB serum.

[0025] Further, after step 4, it further includes:

[0026] Step 5: In vitro expand the γδt cells co-cultured with the PD-1 monoclonal antibody-coated TLR8 agonist:

[0027] Within 0-3 days, the amount of TLR8 agonist added per day is 0.2-0.6 ml;

[0028] Within 3-5 days, the amount of TLR8 agonist added per day is 1.2 ml;

[0029] Within 5-7 days, the amount of TLR8 agonist added per day is 4 ml;

[0030] Within 7-9 days, the amount of TLR8 agonist added per day is 12 ml;

[0031] Within 9-11 days, the amount of TLR8 agonist added per day is 26 ml;

[0032] Within 11-13 days, the amount of TLR8 agonist added per day is 44 ml.

[0033] Further, the culture medium used for in vitro expansion is 581 complete medium.

[0034] In a second aspect, the present invention provides γδt cells cultured by the above-described method for culturing γδt cells.

[0035] Further, the killing efficiency of the γδt cells against K562 target cells is greater than or equal to 82.12%.

[0036] In a third aspect, the present invention provides the use of the γδ T cells according to the above in the preparation of a medicament for treating liver cancer.

[0037] Compared with the prior art, the present invention has the following advantages:

[0038] 1. By combining in vitro PD-1 monoclonal antibody coating and TLR8 agonist to reverse the immunosuppressive effect of γδ T cells, and then activating and massively amplifying γδ T cells in vitro, and taking advantage of the characteristic that γδ T cells can highly infiltrate into the tumor microenvironment, the killing effect on solid tumors is exerted, providing a new solution for using γδ T cells to treat tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is the in vitro amplification growth curve of γδ T cells in an embodiment of the present invention;

[0040] Figure 2 It is the culture diagram of γδ T cells in an embodiment of the present invention;

[0041] Figure 3 It is the flow cytometry phenotype detection result diagram of γδ T cells in an embodiment of the present invention;

[0042] Figure 4 It is the result diagram of detecting the levels of IFN-γ and TNF-α produced by γδ T cells in an embodiment of the present invention;

[0043] Figure 5 It is the in vitro killing ability detection result diagram of γδ T cells in an embodiment of the present invention;

[0044] Figure 6 It is the comparison diagram of the body shape and weight between the group receiving γδ T cell reinfusion and the control group after treating liver cancer mice in an embodiment of the present invention;

[0045] Figure 7 It is the comparison diagram of the tumor lesion size between the group receiving γδ T cell reinfusion and the control group after treating liver cancer mice in an embodiment of the present invention;

[0046] Figure 8 It is the comparison diagram of the number of lesion metastases between the group receiving γδ T cell reinfusion and the control group after treating liver cancer mice in an embodiment of the present invention;

[0047] The following specific embodiments will further illustrate the present invention in conjunction with the above drawings. DETAILED DESCRIPTION OF THE INVENTION

[0048] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein shall have the ordinary meanings understood by those of ordinary skill in the art to which the present invention pertains. The words such as "including" used herein are intended to mean that the elements or items appearing before this word cover the elements or items listed after this word and their equivalents, without excluding other elements or items.

[0049] The applicant found that: the expression of inhibitory receptors related to tumor-infiltrating γδ T cell immunosuppression was examined by fluorescence-activated cell sorting (FACS). The results showed that the inhibitory receptor PD-1 of γδ T cells was highly expressed, resulting in a decrease in the ability to produce IFN-γ and TNF-α. Therefore, the immunosuppression of γδ T cells can be reversed by regulating the inhibitory receptor, enhancing the anti-tumor potential of γδ T cells. In addition, the immunosuppressive activity of γδ T cells is closely related to human Toll-like receptor (TLR) 8. The TLR8 ligand polyguanylic acid (Poly-G) oligonucleotide can directly reverse the inhibitory function of CD4+CD25+ Treg cells, but it is still unclear whether the TLR8 ligand can reverse the inhibitory function of γδ T cells. It was determined by real-time PCR that TLR8 was highly expressed in γδ T cells, even 15 times higher than that in 293 cells. These research results provide new insights into regulating the immunosuppression of γδ T cells. Therefore, we hope to reverse the immunosuppressive effect of γδ T by combining in vitro PD-1 monoclonal antibody coating and TLR8 agonist, activate γδ T cells and expand them in large numbers, and then reinfuse them into the body to play an anti-tumor role, providing a new solution for using γδ T cells to treat tumors.

[0050] Example 1

[0051] Step S01: Collect the cells in malignant pleural and peritoneal effusions and isolate PBMC (human peripheral blood mononuclear cells)

[0052] Transfer the malignant pleural and peritoneal effusions of the collected solid tumor patients into 50-ml centrifuge tubes, with the volume in each tube not exceeding 45 ml. Then centrifuge at 1800 rpm, 4 °C, up9, down9 for 10 min. After centrifugation, discard the supernatant to obtain the first precipitate. Resuspend the first precipitate with 20 ml of normal saline to obtain the first resuspended solution. Filter the first resuspended solution through a filter membrane with a pore size of 70 μm to collect the filtrate, and centrifuge the filtrate at 1800 rpm, 4 °C, up9, down9 for 10 min. Discard the supernatant to obtain the second precipitate. Resuspend the second precipitate with 10 ml of normal saline, and count using a hematology analyzer. Adjust the cell density to 1*10^ 6 cells / ml.

[0053] Take 50-ml centrifuge tubes, and add 20 ml of human lymphocyte separation medium restored to room temperature to each tube to prepare Ficoll separation medium tubes (density: 1.077 g / ml). Use a pipette to slowly add 20 ml of the second resuspended solution to the upper layer of the Ficoll separation medium. Then centrifuge at 800 rpm, up7, down0 for 20 min. After centrifugation, use a Pasteur pipette to aspirate the PBMC cells in the white film layer into a new 50-ml centrifuge tube, add sufficient normal saline to obtain the third resuspended solution, and centrifuge at 1800 rpm, 4 °C, up9, down9 for 10 min. Discard the supernatant to obtain the cell precipitate.

[0054] Step S02: Immunomagnetic negative selection to isolate γδt cells

[0055] Resuspend the cell precipitate with 3 ml of γδt cell sorting buffer to obtain the fourth resuspended solution. Then take 300 μl of γδt magnetic bead particles into a 15-ml centrifuge tube, wash the magnetic beads with the sorting buffer, and use a Pasteur pipette to aspirate the fourth cell suspension and add it to the tube with the washed magnetic bead particles. Place it in the refrigerator for incubation for 30 min, and gently mix and shake every 5 min. After incubation, place the centrifuge tube on a magnetic rack and let it stand for 2 min. Vertically insert a Pasteur pipette along the side wall without magnetic beads and aspirate all the supernatant. At this time, the γδt cells are in the supernatant. Centrifuge the supernatant at 1800 rpm, 4 °C, up9, down9 for 10 min. Discard the supernatant to obtain the third precipitate. Resuspend the third precipitate with 581 complete medium (the first medium) containing 100 IU / ml IL-2 and 3% human AB serum to obtain the γδt cell suspension. Take 0.5 ml of the γδt cell suspension to count the cell density and viability, and adjust the cell density to 1*10^ 6 cells / ml.

[0056] Step S03: Coating of culture flasks

[0057] In a T-175 cm 2In a suspension culture flask, add 4 ml of PBS and 1 ml of PD-1 monoclonal antibody solution, and gently shake to allow the solution to spread on the bottom of the culture flask, covering the bottom completely. Let it stand overnight at 4 °C. Remove the coating solution, wash the bottom of the flask once with 10 ml of PBS, and add the γδt cell suspension to the T-175 culture flask coated with PD-1 monoclonal antibody.

[0058] Step S04: In vitro expansion culture of γδt cells

[0059] In the culture flask coated with the antibody, add 0.1 mg / mL TLR8 agonist at 2% of the cell volume. The operation should be gentle, and the pipette should not scratch the bottom of the flask. Place the T-175 culture flask in an environment of 3%-7% CO 2 25 - 50 °C and culture for 3 days. Detect the phenotype of γδt cells by flow cytometry, and then carry out in vitro expansion culture.

[0060] It should be noted that for in vitro expansion culture, within 0 - 3 days, the amount of TLR8 agonist added per day is 0.2 - 0.6 ml; within 3 - 5 days, the amount of TLR8 agonist added per day is 1.2 ml; within 5 - 7 days, the amount of TLR8 agonist added per day is 4 ml; within 7 - 9 days, the amount of TLR8 agonist added per day is 12 ml; within 9 - 11 days, the amount of TLR8 agonist added per day is 26 ml; within 11 - 13 days, the amount of TLR8 agonist added per day is 44 ml. In addition, the culture medium used for in vitro expansion is 581 complete medium, as shown in Table 1 below:

[0061] Table 1

[0062]

[0063]

[0064] It should also be noted that since the culture system is small within 0 - 7 days, culture flasks are selected. At 7 - 13 days, the bottles can no longer hold the cells, so culture bags are used instead. Since two culture bags are selected, the amount of TLR8 agonist added subsequently is half of the original amount, and 6 / Bag means adding 6 ml per bag.

[0065] As Figure 1 and Figure 2 shown, the initial cell number is 2 * 10^6 cells. After 13 days of expansion culture, the total cell number is 4 * 10^9 cells, with a 2000-fold expansion. The cells grow semi-adherently and partially in clusters, reflecting the change trend and growth characteristics of γδt cell expansion.

[0066] Test Example 1: Flow cytometry phenotype detection of γδt cells

[0067] When culturing to D5, take 6 ml of cell suspension and divide it equally into 6 flow cytometry tubes. Add 3 ml of normal saline, centrifuge at 1800 rpm for 5 min, Up 9 Down 9. After centrifugation, discard the supernatant, resuspend with 100 ul of normal saline, and add the corresponding antibodies. The grouping is as shown in Table 2 below:

[0068] Table 2

[0069]

[0070]

[0071] According to the above grouping, add the corresponding antibodies or proteins, incubate in the dark at room temperature for 15 min. After incubation, add 2 mL of PBS to mix the cells, centrifuge at 1800 rpm for 5 min, UP 9 Down 9. After centrifugation, discard the supernatant, resuspend the cell pellet with 100 ul of PBS, and perform on-machine detection. The results are as Figure 3 shown. The surface of γδT cells expresses CD3 and does not express CD4 and CD8, which is in line with the characteristics of γδt cells.

[0072] Test Example 2: Detection of the levels of IFN-γ and TNF-α produced by γδt cells using an ELISA kit

[0073] Take suspensions of γδt cells cultured by co-culturing with in vitro PD-1 monoclonal antibody coating + TLR8 agonist and normally cultured γδt cells (Con) with the same number of cells. Centrifuge both at 1800 rpm, 5 min, UP 9, Down 9. Collect the cell supernatants and use an ELISA kit to detect the levels of IFN-γ and TNF-α produced by γδt cells. Set standard wells, sample wells, and blank wells respectively. Add 50 ul of standards with different concentrations to the standard wells, and add 10 ul of supernatant and 40 ul of diluent to the sample wells. Except for the blank wells, add 100 ul of enzyme-labeled reagent to each well. Incubate at 37 °C for 1 hour, then wash all wells with washbuffer, and then add the chromogenic reagent. Develop color in the dark at 37 °C for 15 minutes. Finally, add the stop solution to each well, zero with the blank well, measure the OD value of each well at a wavelength of 450 nm, and calculate the concentrations of IFN-γ and TNF-α in the supernatant. The experimental results are as Figure 4 shown, and from Figure 4It can be seen that the average level of IFN-γ in the γδt cell suspension cultured by the combination of in vitro PD-1 monoclonal antibody coating and TLR8 agonist is 146 pg / ml, and the average level of TNF-a is 6.75 pg / ml. The average level of IFN-γ in the control group cell suspension is 118 pg / ml, and the average level of TNF-a is 6.4 pg / ml. Thus, it can be known that the γδt cells cultured by the combination of in vitro PD-1 monoclonal antibody coating and TLR8 agonist can significantly increase the levels of IFN-γ and TNF-a they secrete.

[0074] Test Example 3: Detection of in vitro killing ability of γδt cells

[0075] Using CellTrace TM Cell Proliferation Kits to label K562 target cells. After co-culturing with the γδt cells obtained in Example 1, use LIVE / DEAD TM Fixable Dead Cell Stain Kits to label dead cells. Analyze the killing effect of γδt cells on K562 target cells by flow cytometry. Flow analysis logic: The K562 spontaneous death group (Con) is used as the reference for setting the positive Cell Trace and positive LIVE / DEAD cell populations. First, the K562 spontaneous death group (Test) is gated to circle out the positive Cell Trace cell population, and the proportion of the positive LIVE / DEAD cell population is analyzed as the K562 spontaneous death ratio. Finally, the K562 spontaneous death ratio is deducted to obtain the final killing ratio. The results of the in vitro flow killing ability detection are as Figure 5 shown. The γδt cells cultured by the combination of in vitro PD-1 monoclonal antibody coating and TLR8 agonist for 13 days have an obvious killing effect on K562 target cells. After co-culturing for 2.5 hours, the killing efficiency on K562 target cells is 82.12%.

[0076] Test Example 4: Detection of in vivo anti-tumor ability

[0077] Purchase a C57 liver cancer orthotopic transplantation mouse model that can better simulate the human liver cancer microenvironment (injecting liver cancer cell cancer fluid or liver cancer tissue fragments into the liver of mice to form tumors), and divide them into two groups, with 5 mice in each group. The experimental group is intravenously injected with γδt cells cultured by the combination of PD-1 monoclonal antibody coating and TLR8 agonist obtained in Example 1 at a dose of 1*10^7 cells / kg, and the control group (Con) is intravenously injected with ordinary γδt cells at a dose of 1*10^7 cells / kg. Three months after the infusion, anatomical sampling is performed. The average body weight of the experimental group mice is 22.3 g, and the average body weight is significantly higher than that of the control group (18.2 g) ( Figure 6 ), and the average tumor size of the experimental group mice is 0.24 cm 2, the average tumor size was significantly lower than that of the control group (0.61 cm 2 )( Figure 7 ). The average number of metastatic lesions in the experimental group was 1.8, and the average number of metastatic lesions was significantly lower than that of the control group (3.1)( Figure 8 ).

[0078] In summary, by coating with anti-PD-1 monoclonal antibody in vitro + using TLR8 agonist in combination to reverse the immunosuppressive effect of γδt, then activating and amplifying γδt cells in large numbers in vitro, and taking advantage of the property that γδt cells can highly infiltrate into the tumor microenvironment, the killing effect on solid tumors is exerted, providing a new solution for using γδt cells to treat tumors.

[0079] Although the embodiments of the present invention have been described in detail above, it is obvious to those skilled in the art that various modifications and changes can be made to these embodiments. However, it should be understood that such modifications and changes are all within the scope and spirit of the present invention described in the claims. Moreover, the present invention described herein can have other embodiments and can be implemented or realized in various ways.

Claims

1. A method for culturing γδt cells, characterized in that: The method comprises: Step 1, collecting malignant pleural effusion and ascites from tumor patients, separating human peripheral blood mononuclear cells from the malignant pleural effusion and adding physiological saline to the separated human peripheral blood mononuclear cells, centrifuging and obtaining cell precipitates; Step 2, separating γδt cells from the cell precipitate by immunomagnetic negative selection, and resuspending the γδt cells in a pre-configured first culture medium to obtain a γδt cell suspension; Step 3, adding the γδt cell suspension to the PD-1 monoclonal antibody-coated T-175 culture flask, adding the pre-prepared second culture medium to the T-175 culture flask, and then adding 0.05-0.2 mg / mL of TLR8 agonist at 1%-3% of the cell volume; Step 4: Place the T-175 culture flask in an environment of 3%-7% CO2 and 25-50°C for 2-4 days to obtain γδt cells cultured in combination with PD-1 monoclonal antibody-coated TLR8 agonist.

2. The method for culturing γδt cells according to claim 1, characterized in that: Step 1 includes: Performing a first centrifugation on the malignant pleural and ascites fluid, discarding the supernatant to obtain a first precipitate, resuspending the first precipitate with 15-25 ml of physiological saline to obtain a first resuspension, filtering the first resuspension and collecting the filtrate; The filtrate is subjected to a second centrifugation treatment, the supernatant is discarded to obtain a second precipitate, and the second precipitate is resuspended with physiological saline to obtain a second resuspended liquid; Add 10-30 ml of human lymphocyte separation solution to a 50 ml centrifuge tube, and slowly add 10-30 ml of the second resuspension solution to the upper layer of the human lymphocyte separation solution, then centrifuge, and use a Pasteur pipette to draw the PBMC cells in the buffy coat layer into a new 50 ml centrifuge tube, add physiological saline, and obtain a third resuspension solution; The third resuspension was subjected to a third centrifugation treatment, and the supernatant was discarded to obtain a cell pellet.

3. The method for culturing γδt cells according to claim 1, characterized in that: Step 2 includes: Resuspend the cell pellet with 2-10 ml of γδt cell sorting buffer to obtain a fourth resuspension, and use a Pasteur pipette to draw the fourth cell suspension and add it to the centrifuge tube containing the magnetic bead particles; Refrigerate and incubate the centrifuge tube for 20-40 minutes, gently mix and shake every 2-8 minutes. After the incubation, place the centrifuge tube in a magnetic rack and let it stand for 1-3 minutes. Insert a Pasteur pipette vertically along the side wall without magnetic beads to suck out the supernatant, and perform a fourth centrifugation on the supernatant, discard the supernatant, and obtain a third precipitate; The third pellet is resuspended with the first culture medium to obtain a γδt cell suspension.

4. The method for culturing γδt cells according to claim 1, characterized in that: Step 3 includes: In a T-175 culture flask, add PBS and PD-1 monoclonal antibody solution at a volume ratio of 3-5:1, shake gently to allow the solution to spread on the bottom of the culture flask and cover the bottom of the flask, and let it stand overnight at 4°C; The bottom of the T-175 culture flask was washed with PBS, and then the γδt cell suspension was added to the T-175 culture flask coated with PD-1 monoclonal antibody.

5. The method for culturing γδt cells according to claim 3, characterized in that: The first culture medium is a 581 complete culture medium containing 100 IU / ml IL-2 and 3% human AB serum.

6. The method for culturing γδt cells according to claim 1, characterized in that: After step 4, also include: Step 5: Coat the PD-1 monoclonal antibody with the TLR8 agonist and culture the γδt cells in vitro for expansion: During days 0-3, the amount of TLR8 agonist added daily was 0.2-0.6 ml; During 3-5 days, the amount of TLR8 agonist added daily was 1.2 ml; During 5-7 days, the amount of TLR8 agonist added daily was 4 ml; During the 7-9 days, the amount of TLR8 agonist added daily was 12 ml; During days 9-11, the amount of TLR8 agonist added daily was 26 ml; During days 11-13, the amount of TLR8 agonist added per day was 44 ml.

7. The method for culturing γδt cells according to claim 6, characterized in that: The culture medium used for in vitro expansion was 581 complete medium.

8. A γδt cell cultured according to the γδt cell culture method according to any one of claims 1 to 7.

9. The γδt cell according to claim 8, characterized in that The killing efficiency of the γδt cells on K562 target cells is greater than or equal to 82.12%.

10. Use of the γδt cells according to claim 8 in preparing a drug for treating liver cancer.

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