Gamma delta T cell efficient amplification method and application

Abstract

The invention discloses a gamma delta T cell efficient amplification method and application, and belongs to the technical field of cell biology. The amplification method comprises the following steps: separating PBMC (peripheral blood mononuclear cells) by adopting a density gradient centrifugation method, carrying out initial culture through a polylysine coated container, carrying out three-stage dynamic stimulation, combining with an X-VIVO 15 culture medium of 5-8% autoserum, and finally carrying out anti-gamma delta TCR magnetic bead separation to obtain high-purity cells. Through collaborative optimization of stepped factor combination, staged container coating and a low-serum system, the amplification multiple of the gamma delta T cells reaches 150-180 times, the purity is larger than or equal to 90% after purification, the cytotoxicity and the survival ability are remarkably improved, the gamma delta T cells can be efficiently used for immunotherapy of tumors and infectious diseases, and stable technical support is provided for clinical transformation of the gamma delta T cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cell biology, in particular to a method for efficient expansion of γδ T cells and application thereof. BACKGROUND

[0002] γδ T cells are a special subset of T lymphocytes, whose T cell receptor (TCR) is composed of γ chain and δ chain. Unlike traditional αβ T cells, γδ T cells do not require processing by antigen presenting cells and major histocompatibility complex (MHC) restriction, and can directly recognize and kill tumor cells, virus-infected cells and other abnormal cells, and play an important role in immune surveillance and immune defense of the body.

[0003] The content of γδ T cells in peripheral blood is low, accounting for only 1-5% of the total number of T cells, which is difficult to meet the needs of clinical treatment, and thus needs to be expanded in vitro. At present, the commonly used method for expansion of γδ T cells mainly uses zoledronate combined with IL-2 stimulation, but there are problems such as low expansion multiple, low purity, insufficient cell activity and cytotoxicity, which limit its clinical application.

[0004] Therefore, it is of great clinical significance and application value to develop a method for efficiently expanding γδ T cells while ensuring high purity, high activity and strong cytotoxicity. SUMMARY

[0005] The present application aims to provide a method for efficient expansion of γδ T cells and application thereof, so as to overcome the defects of low expansion efficiency, low purity, insufficient activity and cytotoxicity of γδ T cells in the prior art.

[0006] To achieve the above-mentioned purpose, the present application provides a method for efficient expansion of γδ T cells, comprising the following steps: S1, isolation of peripheral blood mononuclear cells (PBMCs): collecting peripheral blood, and separating PBMCs by density gradient centrifugation; S2, initial culture: inoculating the PBMCs obtained in S1 into a culture flask coated with polylysine, adding X-VIVO 15 medium containing 5-8% autologous serum, adjusting the cell density to 1-2×10 6 / mL, and placing in a 37℃, 5% CO2 incubator for culture for 24-48h; S3, stepwise stimulation culture: S31, first stimulation: after culture for 24-48h, adding zoledronate to a final concentration of 5-10μM and IL-2 to a final concentration of 500-800IU / mL to the culture system, and continuing to culture for 3-4 days; S32, secondary stimulation: after the first stimulation, the cells were collected by centrifugation, resuspended in X-VIVO 15 medium containing 5-8% autologous serum, and adjusted to a cell density of 0.5-1 x 10 6 6 / mL, IL-15 was added to a final concentration of 10-20 ng / mL, IL-21 was added to a final concentration of 5-10 ng / mL, and the cells were transferred to culture bottles coated with anti-CD3 monoclonal antibody and cultured for 4-5 days. S33, tertiary stimulation: after the secondary stimulation, the cells were collected by centrifugation, resuspended in X-VIVO 15 medium containing 5-8% autologous serum, and adjusted to a cell density of 0.3-0.5 x 10 6 6 / mL, IL-2 was added to a final concentration of 300-500 IU / mL, IL-15 was added to a final concentration of 5-10 ng / mL, and the cells were cultured for 3-4 days. S4, purification: after the S3 culture, high-purity γδ T cells were obtained by magnetic bead sorting with anti-γδ TCR monoclonal antibody.

[0007] Preferably, in S1, the centrifugation liquid used in the density gradient centrifugation method is Ficoll-Hypaque, and the centrifugation conditions are 2000 rpm for 20 min.

[0008] Preferably, in S2, the specific operation of polylysine coating is as follows: 0.01% polylysine solution is added to the culture bottle to cover the bottom of the bottle, and after standing at room temperature for 30 min, the solution is discarded, and the bottle is washed with sterile PBS for 2-3 times and dried for standby.

[0009] Preferably, in S32, the specific operation of anti-CD3 monoclonal antibody coating is as follows: the anti-CD3 monoclonal antibody is diluted with PBS to 5-10 μg / mL, added to the culture bottle to cover the bottom of the bottle, and placed at 4°C overnight, then the solution is discarded, and the bottle is washed with sterile PBS for 2-3 times and dried for standby.

[0010] Preferably, in S3, during each stimulation and culture, when the culture medium turns yellow or the cell density exceeds 2 x 10 6 6 / mL, fresh culture medium is supplemented to maintain the cell density at 0.3-2 x 10 6 6 / mL.

[0011] Preferably, in S4, the purity of γδ T cells after magnetic bead sorting with anti-γδ TCR monoclonal antibody is ≥ 90%.

[0012] In another aspect, the present application also provides a γδ T cell obtained by the above method.

[0013] In another aspect, the present application also provides the use of the above-mentioned γδ T cell in the preparation of a tumor treatment drug.

[0014] In another aspect, the present application also provides a use of the above-mentioned γδ T cell in the preparation of a medicament for treating infectious diseases.

[0015] In another aspect, the present application also provides a medicament for treating tumors, comprising the above-mentioned γδ T cell and a pharmaceutically acceptable carrier.

[0016] Therefore, the present application has the following beneficial effects. (1) The efficiency of expansion is significantly improved: the three-stage dynamic stimulation strategy combined with the stepwise cytokine combination makes the expansion multiple of γδ T cells reach 100-200 times, which is 3-4 times higher than the traditional single-factor stimulation method (30-50 times), and the logarithmic growth period is extended to 8-9 days, solving the problem that the cells easily enter the plateau phase in the late stage of cell proliferation in the prior art.

[0017] (2) The purity of cells is greatly improved: through the synergistic effect of initial coating with polylysine and stage cross-linking with anti-CD3 monoclonal antibody, combined with anti-γδTCR magnetic bead sorting, the purity of cells after purification is ≥90%, which is significantly higher than that of the traditional method (70-80%), and the impurity cell pollution caused by heterologous serum is avoided.

[0018] (3) The function and survival ability of cells are enhanced: the low serum (5-8% autologous serum) culture system reduces the inhibitory components, combined with precise cytokine time sequence regulation, so that the killing rate of expanded cells to tumor cells reaches 75%-82% (about 50% by traditional method), and the secretion amount of IFN-γ is increased by 2.5 times; at the same time, the in vitro survival time of cells is prolonged to more than 14 days, and the apoptosis rate is reduced to 10%-12%, solving the defect that the cell activity decreases with the culture time in the prior art.

[0019] (4) The operation stability and clinical applicability are excellent: the whole process of the method adopts standardized operation, the difference coefficient between donors is ≤15%, and the use of heterologous serum is avoided, reducing the risk of immune rejection, and being easy to scale production, which provides efficient and stable technical support for the clinical application of γδ T cells in tumor immunotherapy and treatment of infectious diseases.

[0020] The technical solutions of the present application are further described in detail through the following examples. DETAILED DESCRIPTION

[0021] The technical solutions of the present application are further described in detail through the following examples.

[0022] In order to make the purposes, technical solutions and advantages of the present application more clear, thorough and complete, the technical solutions of the present application are described clearly and completely by examples below. The following detailed descriptions are the descriptions of examples, which are intended to provide further detailed descriptions of the present application. Unless otherwise specified, all technical terms used in the present application have the same meanings as generally understood by those skilled in the art to which the present application belongs.

[0023] The instrument equipment and reagent materials used in the examples are obtained through commercial channels.

[0024] Example 1 A method for efficient expansion of γδ T cells, comprising the following steps: S1, isolation of PBMC: take 50 mL of peripheral blood, dilute with an equal amount of sterile PBS, slowly stack on the upper layer of 20 mL Ficoll-Hypaque centrifugal liquid, centrifuge at 2000 rpm for 20 min. After centrifugation, the middle white membrane layer is aspirated and washed with sterile PBS twice (1500 rpm, 10 min) to obtain PBMC.

[0025] S2, initial culture: resuspend PBMC with X-VIVO 15 medium containing 5% autologous serum, adjust the cell density to 1×10 6 6 / mL, inoculate into a T25 culture flask coated with polylysine, and incubate in a 37℃, 5% CO2 incubator for 48 h.

[0026] Polylysine coating operation: add 0.01% polylysine solution to the T25 culture flask to cover the bottom of the flask, place at room temperature for 30 min, then discard the solution, wash with sterile PBS for 3 times, and dry for standby.

[0027] S3, stepwise stimulation culture: S31, first stimulation: after 48 h of culture, add zoledronic acid to a final concentration of 5 μM and IL-2 to a final concentration of 500 IU / mL to the culture flask, and continue to culture for 3 days.

[0028] S32, second stimulation: after the first stimulation, centrifuge at 1500 rpm for 10 min to collect the cells, resuspend with X-VIVO 15 medium containing 5% autologous serum, adjust the cell density to 0.5×10 6 6 / mL, add IL-15 to a final concentration of 10 ng / mL and IL-21 to a final concentration of 5 ng / mL, transfer to a T75 culture flask coated with anti-CD3 monoclonal antibody, and continue to culture for 4 days.

[0029] Anti-CD3 monoclonal antibody coating operation: anti-CD3 monoclonal antibody was diluted with PBS to 5 μg / mL, added to a T75 culture flask to cover the bottom of the flask, and placed at 4°C overnight. The solution was discarded, and the flask was washed with sterile PBS three times and air-dried for standby use.

[0030] S33, three times of stimulation: after the second stimulation, the cells were collected by centrifugation at 1500 rpm for 10 min, resuspended with X-VIVO 15 medium containing 5% autologous serum, and the cell density was adjusted to 0.3×10 6 IU / mL, IL-15 to a final concentration of 5 ng / mL, and cultured for 3 days.

[0031] During the culture process, the cell state was observed every day, and when the culture medium turned yellow or the cell density exceeded 2×10 6 IU / mL, fresh medium was supplemented.

[0032] S4, purification: after the culture ended, the cells were collected and sorted by anti-γδTCR monoclonal antibody magnetic beads according to the instructions to obtain γδT cells.

[0033] Example 2 A method for efficient expansion of γδT cells, comprising the following steps: S1, isolation of PBMC, the specific method is the same as that in Example 1, and PBMC is obtained.

[0034] S2, initial culture: PBMC was resuspended with X-VIVO 15 medium containing 5% autologous serum, and the cell density was adjusted to 1×10 6 IU / mL, inoculated into a T25 culture flask coated with polylysine, and cultured in a 37°C, 5% CO2 incubator for 48 h.

[0035] Polylysine coating operation: 0.01% polylysine solution was added to a T25 culture flask to cover the bottom of the flask, and the flask was placed at room temperature for 30 min. The solution was discarded, and the flask was washed with sterile PBS three times and air-dried for standby use.

[0036] S3, stepwise stimulation culture: S31, first stimulation: after 48 h of culture, zoledronate was added to a final concentration of 5 μM, and IL-2 was added to a final concentration of 800 IU / mL, and the culture was continued for 3 days.

[0037] S32, second stimulation: after the first stimulation, the cells were collected by centrifugation at 1500 rpm for 10 min, resuspended with X-VIVO 15 medium containing 5% autologous serum, and the cell density was adjusted to 0.5×10 6Add IL-15 to a final concentration of 20 ng / mL and IL-21 to a final concentration of 10 ng / mL, then transfer to a T75 culture flask coated with anti-CD3 monoclonal antibody and continue culturing for 4 days.

[0038] Anti-CD3 monoclonal antibody coating procedure: Dilute the anti-CD3 monoclonal antibody to 5 μg / mL with PBS, add it to a T75 culture flask, cover the bottom of the flask, incubate at 4°C overnight, discard the solution, wash 3 times with sterile PBS, and air dry for later use.

[0039] S33. Triple stimulation: After the second stimulation, cells were collected by centrifugation at 1500 rpm for 10 min, resuspended in X-VIVO 15 medium containing 5% autologous serum, and the cell density was adjusted to 0.3 × 10⁻⁶ cells / min. 6 Add IL-2 to a final concentration of 500 IU / mL and IL-15 to a final concentration of 10 ng / mL, and continue culturing for 3 days.

[0040] During the culture process, observe the cell status daily. The cells should be identified when the culture medium turns yellow or the cell density exceeds 2 × 10⁻⁶. 6 When the number of cells / mL reaches a certain level, replenish with fresh culture medium.

[0041] S4. Purification: After culture, collect the cells and sort them using magnetic beads containing anti-γδTCR monoclonal antibody. Follow the instructions for specific procedures to obtain γδT cells.

[0042] Comparative Example 1 A method for expanding γδT cells includes the following steps: S1, PBMC isolation: The density gradient centrifugation operation was the same as in Example 1. After obtaining PBMC, it was resuspended in RPMI-1640 medium containing 10% fetal bovine serum (FBS).

[0043] S2. Culture system: Use uncoated T25 culture flasks, inoculation density 1×10⁻⁶. 6 Cells / mL, culture medium containing 10% FBS, 100U / mL penicillin-streptomycin, and 2mM L-glutamine, cultured at 37℃ and 5% CO2.

[0044] S3. Stimulation method: Zoledronic acid 5μM + IL-2 300IU / mL was added at the start of culture. The culture medium type and culture container were not changed throughout the entire process. When the cell density exceeded 2×10⁻⁶, the stimulation was initiated. 6 When the number of cells / mL is reached, only an equal volume of RPMI-1640 medium containing the same factor is added, and the cells are cultured continuously for 10 days.

[0045] S4. Purification steps: Same as the magnetic bead sorting method in Example 1, but sorting is performed on the 10th day of culture.

[0046] Comparative Example 2 A method for expanding γδT cells includes the following steps: S1. PBMC isolation and initial culture: Same as the steps in Example 1.

[0047] S2. Stimulation method: From day 1 of culture, a mixed factor (zoledronic acid 5 μM + IL-2 500 IU / mL + IL-15 10 ng / mL) was continuously added. The same polylysine-coated culture flask was used throughout the process. No container replacement or factor combination adjustment was performed for secondary stimulation. Fresh culture medium was added according to the density standard of Example 1 during the culture period. The total culture time was 10 days.

[0048] S3. Purification steps: Same as in Example 1.

[0049] Comparative Example 3 A method for expanding γδT cells includes the following steps: S1. PBMC isolation and initial culture: Same as the steps in Example 1.

[0050] S2. Culture container treatment: Culture flasks coated with 0.01% poly-L-lysine were used throughout the process (the same coating method was used when changing from T25 to T75), and no anti-CD3 monoclonal antibody coating treatment was performed.

[0051] S3. Stimulation protocol: Strictly follow the three-stage factor addition sequence of Example 1 (zoledronic acid 5μM + IL-2 500IU / mL → IL-15 10ng / mL + IL-2 1 5ng / mL → IL-2 300IU / mL + IL-15 5ng / mL), and the cell density control and culture medium replenishment method are the same as in Example 1.

[0052] S4. Purification steps: Same as in Example 1.

[0053] Test Example 1 Amplification efficiency detection: Cell counts were monitored daily using a Countstar cell counter to calculate fold expansion, and the duration of the logarithmic growth phase was determined by daily cell density monitoring. The results are shown in Table 1 below. Table 1 Amplification Efficiency

[0054] The results showed that the amplification fold of the embodiment of the present invention was significantly higher than that of the comparative example. p <0.01), among which Example 2, which used a higher concentration of factor combination, had the best amplification effect, indicating that the stepwise high concentration factor combination can effectively prolong the logarithmic growth phase of cells.

[0055] Test Example 2 Purity testing: Flow cytometry assay: Cells were labeled with anti-γδTCR-PE and anti-CD3-FITC antibodies, and γδTCR was analyzed by flow cytometry. + CD3 + The percentage of double-positive cells was calculated, and the purity before and after purification was determined. The results are shown in Table 2 below. Table 2 Purity Testing

[0056] The results showed that the purity of the purified samples in the embodiments of the present invention all exceeded 90%, which was significantly higher than that of the comparative example. p <0.01), demonstrating that staged cross-linked antibody coating can effectively enrich γδT cells.

[0057] Test Example 3 Cytotoxicity test: LDH release assay: Expanded cells were co-cultured with HepG2 cells at a 20:1 effector-to-target ratio for 4 h. The killing rate was calculated by detecting LDH activity in the supernatant. The amount of IFN-γ secreted in the culture supernatant was determined by ELISA. The results are shown in Table 3 below. Table 3 Cytotoxicity Detection

[0058] The results showed that the cytotoxicity of the example was significantly higher than that of the control group ( p The cell count is <0.01), and the cell secretion capacity is stronger, indicating that the cell function expanded by the method of the present invention is superior.

[0059] Test Example 4 Cell viability assay: CFSE labeling method: Cells were cultured for 14 days after CFSE staining, and the proportion of viable cells was detected by flow cytometry (CFSE). + PI - ) and apoptosis rate (Annexin V) + PI + The results are shown in Table 4 below: Table 4 Cell viability test

[0060] The results showed that the cell survival time was significantly prolonged and the apoptosis rate was reduced in the embodiments of the present invention, proving that stepwise stimulation can effectively maintain cell viability.

[0061] Therefore, the present invention provides a method and application for efficient expansion of γδT cells. Through the synergistic effect of a stepwise combination of stimulating factors, staged cross-linking coating, and optimization of a low serum system, the method achieves efficient expansion of γδT cells. The expansion fold, purity, cytotoxicity, and survival ability are significantly superior to existing technologies.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for efficient expansion of γδT cells, characterized in that, Includes the following steps: S1. Isolation of peripheral blood mononuclear cells (PBMCs): Peripheral blood was collected, and PBMCs were isolated using density gradient centrifugation. S2, Initial Culture: The PBMCs obtained in S1 were seeded into poly-L-lysine-coated culture flasks, and X-VIVO 15 medium containing 5-8% autologous serum was added to adjust the cell density to 1-2 × 10⁶ cells / year. 6 The cells / mL were incubated at 37℃ in a 5% CO2 incubator for 24-48 hours. S3, Stepwise Stimulation Culture: S31. Initial stimulation: After culturing for 24-48 hours, add zoledronic acid to the culture system to a final concentration of 5-10 μM and IL-2 to a final concentration of 500-800 IU / mL, and continue culturing for 3-4 days. S32. Secondary stimulation: After the first stimulation, collect the cells by centrifugation, resuspend them in X-VIVO 15 medium containing 5-8% autologous serum, and adjust the cell density to 0.5-1×10⁶ cells / year. 6 Add IL-15 to a final concentration of 10-20 ng / mL and IL-21 to a final concentration of 5-10 ng / mL, then transfer to a culture flask coated with anti-CD3 monoclonal antibody and continue culturing for 4-5 days; S33. Triple stimulation: After the second stimulation, collect the cells by centrifugation, resuspend them in X-VIVO 15 medium containing 5-8% autologous serum, and adjust the cell density to 0.3-0.5 × 10⁶ cells / year. 6 Add IL-2 to a final concentration of 300-500 IU / mL and IL-15 to a final concentration of 5-10 ng / mL, and continue culturing for 3-4 days; S4. Purification: After S3 culture, high-purity γδT cells were obtained by magnetic bead sorting using anti-γδTCR monoclonal antibody.

2. The method for high-efficiency expansion of γδT cells according to claim 1, characterized in that: In S1, the density gradient centrifugation method used Ficoll-Hypaque as the centrifuge fluid, and the centrifugation conditions were 2000 rpm and 20 min.

3. The method for high-efficiency expansion of γδT cells according to claim 1, characterized in that, In S2, the specific procedure for poly-L-lysine coating is as follows: add 0.01% poly-L-lysine solution to the culture flask, cover the bottom of the flask, place at room temperature for 30 minutes, discard the solution, wash 2-3 times with sterile PBS, and air dry for later use.

4. The method for high-efficiency expansion of γδT cells according to claim 1, characterized in that, In S32, the specific procedure for coating with anti-CD3 monoclonal antibody is as follows: dilute the anti-CD3 monoclonal antibody with PBS to 5-10 μg / mL, add it to the culture flask, cover the bottom of the flask, place it at 4°C overnight, discard the solution, wash 2-3 times with sterile PBS, and air dry for later use.

5. The method for high-efficiency expansion of γδT cells according to claim 1, characterized in that, In S3, during each stimulation culture process, when the culture medium turns yellow or the cell density exceeds 2 × 10⁻⁶, 6 When the cell density reaches 0.3-2 × 10⁶ cells / mL, replenish with fresh culture medium to maintain a cell density of 0.3-2 × 10⁶ cells / mL. 6 per mL.

6. The method for high-efficiency expansion of γδT cells according to claim 1, characterized in that, In S4, the purity of γδT cells after sorting with anti-γδTCR monoclonal antibody magnetic beads is ≥90%.

7. A type of γδT cell amplified by the method according to any one of claims 1-6.

8. The use of the γδT cells according to claim 7 in the preparation of tumor therapeutic drugs.

9. The use of the γδT cells according to claim 7 in the preparation of drugs for treating infectious diseases.

10. A tumor treatment drug, characterized in that: It comprises the γδT cells as described in claim 7 and a pharmaceutically acceptable carrier.

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