CN109251891B

CN109251891B - Method for expanding PBMC by combining CD40 with PD-L1 and cytokines

Info

Publication number: CN109251891B
Application number: CN201811107312.7A
Authority: CN
Inventors: 古彦铮; 张学光; 黄子逸; 刘翠平
Original assignee: First Affiliated Hospital of Suzhou University
Current assignee: First Affiliated Hospital of Suzhou University
Priority date: 2018-09-21
Filing date: 2018-09-21
Publication date: 2021-10-22
Anticipated expiration: 2038-09-21
Also published as: CN109251891A

Abstract

The invention discloses a method for expanding PBMC by combining CD40 with PD-L1 and cytokines, which comprises the following steps: 1) taking normal human peripheral blood, mixing and diluting with PBS, and then carrying out Ficoll density gradient centrifugation; 2) after the centrifugation is finished, 4 layers of liquid are obtained; sucking the middle haze layer cells with a pipetteWashing and centrifuging with 10 times volume of PBS to obtain PBMC; 3) preparing PBMC cells into cell suspension by using an RMPI1640 complete culture medium, transferring the cell suspension into a 6-well plate and a 24-well plate, and then adding corresponding cytokine liquid into each group of cell culture liquid: placing the orifice plate in the CO₂Culturing in an incubator; 4) and supplementing the cell factor solution every 2 to 3 days according to the cell state. The invention provides a novel in vitro induction scheme of adoptive immune cells, which is used as an entry point for preparing novel adoptive immune cells by expanding PBMCs (peripheral blood mononuclear cells) through the combination of CD40 signals and PD-L1 as well as cytokines IL-15 and IL-7. In addition, the method provided by the invention has the advantages of low requirement on experimental equipment, simple and time-saving operation, high adoptive immune cell collection rate and high purity.

Description

Method for expanding PBMC by combining CD40 with PD-L1 and cytokines

Technical Field

The application relates to the technical field of biology, in particular to a method for expanding PBMC by combining CD40 with PD-L1 and cytokines.

Background

Malignant tumors are one of the important diseases that seriously endanger human health. At present, surgery, radiotherapy and chemotherapy are still the main treatment methods. But the recurrence rate and mortality rate are still high. Adoptive cell immunotherapy (ACT) is becoming a new treatment means for treating malignant tumor after radiotherapy and chemotherapy at present, and is mainly infused to tumor patients through sensitized lymphocytes (with specific immunity) to ensure that the tumor patients obtain anti-tumor immunity.

The effector cells of adoptive immunotherapy have heterogeneity, such as cytotoxic T Cells (CTL), natural killer cells (NK), macrophages, lymphokine-activated killer cells (LAK), and tumor-infiltrating lymphocytes (TIL), all play a role in tumor cell killing.

Peripheral Blood Mononuclear Cells (PBMC) refer to cells having a mononuclear nucleus in peripheral blood, including lymphocytes, monocytes, dendritic cells and other small numbers of cells (hematopoietic stem cells, etc.).

A novel adoptive immune cell therapy technique is to co-culture human peripheral blood mononuclear cells in vitro with various cytokines (CD3McAb, IL-2, IFN-gamma, IL-1 alpha, etc.) for a period of time to obtain a population of CD3⁺CD56⁺、CD3⁺CD8⁺The heterogeneous cell group mainly containing cells has the advantages of high proliferation speed, high tumor killing activity, high tumor killing spectrum, sensitivity to multi-drug resistant tumor cells, killing effect on G0 phase cells, no influence of immunosuppressive agents such as surimin A (CsA) and FKSO6 on the tumor killing activity, low toxicity to normal bone marrow hematopoietic precursor cells, and resistance to apoptosis of effector cells induced by the tumor cells through Fas-FasL molecular pathway.

The conventional novel adoptive immune cell therapy induced by CD3McAb combined with cytokines IL-2, IFN-gamma and IL-1 alpha still has a plurality of problems to be solved: since proliferation of late T cells is maintained with IL-2, the proportion of the negative co-stimulatory molecule PD-1/PD-L1 in IL-2-induced lymphocytes is quite high. And the application of large doses of IL-2 induces a certain number of regulatory T cells (tregs), which may be a key factor affecting the efficacy of novel adoptive immune cells.

Disclosure of Invention

The main objective of the present application is to provide a novel method for expanding PBMCs with CD40 in combination with PD-L1 and cytokines. The method provided by the present invention is an in vitro induction method of adoptive immune cells, and is not intended for in vivo studies, nor is it intended for the direct treatment of diseases.

Negative co-stimulatory molecules play an important role in T cell-mediated tumor immune escape through T cell expression. PD-1 is a 55KD transmembrane protein consisting of an extracellular IgV-like motif, a transmembrane region, an intracellular ITIM (immunoglobulin receptor type-based inhibition motif) and an ITSM (immunoglobulin receptor type-based switch motif) motif. It is expressed in T cells, B cells, thymocytes, etc., and up-regulates expression upon cell activation. After PD-1 is combined with a ligand PD-L1 thereof, T cell apoptosis and failure, T cell tolerance, cytokine secretion change, proliferation weakening and cytotoxicity reduction of effector T cells can be caused, and effective anti-tumor immune response can not be generated, so that the tumor cells have immune escape.

Blocking the PD-L1/PD-1 signaling pathway may partially restore the function of tumor-specific T lymphocytes. Therefore, the tumor-derived T cell response is enhanced by blocking a PD-1/PD-L1 signal transduction pathway, so that the effect of killing tumor cells can be achieved, and the method becomes an important means for reversing tumor immune escape.

Therefore, the main research content of the invention is as follows: 1. CD40 signal was combined with a comparative study of the cellular biology of adoptive immune cells induced by PD-L1 and cytokines IL-15 and IL-7 and conventional novel adoptive immune cells. The PBMC of normal people is amplified through the combination of two groups of cytokines, the vitality of induced cells, the activation and the proliferation of cells, the expression difference of lymphocyte subsets, the expression difference of negative co-stimulatory molecules PD-1, Tim-3 and the like in lymphocytes and the expression of IFN-gamma in regulatory T cells and memory T cells are researched. 2. CD40 signal in combination with IFN-alpha, IL-7 induced adoptive immune cells and conventional novel adoptive immune cells in vitro anti-tumor effect comparative study. Mitomycin is used for treating several different tumor cell strains, two groups of adoptive immune cells are placed in a 96-well plate according to an effect-target ratio gradient for culture, and after 3 days, CCK-8 is added for detection.

Specifically, the method for expanding PBMCs by combining CD40 with PD-L1 and cytokines comprises the following steps:

1) taking normal human peripheral blood, mixing and diluting with PBS, and then carrying out Ficoll density gradient centrifugation;

2) after the centrifugation is finished, 4 layers of liquid are obtained; sucking the middle haze layer cells by a pipette, washing and centrifuging by using PBS with 10 times of volume, and finally obtaining PBMC; (the 4 layers of liquid are platelets, mononuclear cells, granulocytes and erythrocytes respectively, and the middle cloud layer is a mononuclear cell layer.)

3) Preparing a cell suspension from PBMC cells by using an RMPI1640 complete culture medium, then transferring the cell suspension into a 6-well plate and a 24-well plate, and then adding corresponding cytokine liquid into each group of cell culture liquid: the orifice plate was then placed in the CO₂Culturing in an incubator;

4) and supplementing the cell factor solution every 2 to 3 days according to the cell state.

In the above steps, the cell culture solution is added with corresponding cell factor solution, and the process is as follows: the CD3 challenge group was initiated by adding IFN-. gamma.at day 0 (1000U/ml), 24h later by adding IL-2 (1000U/ml), IL-1. alpha. (100U/ml) and anti-CD 3mAb (100 ng/ml).

The CD40 challenge group was initially, i.e., day 0, supplemented with 5C11 (2. mu.g/ml), PD-L1 (1. mu.g/ml), anti-CD 3mAb (30 ng/ml), IL-2 (300U/ml), IL-7 (5 ng/ml), IL-15 (5 ng/ml).

Furthermore, when cytokine solution was added, the CD3 stimulated group was supplemented with IL-2 alone (1000U/ml).

When cytokine solution was added, the CD40 stimulated group, which was supplemented with IL-2 (200U/ml), IL-7 (2 ng/ml), and IL-15 (2 ng/ml).

Specifically, the Ficoll density gradient centrifugation is as follows: the Ficoll, which was previously kept at room temperature, was added to a fahrenheit tube, and then the ratio of Ficoll: diluted blood = 1:2, slowly adding diluted blood along the tube wall, and then centrifuging in a temperature-controlled centrifuge. When diluted blood is added, the Fahrenheit tube is tilted by 45 degrees to avoid strong shaking of the Ficoll and diluted blood interface.

In particular, said CO₂The culture conditions in the incubator are as follows: CO 2₂The concentration is 5%, the temperature is 37 ℃, and the saturation humidity is 95%.

Specifically, the centrifugation conditions are 1800rpm/min, 30min and 20-24 ℃.

Specifically, the RMPI1640 complete culture medium contains 10% calf serum.

Specifically, the 6-hole plate is as follows: 5.0X 10⁶A hole; the 24-hole plate comprises: 2.0X 10⁵A hole.

Has the advantages that: the invention provides a novel optimized in-vitro induction scheme of adoptive immune cells, which is used for preparing novel adoptive immune cells by expanding PBMCs (peripheral blood mononuclear cells) from CD40 signals in combination with PD-L1 and cytokines IL-15 and IL-7 and serving as entry points and serving as controls of novel adoptive immune cells cultured by a conventional method.

The method provided by the invention has the advantages of low requirement on experimental equipment, simple and time-saving operation, high adoptive immune cell collection rate and high purity.

Drawings

FIG. 1 is a schematic diagram showing the proliferation of cell numbers of the adoptive immune cells cultured in the conventional manner and in the adoptive immune cell culture manner according to the present invention;

FIG. 2 is a diagram showing the analysis of cell subpopulations of the cultured adoptive immune cells according to the present invention in a culture mode of adoptive immune cells and a conventional mode of culture;

FIG. 3 shows CD8 in the CD 40-stimulated group of adoptive immune cells cultured in the present invention and conventional manner⁺Schematic scale of Tcm cells;

FIG. 4 is a schematic diagram showing the ratio of the culture mode of the adoptive immune cells of the present invention to the culture mode of the adoptive immune cells of the conventional mode of culturing CD40 stimulated group NK-T cells;

FIG. 5 is a schematic diagram showing the proportion of the cultured adoptive immune cells of the present invention and the cultured adoptive immune cells of the conventional manner, namely CD 40-stimulated group Treg cells;

FIG. 6 is a schematic diagram showing the ratio of the culture mode of the adoptive immune cells of the present invention to the traditional mode of cultured adoptive immune cells CD 40-stimulated group IFN-gamma cells;

FIG. 7 is a schematic diagram showing the killing ability of the cultured adoptive immune cells CD40 of the present invention in combination with the cultured adoptive immune cells CD-L1 of the conventional manner to kill tumor cells HTC 116;

FIG. 8 is a schematic diagram of the cytokine secretion of the tumor cell HTC116 killed by the adoptive immune cell culture mode of the present invention in combination with the traditional cultured adoptive immune cell CD40 and PD-L1;

Detailed Description

In order that those skilled in the art will better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings, and it is obvious that the described embodiments are only partial embodiments of the present application,

the experimental results of the specific embodiment of the invention all adopt the graphpad 5.0 statistical software, and the measured data is x

s represents, the count data is represented by a ratio (P), X²Inspection with P<0.05 indicates that the difference is statistically significant. The main reagents used in the specific examples of the present invention are: human-mouse chimeric antibody CD40(5C11), human-mouse chimeric antibody PD-L1(2H11), anti-human CD3 monoclonal antibody (OKT-3), IL-2 is commercially available anti-human CD3 monoclonal antibody (OKT-3), IL-2 is commercially available; CD4-FITC, CD8-PE, CD8-PE-CY5, CD3-PE-CY7, CD3-PE-CY5, CD19-FITC, CD28-PE, PD-1-PE-CY5.5, CD4-PE-CY5, CD25-PE-CY5, CD14-FITC, CD4-PE-Cy7, CD3-PE, CD8-PE, CD19-PE and PD-L1-PE, PD-1-PE, Tim-3-PE fluorescent antibodies are all commercially available; PBS, cck-8, CFSE, 1640 culture medium, fetal bovine serum, lymphocyte separation medium (ficoll, specific gravity 1.077 g/ml), OKT-3, CFSE, CD3⁺Magnetic Bead sorting reagents and Cytometric Bead Array (CBA) kits are all commercially available products, and reagents, operation tools, instruments and the like which are not particularly mentioned in the following specific examples are all conventional reagents, operation tools and instruments in the field, and are not described again.

Example 1: in vitro amplification culture of adoptive immunotherapy cells

PBMC isolation, extraction and amplification

Taking normal human peripheral blood, diluting with PBS according to the proportion of 1:2, and then carrying out Ficoll density gradient centrifugation. The Ficoll, which was previously kept at room temperature, was added to a fahrenheit tube, and then the ratio of Ficoll: diluted blood = 1:2, adding diluted blood slowly along the tube wall (45 degrees Fahrenheit tube inclination, strong shaking of Ficoll and diluted blood interface is avoided), and centrifuging in a temperature-controlled centrifuge (1800 rpm/min, 30min, 20-24 ℃). After centrifugation was complete, 4 layers of fluid were obtained as shown in the following figure, with our desired PBMC from the middle haze layer. The cloud layer cells were carefully pipetted and then centrifuged by washing with 10 volumes of PBS to obtain PBMCs.

PBMC cells were made into cell suspension in RMPI1640 complete medium (containing 10% calf serum) and transferred to 6-well plates (5.0X 10)⁶Perwell) and 24-well plate (2.0X 10)⁵Per well), then the corresponding cytokines were added to each set of cell culture: IFN-gamma (1000U/ml) was added at the beginning of the CD3 challenge group (i.e., day 0), and IL-2 (1000U/ml), IL-1 alpha (100U/ml), and anti-CD 3mAb (100 ng/ml) were added after 24 h; the CD40 challenge group was initiated by adding 5C11 (2. mu.g/ml), PD-L1 (1. mu.g/ml), anti-CD 3mAb (30 ng/ml), IL-2 (300U/ml), IL-7 (2 ng/ml), IL-15 (2 ng/ml), and the plates were placed at 37 ℃ in a 5% CO format₂CO with a saturation humidity of 95%₂Culturing in an incubator. Then, the cytokine solution was supplemented every 2-3 days, i.e., every 3 days depending on the cell status, wherein the CD 3-stimulated group was supplemented with IL-2 only (1000U/ml), and the CD 40-stimulated group was supplemented with IL-2 (200U/ml), IL-7 (5 ng/ml), IL-15 (5 ng/ml). Observing every day, and taking pictures and recording as figure 1

During the in vitro culture of adoptive immunotherapy cells, we observed the growth status of the cells in the well plate using an inverted microscope, and recorded the growth morphology by photographing the cells of the CD 3-stimulated group and the CD 40-stimulated group using a cell imager on days 6, 9, and 12.

Trypan blue assay for in vitro proliferation of adoptive immunotherapy cells

Covering with a cover glass: a set of blood counting chamber is taken, and a specially-made cover glass sheet is covered on the blood counting chamber.

Preparation of cell suspensions for counting: 10 mul of cell suspension is sucked into a centrifuge tube by a pipette gun, 10 mul of trypan blue staining solution (0.4%) is added, and the dead cells are stained blue, so that the living cells and the dead cells are distinguished.

After 4 minutes the cell suspension was dropped into the counting plate: blowing the cell suspension to be detected uniformly, and then sucking a small amount of suspension along the edge of the cover plateSlowly dripping into the suspension along the edge, ensuring that the suspension is filled under the cover plate, paying attention to the fact that no air bubbles exist under the cover plate, and preventing the suspension from flowing into the side groove.

The cell number of four big lattices was counted: the blood counting plate is placed under a low power lens of a microscope for observation, the counting plate is moved, and after counting squares appear in the lens, the number of cells which are not stained by staining solution in four big lattices (each big lattice contains 16 middle lattices) at four corners is counted.

Calculating the cell number of the original cell suspension: the cell density was calculated according to the following formula.

(cell number of cell suspension)/ml ═ four large lattice cell number/4) × dilution factor × 10⁴。

Labeled PBMC detection in vitro proliferation assay

Detection of human peripheral blood T cell expansion in vitro with CFSE: mixing appropriate amount of separated human Peripheral Blood Mononuclear Cells (PBMC) in appropriate amount of CFSE working solution, gently mixing, water bathing at 37 deg.C for 10 min, centrifuging, removing supernatant, adding 5 ml 1640 solution of 10% calf serum, centrifuging, removing supernatant, repeating the operation for 1 time, adding appropriate culture medium to obtain cell suspension, and plating (24-well plate, 1 × 10 per well)⁶) The corresponding cytokine was added at 37 ℃ in 5% CO as above₂CO with a saturation humidity of 95%₂Culturing in an incubator, observing the cell morphology every day, and taking pictures for recording.

Adoptive immunotherapy cells labelled with CFSE fluorescence were collected on day 10 of culture and tested for CD4⁺T ,CD8⁺T proliferation status. As can be seen from FIG. 1, CD3 in the CD40 challenge group⁺T amplification was higher than CD3 challenge; CD8 of CD40 challenge group⁺T ratio of 78.9%, CD8 of CD3 challenge group⁺The T ratio was 54.4%, from which it can be seen that CD8 of the CD3 challenge group⁺The number of T amplifications was significantly lower than for the CD40 challenge group (see figure 1).

Example 2: flow cytometry detection of immunophenotype of adoptive immunotherapy cells

Flow detection of membrane surface molecules of cells

The cell suspension was transferred to a centrifuge tube, centrifuged at 1500 rpm/min for 3 min and the supernatant discarded.

Adding PBS (containing 1% calf serum), centrifuging at 1500 rpm/min for 3 min, removing supernatant, and repeating the above steps once.

Setting a comparison group: negative control tubes (only unstained cells added), single compensation tubes.

Adding PBS (containing 1% calf serum) to make final concentration of 1 × 10⁶cells/ml cell suspension.

Mu.l of the cell suspension from step 4 was taken and added to a fresh tube.

Adding fluorescent antibody into the cell suspension, and culturing at room temperature in dark for 30 min.

Washing, centrifuging, adding 500 μ l of sheath liquid, and detecting on a machine.

Collecting the adoptive immunity of the culture on the 14 th day of the cultureTreating cells, detecting two sets of CD3⁺CD4⁺T and CD3⁺CD8⁺Variation in T cell population. The results show that CD3 of the CD3 challenge group⁺CD4⁺ T cell ratio 25%, CD3 of CD40 challenge group⁺CD4⁺T cells 13.7%; CD3 of CD3 challenge group⁺CD8⁺The T cell ratio was 66.4%, while CD3 of the CD40 challenge group⁺CD8⁺The T cell proportion was 79.1%; CD4 of CD3 challenge group⁺T cell ratio higher than CD8⁺T cells, and CD40 challenge group of CD8⁺T cell ratio higher than CD4⁺T cells. Meanwhile, by comparison, the expression of negative co-stimulatory molecules PD-1, PD-L1 and Tim-3 of the CD40 stimulated group T lymphocytes is obviously reduced. (see FIG. 2)

Adoptive immunotherapy cells were collected on day 14 of culture and tested for T for CCR7 and CD62L_CMAnd CD3⁺CD56⁺A changing condition of the cell population. The results show that T of the CD3 challenge group_CM(see FIG. 2) and CD3⁺CD56⁺The proportion of NK-T (as shown in FIG. 4) cells was lower than that of CD 40-stimulated group.

1.2.2.2 flow assay of Treg cells

Add 100. mu.l of the prepared cell suspension to each flow loading tube, approximately 1X10 cells⁶And (4) respectively.

Surface antigens were labeled according to the cell surface antigen staining method (CD 4, CD 25).

Cells were washed with pre-cooled PBS.

Vortex oscillation heavy suspension fineAfter the cells, 100. mu.l of a Fixation/rupture working solution (Fixation/Permeabilization) was added and vortexed again.

Incubate for 90 minutes at 4 ℃ in the dark.

Cells were washed by centrifugation with 1ml of Permeabilization Buffer working solution and the supernatant was discarded.

The 6 th operation was repeated to wash the cells.

Mu.l of diluted fluorescently labeled Foxp3 antibody (diluted with Permeabilisation Buffer working solution) was added directly and incubated for 30min at 4 ℃ in the absence of light.

After repeating the previous step of washing the cells, the cells were resuspended in 500. mu.l of sheath fluid and analyzed by machine detection.

CD4 of CD40 challenge group⁺CD25⁺Foxp3⁺Cells were significantly lower than the CD3 challenge group. Foxp3 of CD40 excitation group⁺Cell proportion was 2.67%, compared to Foxp3 of the CD3 challenge group⁺The proportion of cells was 10.0%. That is, the proportion of Treg cells in the CD40 challenge group was significantly lower than that in the CD3 challenge group (see fig. 3).

Example 3: in vitro killing function detection

Flow assay for IFN-gamma expression by T lymphocytes

100. mu.l of the prepared cell suspension was added to each flow loading tube, and the number of cells was about 1X 106.

Surface antigens (CD3, CD4, CD 8) were labeled according to the cell surface antigen staining method, and the amount of fluorescent antibody used was 2. mu.l/test.

Cells were washed with pre-cooled PBS.

After resuspending the cells by vortexing, 200. mu.l of a fixative/rupture solution (Fixation/Permeabilization) was added and mixed again.

Incubate for 12 min at 4 ℃ in the dark.

Add 500 u l Permeabilization Buffer working solution centrifugal washing cells and discard the supernatant.

The 6 th operation was repeated to wash the cells.

Diluted fluorescently labeled IFN-. gamma.antibody was added directly (working with Permeabilization Buffer)Diluted with solution) 100. mu.l, and incubated for 20 minutes at 4 ℃ in the absence of light.

Add 500 u l Permeabilization Buffer working solution centrifugal washing cells and discard the supernatant and repeat washing cells once.

Cells were resuspended with 500. mu.l of sheath fluid and analyzed by on-machine detection.

CD8 of CD40 challenge group⁺IFN-γ⁺Cells were significantly lower than the CD3 challenge group. CD8 of CD40 challenge group⁺IFN-γ⁺The cell proportion was 17.2%, whereas CD8 of the CD3 challenge group⁺IFN-γ⁺The proportion of cells was 13.8%. I.e., CD8 of CD40 challenge group⁺IFN-γ⁺The cell proportion was significantly higher than that of the Treg cells of the CD3 challenge group (see fig. 6).

Comparison of killing experiments:

the cells were cultured to day 10, and the tumor cells HCT116 were seeded into 6-well plates to be completely adherent, followed by treatment with mitomycin (100. mu.l per ml of medium) for 2 hours, followed by washing twice with PBS, digestion of the tumor cells with appropriate concentrations of pancreatin, counting, and seeding into 96-well plates at 1X10 per well⁴Individual cells (100. mu.l medium); after two hours, the adoptive immunotherapy cells which are cultured in vitro are started to be completely attached to the wall, and are respectively placed at the temperature of 37 ℃ and the content of 5 percent CO according to the proportion of 1:20, 1:40, 1:60 and 1:80₂CO with a saturation humidity of 95%₂Culturing in an incubator, and adding 20 mul cck-8 per hole after three days for detection. (see fig. 7)

From the killing results it can be demonstrated that: the killing effect of the adoptive immunotherapy cells of the CD40 stimulated group on tumor cells is not much different from that of the CD3 stimulated group.

Comparison of in vitro cytokine secretion experiments

The experiment uses CBA kit of BD company, and the experimental procedures are shown as the experimental results of the experimental specification: compared with the single CD3 challenge group, the CD40 combined with PD-L1 secretes more tumor killing cytokines. (see fig. 8)

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for expanding PBMCs by combining CD40 with PD-L1 and cytokines, which is characterized by comprising the following steps:

2) after the centrifugation is finished, 4 layers of liquid are obtained; sucking the middle haze layer cells by a pipette, washing and centrifuging by using PBS with 10 times of volume, and finally obtaining PBMC;

4) supplementing a cell factor solution every 2-3 days according to the cell state;

wherein, corresponding cell factor liquid is added into the cell culture solution, and the process is as follows:

CD40 challenge group, initially, day 0, 5C11 at a concentration of 2. mu.g/ml, PD-L1 at a concentration of 1. mu.g/ml, anti-CD 3mAb at a concentration of 30ng/ml, IL-2 at a concentration of 300U/ml, IL-7 at a concentration of 5ng/ml, IL-15 at a concentration of 5 ng/ml;

wherein, when the cytokine solution is supplemented, the CD40 stimulating group is supplemented with IL-2 with the concentration of 200U/ml, IL-7 with the concentration of 2ng/ml and IL-15 with the concentration of 2 ng/ml.

2. The method of claim 1, wherein the Ficoll density gradient centrifugation is performed by combining CD40 with PD-L1 and cytokines to expand PBMCs: the Ficoll, which was previously kept at room temperature, was added to a fahrenheit tube, and then the ratio of Ficoll: diluted blood = 1:2, slowly adding diluted blood along the tube wall, and then centrifuging in a temperature-controlled centrifuge.

3. The method of claim 1, wherein the CO is present in CD40 in combination with PD-L1 and cytokines to expand PBMCs₂The culture conditions in the incubator are as follows: CO 2₂The concentration is 5%, the temperature is 37 ℃, and the saturation humidity is 95%.

4. The method for expanding PBMCs according to claim 1, wherein the CD40 is combined with PD-L1 and cytokines, and the centrifugation is performed at 1800rpm/min for 30min at 20-24 ℃.

5. The method for expanding PBMCs according to claim 1 by combining CD40 with PD-L1 and cytokines, wherein the RMPI1640 complete medium contains 10% calf serum.

6. The method for expanding PBMCs according to claim 1, wherein the CD40 is combined with PD-L1 and cytokines in a 6-well plate: 5.0X 10⁶A hole; the 24-hole plate comprises: 2.0X 10⁵A hole.