An iNKT cell and an in vitro expansion method thereof

Abstract

The present application relates to the technical field of cell culture, in particular to an iNKT cell and an in vitro expansion method thereof. The method comprises the following steps: separating peripheral blood PBMC, obtaining purified iNKT cells and negative component PBMC by magnetic bead sorting with anti-Vα24-Jα18 antibody, irradiating the negative component PBMC, and then incubating the negative component PBMC with α-GalCer so that antigen presenting cells in the negative component PBMC are loaded with α-GalCer; after centrifugation to remove free α-GalCer, the negative component PBMC loaded with α-GalCer is co-cultured with the purified iNKT cells, and cytokines are added in stages: rhIL-2 is added on the 1st-7th day, rhIL-2 and rhIL-15 are added on the 8th-14th day, and rhIL-2, rhIL-15 and rhIL-12 are added on the 15th-21st day; and the culture is harvested on the 22nd day. After expansion, the purity of the iNKT cells reaches more than 95%, and the proportion of early memory phenotype is more than 50%.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, specifically to an iNKT cell and its in vitro expansion method. Background Technology

[0002] iNKT cells are a subset of T cells that express semi-constant T cell receptors (TCRs). Their TCRα chains consist of Vα24-Jα18 and can specifically recognize lipid antigens presented by CD1d molecules. While iNKT cells are present in very low concentrations in human peripheral blood, accounting for only 0.01%-1% of lymphocytes, they play a crucial role in anti-tumor immunity. They can directly kill target cells by secreting perforin and granzymes, and activate CD8+ cells by secreting cytokines such as IFN-γ and rhIL-2. + T cells, NK cells, and DC cells promote Th1 immune responses.

[0003] CN106566807A discloses a concentration gradient rhIL-2-dependent iNKT cell expansion method. This method involves extracting peripheral blood mononuclear cells (PBMCs), directly stimulating them with α-GalCer, adding autologous induced dendritic cells (DCs) on day 7 of culture, purifying them with magnetic beads on day 14, and then transferring them into a CD3 / CD28 antibody-coated system for secondary expansion. This method has the following drawbacks: First, this method employs a "amplification followed by purification" process. The initial proportion of iNKT cells in PBMCs is extremely low, approximately 0.01%-0.1%. During the first 14 days of expansion, a large amount of cytokines and culture materials are used to support the growth of non-target cells. Simultaneously, iNKT cells in the unpurified mixed culture system are subject to interference from other cell subpopulations, limiting expansion efficiency.

[0004] Second, this method involves directly adding α-GalCer to a PBMC culture system containing iNKT cells. α-GalCer provides continuous stimulation to iNKT cells, potentially leading to overactivation of TCR signaling and causing iNKT cells to differentiate prematurely into terminal effector cells, which is detrimental to the maintenance of early memory phenotypes.

[0005] Third, this method requires the separate induction and culture of dendritic cells (DCs) as antigen-presenting cells in vitro. DC preparation involves inducing and culturing PBMCs in a medium containing GM-CSF and rhIL-4 for 5-7 days, followed by irradiation or mitomycin C treatment to prevent proliferation, and then loading α-GalCer. This process increases the number of steps, prolongs the culture period, raises costs, and results in significant individual variations in DC preparation, making standardization difficult.

[0006] Fourth, this method requires a second forced amplification using CD3 / CD28 antibodies after magnetic bead sorting on day 14 of culture, which further increases the number of steps involved. Summary of the Invention

[0007] The purpose of this invention is to provide iNKT cells and their in vitro expansion method to solve the problems of complex operation, long cycle, high cost, high safety risk and difficulty in standardization caused by the need to separately prepare dendritic cells (DCs) from different donors as antigen-presenting cells in the prior art; and to further solve the problem of low proportion of early memory phenotype (Tnaive / TCM) of iNKT cells due to forced expansion using CD3 / CD28 antibodies and continuous addition of cytokines in the prior art, which is not conducive to long-term in vivo survival after adoptive infusion.

[0008] The technical solution of the present invention is as follows: On the one hand, a method for in vitro expansion of iNKT cells is provided, comprising the following steps: (1) PBMCs were isolated from peripheral blood of healthy donors and magnetically sorted using anti-Vα24-Jα18 antibody to obtain purified iNKT cells and negative fraction PBMCs; (2) Irradiate the negative component PBMC and then co-incubate it with α-GalCer to load the antigen-presenting cells in the negative component PBMC with α-GalCer, thereby obtaining negative component PBMC containing antigen-presenting cells loaded with α-GalCer. (3) After centrifugation to remove free α-GalCer, the iNKT cells and the negative component PBMCs of the antigen-presenting cells loaded with α-GalCer were mixed in proportion and co-cultured in the culture medium. (4) Add cytokines in stages during co-culture: add rhIL-2 to the culture medium in the first time period, add rhIL-2 and rhIL-15 to the culture medium in the second time period, and add rhIL-2, rhIL-15 and rhIL-12 to the culture medium in the third time period. (5) Culture until the predetermined time and collect the expanded iNKT cells.

[0009] Preferably, in step (4), the first time period is day 1-7, the second time period is day 8-14, and the third time period is day 15-21.

[0010] Preferably, the predetermined time in step (5) is the 22nd day.

[0011] Preferably, the irradiation dose in step (2) is 20 Gy.

[0012] Preferably, in step (2), the negative component PBMC is incubated with the α-GalCer at a temperature of 37°C for 1-4 hours.

[0013] Preferably, the ratio of the iNKT cells in step (3) to the negative component PBMCs of the antigen-presenting cells loaded with α-GalCer is 1:5.

[0014] Preferably, the concentration of rhIL-2 in step (4) is 20 ng / mL, the concentration of rhIL-15 is 25 ng / mL, and the concentration of rhIL-12 is 10 ng / mL.

[0015] Preferably, the culture medium in the first time period of step (4) does not contain rhIL-15 and rhIL-12.

[0016] On the other hand, an iNKT cell is provided, obtained by the in vitro expansion method described in any of the above.

[0017] Compared with the prior art, the advantages of the present invention are: (1) By placing the magnetic bead sorting step before amplification culture, i.e. "purify first, then amplify", the magnetic bead sorting is carried out when the total number of cells is at its minimum at the beginning of the culture stage, and there is no need to sort after cell amplification; at the same time, it avoids the interference of other cell subpopulations on the amplification efficiency of iNKT cells in the unpurified PBMC mixed culture system.

[0018] (2) By loading α-GalCer onto negative PBMCs that do not contain iNKT and removing free α-GalCer before mixing them with purified iNKT cells, i.e., “indirect stimulation”, the non-specific continuous direct stimulation of iNKT cells by α-GalCer caused by directly adding α-GalCer to the PBMC culture system containing iNKT cells in the prior art is avoided, thereby preventing iNKT cells from prematurely differentiating and functionally exhausted due to overstimulation.

[0019] (3) By using negative PBMCs derived from the same healthy donor as iNKT cells as antigen-presenting cells, the operation method of using DC cells from different donors is different from the existing technology. APCs and iNKT cells are derived from the same donor, and there is no need to induce and culture DC cells in vitro, which simplifies the production process.

[0020] (4) By using healthy donors instead of patients’ own blood as cell source, iNKT cells can be prepared in a “universal” batch, which is different from the existing technology that requires individualized preparation by temporarily collecting autologous blood from each patient, and allows the amplification products to be prepared in advance. Attached Figure Description

[0021] The present invention will be further described below with reference to the accompanying drawings and embodiments: Figure 1 This is a fold increase diagram of iNKT cells from Day 0 to Day 21 as described in this invention; Figure 2 This is a flow cytometry image of iNKT cells cultured on day 8 in Example 1 of this invention. Figure 3 This is a flow cytometry image of iNKT cells cultured on day 15 in Example 1 of this invention. Figure 4 This is a flow cytometry graph of iNKT cells cultured on day 22 in Example 1 of the present invention. Figure 5 This is a diagram showing the in vitro killing activity of the iNKT cells expanded in Example 3 of the present invention against the Jurkat-luc tumor cell line. Figure 6 This is a diagram showing the in vitro killing activity of the iNKT cells expanded in Example 3 of the present invention against the Raji-luc tumor cell line; Figure 7 This is a diagram showing the in vitro killing activity of the iNKT cells expanded in Example 3 of the present invention against the Ramos-luc tumor cell line. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to specific embodiments: The main reagents and materials used in the examples are as follows: Anti-Vα24-Jα18 antibody; anti-iNKT magnetic beads (Anti-iNKT MicroBeads); α-galactosylceramide (α-GalCer); rhIL-2, rhIL-12, rhIL-15; 1640 medium; lymphocyte separation medium (Ficoll-Hypaque); fetal bovine serum (FBS) or serum substitutes.

[0023] Example 1

[0024] Perform in vitro expansion of iNKT cells according to the following steps.

[0025] (a) Separation of PBMC Peripheral venous blood was collected from healthy donors in sterile blood collection tubes containing heparin sodium anticoagulant, stored at 4°C, and processed within 4 hours.

[0026] Mix the anticoagulated blood with an equal volume of sterile PBS buffer (pH 7.2-7.4) and gently invert to mix. Take a 50 mL centrifuge tube, pre-fill it with lymphocyte separation medium (Ficoll-Hypaque), and slowly add the diluted blood along the tube wall to the top layer of the lymphocyte separation medium, keeping the interface between the two liquids clear. Centrifuge at 400 g for 30 minutes at 20°C, with the acceleration and deceleration settings set to low.

[0027] After centrifugation, the liquid separated into four layers: an upper plasma layer, a middle white membrane layer (containing PBMCs), a lower lymphocyte separation medium layer, and a bottom erythrocyte layer. Carefully aspirate the white membrane layer cells using a sterile pipette and transfer them to a new 50mL centrifuge tube. Add PBS buffer to 50mL, mix well by pipetting, centrifuge at 200g for 10 minutes, discard the supernatant, and repeat the washing once. Resuspend the cell pellet in PBS buffer and count the cells.

[0028] (ii) Obtaining iNKT cells and negative fraction PBMCs by magnetic bead sorting The PBMCs were resuspended in PBS containing 0.5% bovine serum albumin and 2 mM EDTA, and the cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 8 cells / mL. Following the Anti-iNKT MicroBeads kit instructions, each 1×10⁶ cells / mL... 7 Add 20 μL of anti-Vα24-Jα18 antibody-conjugated magnetic beads to each cell, mix thoroughly, and incubate at 4°C in the dark for 15 minutes.

[0029] After incubation, add 10 volumes of MACS buffer, centrifuge at 300g for 10 minutes, discard the supernatant, and resuspend the cell pellet with an appropriate amount of MACS buffer. Place the MS magnetic bead sorting column on a magnetic rack and rinse the column with 0.5 mL of MACS buffer. Slowly add the cell suspension to the sorting column and collect the flow-through, which is the negative fraction PBMC. Wash the sorting column three times with 0.5 mL of MACS buffer, and collect all the flow-through and combine it with the negative fraction PBMC. Remove the sorting column from the magnetic rack, add 1 mL of MACS buffer, and use the stopcock to push the cells bound to the sorting column into the collection tube; this portion is the purified iNKT cells. Take a small number of cells for counting and flow cytometry analysis to calculate the iNKT cell purity.

[0030] (III) Obtaining negative component PBMCs containing antigen-presenting cells loaded with α-GalCer The negative PBMCs collected in step (II) above were resuspended in 1640 medium, and the cell density was adjusted to 1×10⁻⁶. 7The cell suspension was transferred to sterile centrifuge tubes and irradiated with an X-ray irradiator at a dose of 20 Gy.

[0031] After irradiation, the negative fraction PBMC was centrifuged at 300g for 10 minutes, the supernatant was discarded, and the PBMC was resuspended in 1640 medium containing 10% fetal bovine serum or a serum substitute. α-GalCer was added to a final concentration of 100ng / mL, the mixture was pipetted and incubated in a 37℃, 5% CO2 incubator for 2 hours.

[0032] During this incubation process, antigen-presenting cells, including monocytes and dendritic cell precursors in the negative fraction PBMCs, all express the CD1d molecule. α-GalCer is a glycolipid antigen whose lipid tail is embedded in the hydrophobic binding groove of the CD1d molecule, while its glycosyl head is exposed on the CD1d molecule surface, forming the CD1d-α-GalCer complex. This complex can be specifically recognized by the semi-constant TCR (Vα24-Jα18 / Vβ11) on the surface of iNKT cells, thereby activating iNKT cells. The negative fraction PBMCs naturally contain monocytes and dendritic cell precursors, all of which express the CD1d molecule, and therefore can serve as antigen-presenting cells without additional induction of differentiation.

[0033] After incubation, centrifuge at 300g for 10 minutes, discard the supernatant to remove free α-GalCer, and resuspend in 1640 medium to obtain the negative fraction PBMC containing antigen-presenting cells loaded with α-GalCer. Compared with directly adding free α-GalCer to a culture system containing iNKT cells, this invention ensures that iNKT cells receive activation signals only through TCR recognition of the CD1d-α-GalCer complex presented on the surface of antigen-presenting cells, avoiding continuous non-specific stimulation of the TCR by free α-GalCer by antigen-presenting cells in the negative fraction PBMC by first loading the antigen-presenting cells with α-GalCer and then centrifuging to remove free α-GalCer.

[0034] (iv) Co-culture of iNKT cells with PBMCs, a negative component of antigen-presenting cells loaded with α-GalCer, and phased addition of cytokines During the culture process, cytokines were added in stages according to the following protocol. Each stage was sequential, with each stage lasting approximately 7 days, for a total culture time of approximately 21 days. The purified iNKT cells obtained in step (II) were resuspended in 1640 medium containing 10% FBS or a serum substitute, and the concentration was adjusted to 1×10⁻⁶. 6cells / mL. The negative PBMCs containing α-GalCer-loaded antigen-presenting cells obtained in step (III) were added at a ratio of 1:5 (iNKT cells: PBMCs containing α-GalCer-loaded antigen-presenting cells), mixed well, and then seeded into cell culture flasks. The culture flasks were placed in a 37℃, 5% CO2 incubator and incubated for day 0.

[0035] During the culture process, cytokines were added in stages according to the following protocol: Days 1-7: Only rhIL-2 was added to the culture medium, with a final concentration of 20 ng / mL. Cell status was observed every 2 days, and fresh medium containing 20 ng / mL rhIL-2 was added as needed based on changes in the medium color, maintaining cell density at 0.5-1.5 × 10⁻⁶ cells / day. 6 cells / mL.

[0036] Days 8-14: Change the culture medium and add rhIL-2 and rhIL-15 to a final concentration of 20 ng / mL and 25 ng / mL, respectively. Thereafter, replenish with fresh culture medium containing the above concentrations of rhIL-2 and rhIL-15 every two days to maintain a cell density of 0.5-1.5 × 10⁻⁶ cells / day. 6 cells / mL.

[0037] Days 15-21: Change the culture medium and add rhIL-2, rhIL-15, and rhIL-12, with final concentrations of 20 ng / mL, 25 ng / mL, and 10 ng / mL, respectively. Thereafter, replenish with fresh culture medium containing the above concentrations of the three cytokines every two days to maintain a cell density of 0.5-1.5 × 10⁻⁶ cells / day. 6 cells / mL.

[0038] It should be noted that no additional APCs are added during the co-culture process; the entire expansion cycle is supported solely by the antigen-presenting cells in the initially added irradiated negative PBMC component.

[0039] The design principle of the staged cytokine addition strategy described above is as follows: rhIL-2, by binding to the rhIL-2 receptor (rhIL-2R), activates the signaling pathway, promoting T cell survival and basal proliferation, and is a key cytokine for T cell survival. In the early culture stage, adding only rhIL-2 on days 1-7 allows iNKT cells to maintain basic survival while receiving TCR activation signals, but does not drive massive cell proliferation and differentiation. rhIL-15 shares the rhIL-2Rβ and γc chains with rhIL-2, promoting T cell proliferation through the same signaling pathway. However, rhIL-15 has a unique role in maintaining the memory T cell phenotype, maintaining the long-term survival of TCM cells by promoting the expression of anti-apoptotic proteins Bcl-2 and Bcl-xL. In the middle culture stage, rhIL-15 is introduced on days 8-14. At this point, the cells have a certain level of activation, and the addition of rhIL-15 effectively drives massive proliferation of iNKT cells. rhIL-12 activates the signaling pathway by binding to its receptor, promoting T cell polarization towards a Th1-like orientation and enhancing IFN-γ secretion and cytotoxic activity. Introducing rhIL-12 during the later stages of culture, from days 15 to 21, guides iNKT cells towards a Th1-like polarization, enhancing their cytotoxic function. This phased, switching addition strategy ensures that iNKT cell activation, proliferation, and polarization receive appropriate signal support at suitable times.

[0040] (v) Cell harvesting and detection All cells were collected on day 22, the day after the three time periods ended. 1 mL of cell suspension was used for cell counting and flow cytometry, and the remaining cells were collected by centrifugation at 300g for 10 minutes.

[0041] The flow cytometry detection method is as follows: Take approximately 1×10 6 Cells were washed once with PBS buffer containing 2% FBS and centrifuged at 300g for 5 minutes. The cell pellet was resuspended in 100 μL of wash buffer, and the following fluorescently labeled antibodies were added: FITC-labeled anti-human CD3 antibody, PE-labeled anti-human TCR Vα24-Jα18 antibody, PerCP-Cy5.5-labeled anti-human CD8 antibody, PE-Cy7-labeled anti-human CD4 antibody, APC-labeled anti-human CD45RA antibody, and BV421-labeled anti-human CCR7 antibody. After mixing, the cells were incubated at 4°C in the dark for 30 minutes. After incubation, 1 mL of wash buffer was added, and the cells were centrifuged at 300g for 5 minutes. The supernatant was discarded, and the washing was repeated once. The cells were resuspended in 500 μL of wash buffer and analyzed by flow cytometry. The gating position was set using the corresponding isotype control antibody.

[0042] Test results as follows Figures 1 to 4 As shown. The amplification fold determination results are as follows. Figure 1 As shown, from day 0 to day 22, the number of iNKT cells continued to increase, expanding more than 10,000-fold. Flow cytometry results are as follows... Figures 2-4 As shown, on the 8th day of cultivation ( Figure 2 Day 15 Figure 3 Day 22 Figure 4 iNKT cell purity (CD3) + Vα24-Jα18 + The purity continued to rise, reaching over 95% by day 22. Flow cytometry data from days 8, 15, and 22 of culture were compiled, including CD3... + Cell percentage, iNKT cell purity (CD3) + Vα24-Jα18 + CD4 + / CD8 + Cell percentage and CD45RA - CCR7 + (TCM) cell ratio.

[0043] The results showed that after 22 days of in vitro amplification, flow cytometry identified it as Tnaive (CD45RA). + CCR7 + ) and TCM (CD45RA) - CCR7 + Early differentiated cells accounted for more than 50% of iNKT cells. The differentiation phenotypes of representative donors are shown in Table 1, where donor A's CD4... + Tnaive+TCM accounted for 79.02% of T cells, CD8 + Tnaive+TCM accounted for 88.25% of T cells, indicating that the amplification products were mainly early-differentiated cell phenotypes.

[0044] Tnaive and TCM are cells in the early stages of differentiation, possessing stronger proliferation and self-renewal capabilities; while TEM and TEF are cells in the late stages of differentiation, gradually differentiating towards terminal effects and exhibiting limited proliferation capacity. The high proportion of early-differentiated cells (Tnaive + TCM) in donors A and B indicates that the iNKT cells expanded by the method of this invention are predominantly of the early-differentiation phenotype, possessing good proliferative potential and long-term viability in vivo.

[0045] Table 1: Differentiation phenotypes of iNKT cells 3 weeks after expansion (donors A and B were under the same experimental conditions)

[0046] Example 2 This embodiment is used to compare the effects of different cytokine addition regimens on the expansion fold and purity of iNKT cells.

[0047] The experiment was conducted according to the method in Example 1, with the only difference being the cytokine addition protocol. Specifically, peripheral blood cells (PBMCs) were isolated from the same donor's peripheral blood, and iNKT cells and negative fraction PBMCs were obtained by magnetic bead sorting. The negative fraction PBMCs were irradiated with 20 Gy and then loaded with α-GalCer (100 ng / mL, incubated at 37°C for 2 hours) to prepare APCs. The purified iNKT cells and APCs were co-cultured at a 1:5 ratio, and the following four different cytokine addition protocols were established: Group A (Scheme of this invention): rhIL-2 (20 ng / mL) was added on days 1-7, rhIL-2 (20 ng / mL) + rhIL-15 (25 ng / mL) was added on days 8-14, and rhIL-2 (20 ng / mL) + rhIL-15 (25 ng / mL) + rhIL-12 (10 ng / mL) was added on days 15-21.

[0048] Group B (simultaneous addition throughout the entire process): rhIL-2 (20 ng / mL) + rhIL-15 (25 ng / mL) + rhIL-12 (10 ng / mL) were added simultaneously throughout the entire process from day 1 to day 21.

[0049] Group C (full rhIL-2 + rhIL-15): rhIL-2 (20 ng / mL) + rhIL-15 (25 ng / mL) were added throughout the 1-21 days, without adding rhIL-12.

[0050] All three groups used cells from the same donor. Cells were harvested on day 22, counted, and their purity was tested. The results are shown in Table 2.

[0051] Table 2: Effects of different cytokine addition regimens on iNKT cell expansion

[0052] Table 2 shows that the phased addition scheme of this invention (Group A) is superior to other schemes in terms of both fold increase and purity. Group B (simultaneous addition of all three factors throughout the process) has lower fold increase and purity than Group A. Group C (rhIL-2 + rhIL-15 throughout the process) has all indicators between Group B and Group A. Although the presence of rhIL-15 throughout the process promotes proliferation, the lack of Th1 polarization signal provided by rhIL-12 in the later stages results in lower amplification efficiency and purity than Group A.

[0053] Group A (the scheme of this invention) avoids premature differentiation induced by rhIL-15 and rhIL-12 in the early stage of culture (days 1-7) by adding only rhIL-2, thus preserving the early memory phenotype of cells; in the middle stage (days 8-14), rhIL-15 is introduced to promote massive proliferation. At this time, the cells already have a certain activation basis, and the addition of rhIL-15 effectively drives the expansion; in the late stage (days 15-21), rhIL-12 is introduced to guide Th1-like polarization and enhance the killing function. The three-stage signals are progressively advanced, corresponding to the three biological processes of activation, proliferation, and polarization, respectively, thus achieving an optimal balance between expansion fold and purity.

[0054] The above results indicate that the phased switching addition of cytokines can better coordinate the proliferation and differentiation of iNKT cells, resulting in a higher expansion rate.

[0055] Example 3

[0056] This embodiment is used to detect the effect of iNKT cells expanded in Example 1 on CD1d. + Tumor cells and CD1d - Killing activity of tumor cells.

[0057] The detection was performed using the lactate dehydrogenase (LDH) release assay. Target cells were human CD1d cells. + Tumor cells (Ramos cells, Jurkat cells) and human CD1d - Tumor cells (Raji cells). Target cells in logarithmic growth phase were collected and the cell concentration was adjusted to 1×10⁶ cells / cells using RPMI 1640 medium containing 10% FBS or serum substitute. 5 cells / mL.

[0058] The effector cells were iNKT cells harvested on day 22 of Example 1, and were adjusted to different concentrations using RPMI 1640 medium containing 10% FBS or serum substitutes.

[0059] In 96-well U-shaped culture plates, 100 μL each of effector cells and target cells were added according to the effector-to-target ratio of 1:4-2:1 as shown in the figure, with 3 replicates per group. Simultaneously, wells for spontaneous release of target cells (target cells + culture medium), maximum release of target cells (target cells + lysis buffer), and spontaneous release of effector cells (effector cells + culture medium) were set up. α-GalCer was added to each well to a final concentration of 100 ng / mL. The culture plates were incubated at 37°C in a 5% CO2 incubator for 4 hours.

[0060] After incubation, centrifuge the culture plate at 250g for 5 minutes. Transfer 100μL of supernatant from each well to a new 96-well plate, add 100μL of LDH detection working solution, and incubate at room temperature in the dark for 30 minutes. Add 50μL of stop solution and measure the absorbance (OD value) at 490nm using a microplate reader.

[0061] The lethality rate is calculated using the following formula: Kill rate (%) = (OD value of experimental wells - OD value of spontaneously released effector cells - OD value of naturally released target cells) / (OD value of maximum release from target cells - OD value of naturally released target cells) × 100% The results are as follows Figure 5-7 As shown, the iNKT cells expanded in Example 1 showed their effect on CD1d. + Tumor cells (Ramos, Jurkat) exhibited significantly higher cytotoxic activity compared to T cells, and the killing rate increased with increasing effector-to-target ratio. However, for CD1d... - The low killing activity of tumor cells (Raji) indicates that the expanded iNKT cells mainly recognize and kill target cells through the CD1d-dependent pathway.

[0062] Example 4

[0063] This embodiment is used to verify the stability and reproducibility of the method of the present invention in PBMCs from different donor sources.

[0064] PBMCs were isolated from peripheral blood of at least three healthy donors and iNKT cells were expanded according to the method in Example 1. Each donor independently completed a full expansion experiment, including PBMC isolation, magnetic bead sorting, treatment of negative PBMC components (irradiation and α-GalCer loading), co-culture, and staged factor addition. The results are shown in Table 3.

[0065] Table 3: Expansion results of iNKT cells from different donors

[0066] Table 3 shows that the initial proportion of iNKT cells in PBMCs from different donors differed. However, after 22 days of expansion using the method of this invention, the expansion fold of each donor was over 10,000 times, and the purity was over 95%. This indicates that the method of this invention can stably achieve efficient expansion of iNKT cells in cells from different donors, and has good universality and reproducibility.

[0067] The above embodiments are merely illustrative of the technical concept and features of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects. The scope of the present invention is defined by the appended claims rather than the foregoing description, and thus all changes falling within the meaning and scope of the equivalents of the claims are intended to be included within the present invention.

Claims

1. A method for in vitro expansion of iNKT cells, characterized in that, Includes the following steps: (1) PBMCs were isolated from peripheral blood of healthy donors and magnetically sorted using anti-Vα24-Jα18 antibody to obtain purified iNKT cells and negative fraction PBMCs; (2) Irradiate the negative component PBMC and then co-incubate it with α-GalCer to load the antigen-presenting cells in the negative component PBMC with α-GalCer, thereby obtaining negative component PBMC containing antigen-presenting cells loaded with α-GalCer. (3) After centrifugation to remove free α-GalCer, the iNKT cells and the negative component PBMCs of the antigen-presenting cells loaded with α-GalCer were mixed in proportion and co-cultured in the culture medium. (4) Add cytokines in stages during co-culture: add rhIL-2 to the culture medium in the first time period, add rhIL-2 and rhIL-15 to the culture medium in the second time period, and add rhIL-2, rhIL-15 and rhIL-12 to the culture medium in the third time period. (5) Culture until the predetermined time and collect the expanded iNKT cells.

2. The method for in vitro expansion of iNKT cells according to claim 1, characterized in that, In step (4), the first time period is days 1-7, the second time period is days 8-14, and the third time period is days 15-21.

3. The method for in vitro expansion of iNKT cells according to claim 1, characterized in that, The scheduled time mentioned in step (5) is the 22nd day.

4. The method for in vitro expansion of iNKT cells according to claim 1, characterized in that, The irradiation dose in step (2) is 20 Gy.

5. The method for in vitro expansion of iNKT cells according to claim 1, characterized in that, In step (2), the negative component PBMC is incubated with the α-GalCer at a temperature of 37°C for 1-4 hours.

6. The method for in vitro expansion of iNKT cells according to claim 1, characterized in that, The ratio of iNKT cells to the negative component PBMCs of antigen-presenting cells loaded with α-GalCer in step (3) is 1:

5.

7. The method according to claim 1, characterized in that, The concentration of rhIL-2 in step (4) is 20 ng / mL, the concentration of rhIL-15 is 25 ng / mL, and the concentration of rhIL-12 is 10 ng / mL.

8. The method for in vitro expansion of iNKT cells according to claim 1, characterized in that, In step (4), the culture medium in the first time period does not contain rhIL-15 and rhIL-12.

9. An iNKT cell, characterized in that, Obtained by the in vitro amplification method according to any one of claims 1-8.

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