Method for in-vitro expansion of primary cytotoxic t cell subset

Abstract

The present invention relates to the technical field of biology and new drugs, and provides a method for in-vitro expansion of a primary cytotoxic T cell subset. The method comprises the following steps: coculturing CD8+T cells and adult mouse microglia cells in a culture medium containing fetal bovine serum, double antibodies and DMEM / F12 of Aβ; and using CD3 / CD28 immunomagnetic beads to perform magnetic cell separation. The embodiments of the present invention indicate that the primary CD8+T cells separated from the peripheral blood and spleen of an AD mouse and intracerebral microglia cells are cocultured in vitro, so as to simulate a glial cell-mediated antigen presentation system (APC), so that the in-vitro expansion speed of the cytotoxic T cells can be rapidly increased and reach 1.5 times a conventional T cell expansion speed. Moreover, in this process, an APC does not need to be manually synthesized, thereby greatly saving the cost of in-vitro expansion of cytotoxic T cells.

Description

A method for expanding primary cytotoxic T cell subsets in vitro Technical Field

[0001] The present invention belongs to biology and new drug technology, and specifically relates to a method for amplifying primary cytotoxic T cell subsets in vitro. Background Art

[0002] Cytotoxic T cells, also known as CD8+ T cells, are a key component of the acquired immune system and play an important role in the immune system's defense against pathogens such as viruses, bacteria, and tumors. CD8+ T cells are widely known for being key effector cells in the primary anti-tumor response, capable of directly killing tumor cells. They are key immune surveillance cells in cell-based tumor therapy. Therefore, in current research on cell therapy for tumors, a high concentration of CD8+ T cells with tumor-killing function in tumor tissue is an important indicator for improving patients' ability to inhibit tumor progression or even ultimately eliminate the tumor.

[0003] In recent years, a growing number of studies have revealed that peripheral immune cells play a crucial role in driving AD disease progression. Further research has revealed that peripheral CD8+ T cells can infiltrate the AD brain and, by activating microglia, exacerbate AD disease progression. This finding confirms the crucial role of peripheral circulating immune cells in immune surveillance during the development of AD and provides theoretical and preclinical evidence for the search for biomarkers for the diagnosis and treatment of AD from peripheral blood. Current clinical diagnostic methods for AD include measuring Aβ and tau levels in cerebrospinal fluid and imaging techniques such as amyloid β-PET and tau-PET pathology scans. These methods are either invasive and carry a high risk of intracranial infection, or are extremely expensive, or both. Therefore, blood-based biomarkers or drug targets offer the potential for safe, minimally invasive, and low-cost clinical diagnosis and treatment of AD.

[0004] Based on this, CD8+ T cells, whether as key immune cells in the primary anti-tumor response or as effector cells regulating neuroinflammation in the brain during AD progression, are one of the future development approaches for treating various diseases. Unprecedented clinical success has been demonstrated in the use of CAR-T in the treatment of solid tumors and B-cell acute lymphoblastic leukemia. However, accurate functional subpopulation typing, in vitro proliferation capacity, and expansion rate of functional T cells remain a challenge. T cell activation requires three signals: (1) T cell receptor (TCR) stimulation, (2) co-stimulation, and (3) pro-survival cytokines. In vivo, these signals are provided by antigen-presenting cells (APCs), which present these cues to T cells in a specific spatiotemporal pattern. Among them, synthetic artificial APCs (aAPCs) are particularly convenient for polyclonal T cell expansion. Currently, commercial microbeads functionalized with activating antibodies to CD3 (αCD3; TCR stimulation) and CD28 (αCD28; co-stimulatory signal) represent one of the most commonly used and clinically relevant synthetic systems. These strains promote polyclonal T cell activation with exogenous interleukin-2 (IL-2) supplementation. Although these cultures provide three key signals to T cells, the context in which these signals are presented does not represent how they are naturally presented by antigen-presenting cells. This can result in suboptimal T cell expansion rates and functionally limited or dysfunctional T cell products.

[0005] Summary of the Invention

[0006] The present invention provides a low-cost, short-time and function-specific in vitro cytotoxic T cell proliferation method, that is, a method for expanding primary cytotoxic T cell subsets in vitro.

[0007] A method for expanding primary cytotoxic T cell subsets in vitro comprises the following steps:

[0008] CD8+ T cells and adult mouse microglia were co-cultured in DMEM / F12 medium containing fetal bovine serum, double-antibody and Aβ, and magnetic cell separation was performed using CD3 / CD28 immunomagnetic beads.

[0009] Furthermore, the method for expanding primary cytotoxic T cell subsets in vitro comprises the following steps:

[0010] S1: Load CD3 / CD28 immunomagnetic beads and place them in DMEM / F12 culture medium containing fetal bovine serum, double antibody and Aβ, and adjust the final concentration of immunomagnetic beads to 4×10 6 pcs / ml;

[0011] S2: Use DMEM / F12 culture medium containing fetal bovine serum, double antibody and Aβ to adjust the CD8+ T cell concentration to 4×10 6cells / ml;

[0012] S3: Adult mouse microglia were cultured at a rate of 1.5 × 10 6 The cells were inoculated at a concentration of 10 cells / ml into fresh DMEM / F12 culture medium containing fetal bovine serum and double-antibody, cultured at 37°C and 5% CO2 for 3 days, and the mixture of steps S1 and S2 was added. Thereafter, the cells were passaged and expanded at a ratio of 1:3 every 2-3 days until the 10th day.

[0013] Furthermore, in the DMEM / F12 culture medium containing fetal bovine serum, double antibody and Aβ in step S1 and step S2, the concentration of fetal bovine serum is 10%, the concentration of double antibody is 1%, and the concentration of Aβ is 0.5 mM / ml;

[0014] In step S3, the concentration of fetal bovine serum in the DMEM / F12 culture medium containing fetal bovine serum and double antibody is 10%, and the concentration of double antibody is 1%;

[0015] In step S3, the mixture of step S1 and step S2 is added in an amount of 2 ml / well at a volume ratio of 1:1.

[0016] Further, the following steps are included: loading CD3 / CD28 immunomagnetic beads in step S1 includes the following steps:

[0017] Add 100 μl of CD3ε-biotin and 100 μl of CD28-biotin antibody, 300 μl of PBS buffer containing 2 mM EDTA, and 500 μl of mixed anti-biotin MACSiBead magnetic beads to a 2 ml sterile low-adhesion centrifuge tube and incubate at 2-8°C for 2 hours.

[0018] One purpose of the present invention is to provide a method for specifically regulating the expansion of cytotoxic T cells that cause neuroinflammation in the AD brain: using Aβ to activate microglia to induce the chemotactic effect of the expanded cytotoxic T cell subsets on neuroinflammation in the AD brain.

[0019] One object of the present invention is to provide a cytotoxic T cell model for regulating neuroinflammation in AD brain, wherein the model is prepared by the above-mentioned method for specifically regulating the expansion of cytotoxic T cells for neuroinflammation in AD brain.

[0020] One object of the present invention is to provide a microglia-mediated antigen presentation system, comprising Aβ and microglia.

[0021] The present invention provides examples of primary CD8+ T cells isolated from the peripheral blood and spleen of AD mice and brain microglia. Through in vitro cell co-culture, a glial cell-mediated antigen presentation system was simulated to rapidly increase the in vitro expansion rate of cytotoxic T cells, which was 1.5 times the expansion rate of conventional T cells. Furthermore, in this process, artificially synthesized APCs were not required, significantly reducing the cost of in vitro expansion of cytotoxic T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG1 shows the efficiency of inducing CD8+ T cell activation and proliferation by the in vitro simulated microglia-mediated antigen presenting cell system method provided in Example 1;

[0023] FIG2 shows the efficiency of inducing CD8+ T cell activation and proliferation by the in vitro simulated microglia-mediated antigen presenting cell system method provided in Example 2;

[0024] FIG3 shows the chemotaxis of CD8+ T cells activated by an Aβ-simulated in vitro antigen presentation system on neuroinflammation provided in Example 3. DETAILED DESCRIPTION

[0025] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings, but they should not be understood as limiting the scope of implementation of the present invention.

[0026] The materials involved in the present invention are as follows:

[0027] Wild-type littermates and the APP / PS1 mouse model were obtained from the Jackson Laboratory in the United States;

[0028] Red blood cell lysis buffer was purchased from BD Biosciences, catalog number: 555899;

[0029] DPBS was purchased from Sigma, product number: D8662-24*500ML;

[0030] Horse serum was purchased from Gibco, catalog number: 26050088;

[0031] Fetal bovine serum was purchased from Life Technologies, catalog number: 16050-122;

[0032] Cell culture dishes were purchased from Thermo Scientific Nunc EasYDish 100 mm, catalog number: 150466;

[0033] 40 μm cell strainer (e.g. Fisherbrand sterile cell strainer, Cat. No. 22-363-548)

[0034] Hanks' balanced salt solution was purchased from Gibco, catalog number: 14060040;

[0035] F12 medium;

[0036] RPIM1640 medium;

[0037] Ms CD45FITC 30-F11, Cat. No. 553079;

[0038] Ms CD3MolCpx PerCP-Cy5.5 17A2, catalog number: 560527;

[0039] Ms CD8a PE 53-6.7, catalog number: 553032;

[0040] CD8 primary antibody was purchased from Invitrogen, catalog number: 14-0195-82;

[0041] Fluorescent secondary antibodies were purchased from Thermo scientific;

[0042] DAPI was purchased from Thermo Scientific, catalog number: 62248;

[0043] MgniSort TM Mouse CD8 + T cell enrichment kit was purchased from ThermoFisher, catalog number: 8802-6842;

[0044] T cell activation and expansion kit was purchased from Miltenyi Biotec, catalog number: 130-093-627;

[0045] Ficoll was purchased from Cytia, product number: 17144002;

[0046] Percoll was purchased from Merck, product number P1644-100ML;

[0047] Trans-well was purchased from Corning, catalog number: 3422;

[0048] Aβ was purchased from rPeptide, catalog number: A-1163-2;

[0049] LPS was purchased from InvivoGen, catalog number: tlrl-eblps.

[0050] Example 1: Isolation, activation and in vitro expansion of primary CD8+ T cells from peripheral blood

[0051] This example uses peripheral blood mononuclear cell (PBMC) separation technology and a CD8+ T cell magnetic bead separation kit to isolate CD8+ T cells from peripheral blood, and then culture, activate, and proliferate them in vitro. The specific steps are as follows:

[0052] 1.1 Isolation of PBMCs by density gradient centrifugation

[0053] (1) Take about 1 ml of fresh anticoagulated whole blood from mice, add DPBS buffer at a ratio greater than 1:2, and dilute the total volume of the diluted anticoagulated blood to 4 ml. Mix by inverting and set aside.

[0054] (2) Add 4 ml of Ficoll concentration gradient separation solution to a 15 ml sterile centrifuge tube, and slowly add the diluted anticoagulated blood to the surface of the Ficoll separation solution (separation solution: diluted whole blood = 1:1), keeping the interface between the two liquid surfaces clear.

[0055] (3) Density gradient centrifugation: centrifuge at room temperature and 800 × g for 40 min in a horizontal centrifuge (high-speed refrigerated centrifuge purchased from ThermoFisher, model: Sorvall AT8R).

[0056] In this step, the centrifuge startup and deceleration process are set to slow increase and slow decrease mode.

[0057] (4) After centrifugation, carefully remove the centrifuge tube and use a 1 ml pipette tip to carefully aspirate the PBMCs layer (i.e., the buffy coat layer) between the top light yellow plasma layer and the middle transparent separation solution, and transfer it to a 15 ml centrifuge tube.

[0058] The above four steps are all carried out at room temperature of 18°C-22°C.

[0059] (5) Add 10 ml of cold (2°C-8°C) DPBS to the centrifuge tube to resuspend the cells, centrifuge at 400g (2°C-8°C) for 5 min, and discard the supernatant.

[0060] (6) Red blood cell lysis: After centrifugation, discard the supernatant, add 5 ml of red blood cell lysis buffer, lyse at room temperature for 5 min, then add 10 ml of PBS, centrifuge at 400g for 5 min.

[0061] (7) PBMCs washing: Add 10 ml of cold (2°C-8°C) DPBS to the centrifuge tube to resuspend the cells, centrifuge at 400g (2°C-8°C) for 5 min, and discard the supernatant.

[0062] (8) PBMCs purification: Add 10 ml of cold (2°C-8°C) DPBS to the centrifuge tube and resuspend the cells. Centrifuge at 250g (2°C-8°C) for 5 min, discard the supernatant, and repeat this step once. The cells can be used for subsequent experiments.

[0063] Reducing the centrifugal force at this step can remove cell debris from PBSCs.

[0064] 1.2 Isolation and purification of CD8+ T cells from PBMCs

[0065] This experiment was performed using Invitrogen TM The CD8+ T cell separation and purification kit was used, and the specific steps are briefly described as follows:

[0066] (1) Resuspend the PBMCs prepared in step 1 in sorting buffer and adjust the cell density to 1×10 8 cells / ml.

[0067] The sorting buffer is DPBS containing 2 mM EDTA and 2% fetal bovine serum (FBS). The prepared buffer needs to be sterilized by filtering through a 0.22 μm filter membrane in advance.

[0068] (2) 100 μL cell suspension (1×10 7 cells) were added into a low-adsorption centrifuge tube, and then 10 μl of CD8 antibody was added, mixed and incubated at 4°C for 10 min.

[0069] (3) After incubation, add 10 μL of washed MgniSort magnetic beads to the centrifuge tube (the beads need to be vortexed and resuspended before use) and incubate at 4°C for 10 min.

[0070] (4) After incubation, add 2.5 ml of sorting buffer to the centrifuge tube and mix by pipetting up and down 5 times (avoid vigorous shaking or inverting to mix).

[0071] (5) Place the centrifuge tube containing cells on a magnetic rack and let it stand for 10 minutes.

[0072] (6) Slowly discard the liquid in the centrifuge tube with a pipette (do not remove the flow tube from the magnetic stand during the liquid aspiration process), and the adsorbed magnetic beads contain purified mouse CD8+ T cells.

[0073] (7) Washing: Remove the centrifuge tube from the magnetic stand, add 2.5 ml of sorting buffer, pipette up and down 5 times to mix thoroughly, place on the magnetic stand, and let it stand for 10 minutes. Slowly discard the liquid in the centrifuge tube with a pipette (do not remove the flow tube from the magnetic stand during the liquid aspiration process).

[0074] (8) Discard the supernatant and collect the cells.

[0075] (9) After washing the cells, resuspend them in the desired culture medium and use them for subsequent T cell activation and proliferation experiments.

[0076] 1.3 Isolation and culture of primary microglia from adult mice

[0077] The primary microglia used in this example were derived from microglia in the cerebral cortex of 6-month-old wild-type mice and APP / PS1 mice, and the steps were as follows:

[0078] (1) Under sterile conditions, the whole brain of the mouse was removed, the medulla oblongata, pons, cerebellum, etc. were removed, and the left and right hemispheres were carefully separated. The olfactory bulb, striatum, hippocampus, and basal tissues were removed. The remaining cortical tissue was carefully transferred to another small culture dish (D = 1 cm) containing ice-cold dissection fluid, and the pia mater and surface blood vessels were gently removed.

[0079] (2) Mince the cortical tissue with a surgical blade, add trypsin containing EDTA, place it in a cell culture dish, and digest it at 37°C for 30-45 minutes.

[0080] (3) After digestion, add three times the volume of 10% fetal bovine serum and 1% double antibody to terminate the digestion.

[0081] (4) The digested cerebral cortex mixture was passed through a 40-μm filter.

[0082] (5) Centrifuge the filtered cell suspension at 300 g for 7 minutes at 18-22°C.

[0083] (6) Microglial cell density gradient centrifugation: After discarding the supernatant, resuspend the cells in 4 ml of 37% Percoll separation buffer. Slowly layer the cells in a 15 ml centrifuge tube containing 4 ml of 70% Percoll separation buffer. Then, slowly layer 4 ml of 30% Percoll on the cell suspension. Finally, add 2 ml of HBSS. In this step, the gradient layers must be clear.

[0084] (7) Centrifuge at 18-22°C, 300 g for 40 min.

[0085] In this step, the centrifuge startup and deceleration process are set to slow increase and slow decrease mode.

[0086] (8) After centrifugation, collect 37-70% of the cells into a 15 ml centrifuge tube.

[0087] (9) Add 10 ml of HBSS, mix well, and centrifuge at 400 g for 7 minutes at 18-22°C. Repeat the washing process once.

[0088] (10) The obtained microglial cells were resuspended in DMEM / F12 containing 10% fetal bovine serum and 1% double antibody for later use.

[0089] 1.4 C8+ T cell activation and proliferation

[0090] This experiment used the Miltenyi Biotec CD8+ T cell activation and expansion kit and the in vitro simulated microglia-mediated antigen presentation system established by the present invention to compare the advantages of the two methods. The specific implementation plan is as follows:

[0091] 1.4.1 Loading CD3 / CD28 mAb-coupled magnetic beads

[0092] (1) Fully resuspend the anti-biotin magnetic beads.

[0093] (2) Pipette 100 μl of CD3ε-biotin and 100 μl of CD28-biotin antibody into a 2 ml sterile low-adsorption centrifuge tube and mix thoroughly.

[0094] (3) Add 300 μL of PBS buffer containing 2 mM EDTA and mix well.

[0095] (4) Pipette 500 μl of the anti-biotin MACSiBead magnetic beads mixed in step (1) and add it to the antibody mixture and mix well.

[0096] (5) Incubate at 2-8°C on a tube rotator for 2 hours.

[0097] (6) Place the loaded CD3 / CD28 monoclonal antibody-coupled magnetic beads at 2-8°C for later use.

[0098] 1.4.2 MACSiBead combined with IL-2 induces CD8+ T cell activation and proliferation

[0099] According to the instructions, the steps are briefly described as follows:

[0100] 1) Pipette 40 μl of the CD3 / CD28 monoclonal antibody-coupled magnetic beads loaded above and add them to a 2 ml centrifuge tube.

[0101] 2) Add 1 ml of RPIM160 culture medium, mix thoroughly, and centrifuge at 300 g for 5 minutes.

[0102] 3) Discard the supernatant and resuspend the CD3 / CD28 monoclonal antibody-coupled magnetic beads in RPIM160 culture medium containing 10% fetal bovine serum, 1% double antibody and 50 U / ml IL-2.

[0103] 4) Adjust the concentration of CD8+ T cells prepared in step 1.1 to 4×10 cells using RPIM160 culture medium supplemented with 10% fetal bovine serum, 1% double antibody, and 50 U / ml IL-2. 6 cells / ml.

[0104] 5) Mix the CD3 / CD28 monoclonal antibody-coupled magnetic bead mixture from step 3) and the CD8+ T cell suspension from step 4) and add 2 ml / well to a 24-well plate.

[0105] 6) Incubate at 37°C and 5% CO2 for 2 days.

[0106] 7) On the second day, gently pipette up and down the 24-well plate with a 1 ml pipette to evenly resuspend the aggregated cells.

[0107] 8) Every 2-3 days, passage the expanded CD8+ T cells at a 1:2 ratio.

[0108] 9) On the 10th day, cells were collected and set aside.

[0109] 1.4.3 The present invention provides an in vitro simulated microglia-mediated antigen presentation system method to induce CD8+ T cell activation and proliferation

[0110] 1) The adult mouse microglia prepared in step 1.3 were cultured at a volume of 1.5 × 10 6 The cells were cultured at a concentration of 10 cells / ml in a fresh DMEM / F12 culture medium containing 10% fetal bovine serum and 1% double antibody at 37°C and 5% CO2 for 3 days before use.

[0111] 2) The loaded CD3 / CD28 monoclonal antibody-coupled magnetic beads prepared in step 1.4.1 were added to DMEM / F12 culture medium containing 10% fetal bovine serum, 1% bispecific antibody, and 500 ng / ml LPS or 0.5 mM / ml Aβ for later use.

[0112] 3) Adjust the concentration of CD8+ T cells prepared in step 1 to 4×10 cells in DMEM / F12 culture medium containing 10% fetal bovine serum, 1% double antibody and 500 ng / ml LPS or 0.5 mM / ml Aβ. 6 cells / ml.

[0113] 4) The CD3 / CD28 monoclonal antibody-coupled magnetic bead mixture from step 2) and the CD8+ T cell suspension from step 3) were mixed and added to the microglia culture plate cultured on the third day at 2 ml / well.

[0114] 5) Every 2-3 days, passage the expanded CD8+ T cells at a 1:3 ratio.

[0115] 6) On day 10, CD8+ T cells were collected for future use.

[0116] 1.5 Experimental Results

[0117] To compare the efficiency of commercial primary CD8+ T cell activation and expansion with the in vitro simulated microglia-mediated antigen presenting cell (APC) system method established by the present invention in inducing CD8+ T cell activation and proliferation, the present invention compared the commercial group, APC-LPS group, and APC-Aβ group (Figure 1). Among them, commercial-mock is a control group for commercial cell activation and expansion of T cells; APC-mock is a control group for in vitro simulated antigen presentation system activation and expansion of T cells (basically the same as the method provided in 1.4.3, except that LPS or Aβ is not added in step 2); Commercial is a commercial activation and expansion T cell group; APC-LPS is a group for LPS-induced in vitro simulated antigen presentation system activation and expansion of T cells; APC-Aβ is a group for Aβ-induced in vitro simulated antigen presentation system activation and expansion of T cells.

[0118] The results showed that the in vitro simulated antigen-presenting cell (APC) system exhibited a faster expansion rate in both the LPS and Aβ groups. This experiment proves that the in vitro simulated antigen-presenting cell (APC) system established by the present invention can induce the activation and expansion of CD8+ T cells more rapidly. Because the present invention uses the antigen stimulators LPS and Aβ instead of the recombinant protein IL-2, the in vitro expansion technology of CD8+ T cells is significantly reduced in cost and more economical and applicable.

[0119] Example 2: Isolation, activation and in vitro expansion of primary CD8+ T cells from the spleen

[0120] In this example, a CD8+ T cell magnetic bead separation kit was used to isolate splenic CD8+ T cells, and the cells were cultured, activated, and proliferated in vitro. The specific steps are as follows:

[0121] 2.1 Preparation of spleen single cell suspension

[0122] 1) Obtain fresh mouse spleen.

[0123] 2) Place the mouse spleen in a culture dish containing 5 ml of HBSS (Hanks' Balanced Salt Solution) buffer.

[0124] 3) Using a razor or surgical blade, carefully cut the spleen into small pieces (~0.2 cm 2 ).

[0125] 4) Myeloid cells were prepared as follows (proceed to step 5 for crude isolation): small pieces of spleen were digested with 5 ml of HBSS solution containing type IV collagenase (100 U / ml) and DNase (20 μg / ml) in 1% FBS at 37°C for 20–30 min.

[0126] 5) Add 1 mM / ml EDTA and incubate at room temperature for 5 minutes to terminate the enzymatic reaction.

[0127] 6) Place the cell strainer over the 50 ml conical tube.

[0128] 7) Using a disposable pipette, transfer the digested spleen to the cell strainer.

[0129] 8) Using the plunger end of a syringe, mash or crush the spleen to force it through the mesh. If necessary, rinse with 5–10 ml of PBS.

[0130] 9) Rinse the cells with plenty of PBS to allow them to pass through the mesh. Repeat steps 5 and 6 as needed.

[0131] 10) Centrifuge the cells at 400-600 x g for 5 minutes at 4°C and discard the supernatant.

[0132] 11) Resuspend cells in 2–5 ml of ice-cold 1x RBC lysis buffer.

[0133] 12) Place the resuspension on ice for 5 minutes. Wash the cell suspension with 10–20 ml of ice-cold PBS.

[0134] 13) Centrifuge the cells at 400-600 x g for 5 minutes at 4°C and discard the supernatant.

[0135] 14) Resuspend the cells in PBS to a cell concentration of 2 x 10 6 cells / ml.

[0136] 2.2 The methods for magnetic bead separation of spleen CD8+ T cells, isolation and culture of microglia, loading of CD3 / CD28 monoclonal antibody-coupled magnetic beads, commercial CD8+ T cell activation and expansion, and in vitro simulated antigen presenting cell (APC) system-induced CD8+ T cell activation and expansion were the same as in Example 1. They will not be described in detail here.

[0137] 2.3 Experimental Results

[0138] To further demonstrate the method for activating and expanding CD8+ T cells induced by an in vitro simulated antigen-presenting cell (APC) system, the present invention conducted a one-step validation in CD8+ T cells isolated from the spleen. The results are shown in Figure 2, where "commercial-mock" represents a control group for commercial cell activation and expansion of T cells; "APC-mock" represents a control group for in vitro simulated antigen presentation system activation and expansion of T cells; "Commercial" represents a commercial activated and expanded T cell group; "APC-LPS" represents a group activated and expanded T cells induced by LPS in vitro simulated antigen presentation system; and "APC-Aβ" represents a group activated and expanded T cells induced by Aβ in vitro simulated antigen presentation system.

[0139] The results showed that the in vitro simulated antigen-presenting cell (APC) system exhibited faster expansion rates in both the LPS and Aβ groups. This experiment demonstrates that the in vitro simulated antigen-presenting cell (APC) system established by the present invention can more rapidly induce the activation and expansion of CD8+ T cells. This further demonstrates that the present invention provides a faster, more efficient, and more economical CD8+ T cell activation and expansion technology.

[0140] Example 3: Aβ-activated microglia-induced CD8+ T cells have a chemotactic effect on neuroinflammation in the brain

[0141] The primary CD8+ T cells in this example were derived from peripheral blood. The commercialized, LPS-induced, and Aβ-induced CD8+ T cell activation and expansion CD+ T cells were subjected to T cell chemotaxis assays. The specific steps were as follows:

[0142] 3.1 The separation and activation of CD8+ T cells and the isolation and culture of microglia in this example are the same as those in Example 1 and will not be repeated here.

[0143] 3.2 CD8+ T cell chemotaxis assay

[0144] (1) 1x10 6 Microglial cells at a concentration of 10 cells / ml were added into a 24-well Trans-well cell culture plate at a volume of 1 ml / well.

[0145] (2) Culture at 37°C and 5% CO2 for 6 days, changing the medium every 3 days.

[0146] (3) On day 6, serum-free medium was added to the 24-well plate for 24 hours of starvation.

[0147] (4) On day 7, 1 ml of DMEM / F12 medium containing 10% fetal bovine serum and 1 μM / ml of β-catenin was added to a 24-well plate.

[0148] (5) Take 100 μl of 2x10 6 Commercial activated and expanded CD8+ T cells with a concentration of 10 cells / mL, and CD8+ T cells activated and expanded by LPS and Aβ-induced simulating in vitro antigen presentation system were added into the Trans-well chamber.

[0149] (6) Incubate the 24-well plate at 37°C and 5% CO2 for 24-48 hours.

[0150] 3.3 Cell immunofluorescence staining

[0151] (1) Cell fixation: Incubate with 4% paraformaldehyde at room temperature for 30 minutes and shake slowly on a shaker.

[0152] (2) Punching and blocking: 0.2% Triton X-100 (diluted with PBS), 0.1% BSA and 5% horse serum (diluted with PBS), incubate at room temperature for 40 minutes, and shake slowly on a shaker.

[0153] (3) Wash three times with PBS at room temperature, 5 min each time.

[0154] (4) Primary antibody incubation: dilute the plate with antibody diluent (PBS containing 0.01% BSA and 5% horse serum) at a ratio of 1:100, add 200 μL to each well, and incubate at 4°C with slow shaking overnight.

[0155] (5) Recover the primary antibody and wash three times with PBS at room temperature, each time for 10 min.

[0156] (6) Block the brain slices with 3% horse serum at room temperature for 30 minutes.

[0157] (7) Secondary antibody incubation and DAPI staining: dilute the secondary antibody in PBS at 1:5000 and incubate at room temperature in the dark for 2 h; dilute the DAPI storage solution at 1:5000 and incubate at room temperature for 15 min.

[0158] (8) Wash with PBS three times at room temperature, 15 minutes each time.

[0159] (9) Sealing: Take a sticky slide, mark the specific information on the right frosted surface with a pencil, drop a drop of PBS in the middle of the slide, dip the slice and place it on the PBS drop, remove the PBS solution, spread 160 μL of mounting medium horizontally in the center of the slide, and cover the slice with a long cover slip to avoid bubbles and wrinkles.

[0160] 3.4 Experimental Results

[0161] To verify the functional activity of CD8+ T cells activated and expanded by different methods, the present invention used Aβ to activate primary microglia to simulate brain neuroinflammation and brain neuroinflammation under AD pathological conditions. The results are shown in Figure 3. The CD8+ T cells induced by Aβ-activated microglia have a chemotactic effect on brain neuroinflammation. The results of immunofluorescence staining are shown. In the figure, Aβ represents the activation and expansion of CD8+ T cells by an Aβ-induced in vitro simulated antigen presentation system; LPS represents the activation and expansion of CD8+ T cells by an in vitro simulated antigen presentation system; and Commercial represents the activation and expansion of CD8+ T cells by commercial cells. The T cell surface marker is CD8, the microglia marker is Iba1, and the cell nuclear marker is DAPI.

[0162] The results showed that CD8 cells exhibited yellow-green fluorescence, Iba1 cells exhibited red fluorescence, and DAPI cells exhibited blue fluorescence. The Aβ-induced CD8+ T cell activation model simulated by an in vitro antigen presentation system demonstrated enhanced chemotaxis against microglia-mediated neuroinflammation. This demonstrates that the Aβ-induced CD8+ T cell activation model simulated by an in vitro antigen presentation system is an ideal cell model for studying neuroinflammation in the AD brain, providing a theoretical basis for clinical research on AD and cell therapy for AD patients.

Claims

1. A method for in vitro expansion of primary cytotoxic T cell subsets, characterized in that, Comprising the following steps: Co-culture CD8+ T cells and adult mouse microglia in a DMEM / F12 medium containing fetal bovine serum, double antibodies, and Aβ, and perform magnetic cell separation using CD3 / CD28 immunomagnetic beads.

2. The method for in vitro expansion of primary cytotoxic T cell subsets according to claim 1, characterized in that, Comprising the following steps: S1: Load CD3 / CD28 immunomagnetic beads, place them in a DMEM / F12 medium containing fetal bovine serum, double antibodies and Aβ, and adjust the final concentration of the immunomagnetic beads to 4×10 6 beads / mL; S2: Adjust the concentration of CD8+ T cells to 4×10 6 cells / ml with a culture medium of DMEM / F12 containing fetal bovine serum, double antibodies and Aβ; S3: Seed adult mouse microglia into fresh medium of DMEM / F12 containing fetal bovine serum and double antibody at a concentration of 1.5×10 6 cells / ml, culture for 3 days at 37°C and 5% CO2 concentration, add the mixtures of step S1 and step S2, and then passage and amplify at a ratio of 1:3 every 2-3 days until the 10th day.

3. The method for in vitro expansion of primary cytotoxic T cell subsets according to claim 1, characterized in that, In the DMEM / F12 medium containing fetal bovine serum, double antibodies, and Aβ in steps S1 and S2, the concentration of fetal bovine serum is 10%, the concentration of double antibodies is 1%, and the Aβ concentration is 0.5 mM / ml; In the DMEM / F12 medium containing fetal bovine serum and double antibodies in step S3, the concentration of fetal bovine serum is 10%, and the concentration of double antibodies is 1%; In step S3, the addition amount of the mixture of steps S1 and S2 is 2 ml / well according to a volume ratio of 1:

1.

4. The method for in vitro expansion of primary cytotoxic T cell subsets according to claim 1, characterized in that, Comprising the following steps: Loading CD3 / CD28 immunomagnetic beads in step S1 comprises the following steps: Add 100 μl of CD3ε-biotin and 100 μl of CD28-biotin antibodies, 300 μl of PBS buffer containing 2 mM EDTA, and 500 μl of mixed anti-biotin MACSiBead magnetic beads to a 2-ml sterile low-attachment centrifuge tube, and incubate at 2-8°C on a tube rotator for 2 hours.

5. A method for specifically regulating the expansion of cytotoxic T cells that mediate neuroinflammation in the brain of AD patients, characterized in that, Activate microglia with Aβ to induce the chemotaxis of activated cytotoxic T cell subsets to neuroinflammation in the AD brain.

6. A cytotoxic T cell model for regulating neuroinflammation in the brain of AD patients, characterized in that, Prepared by the method for specifically regulating the expansion of cytotoxic T cells for neuroinflammation in the AD brain as described in claim 5.

7. A microglia-mediated antigen presentation system, characterized in that, Comprising Aβ and microglia.

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