Method for in-vitro amplification of primary cytotoxic T cell subpopulation

Abstract

The invention belongs to a biological and new drug technology, and provides a method for in vitro amplification of a primary cytotoxic T cell subset. The method comprises the following steps: co-culturing CD8 + T cells and adult mouse microglia cells in a DMEM / F12 culture medium containing fetal calf serum, double antibodies and A beta, and carrying out magnetic cell separation by using CD3 / CD28 immunomagnetic beads. According to the embodiment provided by the invention, primary CD8 + T cells separated from peripheral blood and spleen of a surface AD mouse and intracerebral microglial cells are subjected to in-vitro cell co-culture, a glial cell mediated antigen presentation system is simulated, and the in-vitro amplification speed of cytotoxic T cells can be quickly increased and is 1.5 times that of conventional T cell amplification speed. Moreover, in the process, APC does not need to be artificially synthesized, so that the cost of in-vitro amplification of the cytotoxic T cells is greatly saved.

Description

Technical Field

[0001] The present invention belongs to the field of biology and new drugs, and particularly relates to a method for in vitro expanding primary cytotoxic T cell subsets. Background Art

[0002] Cytotoxic T cells, also known as CD8+ T cells, are key components 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 as the key effector cells of the main anti-tumor response, capable of directly killing tumor cells, and they are key immune surveillance cells for cell therapy of tumors. Therefore, at the present stage in the research of tumor cell therapy, a high peak level of CD8+ T cells with tumor-killing function in tumor tissues is an important indicator for improving the patient's ability to inhibit tumor progression and even ultimately eliminate tumors.

[0003] In recent years, more and more studies have found that peripheral immune cells play an important role in promoting the progression of AD disease. Further research has found that peripheral CD8+ T cells can infiltrate into the AD brain and, by activating microglia, further exacerbate the progression of AD disease. This finding confirms that peripheral circulating immune cells play an important role in immune surveillance in the occurrence and development of AD, providing a theoretical and preclinical basis for finding biomarkers for the diagnosis and treatment of AD from peripheral blood. Currently, the methods for clinically diagnosing AD include detecting the content of Aβ and tau in cerebrospinal fluid and imaging techniques such as amyloid β-PET and tau-PET pathological scans. These methods are either invasive, with a high risk of intracranial infection; or the detection costs are very expensive, or both. Therefore, blood-based biomarkers or drug targets will provide the possibility of safe, minimally invasive, and low-cost clinical diagnosis and treatment of AD.

[0004] Based on this, whether CD8+ T cells are key immune cells in the main anti-tumor response or effector cells that regulate neuroinflammation in the brain during the AD process, T cell therapy is one of the future development methods for treating various diseases, and has shown unprecedented clinical success cases in the treatment of solid tumors and B cell acute lymphoblastic leukemia using CAR-T. However, the precise functional subset classification, in vitro proliferation ability, and expansion speed 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 spatio-temporal pattern. Among them, synthetic artificial APCs (aAPCs) are particularly convenient for polyclonal T cell expansion. Currently, commercial microbeads (Dynabeads) functionalized with activating antibodies against CD3 (αCD3; TCR stimulation) and CD28 (αCD28; co-stimulation signal) represent one of the most commonly used and clinically relevant synthetic systems. These beads promote polyclonal T cell activation with exogenous interleukin-2 (IL-2) supplementation. Although these cultures provide the three key signals for T cells, the presentation context of these signals does not represent how they are naturally presented by antigen-presenting cells. This may lead to suboptimal T cell expansion rates, limited function, or dysfunctional T cell products. Summary of the Invention

[0005] The present invention provides a method for proliferating in vitro cytotoxic T cells with low cost, short time consumption, and specific function, that is, a method for expanding primary cytotoxic T cell subsets in vitro.

[0006] A method for expanding primary cytotoxic T cell subsets in vitro includes the following steps:

[0007] 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.

[0008] Furthermore, the method for expanding primary cytotoxic T cell subsets in vitro includes the following steps:

[0009] 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 cells / ml;

[0010] S2: Adjust the concentration of CD8+ T cells to 4×10 6 cells / ml with a DMEM / F12 medium containing fetal bovine serum, double antibodies, and Aβ;

[0011] S3: Seed adult mouse microglia into a fresh medium of DMEM / F12 containing fetal bovine serum and double antibodies at a concentration of 1.5×10 6 cells / ml, culture for 3 days at 37 °C and 5% CO2 concentration, add the mixture 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.

[0012] Furthermore, in the medium of DMEM / F12 containing fetal bovine serum, double antibodies and Aβ in Step S1 and Step S2, the concentration of fetal bovine serum is 10%, the concentration of double antibodies is 1%, and the concentration of Aβ is 0.5 mM / ml;

[0013] In the medium of DMEM / F12 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%;

[0014] In Step S3, the addition amount of the mixture of Step S1 and Step S2 at a volume ratio of 1:1 is 2 ml / well.

[0015] Furthermore, it includes the following steps: Loading CD3 / CD28 immunomagnetic beads in Step S1 includes the following steps:

[0016] Add 100 μl of CD3ε-biotin, 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 into a 2 ml sterile low-attachment centrifuge tube, and incubate at 2 - 8 °C on a tube rotator for 2 hours.

[0017] An object of the present invention is to provide a method for specifically regulating the expansion of cytotoxic T cells in AD brain neuroinflammation: the chemotactic effect of the cytotoxic T cell subset amplified by the method of activating microglia with Aβ to induce the activation of the cytotoxic T cell subset on AD brain neuroinflammation.

[0018] An object of the present invention is to provide a cytotoxic T cell model for regulating AD brain neuroinflammation, which is prepared by the method for specifically regulating the expansion of cytotoxic T cells in AD brain neuroinflammation as described above.

[0019] An object of the present invention is to provide a microglia-mediated antigen presentation system, including Aβ and microglia.

[0020] The primary CD8+ T cells isolated from the peripheral blood and spleen of surface AD mice and microglial cells in the brain provided by the embodiments of the present invention can rapidly increase the in vitro amplification rate of cytotoxic T cells, which is 1.5 times that of the conventional T cell amplification rate, by simulating the glial cell-mediated antigen presentation system through an in vitro cell co-culture method. Moreover, during this process, artificial synthesis of APC is not required, greatly saving the cost of in vitro amplification of cytotoxic T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Efficiency of inducing CD8+ T cell activation and proliferation by the in vitro simulated microglial cell-mediated antigen presenting cell system method provided in Example 1;

[0022] Figure 2 Efficiency of inducing CD8+ T cell activation and proliferation by the in vitro simulated microglial cell-mediated antigen presenting cell system method provided in Example 2;

[0023] Figure 3 Chemotaxis of Aβ-induced CD8+ T cells activated by the simulated in vitro antigen presenting system to neuroinflammation provided in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0024] In order to make the above objects, features and advantages of the present invention more obvious and understandable, the following detailed description of the specific embodiments of the present invention is made in conjunction with the accompanying drawings, but it should not be construed as a limitation on the implementable scope of the present invention.

[0025] The materials involved in the present invention are as follows:

[0026] Littermate wild-type mice and APP / PS1 mouse models are from Jackson Laboratory, USA;

[0027] Red blood cell lysate is purchased from BD Biosciences, product number: 555899;

[0028] DPBS is purchased from Sigma, product number: D8662-24*500ML;

[0029] Horse serum is purchased from Gibco, product number: 26050088;

[0030] Fetal bovine serum is purchased from Life Technologies, product number: 16050-122;

[0031] Cell culture dishes are purchased from Thermo Scientific Nunc EasYDish 100mm, product number: 150466;

[0032] 40 μm cell strainer (e.g., Fisherbrand sterile cell strainer, catalog number: 22-363-548)

[0033] Hanks' Balanced Salt Solution was purchased from Gibco, catalog number: 14060040;

[0034] F12 medium;

[0035] RPIM1640 medium;

[0036] Ms CD45 FITC 30-F11, catalog number: 553079;

[0037] Ms CD3 MolCpx PerCP-Cy5.5 17A2, catalog number: 560527;

[0038] Ms CD8a PE 53-6.7, catalog number: 553032;

[0039] CD8 primary antibody was purchased from Invitrogen, catalog number: 14-0195-82;

[0040] Fluorescent secondary antibody was purchased from Thermo scientific;

[0041] DAPI was purchased from Thermo scientific, catalog number: 62248;

[0042] MgniSort TM Mouse CD8+ T cell enrichment kit was purchased from ThermoFisher, catalog number: 8802-6842;

[0043] T cell activation and expansion kit was purchased from Miltenyi Biotec, catalog number: 130-093-627;

[0044] Ficoll was purchased from Cytia, catalog number: 17144002;

[0045] Percoll was purchased from Merck, catalog number P1644-100ML;

[0046] Trans-well was purchased from Corning, catalog number: 3422;

[0047] Aβ was purchased from rPeptide, catalog number: A-1163-2;

[0048] LPS was purchased from InvivoGen, catalog number: tlrl-eblps.

[0049] Example 1: Isolation, activation, and in vitro expansion of primary CD8+ T cells from peripheral blood

[0050] In this example, CD8+ T cells in peripheral blood were isolated using peripheral blood mononuclear cell (PBMCs) isolation technology and a CD8+ T cell magnetic bead isolation kit, and then cultured, activated, and proliferated in vitro. The specific steps are as follows:

[0051] 1.1 Isolation of PBMCs by density gradient centrifugation

[0052] (1) Take about 1 ml of fresh anticoagulated whole blood from mice, add DPBS buffer at a ratio greater than 1:2, and fix the total volume of the diluted anticoagulated blood to 4 ml for dilution. Mix well by inverting up and down and set aside.

[0053] (2) Add 4 ml of Ficoll density gradient separation solution to a 15-ml sterile centrifuge tube, and slowly add the diluted anticoagulated blood above the liquid surface of the Ficoll separation solution (separation solution: diluted whole blood = 1:1), keeping the interface between the two liquid surfaces clear.

[0054] (3) Density gradient centrifugation: Centrifuge at 800×g at room temperature for 40 min using a horizontal centrifuge (the high-speed refrigerated centrifuge is purchased from ThermoFisher, model: Sorvall AT8R).

[0055] In this step, the centrifuge is set to start and decelerate in a slow acceleration and slow deceleration mode.

[0056] (4) After centrifugation, carefully take out the centrifuge tube, and carefully aspirate the PBMCs layer (i.e., the buffy coat) between the uppermost light yellow plasma layer and the middle transparent separation solution with a 1-ml pipette tip, and transfer it to a 15-ml centrifuge tube.

[0057] The above 4 steps are all carried out at room temperature of 18°C - 22°C.

[0058] (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 then discard the supernatant.

[0059] (6) Erythrocyte lysis: After centrifugation, discard the supernatant, add 5 ml of erythrocyte lysis solution, lyse at room temperature for 5 min, then add 10 ml of PBS, and centrifuge at 400g for 5 min.

[0060] (7) PBMCs washing: Add 10 ml of cold (2°C - 8°C) DPBS to the centrifuge tube to resuspend the cells, centrifuge at 400g,

[0061] (2°C - 8°C) for 5 min, and then discard the supernatant.

[0062] (8) PBMC purification: Add 10 mL of cold (2°C - 8°C) DPBS to a centrifuge tube to resuspend the cells, centrifuge at 250 g,

[0063] (2°C - 8°C) for 5 min, then discard the supernatant. Repeat this step once, and it can be used for subsequent experiments.

[0064] Reducing the centrifugal force of the centrifuge in this step can remove cell debris in PBSCs.

[0065] 1.2 Isolation and purification of CD8+ T cells in PBMCs

[0066] This experiment was carried out using an Invitrogen TM CD8+ T cell isolation and purification kit. The specific steps are briefly described as follows:

[0067] (1) Resuspend the PBMCs prepared in step 1 in sorting buffer and adjust the cell density to 1×10 8 cells /

[0068] mL.

[0069] The sorting buffer is DPBS containing 2 mM EDTA and 2% fetal bovine serum (FBS). The prepared buffer needs to be pre-filtered and sterilized through a 0.22 μm filter membrane.

[0070] (2) Add 100 μL of cell suspension (1×10 7 cells) to a low-attachment centrifuge tube, then add 10 μL of CD8 antibody, mix well and incubate at 4°C for 10 min.

[0071] (3) After incubation, add 10 μL of washed MgniSort magnetic beads (the magnetic beads need to be vortexed and resuspended before use) and incubate at 4°C for 10 min.

[0072] (4) After incubation, add 2.5 mL of sorting buffer to the centrifuge tube and mix well by pipetting up and down 5 times (avoid vigorous shaking or inverting to mix).

[0073] (5) Place the centrifuge tube containing the cells on a magnetic stand and let it stand for 10 min.

[0074] (6) Slowly discard the liquid in the centrifuge tube with a pipette (the flow tube should not be removed from the magnetic stand during the liquid aspiration process). The adsorbed magnetic beads contain purified mouse CD8+ T cells.

[0075] (7) Washing: Detach the centrifuge tube from the magnetic stand, add 2.5 mL of sorting buffer, mix well by pipetting up and down 5 times, place it on the magnetic stand, and let it stand for 10 min. Slowly discard the liquid in the centrifuge tube using a pipette (during the process of aspirating the liquid, the flow cytometry tube should not be detached from the magnetic stand).

[0076] (8) Discard the supernatant and collect the cells.

[0077] (9) After washing the cells, resuspend the cells in the required culture medium, which can be used for subsequent T cell activation and proliferation experiments.

[0078] 1.3 Isolation and culture of primary microglia from adult mice

[0079] The primary microglia used in this example are derived from the microglia of the cerebral cortex of 6-month-old wild-type mice and APP / PS1. The steps are as follows:

[0080] (1) Under sterile conditions, take out the whole brain of the mouse, remove the medulla oblongata, pons, cerebellum, etc., carefully separate the left and right cerebral hemispheres, remove the olfactory bulb, striatum, hippocampus, basal brain tissue, etc., and carefully transfer the remaining cortical tissue to another small culture dish (D = 1 cm) containing ice-cold dissection solution, and gently remove the pia mater and surface blood vessels.

[0081] (2) Chop the cortical tissue with a surgical blade, add trypsin containing EDTA, place it in a cell culture dish, and digest at 37 °C

[0082] for 30 - 45 minutes.

[0083] (3) After digestion, add three volumes of the solution containing 10% fetal bovine serum and 1% double antibody to terminate the digestion.

[0084] (4) Pass the digested cerebral cortex mixture through a 40-μm filter.

[0085] (5) Centrifuge the filtered cell suspension at 300 g for 7 minutes at 18 - 22 °C.

[0086] (6) Microglia density gradient centrifugation: After discarding the supernatant, resuspend the cells in 4 mL of 37% Percoll separation solution.

[0087] And slowly spread it evenly in a 15-mL centrifuge tube containing 4 mL of 70% Percoll separation solution, then slowly spread 4 mL of 30% Percoll on the cell suspension, and finally add 2 mL of HBSS. This step must have a clear gradient stratification.

[0088] (7) Centrifuge in a horizontal centrifuge at 18 - 22 °C and 300 g for 40 min.

[0089] In this step, the centrifuge startup and spin-down process are set to slow speed up and slow speed down mode.

[0090] (8) After centrifugation, collect 37-70% of the cells into a 15 ml centrifuge tube.

[0091] (9) Add 10 ml of HBSS, mix well, and centrifuge at 18-22°C, 400 g for 7 minutes. Repeat the washing process once.

[0092] (10) The obtained microglial cells were resuspended in DMEM / F12 containing 10% fetal bovine serum and 1% double antibody for later use.

[0093] 1.4C8+ T cell activation and proliferation

[0094] This experiment uses the Miltenyi Biotec CD8+ T cell activation and expansion kit and the in vitro simulated microglia-mediated antigen presentation system method established by the present invention to compare the advantages of the two methods. The specific implementation plan is as follows:

[0095] 1.4.1 Loading CD3 / CD28 mAb-coupled magnetic beads

[0096] (1) Fully resuspend the anti-biotin magnetic beads.

[0097] (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.

[0098] (3) Add 300 μl of PBS buffer containing 2 mM EDTA and mix well.

[0099] (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.

[0100] (5) Incubate at 2-8°C on a test tube rotator for 2 hours.

[0101] (6) Place the loaded CD3 / CD28 monoclonal antibody-coupled magnetic beads at 2-8°C for later use.

[0102] 1.4.2MACSiBead combined with IL-2 induces CD8+ T cell activation and proliferation

[0103] According to the instructions, the steps are briefly described as follows:

[0104] 1) Pipette 40 μl of the above loaded CD3 / CD28 monoclonal antibody-coupled magnetic beads into a 2 ml centrifuge tube.

[0105] 2) Add 1 mL of RPMI 160 medium, mix well, and centrifuge at 300 g for 5 minutes.

[0106] 3) Discard the supernatant, and resuspend the CD3 / CD28 monoclonal antibody-conjugated magnetic beads with RPMI 160 medium containing 10% fetal bovine serum, 1% double antibody, and 50 U / mL of IL-2.

[0107] 4) Adjust the concentration of the CD8+ T cells prepared in step 1.1 to 4×10 6 cells / mL with RPMI 160 medium containing 10% fetal bovine serum, 1% double antibody, and 50 U / mL of IL-2.

[0108] 5) Mix the CD3 / CD28 monoclonal antibody-conjugated magnetic bead mixture in step 3) and the CD8+ T suspension in step 4), and add 2 mL / well to a 24-well plate.

[0109] 6) Incubate for 2 days at 37 °C and 5% CO2 concentration.

[0110] 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.

[0111] 8) Passage the amplified CD8+ T cells at a ratio of 1:2 every 2 - 3 days.

[0112] 9) Collect the cells on the tenth day for standby.

[0113] 1.4.3 Method for inducing CD8+ T cell activation and proliferation by the in vitro simulated microglia-mediated antigen presentation system provided by the present invention

[0114] 1) Culture the adult mouse microglia prepared in step 1.3 at a concentration of 1.5×10 6 cells / mL in fresh DMEM / F12 medium containing 10% fetal bovine serum and 1% double antibody at 37 °C and 5% CO2 concentration for 3 days for standby.

[0115] 2) Place the loaded CD3 / CD28 monoclonal antibody-conjugated magnetic beads prepared in step 1.4.1 in DMEM / F12 medium containing 10% fetal bovine serum, 1% double antibody, and 500 ng / mL of LPS or 0.5 mM / mL of Aβ for standby.

[0116] 3) Adjust the concentration of the CD8+ T cells prepared in step 1 to 4×10 6 cells / mL with DMEM / F12 medium containing 10% fetal bovine serum, 1% double antibody, and 500 ng / mL of LPS or 0.5 mM / mL of Aβ.

[0117] 4) Mix the CD3 / CD28 monoclonal antibody conjugated magnetic bead mixture in step 2) and the CD8+ T suspension in step 3), and add 2 mL per well to the microglial cell culture plate on the third day of culture.

[0118] 5) Passage and amplify the CD8+ T cells at a ratio of 1:3 every 2 - 3 days.

[0119] 6) Collect the CD8+ T cells on the 10th day for standby.

[0120] 1.5 Experimental results

[0121] To compare the efficiency of activating and amplifying commercial primary CD8+ T cells with that of inducing the activation and proliferation of CD8+ T cells by the in vitro simulated microglia-mediated antigen-presenting cell (APC) system method established in the present invention, the present invention makes a comparison among the commercial group, the APC-LPS group and the APC-Aβ group ( Figure 1 ). Among them, commercial-mock is the control group for activating and amplifying T cells with commercial cells; APC-mock is the control group for activating and amplifying T cells with the in vitro simulated antigen-presenting system (basically the same as the method provided in 1.4.3, the difference is that LPS or Aβ is not added in step 2)); Commercial is the group for activating and amplifying T cells with commercial cells; APC-LPS is the group for activating and amplifying T cells with the in vitro simulated antigen-presenting system induced by LPS; APC-Aβ is the group for activating and amplifying T cells with the in vitro simulated antigen-presenting system induced by Aβ.

[0122] The results show that the in vitro simulated antigen-presenting cell (APC) system exhibits a faster amplification rate in both the LPS and Aβ groups. This experiment proves that the in vitro simulated antigen-presenting cell (APC) system established in the present invention can more rapidly induce the activation and amplification of CD8+ T cells. Since the present invention uses the antigen stimulants LPS and Aβ to replace the recombinant protein IL-2, the cost of the in vitro amplification technology of CD8+ T cells is greatly reduced, making it more economical and applicable.

[0123] Example 2: Isolation, activation and in vitro amplification of primary CD8+ T cells in the spleen

[0124] In this example, the CD8+ T cells in the spleen are isolated using a CD8+ T cell magnetic bead isolation kit from the spleen, and are cultured, activated and proliferated in vitro. The specific steps are as follows:

[0125] 2.1 Preparation of single cell suspension from the spleen

[0126] 1) Obtain fresh mouse spleens.

[0127] 2) Put the mouse spleens into a culture dish containing 5 mL of HBSS (Hanks' balanced salt solution) buffer.

[0128] 3) Using a razor or scalpel, carefully cut the spleen into small pieces (~0.2 cm 2 ).

[0129] 4) Myeloid cell preparation is as follows (continue with step 5 for crude separation): At 37 °C, digest the small pieces of spleen with 5 ml of HBSS solution containing collagenase type IV (100 U / ml) and DNase (20 μg / ml) with 1% FBS for 20–30 minutes.

[0130] 5) Add 1 mM / ml EDTA and let it stand at room temperature for 5 minutes to terminate the enzymatic reaction.

[0131] 6) Place the cell strainer above a 50-ml conical tube.

[0132] 7) Using a disposable pipette, transfer the digested spleen to the cell strainer.

[0133] 8) Using the plunger end of a syringe, mash or crush the spleen to pass it through the strainer. If necessary, add 5–10 ml of PBS for rinsing.

[0134] 9) Rinse the cells with a large amount of PBS to pass them through the strainer. If needed, repeat steps 5 and 6.

[0135] 10) Centrifuge the cells at 400 - 600 x g for 5 minutes at 4 °C and discard the supernatant.

[0136] 11) Resuspend the cells in 2–5 ml of pre-cooled 1x RBC lysis buffer.

[0137] 12) Place the resuspended solution on ice for 5 minutes. Wash the cell suspension with 10–20 ml of ice-cold PBS.

[0138] 13) Centrifuge the cells at 400 - 600 x g for 5 minutes at 4 °C and discard the supernatant.

[0139] 14) Resuspend the cells in PBS to a cell concentration of 2x10 6 cells / ml.

[0140] 2.2 The methods of magnetic bead separation of spleen CD8+ T cells, microglia separation and culture, loading of CD3 / CD28 monoclonal antibody-conjugated magnetic beads, and commercial CD8+ T cell activation and expansion, and the method of inducing CD8+ T cell activation and expansion by an in vitro simulated antigen-presenting cell (APC) system are the same as those in Example 1. They will not be elaborated here.

[0141] 2.3 Experimental results

[0142] To further prove the method for inducing CD8+ T cell activation and expansion by the in vitro simulated antigen-presenting cell (APC) system involved in the present invention, the present invention conducts a further verification in CD8+ T cells isolated from the spleen. The results are as Figure 2 shown, where commercial-mock is the control group for commercial cell activation and expansion of T cells; APC-mock is the control group for activation and expansion of T cells by the in vitro simulated antigen-presenting system; Commercial is the group for commercial activation and expansion of T cells; APC-LPS is the group for activation and expansion of T cells by the in vitro simulated antigen-presenting system induced by LPS; APC-Aβ is the group for activation and expansion of T cells by the in vitro simulated antigen-presenting system induced by Aβ.

[0143] The results show that: The in vitro simulated antigen-presenting cell (APC) system shows 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 more rapidly induce the activation and expansion of CD8+ T cells. It further proves that the present invention provides a faster, more efficient, and more economical technology for CD8+ T cell activation and expansion.

[0144] Example 3: CD8+ T cells induced by Aβ-activated microglia have chemotactic effects on intracerebral neuroinflammation

[0145] The primary CD8+ T cells in this example are derived from peripheral blood. T cell chemotaxis experiments are performed on the above-mentioned CD+ T cells after commercial, LPS-induced, and Aβ-induced CD8+ T cell activation and expansion. The specific steps are as follows:

[0146] 3.1 The isolation, 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 elaborated here.

[0147] 3.2 CD8+ T cell chemotaxis experiment

[0148] (1) Add microglia at a density of 1x10 6 cells / ml at a volume of 1 ml / well into a 24-well Trans-well cell culture plate.

[0149] (2) Culture at 37°C and 5% CO2 concentration for 6 days, and change the medium every 3 days.

[0150] (3) On the 6th day, add serum-free medium to the 24-well plate and starve for 24 hours.

[0151] (4) On the 7th day, add 1 ml of DMEM / F12 medium containing 10% fetal bovine serum and 1 μM / ml to the 24-well plate.

[0152] (5) Take 100 μl of 2x106 Commercially activated and expanded CD8+ T cells at [X] cells / mL, LPS, and CD8+ T cells induced to activate and expand by simulating an in vitro antigen presentation system with Aβ were added to the Trans-well chambers.

[0153] (6) Incubate the 24-well plate at 37°C and 5% CO2 for 24 - 48 hours.

[0154] 3.3 Cellular immunofluorescence staining

[0155] (1) Cell fixation: Incubate with 4% paraformaldehyde at room temperature for 30 minutes and place on a shaker for slow shaking.

[0156] (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 min and place on a shaker for slow shaking.

[0157] (3) Wash 3 times with PBS at room temperature for 5 minutes each time.

[0158] (4) Primary antibody incubation: Dilute the antibody with antibody diluent (containing 0.01% BSA and 5% horse serum in PBS) at 1:100, add 200 μL to each well, and incubate at 4°C with slow shaking overnight.

[0159] (5) Recover the primary antibody, wash 3 times with PBS at room temperature for 10 minutes each time.

[0160] (6) Block the brain slices with 3% horse serum at room temperature for 30 minutes.

[0161] (7) Secondary antibody incubation and DAPI staining: Dilute the secondary antibody with PBS at 1:5000, incubate at room temperature in the dark for 2 h; dilute the DAPI stock solution at 1:5000 and incubate at room temperature for 15 min.

[0162] (8) Wash 3 times with PBS at room temperature for 15 minutes each time.

[0163] (9) Mounting: Take a sticky glass slide, mark the specific information with a pencil on the frosted side on the right, place a drop of PBS in the middle of the slide, pick up the section and place it on the PBS droplet, suck out the PBS solution, spread 160 μL of mounting medium horizontally in the center of the slide, cover the section with a long coverslip, avoiding the formation of air bubbles and wrinkles.

[0164] 3.4 Experimental results

[0165] To verify the functional activities of CD8+ T cells activated and expanded by different methods, the present invention simulated the in-brain neuroinflammation and the in-brain neuroinflammation under the AD pathological state by activating primary microglia with Aβ. The results are as Figure 3As shown in the figure, it is a result picture of immunofluorescence staining showing that CD8+ T cells induced by Aβ-activated microglia have chemotactic effects on neuroinflammation in the brain. In the figure, Aβ represents the activation and amplification of CD8+ T cells by an in vitro simulated antigen presentation system induced by Aβ; LPS represents the activation and amplification of CD8+ T cells by an in vitro simulated antigen presentation system induced by LPS; Commercial represents the activation and amplification of CD8+ T cells by a commercial cell activation system. Among them, the surface marker of T cells is CD8, the marker of microglia is Iba1, and the nuclear marker is DAPI.

[0166] The results show that the CD8+ T cells activated by the Aβ-induced in vitro simulated antigen presentation system involved in the present invention have better chemotactic effects on microglia-mediated neuroinflammation. Furthermore, it shows that the CD8+ T cell model activated by the Aβ-induced in vitro simulated 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 expanding a primary cytotoxic T cell subset, 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 expanding a primary cytotoxic T cell subset as described in claim 1, wherein, 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 using a 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 antibiotics 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 expanding primary cytotoxic T cell subsets according to claim 1, characterized in that, In the medium of DMEM / F12 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 concentration of Aβ is 0.5 mM / ml; In the medium of DMEM / F12 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 according to a volume ratio of 1:1 is 2 ml / well.

4. The method for in vitro amplifying 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, 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 into 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 regulate neuroinflammation in the brain of patients with Alzheimer's disease, characterized in that, Use Aβ to activate microglia 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 patients with Alzheimer's disease, characterized in that, Prepared by the method for specifically regulating the expansion of cytotoxic T cells for neuroinflammation in the AD brain according to claim 5.

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

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