Construction method and application of in-vitro depletion type CD8 + T cell mouse model

Abstract

The invention discloses a construction method and application of an in-vitro depletion type CD8 + T cell mouse model. The construction method comprises the following steps: S1, preparing antibody coating liquid and pre-coating plate holes; s2, separating, extracting and culturing mouse CD8 + T cells; s3, carrying out induction establishment on early-stage depletion type CD8 + T cells; s4, establishment of induction of mid-term depletion type CD8 + T cells; and S5, induction establishment of the late exhaustion type CD8 + T cells. The three-high characteristics of the in-vitro depletion CD8 + T cell model, namely high phenotype fidelity, high cell survival rate and high data reproducibility, are realized for the first time, a standardized platform is provided for immune depletion mechanism research, immune checkpoint drug development and combined treatment strategy optimization, and the method has remarkable industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of immunological research technology, and in particular relates to a method for constructing an in vitro depleted CD8+ T cell mouse model and its application. Background Technology

[0002] CD8+ T cell exhaustion is a core mechanism of immune response failure in pathological states such as chronic infections (e.g., HIV, HCV), tumors, and autoimmune diseases. Exhausted CD8+ T cells gradually lose their effector function and persistently overexpress multiple inhibitory receptors (e.g., PD-1, LAG-3, TIM-3), making them the primary targets of current tumor immunotherapy (e.g., PD-1 / PD-L1 inhibitors). Therefore, a thorough understanding of the mechanisms underlying T cell exhaustion is crucial for developing new immunotherapies.

[0003] Currently, research in this field heavily relies on two classic in vivo induction models: one is a chronic infection model based on clonal strain 13 of lymphocytic choriomeningitis virus (LCMV); the other is a tumor microenvironment model constructed by transplanting tumor cells. While these in vivo models can effectively simulate the exhaustion process under physiological conditions, their inherent limitations are also quite prominent: First, the experimental cycle can last for weeks or even months, resulting in high time and economic costs; second, due to differences in the immune systems of individual experimental animals, the models have poor reproducibility and comparability, making it difficult to conduct standardized, high-throughput drug screening; third, the in vivo environment is complex, with intertwined factors, making it difficult to accurately analyze the independent role of specific signaling pathways in the exhaustion process.

[0004] To overcome the limitations of in vivo models, some studies have attempted to induce T cell exhaustion through short-term in vitro antigen stimulation. However, such methods often fail to mimic the key microenvironmental characteristic of "continuous antigen stimulation" in vivo, which leads to exhaustion. This results in unstable cell states and an inability to reliably and synchronously express multiple exhaustion markers such as PD-1, LAG-3, and TIM-3 at high levels, leading to significant differences in functional phenotypes compared to real exhausted T cells.

[0005] In summary, the core technical challenges faced by existing technologies lie in: 1. How to effectively simulate and maintain the key microenvironment of "continuous antigen stimulation" in an in vitro environment; 2. How to achieve a stable, high-expression phenotype of multiple exhaustion markers that is highly consistent with in vivo exhausted T cells; 3. How to maintain basic cell survival and functional detectability while inducing deep exhaustion, so as to meet the needs of subsequent mechanism research and drug screening.

[0006] Therefore, developing an in vitro CD8+ T cell exhaustion model that is easy to construct, cost-effective, phenotypically stable, and highly simulates the in vivo state is of vital importance for accelerating the study of the basic mechanisms of T cell exhaustion and the development of innovative drugs. Summary of the Invention

[0007] To address the problems of long construction cycles, incomplete depletion phenotypes, and low cell survival rates in existing models, this invention provides a method for constructing an in vitro depleted CD8+ T cell mouse model and its application. For the first time, it achieves the "three high characteristics" of an in vitro depleted CD8+ T cell model—high phenotypic fidelity, high cell survival rate, and high data reproducibility. This provides a standardized platform for the study of immune depletion mechanisms, the development of immune checkpoint drugs, and the optimization of combination therapy strategies, and has significant industrial application value.

[0008] To achieve the above objectives, this application adopts the following technical solution: In a first aspect, the present invention provides a method for constructing an in vitro depleted CD8+ T cell mouse model, comprising the following steps: S1, Antibody coating solution preparation and plate well pre-coating: Prepare antibody coating solution, then pre-coat the antibody onto the plate wells, aspirate the liquid in the wells, and prepare a coated antibody plate. The antibody coating solution includes anti-CD3 antibody, anti-CD28 antibody and PBS solution. S2, Isolation, extraction and culture of mouse CD8+ T cells: Mouse CD8+ T cells were extracted to obtain cell slurry, and then T cell complete culture medium and IL-2 were added to prepare a cell culture suspension. The cell culture suspension was added to the antibody-coated plate prepared in S1 and then cultured. S3, Induction and establishment of early exhausted CD8+ T cells: On the 3rd day of cell culture, the liquid in the well was aspirated and transferred to a centrifuge tube. Complete T cell culture medium was added to the well, and the bottom of the plate was gently blown until the cells detached. Then, the liquid in the well was aspirated and collected into the same centrifuge tube. IL-2 was added to the centrifuge tube to make a cell suspension. The cell suspension was added to the coated antibody plate prepared in S1. Centrifugation was performed to allow the cells to settle and make full contact with the coating surface, thus obtaining early exhausted CD8+ T cells, which were then cultured for a period of time. S4, Induction and establishment of mid-stage exhausted CD8+ T cells: On day 5 of cell culture, the liquid in the wells was aspirated and transferred to a centrifuge tube. Complete T cell culture medium was added to the wells, and the bottom of the wells was gently blown until the cells detached. The liquid in the wells was then aspirated and collected into the same centrifuge tube. IL-2 was added to the centrifuge tube to prepare a cell suspension. The cell suspension was added to the antibody-coated well plate prepared in S1. The plate was centrifuged to allow the cells to settle and make full contact with the coating surface, thus obtaining mid-stage exhausted CD8+ T cells, which were then cultured for a period of time. S5, Induction and establishment of late-stage exhausted CD8+ T cells: On day 7 of cell culture, cells are collected to obtain late-stage exhausted CD8+ T cells.

[0009] In the above technical solution, in S1, the final concentrations of both anti-CD3 antibody and anti-CD28 antibody in the PBS solution are 5 μg / mL.

[0010] In the above technical solution, the preparation method of T cell complete culture medium is as follows: taking 50 mL as an example, take 44 mL of RPMI-1640 basal culture medium, add 5 mL of fetal bovine serum, 500 μL of 200 mM L-glutamine, 500 μL of penicillin-streptomycin solution and 0.05 mM β-mercaptoethanol, mix thoroughly, filter through a 0.22 μm filter membrane for sterilization, and store at 4 ℃ for later use.

[0011] Secondly, the present invention provides an in vitro depleted CD8+ T cell mouse model, which is constructed using the above-described construction method.

[0012] Thirdly, this invention provides the application of the above-mentioned mouse model in the study of CD8+ T cell exhaustion mechanisms.

[0013] Fourthly, the present invention provides the application of the above-mentioned cell mouse model in screening or evaluating synergistic drugs or gene targets for preventing or reversing the depletion of therapeutic T cells in vivo.

[0014] Fifthly, this invention provides the application of the aforementioned mouse model in studying the specific function of the CD8 gene during exhaustion. This mouse model is used to study the effects of conditional knockout of the CD8 gene in vivo on the occurrence, development, and functional recovery of T cell exhaustion, thereby verifying the specific function of the CD8 gene during exhaustion.

[0015] Sixthly, this invention provides the application of the aforementioned mouse model in identifying key genes or therapeutic targets that regulate T cell exhaustion. This mouse model is used in conjunction with Cas9 transgenic mice and an AAV-delivered sgRNA library to identify key genes or therapeutic targets that regulate T cell exhaustion through in vivo screening techniques.

[0016] The beneficial effects of this invention are as follows: Through an innovative in vitro induction system and metabolic regulation strategy, this invention achieves the following breakthrough advantages: 1. Advantages in efficient construction and time cost (1) Shorter cycle: Stable exhaustion phenotype can be obtained in 5-7 days of in vitro induction (traditional in vivo model requires 4-8 weeks, and short-term in vitro model requires repeated stimulation 3-4 times).

[0017] (2) Efficiency improvement: (2-3)×10 can be obtained in a single experiment. 6 High-purity depleted CD8+ T cells (4.2 times higher cell yield compared to traditional methods).

[0018] 2. Breakthrough in the integrity of the exhausted phenotype (1) Dynamic changes in the expression of key proteins: During three consecutive stimulations of CD8+ T cells, exhaustion-related molecules exhibited a dynamic change characterized by a gradual increase: PD-1: Day 3 showed a significant increase compared to Day 1 (Naïve), with a relative increase of approximately 2.0 times compared to MFI. Days 5 and 7 remained at a high level of approximately 3.0 times.

[0019] LAG-3: No significant change on day 3, significant increase on day 5, reaching approximately 2.8 times the MFI, and maintained at a high level on day 7.

[0020] TIM-3: It slightly increases on day 3 (about 1.5 times), slightly decreases on day 5, and then significantly increases on day 7, nearly 2.3 times that of MFI.

[0021] This trend suggests that three biomarkers are activated sequentially: PD-1 is the first to rise, followed by LAG-3 in the middle stage, and finally TIM-3 is significantly upregulated in the late stage, mimicking the dynamic process of CD8+ T cell depletion in vivo.

[0022] (2) Effect function continues to decline: The functional indicators of CD8+ T cells gradually declined from day 3 onwards: IFN-γ⁺ cell percentage: approximately 24% on day 3, decreasing to approximately 18% on day 5, and further decreasing to approximately 14% on day 7, with an overall decrease of approximately 42% (Day 7 vs. Day 3).

[0023] GzmB⁺ cell percentage: approximately 22% on day 3, decreasing to approximately 15% on day 5, and further decreasing to approximately 13% on day 7, with an overall decrease of approximately 41% (Day 7 vs. Day 3).

[0024] These results indicate that with increasing stimulation frequency, the cytokine secretion and killing function of CD8+ T cells are significantly weakened, exhibiting a typical state of functional exhaustion.

[0025] 3. Cell viability and model stability Survival control: Through three rounds of continuous stimulation, the proportion of viable CD8+ T cells (Annexin V⁻ / PI⁻, Q4 quadrant) at different time points was as follows: Day 3: approximately 84.7%; Day 5: approximately 85.1%; Day 7: decreased to 58.7%.

[0026] Statistical results showed that the proportion of viable cells was 81.2±2.4% on Day 3 and 82.6±2.7% on Day 5. Day 7 decreased to 61.4±3.1%.

[0027] Compared with the traditional long-term culture model, which has a survival rate of less than 30% after 120 hours, this model maintains a survival rate of >80% for the first 5 days and remains at about 60% on the 7th day, which is significantly better than the traditional model and ensures the operability and reproducibility of the experiment.

[0028] 4. Cost and resource conservation (1) Reduction of experimental animals: Only 1 mouse is needed for a single model (5 mice are needed per group for traditional in vivo models).

[0029] (2) Reduced reagent consumption: Antibody usage reduced by 60% (optimized by pre-coating technology). Attached Figure Description

[0030] Figure 1 This is an experimental design flowchart.

[0031] Figure 2 This is a graph showing the dynamic changes of depleted proteins at different time points and stages as detected by flow cytometry.

[0032] Figure 3 This is a graph showing the changes in effector function at different time points and stages as detected by flow cytometry.

[0033] Figure 4 This is a graph showing the results of transcriptome sequencing analysis of cells collected at different time points.

[0034] Figure 5 This is a graph showing the results of flow cytometry analysis of CD8+ T cell survival at different time points during continuous stimulation. Detailed Implementation

[0035] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. This invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art. This invention will be defined only by the claims.

[0036] Unless otherwise specified, the following test methods or experimental methods are conventional methods; reagents and materials, unless otherwise specified, are obtained from conventional commercial sources or prepared using conventional methods. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] This invention belongs to the field of immunological research technology, specifically relating to a method for establishing a CD8+ T cell model with a typical exhaustion phenotype through in vitro antibody and cytokine synergistic induction, and its application in the analysis of T cell exhaustion mechanisms, development of immunotherapy strategies, and construction of drug screening platforms. The method includes: isolating CD8+ T cells from mice, and synergistically inducing and culturing them in vitro using a specific combination of antibodies and cytokines to differentiate them into a CD8+ T cell population exhibiting typical exhaustion characteristics, thereby constructing a mouse T cell model with a stable exhaustion phenotype. This invention provides a phenotypically stable and reproducible CD8+ T cell exhaustion model by simulating the key microenvironmental conditions of in vivo T cell exhaustion. This model is highly practical and overcomes the limitations of existing models, such as atypical exhaustion characteristics and inconsistent induction, enabling a more realistic simulation of the T cell exhaustion state and providing a reliable platform for mechanism research, immunotherapy strategy screening, and related drug development.

[0038] Example 1 A method for constructing an in vitro depleted CD8+ T cell mouse model includes the following steps: Step 1: Preparation of antibody coating solution and pre-coating of plate wells 1.1) Prepare a clean bench, sterile 12-well cell culture plates, pre-cooled sterile phosphate-buffered saline (PBS, e.g., Servicebio), and sealing film (e.g., Parafilm M); and prepare antibody stock solutions according to the product instructions: InVivoMAb anti-mouse CD3 (concentration 1 mg / mL, Bio X Cell) and InVivoMAb anti-mouse CD28 (concentration 1 mg / mL, Bio XCell).

[0039] 1.2) Add 12 mL of PBS to a 15 mL sterile centrifuge tube, then add 60 μL of... InVivoMAb anti-mouseCD3 (1 mg / mL) and 60 μL InVivoMAb Anti-mouse CD28 (1 mg / mL) was gently pipetted or vortexed to mix thoroughly until both antibodies reached a final concentration of 5 μg / mL in PBS; then 500 μL of the antibody-containing PBS solution was added to each well of a 12-well plate.

[0040] 1.3) Seal the entire 12-well plate with sealing film, place it on a horizontal shaker and gently mix at an appropriate speed, then incubate at 4°C for no less than 24 hours.

[0041] Step 2: Isolation and extraction of mouse CD8+ T cells: 2.1) Prepare sterile surgical instruments, a laminar flow hood (or biosafety cabinet), sterile erythrocyte lysis buffer (e.g., Servicebio), sterile CD8+ T cell sorting kits (e.g., MojoSort™ Mouse CD8+ T Cell Isolation Kit, BioLegend), sterile flow cytometry tubes, sterile 40 μm cell filters, sterile 1 mL syringes, sterile culture dishes, sterile 15 mL and 50 mL centrifuge tubes, and a cell counting chamber (or automated cell counter). Prepare the culture medium for T cell manipulation (hereinafter referred to as "T cell complete medium") and magnetic cell sorting buffer (hereinafter referred to as "MACS Buffer") in advance.

[0042] The preparation method of T cell complete culture medium is as follows (taking 50 mL as an example): Take 44 mL of RPMI-1640 basal culture medium (e.g., Servicebio), add 5 mL of fetal bovine serum (e.g., Gibco), 500 μL of 200 mM L-glutamine (e.g., Gibco), 500 μL of penicillin-streptomycin solution (e.g., 5,000 U / mL, Gibco), and 0.05 mM β-mercaptoethanol (e.g., Sigma). Mix thoroughly, filter through a 0.22 μm filter membrane for sterilization, and store at 4 °C for later use.

[0043] The preparation method of MACS Buffer is as follows (taking 500 mL as an example): Take 400 mL of 1x PBS buffer (sterile, containing calcium and magnesium ions), add 2.5 g to 5.0 g of bovine serum albumin (BSA, sterile grade) and stir to dissolve, then add 2 mL of 0.5 MEDTA solution (pH 8.0), and finally adjust the volume to 500 mL with 1x PBS. Filter through a 0.22 μm filter membrane for sterilization and store at 4 ℃ for later use.

[0044] 2.2) Lymphoid tissue acquisition: One 6-8 week old male C57BL / 6 mouse was euthanized by cervical dislocation or other methods. Under aseptic conditions, the inguinal lymph nodes, axillary lymph nodes, cervical lymph nodes, and spleen were harvested and immediately placed in pre-cooled T-cell complete culture medium to keep them moist.

[0045] 2.3) Preliminary preparation of single-cell suspension: The lymphoid tissue obtained in step 2.2 was placed on a 40 μm sterile cell filter and transferred to the opening of a 50 mL sterile centrifuge tube containing an appropriate amount of RPMI-1640 basal medium. The tissue was gently abraded using the end of a 1 mL syringe plunger while simultaneously rinsing with RPMI-1640 medium to allow cells to pass through the filter into the medium. The cell suspension was collected into a 50 mL centrifuge tube. The tube was then centrifuged at 1500 rpm for 5 minutes (approximately 300 × g), and the supernatant was discarded.

[0046] 2.4) Red blood cell lysis: Add 3 mL of sterile red blood cell lysis buffer to the cell pellet obtained in step 2.3, gently pipette to resuspend the cells, and let stand at room temperature for 3 minutes to lyse the red blood cells.

[0047] 2.5) Termination of lysis reaction and cell washing: Add 3 mL of complete T cell culture medium to the centrifuge tube from step 2.4 to terminate the lysis reaction. After mixing, centrifuge at 1500 rpm for 5 minutes (approximately 300 × g) and discard the supernatant. This step can be repeated once to thoroughly remove lysis buffer and cell debris.

[0048] 2.6) Magnetic bead antibody incubation: Resuspend the cell pellet obtained in step 2.5 with 1 mL of MACS Buffer. Follow the proportions recommended in the instructions of the sorting kit used (e.g., per 10^6 cells). 7 Use 60 μL per cell line. Add an appropriate amount of anti-mouse CD8 antibody mixture (from the kit described in step 2.1), mix gently, and incubate at 2–8 °C (on ice) for 15 minutes. After incubation, without washing, add the magnetic beads provided in the kit in the same proportion, mix gently, and continue incubating at 2–8 °C (on ice) for 15 minutes.

[0049] 2.7) Magnetic sorting: After incubation, transfer the cell suspension from the centrifuge tube to a sterile flow cytometry tube. Add MACS Buffer until the level is slightly above the top of the sorting magnet. Place the flow cytometry tube in the sorting magnet and let it stand for 5 minutes to allow the CD8+ T cells bound to the magnetic beads to adhere to the tube wall. Holding the magnet, slowly tilt the flow cytometry tube to collect the unbound cell suspension (i.e., the negative sorting fraction rich in CD8+ T cells) into a new 15 mL sterile centrifuge tube.

[0050] 2.8) Cell washing after sorting: Add sufficient MACS Buffer to a 15 mL centrifuge tube containing the cell suspension, centrifuge at 1500 rpm for 5 minutes (approximately 300 × g), and discard the supernatant. This washing step can be repeated once.

[0051] 2.9) Cell counting and resuscitation: Resuspend the cell pellet obtained in step 2.8 in 1 mL of complete T cell culture medium. Take 20 μL of the cell suspension for counting, and determine the cell concentration using a cell counting chamber or automated cell counter. Based on the counting results, adjust the cell suspension volume to ensure the total number of CD8+ T cells is approximately 1.44 × 10⁻⁶. 7 indivual.

[0052] 2.10) Establishment and initialization of cell culture system: Add 24 mL of complete T cell culture medium and 24 μL of interleukin-2 (IL-2, for example, a working concentration of 1 U / mL) to the cell suspension prepared in step 2.9, and gently mix by pipetting to prepare a cell density of approximately 0.6 × 10⁻⁶. 6 Add 1 mL of the cell culture suspension per mL. Discard the liquid in the 12-well plate containing the antibody-coated cells from step 1.3, and add 1 mL of this cell culture suspension to each well. Centrifuge the plate at 1500 rpm for 3 minutes (approximately 300 × g) to ensure contact between the cells and the coated substrate. Finally, transfer the cell culture plate to a 37 °C, 5% CO2 incubator for further culture.

[0053] Step 3: Induction and establishment of early exhausted CD8+ T cells 3.1) On the second day of cell culture (early exhaustion), repeat the operations described in steps 1.1 to 1.4 to prepare two 12-well cell culture plates coated with anti-CD3 / CD28 antibody, and prepare 24 mL of complete T cell culture medium in advance for later use.

[0054] 3.2) On day 3 of cell culture, observe cell status and growth density under a microscope. Taking any well in a 12-well plate as an example, perform replenishment and cell collection as follows: Aspirate the culture medium from the well and transfer it to a 50 mL centrifuge tube for temporary storage; add 1 mL of fresh, pre-warmed T-cell complete culture medium to the well, and gently pipette the bottom of the plate until the bottom layer changes from a frosty texture to a smooth surface, indicating that the cells have detached; then aspirate the liquid from the well and collect it into the same 50 mL centrifuge tube. Throughout the pipetting process, avoid generating air bubbles and ensure that no liquid from the well is discarded directly, in order to preserve all cells and the cytokines secreted by the cells.

[0055] 3.3) Collect all the cell suspensions from all wells of the current 12-well plate into the same 50 mL centrifuge tube as in step 3.2, add 24 μL of IL-2 cytokine to the centrifuge tube, and mix gently.

[0056] 3.4) Discard the liquid from each well of the two new coated plates prepared in step 3.1, and add 1 mL of the cell suspension prepared in step 3.3 to each well. Place the culture plate in a centrifuge and centrifuge at 1500 rpm for 3 minutes (approximately 300×g) to allow the cells to settle and fully contact the coated surface. Then transfer the plate to a 37°C, 5% CO2 incubator for further culture.

[0057] Step 4: Induction and establishment of mid- and late-stage exhausted CD8+ T cells 4.1) On day 4 of cell culture, repeat steps 1.1 to 1.4 to prepare two 12-well cell culture plates coated with anti-CD3 / CD28 antibody and prepare 24 mL of complete T cell culture medium in advance.

[0058] 4.2) On day 5 of cell culture (mid-stage exhaustion), observe cell state and growth density under a microscope. Perform medium change and cell transfer as follows: aspirate the culture medium from the original wells of the culture plate and transfer it to a 50 mL centrifuge tube for temporary storage; add 1 mL of fresh, pre-warmed T-cell complete culture medium to each well, and gently pipette the bottom of the plate to detach the attached cells (the bottom layer will change from a frosted texture to a smooth texture); then aspirate the liquid from the wells and collect it into the same 50 mL centrifuge tube. The entire procedure should avoid generating air bubbles and retain all cells and cytokines.

[0059] 4.3) Collect all cell suspensions from all wells of the current 12-well plate into the same 50 mL centrifuge tube as in step 5.2, add 24 μL of IL-2 cytokine to the centrifuge tube, and mix gently.

[0060] 4.4) Discard the liquid from each well of the two new coated plates prepared in step 5.1, and add 1 mL of the cell suspension prepared in step 5.3 to each well. Place the culture plate in a centrifuge and centrifuge at 1500 rpm for 3 minutes (approximately 300×g) to ensure full contact between the cells and the coated surface. Then transfer the plate to a 37°C, 5% CO2 incubator for further culture.

[0061] 4.5) On day 7 of cell culture (late exhaustion), collect cells according to steps 5.2 and 5.3 to obtain late exhaustion CD8+ T cells for subsequent experimental analysis.

[0062] Example 2: Flow cytometry identification of early, middle, and late-stage exhausted CD8+ T cells Step 5: Flow cytometry identification 5.1) On days 3, 5, and 7 of cell culture, take one 12-well plate from the incubator, observe the cell state under a microscope, collect the cells from the wells into flow cytometry tubes, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant. Prepare flow cytometry tubes and the following cell flow cytometry detection antibodies: APC-labeled mouse anti-CD8 monoclonal antibody, BV421-labeled mouse anti-PD-1 (CD279) monoclonal antibody, BV605-labeled mouse anti-TIM-3 monoclonal antibody, BV785-labeled mouse anti-LAG-3 monoclonal antibody, PE-labeled mouse anti-IFN-γ monoclonal antibody, and FITC-labeled mouse anti-granzyme B (GZMB) monoclonal antibody.

[0063] 5.2) After digestion and resuspending, the cells were centrifuged at 1500 rpm and 4 ℃ for 5 minutes, the supernatant was discarded, and the cells were washed once with PBS (containing 1% BSA). The cells were then centrifuged with (0.25-1)×10⁻⁶ cells / mL. 7 Cells were resuspended in 1 mL PBS (containing 1% BSA) at a concentration of 1 cell / mL.

[0064] 5.3) Set up blank control tubes, isotype control tubes, and test sample tubes, and take 100 μL of the above cell suspension in each tube. No antibody was added to the blank control tubes; 0.5 μL of FVS dye (564406, BD Bioscience) was added to the isotype control tubes and test sample tubes, gently mixed, and incubated at 4 ℃ in the dark for 20 minutes, gently mixing every 10 minutes during incubation. After incubation, centrifuge at 1500 rpm at 4 ℃ for 5 minutes and discard the supernatant.

[0065] 5.4) Resuspend cells in 100 μL PBS (containing 1% BSA), and do not add antibody to blank control tubes; add the corresponding flow cytometry antibody to isotype control tubes and test sample tubes respectively (it is recommended to refer to the antibody instruction manual for the recommended dosage, such as 1 μL / test), and incubate at 4 ℃ in the dark for 30 minutes, gently mixing once every 10 minutes during the process.

[0066] 5.5) If it is necessary to detect intracellular factors (such as IFN-γ, GZMB), after completing the surface staining, fixation, membrane perforation and intracellular antibody staining should be performed according to the instructions of the intracellular fixation and perforation kit.

[0067] 5.6) Add 100 μL PBS (containing 1% BSA), centrifuge at 1400 rpm and 4 ℃ for 5 minutes, and wash 1–2 times; resuspend the cells in 500 μL PBS (containing 1% BSA), filter through a cell sieve, and transfer to a flow cytometry tube for flow cytometry detection.

[0068] Step 6, Test Results This invention extracts CD8+ T cells from mouse spleen and peripheral lymph nodes, and uses Anti-CD3 / 28 antibody and cytokine IL-2 to continuously stimulate and induce CD8+ T cells. Flow cytometry was used to detect changes in CD8+ T cell function. It was found that the expression levels of exhaustion markers PD-1 and LAG-3 were continuously enhanced, but the killing ability of GZMB was weakened, suggesting that continuous stimulation by antibodies and cytokines can establish a stable exhaustion state CD8+ T cell model.

[0069] like Figure 1 The diagram shown is a flowchart of the experimental design.

[0070] like Figure 2 The graph shows flow cytometry data of exhaustion indicators. Flow cytometry was used to detect the dynamic changes in the expression of typical exhaustion-related proteins PD-1, LAG-3, and TIM-3 in CD8+ T cells at different stimulation time points (Day 3, Day 5, Day 7) and different stages. The results showed that PD-1 rapidly increased in the early stage (Day 3), reaching approximately 2.0-fold relative to the MFI, and then further increased on Day 5 and Day 7, stabilizing at a high level of approximately 3.0-fold. LAG-3 showed no significant change on Day 3, but significantly increased on Day 5, reaching nearly 3.0-fold relative to the MFI, and maintained high expression levels on Day 7. TIM-3 was only slightly upregulated on Day 3 (approximately 1.5-fold), briefly decreased on Day 5, and then significantly increased to approximately 2.2-fold on Day 7; all differences were statistically significant.

[0071] Overall, the three exhaustion markers exhibited a progressive expression pattern: PD-1 was activated first, followed by a significant upregulation of LAG-3 in the intermediate stage, while TIM-3 peaked in the late stage. This dynamic trend closely mimics the natural process of CD8+ T cell exhaustion in vivo, suggesting that this in vitro model can reproduce the process of T cells from early functional impairment to late exhaustion at different stages.

[0072] like Figure 3 The figure shows flow cytometry data of functional indicators, detecting changes in the expression of CD8+ T cell effector function-related molecules IFN-γ and GzmB at different time points using flow cytometry. The results showed that the proportion of IFN-γ⁺ cells continuously decreased with increasing stimulation frequency: the proportion of IFN-γ⁺ cells was 23.8±0.4% on day 3, decreased to 17.9±2.2% on day 5, and further decreased to 13.8±1.2% on day 7, with an overall decrease of approximately 42% (Day 7 vs. Day 3), and the difference was statistically significant.

[0073] Similarly, the proportion of GzmB⁺ cells showed a significant decreasing trend: 21.3±2.1% on day 3, 11.4±1.6% on day 5, and further decreased to 11.4±0.6% on day 7, with an overall decrease of about 46% (Day 7 vs. Day 3), and the difference was also statistically significant.

[0074] Overall, with prolonged stimulation, the IFN-γ secretion capacity and the expression of the cytotoxic factor GzmB in CD8+ T cells gradually decreased, indicating a continuous decline in their effector function and exhibiting typical exhaustion characteristics. This result is consistent with the aforementioned trend of gradually increasing exhaustion markers, further validating that the model accurately reproduces the dynamic process of CD8+ T cells from early activation to functional exhaustion in vitro.

[0075] like Figure 4 The image shows transcriptome sequencing analysis performed on cells collected at different time points to further validate the molecular characteristics of this in vitro constructed CD8+ T cell exhaustion model. Figure 4 A showed that KEGG pathway enrichment analysis of differentially expressed genes revealed significant enrichment of several classical signaling pathways associated with T cell activation and exhaustion, including the HIF-1 signaling pathway, JAK-STAT signaling pathway, p53 signaling pathway, and MAPK signaling pathway (P<0.05). Further analysis of the expression of key exhaustion-related genes... Figure 4 As shown in Figure B, the expression levels of exhaustion marker genes (such as Pdcd1, Lag3, and Havcr2) significantly increased, while the expression levels of effector function-related genes (such as Ifng and Gzmb) gradually decreased with increasing stimulation frequency. This result is highly consistent with the protein expression trends detected by flow cytometry, indicating that CD8+ T cells gradually transition from early activation to deep exhaustion. A stable and reproducible in vitro exhaustion model was successfully established, providing a reliable molecular basis for subsequent research on T cell exhaustion mechanisms and intervention strategies.

[0076] like Figure 5The image shows the flow cytometry results of apoptosis, used to detect the cell survival of CD8+ T cells at different time points during continuous stimulation. The results showed that on Day 3, the proportion of viable cells (Annexin V⁻ / PI⁻) was 79.4±4.2%; on Day 5, it remained at 77.4±5.6%; and on Day 7, it decreased to 60.0±3.0%. These results indicate that the model maintains high cell activity and stability in the first five days. Although cell survival decreased somewhat on Day 7, it still remained around 60%, significantly better than the traditional long-term culture model where survival is below 30%. This characteristic ensures the feasibility of conducting phenotypic and functional studies at each stage and provides an ideal time window for subsequent experiments.

[0077] Compared with the prior art, the present invention has the following advantages: 1. Stimulate the application of soluble microbeads: Existing technologies employ non-coated stimuli such as soluble microbeads, which result in dispersed, unstable, and easily endocytosed and degraded stimuli. This leads to insufficient stimulation intensity and short signal duration, failing to achieve sufficient and synchronous activation of T cells. Consequently, cell activation processes are slow and uneven, with a large number of cells failing to cross the activation threshold for differentiation into a deeply exhausted state.

[0078] The plate antibody coating technology used in this invention provides a powerful, uniform and continuous antigen signal simulation environment, ensuring that CD8+ T cells are rapidly and powerfully activated in the early stage, laying a solid foundation for the precise and synchronous initiation of the subsequent exhaustion program.

[0079] 2. Regarding the maintenance of the cultivation system: Traditional "partial fluid replacement" or "full fluid replacement" operations have long been a technical pain point in this field, directly restricting the yield and quality of depleted cells.

[0080] "Partial culture medium replacement" inevitably causes a large physical loss of target cells while removing the old culture medium, resulting in a significant reduction in the yield of exhausted T cells. Furthermore, the operation is difficult to standardize and has poor reproducibility.

[0081] "Complete medium replacement" more radically interrupts the cell growth microenvironment. While removing metabolic waste, it also clears out various cytokines (such as IL-2) that are essential for cell survival and proliferation, artificially creating "growth factor starvation," which ultimately leads to cell proliferation arrest or even apoptosis, and cannot support the complete induction of the depleted phenotype.

[0082] This invention achieves a dynamic and precise balance between nutrient replenishment and cytokine concentration through an optimized phased fluid replenishment strategy and culture medium formulation. Without the need for large-scale physical disturbance of cells, it maintains the optimal environment required for cell health and differentiation for a long time, thereby ensuring the successful production of high quantity, high quality, and phenotypically consistent exhausted T cells.

[0083] 3. Regarding the core inducement process: Existing methods lack clear and unified induction standards, and the timing and frequency of stimulation are highly arbitrary, resulting in huge differences in cell state between different batches, making it impossible to achieve precise control and phased research on the exhaustion process.

[0084] The core innovation of this invention lies in establishing a clear and repeatable "staged restimulation procedure." By repeatedly stimulating cells with CD3 / CD28 antibodies and IL-2 cytokines at key time points such as days 3, 5, and 7, we precisely simulated continuous antigen attack in vivo. This design allows us to controllably and systematically obtain phenotypically defined early, mid, and late-stage exhausted CD8+ T cells at specific time windows (such as days 3, 5, and 7), achieving for the first time the "staged" and "standardized" exhaustion process in vitro. This provides an unprecedentedly precise tool for studying the biological characteristics and reversal strategies of exhausted T cells at different stages.

[0086] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing an in vitro depleted CD8+ T cell mouse model, characterized in that: Includes the following steps: S1, Antibody coating solution preparation and plate well pre-coating: Prepare antibody coating solution, then pre-coat the antibody onto the plate wells, aspirate the liquid in the wells, and prepare a coated antibody plate. The antibody coating solution includes anti-CD3 antibody, anti-CD28 antibody and PBS solution. S2, Isolation, extraction and culture of mouse CD8+ T cells: Mouse CD8+ T cells were extracted to obtain cell slurry, and then T cell complete culture medium and IL-2 were added to prepare a cell culture suspension. The cell culture suspension was added to the antibody-coated plate prepared in S1 and then cultured. S3, Induction and establishment of early exhausted CD8+ T cells: On the 3rd day of cell culture, the liquid in the well was aspirated and transferred to a centrifuge tube. Complete T cell culture medium was added to the well, and the bottom of the plate was gently blown until the cells detached. Then, the liquid in the well was aspirated and collected into the same centrifuge tube. IL-2 was added to the centrifuge tube to make a cell suspension. The cell suspension was added to the coated antibody plate prepared in S1. Centrifugation was performed to allow the cells to settle and make full contact with the coating surface, thus obtaining early exhausted CD8+ T cells, which were then cultured for a period of time. S4, Induction and establishment of mid-stage exhausted CD8+ T cells: On day 5 of cell culture, the liquid in the wells was aspirated and transferred to a centrifuge tube. Complete T cell culture medium was added to the wells, and the bottom of the wells was gently blown until the cells detached. The liquid in the wells was then aspirated and collected into the same centrifuge tube. IL-2 was added to the centrifuge tube to prepare a cell suspension. The cell suspension was added to the antibody-coated well plate prepared in S1. The plate was centrifuged to allow the cells to settle and make full contact with the coating surface, thus obtaining mid-stage exhausted CD8+ T cells, which were then cultured for a period of time. S5, Induction and establishment of late-stage exhausted CD8+ T cells: On day 7 of cell culture, cells are collected to obtain late-stage exhausted CD8+ T cells.

2. The construction method according to claim 1, characterized in that: In S1, the final concentrations of both anti-CD3 antibody and anti-CD28 antibody in the PBS solution were 5 μg / mL.

3. The construction method according to claim 1, characterized in that: The preparation method of T cell complete culture medium is as follows: Taking 50 mL as an example, take 44 mL of RPMI-1640 basal culture medium, add 5 mL of fetal bovine serum, 500 μL of 200 mM L-glutamine, 500 μL of penicillin-streptomycin solution and 0.05 mM β-mercaptoethanol, mix thoroughly, filter through a 0.22 μm filter membrane for sterilization, and store at 4 ℃ for later use.

4. An in vitro depleted CD8+ T cell mouse model, characterized in that: It is constructed using the construction method described in any one of claims 1-3.

5. The application of the mouse model described in claim 4 in the study of CD8+ T cell exhaustion mechanisms.

6. The use of the cell mouse model of claim 4 in screening or evaluating synergistic drugs or gene targets for preventing or reversing the depletion of therapeutic T cells in vivo.

7. The application of the mouse model of claim 4 in studying the specific function of the CD8 gene during depletion.

8. The use of the mouse model of claim 4 in identifying key genes or therapeutic targets that regulate T cell exhaustion.

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