Methods of establishing an in vitro CD8 + T cell exhaustion model and uses thereof

Abstract

The embodiments of the present application provide a method for establishing in vitro CD8 + T cell exhaustion model and application thereof. The method comprises: using T cell receptor signal stimulator combined with interleukin-10 to continuously stimulate CD8 + T cells under in vitro culture conditions. The cells induced by the method not only highly express exhaustion key transcription factor TOX and various surface inhibitory receptors (such as PD1, TIM3, LAG3 and the like), but also significantly reduce the secretion ability of effector cytokines, highly reduce the real exhaustion performance in in vivo chronic infection or tumor microenvironment in vitro, and effectively overcome the defect of few CD8 + T cells in traditional in vivo animal model, and high-quality exhaustion cells can be obtained in vitro in large scale in only 6 to 8 days.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and more particularly to a method for establishing in vitro CD8 + Methods and applications of T cell exhaustion models. Background Technology

[0002] In the adaptive immune response, CD8 + T cells (Cytotoxic T lymphocytes, CTLs) are the main effector cells for clearing viruses and tumors. Under normal circumstances, the body's CD8+ cells... + After T cells clear the antigen, the body forms protective immune memory. When the body encounters the same virus again, memory cells can quickly initiate an immune response, thereby preventing reinfection.

[0003] However, during chronic viral infections and tumor development, the continuous stimulation of CD8 by antigens... + T cells gradually enter a state of loss of function and inability to proliferate, eventually reaching a state of exhaustion, known as CD8. + T cell exhaustion (CD8+ T cell exhaustion) is characterized by impaired proliferation, weakened secretion of effector cytokines (IL-2, TNFα, IFNγ, etc.) and sequential reduction in cytotoxic function, while simultaneously exhibiting high expression of a series of inhibitory receptors (Tim-3, LAG-3, CD160, 2B4, TIGIT, and CTLA-4, etc.) and an inability to differentiate into an immunological memory phenotype. CD8+ T cell exhaustion gradually leads to the body's loss of the ability to control and clear chronic viral infections and tumors. Therefore, reversing and preventing CD8+ T cell exhaustion is crucial. + T-cell depletion is key to curing and preventing chronic viral infections and tumors.

[0004] CD8 + In T cell exhaustion studies, establishing a chronic viral infection mouse model using LCMV-CL13 (lymphocytic choriomeningitis virus) is crucial for investigating virus-specific CD8+. + The classic model of T cell exhaustion.

[0005] Tumor cells carrying specific antigens, such as MC38-OVA, B16-OVA, and B16-gp, are inoculated into mice via subcutaneous inoculation, followed by the reinfusion of antigen-specific CD8+ cells such as OT-1 cells or P14 cells. + T cells are used to study CD8+ cells in tumor research. +T cell exhaustion model. In the LCMV-CL13 model, CD8+ was reinfused to effectively establish chronic infection. + The number of T cells should not exceed 2000. However, in tumor models, as the tumor grows, the number of infiltrated CD8+ cells increases. + The number of T cells (Tumor Infiltrating Lymphocytes, TILs) will continue to decline; for example, in the B16-gp model, there may be fewer than 100 CD8 cells. + T cells. In other words, exhausted CD8 cells obtained through in vivo models. + The number of T cells is extremely limited.

[0006] This limitation in quantity poses a significant obstacle to the application of high-throughput screening (HTS) technology. Therefore, there is an urgent need to develop a highly efficient in vitro CD8 assay. + A method for establishing T-cell exhaustion models aims to overcome the limitations of in vivo model production, providing a sufficient cell source and a reliable in vitro platform for high-throughput screening and related mechanism research. Summary of the Invention

[0007] This application aims to address the exhaustive CD8 content in existing in vivo model acquisition methods. + The problem of a limited number of T cells.

[0008] Firstly, this application provides a method for establishing in vitro CD8 + A method for a T-cell exhaustion model. This method includes: under in vitro culture conditions, using a T-cell receptor signaling stimulator in combination with interleukin-10 to stimulate CD8+ receptor antagonists. + T cells are continuously stimulated.

[0009] Optionally, the TCR signaling stimulant includes a combination of anti-CD3 antibody and anti-CD28 antibody, or a specific antigenic peptide.

[0010] Optionally, the specific antigenic peptide includes: gp33-41 antigenic peptide.

[0011] Optionally, when the TCR signaling stimulant is a combination of anti-CD3 antibody and anti-CD28 antibody, the specific method for continuous stimulation includes: injecting CD8 every 2 days. + T cells were transferred to newly coated culture plates containing anti-CD3 antibodies for stimulation, and supplemented with culture medium containing anti-CD28 antibodies and IL-10, for a total of 3 to 4 rounds of stimulation; or CD8 cells were transferred to new culture plates containing anti-CD3 antibodies for stimulation. + T cells were placed in a culture plate coated with anti-CD3 antibody, and cultured with medium containing anti-CD28 antibody and IL-10 for 8 days without replacing the culture plate.

[0012] Optionally, when the TCR signaling stimulant is the gp33-41 antigenic peptide, the specific operation method for continuous stimulation includes: changing the culture medium containing fresh gp33-41 antigenic peptide every 2 days, with a total stimulation time of 8 days; or continuously culturing CD8 in a culture medium containing gp33-41 antigenic peptide. + T cells were collected for 8 days without changing the culture medium.

[0013] Optionally, the continuous stimulation is performed in a basal culture medium containing interleukin-2.

[0014] Optionally, the basal culture medium is RPMI-1640 medium containing 10% fetal bovine serum.

[0015] Optionally, the in vitro culture conditions do not contain transforming growth factor-β.

[0016] Optionally, after continuous stimulation of CD8 culture + When T cells possess the following characteristics (a), (b), and (c), the CD8+ is determined. + A model of T cell exhaustion has been established; (a) High expression of inhibitory receptor markers, said inhibitory receptor markers including one or more of PD1, TIM3, LAG3 and CD39; (b) High expression of transcription factor TOX; (c) Low expression of effector cytokines, including one or more of IL-2, TNFα and IFNγ.

[0017] Secondly, this application also provides an in vitro CD8 genome established by the method described above. + T cell exhaustion model in preparation for screening CD8 regulation + Application in high-throughput screening platforms for drugs or compounds that have exhausted T cells. At least one advantage provided by the embodiments of this application is that the method, by combining interleukin-10 (IL-10) with continuous TCR signaling stimulation, can significantly synergistically promote CD8. + The formation of T cell exhaustion. The induced cells not only highly express the key exhaustion transcription factor TOX and various surface inhibitory receptors (such as PD1, TIM3, LAG3, etc.), but also have a significantly reduced ability to secrete effector cytokines, thus highly replicating the true exhaustion manifestation in vivo in chronic infection or tumor microenvironment in vitro.

[0018] Furthermore, this method effectively overcomes the limitations of traditional in vivo animal models for obtaining depleted CD8. +The limitation of extremely low T cell numbers allows for the large-scale, stable acquisition of high-quality exhausted cells in vitro in just 6 to 8 days. This not only significantly shortens the experimental cycle and reduces construction costs, but also provides ample cell sources and reliable platform support for high-throughput screening technologies such as small molecule drug screening and CRISPR gene editing, greatly enhancing the CD8 cell count. + The efficiency of research on T cell depletion mechanisms and the development of targeted immunotherapies. Attached Figure Description

[0019] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, which are not intended to limit the embodiments. Statistical data in the figures are analyzed using unpaired t-tests, and the results are presented in Mean ± SD format.

[0020] Figure 1 This is a schematic diagram of the experimental design for Embodiment 7-1 of this application; Figure 2 Flow cytometry detection of CD8 under different stimulation modes in Example 7-1 of this application + Flow cytometry contour plot of expression levels of T cell surface exhaustion markers (TIM3, PD1, LAG3); Figure 3 for Figure 2 PD1 + TIM3 + A bar chart showing the percentage of double-positive cells; Figure 4 for Figure 2 PD1 + LAG3 + A bar chart showing the percentage of double-positive cells; Figure 5 Flow cytometry detection of CD8 under different stimulation modes in Example 7-1 of this application + Flow cytometry histogram of TOX expression levels in T cells; Figure 6 A statistical bar chart showing the average fluorescence intensity (MFI) of five TOX samples; Figure 7 Flow cytometry detection of CD8 under different stimulation modes in Example 7-1 of this application + Flow cytometry contour plot of T cell secretion levels of effector cytokines (IL-2, TNF-α, IFN-γ); Figure 8 for Figure 7 IL-2 + IFN-γ + A bar chart showing the percentage of double-positive cells; Figure 9 for Figure 7 TNF-α+ IFN-γ + A bar chart showing the percentage of double-positive cells; Figure 10 This is a schematic diagram of the experimental design for stimulating spleen cells in Example 7-2 of this application; Figure 11 For example, in Examples 7-2 of this application, flow cytometry was used to detect CD8 antigen peptide gp33-41 under different stimulation modes. + Flow cytometry contour plot of expression levels of T cell surface exhaustion markers (TIM3, PD1, LAG3); Figure 12 for Figure 11 PD1 + TIM3 + A bar chart showing the percentage of double-positive cells; Figure 13 for Figure 11 PD1 + LAG3 + A bar chart showing the percentage of double-positive cells; Figure 14 For example, in Examples 7-2 of this application, flow cytometry was used to detect CD8 antigen peptide gp33-41 under different stimulation modes. + Flow cytometry histogram of TOX expression levels in T cells; Figure 15 for Figure 14 Statistical histogram of mean fluorescence intensity (MFI) of TOX in medium; Figure 16 For example, in Examples 7-2 of this application, flow cytometry was used to detect CD8 antigen peptide gp33-41 under different stimulation modes. + Flow cytometry contour plot of T cell secretion levels of effector cytokines (IL-2, TNF-α, IFN-γ); Figure 17 for Figure 16 IL-2 + IFN-γ + A bar chart showing the percentage of double-positive cells; Figure 18 for Figure 16 TNF-α + IFN-γ + A bar chart showing the percentage of double-positive cells; Figure 19 (Examples 7-3 of this application) IL-10 combined with continuous TCR signaling stimulation of mouse CD8 + Schematic diagram of the experimental design for T cells; Figure 20 For example, in embodiments 7-3 of this application, flow cytometry was used to detect CD8 under different stimulation methods. +Flow cytometry contour plot of expression levels of T cell surface exhaustion markers (TIM3, PD1, LAG3); Figure 21 for Figure 20 PD1 + TIM3 + A bar chart showing the percentage of double-positive cells; Figure 22 for Figure 20 PD1 + LAG3 + A bar chart showing the percentage of double-positive cells; Figure 23 Flow cytometry detection of CD8 under different stimulation modes in Examples 7-3 of this application + Flow cytometry histogram of TOX expression levels in T cells; Figure 24 for Figure 23 A statistical bar chart of the mean fluorescence intensity (MFI) of TOX in the medium. Detailed Implementation

[0021] To facilitate understanding of this application, a more detailed description is provided below with reference to the accompanying drawings and specific embodiments. It should be noted that when an element is described as being "fixed to" another element, it can be directly on the other element, or one or more intermediate elements may exist between them. When an element is described as being "connected" to another element, it can be directly connected to the other element, or one or more intermediate elements may exist between them. The terms "upper," "lower," "inner," "outer," "bottom," etc., used in this specification indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application. Furthermore, the terms "first," "second," "third," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0022] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. The term "and / or" as used in this specification includes any and all combinations of one or more of the associated listed items.

[0023] To further illustrate the technical solution, purpose, and technical effects of this application in detail, the following will describe this application in detail with reference to specific experimental materials and operating procedures. It should be noted that experimental methods in the following embodiments without specified conditions are generally performed according to conventional conditions in the field or conditions recommended by reagent and instrument manufacturers. Unless otherwise specified, the materials, reagents, and instruments used in the following embodiments are commercially available.

[0024] 1) Experimental materials: RPMI-1640 medium, fetal bovine serum (FBS), penicillin-streptomycin, sodium pyruvate, and non-essential amino acids (NEAA) were purchased from Thermo Fisher Scientific (Gibco); aCD3 & CD28 antibodies were purchased from Biolegend; fluorescently labeled anti-mouse antibodies were purchased from Biolegend; BD magnetic bead sorting kit, intracellular staining kit, and BD Fortessa flow cytometer were all products of BD.

[0025] P14 mice are CD8 + Mice that use T cells to specifically recognize the gp33-41 site of LCMV.

[0026] 2) BD negative selection magnetic beads for sorting mouse CD8 + T lymphocytes: Spleens from 6-8 week old C57BL / 6 mice were collected, ground, and filtered through a 70-micron screen to obtain a single-cell suspension. Mouse CD8 cells were then enriched using the BDIMag™ Mouse CD8 T Lymphocyte Enrichment Set – DM kit according to the manufacturer's instructions. + T cells.

[0027] 3) Primary CD8 in mice + T cell culture: mouse primary CD8 + T cells were cultured in R10 medium (RPMI 1640 + 10% FBS + 1 mM sodiumpyruvate + 10 mM HEPES + 50 M β-mercaptoethanol + penicillin (50 U / ml), and streptomycin (50 μg / ml). Cells were cultured in a clean incubator at 37°C and 5% CO2.

[0028] 4) CD8 + T cell stimulation culture: 4-1) Stimulation with aCD3 & CD28 antibodies: Add 0.5 ml of PBS containing 6 μg / ml aCD3 antibody to each well of a 12-well plate and incubate overnight at 4°C to coat the aCD3 antibody. Before use, discard the PBS, then gently add 1 ml of PBS along the plate wall to wash away any uncoated antibody. Finally, incubate each well with 2 ml of R10 buffer containing 100 active units of IL-2 and 0.5 μg / ml aCD28 antibody. + T cells, cell density 1×10⁶ 6 cells / ml.

[0029] 4-2) Stimulation by antigenic peptide gp33-41: Add antigen peptide gp33-41 directly to R10 culture containing 100 active units of IL-2 to culture CD8 + The cell density in T cells is 0.5 × 10⁻⁶. 6 cells / ml.

[0030] 5) Flow cytometry detection: 5-1) Flow cytometry antibody staining of cell membrane surface: After centrifuging and discarding the supernatant, add an appropriate amount of flow cytometry antibody to an appropriate amount of buffer solution to resuspend the cells. Incubate at 4 °C for 30 minutes, wash twice, and resuspend in an appropriate amount of 2% PFA before testing.

[0031] 5-2) Intracellular molecular flow cytometry antibody staining: After centrifuging an appropriate amount of cells and discarding the supernatant, stain the molecules on the cell surface, fix and permeate the cells (refer to the BD Cytofix / Cytoperm™ Fixation / Permeabilization Kit instructions), then stain the intracellular or nuclear cells. After incubation, discard the supernatant and wash, resuspend in an appropriate amount of 2% PFA, and then it is ready for flow cytometry detection.

[0032] 6) Statistical analysis: FlowJo V10 software was used to analyze the stream cytometry data, and GraphPad Prism 9.0 was used for statistical analysis. Unpaired t-tests were used to compare the two groups, and P < 0.05 was considered statistically significant.

[0033] Based on the experimental materials and procedures described above, this application aims to provide and validate an efficient method for in vitro induction of CD8+ T cell exhaustion, and further explore the regulatory role of specific microenvironmental factors in this exhaustion process. The setup and specific results of each experiment are described below.

[0034] 7-1) In vitro αCD3 & CD28 antibody continuous stimulation experiment: like Figure 1As shown, different αCD3 & CD28 antibody stimulation conditions were set to treat in vitro cultured CD8 cells. + T cells. Specific grouping and treatment methods include: (1) Non-stimuli control group; (2) Multi-round passage stimulation group: Every 2 days, cells were transferred to a new culture plate coated with αCD3 antibody for stimulation, and cells were collected after 1 round of stimulation (S1, i.e., stimulation for 2 days), 2 rounds (S2, i.e., stimulation for 4 days), 3 rounds (S3, i.e., stimulation for 6 days) and 4 rounds (S4, i.e., stimulation for 8 days). (3) Continuous in situ stimulation group: Cells were placed in culture plates coated with αCD3 antibody and the culture plates were not replaced with new ones. The cells were directly stimulated for 8 days.

[0035] like Figures 2 to 8 As shown: Unstimulated cells, cells stimulated for only 1 round, and cells stimulated for 2 rounds (i.e., cells stimulated for less than 4 days) exhibited antigen-specific CD8 expression associated with acute viral infection. + T cells are characterized by minimal expression of cell surface depletion markers TIM3 and LAG3 and low expression of PD1, while possessing the potential to overexpress effector cytokines (IL-2, TNFα, and IFNγ).

[0036] Cells stimulated for 3 rounds, 4 rounds, and continuous stimulation for 8 days (i.e., cells stimulated for more than 6 days) showed high expression of TIM3, LAG3, and PD1, and impaired secretion of effector cytokines (IL-2, TNFα, and IFNγ). This characteristic is associated with chronic viral infection of CD8 cells. + T-cell consistency.

[0037] TOX is a driver for CD8. + Key cytokines for T cell exhaustion, when CD8 is continuously stimulated by αCD3 & CD28 antibodies. + T cells showed significantly upregulated TOX expression after 6 days.

[0038] The above results show that: In vitro αCD3 & CD28 antibodies continuously stimulate CD8 + T cells can fully induce CD8 after 6 days or more. + T cell depletion.

[0039] 7-2) In vitro continuous stimulation experiment with antigen peptide gp33-41: like Figure 10 As shown, the specific antigenic peptide gp33-41 was used to target CD8 antibodies enriched from mouse spleen. + T cells (represented as spleen cells in the diagram) were stimulated in vitro. Specific grouping and treatment methods included: (1) Non-stimuli control group; (2) Short-term stimulation group (Acute): Stimulated with gp33-41 for 2 days; (3) Repeat stimulation group: gp33-41 was used for continuous stimulation for 8 days, and the culture medium containing fresh antigen peptides was changed every 2 days during the period; (4) Continuous in situ stimulation group: cultured in a medium containing gp33-41 for 8 days without changing the medium.

[0040] like Figures 11 to 18 As shown, in the short-term stimulation group (stimulated for only 2 days), the cells did not express exhaustion markers (PD1, TIM3, LAG3) and the key transcription factor TOX, and the secretion function of effector cytokines (IL-2, TNFα and IFNγ) was normal, showing typical acute activation characteristics.

[0041] For the continuous stimulation groups (i.e., the in situ continuous stimulation group and the in situ continuous stimulation group for 8 days), CD8 was effectively induced regardless of whether the antigen peptide culture medium was changed to fresh during the period. + T cells are exhausted. These cells significantly overexpress surface exhaustion markers (PD1, TIM3, LAG3) and the transcription factor TOX, while their ability to secrete effector cytokines (IL-2, TNFα, and IFNγ) is severely impaired.

[0042] The above results show that in vitro specific antigen stimulation can also effectively induce CD8. + T cell depletion.

[0043] 7-3) IL-10 and in vitro CD8 + The relationship with T cell exhaustion: like Figure 19 As shown, in order to investigate the effects of immune microenvironment factors on CD8 + The effect of T cell exhaustion: This experiment investigated the effects of CD8 cell depletion isolated from C57BL / 6 mice. + T cells underwent different in vitro stimulation treatments. Specific groupings and treatment methods included: (1) Acute activation group: After stimulation with αCD3 & CD28 antibodies for 2 days, the cells were transferred to R10 medium containing IL-2 and cultured for another 6 days; (2) Continuous stimulation group: αCD3 & CD28 antibodies were used for continuous stimulation for 8 days (i.e., continuous stimulation of TCR signal alone). (3) IL-10 combined stimulation group (IL10 & Continuous): 20 ng / mL of IL-10 was added for combined treatment while αCD3 & CD28 antibody was continuously stimulated for 8 days.

[0044] like Figures 20 to 24 As shown, flow cytometry was used to detect CD8 in each group. + Indicators related to T cell exhaustion show: On the one hand, compared to continuous stimulation of TCR signals alone, continuous stimulation in combination with IL-10 can enhance CD8 activity. + The expression levels of T cell surface exhaustion markers PD1, TIM3, and LAG3 were higher, and TOX expression was also significantly higher than in cells stimulated by TCR signaling alone, indicating that IL-10 can further promote CD8+ expression in vitro. + The formation of T cell exhaustion.

[0045] The experimental results above show that continuous antigen stimulation is effective against CD8. + The root cause of T cell exhaustion is the continuous stimulation of antigens by CD8 cells. + The occurrence and maintenance of T cell exhaustion are determined by both intrinsic and extrinsic factors. Sustained TCR signaling stimulation is a method for in vitro induction of CD8... + The basic strategy for T cell exhaustion.

[0046] Transcription factor TOX (Thymocyte selection-associated HMG box protein) is a driver of CD8. + A key intrinsic factor in T cell exhaustion. High TOX expression maintains CD8. + T cell exhaustion state. This application demonstrates that continuous stimulation with αCD3 & CD28 antibodies and the gp33-41 peptide effectively induces high TOX expression, indicating that potent and effective TCR signaling stimulation induces CD8+ expression. + The root cause of T cell depletion.

[0047] In the process of developing this application, the applicant discovered that continuous TCR signaling stimulation combined with IL-10 treatment can further induce CD8+ in vitro. + T cell depletion.

[0048] IL-10 is a classic immunosuppressive cytokine that inhibits CD8+. + The regulatory role of T cell exhaustion is influenced by CD8. + The microenvironment in which T cells reside has a significant impact. In the aforementioned in vitro model experiments, it was confirmed that IL-10 can synergistically promote CD8 cell growth with sustained TCR signaling stimulation. +T cell exhaustion, particularly compared to continuous TCR signaling stimulation alone, was significantly upregulated by IL-10 combined stimulation, leading to TOX expression. This result provides a basis for further research on CD8. + This study provides an excellent in vitro induction method for regulating TOX expression in T cell exhaustion.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and not to limit them; under the concept of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of this application as described above, which are not provided in detail for the sake of brevity; although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A method for establishing in vitro CD8 + The method for the T cell exhaustion model is characterized by... include: Under in vitro culture conditions, T-cell receptor signaling stimulants combined with interleukin-10 were used to target CD8+. + T cells are continuously stimulated.

2. The method according to claim 1, characterized in that, The TCR signaling stimulants include: a combination of anti-CD3 antibody and anti-CD28 antibody, or a specific antigenic peptide.

3. The method according to claim 2, characterized in that, The specific antigenic peptides include: gp33-41 antigenic peptide.

4. The method according to claim 2, characterized in that, When the TCR signaling stimulant is a combination of anti-CD3 antibody and anti-CD28 antibody, the specific operation method for continuous stimulation includes: CD8 every 2 days + T cells were transferred to new culture plates coated with anti-CD3 antibodies for stimulation, and supplemented with culture medium containing anti-CD28 antibodies and IL-10. The total number of stimulation rounds was 3 to 4. or CD8 + T cells were placed in a culture plate coated with anti-CD3 antibody, and cultured with medium containing anti-CD28 antibody and IL-10 for 8 days without replacing the culture plate.

5. The method according to claim 3, characterized in that, When the TCR signaling stimulant is the gp33-41 antigen peptide, the specific operation method for continuous stimulation includes: The culture medium containing fresh gp33-41 antigen peptide was replaced every 2 days, with a total stimulation time of 8 days; or CD8 cells were continuously cultured in a medium containing the gp33-41 antigenic peptide. + T cells were collected for 8 days without changing the culture medium.

6. The method according to claim 1, characterized in that, The continuous stimulation was performed in a basal culture medium containing interleukin-2.

7. The method according to claim 6, characterized in that, The basal culture medium is RPMI-1640 medium containing 10% fetal bovine serum.

8. The method according to any one of claims 1-7, characterized in that, CD8 after continuous stimulation culture + When T cells possess the following characteristics (a), (b), and (c), the CD8+ is determined. + A model of T cell exhaustion has been established; (a) High expression of inhibitory receptor markers, said inhibitory receptor markers including one or more of PD1, TIM3, LAG3 and CD39; (b) High expression of transcription factor TOX; (c) Low expression of effector cytokines, including one or more of IL-2, TNFα and IFNγ.

9. An in vitro CD8 assay established by the method of any one of claims 1-8 + T cell exhaustion model in preparation for screening CD8 regulation + Application in high-throughput screening platforms for drugs or compounds with T-cell depletion.

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