Method for evaluating t cell exhaustion function in vitro and application thereof in predicting efficacy of immunotherapy

Abstract

The present application relates to a kind of in vitro T cell exhaustion function evaluation method and its application in immunotherapy efficacy prediction, belong to biological medicine technical field.The present application is by obtaining the T cell of subject source, constructs sustained antigen stimulation system in vitro, makes T cell form exhaustion related phenotype and functional change within a certain culture period, detects exhaustion related molecular marker expression and functional index change condition at preset time point, and based on HACD3 positive / negative subpopulation same sample in part layer comparison, establishes quantitative scoring model, for evaluating the exhaustion degree of T cell, to improve the accuracy and repeatability of immunotherapy efficacy prediction.The quantitative scoring model of the present application can be applied to the efficacy prediction and dynamic monitoring in treatment process of immune checkpoint inhibitor treatment, provide auxiliary basis for individualized treatment decision.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an in vitro T cell exhaustion function evaluation method and its application in predicting the efficacy of immunotherapy. In particular, it relates to a quantitative evaluation method of T cell exhaustion function based on continuous in vitro stimulation and HACD3 positive / negative internal stratification, and its application in predicting the efficacy of immune checkpoint inhibitors and monitoring drug use. Background Technology

[0002] Immune checkpoint inhibitors, especially anti-PD-1 / PD-L1 antibodies, have been widely used in the treatment of various malignant tumors, but significant individual differences exist in clinical efficacy. Current technologies used for efficacy prediction and treatment monitoring mainly include tumor tissue-based detection methods, such as PD-L1 immunohistochemical scoring, tumor mutational burden (TMB), and microsatellite instability (MSI) detection, as well as detection methods based on peripheral blood immune cell phenotypes or transcriptional characteristics. In addition, some studies observe changes in T cell function through short-term in vitro stimulation or co-culture systems for mechanistic studies or efficacy evaluation.

[0003] In general, existing technologies mainly rely on static expression levels or short-term functional indicators for detection, which mainly reflect the immune status at a certain point in time. There is still a lack of standardized in vitro modeling and quantitative evaluation systems for T cells to gradually become exhausted under continuous antigen stimulation.

[0004] Existing detection methods are mostly based on molecular or phenotypic expression results at a single time point, which makes it difficult to reflect the dynamic functional evolution of T cells under chronic antigen stimulation conditions, thus limiting their ability to predict individual immune reserve and potential response capacity. In addition, peripheral blood flow cytometry results are greatly affected by experimental conditions and gating strategies, and the consistency between different batches or different experimental platforms is limited.

[0005] Meanwhile, existing methods mostly focus on expression level detection and lack a standardized system for constructing a continuous antigen stimulation environment in vitro to simulate the exhaustion process. Therefore, it is difficult to quantitatively integrate the T cell exhaustion capacity, stability and functional decline, which limits its application value in efficacy prediction and drug monitoring. Summary of the Invention

[0006] To address the shortcomings of existing methods for predicting the efficacy of immunotherapy, such as reliance on static molecular expression detection, lack of in vitro functional exhaustion modeling systems, limited predictive ability, and insufficient reproducibility, this invention provides an in vitro T cell exhaustion function evaluation method and its application in predicting immunotherapy efficacy. This invention induces stable exhaustion-related phenotypes and functional states in T cells in vitro by constructing continuous antigen stimulation conditions. Based on stratified comparisons within the same sample of HACD3 positive / negative subsets, a corresponding quantitative evaluation system is established to improve the accuracy and reproducibility of immunotherapy efficacy prediction. Furthermore, this invention aims to apply the above-mentioned in vitro evaluation method to the efficacy prediction and dynamic monitoring of immune checkpoint inhibitor therapy, providing auxiliary evidence for personalized treatment decisions.

[0007] The objective of this invention can be achieved through the following methods: In a first aspect, the present invention provides a method for evaluating the exhaustive function of T cells in vitro, comprising the following steps: S1. Collect peripheral venous blood from the patient and separate peripheral blood mononuclear cells (PBMCs). S2. Sorting peripheral blood mononuclear cells to obtain CD8+ + T cells; S3. Add recombinant human IL-2 and human CD3 / CD28 T cell activator to the culture medium, and add CD8... + T cells were cultured in vitro in a culture medium to complete the initial stimulation; S4. Construct the target fragment containing the reporter gene and the persistent antigen-stimulating element HA-CD3ζ, and insert the target fragment into the lentiviral transfer vector backbone to obtain the recombinant expression vector; S5. Transducing the recombinant expression vector into CD8 after the initial stimulation in step S3. + T cells were cultured in vitro and the cells were collected. S6. First, stain the cells collected in step S5 with a live-cell dye, then stain the surface with an antibody against depletion-related molecular markers. After staining, perform flow cytometry to distinguish between HACD3+ and HACD3+ cells. Two subgroups; S7. Statistically analyze the positive rate and geometric mean fluorescence intensity of depletion-related molecular markers in the two subgroups, and establish a quantitative scoring model.

[0008] As one embodiment of the present invention, in step S1, the separation method includes density gradient centrifugation.

[0009] As one embodiment of the present invention, in step S2, the sorting method includes either magnetic bead sorting or flow sorting.

[0010] In one embodiment of the present invention, in step S3, the culture medium includes a T cell amplification culture medium.

[0011] As one embodiment of the present invention, in step S3, the final concentration of recombinant human IL-2 in the culture medium is 95-105 IU / mL, and the final concentration of human CD3 / CD28 T cell activator is 20-30 μL / mL.

[0012] In one embodiment of the present invention, in step S4, the reporter gene includes eGFP; and the lentiviral transfer vector backbone includes the pWPT vector.

[0013] As one embodiment of the present invention, in step S4, the nucleotide sequence of the continuous antigen-stimulating element HA-CD3ζ is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0014] As one embodiment of the present invention, in step S4, the nucleotide sequence of the target fragment is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0015] As one embodiment of the present invention, in step S6, the exhaustion-related molecular marker antibodies include anti-PD1 antibody, TIM3 antibody, SLAMF6 antibody, and CD39 antibody.

[0016] As one embodiment of the present invention, in step S7, the quantitative scoring model includes Target Score and Exhaustion Score; The formula for calculating the Target Score is as follows: ; Wherein, PD1 Pos Rate is the PD1 positivity rate, and GM PD1+ GM represents the geometric mean fluorescence intensity of PD1 in the HACD3+ subset. PD1- The geometric mean fluorescence intensity of PD1 in the HACD3- subgroup; The formula for calculating the Exhaustion Score is: ; Wherein, TIM3 is the geometric mean fluorescence intensity of TIM3, CD39 is the geometric mean fluorescence intensity of CD39, and SLAMF6 is the geometric mean fluorescence intensity of SLAMF6.

[0017] In this invention, the Target Score characterizes the PD-1 targetability / potential immunotherapy benefit space formed by T cells under continuous stimulation; the Exhaustion Score characterizes the degree of T cell exhaustion, especially the burden of progressing to terminal exhaustion. Based on these quantifiable indicators, the degree of T cell exhaustion, functional potential, and potential responsiveness to immune checkpoint interventions in a sample can be assessed, thereby providing a basis for efficacy prediction, sample stratification, and functional assessment.

[0018] This invention reveals that sustained TCR / CD3 signaling input is a key factor driving the gradual transition from an activated state to a state of functional decline and exhaustion during T cell exhaustion. In chronic infection or tumor microenvironments, T cells are subjected to prolonged antigen stimulation. The pathophysiological process initially manifests as sustained and repetitive TCR / CD3 axis activation, followed by progressive upregulation of inhibitory receptors, decreased effector function, and exhaustion-related program remodeling. Compared to the traditional HA-28Z conformation, which also contains co-stimulatory domains, HACD3 primarily provides sustained CD3ζ signaling input, reducing the amplifying effect of additional co-stimulatory signals on activation intensity, amplification capacity, and short-term effector output. Therefore, it more closely approximates the basal state of T cells under sustained receptor stimulation under chronic antigen exposure conditions. Based on this, the HACD3 of this invention is more suitable for simulating the dynamic process of T cell exhaustion in vitro and for subsequent quantitative evaluation of exhausted functional states.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. First, this invention is no longer limited to static expression detection at a single time point, but rather simulates the T cell exhaustion process under chronic antigen stimulation in vitro, evaluating the functional potential of individual T cells from a dynamic perspective. Second, by constructing a standardized stimulation system, this invention improves the consistency and repeatability of experimental conditions, reducing operational differences between different batches and different experimenters. Third, this invention integrates multiple exhaustion-related indicators to form a quantifiable scoring system, enhancing the comparability and interpretability of the results. In addition, the method of this invention can be implemented before or in the early stages of treatment, and is characterized by relatively simple operation and high sample availability, making it suitable for clinical decision support and dynamic monitoring applications.

[0020] 2. This invention simulates the gradual exhaustion of T cells under chronic antigen exposure by constructing continuous antigen stimulation conditions in vitro, thus evaluating the exhaustion tendency and functional potential of individual T cells from a dynamic functional perspective. Compared to existing prediction methods that primarily rely on single-timepoint molecular expression detection, this invention can more comprehensively reflect the evolution of T cell function and improve the accuracy of predicting the efficacy of immune checkpoint inhibitors.

[0021] 3. This invention establishes a relatively standardized in vitro stimulation system and quantitative scoring method, which helps to reduce the impact of experimental operation differences on test results and improve the repeatability and comparability of test results. At the same time, by integrating and analyzing multiple exhaustion-related phenotypes and functional indicators, quantifiable and traceable functional evaluation results can be formed, enhancing the stability and consistency of result interpretation.

[0022] 4. The sample source required for this invention is peripheral blood T cells, which are relatively easy to obtain and have good clinical operability. It can be implemented before or early in treatment for efficacy prediction or dynamic monitoring during treatment. This method helps provide auxiliary evidence for the development of personalized immunotherapy strategies and has potential clinical translational value. Attached Figure Description

[0023] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 A flowchart illustrating the in vitro T cell exhaustion function evaluation method of this invention and a schematic diagram illustrating the structure of the lentiviral vector used; wherein, Figure 1 A is a flowchart illustrating the in vitro T cell exhaustion function evaluation method; Figure 1 B is a schematic diagram of a lentiviral vector structure; Figure 2 This is a schematic diagram illustrating the flow cytometry gating strategy and PD1 detection; among which... Figure 2 A represents the test results of the gated process and the PD1 positive group; Figure 2 B represents the statistical results of PD1 positivity rate in HACD3 positive and negative populations; Figure 2 C represents the statistical result of the geometric mean fluorescence intensity (GeoMFI) of PD1; Figure 3 This is a schematic diagram illustrating the detection results of depletion-related molecular markers; among which, Figure 3 A is a flow cytometry histogram of TIM3, CD39, and SLAMF6 expression in HACD3 positive and negative populations; Figure 3 B represents the statistical result of the geometric mean fluorescence intensity of the corresponding marker; Figure 3 C is the flow cytometry histogram for further analysis of TIM3, CD39 and SLAMF6 expression within the PD1 positive subset; Figure 3 D represents the corresponding statistical results; Figure 4 This is a schematic diagram illustrating the construction and calculation results of the scoring model; where, Figure 4 A is the formula for calculating the Target Score; Figure 4 B represents the Target Score results for different groups; Figure 4 C is the formula for calculating the Exhaustion Score; Figure 4 D represents the Exhaustion Score results for different groups. Detailed Implementation

[0024] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are implemented under the premise of the technical solution of the present invention, providing detailed implementation methods and specific operating procedures, which will help those skilled in the art to further understand the present invention. It should be noted that the scope of protection of the present invention is not limited to the following embodiments; any adjustments and improvements made under the concept of the present invention are all within the scope of protection of the present invention.

[0025] The HACD3+ subset refers to a population of T cells expressing the persistent antigen-stimulating element, preferably identified by a reporter signal co-expressed with the persistent antigen-stimulating element; HACD3 Subpopulation refers to a population of negative T cells in the same sample that do not express the continuously stimulating antigen. This embodiment distinguishes between HACD3+ and HACD3+ cells based on eGFP signaling. Subgroups, but not limited to this approach.

[0026] Example like Figure 1 As shown in Figure A, peripheral blood samples were first obtained from the subjects. Peripheral blood mononuclear cells (PBMCs) were then isolated using density gradient centrifugation. The specific procedures were as follows: 5 mL of peripheral venous blood was collected from a gastric cancer patient and placed in an EDTA anticoagulant blood collection tube. The blood was processed within 2 hours of collection. The blood was slowly spread on top of 5 mL of Ficoll separation solution and centrifuged vertically at 2000 rpm for 20 min at room temperature, without braking during the deceleration phase. After centrifugation, the cells in the middle white membrane layer were aspirated and transferred to a new 15 mL centrifuge tube. 10 mL of PBS was added, and the tube was centrifuged at 400 g for 5 min. The supernatant was discarded, and the cells were washed twice. Finally, the cells were resuspended in sorting buffer (STEMCELL EasySep™ Buffer) for subsequent counting and sorting.

[0027] CD8 was then separated using a magnetic bead sorting method. + T cells were prepared by taking PBMCs obtained using the aforementioned method and adjusting the cell concentration to 5 × 10⁻⁶. 7 Cells / mL. Place the cell suspension in a 5mL round-bottom sorting tube and add 50μL / mL of LEasySep™ Human CD8. +T Cell Isolation Cocktail: Gently mix and incubate at room temperature for 5 min. Vortex EasySep™ Dextran RapidSpheres™ for 30 s, then add 50 μL / mL magnetic beads and gently mix. Add sorting buffer to bring the total volume to 2.5 mL, remove the cap, and place in an EasySep™ Magnet. Incubate at room temperature for 3 min. After incubation, invert the magnetic rack along with the sorting tube and continuously transfer the supernatant into new sterile centrifuge tubes. The resulting supernatant is the isolated CD8. + T cells can be directly used for subsequent culture, stimulation, and flow cytometry detection. The above procedure is based on the manual sorting conditions for PBMCs listed in the official manual of this kit.

[0028] CD8 obtained by sorting + T cells were initially activated under in vitro culture conditions to improve cell viability and transduction efficiency. The specific steps were as follows: CD8 cells obtained from the above isolation were... + T cells were resuspended in ImmunoCult™-XF T Cell Expansion Medium and the viable cell density was adjusted to 1×10⁻⁶. 6 Cells / mL. Recombinant human IL-2 was added to the culture medium to a final concentration of 100 IU / mL. Then, 25 μL / mL of ImmunoCult™ Human CD3 / CD28 T CellActivator was added, gently mixed, and incubated at 37°C in a 5% CO2 incubator for 3 days to complete initial activation. After activation, the culture system was continued, with cell counts every 2–3 days, fresh culture medium added, and cell density adjusted.

[0029] Following initial stimulation, a continuous antigen-stimulating element was introduced into CD8 via lentiviral transduction. + In T cells, the specific steps were as follows: RetroNectin was diluted to 5 μg / mL with sterile PBS and added to a culture plate for coating, then incubated overnight at 4°C. The next day, the coating solution was discarded, and the plate was washed once with PBS and once with culture medium. Subsequently, initially activated CD8+ was added to the coated plate. +T cells and lentiviral solution containing a continuous antigen-stimulating element were used. Cells were resuspended in ImmunoCult™-XF T Cell Expansion Medium, and the culture system contained 100 IU / mL IL-2. After adding the samples, the culture plates were centrifuged at 4000 rpm for 90 min to promote cell-lentiviral particle contact. After centrifugation, the plates were incubated at 37°C in a 5% CO2 incubator. On the second day, the culture medium was replaced with fresh medium for further amplification. The coating concentration, blocking conditions, and culture conditions of RetroNectin can be set according to Takara's official documentation; the centrifugation-induced transduction step can be optimized according to viral titer, cell state, and plate size.

[0030] The continuous antigen-stimulating element described in this embodiment includes a domain capable of continuously transmitting activation signals within the cell, and its expression can be monitored using a fluorescent reporter gene. For example... Figure 1 As shown in Figure B, using pWPT as the lentiviral transfer vector backbone, and following the expression cassette construction principles, the target fragment eGFP-F2A-HA-CD3ζ (synthesized by Suzhou Anshengda Gene Technology Co., Ltd., its nucleotide sequence is shown in SEQ ID NO.3, and its amino acid sequence is shown in SEQ ID NO.4) was directionally inserted into the pWPT vector to obtain the recombinant expression vector pwpt-eGFP-F2A-HA-CD3ζ. The expression cassette is driven by the EF-1α core promoter, eGFP serves as a reporter gene to indicate transduction efficiency, and the F2A sequence mediates the co-expression of eGFP and the HA-CD3ζ element. The HA-CD3ζ element includes a CD8 signal peptide, an HA-related antigen recognition sequence, a CD8 hinge, a CD8 transmembrane region, and a CD3ζ intracellular domain, used for expression of CD8+ after transduction. + Continuous antigen stimulation signals are generated in T cells. The nucleotide sequence of the HA-CD3ζ element is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2. After the recombinant vector is constructed, positive clones are identified, and the target insert fragment is sequenced to confirm its correctness in terms of insertion sequence, orientation, and reading frame. The correctly identified recombinant plasmid is used for subsequent lentiviral packaging. The constructed pWPT-eGFP-F2A-HA-CD3ζ transfer vector plasmid, along with helper packaging plasmids and envelope plasmids, is introduced into 293T packaging cells, and viral packaging is performed using PEI-mediated transient transfection. After transfection, packaging cells are cultured, and the culture supernatant containing recombinant lentiviral particles is collected. The obtained viral supernatant is purified and concentrated for subsequent use on initially activated CD8 cells. +T cells are used for transduction. The PEI can be a cationic polymer transfection reagent capable of mediating transient transfection of eukaryotic cells; the specific types, sources and proportions of each plasmid in the three-plasmid system can be selected and optimized according to conventional techniques in the art. As long as the viral packaging of the target expression cassette can be achieved and recombinant lentiviral particles that can be used for transduction of target cells are obtained, they are all within the scope of protection of this invention.

[0031] After transfection, cells were recovered and cultured in ImmunoCult™-XF T Cell Expansion Medium supplemented with 100 IU / mL IL-2. Cells were incubated at 37°C in a 5% CO2 incubator, with the medium changed every 2-3 days and fresh medium added as needed based on cell condition. After 10-15 days of continued culture, GFP expression and related phenotypic changes were detected, and cells expressing the target element were used for subsequent continuous antigen stimulation and exhaustion evaluation experiments. The culture medium was a serum-free, heterologous-free medium suitable for human T cell expansion, containing the exogenous cytokine IL-2 but without other exogenous recombinant cytokines. In vitro cultured GFP-positive T cells gradually developed exhaustion-related phenotypes under continuous signal stimulation.

[0032] like Figure 2 As shown in Figure A, cells were collected between days 10 and 15 of culture, with testing preferably performed on day 15. Approximately 5 × 10⁶ cells were collected from each sample. 5 Cells were resuspended in flow cytometry staining buffer. Live cells were first stained with a live-dead stain, washed, and then surface stained with anti-PD1 antibody. Other marker-related antibodies, such as TIM3, SLAMF6, and CD39, were added simultaneously if necessary. After staining, cells were washed and resuspended for flow cytometry analysis. For flow cytometry results analysis, gating was performed sequentially as follows: first, lymphocyte populations were delineated on the FSC-A / SSC-A map; then, single-cell populations were delineated on the FSC-H / FSC-A map; next, live cell populations were delineated based on live-dead staining signals; finally, within the live single-cell population, HACD3+ and HACD3+ were distinguished based on eGFP signals. Two subgroups. Within each subgroup, the PD1 positivity rate and PD1 GeoMFI were calculated separately. For example... Figure 2 B and Figure 2 As shown in C ( Figure 2 B and Figure 2 The straight lines in C represent the peripheral blood sample test results of the five patients, with P values ​​of 0.0133 and 0.0011, respectively. The eGFP positivity threshold and PD1 positivity threshold are preferably set based on the non-transduced negative control and / or FMO control. Each sample should ideally contain no fewer than 10,000 live single-cell events.

[0033] Furthermore, such as Figure 3 As shown in Figure A, the expression of exhaustion-related or function-related molecular markers such as TIM3, CD39, and SLAMF6 was detected in continuously stimulated positive and negative populations. Specifically, during flow cytometry analysis, gating was performed in the following order: first, lymphocyte populations were delineated on the FSC-A / SSC-A map; then, single-cell populations were delineated on the FSC-H / FSC-A map; next, live cell populations were delineated based on live / dead staining signals; finally, within the live single-cell population, HACD3+ and HACD3+ were distinguished based on eGFP signals. Two subgroups were identified. The expression levels of TIM3, CD39, and SLAMF6 were detected within each of these two subgroups. In this embodiment, the geometric mean fluorescence intensity (GeoMFI) was calculated (in other embodiments, the positive rate can also be calculated as an auxiliary analytical indicator). Figure 3 A and Figure 3 As shown in B. The positive thresholds for each marker are preferably set based on the unstained control and / or FMO control. Each sample preferably contains no fewer than 10,000 live single-cell events. In another preferred embodiment of the invention, the expression of TIM3, CD39, and SLAMF6 can be further analyzed within the PD1-positive cell population. Specifically, after completing the aforementioned lymphocyte, single-cell, live cell, and eGFP positive / negative populations, PD1-positive cell populations are further delineated within each population based on PD1 staining signals, and the expression levels of TIM3, CD39, and SLAMF6 in the continuously stimulated positive / PD1-positive population and the continuously stimulated negative / PD1-positive population are compared respectively. The preferred result reading parameter is GeoMFI (in other embodiments, its positive rate can also be further calculated as an auxiliary analytical indicator). Figure 3 C and Figure 3 As shown in D, this is to improve the precision of the evaluation. Among them, Figure 3 B and Figure 3 The straight lines in D represent the test results of the four patients.

[0034] In the data processing stage, such as Figure 4 A and Figure 4 As shown in C, a quantitative scoring model can be established based on the positive rate and fluorescence intensity parameters obtained from the detection. For example, the Target Score can be calculated from the PD1 positive rate and its fluorescence intensity ratio (the Target Score is mainly used to reflect the "targetability" or "potential benefit space" of T cells in intervention on the PD-1 axis, and its core should be calculated based on PD1-related indicators); the Exhaustion Score can be calculated by integrating the expression levels of exhaustion-related molecules and function-related molecules. Through the above scoring methods, multi-dimensional detection results are transformed into quantifiable indicators for evaluating the degree of T cell exhaustion and functional potential, such as... Figure 4 B and Figure 4As shown in D (where, Figure 4 The straight line in B represents the test results of 5 patients, with a P-value of 0.00973; Figure 4 The straight line in D represents the test results of 4 patients, with a P-value of 0.0194.

[0035] In this embodiment, the duration of continuous antigen stimulation, the detection time point, and the scoring calculation method can be adjusted according to different application requirements. The method can be used for baseline evaluation before treatment and for dynamic monitoring during treatment. The above embodiments demonstrate that the present invention can simulate the T-cell exhaustion process under chronic antigen stimulation in vitro and form a standardized, quantifiable functional evaluation system.

[0036] The purpose of this embodiment is not to directly complete a clinical efficacy prediction model validated by a large sample size using an existing small sample size, but rather to demonstrate the feasibility, quantifiability, and reproducible observable technical effects of the in vitro T cell exhaustion function evaluation method, the stratified comparison strategy within the same sample for HACD3 positive / negative samples, and the Target Score and Exhaustion Score scoring system described in this application. This embodiment is used to demonstrate the feasibility and quantifiability of the in vitro continuous stimulation system and scoring method, rather than limiting a single clinical prediction threshold. The stratification threshold and prediction model can be further optimized in a larger cohort by incorporating clinical efficacy endpoints.

[0037] Those skilled in the art can make various modifications or substitutions to the above embodiments without departing from the spirit and essence of the present invention, and all such modifications or substitutions should fall within the protection scope of the present invention.

[0038] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for evaluating the exhaustive function of T cells in vitro, characterized in that, Includes the following steps: S1. Collect peripheral venous blood from the patient and separate peripheral blood mononuclear cells; S2. Sorting peripheral blood mononuclear cells to obtain CD8+ + T cells; S3. Add recombinant human IL-2 and human CD3 / CD28 T cell activator to the culture medium, and add CD8... + T cells were cultured in vitro in a culture medium to complete the initial stimulation; S4. Construct the target fragment containing the reporter gene and the persistent antigen-stimulating element HA-CD3ζ, and insert the target fragment into the lentiviral transfer vector backbone to obtain the recombinant expression vector; S5. Transducing the recombinant expression vector into CD8 after the initial stimulation in step S3. + T cells were cultured in vitro and the cells were collected. S6. First, stain the cells collected in step S5 with a live-cell dye, then stain the surface with an antibody against depletion-related molecular markers. After staining, perform flow cytometry to distinguish between HACD3+ and HACD3+ cells. Two subgroups; S7. Statistically analyze the positive rate and geometric mean fluorescence intensity of depletion-related molecular markers in the two subgroups, and establish a quantitative scoring model.

2. The evaluation method according to claim 1, characterized in that, In step S1, the separation method includes density gradient centrifugation.

3. The evaluation method according to claim 1, characterized in that, In step S2, the sorting method includes either magnetic bead sorting or flow sorting.

4. The evaluation method according to claim 1, characterized in that, In step S3, the culture medium includes a T cell expansion culture medium.

5. The evaluation method according to claim 1, characterized in that, In step S3, the final concentration of recombinant human IL-2 in the culture medium is 95-105 IU / mL, and the final concentration of human CD3 / CD28 T cell activator is 20-30 μL / mL.

6. The evaluation method according to claim 1, characterized in that, In step S4, the reporter gene includes eGFP; the lentiviral transfer vector backbone includes the pWPT vector.

7. The evaluation method according to claim 1, characterized in that, In step S4, the nucleotide sequence of the continuous antigen-stimulating element HA-CD3ζ is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

2.

8. The evaluation method according to claim 1, characterized in that, In step S4, the nucleotide sequence of the target fragment is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.

4.

9. The evaluation method according to claim 1, characterized in that, In step S6, the exhaustion-related molecular marker antibodies include anti-PD1 antibody, TIM3 antibody, SLAMF6 antibody, and CD39 antibody.

10. The evaluation method according to claim 1, characterized in that, In step S7, the quantitative scoring model includes Target Score and Exhaustion Score; The formula for calculating the Target Score is as follows: ； Wherein, PD1 Pos Rate is the PD1 positivity rate, and GM PD1+ GM represents the geometric mean fluorescence intensity of PD1 in the HACD3+ subset. PD1- The geometric mean fluorescence intensity of PD1 in the HACD3- subgroup; The formula for calculating the Exhaustion Score is: ； Wherein, TIM3 is the geometric mean fluorescence intensity of TIM3, CD39 is the geometric mean fluorescence intensity of CD39, and SLAMF6 is the geometric mean fluorescence intensity of SLAMF6.

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