A Single-Cell In Situ Imaging Method for T-Cell Immune Status Based on Rolling Circle Amplification Reaction and Its Application

Abstract

This invention relates to a single-cell in situ imaging method for T-cell immune status based on rolling circle amplification (RCA) and its applications. The method is as follows: 1) Cell samples are fixed and permeabilized; 2) A lock-type probe composed of a recognition sequence, a binding sequence, and a structure-modification sequence is used to specifically identify characteristic mRNAs of T-cell immune status; 3) A circular template is formed under the action of T4 DNA ligase, and the circular template is amplified in situ using trigger primers and phi29 DNA polymerase to obtain RCA amplification products; fluorescent detection probes bind to the RCA amplification products to generate fluorescent spots for single-cell in situ imaging analysis. This method has high selectivity and specificity, enabling in situ visualization analysis of T-cell immune activation status, and visualization of the immune activation status, quantity, and location information of tumor-infiltrating T cells, laying the foundation for constructing an immunophenotyping index system based on molecular imaging.

Description

Technical Field

[0001] This invention belongs to the field of cell imaging, specifically relating to a single-cell in situ imaging method for T cell immune status based on rolling circle amplification reaction and its application. Background Technology

[0002] The World Health Organization (WHO) predicts that the number of cancer cases worldwide may increase by 60% in the next 20 years, making the fight against cancer an urgent priority. In recent years, tumor immunotherapy has become a cutting-edge field in cancer treatment. It activates the body's immune system to recognize and eliminate tumor cells, offering advantages such as low toxicity, high specificity, long-lasting effectiveness, and the ability to generate immune memory. This provides a new direction for cancer treatment and has been clinically applied to the treatment of various malignant tumors. However, sustained immune responses induced by immunotherapy in cancer patients are not common, with an overall efficacy rate of less than 20% in various malignant tumors. Tumor-infiltrating lymphocytes (TILs) play a crucial role in tumor immunotherapy. Studies have shown a positive correlation between the content of TILs in various primary solid tumors and the patient's clinical prognosis. The immune status and quantity of tumor-infiltrating CD8+ T cells vary significantly in the tumor microenvironment of different types of cancer, which may fundamentally affect the drug response to immunotherapy. Therefore, accurate and systematic analysis of cell type, number, location and immune status of TILs is of great scientific and clinical value for predicting clinical response, treatment effect and prognostic assessment of tumor immunotherapy.

[0003] Currently, the most widely used routine diagnostic methods in clinical practice include magnetic resonance imaging (MRI), positron emission tomography (PET), flow cytometry (FCM), and immunohistochemistry (IHC). While MRI and PET can achieve specific targeted imaging of cells or tumors, their low specificity, sensitivity, and spatiotemporal resolution make it difficult to accurately assess the immune status of CD8+ T cells. FCM, due to its high quantitative accuracy, strong anti-interference ability, and multiplexing capabilities, is widely used for cell subset typing and functional status analysis. However, FCM requires the preparation of single-cell suspensions, which involves detaching cells from their immune microenvironment, resulting in the loss of spatial location information regarding cell-microenvironment interactions and potentially altering cell state. Immunohistochemistry can provide information on immune cell types, cell boundaries, and tissue environment, enabling immunophenotyping of the tumor microenvironment. However, its quantitative accuracy is not high, its throughput is low, and it is limited to certain specific proteins, thus lacking in comprehensive and accurate analysis of cell types and states. Single-cell RNA sequencing (scRNA-seq) results indicate that mRNA can serve as a biomarker for TIL cell type and immune status, and can also characterize certain specific T cell states (such as the "pre-exhausted" state). However, due to the lack of necessary information on the location and number of tumor-infiltrating T cells, scRNA-seq still faces significant challenges in accurately assessing the immune microenvironment. Therefore, there is an urgent need to develop a precise in-situ immune status typing method for tumor-infiltrating T cells to meet the clinical need for comprehensive and accurate analysis of the number, immune status, and location (depth of invasion) of tumor-infiltrating T cells. Summary of the Invention

[0004] The purpose of this invention is to propose a single-cell in situ imaging method for T-cell immune status based on rolling circle amplification reaction, which can be used for in situ single-cell analysis of the immune status, number and location of tumor-infiltrating T cells in clinical specimens.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A single-cell in situ imaging method for T-cell immune status based on rolling circle amplification (RCA) involves fixing and permeabilizing cell samples; using lock-lock probes to specifically identify characteristic mRNAs of T-cell immune status; forming a circular template under the action of T4 DNA ligase; and using trigger primers and phi29 DNA polymerase to perform an in situ amplification reaction on the circular template to obtain RCA amplification products; and generating fluorescent spots by binding to the RCA amplification products with fluorescent detection probes for single-cell in situ imaging analysis.

[0006] The above-mentioned single-cell in situ imaging method for T cell immune status based on rolling circle amplification reaction includes the following steps: 1) Fix cell samples with 4% paraformaldehyde for 10-25 min, then permeabilize with 0.5% Triton-X 100 for 5 min, and wash cells with PBS; 2) Add 20×SSC buffer, dithiothreitol, RiboLock RNase inhibitor, yeast tRNA, DEPC-treated water, and lock probe to the cells treated in step 1). Incubate overnight at 37°C, and wash cells with PBS. The lock probe comprises three functional regions: a recognition sequence, a binding sequence, and a structure adjustment sequence. The recognition sequence is divided into two segments and can specifically recognize the target nucleic acid sequence. The binding sequence can specifically bind to the fluorescent probe to output a detection signal. The structure adjustment sequence is designable and can alter the secondary structure of the lock probe, reducing intramolecular hybridization and improving the recognition efficiency of the target nucleic acid. 3) Add T410×Ligation buffer, T4 DNA ligase, RiboLock RNase inhibitor, and DEPC-treated water to the cells treated in step 2), react at 37°C for 2 h, and wash the cells with PBS; then add 20×SSC buffer, deionized formamide, trigger primer, and DEPC-treated water, react at 37°C for 60 min, and wash the cells with PBS; then add 10×phi29 buffer, phi29 DNA polymerase, dNTP Mix, and DEPC-treated water, react at 37°C for 2 h, and wash the cells with PBS. 4) Add 20×SSC buffer, deionized formamide, salmon sperm DNA, fluorescent detection probe and DEPC-treated water to the cells treated in step 3), and react at 37°C for 30 min; wherein, the fluorescent detection probe can bind to the RCA amplification products generated in the cells to produce fluorescent spots, thereby realizing single-cell in situ imaging of T cell immune status. The locking probe is 50-120 nt in length. The length of both identification sequences is 10-40 nt; The trigger primer sequence is: 5'-TGAGGTAGTATGTTGTATAGTT-3'; The fluorescent detection probe sequence is: 5'-AACTATACAACATACTACCTCA-Alexa Fluor488-3'.

[0007] The above method is applied to the in situ visualization of T cells in the immune-activated state.

[0008] The significant advantages of this invention are: 1) Compared with PCR, RCA does not require repeated heat denaturation and can be amplified at a constant temperature (37℃), which reduces its dependence on precision instruments. Compared with in situ sequencing / spatial transcriptomics technology (4-5 days), the entire RCA experimental process can be completed within 24 hours, making it simpler to operate, lower in detection cost, and more suitable for clinical applications.

[0009] 2) Specificity and sensitivity: RCA has single-molecule recognition capability (a single target molecule produces a single amplification product), and compared with magnetic resonance imaging (MRI) and positron emission tomography (PET), RCA exhibits higher specificity and sensitivity.

[0010] 3) Spatial Information: Flow cytometry (FCM) and single-cell RNA sequencing (scRNA-seq) require the preparation of single-cell suspensions, which removes cells from their immune microenvironment. This not only results in the loss of spatial location information regarding cell-microenvironment interactions but may also alter cell state. In contrast, RCA is an in situ amplification technique for characteristic genes, providing precise spatial location information for target genes and offering a technological advantage in assessing the spatial distribution of tumor-infiltrating T cells.

[0011] 4) Versatility: Antibodies used in immunohistochemistry / fluorescence techniques require strict screening, and the number of antibodies that can specifically identify T cell types and immune status markers is limited; while this invention uses characteristic genes as markers of T cell types and immune status, making the identification probe design simpler and more versatile. Attached Figure Description

[0012] Figure 1 : A schematic diagram of the structure of the padlock probe, which is a T-cell type and immune status specific recognition probe of the present invention.

[0013] Figure 2 : Schematic diagram of the rolling circle amplification reaction principle of this invention.

[0014] Figure 3 To verify the feasibility of a single-cell in situ imaging method based on rolling circle amplification reaction.

[0015] Figure 4 Investigating the effect of lock probe recognition length on the efficiency of rolling circle amplification reaction.

[0016] Figure 5 To verify the specificity of a single-cell in situ imaging method based on nucleic acid rolling circle amplification.

[0017] Figure 6 : To verify the single-cell in situ imaging of the T-cell immune activation state characteristic gene 4-1BB.

[0018] Figure 7Single-cell in situ imaging of the immune activation status of tumor-infiltrating T cells in clinical tissue specimens.

[0019] Figure 8 In situ visualization and statistical analysis of the depth and number of T-cell tumor infiltration in the immune-activated state in clinical tissue specimens. Detailed Implementation

[0020] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0021] This invention uses the characteristic gene of CD8+ T cells (CD8a mRNA) as the target gene, designs a lock-lock probe that specifically recognizes CD8a mRNA, establishes a single-cell in situ imaging method based on rolling circle amplification technology, and verifies that the method has high selectivity and specificity. Using 4-1BB mRNA as the characteristic gene of T cell immune activation status, the above-constructed single-cell in situ fluorescence imaging method based on rolling circle amplification reaction is applied to actual clinical tumor tissue sections to achieve visualization analysis of the immune activation status, quantity, and location information of tumor-infiltrating T cells. This lays the foundation for constructing an immunophenotyping index system based on molecular imaging and provides data support for the accurate prediction, prognostic assessment, and clinical application of tumor immunotherapy efficacy.

[0022] The nucleic acid primers used in the following examples were all purchased from General Biotech (Anhui) Co., Ltd., and purified by PAGE / HPLC. The CD8+T cell isolation kit (CD8+T Cell Isolation Kit, 130-096-495) was purchased from Miltenyi Biotec. 4% paraformaldehyde fixative (E672002-0100), Triton X-100 (A600198), PBS buffer (B540626-0500), dithiothreitol (DTT, A620058), deionized formamide (A600211), dNTP Mix (10mM, B500056-0500), and DEPC-treated water (B501005) were purchased from Sangon Biotech (Shanghai) Co., Ltd. Human whole blood mononuclear cell separation medium (Ficoll preparation, P9011), 20×SSC buffer (pH=7.4, S1090), yeast tRNA (10 mg; T8630), Tween-20 (T8220), salmon sperm DNA (H1060), and anti-fluorescence decay mounting medium (containing DAPI, S2110) were purchased from Solarbio Science & Technology Co., Ltd. RiboLock RNase inhibitor (EO0382), T4 DNA ligase (EL0011), and phi29 DNA polymerase (EP0092) were purchased from Thermo Fisher Scientific. Tissue autofluorescence quencher ( Lipofuscin Autofluorescence Quencher (23007) was purchased from Biotium.

[0023] Example 1: Design of Lock-on Probes for Specific Recognition of T Cell Type and Immune Status The lock-on probe designed in this application comprises three functional regions: a recognition sequence, a binding sequence, and a structure-modifying sequence. Figure 1 The recognition sequence is divided into two segments, which can specifically recognize the target nucleic acid sequence; the binding sequence can specifically bind to the fluorescent probe to output a detection signal; the structure adjustment sequence is designable and can change the secondary structure of the lock probe, reduce its intramolecular hybridization, and improve the recognition efficiency of the target nucleic acid.

[0024] Using CD8a mRNA specifically expressed by CD8+ T cells as the target nucleic acid, intramolecular hybridization and secondary structure of the lock-in probe were reduced through nucleic acid hybridization thermodynamic-assisted calculations (NUPACK, http: / / www.nupack.org; IDT-OligoAnalyzer, https: / / sg.idtdna.com / calc / analyzer). Based on this, the designed specific recognition lock-in probe nucleic acid sequence is shown in the table below: Example 2: Construction of a single-cell in situ imaging method based on nucleic acid rolling circle amplification Using the characteristic gene of CD8+ T cells (CD8a mRNA) as the target nucleic acid, a lock-lock probe that specifically recognizes CD8a mRNA was designed, and a single-cell in situ imaging method based on rolling circle amplification technology was established. The technical process is as follows: Figure 2 As shown: The recognition process involves the padlock probe specifically recognizing characteristic mRNAs of T cells; the amplification process involves forming a circular template using T4 DNA ligase, followed by in-situ amplification using a trigger primer and Phi29 DNA polymerase, yielding a large number of DNA repeat sequences carrying the detection probe binding sequence, i.e., the RCA amplification product; the imaging process involves the fluorescent detection probe binding to the RCA amplification product, producing fluorescent spots for single-cell in-situ imaging analysis. The specific experimental steps are as follows: 1) Cell pretreatment: Human blood samples (approved by the Medical Ethics Committee) were obtained from the biobank of Mengchao Hepatobiliary Hospital, Fujian Medical University, and placed in centrifuge tubes. An equal volume of human whole blood mononuclear cell separation solution was added and mixed thoroughly. The tubes were centrifuged at 800×g (slow deceleration mode) to collect peripheral blood mononuclear cells from the whole blood. CD8+ T cells (10T) were isolated from the peripheral blood mononuclear cells using a CD8+ T cell separation kit via magnetic bead sorting. 6 (3 mL, per 100 cells / mL); take 5 × 10 5 CD8+ T cells were fixed in a confocal microplate at 25°C for 10 min with 200 μL of 4% w / v paraformaldehyde. The surface liquid in the microplate was aspirated and the cells were washed once with PBS. Then, the cells were permeabilized in the microplate with 20 μL of 0.5% v / v Triton-X 100 at room temperature for 5 min. The surface liquid in the microplate was aspirated and the cells were washed once with PBS to obtain the pretreated cells.

[0025] 2) Identification process: Add a 20 μL lock-probe hybridization system to the cells pretreated in step 1). The 20 μL lock-probe hybridization system comprises: 2 μL 20×SSC buffer, 2 μL 10 μM Padlock probe (CD8a), 1 μL 100 mM dithiothreitol (DTT), 0.5 μL 40 U / mL RiboLock RNase inhibitor, 4 μL 10 mg / mL yeast tRNA, and 10.5 μL DEPC-treated water; incubate overnight at 37°C. After the reaction, aspirate the surface liquid from the microwells and wash the cells once with PBS.

[0026] 3) Amplification process: Add 2 μL T410×Ligation buffer, 2 μL 5 U / μL T4 ligase, 0.5 μL 40 U / mL RiboLock RNase inhibitor, and 15.5 μL DEPC-treated water to the cells treated in step 2). Incubate at 37°C for 2 h. After the reaction, aspirate the surface liquid from the wells and wash the cells once with PBS. Then add 2 μL 20×SSC buffer, 2 μL deionized formamide, 1 μL 5 μM trigger primer, and 15 μL LDEPC-treated water. Incubate at 37°C for 60 min. After the reaction, aspirate the surface liquid from the wells and wash the cells once with PBS. Finally, add 2 μL 10×phi29 buffer, 2 μL 10 U / μL phi29 DNA polymerase, 6 μL 10 mM dNTP Mix, and 10 μL DEPC-treated water. Incubate at 37°C for 2 h. After the reaction, aspirate the surface liquid from the wells and wash the cells once with PBS.

[0027] 4) Imaging process: Add 2 μL of 20×SSC, 2 μL of deionized formamide, 2 μL of 1 μg / μL salmon sperm DNA, 0.5 μL of 10 μM fluorescent detection probe, and 13.5 μL of DEPC-treated water to the cells treated in step 3). Incubate at 37°C for 30 min. After the reaction, aspirate the surface liquid from the microwells, wash the cells three times with PBS-T (PBS solution containing 0.05% Tween-20, pH = 7.4), and finally add 30 μL of anti-fluorescence attenuation mounting medium (containing DAPI) to the microwells. Seal with a coverslip and store at room temperature in the dark. After 1 h, perform single-cell in situ imaging analysis using a laser confocal fluorescence microscope.

[0028] To verify the ability of the single-cell in situ imaging method based on nucleic acid rolling circle amplification to specifically identify target nucleic acids within cells, this application designed a series of control experiments. For example... Figure 3As shown: Using a padlock probe (CD8a, 30nt) that specifically recognizes CD8a mRNA, bright green spots are generated in cells, easily distinguishable from the background. In contrast, using water to replace an equal amount of the trigger primer and the padlock probe (CD8a) (i.e., without the trigger primer and without the padlock probe) as a control group, no obvious fluorescent signal is generated in cells, indicating that the fluorescent bright spots originate from the amplification products of RCA. To verify the specificity of this method, random padlock probes and mismatched padlock probes (CD8a) were used instead of the padlock probe (CD8a), and again, no obvious fluorescent bright spots were observed in cells. Therefore, the single-cell in situ imaging method based on nucleic acid rolling circle amplification proposed in this application can achieve specific recognition of target nucleic acids within cells.

[0029] Due to the complexity of the intracellular environment and the existence of secondary structures and potential protein binding sites in the target nucleic acid, the recognition and amplification efficiency of the target nucleic acid may be affected by the above factors, resulting in deviations in target nucleic acid amplification. This application investigates the effects of lock probe length (base number, 66nt / 88nt / 110nt), lock probe recognition sequence length (binding number, 20nt / 30nt / 40nt), and target nucleic acid recognition sites (different regions of the CD8a gene, 5'-UTR / CDS / 3'-UTR) on the efficiency of rolling circle amplification reaction, optimizing probe design strategies and improving the recognition efficiency of target nucleic acids. Figure 4 As shown, when the lock probe recognition sequence length is 20 nt and 30 nt, it can trigger rolling circle amplification, producing bright green fluorescent spots in the cell. However, when the lock probe recognition sequence length reaches 40 nt, there are almost no bright fluorescent spots in the cell, indicating that rolling circle amplification does not occur. This may be because the recognition sequence accounts for too high a proportion of the lock probe nucleic acid sequence (40 nt / 88 nt), affecting the formation of the lock probe's circular structure. Alternatively, it may be because the increased recognition sequence leads to the formation of a complex secondary structure in the lock probe, affecting its specific recognition of the target gene. Considering the relationship between recognition sequence length and target gene recognition efficiency, this application selected a lock probe recognition sequence length of 30 nt for subsequent experimental verification.

[0030] Furthermore, to verify the specificity and selectivity of the single-cell in situ imaging method based on nucleic acid rolling circle amplification, this application further performed single-cell in situ imaging on CD8a mRNA in Jurkat cells, HepG2 cells, and HL7702 cells, respectively. The specific operation steps were basically the same as steps 1) to 4) of the aforementioned embodiment, except that CD8+ T cells were replaced with Jurkat cells, HepG2 cells, or HL7702 cells. Figure 5 As shown, obvious green fluorescent bright spots (CD8a amplification products) appeared in CD8+ T cells. Since Jurkat cells, HepG2 cells and HL7702 cells do not express CD8a mRNA, no fluorescent bright spots appeared in the cells, indicating that this method has the ability to specifically recognize target cells.

[0031] The above experimental results have preliminarily confirmed that this single-cell in situ imaging method based on nucleic acid rolling circle amplification can achieve specific recognition of target nucleic acids in CD8+ T cells, with high selectivity and specificity.

[0032] Example 3: Single-cell in situ imaging of T-cell immune activation state Human T cells are diverse in type and complex in function. Based on their immune status, they can be categorized into: naive, effective, pre-exhausted, and exhausted states. Naive T cells are generally found in the blood, while mature T cells in the thymus are typically in an effective state, and tumor-infiltrating T cells may be in an exhausted state. Immuneally activated T cells possess anti-tumor immune activity; therefore, accurately identifying the immune activation state of T cells is of significant research value and clinical importance for predicting clinical response, treatment efficacy, and prognostic assessment of tumor immunotherapy.

[0033] This application uses 4-1BB mRNA as a characteristic gene of T cell immune activation state, and applies the single-cell in situ fluorescence imaging method based on rolling circle amplification reaction constructed above to the in situ visualization analysis of T cell immune activation state. The specific experimental operation steps are basically the same as steps 1) to 4) of Example 2 above, except that the Padlock probe (CD8a) is replaced with the Padlock probe (4-1BB). Figure 6 As shown, after the fluorescent detection probe hybridizes with the RCA amplification product, green fluorescent bright spots appear in T cells, which represent the expression of the T cell immune activation characteristic gene 4-1BB.

[0034] The above results indicate that, using 4-1BB mRNA as a characteristic gene of T cell immune activation, in situ visualization analysis of T cell immune activation can be achieved using a single-cell in situ imaging method based on rolling circle amplification reaction.

[0035] Example 4: Visualization of the Immune Activation Status, Number, and Location of Tumor-Infiltrating T Cells. Using the single-cell in situ imaging method established above, this application selected tumor tissue specimens from hepatocellular carcinoma (HCC) patients to perform in situ visualization analysis of the immune activation status, number, and invasion depth of tumor-infiltrating T cells. The specific experimental procedures are as follows: 1) Pretreatment of tumor tissue specimens: In order to reduce the degradation of T cell immune activation characteristic genes during the operation, tumor tissue specimens were processed by frozen sectioning to obtain tissue sections, fixed with 4% paraformaldehyde for 25 min, and permeabilized with 0.5% Triton-X100 at room temperature for 5 min.

[0036] 2) Rolling circle amplification reaction (RCA): Consistent with the single-cell in situ imaging method established above, see steps 2) to 3) of Example 2 for details.

[0037] 3) In situ imaging: Before hybridization of the fluorescent detection probe, the tissue was treated with an autofluorescence quencher for 1 min, and then the operation was performed according to step 4) of Example 2. Finally, the slide was mounted with an antifluorescence quencher (containing DAPI) and imaged by fluorescence confocal microscopy.

[0038] like Figure 7 As shown, this application uses DAPI to label cell nuclei and a fluorescent detection probe (Alexa-488) to label the amplification product of the immune activation state characteristic gene 4-1BB. Therefore, the green fluorescent bright spots in the tumor tissue section represent immune-activated T cells. Immune-activated T cells are mainly distributed in the peripheral region of the tumor tissue, while fewer activated T cells infiltrate the inner region of the tumor. Furthermore, statistical analysis was performed on the tumor invasion depth and number of immune-activated T cells in different regions of the tumor tissue. Figure 8 The results showed that in the tumor periphery, activated T cells were located at the superficial edge of the tumor tissue, and the number of activated T cells decreased rapidly with increasing invasion depth. Some activated T cells infiltrated relatively uniformly in the tumor periphery, and their number remained essentially unchanged with increasing invasion depth, but there were almost no activated T cells in the superficial area. The number of activated T cells infiltrating the tumor interior was relatively small, with only a small number present in a specific internal region. In summary, this invention utilizes a single-cell in situ imaging method based on rolling circle amplification reaction to visualize the immune activation status, number, and location information of tumor-infiltrating T cells, laying the foundation for constructing an immunophenotyping index system based on molecular imaging.

[0039] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A method for single-cell in situ imaging of T cell immune status based on rolling circle amplification reaction for non-disease diagnosis and treatment purposes, characterized in that: Cell samples are fixed and permeabilized; lock-type probes are used to specifically identify characteristic mRNAs of T cell immune status; a circular template is formed under the action of T4 DNA ligase, and the circular template is amplified in situ using trigger primers and phi29 DNA polymerase to obtain RCA amplification products; fluorescent detection probes are used to bind to the RCA amplification products to generate fluorescent spots for single-cell in situ imaging analysis. The method includes the following steps: 1) Fix cell samples with 4% paraformaldehyde for 10-25 min, then permeabilize with 0.5% Triton-X 100 for 5 min, and wash cells with PBS; 2) Add 20×SSC buffer, dithiothreitol, RiboLock RNase inhibitor, yeast tRNA, DEPC-treated water, and lock probe to the cells treated in step 1). Incubate overnight at 37°C, then wash the cells with PBS. The lock probe comprises three functional regions: a recognition sequence, a binding sequence, and a structure adjustment sequence. The recognition sequence is divided into two segments and can specifically recognize the target nucleic acid sequence. The binding sequence can specifically bind to the fluorescent probe to output a detection signal. The structure adjustment sequence is designed to alter the secondary structure of the lock probe, reducing intramolecular hybridization and improving the recognition efficiency of the target nucleic acid. 3) Add T4 10×Ligation buffer, T4 DNA ligase, RiboLockRNase inhibitor and DEPC-treated water to the cells treated in step 2), react at 37°C for 2 h, and wash the cells with PBS; then add 20×SSC buffer, deionized formamide, trigger primer and DEPC-treated water, react at 37°C for 60 min, and wash the cells with PBS; then add 10×phi29 buffer, phi29 DNA polymerase, dNTP Mix and DEPC-treated water, react at 37°C for 2 h, and wash the cells with PBS. 4) Add 20×SSC buffer, deionized formamide, salmon sperm DNA, fluorescent detection probe and DEPC-treated water to the cells treated in step 3), and react at 37°C for 30 min; wherein, the fluorescent detection probe can bind to the RCA amplification products generated in the cells to produce fluorescent spots, thereby realizing single-cell in situ imaging of T cell immune status. The lock-type probe has a total length of 50~120nt; Both of the identification sequences are 10~40 nt in length; The trigger primer sequence is: 5'-TGAGGTAGTATGTTGTATAGTT-3'; The fluorescent detection probe sequence is: 5'-AACTATACAACATACTACCTCA-Alexa Fluor 488-3'.

2. Use of the method according to claim 1 for in situ visualization of the state of T cell immune activation, characterized in that: The application is for purposes other than disease diagnosis and treatment.

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