Method for screening antigen-specific T cells based on intercellular interaction screening platform

Abstract

The invention discloses an antigen-specific T cell screening method based on an intercellular interaction screening platform, and belongs to the technical field of biological immunodetection. Drops wrap DNA molecular tags and antigen-loaded presenting cells (APCs), and meanwhile, T cells subjected to click chemical modification are injected into the drops to capture free molecular tags and obtain corresponding antigen information. After interaction between cells, demulsification is carried out to recover the cells, nano magnetic beads of which the surfaces are modified with streptavidin are added, APC is combined with the nano magnetic beads through biotin on the surfaces of APC, and APC is removed under the action of a magnetic field, so that pollution is prevented. The obtained T cells are subjected to unicellular omics sequencing, so that the antigen specificity, transcriptome and genomics of the T cells are accurately and comprehensively analyzed. The click chemistry method allows detection of antigen-specific T cells under the condition of not splitting APC, avoids cross contamination of cells, and realizes low-noise multi-modal analysis of primary T cells. And the completion time is within 12 hours, and complicated equipment is not needed.

Description

Technical Field

[0001] This invention belongs to the field of biological immunoassay technology, specifically relating to a screening method for antigen-specific T cells based on an intercellular interaction screening platform. Background Technology

[0002] Cancer has become a major threat to human health. If the 20th century was the era of drug therapy, then the 21st century is the era of cell therapy. Adoptive immunotherapy is an important immunotherapy method, which involves infusing a large number of autologous or allogeneic immune effector cells, activated through various stimuli or genetic engineering in vitro, into the patient to effectively kill tumor cells. T-cell therapy, based on gene modification technology to construct T-cells, has received considerable attention in recent years. T-cells can effectively recognize solid tumor antigens, thus enhancing tumor recognition and killing effects when reinfused into the patient. Preclinical studies have shown that adoptive transfer of T cells modified with neoantigen-specific TCRs can effectively inhibit solid tumors in animal tumor experiments. In the human body, T cells specifically recognize the major histocompatibility complex (MHC) and antigen-peptide complexes through their surface-expressed receptors (T cell receptors, TCRs), thereby activating the T cells themselves. Activated T cells, depending on their functional differences, regulate immune responses by expressing cytokines or directly kill target cells, thereby maintaining normal bodily functions and achieving homeostasis. Therefore, identifying TCRs that are specific and functional in solid tumors is crucial.

[0003] With the application of sequencing technology in TCR sequence detection and the development of immunotherapy in cancer and autoimmune diseases, research on TCR antigen specificity has become increasingly urgent. In early studies, researchers utilized the characteristic that T cell activation requires TCR signals to develop antigen-specific TCR identification strategies based on cell proliferation. However, this technique has significant drawbacks: 1) it requires a large number of samples, placing a burden on research subjects; 2) the experimental procedures are cumbersome, requiring 2-3 rounds of stimulation for cell culture, with a culture cycle of 1-2 months; 3) the bystander effect is significant during culture, affecting the final true positive rate of cells. To study antigen-specific TCRs, in the past thirty years, researchers have developed various methods, such as major histocompatibility complex tetramer labeling and flow cytometry sorting of antigen-reactive T cells. These methods are now widely used in immunotherapy-related research and clinical practice, but these techniques still have significant limitations. The MHC tetramer technique (Altman, JD; Moss, PA; Goulder, PJ et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 1996, 274(5284), 94-96.) was invented by Mark Davis's team in the late 1990s. The advantages of this technique are: rapid detection, fewer cells required than amplification methods, and high specificity. However, the disadvantages are: high cost of tetramer preparation, cumbersome experimental operation, and limited TCR-MHC affinity, resulting in limited fluorescent intensity of the label and failure to detect trace amounts of antigen T cells.After 2000, with the discovery of molecular markers for T cell activation, CD154 (Kirchhoff, D.; Frentsch, M.; Leclerk, P. et al. Identification and isolation of murineantigen‐reactive T cells according to CD154 expression. European journal of immunology 2007, 37 (9), 2370-2377.) and CD137 (Schoenbrunn, A.; Frentsch, M.; Kohler, S. et al. Aconverse 4-1BB and CD40 ligand expression pattern delineates activated regulatory T cells (Treg) and conventional T cells enabling direct isolation of alloantigen-reactive natural Foxp3+ Treg. The Journal of Immunology 2012, 189 (12), 5985-5994.) were used as markers for TCR-activated cells to sort antigen-specific T cells. Although this method is convenient to use, it is greatly affected by the bystander effect (activated T cells will activate their neighboring non-specific T cells), which leads to a high false positive rate of TCR screening by this method.

[0004] Since 2010, with the development of molecular biology techniques, methods have emerged for expanding antigen-specific T cells by transfecting MHC into tool cells and loading them with peptides. This method, through co-transfection of a second signaling molecule, enables antigen-presenting cells to activate intracellular signals in T cells while being readily available, significantly improving the expansion efficiency of therapeutic cells. Regarding the validation of antigen specificity, in 2020, Mark Davis's team constructed cells expressing TCR and Luciferase (luciferase) based on MHC tool cells. After specific recognition of the two cell types, the TCR signal activated intracellular Luciferase. After adding the enzyme substrate, the fluorescence released by the enzyme chemical reaction was detected (Huang, H.; Wang, C.; Rubelt, F. et al. Analyzing the Mycobacterium tuberculosis immune response by T-cell receptorclustering with GLIPH2 and genome-wide antigen screening. Nature biotechnology 2020, 1-9.). The advantages of the above-mentioned technology are: compared to MHC tetramer, it utilizes enzyme expression to amplify the fluorescence signal, enabling the detection of trace amounts of antigen-specific cells; and it can demonstrate the physiological function of intracellular TCR signaling. However, this method relies on the presence of enzyme substrates, can only be performed within a well plate, and can only add one type of TCR to each well to identify the functional TCR. Therefore, this method has significant throughput limitations. Thus, there is an urgent need for a method that can rapidly detect and screen antigen-specific TCRs to accelerate the research progress of adoptive T-cell therapy.

[0005] The principle of droplet microfluidics is to rapidly prepare micron-scale droplets through a special microchannel design, enabling the separation of single cells into individual droplets with good biocompatibility and avoiding the influence of shear forces. This technology allows for the convenient preparation of millions of droplets in a short time, as well as high-throughput droplet sorting, an advantage not found in other methods. Segaliny et al. (Segaliny, AI; Li, G.; Kong, L. et al. Functional TCR T cell screening using single-cell droplet microfluidics. Labon a Chip 2018, 18(24), 3733-3749.) used droplet microfluidics and microwell array chips to screen functional TCR-T cells and monitored the activation process of individual cells in real time. Specificity verification was completed by combining single-cell RT-PCR and TCR sequencing, demonstrating that the platform achieves 100% specific TCR-T screening. However, the low cell throughput of this method is a key limitation to its further application.

[0006] Single-cell sequencing is a widely used method for studying cell function. Recently, researchers developed a multimodal model combining single-cell RNA sequencing and single-cell TCR sequencing to predict the anti-tumor function of T cells in melanoma, highlighting the potential of single-cell sequencing in disease prognosis and treatment (Tan, CL; Lindner, K.; Boschert, T. et al. Prediction of tumor-reactive T cell receptors from scRNA-seq data for personalized T cell therapy[J]. Nature biotechnology, 2024: 1-9.). DNA barcoding technology has been incorporated into single-cell sequencing to detect TCR specificity at single-cell resolution. By binding DNA barcodes to MHC tetramers, cells binding to MHC tetramers can be detected via single-cell sequencing (Ma, KY; Schonnesen, AA; He, C. et al. High-throughput and high-dimensional single-cell analysis of antigen-specific CD8+ T cells[J]. Nature immunology, 2021, 22(12): 1590-1598.). However, the limitations of MHC tetramers still restrict this approach.

[0007] Qi et al. considered two different types of interacting cells, namely Jurkat reporter cells and antigen-presenting cells, and designed corresponding traceable molecular tags for these antigen-presenting cells (Qi, J.; Zhu, H.; Li, Y. et al. Creation of a High‐Throughput Microfluidic Platform for Single‐CellTranscriptome Sequencing of Cell‐CellInteractions[J]. Small Methods, 2023, 7(11): 2300730.). The molecular tags were pre-labeled on the interacting cells before co-incubation. To prevent data loss, the researchers injected the lysis buffer into droplets through an injection chip, releasing RNA and molecular tags, which were then captured by a pre-defined capture sequence. After a series of steps, including library construction and sequencing, the specific gene expression in the Jurkat reporter cell and antigen-presenting cell interaction system was analyzed and decomposed, and the molecular tags were traced back to their corresponding relationships. However, the simultaneous lysis of reporter cells and antigen-presenting cells may lead to nucleic acid contamination, which is an important issue that needs to be considered when studying primary T cells.

[0008] To address this issue, this application develops a droplet-based intercellular interaction method to identify and screen antigen-specific T cell functions, achieving contamination-free operation at single-cell resolution. When these cells are co-encapsulated in microdroplets, the method utilizes click chemistry-based capture structures on the target cells to bind DNA barcodes from APC cells. Subsequently, single-cell omics sequencing is used to detect the presence of molecular tags and the genetic and proteomic profiles of the target cells. Summary of the Invention

[0009] To overcome the problems in the prior art, the present invention provides a method for screening antigen-specific T cells based on an intercellular interaction screening platform.

[0010] To achieve the above objectives, the present invention adopts the following technical solution: A method for screening antigen-specific T cells based on a cell-cell interaction screening platform, the process of which is as follows: (1) Surface modification of APC cells and T cells T cell sap was incubated with DBCO-sulfonyl-NHS ester (dibenzylcyclooctyl-sulfonyl-N-hydroxysuccinimide ester), and APC cell sap was incubated with Biotin-sulfonyl-NHS ester (biotin-sulfonyl-N-hydroxysuccinimide ester) to obtain DBCO-modified T cell sap and biotin-modified APC cell sap.

[0011] (2) Droplet formation A cell suspension containing a free molecular tag is prepared by mixing a 5'-terminal azide-modified molecular tag with biotin-modified APC cell fluid at a density of 10,000-30,000 cells / μL (cell density depends on droplet diameter; larger droplets have lower density). The cell suspension containing the free molecular tag and droplet-generating oil are then placed in a droplet-generating chip to form water-in-oil droplets. The microfluidic channel of the droplet-generating chip can be driven by either positive or negative pressure. The final droplets contain both the free molecular tag and APC cells, aiming to ensure that each droplet contains at least one APC cell. Multiple different tags and corresponding antigenic peptides can be set according to experimental needs. Finally, the generated droplets containing different tags are mixed together for later use.

[0012] (3) T cell injection The droplets generated in the previous step are transferred to the droplet inlet of the droplet injection chip, and then DBCO-modified T cell fluid is injected through the injection port. The T cell density is 2,000-8,000 cells / μL (the cell density depends on the diameter of the droplet after injection; the larger the droplet, the lower the density). By adjusting the cell density, the injected T cell is kept at least one per cell. At the droplet injection port, under the application of an external 200V voltage, the injection fluid and droplet fuse, thus completing the T cell injection. Different types of T cells can be injected as needed to achieve high-throughput detection.

[0013] (4) Droplet incubation and demulsification recovery First, place the injected droplets in an incubator and incubate for 5-7 hours. For example... Figure 2 As shown, during incubation, the free 5'-terminal azide-modified molecular tag in the droplet undergoes a click chemical reaction with DBCO on the surface of T cells, thereby achieving T cell molecular tagging. Simultaneously, specific T cells interacting with APC cells activate the expression of specific genes.

[0014] Then, PFO (1H,1H,2H,2H-perfluoro-1-octanol) was mixed with the droplets. After centrifugation, the cell suspension was collected from the supernatant. Figure 3 As shown, cells were mixed with streptavidin nanobeads and incubated at 37°C for 20 minutes. During this time, the biotin on the APC cells reacted with the streptavidin on the magnetic beads, thus achieving magnetic bead labeling of the APC cells. Finally, the collection tube containing the cells was placed on a magnetic rack for 3-8 minutes. The magnet attracted the bead-labeled APC cells to the wall of the collection tube adjacent to the magnetic rack, while the T cells remained free in the supernatant. The purified T cells were obtained by collecting the supernatant.

[0015] The recovered cells underwent single-cell sequencing analysis to obtain transcriptome libraries, molecular tag libraries, and TCR libraries. Pairing relationships between T cells and APC cells were determined by comparing molecular tags. If a match was found, the corresponding T cell genes were activated and expressed; otherwise, gene expression remained unchanged. Analyzing the transcriptome and TCR libraries may provide a more comprehensive and in-depth understanding of the multimodal information of T cells.

[0016] Furthermore, in step (1), the concentrations of DBCO-sulfonyl-NHS ester and Biotin-sulfonyl-NHS ester are both 1 mg / mL, and the concentration of T cell sap APC cell sap is 10 mg / mL. 4 ~10 6 The volume ratio of T cell sap to DBCO-sulfonyl-NHS ester was 100:(0.4~0.6), and the volume ratio of APC cell sap to Biotin-sulfonyl-NHS ester was 100:(0.4~0.6).

[0017] Further, in step (2), the 5' end azide-modified molecular tag is diluted with TE buffer to a concentration of (0.5~2) μmol / L, and the (0.5~2) μmol / L 5' end azide-modified molecular tag is mixed with the culture medium at a ratio of 1:100, and then biotin-modified APC cell lysate with the same volume as the culture medium is added.

[0018] Furthermore, in step (2), the volume ratio of the cell suspension containing the free molecular tag to the droplet-generated oil is 1:(1~2).

[0019] Furthermore, in step (3), the volume ratio of the droplet to the DBCO-modified T cell fluid is 1:(3~5).

[0020] Furthermore, in step (4), 5~15 μL of streptavidin nanomagnetic beads with a concentration of 10 mg / mL need to be added to every 200 μL of incubated droplets.

[0021] Furthermore, in step (3), the width of the injection port of the droplet injection chip is 10~20 micrometers (the injection port width is determined according to the cell size) to ensure that T cells can be successfully injected into the droplet.

[0022] This invention designs a capture structure on the surface of T cells to bind free barcodes encapsulated in droplets. This method transmits APC information to T cells, enabling us to analyze information from both APCs and T cells simultaneously.

[0023] This invention provides a novel method for delivering antigen information to co-incubated T cells for high-throughput, contamination-free multimodal analysis. In this method, droplets encapsulate DNA molecular tags and antigen-presenting cells (APCs), while click-chemically modified T cells are injected into the droplets to capture free molecular tags and acquire corresponding antigen information. After cell-cell interactions, the cells are demulsified and recovered, and streptavidin-modified magnetic nanobeads are added. APCs bind to these nanobeads via biotin on their surface and are removed under a magnetic field to prevent contamination. The resulting T cells are then subjected to single-cell omics sequencing for accurate and comprehensive analysis of their antigen specificity, transcriptome, and genomics. This click-chemistry method allows for the detection of antigen-specific T cells without APC lysis, avoids cell cross-contamination, and enables low-noise multimodal analysis of primary T cells. The process is completed within 12 hours, requires no complex equipment, and provides unbiased single-cell sequencing results, offering a comprehensive understanding of anti-tumor T cell responses. Attached Figure Description

[0024] Figure 1 This is a process flow diagram of the present invention; Figure 2 The process for preparing molecularly labeled T cells; Figure 3 A flowchart for removing APC cells; Figure 4 This is a schematic diagram of droplet formation; Figure 5 This is a schematic diagram of droplet injection; Figure 6 Images before and after droplet injection, scale bar is 100 μm; Figure 7 Figures for sequencing data analysis: (A) tSNE scatter plot of cell clusters identified by DNA tags. (C) Expression of the top 15 differentially expressed genes between T cells (Jurkat cells) and APCs (T2 cells). (D) Percentage of TCR clones expressed in T cells (Jurkat cells). Detailed Implementation

[0025] The technical solution of the present invention will be further described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] In this embodiment, T2 cells are used as APC cells and Jurkat cells are used as T cells.

[0027] Example 1 A method for screening antigen-specific T cells based on an intercellular interaction screening platform, such as Figure 1 As shown, the process is as follows: 1. Surface modification of T2 cells and Jurkat cells DBCO and azides can react in biological systems without disrupting normal biochemical processes. In this method, three tubes of cell suspension are taken, 10 μL per tube. 7 One tube of Jurkat cells and two tubes of T2 cells were cultured (both cell cultures were cultured in 90% RPMI 1640 + 10% FBS + 1% P / S). The cells were centrifuged at 300g for 3 min, and the supernatant was discarded. Then, each of the three cell lines was resuspended in 3 mL of PBS and centrifuged at 300g for 3 min, and the supernatant was discarded. Next, 100 μL of PBS was added to each cell line. 0.5 μL of 1 mg / mL DBCO-sulfonyl-NHS ester (dibenzylcyclooctyl-sulfonyl-N-hydroxysuccinimide) was added to one tube of Jurkat cells and one tube of T2 cells, while 0.5 μL of 1 mg / mL Biotin-sulfonyl-NHS ester (biotin-sulfonyl-N-hydroxysuccinimide) was added to the other tube of T2 cells. All cells were mixed thoroughly and incubated at 37°C for 20 min. Finally, 1 mL of culture medium (90% RPMI 1640 + 10% FBS + 1% P / S + 1%) was added to each cell line. (10%) F-68), incubated at room temperature for 5 min, centrifuged at 300g for 5 min, and the supernatant was discarded; repeated once. Finally, DBCO-modified Jurkat cells, DBCO-modified T2 cells, and biotin-modified T2 cells were obtained, respectively.

[0028] 2. Droplet formation Take 1 μL and dilute it with TE buffer to a concentration of 1 μM. Four molecular tags (four specific DNA sequences, as shown in Table 1) with 5' ends modified by azide were mixed with 100 μL of culture medium (90% RPMI 1640 + 10% FBS + 1% P / S + 1% (10%) F-68). Then, mix with 100 μL of biotin-modified T2 cells at a density of 20,000 cells / μL to form the final cell suspension containing free molecular tags. Figure 4As shown, 100 μL of oil was added to the droplet collection tube of the droplet generation chip beforehand, so that the long tube was submerged below the oil layer (QX200 Droplet Generation Oil came from Bio-Rad) to avoid the collected droplets breaking. 150 μL of droplet generation oil (QX200 Droplet Generation Oil came from Bio-Rad) was added to the oil inlet, and 100 μL of cell suspension containing molecular tags was added to the cell inlet. The 30 mL syringe at the tail was pulled from the 15 mL position to the 17.5 mL position and kept fixed. The negative pressure formed at this time can ensure the smooth generation of droplets. The droplets were collected in the recovery tube for later use.

[0029] Table 1 Molecular tag sequence listing 3. Cell injection To verify whether specific T cells can be traced back through molecular tags, the scheme designed in this application is as follows: the injected cells are DBCO-modified Jurkat cells and DBCO-modified T2 cells, respectively. The droplets corresponding to the first three molecular tags are used for the injection of DBCO-modified Jurkat cells, and the droplets containing the last molecular tag are used as the control group for the injection of DBCO-modified T2 cells.

[0030] Taking the injection of DBCO-modified Jurkat cells as an example: 100 μL droplets of one of the three molecular tags generated in the previous step are transferred using a pipette. Figure 5 The droplet inlet of the injection chip shown has a width of 15 micrometers to ensure that Jurkat cells can be successfully injected into the droplet. The Jurkat cell density is approximately 6,000 cells / μL, and the injection volume is approximately 400 μL. Figure 6 The droplet diameter before injection was approximately 65 μm, and after injection, it was approximately 95 μm. At the injection port, under the influence of an applied 200V voltage, the injection solution fused with the droplet, thus completing the injection of Jurkat cells. The control group received T2 cell injection in the same manner. The entire injection process was powered by the negative pressure created by pulling the 30mL syringe from the 18mL position to the 20mL position and holding it fixed. Finally, all injected droplets were collected in a collection tube for later use.

[0031] 4. Droplet incubation and demulsification recovery First, place the injected droplets in an incubator and incubate at 37°C for 6 hours. For example... Figure 2As shown, during incubation, the free 5'-terminal azide-modified molecular tag in the droplet undergoes a click chemical reaction with DBCO on the surface of T cells, thereby achieving T cell molecular tagging. Simultaneously, specific T cells interacting with APC cells activate the expression of specific genes.

[0032] Subsequently, 100 μL of PFO (1H,1H,2H,2H-perfluoro-1-octanol) was mixed with 200 μL of the solution in a proportional manner. After brief centrifugation, the cell suspension was collected from the supernatant. Figure 3 As shown, cells were mixed with 10 μL of streptavidin nanobeads (10 mg / mL) and incubated at 37°C for 20 minutes. During this time, biotin on the APC cells reacted with streptavidin on the magnetic beads, thus achieving magnetic bead labeling of the APC cells. The collection tube containing the cells was placed on a magnetic rack for 5 minutes, and the supernatant of the experimental group Jurkat cells and the control group T2 cells was collected.

[0033] 5. Single-cell sequencing analysis The recovered cells were subjected to single-cell sequencing analysis to obtain sequencing data. Bioinformatics analysis revealed that the cells clustered into four groups, such as... Figure 7 As shown in Figure A, cells identified by molecular tags are T cells and APC cells. Three groups are T cells (corresponding to Jurkat cells tagged with the first three molecular tags), and one group is APC cells (corresponding to T2 cells tagged with molecular tag 4). To confirm correct cell identification, such as... Figure 7 Figure B shows the analysis of differentially expressed genes in these cells. T cells highly expressed marker genes such as TRBV12-3, TRAV8-4, and CD3D, confirming that cells co-coated with different APCs can be recognized through DNA molecular tags. Furthermore, TCR analysis showed ( Figure 7 (C) Approximately 80.2% of cells showed simultaneous detection of both TRA and TRB genes, while approximately 19.8% of cells showed detection of only the TRB strand. The TCR consists of two strands, and the RNA expression levels of the two strands are unequal. The low expression of the TRA gene compared to the TRB gene makes the detection of TRA and TRB in single cells challenging; however, in this application, most cells exhibited both strands, indicating that this method effectively maintained cell viability. Different injected cells can be distinguished using molecular tags.

Claims

1. A method for screening antigen-specific T cells based on a cell-cell interaction screening platform, characterized in that: The process is as follows: (1) Incubating T cell fluid with DBCO-sulfo-NHS ester and APC cell fluid with Biotin-sulfo-NHS ester respectively to obtain DBCO-modified T cell fluid and biotin-modified APC cell fluid; (2) mixing the 5'-end azide-modified molecular tag with a biotin-modified APC cell solution at a density of 10,000-30,000 cells / μL to prepare a cell suspension containing free molecular tags, and forming oil-in-water droplets in a droplet generation chip with the cell suspension containing free molecular tags and droplet generation oil; (3) Transferring the droplets generated in step (2) to the droplet inlet of the droplet injection chip, and then injecting DBCO-modified T cell solution from the injection port, the density of T cells is 2,000-8,000 / μL, and applying a voltage of 150-250V at the droplet injection port to cause the injection solution to fuse with the droplet; (4) The fused droplets in step (3) are placed in an incubator for incubation for 5 to 7 hours. Subsequently, PFO is mixed with the incubated droplets in a volume ratio of 1:(1 to 3). After centrifugation, the cell suspension is taken out from the upper layer, and the cell suspension is mixed with streptavidin nanomagnetic beads and incubated. Finally, the collection tube containing the cells is placed on a magnetic rack for 3 to 8 minutes. At this time, the magnet will adsorb the APC cells labeled with magnetic beads to the wall of the collection tube next to the magnetic rack, while the T cells will be free in the supernatant. The supernatant is collected to obtain purified T cells.

2. The method for screening antigen-specific T cells based on the cell-cell interaction screening platform according to claim 1, characterized in that: In step (1), the concentrations of DBCO-sulfo-NHS ester and Biotin-sulfo-NHS ester were both 1 mg / mL, and the concentrations of T cell suspension and APC cell suspension were 10 4 ~10 6 / μL, the volume ratio of T cell fluid and DBCO-sulfo-NHS ester is 100: (0.4~0.6), and the volume ratio of APC cell fluid Biotin-sulfo-NHS ester is 100: (0.4~0.6).

3. The method for screening antigen-specific T cells based on the cell-cell interaction screening platform according to claim 1, characterized in that: In step (2), the 5'-end azide-modified molecular tag is diluted with TE buffer to a concentration of (0.5~2) μmol / L, and the (0.5~2) μmol / L 5'-end azide-modified molecular tag is mixed with the culture medium at a ratio of 1:100, and then the biotin-modified APC cell fluid with the same volume as the culture medium is added.

4. The method for screening antigen-specific T cells based on the cell-cell interaction screening platform according to claim 1, characterized in that: In step (2), the volume ratio of the cell suspension containing free molecular labels and the droplet-forming oil is 1:(1~2).

5. The method for screening antigen-specific T cells based on the cell-cell interaction screening platform according to claim 1, characterized in that: In step (3), the volume ratio of the droplets to the DBCO-modified T cell solution is 1:(3~5).

6. The method for screening antigen-specific T cells based on the cell-cell interaction screening platform according to claim 1, characterized in that: In step (4), 5-15 μL of 10 mg / mL streptavidin nanomagnetic beads need to be added to every 200 μL of incubated droplets.

7. The method for screening antigen-specific T cells based on the cell-cell interaction screening platform according to claim 1, characterized in that: In step (3), the width of the injection port of the droplet injection chip is 10 to 20 microns.

Images