Method and system for qualitative and quantitative analysis of immune cell secreted proteins based on sequencing technology

Abstract

The application discloses a method and system for qualitatively and quantitatively analyzing immune cell secreted proteins based on sequencing technology. The method comprises the following steps: S1, a double-specific antibody labeled immune cell is co-encapsulated with a stimulant or an antigen presenting cell loaded with a tumor antigen polypeptide into a droplet; S2, the droplet formed in S1 is incubated, and the cytokine secretion of the immune cell is observed in real time by using a droplet micro-well chip; S3, the droplet is broken at different incubation time points, the cell is recovered, and the cell membrane protein and the cytokine captured on the cell surface are analyzed by using flow cytometry; and S4, a target cell population is sorted for single cell transcriptome and secreted protein sequencing and integrated analysis. By using the technical scheme of the application, the cell population of interest can be sorted for single cell transcriptome combined with secreted protein sequencing, and the comprehensive and accurate analysis of the function and transcriptome characteristics of a single T cell is realized with high throughput, high efficiency and low cost.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method and system for qualitative and quantitative analysis of proteins secreted by immune cells based on sequencing technology. Background Technology

[0002] Cells in biological systems exhibit significant heterogeneity, particularly T cells and B cells in the adaptive immune response. Cellular subsets and functions are highly diverse, with marked individual cell differences. While single-cell transcriptome sequencing technology is widely used to analyze cell population differences, RNA information from immune cells cannot fully reflect the differences in protein expression between cells. Cellular proteins play crucial roles in various biological functions, and significant functional differences exist between individual cells. T cells are activated by binding to specific antigenic peptides presented by MHC molecules on antigen-presenting cells via their surface receptors (TCRs). They can kill virus-infected and mutated target cells or secrete various cytokines (such as IFN-γ and TNF-α) to regulate the immune response. Different T cell populations differ in killing efficiency, the types and rates of cytokine secretion, and the high diversity of T cell function and phenotype. Their surface TCRs further determine individual T cell differences. Therefore, developing high-throughput technologies to study cell function and transcriptome characteristics at the single-cell level is essential for accurately interpreting immune responses.

[0003] Most current experimental methods for T cell function, such as enzyme-linked immunosorbent assay (ELISA), only produce overall results for a large population, which may mix in and mask the unique contributions of individual cells. Studies primarily reflect the average level of the cell population, ignoring differences between individual cells. ELISPOT can detect secreted proteins in individual cells, but its low throughput limits its ability to comprehensively analyze T cell functional responses. The development of single-cell sequencing technology plays a crucial role in providing a more comprehensive and accurate high-throughput single-cell analysis of T cell genetic characteristics, but it cannot achieve more precise studies of antigen-specific T cell function and status. Gaining deeper insights into immune cell behavior, especially cytokine secretion, at the single-cell level is essential for defining intercellular differences and better understanding how individual immune cells promote and control the overall immune response. Antibody-based flow cytometry can perform single-cell cytokine readings, but they are designed as endpoint assays and cannot be used to monitor cytokine production at high spatiotemporal resolution.

[0004] Specifically, the commercially available methods for detecting T cell-secreted cytokines mainly include the following:

[0005] Enzyme-linked immunosorbent assay (ELISA): A specific concentration of antigen or antibody is immobilized on the surface of a polystyrene microplate through physical adsorption. The sample to be tested is added, and the intensity of the color development by the enzyme-labeled antibody indirectly reflects the presence or quantity of the antigen or antibody being tested. ELISA is currently the most common and widely used method for detecting cytokines. The sample is typically cell culture supernatant or cell lysate, and the result reflects the average level of the entire cell count.

[0006] Enzyme-linked immunospot (ELISPOT) assay combines cell culture technology with enzyme-linked immunosorbent assay (ELISA) to detect cytokines secreted by individual cells. Simply put, it uses coated antibodies to capture cytokines secreted by cultured cells and displays them as ELISPOT spots. While it can reflect cytokine secretion in individual cells, it has low throughput, is cumbersome to perform, and is time-consuming.

[0007] Intracellular cytokine staining: Intracellular cytokine (e.g., IFNγ) staining methods analyze lymphocyte function at the single-cell level. By combining surface staining and intracellular cytokine staining, this method can obtain the percentage of cells in a given population that are capable of releasing cytokines, which cannot be obtained by enzyme-linked immunospot (ELISA) methods. This method is designed as an endpoint cell assay; fixed cells cannot be used for high spatiotemporal resolution monitoring of cytokine production and other experiments such as single-cell sequencing.

[0008] Luminex Technology: In 1997, the journal *Clinical Chemistry* published a special article introducing Luminex. The technology (liquid-suspended chip technology), on July 27, 2001, INOVA's ENA series products were the first to receive FDA approval in the United States, becoming a multi-indicator parallel detection technology applicable to clinical diagnostics. Its technical principle involves staining 5.6-micrometer-diameter polystyrene microspheres with different ratios of two red fluorescent dyes to obtain up to 100 fluorescently encoded microspheres. Antibody molecules or gene probes targeting different analytes are covalently cross-linked to specific encoded microspheres, each microsphere corresponding to a specific detection item. First, the fluorescently encoded microspheres targeting different analytes are mixed, then the analyte or amplified fragment is added, and the resulting complex reacts with a labeled fluorescein. The microspheres are then sequentially passed through red and green lasers by a flowing sheath fluid. The red laser is used to determine the fluorescent encoding of the microsphere, and the green laser is used to measure the fluorescence intensity of the reporter molecule on the microsphere. This achieves rapid and accurate quantitative detection. Its advantages include high sensitivity, low sample requirements, and the ability to simultaneously detect multiple cytokines, while still detecting the average level of whole cells.

[0009] BD Biosciences' Cytometric BeadsArray (CBA) system is a flow cytometry-based multiplex protein quantification method. It allows for the simultaneous detection of multiple indicators in a single sample. In routine experiments, it is often necessary to quantify soluble proteins in solution systems, such as the quantitative analysis of cytokine levels in cell culture supernatants or serum / plasma. Because these factors are present in small quantities, below the detection limits of conventional methods, they are difficult to detect using traditional protein detection methods. Furthermore, due to the small sample volume, methods that detect only one factor at a time are difficult to implement for detecting multiple factors and are time-consuming. BD Biosciences utilizes the high sensitivity of flow cytometry to fluorescence signals and its ability to simultaneously distinguish multiple fluorescence frequencies and intensities. By attaching the soluble factors to microparticles with a diameter similar to that of cells, multiple soluble factors in the sample can be detected. Therefore, BD Biosciences developed the Cytometric BeadsArray (CBA) system. First, a series of fluorescently labeled microspheres linked to specific capture antibodies are used to capture analytes in the solution system. Then, the different fluorescence intensities on the corresponding analyte-specific microspheres are used to simultaneously quantify and analyze multiple soluble components in the sample. BD CBA technology is used for multiplex analysis, allowing more data to be obtained from a single sample. Especially with small sample volumes, multiplex detection maximizes the number of analyzable proteins. Using BD CBA technology, up to 30 proteins can be analyzed with only 25 to 50 μl of sample. Other methods such as ELISA and Western Blot can only detect one protein with the same sample volume. However, this technology also detects the average level of T cells in a population.

[0010] Isoplexis:16 employs microfluidic technology, simultaneously capturing thousands of single cells through 12,000 independent sub-nanoscale microchambers. Combined with IsoCode chip antibody barcoding technology, it captures multiple proteins secreted by a single live cell in each chamber, or captures multiple phosphorylated proteins released after single-cell lysis. Then, using a fully automated protein detection technology based on ELISA principles, it captures the fluorescence signals of multiple proteins, collecting up to 30 or more functional proteins at a time. During detection, the proteomic signals obtained from each single cell are seamlessly transmitted to IsoSpeak bioinformatics analysis software for fully automated analysis and visualization. This technology enables complete single-cell functional characterization through functional immunoprofiling of each immune cell, thereby helping to discover better biomarkers and accelerate therapy development. However, the throughput of this technology is still limited, analyzing only 1000–2000 cells at a time. Cells need to be pre-stimulated before analysis, and the incubation time within the chip must be fixed. Cells cannot be recovered for further analysis after analysis.

[0011] In addition, the following methods for detecting T cell-secreted cytokines are currently being explored and developed in research:

[0012] In 2013, the team led by Christopher Love in the United States analyzed the secretion of three cytokines (IFNγ, TNFα, and IL2) from thousands of individual T cells by incubating them in droplets with a method that combined flow cytometry with a co-encapsulation of single T cells and beads coated with the cytokine captureAb. However, this method could not precisely locate the secretion of multiple cytokines in the same cell and could not perform further analysis of the target cells using single-cell sequencing.

[0013] Dropmap Platform: In 2020, Andrew Griffiths' team in France developed Dropmap, a single immune cell phenotypic analysis platform based on droplet microfluidics. Dropmap can accurately analyze the rate of protein secretion by a single cell. By encapsulating cells and magnetic beads labeled with capture antibodies in a droplet, and after a certain incubation time, the amount of protein secreted by the cell is quantified by the fluorescence peak generated by the repositioning of fluorescently labeled detection antibodies on the magnetic beads. This enables real-time high-throughput analysis and characterization of single-cell secreted proteins.

[0014] In summary, current methods for detecting T cell-secreted cytokines have the following main drawbacks:

[0015] 1) Reflects the average level of overall T cell cytokine secretion, but the detection cost is relatively high (e.g., ELISA);

[0016] 2) Low throughput, long processing time, and high cost (e.g., ELISPOT);

[0017] 3) It cannot accurately reflect the cytokine secretion of a single cell (such as Luminex, CBA, etc.);

[0018] 4) Inability to monitor cytokine secretion (such as ICS) in real time;

[0019] 5) It cannot be effectively combined with other platform flow cytometry and single-cell sequencing technologies to comprehensively and accurately analyze the functional characteristics and corresponding transcriptome features of individual immune cells (such as Isoplexis). Summary of the Invention

[0020] This invention aims to provide a method and system for qualitative and quantitative analysis of immune cell secreted proteins based on sequencing technology. By sequencing, it is possible to simultaneously detect the transcriptome and secreted proteins of a single immune cell at high throughput, high efficiency and low cost, and to comprehensively and accurately analyze the characteristics of T cell responses.

[0021] To achieve the above objectives, according to one aspect of the present invention, a method for high-throughput single-cell level analysis of immune cell secreted proteins is provided. The method includes: S1, encapsulating bispecific antibody-labeled immune cells and a stimulant or antigen-presenting cells loaded with tumor antigen peptides into a droplet; S2, incubating the droplet formed in S1, and observing the cytokine secretion of immune cells in real time using a droplet microarray; S3, breaking the droplet at different incubation time points, recovering the cells, and analyzing cell membrane proteins and cytokines captured on the cell surface using flow cytometry; and S4, sorting the target cell population for single-cell transcriptome and secreted cytokine sequencing, and performing integrated analysis.

[0022] Furthermore, the immune cells are T cells.

[0023] Furthermore, the bispecific antibody label is CD45-IFNγ bispecific antibody label; and / or the stimulant is magnetic beads conjugated with CD3 / CD28 antibodies; and / or the antigen-presenting cells loaded with tumor antigen peptides are antigen-presenting cells loaded with MART1 peptides.

[0024] Furthermore, the preparation of immune cells includes: isolating T cells from PBMCs by enriching them with CD3 magnetic beads and culturing them for more than 48 hours; and / or the preparation of antigen-presenting cells includes: loading T2 cell culture medium with MART1 peptide and incubating for more than 12 hours.

[0025] Furthermore, S1 also includes: staining the bispecific antibody-labeled immune cells and the antigen-presenting cells loaded with tumor antigen peptides differently before encapsulating them into the droplet; preferably, the immune cells are stained with calcein am and the antigen-presenting cells are stained with Hoechst 33342.

[0026] Furthermore, in S1, a method is used to generate droplets by drawing a sealed syringe to create negative pressure, thereby encapsulating bispecific antibody-labeled immune cells and stimulants or antigen-presenting cells loaded with tumor antigen peptides into the droplets.

[0027] Furthermore, when the droplet diameter is 90 μm, the density of immune cells is 1000 cells / μL, and the density of antigen-presenting cells is 5000 cells / μL.

[0028] Furthermore, in S2, the droplets are incubated in a 37°C 5% CO2 incubator; preferably, the instrument conditions are set to 37°C 5% CO2 when using the droplet microwell chip to observe the cytokine secretion of immune cells in real time.

[0029] Further, S3 includes taking droplets at different incubation time points, adding 1H, 1H, 2H, 2H-perfluoro-1-octanol, letting it stand at room temperature for 3-5 minutes, recovering the cells from the upper aqueous phase, washing, adding fluorescent and oligo-barcode labeled antibodies for staining, flow cytometry detection of cell membrane proteins and cytokines captured on the cell surface, and performing statistical analysis.

[0030] Furthermore, S4 includes: after sorting out cells that secrete cytokines, performing subsequent transcriptome sequencing and oligo sequencing of secreted cytokines detection antibodies to analyze gene expression characteristics and cytokine secretion characteristics.

[0031] According to another aspect of the present invention, a system for qualitative and quantitative analysis of immune cell secreted proteins based on sequencing technology is provided. The system includes: a packaging unit configured to package bispecific antibody-labeled immune cells and stimulants or antigen-presenting cells loaded with tumor antigen peptides into a droplet; an observation unit configured to incubate the droplet formed in the packaging unit and observe the cytokine secretion of immune cells in real time using a droplet microwell chip; an analysis unit configured to break the droplet at different incubation time points, recover the cells, and analyze cell membrane proteins and cytokines captured on the cell surface using flow cytometry; and an integrated analysis unit configured to sort the target cell population for single-cell transcriptome and secreted cytokine sequencing and perform integrated analysis.

[0032] Further, the immune cells are T cells; preferably, the bispecific antibody label is a CD45-IFNγ bispecific antibody label; and / or the stimulant is a magnetic bead conjugated with a CD3 / CD28 antibody; and / or the antigen-presenting cells loaded with tumor antigen peptides are antigen-presenting cells loaded with MART1 peptide; preferably, the preparation of immune cells includes: isolating T cells from PBMCs by enriching them with CD3 magnetic beads and culturing them for more than 48 hours; and / or the preparation of antigen-presenting cells includes: adding MART1 peptide to T2 cell culture medium for loading and incubating for more than 12 hours.

[0033] Furthermore, the system also includes a staining unit, which is positioned before the packaging unit and configured to perform different staining on immune cells labeled with bispecific antibodies and antigen-presenting cells loaded with tumor antigen peptides.

[0034] Preferably, immune cells are stained with calcein am, and antigen-presenting cells are stained with Hoechst 33342.

[0035] Preferably, the encapsulation unit is configured to use a sealed syringe to generate negative pressure to create droplets, thereby encapsulating bispecific antibody-labeled immune cells and stimulants or antigen-presenting cells loaded with tumor antigen peptides into the droplets.

[0036] Alternatively, the sealed syringe can be configured such that when the droplet diameter is 90 μm, the density of immune cells is 1000 cells / μL and the density of antigen-presenting cells is 5000 cells / μL.

[0037] Furthermore, in the observation unit, the droplets are incubated in a 37°C, 5% CO2 incubator. Preferably, in the observation unit, when using the droplet microwell chip to observe the cytokine secretion of immune cells in real time, the instrument conditions are set to 37°C, 5% CO2. Preferably, in the analysis unit, droplets are taken at different incubation time points, 1H, 1H, 2H, 2H-perfluoro-1-octanol is added, and the mixture is allowed to stand at room temperature for 3-5 minutes. Cells in the upper aqueous phase are recovered, washed, stained with antibodies, and flow cytometry is used to detect cell membrane proteins and cytokines captured on the cell surface for statistical analysis. Preferably, in the integrated analysis unit, cells that secrete cytokines are sorted, and subsequent transcriptome sequencing is performed combined with sequencing of secreted cytokines to analyze gene expression characteristics and qualitative and quantitative analysis of cytokines.

[0038] By applying the technical solution of this invention, high-throughput, low-cost, and accurate analysis of immune cells at the single-cell level is achieved based on droplet microfluidic technology. This is combined with the use of bispecific antibodies to label immune cells and simultaneously capture secreted cytokines. Subsequently, the droplets are broken, cells are recovered, and flow cytometry is used to simultaneously analyze cell membrane proteins and cytokines captured on the cell surface. Furthermore, cell populations of interest are sorted for single-cell transcriptome sequencing and qualitative and quantitative detection and analysis of cytokines, achieving a comprehensive and accurate high-throughput, high-efficiency, and low-cost analysis of the function and transcriptome characteristics of individual T cells. Attached Figure Description

[0039] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0040] Figure 1 A schematic flowchart of a method for qualitative and quantitative analysis of immune cell secreted proteins based on sequencing technology according to an embodiment of the present invention is shown.

[0041] Figure 2 A schematic diagram of the droplet generation device and process is shown;

[0042] Figure 3This demonstrates the ability of the catch reagent to capture IFNγ onto the surface of T cells at the Bulk level;

[0043] Figure 4 This demonstrates the ability of the catch reagent to capture IFNγ onto the surface of T cells (CD3 / CD28 antibody-conjugated magnetic beads) within droplets;

[0044] Figure 5 The efficiency of co-encapsulation of primary T cells and antigen-presenting cells (T2 cells) within droplets was demonstrated.

[0045] Figure 6 The study showed the detection of IFNγ secretion after co-encapsulation stimulation of primary T cells and antigen-presenting cells (T2).

[0046] Figure 7 The results of integrated analysis of single-cell sequencing T cell transcriptome and secreted IFNr cytokines are shown; and

[0047] Figure 8 The study demonstrates the detection of IFNγ secretion following activation of primary T cells mediated by bispecific antibodies. Detailed Implementation

[0048] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0049] The development of droplet microfluidics has greatly facilitated research on single cells. Microfluidics can generate water-in-oil droplets with a picoliter capacity, serving as a multifunctional platform for developing high-throughput, low-cost single-cell analysis tools. Although microfluidic systems have been successfully used to distinguish single cells since the 1950s, the lack of easy-to-operate detection techniques, online implementation, high sensitivity, high selectivity, and "signal-on" reporting mechanisms has hindered the development of droplet-based single-cell research. Currently, droplet microfluidics has been applied to the study of single-cell genomics, transcriptomics, proteomics, and metabolomics. In particular, droplet microfluidics has a significant advantage in studying cytokine secretion by single immune cells because it easily provides specific stimulants to single cells and effectively limits the leakage of secreted proteins. Existing studies have demonstrated this advantage by encapsulating immune cells with magnetic beads labeled with cytokine-capturing antibodies within droplets, enabling high-throughput study of multiple cytokines secreted by single immune cells. Alternatively, microfluidic chip technology allows stimulated single immune cells to be cultured in microcells pre-coated with multiple cytokine-capturing antibodies, achieving simultaneous detection of multiple cytokines in thousands of single cells. However, due to the Poisson distribution problem inherent in droplet microfluidics and the fact that droplet sorting instruments are still in their initial stages of development and have limited applications, the screening throughput and detectable parameters of these methods remain very limited. They have also not been combined with single-cell transcriptome sequencing technology to more accurately analyze the characteristics of individual immune cells.

[0050] Based on existing methods for analyzing cytokine secretion using droplet microfluidics, the inventors have further expanded and optimized this approach. By labeling T cells with bispecific antibodies, cytokines secreted by T cells can be captured. T cells and target cells are co-encapsulated within droplets, allowing observation of cytokine secretion after stimulation of target-specific T cells. This method combines a microwell array with fixed droplets to achieve real-time visualization and analysis of cytokine secretion. Cells at different incubation time points are recovered and flow cytometry is used to detect the secreted cytokines and cell membrane surface protein characteristics (such as CD8, TCR, PD1, etc.) captured on the cell surface. This allows for precise analysis of cell characteristics and enables single-cell transcriptome sequencing after sorting different cell populations. Finally, high-throughput analysis of cytokine expression, cell membrane protein detection, and cell transcriptome characteristics at the single-cell level is achieved, providing comprehensive and accurate analysis of individual T cell characteristics. This method can be more widely applied to the in-depth analysis of immune response characteristics under physiological and pathological conditions and to aid in the elucidation of mechanisms related to immunotherapy.

[0051] According to a typical embodiment of the present invention, a method for high-throughput single-cell level analysis of proteins secreted by immune cells is provided. (Reference) Figure 1The method includes: S1, encapsulating bispecific antibody-labeled immune cells and stimulants or antigen-presenting cells loaded with tumor antigen peptides into droplets; S2, incubating the droplets formed in S1, and observing the cytokine secretion of immune cells in real time using droplet microarrays; S3, breaking the droplets at different incubation time points, recovering the cells, and analyzing cell membrane proteins and cytokines captured on the cell surface using flow cytometry; and S4, sorting the target cell population for single-cell transcriptome and secreted cytokine sequencing and performing integrated analysis.

[0052] By applying the technical solution of this invention, high-throughput and low-cost accurate analysis of immune cells at the single-cell level is achieved based on droplet microfluidic technology. This is combined with the use of bispecific antibodies to label immune cells and simultaneously capture secreted cytokines. Subsequently, the droplets are broken up, the cells are recovered, and flow cytometry is used to analyze cell membrane proteins and cytokines captured on the cell surface simultaneously. Furthermore, the cell populations of interest are sorted out for single-cell transcriptome sequencing, achieving a comprehensive and accurate high-throughput, high-efficiency, and low-cost analysis of the function and transcriptome characteristics of individual T cells.

[0053] Typically, in a specific embodiment of the present invention, CD45-IFNγ bispecific antibody-labeled T cells are co-encapsulated in a droplet with a stimulant (CD3 / CD28 antibody-conjugated magnetic beads) or antigen-presenting cells (T2 cells) loaded with tumor antigen peptides. Cytokine secretion is observed in real time using a droplet microarray. Subsequently, the droplet can be broken at different time points, and the cells can be recovered and analyzed simultaneously using flow cytometry to statistically analyze cell membrane proteins and cytokines captured on the cell surface. Furthermore, cell populations of interest can be further sorted for single-cell transcriptome sequencing, achieving high-throughput, high-efficiency, and low-cost comprehensive and accurate analysis of the function and transcriptome characteristics of individual T cells. In other embodiments of the present invention, the stimulant can be a target cell expressing or loaded with a specific antigen, and the tumor antigen can be replaced with other viral antigens of interest.

[0054] In a specific embodiment of the present invention, the experimental steps and principles are as follows:

[0055] (1) Cell preparation:

[0056] Primary T cell preparation: Primary T cells were isolated from PBMCs and cultured. T cells were enriched and isolated using a CD3 T cell sorting kit (Miltenyi) and cultured in H009 + 10% FBS + 1% P / S + IL2 (10 ng / mL) + IL7 (10 ng / mL) + IL15 (10 ng / mL) for more than 48 hours.

[0057] Antigen-presenting cells T2 loaded with peptide: 5 × 10⁻⁶ peptides were administered 12 hours in advance. 6Add 10 μg / mL MART1 peptide loading to T2 cell culture medium;

[0058] Cell staining and catch reagent labeling: Before generating droplets, T2 cells were resuspended in cell culture medium after diluting 1 μg / μL Hoechst 333421:500 and incubated at 37°C for 10 mins, and T2 cells were labeled blue; primary T cells were stained in FACs buffer after diluting 1 μM calcein am (1:200) and catch reagent (1:10) at 4°C for 45 mins; after cell staining, the cells were washed once with ice-cold FACs buffer to remove excess dye, etc., and the cells were resuspended in working solution to make the density of primary T cells 1000 cells / μL and the density of T2 cells 5000 cells / μL.

[0059] (2) Droplet generation and incubation:

[0060] Using droplet generation chip devices (such as...) Figure 2 The device generates droplets based on the principle of negative pressure created by pulling a syringe, such as... Figure 2 The diagram shows two sample inlets: one for the droplet-generating oil and the other for collecting the generated droplets. The top is sealed with a removable sealing ring. A small hole connects to the syringe via a sealing coil. The syringe is rapidly pulled from the 12mL mark to 20mL, producing uniform droplets with a diameter of ~90μm. 200μL of T2 cells loaded with the MART1 antigen peptide and 200μL of primary T cells are added to each well, along with 800μL of droplet-generating oil. Droplets are generated using the principle of negative pressure created by pulling the sealed syringe. The syringe is initially adjusted to the 12mL mark, then pulled to 20mL and fixed to allow droplet generation. The generated droplets are collected in the syringe. Figure 2 The droplet collection tube contains T cells. To achieve single-cell encapsulation of primary T cells as much as possible and to achieve co-encapsulation of T cells and T2 cells in as many droplets as possible, so that T cells can receive antigen stimulation, the sample density of the two types of cells is optimized. For example, in a specific embodiment of this application, the sample density of the two types of cells is adjusted. When the droplet diameter is 90 μm, the T cell density is 1000 cells / μL and the T2 cell density is 5000 cells / μL. This ensures that in the primary T cell encapsulation droplets, more than 90% of the droplets containing T cells contain only a single T cell, and more than 70% of the droplets simultaneously encapsulate both T cells and antigen-presenting cells (T2 cells, such as T cells and T2 cells). Figure 5 In section A, the upper left image shows the bright field, the upper right image shows the dark field, the lower left image shows T cells stained with Calcein AM, and the lower right image shows T2 cells stained with Hoechst 33342; section B shows the cell percentage.

[0061] (3) Real-time observation using a fluorescence microscope:

[0062] Most of the generated droplets were placed in a 37°C, 5% CO2 incubator for incubation, while a portion was loaded into a droplet microwell chip. The microwell chip was then placed in a Cytation5 (Biotek) instrument, and the temperature was set to 37°C and 5% CO2. The droplets were incubated and fluorescence images were observed and captured, and the secretion of the cytokine IFNγ was recorded in real time.

[0063] (4) Cell recovery analysis of cytokine-positive populations:

[0064] Take 200 μL droplets at different incubation time points (3h, 6h, 9h). Slowly add 50 μL of 1H, 1H, 2H, 2H-perfluoro-1-octanol (PFO) dropwise, incubate at room temperature for 3-5 mins, collect the cells from the upper aqueous phase, wash once with cell culture medium, add APC-IFNγdetectionAb and other cell membrane surface fluorescent antibodies (such as PD1, TCR, CD8) for staining, and detect the expression of IFNγ captured on the cell membrane surface and membrane proteins by flow cytometry.

[0065] (5) Single-cell library construction and sequencing of sorted target cells:

[0066] After further sorting and recovery of cells secreting cytokines, subsequent transcriptome enrichment and sequencing were performed according to the DNBelab C4 RNA-seq manual. The FASTQ data obtained from sequencing was used for automated analysis using PISA software. Single-cell library construction and transcriptome sequencing were performed to analyze gene expression characteristics and identify T cell characteristics capable of rapidly and efficiently responding to stimuli and secreting cytokines.

[0067] To facilitate the implementation of the methods described in this application, according to a typical embodiment of the present invention, a system for qualitative and quantitative analysis of immune cell secreted proteins based on sequencing technology is provided. The system includes: a packaging unit configured to package bispecific antibody-labeled immune cells and stimulants or antigen-presenting cells loaded with tumor antigen peptides into a droplet; an observation unit configured to incubate the droplet formed in the packaging unit and observe the cytokine secretion of immune cells in real time using a droplet microwell chip; an analysis unit configured to break the droplet at different incubation time points, recover the cells, and analyze cell membrane proteins and cytokines captured on the cell surface using flow cytometry; and an integrated analysis unit configured to sort the target cell population for single-cell transcriptome and secreted cytokine sequencing and perform integrated analysis.

[0068] By applying the technical solution of this invention, high-throughput and low-cost accurate analysis of immune cells at the single-cell level is achieved based on droplet microfluidic technology. This is combined with the use of bispecific antibodies to label immune cells and simultaneously capture secreted cytokines. Subsequently, the droplets are broken up, the cells are recovered, and flow cytometry is used to analyze cell membrane proteins and cytokines captured on the cell surface simultaneously. Furthermore, the cell populations of interest are sorted out for single-cell transcriptome sequencing, achieving a comprehensive and accurate high-throughput, high-efficiency, and low-cost analysis of the function and transcriptome characteristics of individual T cells.

[0069] In one embodiment of the present invention, the immune cells are T cells; preferably, the bispecific antibody labeling is CD45-IFNγ bispecific antibody labeling; and / or the stimulant is magnetic beads conjugated with CD3 / CD28 antibodies; and / or the antigen-presenting cells loaded with tumor antigen peptides are antigen-presenting cells loaded with MART1 peptides; preferably, the preparation of immune cells includes: isolating T cells from PBMCs by enriching them with CD3 magnetic beads and culturing them for more than 48 hours; and / or the preparation of antigen-presenting cells includes: adding MART1 peptides to T2 cell culture medium for loading and incubating for more than 12 hours.

[0070] Furthermore, the system also includes: a staining unit, disposed before the encapsulation unit, configured to perform different staining on bispecific antibody-labeled immune cells and antigen-presenting cells loaded with tumor antigen peptides; preferably, immune cells are stained with calcein am, and antigen-presenting cells are stained with Hoechst 33342; preferably, the encapsulation unit is configured to use a sealed syringe to generate negative pressure to create droplets, thereby encapsulating the bispecific antibody-labeled immune cells and the stimulant or antigen-presenting cells loaded with tumor antigen peptides into the droplets; more preferably, the sealed syringe is configured such that when the droplet diameter is 90 μm, the density of immune cells is 1000 cells / μL, and the density of antigen-presenting cells is 5000 cells / μL.

[0071] In a preferred embodiment of the present invention, the observation unit is configured to incubate the droplets in a 37°C, 5% CO2 incubator; preferably, the instrument conditions for real-time observation of cytokine secretion by immune cells using the droplet microwell chip are set to 37°C, 5% CO2; preferably, the analysis unit is configured to take droplets at different incubation time points, add 1H, 1H, 2H, 2H-perfluoro-1-octanol, let stand at room temperature for 3-5 mins, recover the cells from the upper aqueous phase, wash, add antibodies for staining, and perform flow cytometry detection of cell membrane proteins and cytokines captured on the cell surface for statistical analysis; preferably, the integrated analysis unit is configured to sort out cells that secrete cytokines and then perform subsequent transcriptome sequencing combined with sequencing of secreted cytokines to analyze gene expression characteristics and qualitative and quantitative analysis of cytokines.

[0072] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0073] Example 1: Detection of IFNγ secreted by CD3 / CD28-stimulated single T cells

[0074] Main experimental materials:

[0075] 1. Primary T cells;

[0076] 2. CD3 T cell enrichment kit (Miltenyi Biotec, 130-050-101);

[0077] 3. Cell stimulant: Transact (Miltenyi Biotec, 130-111-160);

[0078] 4. IFNγ detection kit (Miltenyi Biotec, 130-090-762);

[0079] 5. Cell culture medium HIPP TM -T009 (BIOENGINE, FG0103801), abbreviated as T009;

[0080] 6. Fetal bovine serum FBS (ExCell Bio, FND500);

[0081] 7. IFNγstandard (invitrogen, KHC4021);

[0082] 8. Droplet Generation Oil for EvaGreen (BIO-RAD, 1864006);

[0083] 9.1H, 1H, 2H, 2H-perfluoro-1-octanol (PFO) (ALDRICH, MKCL9941);

[0084] 10.30mL syringe (BD, 302833).

[0085] Experimental steps:

[0086] 1. Cell preparation:

[0087] Primary T cell preparation: Primary T cells were isolated from PBMCs and cultured. T cells were enriched and isolated using a CD3 T cell sorting kit (Miltenyi) and cultured in H009 + 10% FBS + 1% P / S + IL2 (10 ng / mL) + IL7 (10 ng / mL) + IL15 (10 ng / mL) for more than 48 hours.

[0088] Cell staining and catch reagent labeling: Before generating droplets, primary T cells were diluted in FACs buffer with 1 μM calcein am (1:200) and CD45-IFNγ catch reagent (1:10) and stained at 4°C for 45 mins. After cell staining, the cells were washed once with ice-cold FACs buffer to remove excess dye, etc., and the cells were resuspended in working solution to make the primary T cell density 1000 cells / μL.

[0089] 2-Droplet generation incubation:

[0090] Using droplet generation chip devices (such as...) Figure 2 The device generates droplets by creating negative pressure through the extraction of a syringe, such as... Figure 2 The diagram shows two sample wells: one for droplet-generating oil and the other for collecting the generated droplets. The top is sealed with a removable sealing ring. A small hole connects to the syringe via a sealing coil. The syringe is rapidly pulled from the 12mL mark to 20mL (producing uniform droplets with a diameter of ~90μm). 200μL of T2 cells loaded with the antigenic peptide MART1 and 200μL of primary T cells are added to each well, along with 600μL of droplet-generating oil. Droplets are generated using the principle of negative pressure created by pulling the sealed syringe. The syringe is initially adjusted to the 12mL mark, then pulled to 20mL and fixed to allow droplet generation. The generated droplets are collected in the [missing information - likely a container or container]. Figure 2 The primary T cells are collected in droplet collection tubes. To achieve single-cell encapsulation of primary T cells as much as possible and optimize cell injection density, when the droplet diameter is 90 μm, the T cell density is 1000 cells / μL. This ensures that over 90% of the primary T cell-encapsulated droplets contain only a single T cell (e.g., ...). Figure 5 ).

[0091] The experimental groups were prepared according to the table below. The positive control group was given IFN-γ standard, and the negative control group was given no capture antibody. Droplets were generated using a droplet generating device and incubated in a 37°C incubator.

[0092] Table 1

[0093]

[0094] PC represents the positive control; TEST represents the experimental group; and NC represents the negative control.

[0095] 3-Cell recovery analysis of cytokine-positive population:

[0096] Most of the generated droplets were placed in a 37°C, 5% CO2 incubator for incubation, while a portion was loaded into a droplet microwell chip. The microwell chip was then placed in a Cytation5 (Biotek) instrument, and the temperature was set to 37°C and 5% CO2. The droplets were incubated and fluorescence images were observed and captured, and the secretion of the cytokine IFNγ was recorded in real time.

[0097] After droplet formation, 100 μL of droplets were taken out every 2 hours of incubation and demulsified with a demulsifier. The recovered cells were then analyzed by flow cytometry to detect the proportion of IFN-γ positive cells.

[0098] 4-Target cell sorting and single-cell sequencing:

[0099] IFN-γ positive cells were sorted using flow cytometry for single-cell sequencing and downstream analysis. After sorting cells that secrete cytokines, subsequent transcriptome enrichment and sequencing were performed according to the DNBelab C4 RNA-seq manual. The FASTQ data obtained from sequencing was used for automated analysis using PISA software to construct single-cell libraries, analyze gene expression characteristics, and identify T cell characteristics capable of rapidly and efficiently responding to stimuli and secreting cytokines.

[0100] Experimental results:

[0101] Result 1: At the bulk level, the catch reagent (CD45 x IFNγBispicificAb) effectively captured IFNγ to the cell surface. Figure 3The cells used in this test were CD3 T cells enriched from PBMCs. In the experimental group (Test), T cells labeled with catch reagent were stimulated with CD3 / CD28 antibody-conjugated magnetic beads and cultured in 12-well plates. The positive group had IFNγ standard added to the culture medium, while the negative control group consisted of unlabeled T cells. After 6 hours of culture, cells were collected and stained with anti-human-CD8-APC and FITC-IFNγ detection antibody staining, respectively. The results showed that T cells in both the positive and experimental groups were labeled with the catch reagent and captured IFNγ.

[0102] Result 2: Within the test droplet, IFNγ secreted by T cells stimulated by CD3 / CD28 antibody-conjugated magnetic beads could be effectively captured to the cell surface.

[0103] Example 2: Detection of IFNγ secretion after T cell activation following co-encapsulation of antigen-presenting cells T2 and TCR-T (5T-4) droplets loaded with peptide MART-1.

[0104] Experimental materials:

[0105] 1. MART-1 27-35 peptide LAGIGILTV(HLA-A:0201)(synthesized by Genscript);

[0106] 2. Antigen-presenting cells T2;

[0107] 3. T2 cell culture medium: IMDM (Gibco, 12440053);

[0108] 3. T2 cell dye Hoechst 33342 (Invitrogen, H3570);

[0109] 4. TCR-T cell dye Calcein AM (Biolegend, 425201);

[0110] 5. Other materials are the same as in Example 1.

[0111] Experimental steps:

[0112] 1. Cell preparation:

[0113] 1-1. Count T2 cells the day before, taking 5 × 10⁻⁶ cells. 6 Cells, adjust cell concentration to 5×10 6 Add 3 mL of cells / well to each well of a six-well plate, and add 10 μg / mL of LMR1 peptide to each well. Separately, add 1 × 10⁻⁶ cells / well. 6T2 cells without MART1 peptide were seeded in six-well plates and incubated overnight at 37°C.

[0114] 1-2. On the second day, count the TCR-T cells, taking 1×10⁻⁶ cells. 6 Cells were resuspended in 100 μL of T009 complete medium, 10 μL of catch reagent and 1 μL of calcein am were added, and the cells were incubated at 4 °C for 30 min. After incubation, the cells were washed with 10 mL of PBS, passed through a 30 μm sieve before centrifugation, and then resuspended in 1 mL of T009 complete medium to achieve a cell concentration of 1000 cells / μL.

[0115] 1-3. Take T2 cells loaded with peptides, resuspend them in 500 μL T009 complete medium, add 1 μL Hoechst 33342, incubate at 37℃ for 20 minutes, wash the cells with 10 mL PBS after incubation, pass them through a 30 μm sieve before centrifugation, and resuspend them in 1 mL T009 complete medium to a cell concentration of 5000 cells / μL.

[0116] 1-4. Take T2 cells without peptide loading, resuspend them in 100 μL of T009 complete medium, add 1 μL of Hoechst 33342, incubate at 37 degrees for 20 minutes, wash the cells with 5 mL of PBS after incubation, and resuspend them in 200 μL of T009 complete medium to a cell concentration of 5000 cells / μL.

[0117] 2-Droplet generation incubation:

[0118] Three experimental groups were prepared according to the table below. The positive control group was added with IFN-γ standard, and the negative control group was not loaded with peptide in T2. ​​Droplets were generated using a droplet generating device and incubated in a 37°C incubator.

[0119] Table 2

[0120] MART1 peptide loading IFNγ standard CaptureAb PC Yes 10 pg / mL 25μL TEST Yes No 25μL NC No No 25ul

[0121] 3. Real-time observation with a fluorescence microscope: The operation is the same as in Example 1.

[0122] 4. Cell recovery analysis of cytokine-positive populations: The procedure is the same as in Example 1.

[0123] 5. Target cell sorting and single-cell sequencing: The procedure is the same as in Example 1.

[0124] Experimental results:

[0125] Result 1: Co-encapsulation efficiency of 5T-4TCR-T cells and antigen-presenting cells (T2): Figure 4

[0126] Statistical observations showed that the two cell types could co-encapsulate well, with over 70% of the droplets encapsulating T cells (green) also encapsulating T2 cells (blue). Figure 4 (A)

[0127] T cells labeled with catch reagent and T2 cells loaded with peptides were co-encapsulated in droplets and incubated at 37°C. The droplets were removed at different time points (2h and 4h) and the secretion and capture of IFNγ were detected using a fluorescence microscope. After 2h of incubation, some live cells in the experimental group were found to be IFNγ positive. Flow cytometry analysis of the recovered droplets showed that the IFNγ-positive population was 15.4% at 2h and 22.6% at 4h. Future experiments will optimize the detection of IFNγ-positive cell proportions at more time points. Figure 4 (B)

[0128] Result 2: Detection of IFNγ secreted by 5T-4TCR-T cells:

[0129] At 3 and 6 hours of droplet formation and incubation, fluorescence microscopy revealed that some cells within the droplets co-encapsulated by T cells and T2 cells were IFNγ-positive. Figure 6 Flow cytometry analysis of recovered cells (A) revealed that after 3 hours of incubation, 10% of the cells were IFNγ-positive. After 6 hours of incubation, the positive population did not increase significantly, with 11.5% remaining positive. Figure 6 (B). Further analysis revealed that only 12.2% of the 5T-4TCR-T cells were TCR-positive, and a portion of these cells expressed PD1 (…). Figure 6 The presence of C in the incubation time could explain why the IFNγ positivity rate did not continue to increase with incubation time. The experiment needs optimization in using flow cytometry to confirm that the IFNγ-positive population in the recovered cells is indeed a 5T-4TCR-positive cell group.

[0130] Result 3:

[0131] Figure 7 The left image shows T cell transcriptome clustering, and the right image shows the characteristics of T cell IFNγ secretion.

[0132] Example 3: Bispecific antibody-mediated activation and secretion of IFNγ by a single T cell

[0133] Main experimental materials:

[0134] 1. Primary T cells;

[0135] 2. Target cells: LIM1215 cell line;

[0136] 3. Control cells: A2780 cell line;

[0137] 4. CD3 T cell enrichment kit (Miltenyi Biotec, 130-050-101);

[0138] 5. IFNγ detection kit (Miltenyi Biotec, 130-090-762);

[0139] 6. Cell culture medium HIPP TM -T009 (BIOENGINE, FG0103801), abbreviated as T009;

[0140] 7. LIM1215 cell culture medium: RPMI 1640 (11875093);

[0141] 8. Fetal bovine serum FBS (ExCell Bio, FND500);

[0142] 9. CD3-EpCam bispecific antibody;

[0143] 10. IFNγstandard (invitrogen, KHC4021);

[0144] 11. Droplet Generation Oil for EvaGreen (BIO-RAD, 1864006);

[0145] 12.1H, 1H, 2H, 2H-perfluoro-1-octanol (PFO) (ALDRICH, MKCL9941);

[0146] 13.30mL syringe (BD, 302833).

[0147] Experimental steps:

[0148] 1. Cell preparation:

[0149] Primary T cell preparation: Primary T cells were isolated from PBMCs and cultured. T cells were enriched and isolated using a CD3 T cell sorting kit (Miltenyi) and cultured in H009 + 10% FBS + 1% P / S + IL2 (10 ng / mL) + IL7 (10 ng / mL) + IL15 (10 ng / mL) for more than 48 hours.

[0150] Cell staining and catchreagent labeling: Before generating droplets, LIM1215 cells were resuspended in cell culture medium at a concentration of 1 μg / μL Hoechst 333421:500 and incubated at 37°C for 10 mins, at which point LIM1215 cells were labeled blue. Primary T cells were stained and labeled in FAC buffer at 4°C for 45 mins with 1 μM calcein am (1:200) and CD45-IFNγ catch reagent (1:10). After staining, the cells were washed once with ice-cold FAC buffer to remove excess dye, and then resuspended in working solution to achieve a primary T cell density of 1000 cells / μL.

[0151] 2-Droplet generation incubation:

[0152] Three experimental groups were prepared according to the table below. The positive control group was supplemented with IFN-γ standard, and the target cells of the negative control group were replaced with A2780 cell line that does not express EpCcam. Droplets were generated using a droplet generator and incubated in a 37°C incubator.

[0153] Table 3

[0154]

[0155] PC represents the positive control; TEST represents the experimental group; and NC represents the negative control.

[0156] 3. Real-time observation with a fluorescence microscope: The operation is the same as in Example 1.

[0157] 4. Cell recovery analysis of cytokine-positive populations: The procedure is the same as in Example 1.

[0158] 5. Target cell sorting and single-cell sequencing: The procedure is the same as in the example.

[0159] Experimental results:

[0160] Result 1: Within the test droplets, IFNγ secreted by T cells activated under CD3-EpCam bispecific antibody-mediated activation was effectively captured and deposited on the cell surface. After 18 hours of incubation, the proportion of T cells secreting IFNγ in the experimental group increased to 53.8%, while the T cells in the control group basically did not secrete IFNγ. Figure 8 ).

[0161] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects:

[0162] First, this method has a high throughput, generating millions of droplets per hour, enabling the analysis of individual T cells encapsulated within them;

[0163] Second: Low cost, small droplet volume, with each droplet having a diameter of 90μm;

[0164] Third: It can monitor the secretion of cytokines by individual cells in real time;

[0165] Fourth: Combined with flow cytometry, the range of detection parameters has been expanded, enabling the simultaneous analysis of dozens of cell membrane proteins and cytokines captured on the cell membrane surface. The analysis throughput is high, and cells of interest can be sorted for further experiments.

[0166] Fifth: By combining single-cell transcriptome sequencing technology, the gold and silver characteristics of functional cells that secrete cytokines can be further studied.

[0167] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for qualitative and quantitative analysis of proteins secreted by immune cells based on sequencing technology, characterized in that, include: S1 encapsulates bispecific antibody-labeled immune cells and stimulants or antigen-presenting cells loaded with tumor antigen peptides into a droplet; S2, incubate the droplets formed in S1, and use the droplet microwell chip to observe the cytokine secretion of the immune cells in real time; S3: The droplets were broken at different incubation time points, the cells were recovered, and flow cytometry was used to analyze cell membrane proteins and cytokines captured on the cell surface. S4. Select the target cell population, perform single-cell transcriptome and secreted cytokine sequencing, and conduct integrated analysis.

2. The method according to claim 1, characterized in that, The immune cells mentioned are T cells.

3. The method according to claim 2, characterized in that, The bispecific antibody label is a CD45-IFNγ bispecific antibody label; and / or The stimulant is a magnetic bead conjugated with a CD3 / CD28 antibody; and / or The antigen-presenting cells loaded with tumor antigen peptides are antigen-presenting cells loaded with MART1 peptides.

4. The method according to claim 2, characterized in that, The preparation of the immune cells includes: isolating T cells from PBMCs using CD3 magnetic beads for enrichment and culturing them for more than 48 hours; and / or The preparation of the antigen-presenting cells includes: adding MART1 peptide to T2 cell culture medium for loading and incubating for more than 12 hours.

5. The method according to any one of claims 1 to 4, characterized in that, The S1 further includes: staining the bispecific antibody-labeled immune cells and the antigen-presenting cells loaded with tumor antigen peptides differently before encapsulating them into the droplets; Preferably, the immune cells are stained with calcein am, and the antigen-presenting cells are stained with Hoechst 33342.

6. The method according to any one of claims 1 to 4, characterized in that, In step S1, a method of generating droplets by drawing a sealed syringe to create negative pressure is used to encapsulate the bispecific antibody-labeled immune cells and the stimulant or antigen-presenting cells loaded with tumor antigen peptides into the droplets.

7. The method according to claim 6, characterized in that, When the droplet diameter is 90 μm, the density of the immune cells is 1000 cells / μL, and the density of the antigen-presenting cells is 5000 cells / μL.

8. The method according to any one of claims 1 to 4, characterized in that, In step S2, the droplet is incubated in a 37°C, 5% CO2 incubator. Preferably, when using the droplet microwell chip to observe the cytokine secretion of the immune cells in real time, the instrument conditions are set to 37°C and 5% CO2.

9. The method according to any one of claims 1 to 4, characterized in that, S3 includes taking droplets at different incubation time points, adding 1H, 1H, 2H, 2H-perfluoro-1-octanol, letting stand at room temperature for 3-5 minutes, recovering cells from the upper aqueous phase, washing, adding antibodies for staining, flow cytometry detection of cell membrane proteins and cytokines captured on the cell surface, and performing statistical analysis.

10. The method according to any one of claims 1 to 4, characterized in that, S4 includes: sorting out cells that secrete cytokines and then performing subsequent transcriptome sequencing combined with sequencing of secreted cytokines to analyze gene expression characteristics and qualitative and quantitative analysis of cytokines.

11. A system for qualitative and quantitative analysis of proteins secreted by immune cells based on sequencing technology, characterized in that, include: The encapsulation unit is configured to encapsulate bispecific antibody-labeled immune cells together with a stimulant or antigen-presenting cells loaded with tumor antigen peptides into a droplet. An observation unit is configured to incubate the droplets formed in the encapsulation unit and to observe the cytokine secretion of the immune cells in real time using a droplet microwell chip. The analysis unit is designed to break droplets at different incubation time points, recover cells, and analyze cell membrane proteins and cytokines captured on the cell surface using flow cytometry. The integrated analysis unit is configured to sort the target cell population for single-cell transcriptome and secreted cytokine sequencing and perform integrated analysis.

12. The system according to claim 11, characterized in that, The immune cells are T cells; Preferably, the bispecific antibody label is a CD45-IFNγ bispecific antibody label; and / or The stimulant is a magnetic bead conjugated with a CD3 / CD28 antibody; and / or The antigen-presenting cells loaded with tumor antigen peptides are antigen-presenting cells loaded with MART1 peptides. Preferably, the preparation of the immune cells includes: isolating T cells from PBMCs using CD3 magnetic beads for enrichment and culturing them for more than 48 hours; and / or The preparation of the antigen-presenting cells includes: adding MART1 peptide to T2 cell culture medium for loading and incubating for more than 12 hours.

13. The system according to claim 11 or 12, characterized in that, The system further includes a staining unit disposed before the packaging unit, configured to perform different staining on the bispecific antibody-labeled immune cells and antigen-presenting cells loaded with tumor antigen peptides. Preferably, the immune cells are stained with calcein am, and the antigen-presenting cells are stained with Hoechst 33342. Preferably, the encapsulation unit is configured to use a sealed syringe to generate negative pressure to create droplets, thereby encapsulating the bispecific antibody-labeled immune cells and the stimulant or antigen-presenting cells loaded with tumor antigen peptides into the droplets. Alternatively, the sealed syringe is configured such that when the droplet diameter is 90 μm, the density of the immune cells is 1000 cells / μL and the density of the antigen-presenting cells is 5000 cells / μL.

14. The system according to claim 11 or 12, characterized in that, In the observation unit, the droplets are set to be incubated in a 37°C, 5% CO2 incubator. Preferably, in the observation unit, when using the droplet microwell chip to observe the cytokine secretion of the immune cells in real time, the instrument conditions are set to 37°C and 5% CO2. Preferably, the analysis unit is configured to take droplets at different incubation time points, add 1H, 1H, 2H, 2H-perfluoro-1-octanol, let stand at room temperature for 3-5 mins, recover the cells in the upper aqueous phase, wash, add antibody for staining, flow cytometry to detect cell membrane proteins and cytokines captured on the cell surface, and perform statistical analysis. Preferably, the integrated analysis unit is configured to sort out cells that secrete cytokines and then perform subsequent transcriptome sequencing combined with sequencing of secreted cytokines to analyze gene expression characteristics and qualitative and quantitative analysis of cytokines.

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