Cell interaction detection method and application thereof

Abstract

The invention relates to a method for detecting cell interaction and a microfluidic system for performing the method. The method of the invention comprises (1) preparing a first cellular phase comprising a first droplet comprising a surface display cell (SD cell) comprising an outer surface display polypeptide and labeled with a first barcode, and free of other types of cells; (2) preparing a second cell phase which comprises second cells and is marked with a second bar code; and (3) injecting the second cell phase into the first cell phase by means of droplet injection to form a mixed phase containing second droplets. The method disclosed by the invention has the advantages of high flux, short time and high utilization rate of the second cells.

Description

Technical Field

[0001] The present invention relates to the field of cell therapy, and more particularly to the detection of cell interaction in cell therapy. Background Art

[0002] Cells are the basic components of organisms. If an organism is compared to a machine, a complex and sophisticated instrument, then the interaction between cells is like a transmission system. The coordination and communication between them are crucial in the process of immune response, cell differentiation and tissue repair. Very typical cell interaction behaviors in basic immunology research include antigen uptake and presentation between tumor cells and antigen presenting cells (APCs), recognition of APCs and T cells, and activated effector T cells attacking tumor cells.

[0003] Currently, the field has produced several tools focused on cell-cell interactions, which can be roughly divided into the following types: monitoring of interacting cell partners based on microscopic imaging called FAP-DAPA, chemical contact-dependent labeling through ligand-receptor binding partners called FucoID and EXCELL, and new technologies for contact-independent labeling of cell-cell interactions, such as Contact-ID and Split-turboid, which have been shown to have good practical application prospects, such as FucoID, which determines characteristics through interaction-dependent focused biotinylation, and operates the entire endogenous tumor antigen-specific TILs library with simpler procedures and faster turnover. However, in some application scenarios (such as antigen-specific T cells), throughput limitations are common.

[0004] In addition, the combination of cell-cell interactions and single-cell transcriptomics makes it possible to visualize inferred cell-cell communication networks. This cell-cell communication can be inferred by methods such as CellChat, which utilize the intrinsic gene expression information in scRNA-seq data to predict key signaling mechanisms of dermal condensate and melanocyte migration in early hair follicle development, as well as elevated inflammatory signals in brain barrier cells during COVID-19 infection. Current research on cell-cell interactions is mainly based on single-cell datasets, computational analysis, etc.

[0005] Cell-based therapy mainly relies on the transformation and reinfusion of the body's own immune cells to eliminate cancer cells, which is also a typical example of cell-cell interaction to achieve functional effects. Adoptive cell immunotherapy often requires multiple rounds of individualized specific therapeutic TCR screening to better meet the clinical needs of such malignancies. At the same time, the specificity of new antigens is required to enhance the targeting of immunotherapy. At present, yeast display CDR3α mutant libraries have successfully identified high-affinity TCRs that recognize pMHC, but the flux is relatively low. Moreover, the higher the affinity, the higher the risk of cross-reaction and off-target recognition. Therefore, rapid evaluation of the cross-reactivity of high-affinity TCRs is crucial to the safety of TCR-T therapy.

[0006] The technical process of TCR-T cell therapy generally includes: (1) screening suitable tumor-specific antigens and TCR αβ chain sequences that are highly specific to the antigen; (2) isolating T cells from the patient; (3) packaging the above-mentioned TCR αβ chain sequences that can recognize tumor-specific antigens into the viral genome to construct a viral vector; (4) viral transfection of T cells to introduce the target TCR sequence into T cells to obtain TCR-T cells that can specifically recognize tumor antigens; (5) culturing TCR-T cells in vitro and amplifying them in large quantities; (6) injecting a qualified number of TCR-T cells into the patient for treatment.

[0007] Both cancer cells and viruses evolve rapidly to evade clearance by high-affinity T cells. Therefore, a rapid, high-throughput method to identify this type of cell-cell interaction is highly necessary. Single-cell transcriptomics facilitates extensive studies of the heterogeneity of tumor cells and their interactions in the local microenvironment. Related genetic data are also widely used to determine biomarkers for cancer diagnosis, prognosis prediction, and to identify new therapeutic targets in designated clinical cohorts. For example, VanGalen et al. combined single-cell RNA sequencing and genotyping to study acute myeloid leukemia by analyzing cell states, regulators, and biomarkers, and further discovered the relationship between developmental grade and malignant genetics. The combined application of single-cell transcriptomics and tumor immunology can greatly promote clinical changes.

[0008] Intercellular interaction is one of the main ways to transmit information between cells and activate functional cells. The research on cell interactions in the prior art mainly exists in the bioinformatics analysis and prediction level, and the experimental verification platform is relatively scarce. Building high-throughput analysis technology from cytogenomics and focusing on the impact of functional cell interactions on targets has not been explored relatively speaking. Most of the existing experimental platforms have problems of low throughput and high consumption of manpower and material resources, and most of the verification cannot be fully focused on the single-cell level. At present, the study of cell interactions at the single-cell level based on microfluidics has the problem of low cell utilization due to Poisson distribution, which is a difficult-to-solve technical bottleneck for the study of precious samples (such as primary T cells). This field is in urgent need of high-cell utilization, high-throughput and rapid microfluidic-based cell-cell interaction sequencing and in vitro screening technology. Summary of the invention

[0009] The present application relates to a method for detecting cell interactions and its application, wherein the detection method is based on microfluidics-based cell-cell interaction sequencing and in vitro screening technology. The method uses cell molecular tags for pre-labeling and introduces additional single-cell sequencing reagents to quickly form a functional droplet mixture.

[0010] In a first aspect, the present invention provides a method for detecting cell interactions, the method comprising the following steps:

[0011] (1) preparing a first cell phase comprising a first droplet, wherein the first droplet comprises a surface-displayed cell (SD cell) and does not contain other types of cells, wherein the SD cell comprises an external surface-displayed polypeptide and is labeled with a first barcode;

[0012] (2) preparing a second cell phase, wherein the second cell phase includes second cells and is labeled with a second barcode; and

[0013] (3) Injecting the second cell phase into the first cell phase by droplet injection to form a mixed phase containing the second droplet.

[0014] In some embodiments, the first cell phase includes one or more first droplets, each of the one or more first droplets contains a surface-displayed cell (SD cell) and does not contain other types of cells, the SD cell contains an external surface-displayed polypeptide and is labeled with a first barcode.

[0015] In some embodiments, the multiple first droplets respectively contain different types of surface displayed cells, the same SD cell includes the same antigen polypeptide or a nucleic acid sequence encoding the antigen polypeptide and is marked with the same first barcode, and different types of SD cells are respectively marked with different first barcodes.

[0016] In some embodiments, the same first droplet includes a single SD cell or multiple SD cells, preferably multiple SD cells, and the multiple SD cells are the same type of cells. Preferably, 2-6 SD cells are encapsulated in the first droplet; preferably, 2-4 SD cells are encapsulated in the first droplet.

[0017] In some embodiments, the SD cells are antigen presenting cells (APCs).

[0018] In some embodiments, the SD cells are tumor antigen presenting cells, such as T2 cells.

[0019] In some embodiments, the APC is a macrophage, a dendritic cell, or a B cell.

[0020] In some embodiments, the SD cell comprises an antigen polypeptide or a nucleic acid sequence encoding the antigen polypeptide. Preferably, the SD cell comprises a tumor antigen polypeptide or a nucleic acid sequence encoding the tumor antigen polypeptide.

[0021] In some embodiments, the SD cell comprises a nucleic acid sequence encoding an MHC sequence.

[0022] The MHC is MHC-I, MHC-II or MHC-III, preferably MHC-I.

[0023] Encapsulating a plurality of identical SD cells in one of the first droplets can increase the stimulation of SD cells on, for example, T cells and can also eliminate the influence of Poisson distribution.

[0024] Because each type of droplet is prepared separately, it is ensured that the first droplet prepared contains one or more cells of the same type.

[0025] In some embodiments, the first droplets are generated by applying negative pressure to the droplet generation chip.

[0026] In some embodiments, the first droplet further comprises microbeads, and the particle size of the microbeads is 0.5 μm to 500 μm.

[0027] In some embodiments, the microbeads are made of a polymer material, such as polystyrene.

[0028] In some embodiments, the microbeads are magnetic beads, which are magnetic or contain magnetic components.

[0029] In some embodiments, the microbeads have biomolecules attached to their surface, such as antibodies, proteins, antigens, DNA, RNA, streptavidin, or a combination thereof.

[0030] In some embodiments, the microbeads are immunoassay particles, or are RNA capture beads.

[0031] In some embodiments, the SD cells are labeled with a first barcode using CASB.

[0032] According to the number of antigens to be detected, such as the number of types of tumor antigens, a corresponding number of groups of first droplets are generated, and then the corresponding number of groups of first droplets are mixed to form a first cell phase.

[0033] In some embodiments, the first droplet comprises an aqueous fluid in a carrier fluid.

[0034] In some embodiments, the carrier fluid comprises oil.

[0035] In some embodiments, the second cell phase includes one or more second cells, and different types of second cells are labeled with different second barcodes.

[0036] In some embodiments, the second cell comprises a T-cell receptor (TCR). Preferably, the second cell is selected from a T cell and a Jurkat cell containing a T-cell receptor (TCR). The Jurkat cell is a cell that reports a TCR.

[0037] In some embodiments, the second cell further comprises a nucleic acid encoding the TCR.

[0038] In some embodiments, each second cell contains or expresses only one TCR.

[0039] In some embodiments, the TCR is a chimeric antigen receptor.

[0040] In some embodiments, the TCR is a T-cell receptor endogenous to the T cell, or is a synthetic T-cell receptor.

[0041] In some embodiments, the second cell is labeled with a second barcode using CASB.

[0042] The same second cell is labeled with the same second barcode.

[0043] Different types of second cells are respectively labeled with different second barcodes.

[0044] In some embodiments, the second cell phase is injected into the first cell phase by droplet injection to form a mixed phase containing second droplets, and each of the second droplets contains less than or equal to 5 second cells; preferably, each of the second droplets contains only 1, 2, 3 or 4 second cells; more preferably, each of the second droplets contains only 1 second cell.

[0045] In some embodiments, each of the second droplets contains one or more SD cells of the same type and a second cell.

[0046] In some embodiments, the concentration of the second cells contained in the second cell phase is adjusted to ensure that the second cells injected via the droplets of the second cell phase are single cells.

[0047] In some embodiments, the plurality of second cells are injected into the first cell phase in sequence according to cell type by droplet injection to form a mixed phase containing second droplets.

[0048] Because the input amount of the second cell, especially T cells, can be controlled, the waste of T cells can be effectively prevented. The input at an extremely high concentration ensures that the entering T cells are single, thereby improving the utilization rate of T cells.

[0049] In some embodiments, the second droplets are generated by applying negative pressure to the droplet generation chip.

[0050] In some embodiments, droplet injection is performed under a certain injection voltage condition.

[0051] In some embodiments, droplet injection is performed under high voltage conditions.

[0052] Preferably, droplet injection is performed under the conditions of 15 kHz frequency, 1 VPP voltage, and 200-fold voltage amplifier.

[0053] In some embodiments, the second droplets comprise an aqueous fluid in a carrier fluid.

[0054] In some embodiments, the carrier fluid comprises oil.

[0055] In some embodiments, the mixed phase is incubated under suitable conditions to allow cells in the first cell phase to interact with cells in the second cell phase.

[0056] In some embodiments, cell interactions are detected in a microfluidic system, and / or a first cell phase comprising a first droplet is prepared by a microfluidic system.

[0057] In some embodiments, the method further comprises the steps of injecting a lysate into the mixed phase by droplet injection and analyzing the first and second cell barcodes and intracellular mRNA released from the surfaces of the SD cells and the second cells via cell lysis.

[0058] Injecting the lysate into the mixed phase can lyse the cells in the second droplet, thereby releasing the cell barcodes on the cell surface and the mRNA in the cells, facilitating the subsequent capture of the RNA capture magnetic beads.

[0059] Any conventional lysis solution in the art can be used, as long as the lysis solution can break up and lyse cells to release their genome.

[0060] In some embodiments, the methods are used to detect rare cell interactions or screen for functional antigens or T cell receptors.

[0061] In some embodiments, the method further comprises the steps of constructing a cDNA library and performing sequencing.

[0062] Through a series of RT-PCR and PCR reactions, the captured mRNA is first converted into DNA and amplified from a small amount to a large amount, and finally a stable single-stranded circular DNA is obtained to meet the sequencing requirements.

[0063] Preferably, the sequencing is next generation sequencing.

[0064] In some embodiments, the method further comprises the step of analyzing cellular interactions.

[0065] The data containing only two types of label information are filtered out, and then the barcodes (with known sequences) are used to analyze which two cells are interacting.

[0066] In some embodiments, the method further comprises determining whether high expression of T cell activation-related genes is generated when the corresponding antigen is used to stimulate the corresponding T cell.

[0067] In a second aspect, the present invention provides a method for high-throughput screening of functional antigens and their specific TCRs, the method comprising the following steps:

[0068] (1) preparing a first cell phase comprising a first droplet, wherein the first droplet comprises a surface-displayed cell (SD cell) and does not contain other types of cells, wherein the SD cell comprises an external surface-displayed polypeptide and is labeled with a first barcode;

[0069] (2) preparing a second cell phase, wherein the second cell phase includes second cells and is labeled with a second barcode; and

[0070] (3) Injecting the second cell phase into the first cell phase by droplet injection to form a mixed phase containing the second droplet.

[0071] In some embodiments, the first cell phase includes one or more first droplets, each of the one or more first droplets contains a surface-displayed cell (SD cell) and does not contain other types of cells, the SD cell contains an external surface-displayed polypeptide and is labeled with a first barcode.

[0072] In some embodiments, the multiple first droplets respectively contain different types of surface displayed cells, the same SD cell includes the same antigen polypeptide or a nucleic acid sequence encoding the antigen polypeptide and is marked with the same first barcode, and different types of SD cells are respectively marked with different first barcodes.

[0073] In some embodiments, the same first droplet includes a single SD cell or multiple SD cells, preferably multiple SD cells, and the multiple SD cells are the same type of cells. Preferably, 2-6 SD cells are encapsulated in the first droplet; preferably, 2-4 SD cells are encapsulated in the first droplet.

[0074] In some embodiments, the SD cells are antigen presenting cells (APCs).

[0075] In some embodiments, the SD cells are tumor antigen presenting cells, such as T2 cells.

[0076] In some embodiments, the APC is a macrophage, a dendritic cell, or a B cell.

[0077] In some embodiments, the SD cell comprises an antigen polypeptide or a nucleic acid sequence encoding the antigen polypeptide. Preferably, the SD cell comprises a tumor antigen polypeptide or a nucleic acid sequence encoding the tumor antigen polypeptide.

[0078] In some embodiments, the SD cell comprises a nucleic acid sequence encoding an MHC sequence.

[0079] The MHC is MHC-I, MHC-II or MHC-III, preferably MHC-I.

[0080] Encapsulating a plurality of identical SD cells in one of the first droplets can increase the stimulation of SD cells on, for example, T cells and can also eliminate the influence of Poisson distribution.

[0081] Because each type of droplet is prepared separately, it is ensured that the first droplet prepared contains one or more cells of the same type.

[0082] In some embodiments, the first droplets are generated by applying negative pressure to the droplet generation chip.

[0083] In some embodiments, the first droplet further comprises microbeads, and the particle size of the microbeads is 0.5 μm to 500 μm.

[0084] In some embodiments, the microbeads are made of a polymer material, such as polystyrene.

[0085] In some embodiments, the microbeads are magnetic beads, which are magnetic or contain magnetic components.

[0086] In some embodiments, the microbeads have biomolecules attached to their surface, such as antibodies, proteins, antigens, DNA, RNA, streptavidin, or a combination thereof.

[0087] In some embodiments, the microbeads are immunoassay particles, or are RNA capture beads.

[0088] In some embodiments, the SD cells are labeled with a first barcode using CASB.

[0089] According to the number of antigens to be detected, such as the number of types of tumor antigens, a corresponding number of groups of first droplets are generated, and then the corresponding number of groups of first droplets are mixed to form a first cell phase.

[0090] In some embodiments, the first droplet comprises an aqueous fluid in a carrier fluid.

[0091] In some embodiments, the carrier fluid comprises oil.

[0092] In some embodiments, the second cell phase includes one or more second cells, and different types of second cells are labeled with different second barcodes.

[0093] In some embodiments, the second cell comprises a T-cell receptor (TCR). Preferably, the second cell is selected from a T cell and a Jurkat cell containing a T-cell receptor (TCR). The Jurkat cell is a cell that reports a TCR.

[0094] In some embodiments, the second cell further comprises a nucleic acid encoding the TCR.

[0095] In some embodiments, each second cell contains or expresses only one TCR.

[0096] In some embodiments, the TCR is a chimeric antigen receptor.

[0097] In some embodiments, the TCR is a T-cell receptor endogenous to the T cell, or is a synthetic T-cell receptor.

[0098] In some embodiments, the second cell is labeled with a second barcode using CASB.

[0099] The same second cell is labeled with the same second barcode.

[0100] Different types of second cells are respectively labeled with different second barcodes.

[0101] In some embodiments, the second cell phase is injected into the first cell phase by droplet injection to form a mixed phase containing second droplets, and each of the second droplets contains less than or equal to 5 second cells; preferably, each of the second droplets contains only 1, 2, 3 or 4 second cells; more preferably, each of the second droplets contains only 1 second cell.

[0102] In some embodiments, each of the second droplets contains one or more SD cells of the same type and a second cell.

[0103] In some embodiments, the cell concentration of the second cells contained in the second cell phase is adjusted to ensure that the second cells injected via the droplets of the second cell phase are single cells.

[0104] In some embodiments, the plurality of second cells are injected into the first cell phase in sequence according to cell type by droplet injection to form a mixed phase containing second droplets.

[0105] Because the input amount of the second cell, especially T cells, can be controlled, the waste of T cells can be effectively prevented. The input at an extremely high concentration ensures that the entering T cells are single, thereby improving the utilization rate of T cells.

[0106] In some embodiments, the second droplets are generated by applying negative pressure to the droplet generation chip.

[0107] In some embodiments, droplet injection is performed under a certain injection voltage condition.

[0108] In some embodiments, droplet injection is performed under high voltage conditions.

[0109] Preferably, droplet injection is performed under the conditions of 15 kHz frequency, 1 VPP voltage, and 200-fold voltage amplifier.

[0110] In some embodiments, the second droplets comprise an aqueous fluid in a carrier fluid.

[0111] In some embodiments, the carrier fluid comprises oil.

[0112] In some embodiments, the mixed phase is incubated under suitable conditions to allow cells in the first cell phase to interact with cells in the second cell phase.

[0113] In some embodiments, cell interactions are detected in a microfluidic system, and / or a first cell phase comprising a first droplet is prepared by a microfluidic system.

[0114] In some embodiments, the method further comprises the steps of injecting a lysate into the mixed phase by droplet injection and analyzing the first and second cell barcodes and intracellular mRNA released from the surfaces of the SD cells and the second cells via cell lysis.

[0115] Injecting the lysate into the mixed phase can lyse the cells in the second droplet, thereby releasing the cell barcodes on the cell surface and the mRNA in the cells, facilitating the subsequent capture of the RNA capture magnetic beads.

[0116] Any conventional lysis solution in the art can be used, as long as the lysis solution can break up and lyse cells to release their genome.

[0117] In some embodiments, the methods are used to detect rare cell interactions or screen for functional antigens or T cell receptors.

[0118] In some embodiments, the method further comprises the steps of constructing a cDNA library and performing sequencing.

[0119] Through a series of RT-PCR and PCR reactions, the captured mRNA is first converted into DNA and amplified from a small amount to a large amount, and finally a stable single-stranded circular DNA is obtained to meet the sequencing requirements.

[0120] Preferably, the sequencing is next generation sequencing.

[0121] In some embodiments, the method further comprises the step of analyzing the functional antigen and its specific TCR.

[0122] In some embodiments, the method further comprises analyzing the information of the corresponding functional antigen and TCR based on the sequencing results of the barcode to determine whether there is a match.

[0123] In some embodiments, the method further comprises determining whether the matched group has high expression of T cell activation-related genes, and whether the unmatched group satisfies low expression.

[0124] In some embodiments, the method further includes determining which tumor antigen is paired with which TCR to complete activation, thereby achieving screening of tumor antigens and their specific TCR sequences.

[0125] In some embodiments, the present invention can prepare up to millions of second droplets simultaneously, and independently complete cell interactions in the second droplets.

[0126] In a third aspect, the present invention provides a microfluidic system, the microfluidic system comprising:

[0127] (1) one or more first droplet generation elements for generating a first cell phase;

[0128] (2) injecting a first element with one or more droplets containing a second cell phase;

[0129] (3) a second droplet generation element for forming a mixed phase and cell interaction; and

[0130] (4) A second droplet injection element for containing the lysate.

[0131] In some embodiments, each first droplet generating element can generate one first droplet.

[0132] The first cell phase is a mixture of m kinds of first liquid droplets.

[0133] Each of the first droplet generation elements comprises a first aqueous phase, a second aqueous phase and an oil phase, wherein the first aqueous phase comprises a buffer solution containing microbeads, the second aqueous phase comprises a buffer solution containing one or more SD cells of the same type, and the oil phase contains droplet generation oil.

[0134] In some embodiments, each injected first element contains one second cell phase.

[0135] In some embodiments, the second droplet generating element is used to accommodate a second droplet, wherein the second droplet is formed by injecting the second cell phase into the first cell phase.

[0136] Each of the second droplets contains one or more SD cells of the same type and a second cell.

[0137] In some embodiments, the first cell phase includes one or more first droplets, each of the one or more first droplets contains a surface-displayed cell (SD cell) and does not contain other types of cells, the SD cell contains an external surface-displayed polypeptide and is labeled with a first barcode.

[0138] In some embodiments, the multiple first droplets respectively contain different types of surface displayed cells, the same SD cell includes the same antigen polypeptide or a nucleic acid sequence encoding the antigen polypeptide and is marked with the same first barcode, and different types of SD cells are respectively marked with different first barcodes.

[0139] In some embodiments, the same first droplet includes a single SD cell or multiple SD cells, preferably multiple SD cells, and the multiple SD cells are the same type of cells. Preferably, 2-6 SD cells are encapsulated in the first droplet; preferably, 2-4 SD cells are encapsulated in the first droplet.

[0140] In some embodiments, the SD cells are antigen presenting cells (APCs).

[0141] In some embodiments, the SD cells are tumor antigen presenting cells, such as T2 cells.

[0142] In some embodiments, the APC is a macrophage, a dendritic cell, or a B cell.

[0143] In some embodiments, the SD cell comprises an antigen polypeptide or a nucleic acid sequence encoding the antigen polypeptide. Preferably, the SD cell comprises a tumor antigen polypeptide or a nucleic acid sequence encoding the tumor antigen polypeptide.

[0144] In some embodiments, the SD cell comprises a nucleic acid sequence encoding an MHC sequence.

[0145] The MHC is MHC-I, MHC-II or MHC-III, preferably MHC-I.

[0146] Encapsulating a plurality of identical SD cells in one of the first droplets can increase the stimulation of SD cells on, for example, T cells and can also eliminate the influence of Poisson distribution.

[0147] Because each type of droplet is prepared separately, it is ensured that the first droplet prepared contains one or more cells of the same type.

[0148] In some embodiments, the first droplets are generated by applying negative pressure to the droplet generation chip.

[0149] In some embodiments, the first droplet further comprises microbeads, and the particle size of the microbeads is 0.5 μm to 500 μm.

[0150] In some embodiments, the microbeads are made of a polymer material, such as polystyrene.

[0151] In some embodiments, the microbeads are magnetic beads, which are magnetic or contain magnetic components.

[0152] In some embodiments, the microbeads have biomolecules attached to their surface, such as antibodies, proteins, antigens, DNA, RNA, streptavidin, or a combination thereof.

[0153] In some embodiments, the microbeads are immunoassay particles, or are RNA capture beads.

[0154] In some embodiments, the SD cells are labeled with a first barcode using CASB.

[0155] According to the number of antigens to be detected, such as the number of types of tumor antigens, a corresponding number of groups of first droplets are generated, and then the corresponding number of groups of first droplets are mixed to form a first cell phase.

[0156] In some embodiments, the first droplet comprises an aqueous fluid in a carrier fluid.

[0157] In some embodiments, the carrier fluid comprises oil.

[0158] In some embodiments, the second cell phase includes one or more second cells, and different types of second cells are labeled with different second barcodes.

[0159] In some embodiments, the second cell comprises a T-cell receptor (TCR). Preferably, the second cell is selected from a T cell and a Jurkat cell containing a T-cell receptor (TCR). The Jurkat cell is a cell that reports a TCR.

[0160] In some embodiments, the second cell further comprises a nucleic acid encoding the TCR.

[0161] In some embodiments, each second cell contains or expresses only one TCR.

[0162] In some embodiments, the TCR is a chimeric antigen receptor.

[0163] In some embodiments, the TCR is a T-cell receptor endogenous to the T cell, or is a synthetic T-cell receptor.

[0164] In some embodiments, the second cell is labeled with a second barcode using CASB.

[0165] The same second cell is labeled with the same second barcode.

[0166] Different types of second cells are respectively labeled with different second barcodes.

[0167] In some embodiments, the second cell phase is injected into the first cell phase by droplet injection to form a mixed phase containing second droplets, and each of the second droplets contains less than or equal to 5 second cells; preferably, each of the second droplets contains only 1, 2, 3 or 4 second cells; more preferably, each of the second droplets contains only 1 second cell.

[0168] In some embodiments, each of the second droplets contains one or more SD cells of the same type and a second cell.

[0169] In some embodiments, the concentration of the second cells contained in the second cell phase is adjusted to ensure that the second cells injected via the droplets of the second cell phase are single cells.

[0170] In some embodiments, the plurality of second cells are injected into the first cell phase in sequence according to cell type by droplet injection to form a mixed phase containing second droplets.

[0171] Because the input amount of the second cell, especially T cells, can be controlled, the waste of T cells can be effectively prevented. The input at an extremely high concentration ensures that the entering T cells are single, thereby improving the utilization rate of T cells.

[0172] In some embodiments, the second droplets are generated by applying negative pressure to the droplet generation chip.

[0173] In some embodiments, droplet injection is performed under a certain injection voltage condition.

[0174] In some embodiments, droplet injection is performed under high voltage conditions.

[0175] Preferably, droplet injection is performed under the conditions of 15 kHz frequency, 1 VPP voltage, and 200-fold voltage amplifier.

[0176] In some embodiments, the second droplets comprise an aqueous fluid in a carrier fluid.

[0177] In some embodiments, the carrier fluid comprises oil.

[0178] In some embodiments, the mixed phase is incubated under suitable conditions to allow cells in the first cell phase to interact with cells in the second cell phase.

[0179] In the droplet injection second element, injecting the lysate into the mixed phase can lyse the cells in the second droplet, thereby releasing the cell barcode on the cell surface and the mRNA in the cell, which is convenient for subsequent capture by RNA capture magnetic beads.

[0180] Any conventional lysis solution in the art can be used, as long as the lysis solution can break up and lyse cells to release their genome.

[0181] In a fourth aspect, the present invention provides the method of the first aspect, the method of the second aspect or the microfluidic system of the third aspect for screening cross-reactions and off-target recognition of candidate antigens and TCRs in immunotherapy; or for high-throughput screening of new antigen-specific TCRs in the peripheral blood of cancer patients.

[0182] Beneficial Effects

[0183] 1. Improved throughput: The method of the present invention can prepare millions of droplets at the same time with the help of droplet microfluidics technology. Different types of cells independently interact with each other in the droplets to form pairs. At the same time, combined with single-cell sequencing, the pairing relationship is analyzed from the cell omics level, and the throughput is significantly improved compared to traditional pairing detection methods.

[0184] 2. Time reduction: Compared with all current conventional methods, the method of the present invention can significantly shorten the screening time (approximately 1 / 3 of the original time).

[0185] 3. Improve the efficiency of cell interaction, for example, it can realize the screening of weakly stimulating tumor antigens and low-affinity TCRs: by separately forming droplets containing only SD cells such as APC cells, the method of the present invention can control the concentration of the APC cell phase when the droplets are generated to achieve one droplet to encapsulate multiple identical APC cells, thereby increasing the subsequent APC stimulation of T cells, so as to realize the screening of weakly stimulating tumor antigens and low-affinity TCRs, and improve the screening sensitivity and accuracy.

[0186] 4. Improve cell utilization: For rare cells, such as primary T cells, which are scarce in source, studying cell interactions within a limited sample size and improving cell utilization are issues that need to be addressed urgently in this field. For example, if the second cell phase is T cells, by adjusting the cell concentration of the cell phase and injecting it into droplets containing SD cells, the utilization rate of primary T cells can be guaranteed to be as high as possible, effectively eliminating the waste of T cells.

[0187] 5. More potential for clinical application and transformation: The method of the present invention identifies the interaction between TCR and pMHC based on the biological phenomenon of cell-cell interaction. The results are more accurate and reliable, and more potential for clinical application and transformation.

[0188] 6. The influence of Poisson distribution of cell interactions at the single-cell level based on microfluidics is eliminated, and the cell utilization rate of the first cell phase and the second cell phase is improved. DETAILED DESCRIPTION

[0189] A "barcode" is a label or identifier that conveys or is capable of conveying information (e.g., information about an analyte, bead, and / or capture probe in a sample). In this application, barcodes are used interchangeably with cell labels. A barcode can be part of an analyte or can be independent of the analyte. A barcode can be attached to an analyte. A particular barcode may be unique relative to other barcodes.

[0190] Barcodes can have a variety of different formats. For example, barcodes can include polynucleotide barcodes, random nucleic acid and / or amino acid sequences, and synthetic nucleic acid and / or amino acid sequences. Barcodes can be attached to an analyte or another part or structure in a reversible or irreversible manner. For example, a barcode can be added to a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before or during sequencing the sample. Barcodes can allow identification and / or quantification of individual sequencing reads (e.g., a barcode can be or can include a unique molecular identifier or "UMI").

[0191] The T cell receptor (TCR) is a molecule present on the surface of T cells that is generally responsible for recognizing antigen fragments as peptides bound to major histocompatibility complex (MHC) molecules. TCRs are typically heterodimers of two chains, each of which is a member of the immunoglobulin superfamily, with an N-terminal variable (V) domain and a C-terminal constant domain. In humans, in 95% of T cells, the TCR is composed of α and β chains, while in 5% of T cells, the TCR is composed of γ and δ (γ / δ) chains. This ratio varies during individual development, in disease states, and in different species. When the TCR binds to an antigenic peptide and MHC (peptide / MHC or pMHC), the T lymphocyte is activated through signal transduction.

[0192] The term "antigen presenting cell" or "APC" includes a heterogeneous population of immunocompetent cells that mediate a cellular immune response by processing and presenting antigens to T cells. Antigen presenting cells include, but are not limited to, macrophages, dendritic cells, Langerhans cells, B cells, monocyte-derived dendritic cells, artificial APCs, engineered APCs, or other cells expressing MHC class I or MHC class II molecules.

[0193] The APC may be a B cell, in particular an immortalized B cell, such as an Epstein-Barr virus (EBV) immortalized B cell.

[0194] "MHC" or "major histocompatibility complex" refers herein to a complex of genes (and molecules encoded therein) encoding cell surface molecules required for antigen presentation to T cells and rapid transplant rejection. In humans, the MHC complex is also referred to as the HLA complex. The proteins encoded by the MHC complex are referred to as "MHC molecules" and are divided into class I and class II MHC molecules. Class I MHC molecules include membrane heterodimeric proteins consisting of α chains encoded in MHC that are non-covalently bound to β2 microglobulin. Class I MHC molecules are expressed by almost all nucleated cells and have been shown to play a role in antigen presentation to CD8+T cells. Class I molecules include HLA-A, HLA-B, and HLA-C in humans. Class II MHC molecules also include membrane heterodimeric proteins consisting of non-covalently bound α and β chains. Class II MHC is known to interact with CD4+T cells and includes HLA-DP, HLA-DQ, and HLA-DR in humans.

[0195] CASB is a sample encoding strategy (CASB) based on concanavalin A (Con A). Biotinylated Con A and ssDNA used for encoding are bridged with streptavidin (SA) to form a Con A-SA-ssDNA complex. The ability of Con A to bind to glycoproteins can be used to successfully fix the complex to cells / nuclei. Different ssDNA sequences can be designed according to different needs. When applied to sequencing, combined with scRNA-seq or snATAC-seq, it can reveal different transcriptome / epigenome dynamics.

[0196] The term "rare cells" refers to a type of cells that are rare in the blood but have important research value and clinical application value. They are usually associated with specific diseases, including stem cells, circulating endothelial cells, circulating tumor cells and rare immune cells.

[0197] More specifically, rare cells may include, but are not limited to, epithelial cells and their progenitors, mesenchymal cells and their progenitors, mature and immature endothelial cells and their progenitors, fibroblasts and their progenitors, melanocytes and their progenitors; monocytes, macrophages and their progenitors, activated lymphocytes and their progenitors, plasma cells and their progenitors, eosinophils and their progenitors, basophils and their progenitors, megakaryocytes and their progenitors; stem cells of any subtype; fetal cells of any origin and type, including lymphocytes, erythrocytes, myeloid cells, embryonic stem cells, trophoblasts (such as cytotrophoblasts and syncytiotrophoblasts) and embryonic cells; and tumor cells of any origin and type and any degree of differentiation, including tumor stem cells, tumor microemboli, aggregated tumor cells, collective tumor cells of any type, and atypical cells of any origin and type. For example, endothelial cells that are present in pathologically higher numbers in the blood of patients with cancer or cardiovascular diseases such as heart disease; cells carrying intracellular viruses, bacteria or other pathogens; cells carrying mutations associated with genetic diseases, etc.

[0198] The term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or spread to other parts of the body through the bloodstream and lymphatic system. The terms "tumor" and "cancer" are used interchangeably herein, for example, both terms cover solid tumors and liquid tumors, for example, diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes pre-malignant and malignant cancers and tumors. As used herein, the term "cancer" is meant to include all types of cancerous growth or carcinogenic processes, metastatic tissues or malignantly transformed cells, tissues or organs, regardless of the invasive histopathological type or stage. Examples of cancerous conditions include, but are not limited to, breast cancer, central nervous system cancer, endometrial cancer, kidney cancer, colorectal cancer, lung cancer, esophageal cancer, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, head and neck cancer (upper respiratory tract cancer), urinary tract cancer, colon cancer, PARP7 expression amplification cancer, etc. In some embodiments, cancers treatable according to the present invention include hematopoietic malignancies, such as leukemias and lymphomas. Exemplary lymphomas include Hodgkin's lymphoma or non-Hodgkin's lymphoma, multiple myeloma, B-cell lymphoma (e.g., diffuse large B-cell lymphoma (DLBCL)), chronic lymphocytic lymphoma (CLL), T-cell lymphoma, hairy cell lymphoma, and Burkitt's lymphoma. Examples of leukemias include acute lymphocytic leukemia (ALL), acute myeloid leukemia (AMIL), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML).

[0199] In a preferred embodiment, the method of the present invention uses injection to inject a single TCR-related cell into the APC cell phase. The stimulation effect of multiple identical APC cells on a single T cell is significantly better than that of a single APC cell on a single T cell, which significantly improves the efficiency of cell interactions and eliminates the influence of the Poisson distribution of cell interactions at the single-cell level based on microfluidics. This provides new possibilities for the screening of TCRs with weak responses but strong killing, and has obvious optimization in the amount of cell input and the effective utilization rate of cells. For precious samples, this method will be more advantageous, and this method has further improved the accuracy of screening (the number of corresponding activated genes matched) and stability (repeatability of results).

[0200] The following are preferred embodiments of the present invention, and the present invention is not limited to the following preferred embodiments. It should be noted that for those skilled in the art, several modifications and improvements made on the basis of this invention and creation concept all belong to the protection scope of the present invention. The reagents used without indicating the manufacturer are all conventional products that can be obtained through commercial purchase. BRIEF DESCRIPTION OF THE DRAWINGS

[0201] Figure 1 A schematic flow chart of the method of the present invention is shown.

[0202] Figure 2 Shown is a device for injecting TCR-associated cells into droplets.

[0203] Figure 3 It is shown that the use of the embodiments of the present invention can accurately match the expression of marker genes associated with Jurkat activation, and the use of cell barcodes can successfully separate positive and negative groups with strong reproducibility.

[0204] Figure 4 The results of the analysis of tumor antigen pairing with specific TCR are shown.

[0205] Example

[0206] The reagents used in the following examples are shown in Table 1.

[0207] Table 1: Reagents used in the examples of this disclosure

[0208]

[0209]

[0210] Example 1: Construction of cell lines

[0211] 1.1. Tumor antigen-loaded APCs

[0212] In this example, T2 cells were selected as APC cells, and two tumor antigens were loaded onto T2 cells respectively one day before the formal sorting experiment.

[0213] Specifically, take 2×10 6 The T2 cells were centrifuged, washed with PBS, and then added to 2 ml of the pre-prepared IMDM complete medium (containing 79% IMDM, 20% FBS, 1% PS), pipetted to mix and evenly divided into two wells of a 48-well plate (1 ml each), and the pre-synthesized tumor antigens: 1G4 and DMF5 (Beijing Liuhe BGI Co., Ltd, 10 μg / ml) were taken out, 2 μl of each was added to the two wells containing the cell suspension (one well for one antigen), pipetted to mix, and placed in a 37°C incubator for overnight incubation to complete the loading of tumor antigens.

[0214] 1.2. Construction of Jurkat cells with different TCR typing

[0215] Because primary T cells are very precious and not suitable for concept verification, Jurkat cells were selected in this example to replace primary T cells for concept verification, but the amount of cells input was exactly the same as that of primary T cells.

[0216] Specifically, the corresponding TCR sequence was first synthesized (BGI Liuhe synthesis), the TCR sequence was introduced into the plasmid by enzyme ligation for plasmid construction, and then the calcium phosphate transfection kit (Invitrogen TM , K278001) were used to transfect wild-type Jurkat cells to construct Jurkat reporter cells that could stably express the corresponding TCR sequence, and drug screening was performed using the resistance gene carried on the plasmid ring to ensure that the obtained Jurkat reporter cells could express TCR 100%.

[0217] Then, 1.5×10 Jurkat cells of different TCR types (1G4, TCR-5T-3, TCR-DMF5) were taken. 5 The cells were centrifuged, washed with PBS and resuspended in 500 μl 1640 complete medium (containing 89% RPMI 1640, 10% FBS, 1% PS), placed in a well plate, and incubated in a 37°C incubator overnight.

[0218] 1.3. Cell Barcode Marking

[0219] 16 kinds of cell barcodes (100bp ssDNA, Beijing Liuhe BGI Co., Ltd., 100nM) were synthesized in advance for use. First, 2μl of streptavidin was mixed with 8μl of cell barcode, reacted at room temperature for 10 minutes, and then 2μl of concanavalin A was added and mixed, and incubated at room temperature for 10 minutes to obtain the Barcode labeling mixture. The cells treated with overnight incubation in 1.1 and 1.2 were removed, centrifuged and discarded, and then resuspended in 1ml PBS, and 10μl of the above-prepared Barcode labeling mixture was added and placed in a 4℃ refrigerator for 10 minutes before being removed, and then washed twice with PBS for use. Different types of T2 cells and T cells were respectively equipped with different cell labels to facilitate the subsequent single-cell sequencing data to split out which types of cells are contained in the droplets.

[0220] Example 2: Cell-cell pairing using microfluidics

[0221] 2.1 Preparation of Working Buffer

[0222] Prepare the working buffer according to the recipe in Table 2 below.

[0223] Table 2: Working buffer formulation

[0224] Reagent Name Volume (1ml) Volume (5ml) Volume (10ml) IMDM medium 0.605 3.0250 6.05 1M HEPES 0.025 0.1250 0.25 FBS 0.05 0.2500 0.5 100X Pen / strep 0.01 0.0500 0.1 20% ficoll 0.3 1.5000 3 10% Pluronic F68 0.01 0.0500 0.1

[0225] 2.2 APC cell droplet generation

[0226] Two types of T2 cells were cultured at a rate of 1×10 7 / ml were resuspended in the configured working buffer to prepare 100μl; 350,000 RNA capture magnetic beads (homemade for bare magnetic beads) were resuspended in 100μl working buffer to prepare two groups, and then two droplet generation chips (Dropchip droplet generation chips) were used to generate two APC cell droplets respectively, and they were mixed evenly. Specifically, on one chip, a working buffer containing magnetic beads was added to an aqueous phase, a working buffer containing an APC cell was added to the other phase, and droplet generation oil was added to the oil phase. A 30ml syringe was used to pull from the scale line 19 to 20 to provide negative pressure for droplet generation. The same operation was performed on another APC cell on another chip, and finally the droplets of the two APC cells were mixed evenly ( Figure 1 :Step 1).

[0227] 2.3 Injection of TCR-associated cells into droplets

[0228] The Jurkat cells prepared previously were cultured at a rate of 3 × 10 6 / ml resuspended in working buffer, using the droplet injection chip ( Figure 2 , where from left to right are the oil phase, APC cell droplet phase, and T cell phase) from left to right, add 100μl of droplet-generated oil, 50μl of evenly mixed APC droplets, and 10μl of working buffer containing one of the Jurkat cells. Use a 30ml syringe to pull from the scale line 17.5 to 20 to provide negative pressure, and adjust the injection voltage (frequency: 15kHZ; voltage: 1VPP; voltage amplifier: 200 times). Then replace 10μl of the working buffer containing the second type of Jurkat cells, continue the injection, and then replace 10μl of the working buffer containing the third type of Jurkat cells. After all are injected into the APC cell droplets, the injection is completed ( Figure 1 : Step 2). The injected droplets were placed in a 37°C incubator for incubation and taken out after 4 hours.

[0229] Example 3: Cell lysis and sequencing

[0230] 3.1 Injecting Lysis Solution into Droplets

[0231] Using a droplet injection chip ( Figure 2 ) 100 μl of droplet generation oil, 30 μl of the above incubated droplets and 100 μl of lysis solution containing 10% RNase inhibitor (50% 1M Tris-HCl, 20% 0.5M EDTA, 25% 20% Ficoll PM-400, 5% 20% Sarkosyl) were added from left to right, and negative pressure was provided by pulling a 30 ml syringe from the scale line 17.5 to 20, and the injection voltage was adjusted (frequency: 15 kHz; voltage: 1 VPP; voltage amplifier: 200 times) to complete the injection ( Figure 1 : Step 3). Incubate at room temperature for 30 minutes until the cells are fully lysed and the magnetic beads are fully captured.

[0232] 3.2 Single-cell library construction and next-generation sequencing

[0233] Demulsification was performed to recover magnetic beads, and reverse transcription reaction, second-strand synthesis, PCR amplification, cDNA and droplet index product purification, droplet index product secondary amplification and purification, cDNA shearing construction were performed in sequence to obtain a purified library. The single-cell sequencing kit made by MGI: DNBelab C4 and portable single-cell system (MGI, 940-00050) were used according to the supporting C4 scRNA-seq-V2 library construction and sequencing operation process ( Figure 1 : Step 4).

[0234] 3.3 Processing and analysis of sequencing results

[0235] First, the data containing only two label information is filtered out, and then the cell barcode (sequence is known) is used to analyze which two cells are interacting, and the corresponding information of tumor antigen and TCR is returned to determine whether they match. Then, check whether the matched group has high expression of T cell activation-related genes, and whether the unmatched group meets the low expression ( Figure 3 ), and then subdivide the paired groups to see if we can separate which tumor antigen is paired with which TCR to complete the activation ( Figure 4 ), thereby achieving the screening of tumor antigens and their specific TCR sequences.

[0236] Specifically, Figure 3 The results of testing two types of tumor antigen peptides (1G4, DMF5) and three types of Jurkat cells (1G4, DMF5 and 5T-3) using the method and screening platform of the present invention are shown. At the functional level, it was found that TCR and tumor antigen peptides were specifically recognized. Figure 3 The results show that the platform of the present invention can accurately match the expression of marker genes related to Jurkat activation, and successfully separate the positive group from the negative group using cell barcodes. Subsequent studies have proved the repeatability of this method and platform. Due to the limitations of the second generation sequencing, Figure 3 The three genes missing in the second group of experiments may not have been found. The positive group was further divided into 1G4, DMF5 and 5T-3 according to the cell barcode markers, and the expression of their respective marker genes was detected. Figure 4 As shown, the marker genes of the three positive groups were activated to varying degrees, further confirming the accuracy of the platform. The same results can still be obtained by comparing the expression of barcode and marker genes in parallel experiments.

[0237] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0238] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0239] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A method for detecting cell interactions, the method comprising: (1) preparing a first cell phase comprising a first droplet, wherein the first droplet comprises a surface-displayed cell (SD cell) and does not contain other types of cells, wherein the SD cell comprises an external surface-displayed polypeptide and is labeled with a first barcode; (2) preparing a second cell phase, wherein the second cell phase includes second cells and is labeled with a second barcode; as well as (3) Injecting the second cell phase into the first cell phase by droplet injection to form a mixed phase containing the second droplet.

2. The method of claim 1, wherein: The first cell phase includes one or more first droplets, each of the one or more first droplets contains a surface display cell (SD cell) and does not contain other types of cells, the SD cell contains an external surface display polypeptide and is labeled with a first barcode; Preferably, the plurality of first droplets respectively contain different types of surface display cells, the same type of SD cells include the same antigen polypeptide or a nucleic acid sequence encoding the antigen polypeptide, and are marked with the same first barcode, and different types of SD cells are respectively marked with different first barcodes; Preferably, the same first droplet includes a single SD cell or multiple SD cells, preferably multiple SD cells, and the multiple SD cells are the same type of cells. Also preferably, 2-6 SD cells are encapsulated in the first droplet; preferably, 2-4 SD cells are encapsulated in the first droplet.

3. The method of claim 1 or 2, wherein the second cell phase comprises one or more second cells, Different types of second cells are labeled with different second barcodes.

4. A method as claimed in any one of the preceding claims, wherein: Injecting the second cell phase into the first cell phase by droplet injection to form a mixed phase containing a second droplet, wherein the second droplet contains less than or equal to 5 second cells; Preferably, the second droplet contains only 1, 2, 3 or 4 second cells; More preferably, the second droplet contains only one second cell; Preferably, the plurality of second cells are injected into the first cell phase in sequence according to cell types by means of droplet injection to form a mixed phase containing second droplets.

5. A method as claimed in any one of the preceding claims, wherein: The first liquid droplets further include micro beads, and the particle size of the micro beads is 0.5 μm to 500 μm.

6. The method of claim 5, wherein: The microbeads are made of a polymer material, such as polystyrene; or the microbeads are magnetic beads, which are magnetic or contain magnetic components; or the microbeads have biological molecules attached to their surfaces, such as antibodies, proteins, antigens, DNA, RNA, streptavidin or a combination thereof; or the microbeads are immunoassay particles, or are RNA capture beads.

7. The method according to any of the preceding claims, further comprising generating a corresponding number of groups of first droplets according to the number of types of antigens to be detected, and then mixing the corresponding number of groups of first droplets to form the first cell phase.

8. A method as claimed in any one of the preceding claims, wherein: The first droplet or the second droplet comprises an aqueous fluid in a carrier fluid, preferably the carrier fluid comprises oil.

9. The method of any one of the preceding claims, further comprising adjusting the concentration of the second cells contained in the second cell phase to ensure that the second cells injected via the droplets of the second cell phase are single cells.

10. A method as claimed in any preceding claim, wherein: Each of the second droplets contains one or more SD cells of the same type and a second cell.

11. The method of any of the preceding claims, further comprising incubating the mixed phase under suitable conditions to allow cells in the first cell phase to interact with cells in the second cell phase.

12. The method as claimed in any of the preceding claims, further comprising the step of injecting a lysis solution into the mixed phase by droplet injection, and the step of analyzing the first and second cell barcodes and intracellular mRNA released from the surface of the SD cells and the second cells via cell lysis.

13. The method of claim 12, wherein the analyzing step comprises screening out data containing only two types of barcode information, and then using the sequence to analyze which two cells are interacting.

14. A method as claimed in any preceding claim, wherein: The first and / or second barcodes are labeled with CASB.

15. A method as claimed in any preceding claim, wherein: The SD cells are antigen presenting cells (APCs), preferably tumor antigen presenting cells, such as T2 cells; and / or The APC is a macrophage, a dendritic cell or a B cell.

16. A method as claimed in any preceding claim, wherein: The SD cells comprise an antigen polypeptide or a nucleic acid sequence encoding the antigen polypeptide, preferably, the antigen polypeptide is a tumor antigen polypeptide; and / or Wherein, the SD cell comprises a nucleic acid sequence encoding an MHC sequence, preferably, the MHC is MHC-I, MHC-II or MHC-III, preferably MHC-I.

17. A method as claimed in any preceding claim, wherein: The second cell phase includes one or more second cells comprising a T-cell receptor (TCR), such as T cells or Jurkat cells containing a T-cell receptor (TCR).

18. A method as claimed in any preceding claim, wherein: The second cell further comprises a nucleic acid encoding the TCR.

19. A method as claimed in any preceding claim, wherein: Each of the second cells contains or expresses only one TCR.

20. The method according to any one of the preceding claims, wherein cell interactions are detected in a microfluidic system, and / or a first cell phase comprising a first droplet is prepared by a microfluidic system.

21. The method of any preceding claim, wherein the method is used to detect rare cell interactions or to screen for functional antigens or T cell receptors.

22. A microfluidic system for performing the method of any one of claims 1 to 21, the microfluidic system comprising: (1) m first droplet generation elements for generating a first cell phase; (2) a first element for injecting a droplet containing a second cell phase; (3) a second droplet generation element for forming a mixed phase and cell interaction; as well as (4) A second droplet injection element for containing the lysate.

23. The microfluidic system of claim 22, comprising one or more droplet injection first elements for containing a second cell phase, each injection first element containing a second cell phase.

24. The microfluidic system according to claim 22 or 23, wherein the m first droplet generating elements are capable of generating m types of first droplets, and preferably, the first cell phase is a mixture of the m types of first droplets.

25. The microfluidic system of any one of claims 22 to 24, wherein each of the first droplet generating elements comprises a first aqueous phase, a second aqueous phase and an oil phase, the first aqueous phase comprises a buffer containing microbeads, the second aqueous phase comprises a buffer containing one or more SD cells of the same type, and the oil phase contains droplet generating oil.

26. The microfluidic system according to any one of claims 22 to 25, wherein the second droplet generating element is used to contain a second droplet, wherein the second droplet is formed by injecting the second cell phase into the first cell phase.

27. The microfluidic system of any one of claims 22 to 26, wherein the second droplet generation element comprises an aqueous phase and an oil phase before forming the second droplet, the aqueous phase comprising the first droplet and a second cell buffer, and the oil phase contains droplet generation oil.

28. Use of the method of any one of claims 1 to 21, or the microfluidic system of any one of claims 22 to 27 in screening cross-reaction and off-target recognition of candidate antigens and TCRs in immunotherapy; or use of the microfluidic system in high-throughput screening of new antigen-specific TCRs in the peripheral blood of cancer patients.

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