Method for detecting intercellular interaction and application thereof

Abstract

A method for detecting intercellular interaction, the method comprising: (1) preparing a first cell expressing a TCR activation response element and a reporter molecule, the first cell expressing TCR; (2) incubating the antigen peptide and a second cell expressing MHC to obtain a second cell loaded with the antigen peptide; (3) marking the first cell by using a first tag to obtain a first cell carrying the tag, and marking the second cell loaded with the antigen peptide by using a second tag to obtain a second cell carrying the tag; (4) wrapping the first cells carrying the labels, the second cells carrying the labels and the magnetic beads with an oil phase to prepare a plurality of liquid drops; (5) injecting a cell lysis solution into the single liquid drop, and sequencing the nucleic acid after lysis; and (6) determining whether the first cell and the second cell in the single liquid drop interact or not according to a sequencing result.

Description

Methods for detecting cell-cell interactions and their applications Technical Field

[0001] The present invention belongs to the field of biotechnology. More specifically, the present invention provides a method for high-throughput detection of cell-to-cell interactions and its application. Background Art

[0002] In recent years, with the rapid development of tumor immunotherapy research, chimeric antigen receptor T cell therapy (CAR-T) has shown good results in the treatment of hematological tumors. However, due to challenges such as the lack of high-expression antigens specific to solid tumors, the tumor immunosuppressive microenvironment, and endogenous inhibitory signals of T cells, it has been difficult to treat solid tumors.

[0003] TCR-T cell (T Cell Receptor-gene engineered T cell) therapy is also one of the commonly used methods for tumor immunotherapy. Its technical process is: 1) Screening suitable tumor-specific antigens and TCRαβ chain sequences that are highly specific to the antigen; 2) Isolating T cells from the patient's body; 3) Packaging the above-mentioned TCRαβ chain sequence that can recognize tumor-specific antigens into the viral genome to construct a viral vector; 4) Virus transfection of T cells, introducing the target TCR sequence into T cells, and obtaining TCR-T cells that can specifically recognize tumor antigens; 5) TCR-T cells are cultured in vitro and expanded in large quantities; 6) A qualified number of TCR-T cells are injected into the patient's body for treatment.

[0004] Currently, TCR-T technology, as the most primitive cell therapy, has high technical barriers. First, compared to CAR-T, TCR-T has lower antigen affinity. Second, its clinical application is limited by the impact of off-target effects. With the development of neoantigen identification technology, tumor-specific antigens generated by high-frequency tumor mutations have become an important breakthrough in addressing off-target effects. However, TCRs in the human body generally undergo affinity screening in the thymus, and tumor neoantigens are not present in the normal population (non-diseased people), making it even more difficult to find specific TCRs for tumor neoantigens in the human body. In addition, due to the high mutation potential of tumors, it is difficult to find ubiquitous tumor-specific antigens at the population level. At the individual level, there is also heterogeneity within tumors. It is difficult to avoid the occurrence of tumor resistance by only generating T cells for a single antigen. Therefore, the large difference in antigen types between individuals and the heterogeneity within individual tumors lead to the diversity of antigen types. How to efficiently find specific TCRs for a wide variety of tumor antigens is a current technical difficulty.

[0005] In terms of obtaining specific TCRs, the main strategies currently used in conventional T cell therapy are: identifying one or more tumor neoantigens as therapeutic targets, synthesizing MHC peptides through data processing, and then constructing pHLA tetramers for recognition; or utilizing the functions of tumor neoantigen-specific T cells for determination, such as identifying reactive T cells with the help of secreted cytokines and surface protein expression, that is, measuring IFNγ levels through ELISA, ELISPOT, and intracellular cytokine staining followed by FACS analysis. In T-cell therapy combined with single cells, the main strategy adopted is to culture polyclonal tumor-infiltrating lymphocytes (TILs) from fresh tumor fragments and screen them against tandem minigenes (TMGs) or peptide libraries to identify neoantigen-reactive TIL populations; then, the TIL population is co-cultured with neoantigen-transfected / pulsed antigen-presenting cells (APCs) (for example, using the Fluidigm C1 system), and high-throughput deep measurement is performed regardless of whether each reaction chamber contains a single cell; finally, single-cell RNA-seq samples with high expression of T cell activation markers, such as IFN-γ and IL-2, are identified through sequencing data, and paired TCRα / β chain sequences are identified from these samples.

[0006] However, these two methods are time-consuming, labor-intensive, and inefficient. It takes approximately 3-5 months to produce a highly personalized TCR therapy cell product targeting neoantigens that meets the Good Manufacturing Practice (GMP) level, and the pairing success rate is low. Existing single-cell screening methods are often performed in the Fluidigm C1 system, which can process up to 96 samples at a time, with obvious throughput limitations. Existing successful cases can only identify approximately 1-6 TIL cultures containing neoantigen-reactive T cells out of 24 TIL cultures generated from tumor specimens, and the consumption of manpower and material resources is still very large. Therefore, it is particularly important to develop new methods that can detect cell-cell interactions.

[0007] Summary of the Invention

[0008] The purpose of the present invention is to screen TCR-T corresponding to APC in a high-throughput manner. The present invention can quickly and conveniently screen the interacting cells.

[0009] In a first aspect, the present invention provides a method for detecting cell-cell interaction, the method comprising the steps of:

[0010] (1) preparing a first cell expressing a TCR activation response element and a reporter molecule, wherein the TCR activation response element is located upstream of the reporter molecule, and the first cell expresses TCR;

[0011] (2) incubating the antigen peptide with a second cell expressing MHC, so that the antigen peptide is loaded onto the MHC of the second cell to form an antigen peptide-MHC complex, thereby obtaining a second cell loaded with the antigen peptide;

[0012] (3) labeling the first cell with a first tag sequence to obtain a first cell carrying the tag sequence, and labeling the second cell loaded with the antigen peptide with a second tag sequence to obtain a second cell carrying the tag sequence, wherein the first tag sequence and the second tag sequence contain the same common sequence, and the first tag sequence and the second tag sequence contain different specific sequences;

[0013] (4) using an oil phase to encapsulate the labeled first cell, the labeled second cell, and the magnetic beads to prepare a plurality of droplets, wherein a single droplet of at least a portion of the plurality of droplets contains only one first cell, one magnetic bead, and at least one second cell, and the surface of the magnetic bead carries a first capture sequence, a second capture sequence, and a unique coding sequence, wherein the first capture sequence is used to capture a cell target sequence, the second capture sequence is complementary to a common sequence in a cell label sequence, and is used to capture the cell label sequence, and the unique coding sequence of each magnetic bead is different from each other and is used to identify the identity of the magnetic bead;

[0014] (5) injecting a cell lysis solution into a single droplet, lysing the cells, releasing and capturing the cell target sequence and the cell tag sequence through the magnetic beads, and sequencing the sequences obtained after capture to obtain sequencing data;

[0015] (6) Analyzing sequencing data containing the same unique coding sequence to determine whether there is interaction between the first cell and the second cell in the single droplet.

[0016] In one embodiment, in (5), the sequences obtained after capture are amplified and library constructed, and the obtained sequencing library is sequenced.

[0017] In one embodiment, in (5), the sequence obtained after capture comprises mRNA and DNA, the mRNA is subjected to reverse transcription, double-strand synthesis, cDNA product amplification, and library construction is performed on the amplified cDNA product to obtain a first sequencing library; the DNA is subjected to secondary amplification and fragment screening to obtain a second sequencing library; and the first and second sequencing libraries are combined for sequencing.

[0018] In one embodiment, in (1)-(3), a plurality of the first cells are prepared for expressing different TCRs, and a plurality of the second cells are prepared for loading different antigen peptides; the plurality of first cells are respectively labeled with a plurality of different first tag sequences, and the plurality of second cells are respectively labeled with a plurality of different second tag sequences.

[0019] In one embodiment, in (1), the first cell is a T cell, preferably a Jurkat cell or a human-derived T lymphocyte.

[0020] In one embodiment, in (1), a TCR lentiviral vector and a reporter molecule vector are constructed and co-transfected with the TCR sequence into a first cell to prepare a first cell expressing the TCR and the reporter molecule.

[0021] In one embodiment, in (1), the TCR activation response element is nuclear factor of activated T cells (NFAT), AP1 transcription factor (AP1) or NF-κB.

[0022] In one embodiment, in (1), the reporter molecule is a fluorescent protein, preferably green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP) or blue fluorescent protein (BFP).

[0023] In one embodiment, in (2), the second cell is an antigen presenting cell, preferably a T2 cell or a K562 cell.

[0024] In one embodiment, in (2), an MHC lentiviral vector is constructed and transfected into the second cell to prepare a second cell expressing MHC.

[0025] In one embodiment, in (2), the antigenic peptide is a tumor-specific antigenic peptide, preferably a NY-ESO-1 polypeptide with a sequence of SLLMWITQC or a MART-1 polypeptide with a sequence of LAGIGILTV. (27-35) polypeptide.

[0026] In one embodiment, in (3), the first cell and the second cell are labeled using a CASB strategy.

[0027] In one embodiment, the first tag sequence and the second tag sequence are both DNA sequences.

[0028] In one embodiment, the DNA sequence is 20-200 bp in length, preferably 100 bp in length.

[0029] In one embodiment, in (4), the droplets are prepared using droplet microfluidics technology.

[0030] In one embodiment, in (4), a droplet generation chip is used to add the first cells and magnetic beads carrying the label, and the second cells and magnetic beads carrying the label into two sample wells, respectively.

[0031] In one embodiment, in (4), by adjusting the concentrations of the labeled first cells and the labeled second cells, a single droplet of at least a portion of the plurality of droplets contains only one first cell and at least one second cell.

[0032] In one embodiment, the first cell and the second cell are added at a concentration that maximizes the probability of a single cell being present in the droplet.

[0033] In one embodiment, an incubation step is included after (4) droplet generation, and the incubation time is preferably 4-8 hours.

[0034] In one embodiment, in (5), the lysate injection is performed using a droplet injection device.

[0035] In one embodiment, in (6), sequencing data containing the same unique coding sequence and having two cell tag sequences are screened out, and based on the reporter molecule expression level of the first cell in the sequencing data, it is determined whether specific recognition occurs between the TCR expressed by the first cell and the MHC-antigen peptide complex of the second cell. If specific recognition occurs, it is determined that there is interaction between the first cell and the second cell in a single droplet.

[0036] In one embodiment, in (6), it is determined that the first cell and the second cell interact with each other, and the cell tag sequence in the sequencing data is further used to obtain the corresponding first cell and second cell.

[0037] In one embodiment, the interaction includes any one or more of cell-to-cell communication through secretion of chemical signals, contact-dependent communication between cells, and formation of gap junctions between adjacent cells in an animal.

[0038] In a second aspect, the present invention provides an application of the method of the first aspect of the present invention in high-throughput screening of antigens and their paired T cell receptors, wherein the first cell is a T cell expressing a TCR, and the second cell is an APC cell loaded with an antigen peptide. There are multiple types of T cells for expressing different TCRs, and there are multiple types of APC cells for loading different antigen peptides; the multiple T cells are respectively labeled with multiple different first tag sequences, and the multiple APC cells are respectively labeled with multiple different second tag sequences.

[0039] In one embodiment, the TCR is a tumor-specific T cell receptor.

[0040] In a third aspect, the present invention provides a method for high-throughput screening of antigens and their paired T cell receptors, comprising using the method of the first aspect of the present invention to screen antigens and their paired T cell receptors, wherein the first cell is a T cell expressing a TCR, and the second cell is an APC cell loaded with an antigen peptide, and the T cells are of multiple types and are used to express different TCRs, and the APC cells are of multiple types and are used to load different antigen peptides; the multiple T cells are respectively labeled with multiple different first labels, and the multiple APC cells are respectively labeled with multiple different second labels.

[0041] The present invention relies on droplets encapsulating antigen-presenting cells loaded with antigen peptides, T cells expressing different types of TCRs, and magnetic beads. By adding different labels to each cell and using the unique coding sequence on the magnetic beads to distinguish which sequences obtained after sequencing come from the same droplet. The present invention incubates the cells in the droplets after droplet generation and before the addition of lysis solution to provide time for cell-cell interactions. The present invention can not only be used to study interactions between single cells, but also achieve high-throughput detection of multiple antigens and their TCR-specific pairing relationships. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings involved in the embodiments. Obviously, the drawings described below are only some of the embodiments of the present invention. For those skilled in the art, other implementation schemes can be obtained based on these drawings without inventive efforts.

[0043] FIG1 shows a schematic diagram of a method according to one embodiment of the present invention.

[0044] FIG2 shows a physical object (A) and a principle design diagram (B) of a droplet injection device according to one embodiment of the present invention.

[0045] FIG3 shows a diagram of analysis results of sequencing data according to one embodiment of the present invention. DETAILED DESCRIPTION

[0046] The present invention will be described clearly and completely below. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention described in this application, all other embodiments that can be obtained by a person of ordinary skill in the art are within the scope of protection of the present invention.

[0047] The current single-cell sequencing technology cannot provide time for cell interactions due to the direct introduction of lysis solution, which causes cell lysis, and most droplets contain only one cell, making it impossible to study cell interactions. The present invention achieves the encapsulation of multiple cells in a droplet by labeling cells; and when generating droplets, the lysis solution is not directly introduced, but after a period of time, the lysis solution is added by droplet injection, thereby providing time conditions for the occurrence of cell interactions. The present invention uses droplet microfluidics technology to prepare millions of droplets, and cells can independently complete interactions within the droplets. At the same time, combined with single-cell sequencing, the pairing relationship is analyzed from the omics level. Compared with traditional pairing methods, the throughput of the present invention is significantly improved. The present invention innovatively introduces oligonucleotide sequences to label antigen-presenting cells, and accurately determines the new antigen and the TCR information that has a pairing relationship with the new antigen from the single-cell level. Therefore, the scheme of the present invention is particularly suitable for high-throughput blind screening and functional verification of tumor neoantigens and their corresponding TCR sequences.

[0048] In this article, the term "interaction" or "interaction" generally refers to the interaction or interaction between cells, which can be understood as cell communication, that is, the signal substance emitted by one cell is transmitted to another target cell through a medium and combines with its corresponding receptor, producing a series of physiological and biochemical changes in the target cell through cell signal transduction, and ultimately manifesting as the overall biological effect of the target cell. Cell interaction mainly includes three categories: (1) Cells communicate through the secretion of chemical signals; (2) Cell-to-cell contact-dependent signaling, direct contact between cells, through the interaction of transmembrane signal molecules (ligands) of signal cells with receptors on the surface of adjacent target cells; (3) gap junctions are formed between adjacent cells in animals, and metabolic coupling or electrical coupling is achieved by exchanging small molecules, thereby achieving functional regulation. Among them, the secretion of chemical signals can be divided into three different ways: endocrine, paracrine, and transmission of neural signals through chemical synapses.

[0049] As used herein, the term "T cell receptor," or TCR, refers to a molecule or complex of molecules on a T cell that is capable of receiving signals and transmitting them across the cell membrane.

[0050] As used herein, the term "antigen specificity" refers to the property of TCR that it can specifically bind to and recognize its corresponding antigen or antigenic epitope, thereby inducing an immune response.

[0051] As used herein, the term "major histocompatibility complex" or "MHC" refers to a group of tightly linked genes present on a chromosome in most vertebrates (e.g., for mice, on chromosome 17; for humans, on chromosome 6), whose encoded products (major histocompatibility antigens) are closely related to specific immune responses, immune regulation, and transplant rejection. The MHC in mice is called the H-2 complex, and the MHC in humans is called human leukocyte antigen (HLA). In addition to H-2 in mice and HLA in humans, corresponding MHCs also exist in other animals, such as rabbit leukocyte antigen (RLA), guinea pig leukocyte antigen (GPLA), house leukocyte antigen (SLA), dog leukocyte antigen (DLA), or monkey leukocyte antigen (RhLA). MHC can be divided into three types of gene groups, namely MHC-I, MHC-II, and MHC-III. For humans, MHC-I class includes three gene sites, namely A, B, and C, and its encoded products are MHC-I class molecules or antigens; MHC-II class includes three subregions, DP, DQ, and DR, and its encoded classic products are MHC-II class molecules or antigens, as well as LMP and TAP related to endogenous antigen processing; MHC III class genes encode MHC-III class molecules or antigens.

[0052] In this context, antigen-presenting cells (APCs) are cells in the body that have the ability to absorb, process, and transmit antigen information, inducing immune responses from T and B cells. APCs engulf external pathogens such as bacteria and parasites, digest them in lysosomes within the cell, and form peptide fragments that form complexes with MHC-II molecules and are presented on the cell surface. APCs primarily include macrophages, dendritic cells, synaptic cells, Langerhans cells, and B cells.

[0053] In one embodiment, the TCR activation response element can be nuclear factor of activated T cells (NFAT), AP1 transcription factor (AP1), or NF-κB. "Nuclear factor κB" or "NF-κB" is a highly conserved family of multifunctional transcription factors that includes five members: p65 (RelA), RelB, c-Rel, NF-κB1, and NF-κB2. They form different homo- or heterodimers to regulate gene transcription, thereby regulating many important cellular behaviors, particularly inflammatory responses.

[0054] As used herein, the term "nuclear factor of activated T cells" or "NFAT" (Nuclear Factor of Activated T cells) refers to a family of transcription factors that play an important role in inducing gene transcription in immune responses. In addition to T cells, this class of proteins is expressed on many immune cells, such as B lymphocytes, mast cells, and eosinophils, and their activity is regulated by the calcium-dependent enzyme calmodulin C. In the present invention, NFAT is a preferred TCR activation response element.

[0055] In the case where the corresponding element for TCR activation is NFAT, if the TCR can specifically recognize the MHC-antigen peptide complex, the structure of the TCR intracellular segment will change and recruit protein kinase K, including the ZAP70 protein. Downstream of the ZAP70 protein, the hydrolysis of phosphatidylinositol 4,5-bisphosphate produces inositol triphosphate, which binds to endoplasmic reticulum receptors, causing calcium ion release, thereby activating the transcription factor NFAT; when NFAT binds to the NFAT binding region upstream of the transfected NFAT-reporter sequence, the downstream reporter molecule expression gene will be transcribed and translated into a reporter molecule (for example, in one embodiment, GFP protein). Under laser excitation of an appropriate wavelength, the reporter molecule will emit a spectrum of a specific wavelength. A photomultiplier tube (PMT) can be used to capture the emitted light of the reporter molecule, thereby achieving the characterization and detection of antigen-specific TCRs.

[0056] In this article, the CASB (Concanavalin A-based Sample Barcoding) strategy was used for single-cell sequencing. This is a concanavalin A-based oligonucleotide sequence tagging technology that uses concanavalin A to label cells individually. The CASB strategy is described in "CASB: a concanavalin A-based sample barcoding strategy for single-cell sequencing." Mol Syst Biol 17(4):e10060(2021).

[0057] However, during sequencing, due to the limitations of current technology, it is usually only possible to obtain the 3' end sequence information of the cell, and it is impossible to obtain all the sequence information of the cell or the 5' end sequence information (the sequence information of TCR is mainly at the 5' end). Therefore, it is impossible to directly obtain the activation status of TCR and TCR sequence from the sequencing data. Here, the inventors introduced a reporter molecule at the 3' end to assist in judging whether the T cell is activated. In this article, the so-called "reporter molecule" refers to a substance that can indicate the reaction between TCR and MHC-antigen peptide complex. The reporter molecule can be a class of visual reporter gene encoding proteins or fluorescent proteins, which can include: green fluorescent protein (GFP) and its mutants such as mGFP5 protein, EGFP protein, D2EGFP protein, etc.; red fluorescent protein (RFP), such as DsRed protein, DsRed-express protein, mRFP1 protein, mCherry protein, Kaede protein, etc.; yellow fluorescent protein (YFP); or blue fluorescent protein (BFP), etc. This type of protein can be excited to emit bright fluorescence at specific wavelengths. It is easy to detect, non-toxic to cells, has stable fluorescence, is species-specific, and requires no substrate. Therefore, it can be widely used in animal, plant, and fungal research. If a droplet is detected to emit fluorescence at a specific wavelength under laser excitation of a specific wavelength, it indicates that the reporter molecule is expressed, the TCR specifically recognizes the MHC-antigen peptide complex, and the first cell contains an antigen-specific TCR. If a droplet is detected to fail to emit fluorescence at a specific wavelength under laser excitation of a specific wavelength, it indicates that the reporter molecule is not expressed, the TCR does not specifically recognize the MHC-antigen peptide complex, and the first cell does not contain an antigen-specific TCR.

[0058] Thus, in one embodiment, the reporter molecule is a fluorescent protein, such as green fluorescent protein (GFP), red fluorescent protein (RFP), or yellow fluorescent protein (YFP), or blue fluorescent protein (BFP).

[0059] Herein, T lymphoma cells include, but are not limited to, Jurkat cells and human-derived T lymphocytes.

[0060] Herein, antigen presenting cells are capable of expressing MHC molecules, such as T2 cells and K562 cells, but are not limited thereto.

[0061] As used herein, the term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises cis-acting elements sufficient for expression; other elements for expression may be provided by a host cell or in an in vitro expression system. In the present invention, expression vectors may include all expression vectors known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses incorporating recombinant polynucleotides, such as lentiviruses, retroviruses, adenoviruses, herpes simplex viruses, and adeno-associated viruses, but are not limited thereto.

[0062] In one embodiment, the TCR expression vector, the MHC expression vector and the vector expressing the TCR activation response element and the reporter molecule are each independently a viral expression vector, such as a lentiviral expression vector, a retroviral vector, an adenoviral vector, a herpes simplex virus vector or an adeno-associated virus vector.

[0063] As used herein, the term "antigenic peptide" refers to a polypeptide that has an antigenic epitope and is therefore recognizable by a TCR. Furthermore, in the present invention, the term "antigen" generally refers to a tumor antigen, i.e., any molecule expressed or overexpressed solely or primarily by tumor cells or cancer cells, such as a protein, polypeptide, peptide, lipid, carbohydrate, etc. Tumor antigens can also be expressed by normal cells, non-tumor cells, or non-cancerous cells. However, in such cases, the expression of tumor antigens by normal cells, non-tumor cells, or non-cancerous cells is not as robust as the expression of tumor antigens by tumor cells or cancer cells. In this regard, tumor cells or cancer cells can overexpress antigens or express antigens at significantly higher levels compared to the expression of antigens by normal cells, non-tumor cells, or non-cancerous cells. Furthermore, tumor antigens can be expressed by cells in different developmental or maturational states. For example, tumor antigens can also be expressed by cells in the embryonic or fetal stage, which are not typically present in adult hosts. Alternatively, tumor antigens can also be expressed by stem cells or precursor cells, which are not typically present in adult hosts. Tumor antigens are known in the art and include, for example, mesothelin, CD19, CD22, CD276 (B7H3), gp100, MART-1, epidermal growth factor receptor variant III (EGFRVIII), TRP-1, TRP-2, tyrosinase, mutant KRAS, NY-ESO-1 (also known as CAG-3), MAGE-1, MAGE-3, etc., but are not limited thereto. In addition, tumor cells have chromosomal instability and abnormal epigenetic regulation (such as methylation), often producing mutant proteins or fusion proteins. Such proteins can become targets for immunotherapy and are referred to as tumor neoantigens.

[0064] In one embodiment, the antigenic peptide is a tumor antigenic peptide, such as the NY-ESO-1 polypeptide with the sequence SLLMWITQC (SEQ ID NO. 1), or the MART-1 polypeptide with the sequence LAGIGILTV (SEQ ID NO. 2). (27-35) Polypeptides, but not limited thereto. In one embodiment, the method of the present invention may comprise five steps: 1) cell line construction; 2) cell labeling by CASB strategy; 3) cell encapsulation and cell response using droplets; 4) droplet injection; 5) single cell sequencing; and 6) sequencing data analysis. The specific steps are described in Figure 1. Figure 1 shows a schematic diagram of a method according to one embodiment of the present invention. The steps are described below:

[0065] (1) Cell line construction. First, two cell lines are constructed. Those skilled in the art will understand that there is no order in which the two cell lines are constructed: 1) An MHC lentiviral vector is constructed, and the constructed MHC lentiviral vector is used to transfect APC cells (such as T2 cells) to prepare an MHC stably transfected APC cell line, and then the antigen peptide and the stably transfected APC cells are co-incubated to load the polypeptide onto the MHC of the APC cells; 2) A TCR lentiviral vector and an enhanced mCherry vector are constructed, and a TCR library is prepared. The prepared TCR library is used to transfect T cells (such as Jurkat cells) to prepare a T cell line stably expressing TCR and mCherry. Among them, an activated T cell nuclear factor of transcription (NFAT) response element is added upstream of mCherry to detect the activation of TCR signals. The TCR library can contain multiple different TCRs, and each T cell is transfected with one TCR. The antigen peptide comes from: tumor cells are isolated from tumor tissue, and possible tumor antigens are obtained by mass spectrometry analysis of tumor cells, and the antigen peptide is synthesized according to the antigen sequence.

[0066] (2) Labeling cells. Different cell label ssDNAs are added to different types of cells (APC cells and T cells) so that in the later single-cell sequencing data analysis, it is possible to separate which cells are contained in the droplet and which cells interact with each other, thereby realizing the visualization of information in the droplet. For high-throughput detection, different cell labels are used to label first cells (T cells) expressing different TCRs, and to label second cells (APC cells) loaded with different antigen peptides. Therefore, a variety of first cells can be prepared for expressing different TCRs, and a variety of second cells can be prepared for loading different antigen peptides; a variety of first cells are respectively labeled with a variety of different first label sequences, and a variety of second cells are respectively labeled with a variety of different second label sequences. The first label sequence and the second label sequence can both be DNA sequences with a length of 20-200bp, preferably 100bp. The first label sequence and the second label sequence contain the same common sequence, which can be combined with the capture sequence on the magnetic bead through base complementary pairing to achieve the capture of the cell label sequence.

[0067] (3) Droplet generation. Using a droplet generation chip (see PCT / CN2019 / 108536, see Figure 1), APC cells and magnetic beads loaded with antigen peptides, as well as T cells and magnetic beads transfected with mCherry reporter molecules, were added to the two sample wells of the chip respectively; in order to be able to specifically screen TCR, the cells were adjusted to an appropriate concentration. This concentration requires that in most droplets, each droplet contains one APC cell and one T cell. The specific design is: according to the Poisson distribution, the number of cells added is calculated to meet λ = 1 (that is, the probability of a single cell in the generated droplet is the highest). Both cells are added according to λ = 1 to ensure that the probability of generating a target droplet containing one T cell and one APC cell is the highest. Using this design, the utilization rate of APC cells and T cells is maximized. After the droplets are generated, they are placed in a 37°C, 0.5% carbon dioxide incubator and incubated for 4 hours to achieve interaction between cells, and then the droplets are removed.

[0068] (4) Droplet injection. The lysis solution is injected to lyse the cells and release mRNA and cell tag ssDNA. Each magnetic bead carries a unique coding sequence on its surface for marking and identifying the identity of the magnetic bead. Connected to the unique coding sequence is the capture sequence of the magnetic bead. There are two types of capture sequences. One is the first capture sequence, which is used to capture the sample target sequence. For example, the TTTTTTTTTTTT (SEQ ID NO.3) sequence is used as the first capture sequence. It can complement the poly A tail of the sample mRNA, thereby capturing the mRNA released by the cell. The other is the second capture sequence, which is an artificially designed fixed sequence that can complement the common sequence on the cell tag ssDNA (such as the GACGCTGCCGACGA (SEQ ID NO.4) sequence) to capture the cell tag ssDNA. It is precisely because of the existence of the unique coding sequence and the two capture sequences that the unique coding sequence of the magnetic bead can be used to identify the ssDNA and mRNA information from the same droplet, thereby completing the assembly of the mRNA information and the ssDNA information.

[0069] (5) mRNA is reverse transcribed to obtain cDNA, cell-tagged ssDNA is amplified by PCR, and then a sequencing library is constructed and sequenced.

[0070] (6) Data processing and splitting. Sequencing data with the same unique coding sequence are divided into a group, and sequencing data containing only two cell label information (one label from APC and the other label from T cells) are further screened out within the group. Then, the mRNA expression of T cells in the offline data is analyzed, focusing on the expression levels of several marker genes related to T cell activation and the mCherry gene. Since the cell label sequence ssDNA is known, the cell label sequence can be used to analyze which two cells interacted. For activated T cells, the ssDNA information in the data can be used to determine which antigen peptide stimulated it, and the ssDNA information can also be used to determine which first cell (T cell) was activated, so that the antigen peptide and the TCR sequence that has a pairing relationship with the antigen peptide can be screened out.

[0071] Example: Detection of the pairing relationship between two antigenic peptides and their corresponding TCRs.

[0072] 1. Experimental materials.

[0073] 1) Jurkat cells that recognize DMF5 and express mCherry, and Jurkat cells that recognize IG4 and express mCherry. These two cell lines were constructed using the method disclosed in the document "The Targeting Effect of Cetuximab Combined with PD-L1 Blockade against EGFR-Expressing Tumors in a Tailored CD16-CAR T-Cell Reporter System" (DOI: 10.1080 / 07357907.2021.1894570), where the corresponding nuclear factor of activated T cells (NFAT) response elements and mCherry sequences were introduced into wild-type Jurkat cells (ATCC, TIB-152) via lentiviral transfection.

[0074] 2) MHC-expressing T2 cells (Shanghai EK-Bioscience Biotechnology Co., Ltd, CC-Y1650), IG4 polypeptide (Mart-1, LAGIGILTV, SEQ ID NO. 2), and DMF5 polypeptide (NY-ESO-1, SLLMWITQC, SEQ ID NO. 1).

[0075] 3) CASB-related reagents: streptavidin (BBI C600432-0005), concanavalin A (SIGMA L7647-100 mg), and four ssDNAs (synthesized by Beijing Liuhe).

[0076] The four ssDNA sequences are:

[0077]

[0078] The underlined sequence is the cell tag sequence, used to match split cells during offline data analysis. This sequence is used to match reads from the offline sequencing data. A match indicates that the droplet carries the corresponding ssDNA, thus confirming that the droplet contains cells labeled with this ssDNA. The NNNNNNN region is a dark reaction region during sequencing; this portion of the sequence cannot be obtained through sequencing and can therefore be a random sequence. The 3' end, GACGCTGCCGACGA (SEQ ID NO. 9), is the consensus sequence for cell tags. This sequence complements the artificially set fixed sequence on the magnetic beads (i.e., the second capture sequence) for subsequent magnetic bead capture.

[0079] 4) Droplet generation oil (Bio-Rad, Cat. No. 1863005).

[0080] 5) Droplet generation device (see PCT / CN2019 / 108536, CN209144161U).

[0081] 6) Droplet injection device.

[0082] 7) Library construction kit: DNBelab C series high-throughput single-cell RNA library preparation kit (MGI, 94-000047-00

[0083] 2. Experimental steps.

[0084] (1) Preparation of peptide-loaded T2 cells.

[0085] T2 cells were adjusted to 1*10 with IMDM complete medium (IMDM complete medium formula: 89% IMDM medium: GIBCO, 12440053; 10% FBS: HYCLONE SH30084.03HI; 1% PS: HYCLONE, SV30010) 6 At a concentration of 100 cells / ml, 1 ml of the cell suspension was taken and placed in two wells of a 24-well plate (the cell suspension in one well was labeled cell suspension C, and the cell suspension in the other well was labeled cell suspension D). 3 μL of 10 μg / μL IG4 peptide was added to cell suspension C for overnight co-stimulation, and 3 μL of 10 μg / μL DMF5 peptide was added to cell suspension D for overnight co-stimulation. The next day, the cells were centrifuged and the supernatant was discarded. The T2 cells loaded with different peptides were resuspended in 1 ml of DPBS (GIBCO, C14190500BT).

[0086] (2) Use CASB strategy to add labels to cells.

[0087] Jurkat cells that recognize DMF5 and express mCherry were adjusted to 1*10 using 1640 complete medium (1640 complete medium formula: 89% 1640 medium; 10% FBS: 1% PS). 6 The concentration of Jurkat cells that recognize IG4 and express mCherry was adjusted to 1*10 cells / ml using 1640 complete medium. 6The concentration of cells / ml was 400 μg / ml (labeled as cell suspension B). Four different ssDNAs were used as labels to construct four CASBs: 2 μL streptavidin was added to 8 μL ssDNA, and the reaction was carried out at room temperature for 10 minutes. Then 2 μL concanavalin A was added and the reaction was carried out at room temperature for 10 minutes to construct the CASB. Cell suspensions A, B, C and D were added to the four CASBs respectively, and the reaction was carried out at 4°C for 10 minutes. The cells were washed three times with DPBS and centrifuged to discard the supernatant. For the CASB strategy, please refer to "CASB: a concanavalin A-based sample barcoding strategy for single-cell sequencing." Mol Syst Biol 17(4):e10060(2021). The cells in each cell suspension were added with corresponding ssDNA labels. Specifically, T2-IG4 cells were labeled with ssDNA-1, T2-DMF5 cells were labeled with ssDNA-2, Jurkat-IG4 cells were labeled with ssDNA-3, and Jurkat-DMF5 cells were labeled with ssDNA-4.

[0088] (3) Droplet generation.

[0089] A droplet generation device was constructed according to the method disclosed in PCT / CN2019 / 108536 and CN209144161U, and 100 μL of droplet generation oil was added to the droplet collection tube as a buffer. A droplet generation chip was used to add the T2 cell mixture loaded with IG4 and DMF5 and magnetic beads obtained in step (2) to one sample well, and the Jurkat cell mixture expressing IG4 and DMF5 and magnetic beads obtained in step (2) were added to another sample well. The cell input amount was 20,000 cells / μL and the magnetic bead input amount was 10,000 cells / μL. Next, resuspend the magnetic beads and cells in the sample wells in 25 μL of working solution (working solution formula: 5% FBS, 2.5% HEP, 1% PS, 1% F68, 30% Fical, 60.5% pure culture medium). Add 400 μL of droplet generation oil to the oil well. Pull the syringe of the droplet generation device from the 15 to 20 mark to apply pressure, generating a total of 50 μL droplets in the collection tube. Incubate the collection tube in a 37°C incubator for 4 hours.

[0090] (4) Droplet injection.

[0091] Build the droplet injection device as shown in Figure 2. Figure 2A is a physical diagram of the injection device, which consists of three units: the "oil phase channel, the droplet phase channel, and the lysis liquid phase channel." Figure 2B is a schematic diagram of the droplet injection device.

[0092] Aspirate 30 μL of droplets from the collection tube obtained in step (3) into a 200 μL pipette tip and place it in the droplet phase channel of the injection device. Filter the droplets using a 0.22 μm sterile syringe filter (MILLIPORE, SLGP033RB) to generate 100 μL of oil, which is placed in the oil phase channel of the injection device. Add 100 μL of lysis buffer (the lysis buffer provided in the DNBelab C Series High-Throughput Single-Cell RNA Library Preparation Kit) to the lysis phase channel. Adjust the machine voltage (frequency: 15 kHz; voltage: 1 VPP; voltage amplifier: 200x). Complete the injection by pulling the droplet injection device from the 17.5 scale to 20 (providing sufficient pressure). The injected droplets will be placed in a new collection tube.

[0093] (5) Build a database.

[0094] The single-cell sequencing kit, DNBelab C Series High-Throughput Single-Cell RNA Library Preparation Kit, was used. Briefly, the injected droplets were incubated at room temperature for 30 minutes to allow complete cell lysis, followed by emulsion recovery.

[0095] The samples were subjected to reverse transcription reaction, second-strand synthesis, cDNA product amplification, and amplification product sorting in sequence to obtain the first amplification product. The first amplification product includes (1) cDNA products obtained by reverse transcription of cell mRNA captured by magnetic beads; (2) oligo products obtained after amplification of ssDNA used to label cells.

[0096] The cDNA product undergoes library construction, including cDNA shearing, fragment screening, end repair, adapter ligation, product purification, and secondary amplification and purification. This is done to remove adapters and other small fragments that are not reverse-transcribed from cellular mRNA. A sequencing library is then obtained.

[0097] Oligo product library construction: Secondary amplification and fragment screening of the oligo product. Because the previously obtained oligo product contains many linkers on the magnetic beads, the purpose of secondary amplification is to remove short DNA fragments such as linkers that are not amplified from the ssDNA used for cell labeling. A sequencing library is then obtained.

[0098] The two libraries were subjected to high-temperature denaturation, single-strand circularization, and enzyme digestion to transform them into single-strand circularized DNA libraries that meet the requirements of single-cell sequencing.

[0099] (6) Sequencing on a machine.

[0100] The library obtained in step 5 was sequenced using BGI's single-cell sequencing platform DNBSEQ-T7.

[0101] (7) Data processing and analysis.

[0102] The data were grouped according to the unique coding sequence, and the sequencing data containing only two cell label information (one label from T2 and the other label from Jurkat cells) were screened out. Specifically, the droplets with four sequences (ssDNA-1+ssDNA-3), (ssDNA-1+ssDNA-4), (ssDNA-2+ssDNA-3) or (ssDNA-2+ssDNA-4) were focused on. The T cell mRNA expression in the sequencing data was analyzed, focusing on the expression levels of several marker genes and mCherry genes related to T cell activation. Since the cell label ssDNA sequence is known, the cell label sequence can be used to analyze which two cells interacted with each other. For activated T cells, the ssDNA information in the data is used to determine which antigen peptide stimulated it and which first cell (T cell) was activated, thereby screening out the correspondence between the antigen peptide and the TCR. That is, positive droplets carrying the two cell labels (ssDNA-1+ssDNA-3) or (ssDNA-2+ssDNA-4) were screened out, and the high expression of the mCherry gene and activation-related genes in the cells in the droplets were analyzed.

[0103] Furthermore, to demonstrate the feasibility of the process, it was also important to examine whether the unpaired droplets corresponded to the unactivated Jurkat cells. Specifically, the negative droplets bearing the (ssDNA-1+ssDNA-4) and (ssDNA-2+ssDNA-3) tag sequences showed low or no expression of the cellular mCherry gene and activation-related genes.

[0104] The results are shown in Figure 3. Figure 3 shows three types of droplets: positive 1 is an IG4-positive droplet containing ssDNA-1 and ssDNA-3; positive 2 is a DMF5-positive droplet containing ssDNA-2 and ssDNA-4; and negative droplets are (ssDNA-2 + ssDNA-3) and (ssDNA-1 + ssDNA-4). As can be seen in Figure 3, the expression of marker genes for T cell activation and the mCherry gene is exactly as expected: expression in the positive group is significantly higher than in the negative group. This indicates that cells in the positive group droplets are interacting with each other.

Claims

1. A method for detecting cell-cell interactions, comprising the steps of: (1) preparing a first cell expressing a TCR activation response element and a reporter molecule, wherein the TCR activation response element is located upstream of the reporter molecule, and the first cell expresses TCR; (2) incubating the antigen peptide with a second cell expressing MHC, so that the antigen peptide is loaded onto the MHC of the second cell to form an antigen peptide-MHC complex, thereby obtaining a second cell loaded with the antigen peptide; (3) labeling the first cell with a first tag sequence to obtain a first cell carrying the tag sequence, and labeling the second cell loaded with the antigen peptide with a second tag sequence to obtain a second cell carrying the tag sequence, wherein the first tag sequence and the second tag sequence contain the same common sequence, and the first tag sequence and the second tag sequence contain different specific sequences; (4) using an oil phase to encapsulate the first cells carrying the label, the second cells carrying the label, and the magnetic beads to prepare a plurality of droplets, wherein: A single droplet of at least a portion of the multiple droplets contains only one first cell, one magnetic bead, and at least one second cell, and the surface of the magnetic bead carries a unique coding sequence, a first capture sequence, and a second capture sequence, wherein the first capture sequence is used to capture a cell target sequence, and the second capture sequence is complementary to a common sequence in a cell label sequence and is used to capture the cell label sequence, and the unique coding sequence of each magnetic bead is different from each other and is used to identify the identity of the magnetic bead; (5) injecting a cell lysis solution into a single droplet, lysing the cells, releasing and capturing the cell target sequence and the cell tag sequence through the magnetic beads, and sequencing the sequences obtained after capture to obtain sequencing data; (6) Analyzing sequencing data containing the same unique coding sequence to determine whether there is interaction between the first cell and the second cell in the single droplet.

2. The method according to claim 1, wherein in (5), the sequence obtained after capture is amplified and library is constructed, and the obtained sequencing library is sequenced.

3. The method according to claim 2, wherein in (5), the sequence obtained after capture comprises mRNA and DNA, the mRNA is subjected to reverse transcription, double-strand synthesis, and cDNA product amplification, and the amplified cDNA product is subjected to library construction to obtain a first sequencing library; the DNA is subjected to secondary amplification and fragment screening to obtain a second sequencing library; and the first and second sequencing libraries are combined for sequencing.

4. The method according to claim 1, wherein in (1)-(3), a plurality of the first cells are prepared for expressing different TCRs, and a plurality of the second cells are prepared for loading different antigen peptides; the plurality of the first cells are respectively labeled with a plurality of different first tag sequences, and the plurality of the second cells are respectively labeled with a plurality of different second tag sequences.

5. The method according to any one of claims 1 to 4, wherein in (1), the first cell is a T cell, preferably a Jurkat cell or a human-derived T lymphocyte.

6. The method according to any one of claims 1 to 4, wherein in (1), a TCR lentiviral vector and a reporter molecule vector are constructed and co-transfected with the TCR sequence into a first cell to prepare a first cell expressing the TCR and the reporter molecule.

7. The method according to any one of claims 1 to 4, wherein in (1), the TCR activation response element is nuclear factor of activated T cells, AP1 transcription factor or NF-κB.

8. The method according to any one of claims 1 to 4, wherein in (1), the reporter molecule is a fluorescent protein, preferably green fluorescent protein, red fluorescent protein, yellow fluorescent protein or blue fluorescent protein.

9. The method according to any one of claims 1 to 4, wherein in (2), the second cell is an antigen presenting cell, preferably a T2 cell or a K562 cell.

10. The method according to any one of claims 1 to 4, wherein in (2), an MHC lentiviral vector is constructed and transfected into the second cell to prepare the second cell expressing MHC.

11. The method according to any one of claims 1 to 4, wherein in (2), the antigenic peptide is a tumor-specific antigenic peptide, preferably a NY-ESO-1 polypeptide with a sequence of SLLMWITQC or a MART-1 polypeptide with a sequence of LAGIGILTV. (27-35) polypeptide.

12. The method according to any one of claims 1 to 4, wherein in (3), the first cell and the second cell are labeled using a CASB strategy. The method according to claim 12 , wherein the first tag sequence and the second tag sequence are both DNA sequences. The method according to claim 13 , wherein the DNA sequence is 20-200 bp in length, preferably 100 bp in length.

15. The method according to any one of claims 1 to 4, wherein in (4), the droplets are prepared using droplet microfluidics technology. 16 . The method according to claim 15 , wherein a droplet generation chip is used, and the first cells and magnetic beads carrying the label, and the second cells and magnetic beads carrying the label are added to two sample wells respectively.

17. The method according to any one of claims 1 to 4, wherein in (4), by adjusting the concentrations of the labeled first cells and the labeled second cells, a single droplet of at least a portion of the plurality of droplets contains only one first cell and at least one second cell.

18. The method of claim 17, wherein the first cell and the second cell are added at a concentration that maximizes the probability of a single cell in the droplet.

19. The method according to any one of claims 1 to 4, comprising an incubation step after (4) droplet generation, preferably with an incubation time of 4 to 8 hours.

20. The method according to any one of claims 1 to 4, wherein in (5), a droplet injection device is used for injection of the lysate.

21. The method according to any one of claims 1 to 4, wherein in (6), sequencing data containing the same unique coding sequence and having two cell tag sequences are screened, and based on the reporter molecule expression level of the first cell in the sequencing data, it is determined whether specific recognition occurs between the TCR expressed by the first cell and the MHC-antigen peptide complex of the second cell; if specific recognition occurs, it is determined that there is interaction between the first cell and the second cell in the single droplet.

22. The method according to claim 21, wherein in (6), it is determined that the first cell and the second cell interact with each other, and the corresponding first cell and second cell are obtained by further using the cell tag sequence in the sequencing data.

23. The method according to any one of claims 1 to 4, wherein the cell-cell interaction comprises any one or more of cell-cell communication by secreting chemical signals, cell-cell contact-dependent communication, and formation of gap junctions between adjacent cells of an animal.

24. Use of the method of any one of claims 1 to 23 in high-throughput screening of antigens and their paired T cell receptors, wherein the first cell is a T cell expressing a TCR, and the second cell is an APC cell loaded with an antigen peptide, the T cells are of multiple types and are used to express different TCRs, and the APC cells are of multiple types and are used to load different antigen peptides; the multiple T cells are respectively labeled with multiple different first tag sequences, and the multiple APC cells are respectively labeled with multiple different second tag sequences.

25. The use according to claim 24, wherein the TCR is a tumor-specific T cell receptor.

26. A method for high-throughput screening of antigens and their paired T cell receptors, comprising using the method of any one of claims 1-23 to screen antigens and their paired T cell receptors, wherein the first cell is a T cell expressing a TCR, and the second cell is an APC cell loaded with an antigen peptide, and the T cells are of multiple types and are used to express different TCRs, and the APC cells are of multiple types and are used to load different antigen peptides; the multiple T cells are respectively labeled with multiple different first tag sequences, and the multiple APC cells are respectively labeled with multiple different second tag sequences.

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