WO2024092740A1 - 检测细胞间相互作用的方法及其应用 - Google Patents

检测细胞间相互作用的方法及其应用 Download PDF

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WO2024092740A1
WO2024092740A1 PCT/CN2022/129973 CN2022129973W WO2024092740A1 WO 2024092740 A1 WO2024092740 A1 WO 2024092740A1 CN 2022129973 W CN2022129973 W CN 2022129973W WO 2024092740 A1 WO2024092740 A1 WO 2024092740A1
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cell
cells
sequence
tcr
label
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French (fr)
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刘亚
齐婧羽
顾颖
刘杨
李毅坚
吴晶
董旋
刘龙奇
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深圳华大生命科学研究院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds

Definitions

  • the present invention belongs to the field of biotechnology, and more specifically, provides a method for high-throughput detection of cell-to-cell interaction and its application.
  • CAR-T chimeric antigen receptor T cell therapy
  • 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 TCR ⁇ chain sequences that are highly specific to the antigen; 2) Isolating T cells from the patient; 3) Incorporating the above-mentioned TCR ⁇ 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 amplified in large quantities; 6) A qualified number of TCR-T cells are injected into the patient for treatment.
  • TCR-T technology as the most primitive cell therapy, has high technical barriers.
  • TCR-T has lower antigen affinity.
  • this type of tumor-specific antigen produced by high-frequency mutation of tumors has become an important breakthrough in solving off-target effects.
  • TCR in the human body generally needs to undergo affinity screening in the thymus, and tumor neoantigens do not exist in the normal population (non-diseased population), making it more difficult to find specific TCRs for tumor neoantigens in the human body.
  • 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 using the function of tumor neoantigen-specific T cells for measurement, such as identifying reactive T cells with the help of secreted cytokines and surface protein expression, that is, measuring IFN ⁇ levels through ELISA, ELISPOT, and FACS analysis after intracellular cytokine staining.
  • TIL tumor-infiltrating lymphocytes
  • TMGs tandem minigenes
  • APCs neoantigen-transfected/pulsed antigen-presenting cells
  • the purpose of the present invention is to screen TCR-T corresponding to APC in high throughput, and the present invention can quickly and conveniently realize the screening of interacting cells.
  • the present invention provides a method for detecting cell-cell interaction, the method comprising the steps of:
  • the surface of the magnetic beads 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;
  • the sequences obtained after capture are amplified and library constructed, and the obtained sequencing library is sequenced.
  • 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
  • the first and second sequencing libraries are combined for sequencing.
  • 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.
  • the first cell is a T cell, preferably a Jurkat cell or a human-derived T lymphocyte.
  • 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.
  • the TCR activation response element is nuclear factor of activated T cells (NFAT), AP1 transcription factor (AP1) or NF- ⁇ B.
  • 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).
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • YFP yellow fluorescent protein
  • BFP blue fluorescent protein
  • the second cell is an antigen presenting cell, preferably a T2 cell or a K562 cell.
  • an MHC lentiviral vector is constructed and transfected into the second cell to prepare a second cell expressing MHC.
  • the antigenic peptide is a tumor-specific antigenic peptide, preferably a NY-ESO-1 polypeptide with a sequence of SLLMWITQC or a MART-1 (27-35) polypeptide with a sequence of LAGIGILTV.
  • the first cell and the second cell are labeled using a CASB strategy.
  • the first tag sequence and the second tag sequence are both DNA sequences.
  • the DNA sequence is 20-200 bp in length, preferably 100 bp in length.
  • the droplets are prepared using droplet microfluidics technology.
  • 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 loading wells, respectively.
  • a single droplet of at least a portion of the plurality of droplets contains only one of the first cells and at least one of the second cells.
  • the first cell and the second cell are added at a concentration that has the highest probability of being a single cell in the droplet.
  • an incubation step is included after (4) droplet generation, and the incubation time is preferably 4-8 hours.
  • the lysate injection is performed using a droplet injection device.
  • sequencing data containing the same unique coding sequence and having two cell label 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.
  • (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.
  • the interaction includes any one or more of cell-to-cell communication by secretion of chemical signals, contact-dependent communication between cells, and formation of gap junctions between adjacent cells of an animal.
  • 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, and the T cells are of multiple types for expressing different TCRs, and the APC cells are of multiple types for loading different antigen peptides; the multiple T cells are respectively labeled with multiple different first label sequences, and the multiple APC cells are respectively labeled with multiple different second label sequences.
  • the TCR is a tumor-specific T cell receptor.
  • 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 for expressing different TCRs, and the APC cells are of multiple types for loading 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.
  • the present invention relies on antigen presenting cells loaded with antigen peptides, T cells expressing different types of TCRs and magnetic beads encapsulated in droplets, adding different labels to each cell, and distinguishing which sequences obtained after sequencing are from the same droplet through the unique coding sequence on the magnetic beads.
  • the present invention incubates the cells in the droplets after the droplets are generated and before the lysate is added to provide time for cell-to-cell interaction.
  • the present invention can not only be used to study the interaction between single cells, but also can achieve high-throughput detection of multiple antigens and their TCR-specific pairing relationships.
  • FIG1 shows a schematic diagram of a method according to an embodiment of the present invention.
  • FIG. 2 shows a real object (A) and a principle design diagram (B) of a droplet injection device according to an embodiment of the present invention.
  • FIG. 3 shows a diagram of analysis results of sequencing data according to an embodiment of the present invention.
  • the current single-cell sequencing technology cannot provide time for cell interactions due to the direct introduction of lysate, which causes cell lysis, and most droplets contain only one cell, so it is impossible to realize the study of cell-to-cell interactions.
  • the present invention achieves the encapsulation of multiple cells in a droplet by labeling cells; and when generating droplets, the lysate is not directly introduced, but after a period of time, the lysate is added by droplet injection, thereby providing time conditions for the occurrence of cell interactions.
  • the present invention can prepare millions of droplets, and cells can independently complete interactions in droplets. At the same time, combined with single-cell sequencing, the pairing relationship is analyzed from the omics level.
  • 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 antigens and TCR information with 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 new antigens and their corresponding TCR sequences.
  • interaction usually 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 binds to its corresponding receptor, producing a series of physiological and biochemical changes in the target cell through cell signal transduction, and finally manifesting as the overall biological effect of the target cell.
  • Cell interaction mainly includes three categories: (1) Cells communicate by secreting chemical signals; (2) 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 of animals, and metabolic coupling or electrical coupling is achieved by exchanging small molecules, thereby achieving functional regulation.
  • the secretion of chemical signals can be divided into three different ways: endocrine, paracrine, and transmission of neural signals through chemical synapses.
  • T cell receptor refers to a molecule or a complex of molecules on a T cell that is capable of receiving a signal and transmitting the signal across the cell membrane.
  • 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.
  • MHC major histocompatibility complex
  • H-2 complex the MHC in mice
  • HLA human leukocyte antigen
  • MHC human leukocyte antigen
  • RLA rabbit leukocyte antigen
  • GPLA guinea pig leukocyte antigen
  • SLA house leukocyte antigen
  • DLA dog leukocyte antigen
  • RhLA monkey leukocyte antigen
  • 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 class III genes encode MHC-III class molecules or antigens.
  • antigen-presenting cells are cells in the body that have the ability to absorb, process and transmit antigen information, and induce immune responses in T and B cells.
  • APCs engulf external pathogenic microorganisms such as bacteria and parasites, digest them in intracellular lysosomes, form polypeptide fragments, and form complexes with MHC-II molecules, which are presented on the cell surface.
  • APCs mainly include macrophages, dendritic cells, synaptic cells, Langerhans cells, and B cells.
  • the TCR activation response element can be nuclear factor of activated T cells (NFAT), AP1 transcription factor (AP1) or NF- ⁇ B.
  • NFAT nuclear factor of activated T cells
  • AP1 transcription factor AP1 transcription factor
  • NF- ⁇ B nuclear factor of activated T cells
  • "Nuclear factor ⁇ B” or “NF- ⁇ B” is a highly conserved multifunctional transcription factor family, which includes five members, namely p65 (RelA), RelB, c-Rel, NF- ⁇ B1 and NF- ⁇ B2, which form different homologous or heterologous dimers to play a role in gene transcription regulation, thereby regulating many important cell behaviors, especially inflammatory responses.
  • NFAT Nuclear Factor of Activated T cell
  • the TCR activation corresponding element is NFAT
  • the structure of the TCR intracellular segment will change and recruit phosphoprotein kinase K, including ZAP70 protein.
  • the hydrolysis of phosphatidylinositol 4,5-bisphosphate will produce inositol triphosphate, which binds to the endoplasmic reticulum receptor, causing calcium ion release, thereby activating the transcription factor NFAT;
  • the downstream reporter molecule expression gene will be transcribed and translated into a reporter molecule (for example, in one embodiment, GFP protein).
  • the reporter molecule Under laser excitation of an appropriate wavelength, the reporter molecule will emit a spectrum of a specific wavelength.
  • the emission light of the reporter molecule can be captured using a photomultiplier tube (PMT), thereby achieving the characterization and detection of antigen-specific TCRs.
  • PMT photomultiplier tube
  • CASB Concanavalin A-based Sample Barcoding
  • the inventor introduced a reporter molecule at the 3' end to assist in judging whether the T cell is activated.
  • reporter molecule refers to a substance that can indicate the reaction between TCR and the MHC-antigen peptide complex.
  • the reporter molecule can be a class of visualized 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.
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • YFP yellow fluorescent protein
  • BFP blue fluorescent protein
  • a droplet If a droplet is detected to emit fluorescence of a specific wavelength under laser excitation of a specific wavelength, it indicates that the reporter molecule is expressed, specific recognition occurs between the TCR and the MHC-antigen peptide complex, and the first cell contains an antigen-specific TCR; if a droplet is detected to fail to emit fluorescence of a specific wavelength under specific laser excitation, it indicates that the reporter molecule is not expressed, no specific recognition occurs between the TCR and the MHC-antigen peptide complex, and the first cell does not contain an antigen-specific TCR.
  • 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).
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • YFP yellow fluorescent protein
  • BFP blue fluorescent protein
  • T lymphoma cells include, but are not limited to, Jurkat cells and human-derived T lymphocytes.
  • antigen presenting cells are capable of expressing MHC molecules, such as T2 cells and K562 cells, but are not limited thereto.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed.
  • An 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.
  • 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.
  • 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.
  • 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.
  • antigenic peptide refers to a polypeptide having an antigenic epitope and thus being recognized by TCR.
  • antigen refers to tumor antigens, i.e., any molecule expressed or overexpressed by tumor cells or cancer cells alone or mainly, such as proteins, polypeptides, peptides, lipids, carbohydrates, 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.
  • tumor cells or cancer cells can overexpress antigens or express antigens at significantly higher levels.
  • tumor antigens can be expressed by cells in different developmental or mature states.
  • tumor antigens can be expressed by cells in embryonic or fetal stages, which are usually not present in adult hosts.
  • tumor antigens can be expressed by stem cells or precursor cells, which are usually not 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.
  • tumor cells have chromosomal instability and abnormal epigenetic regulation (such as methylation, etc.), often producing mutant proteins or fusion proteins, which can become targets for immunotherapy and are called tumor neoantigens.
  • the antigen peptide is a tumor antigen peptide.
  • the NY-ESO-1 polypeptide with a sequence of SLLMWITQC (SEQ ID NO.1), or the MART-1 (27-35) polypeptide with a sequence of LAGIGILTV (SEQ ID NO.2) but not limited thereto.
  • the method of the present invention may include five steps: 1) cell line construction; 2) labeling of cell tags by CASB strategy; 3) use of droplet encapsulation of cells and cell reactions; 4) droplet injection; 5) single cell sequencing; and 6) sequencing data analysis. The specific steps are described as shown 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:
  • (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) MHC lentiviral vectors are constructed, and APC cells (such as T2 cells) are transfected with the constructed MHC lentiviral vectors to prepare APC cell lines stably transfected with MHC, and then the antigen peptides and the stably transfected APC cells are co-incubated to load the polypeptides onto the MHC of the APC cells; 2) TCR lentiviral vectors and enhanced mCherry vectors are constructed, and TCR libraries are prepared, and T cells (such as Jurkat cells) are transfected with the prepared TCR libraries to prepare T cell lines stably expressing TCR and mCherry.
  • APC cells such as T2 cells
  • the TCR library can contain a variety of different TCRs, and each T cell is transfected with one TCR.
  • the antigen peptides come from: tumor cells are isolated from tumor tissues, and possible tumor antigens are obtained by mass spectrometry analysis of tumor cells, and the antigen peptides are synthesized according to the antigen sequence.
  • first cells can be prepared to express different TCRs
  • second cells can be prepared to load different antigenic peptides
  • a variety of first cells are respectively labeled with a variety of different first label sequences
  • 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.
  • 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 utilization rate of APC cells and T cells is maximized.
  • 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.
  • capture sequences 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.
  • 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.
  • mRNA is reverse transcribed to obtain cDNA, the cell-tagged ssDNA is amplified by PCR, and then a sequencing library is constructed and sequenced.
  • 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.
  • T2 cells expressing MHC (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).
  • CASB-related reagents streptavidin (BBI C600432-0005), concanavalin A (SIGMA L7647-100mg), and four ssDNAs (synthesized by Beijing Liuhe).
  • the four ssDNA sequences are:
  • the underlined sequence is the cell label sequence, which is used to match the split cells when analyzing the offline data. Use this sequence to match the read segment of the offline sequencing data. If the sequence matches, it is considered that the droplet carries the corresponding ssDNA, thereby judging that the droplet contains cells labeled with this ssDNA; the NNNNNNN region is the dark reaction region of sequencing, and this part of the sequence cannot be obtained by sequencing, so it can be a random sequence; the 3' end GACGCTGCCGACGA (SEQ ID NO.9) is the common sequence of the cell label, which can be complementary to the artificially set fixed sequence on the magnetic beads (i.e., the second capture sequence) for subsequent capture of magnetic beads.
  • T2 cells were adjusted to a concentration of 1*10 6 cells/ml with IMDM complete medium (IMDM complete medium formula: 89% IMDM medium: GIBCO, 12440053; 10% FBS: HYCLONE SH30084.03HI; 1% PS: HYCLONE, SV30010), and 1 ml was taken and placed in two wells of a 24-well plate (the cell suspension in one well was marked as cell suspension C, and the cell suspension in the other well was marked as cell suspension D), and 3 ⁇ L of 10 ⁇ g/ ⁇ L IG4 peptide was added to co-stimulate with cell suspension C overnight, and 3 ⁇ L of 10 ⁇ g/ ⁇ L DMF5 peptide was added to co-stimulate with cell suspension D overnight. The next day, the supernatant was discarded by centrifugation, and the T2 cells loaded with different peptides were resuspended in 1 ml DPBS (GIBCO, C14190500BT).
  • IMDM complete medium
  • Jurkat cells that recognize DMF5 and express mCherry were adjusted to a concentration of 1*10 6 cells/ml using 1640 complete medium (1640 complete medium formula: 89% 1640 medium; 10% FBS: 1% PS) (labeled as cell suspension A).
  • Jurkat cells that recognize IG4 and express mCherry were adjusted to a concentration of 1*10 6 cells/ml using 1640 complete medium (labeled as cell suspension B).
  • Four different ssDNAs were used as tags to construct four CASBs: 2 ⁇ L streptavidin was added to 8 ⁇ L ssDNA, reacted at room temperature for 10 minutes, and then 2 ⁇ L concanavalin A was added. The CASB was constructed after reacting at room temperature for 10 minutes.
  • T2-IG4 cells were labeled with ssDNA-1
  • T2-DMF5 cells were labeled with ssDNA-2
  • Jurkat-IG4 cells were labeled with ssDNA-3
  • Jurkat-DMF5 cells were labeled with ssDNA-4.
  • a droplet generation device was constructed according to the method disclosed in PCT/CN2019/108536 and CN209144161U, and 100 ⁇ L 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 well, and the Jurkat cell mixture expressing IG4 and DMF5 and magnetic beads obtained in step (2) were added to another well.
  • the cell input amount was 20,000 cells/ ⁇ L
  • the magnetic bead input amount was 10,000 cells/ ⁇ L.
  • the magnetic beads and cells in the sample wells were resuspended in 25 ⁇ L of working solution (working solution formula: 5% FBS, 2.5% HEP, 1% PS, 1% F68, 30% Fical, 60.5% pure culture medium), 400 ⁇ L of droplet generation oil was added to the oil filling well, and the syringe of the droplet generation device was pulled from the scale 15 to 20 to provide pressure, and a total of 50 ⁇ L droplets were generated in the collection tube.
  • the collection tube was placed in a 37°C incubator and incubated for 4 hours.
  • Figure 2 A is a physical picture of the injection device, which consists of three units: "oil phase channel, droplet phase channel and cracking liquid phase channel”.
  • Figure 2 B is a schematic diagram of the droplet injection device.
  • the single-cell sequencing kit DNBelab C series high-throughput single-cell RNA library preparation kit, was used. In brief, the injected droplets were incubated at room temperature for 30 minutes to allow complete cell lysis, followed by demulsification recovery.
  • the samples are sequentially subjected to reverse transcription reaction, second-strand synthesis, cDNA product amplification and amplification product sorting 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.
  • the cDNA product library is constructed: cDNA shearing, fragment screening of the sheared product, end repair, adapter ligation, product purification, secondary amplification and purification of the cDNA product, the purpose is to screen out the adapter and other small molecule fragments that are not reverse transcribed from the cell mRNA. Then the sequencing library is obtained.
  • oligo products obtained previously contain many connectors on the magnetic beads the purpose of secondary amplification is mainly to remove short DNA fragments such as connectors that are not amplified from ssDNA used for cell labeling. Then the sequencing library is obtained.
  • the two libraries were subjected to high temperature denaturation, single-strand circularization, and enzyme digestion, so that both libraries became single-strand circularized DNA libraries that meet the requirements of single-cell sequencing.
  • the library obtained in step 5 was sequenced using BGI’s single-cell sequencing platform DNBSEQ-T7.
  • the sequencing data containing only two cell label information are grouped. Specifically, the droplets with four sequences (ssDNA-1+ssDNA-3), (ssDNA-1+ssDNA-4), (ssDNA-2+ssDNA-3) or (ssDNA-2+ssDNA-4) are focused on.
  • the expression of T cell mRNA in the sequencing 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 ssDNA sequence is known, the cell label sequence can be used to analyze which two cells interact with each other.
  • 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 the correspondence between the antigen peptide and TCR. That is, positive droplets carrying the two cell labels (ssDNA-1+ssDNA-3) or (ssDNA-2+ssDNA-4) are screened out, and the high expression of the mCherry gene and activation-related genes of the cells in the droplets are analyzed.
  • Figure 3 shows three types of droplets: positive 1 is an IG4 positive droplet of ssDNA-1+ssDNA-3; positive 2 is a DMF5 positive droplet of ssDNA-2+ssDNA-4, and negative is a negative droplet of (ssDNA-2+ssDNA-3) and (ssDNA-1+ssDNA-4).
  • the expression of the marker genes and mCherry genes for T cell activation is completely consistent with expectations: the expression of the positive group (positive) is significantly higher than that of the negative group (negative). It can be judged that the cells in the positive group droplets interact with each other.

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Abstract

一种检测细胞间相互作用的方法，所述方法包括(1)制备表达TCR激活响应元件和报告分子的第一细胞，所述第一细胞表达TCR; (2)将抗原肽与表达MHC的第二细胞孵育，得到加载抗原肽的第二细胞;(3)使用第一标签对所述第一细胞进行标记，得到携带标签的第一细胞，以及使用第二标签对所述加载抗原肽的第二细胞进行标记，得到携带标签的第二细胞;(4)使用油相包裹所述携带标签的第一细胞、所述携带标签的第二细胞和磁珠以制备多个液滴；(5)向单个液滴中注入细胞裂解液，对裂解后核酸进行测序;(6)根据测序结果确定单个液滴中第一细胞与第二细胞是否存在相互作用。

Description

检测细胞间相互作用的方法及其应用 技术领域
本发明属于生物技术领域，更具体而言，本发明提供了一种高通量检测细胞间相互作用的方法及其应用。
背景技术
近年，随着肿瘤免疫治疗研究突飞猛进的发展，嵌合抗原受体t细胞疗法(Chimeric Antigen Receptor-modified T cell，CAR-T)在血液类肿瘤治疗方面表现出良好的效果，但由于存在缺乏实体瘤特异性高表达抗原、肿瘤免疫抑制微环境、T细胞内源性的抑制信号等因素的挑战，导致其在实体瘤的治疗方面举步维艰。
而TCR-T细胞(T Cell Receptor-gene engineered T cell)疗法也是肿瘤免疫治疗常用方法之一，其技术流程为：1)筛选合适的肿瘤特异性抗原，以及对该抗原具有高度特异性的TCRαβ链序列；2)从患者体内分离出T细胞；3)将上述能够识别肿瘤特异性抗原的TCRαβ链序列包入病毒基因组，构建病毒载体；4)病毒转染T细胞，将目标TCR序列引入T细胞中，获得能够特异性识别肿瘤抗原的TCR-T细胞；5)TCR-T细胞体外培养，大量扩增；6)将数量合格的TCR-T细胞输入患者体内，进行治疗。
目前，TCR-T技术作为最原始的细胞疗法，技术壁垒高。首先，相比较于CAR-T，TCR-T的抗原亲和力更低。其次，由于受到脱靶效应的影响，限制了其在临床上的应用。随着新抗原鉴定技术的发展，这类由肿瘤高频突变而产生的肿瘤特异性抗原成为解决脱靶效应的重要突破口。但是，人体中TCR一般需要经过胸腺的亲和力筛选，而肿瘤新抗原在正常群体(未患病人群)中不存在，使得人体中肿瘤新抗原的特异性TCR更难以寻找。此外，由于肿瘤的突变潜力大，人群水平上难以找到普遍存在的肿瘤特异性抗原，在个体水平上，肿瘤内部也存在异质性，仅仅制备单个抗原的T细胞难以避免肿瘤耐药的发生。因此，个体间抗原种类差异大以及单个肿瘤内部异质性，导致了抗原种类的多样性。如何高效地为种类多样的肿瘤抗原寻找特异性TCR是当前的技术难点。
在获得特异性TCR方面，目前常规的T细胞疗法中主要采用的策略为：鉴定出 一种或多种肿瘤新抗原作为治疗靶点，经过数据处理合成MHC多肽，然后构建pHLA四聚体进行识别；或者利用肿瘤新抗原特异性T细胞的功能进行测定，例如借助分泌的细胞因子和表面蛋白的表达来识别反应性的T细胞，即通过ELISA、ELISPOT、细胞内细胞因子染色后进行FACS分析测量IFNγ的水平。而结合单细胞的T细胞疗法中，其主要采用的策略为：从新鲜肿瘤片段中培养多克隆肿瘤浸润淋巴细胞(TIL)，并针对串联小基因(TMG)或肽文库进行筛选，以鉴定新抗原反应性TIL群体；然后，将该TIL群体与新抗原转染/脉冲抗原呈递细胞(APC)共培养(例如使用Fluidigm C1***)，无论每个反应室是否包含单个细胞，均进行高通量深度测；最后通过测序数据鉴定出具有高表达T细胞活化标志物的单细胞RNA-seq样品，如IFN-γ和IL-2，并从这些样品中鉴定出成对的TCRα/β链序列。
然而，这两种方法耗时耗力，效率低，大约需要3-5个月才能制备出针对新抗原的高度个性化的且满足生产规范(GMP)级别的TCR疗法细胞产品，且配对成功率低；而目前已有的结合单细胞的筛选方法常在Fluidigm C1***中进行，最多一次同时处理96个样本，存在明显的通量限制，已有的成功案例也仅可以在肿瘤标本产生的24个TIL培养物中鉴定出大约1-6个含有新抗原反应性T细胞的TIL培养物，人力物力消耗量仍然很大。因此，开发能够检测细胞间相互作用的新方法尤其重要。
发明内容
本发明的目的在于高通量筛选与APC对应的TCR-T，本发明可以快速便捷地实现对相互作用的细胞的筛选。
在第一方面，本发明提供了一种检测细胞间相互作用的方法，所述方法包括步骤：
(1)制备表达TCR激活响应元件和报告分子的第一细胞，所述TCR激活响应元件位于所述报告分子上游，所述第一细胞表达TCR；
(2)将抗原肽与表达MHC的第二细胞孵育，使抗原肽加载到所述第二细胞的MHC上，以形成抗原肽-MHC复合物，得到加载抗原肽的第二细胞；
(3)使用第一标签序列对所述第一细胞进行标记，得到携带标签序列的第一细胞，以及使用第二标签序列对所述加载抗原肽的第二细胞进行标记，得到携带标签序列的第二细胞，其中所述第一标签序列与第二标签序列包含有相同的共有序列，并且 所述第一标签序列与第二标签序列包含有彼此不相同的特异性序列；
(4)使用油相包裹所述携带标签的第一细胞、所述携带标签的第二细胞和磁珠以制备多个液滴，其中，所述多个液滴中至少一部分的单个液滴中仅包含一个所述第一细胞、一个所述磁珠和至少一个所述第二细胞，并且所述磁珠表面携带有第一捕获序列、第二捕获序列和唯一编码序列，所述第一捕获序列用于捕获细胞靶标序列，所述第二捕获序列与细胞标签序列中的共有序列互补，用于捕获所述细胞标签序列，每个磁珠的唯一编码序列彼此不同，用于识别磁珠身份；
(5)向单个液滴中注入细胞裂解液，将细胞裂解，释放并通过所述磁珠捕获细胞靶标序列和细胞标签序列，对捕获后获得的序列进行测序，得到测序数据；
(6)对包含相同唯一编码序列的测序数据进行分析，确定单个液滴中第一细胞与第二细胞是否存在相互作用。
在一个实施方案中，在(5)中，对捕获后获得的序列进行扩增与文库构建，对获得的测序文库进行测序。
在一个实施方案中，在(5)中，捕获后获得的序列包含mRNA和DNA，对所述mRNA进行逆转录、二链合成、cDNA产物扩增，并对扩增的cDNA产物进行文库构建，获得第一测序文库；对所述DNA进行二次扩增及片段筛选，获得第二测序文库；将所述第一和第二测序文库合并进行测序。
在一个实施方案中，在(1)-(3)中，制备多种所述第一细胞，用于表达不同TCR，制备多种所述第二细胞，用于加载不同抗原肽；所述多种第一细胞分别用多种不同第一标签序列进行标记，所述多种第二细胞分别用多种不同第二标签序列进行标记。
在一个实施方案中，在(1)中，所述第一细胞为T细胞，优选为Jurkat细胞或人来源T淋巴细胞。
在一个实施方案中，在(1)中，构建TCR慢病毒载体和报告分子载体，与TCR序列共转染至第一细胞中，制备表达TCR和报告分子的第一细胞。
在一个实施方案中，在(1)中，所述TCR激活响应元件为活化T细胞核因子(NFAT)、AP1转录因子(AP1)或NF-κB。
在一个实施方案中，在(1)中，所述报告分子为荧光蛋白，优选为绿色荧光蛋白(GFP)、红色荧光蛋白(RFP)、黄色荧光蛋白(YFP)或者蓝色荧光蛋白(BFP)。
在一个实施方案中，在(2)中，所述第二细胞为抗原呈递细胞细胞，优选为T2细胞或K562细胞。
在一个实施方案中，在(2)中，构建MHC慢病毒载体，转染至所述第二细胞，制备表达MHC的第二细胞。
在一个实施方案中，在(2)中，所述抗原肽为肿瘤特异性抗原肽，优选为序列为SLLMWITQC的NY-ESO-1多肽或者序列为LAGIGILTV的MART-1 (27-35)多肽。
在一个实施方案中，在(3)中，利用CASB策略对第一细胞和第二细胞进行标记。
在一个实施方案中，所述第一标签序列和所述第二标签序列均为DNA序列。
在一个实施方案中，所述DNA序列长度为20-200bp，优选为100bp。
在一个实施方案中，在(4)中，使用液滴微流控技术制备所述液滴。
在一个实施方案中，在(4)中，使用液滴生成芯片，在两个加样孔内分别加入携带标签的第一细胞和磁珠、以及携带标签的第二细胞和磁珠。
在一个实施方案中，在(4)中，通过调整所述携带标签的第一细胞和所述携带标签的第二细胞的浓度，实现所述多个液滴中至少一部分的单个液滴中仅包含一个所述第一细胞和至少一个所述第二细胞。
在一个实施方案中，按液滴中单项细胞单胞的概率最高的浓度加入所述第一细胞和所述第二细胞。
在一个实施方案中，在(4)液滴生成后包括孵育步骤，优选孵育时间为4-8小时。
在一个实施方案中，在(5)中，使用液滴注射装置进行裂解液注射。
在一个实施方案中，在(6)中，筛选出包含相同的唯一编码序列、且具有两种细胞标签序列的测序数据，根据测序数据中第一细胞的报告分子表达水平，确定第一细胞所表达的TCR与第二细胞的MHC-抗原肽复合物之间是否发生特异性识别，若发生特异性识别则判定单个液滴中第一细胞与第二细胞存在相互作用。
在一个实施方案中，在(6)中，判定第一细胞与第二细胞存在相互作用，进一步利用测序数据中的细胞标签序列得到对应的第一细胞与第二细胞。
在一个实施方案中，相互作用包括细胞通过分泌化学信号进行细胞间的通讯、细胞间接触依赖性通讯和动物相邻细胞间形成间隙连接中的任意一种或多种。
在第二方面，本发明提供了本发明第一方面的方法在高通量筛选抗原及其配对的T细胞受体中的应用，其中所述第一细胞是表达TCR的T细胞，所述第二细胞是加载抗原肽的APC细胞，所述T细胞有多种，用于表达不同TCR，所述APC细胞有多种，用于加载不同抗原肽；所述多种T细胞分别用多种不同第一标签序列进行标记，所述多种APC细胞分别用多种不同第二标签序列进行标记。
在一个实施方案中，所述TCR为肿瘤特异性T细胞受体。
在第三方面，本发明提供了一种高通量筛选抗原及其配对的T细胞受体的方法，包括使用本发明第一方面的方法筛选抗原及其配对的T细胞受体，其中所述第一细胞是表达TCR的T细胞，所述第二细胞是加载抗原肽的APC细胞，所述T细胞有多种，用于表达不同TCR，所述APC细胞有多种，用于加载不同抗原肽；所述多种T细胞分别用多种不同第一标签进行标记，所述多种APC细胞分别用多种不同第二标签进行标记。
本发明依靠液滴内包裹着负载有抗原肽的抗原呈递细胞、表达不同种类TCR的T细胞和磁珠，通过给每种细胞添加不同标签，以及通过磁珠上的唯一编码序列区分测序后获得的序列中哪些是来自于同一个液滴。本发明在液滴生成之后、裂解液加入之前先孵育液滴中的细胞，为细胞间相互作用提供时间。本发明不仅可以用于研究单细胞间的相互作用，而且可以实现多个抗原及其TCR特异性配对关系的高通量检测。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所涉及的附图作简单地介绍。显而易见地，下面描述中的附图仅仅是本发明的一部分实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的实施方案。
图1示出了根据本发明一个实施方案的方法示意图。
图2示出根据本发明一个实施方案的液滴注射装置实物(A)及原理设计图(B)。
图3示出了根据本发明一个实施方案的测序下机数据分析结果图。
具体实施方式
下面将对本发明进行清楚、完整的描述。显然，所描述的实施方案仅仅是本发明的一部分实施方案，而不是全部的实施方案。基于本申请中描述的本发明实施方案，本领域普通技术人员可以获得的所有其他实施方案，且这些实施方案都属于本发明保护的范围。
目前的单细胞测序技术由于直接引入裂解液导致细胞裂解，无法为细胞互作提供时间，且大多数液滴都只含有一个细胞，因而无法实现细胞间的相互作用研究。本发明通过标记细胞的方法实现了一个液滴中包裹多个细胞；且在生成液滴时不直接引入裂解液，而是间隔一段时间后，通过液滴注射的方式再加入裂解液，从而为细胞互作的发生提供了时间条件。本发明借助液滴微流控技术，可以制备上百万个液滴，细胞可以在液滴内独立地完成相互作用，同时结合单细胞测序，从组学层面解析配对关系。相对于传统的配对方法，本发明的通量显著提高。本发明创新性地引入寡核苷酸序列标记抗原呈递细胞，从单细胞层面精准化地确定新抗原以及与该新抗原具有配对关系的TCR信息。因此，本发明方案特别适合用于肿瘤新抗原及其对应的TCR序列的高通量盲筛和功能验证。
在本文中，术语“相互作用”或“互作”，通常指细胞之间的相互作用或互作，可以理解为细胞通讯，即一个细胞发出的信号物质通过介质传递给另一个靶细胞并与其相应的受体相结合，通过细胞信号转导产生靶细胞内一系列生理生化变化，最终表现为靶细胞整体生物学效应的过程。细胞互作主要包括三类：(1)细胞通过分泌化学信号进行细胞间的通讯；(2)细胞间接触依赖性通讯(contact-dependent signaling)，细胞间直接接触，通过信号细胞跨膜信号分子(配体)与相邻靶细胞表面受体相互作用；(3)动物相邻细胞间形成间隙连接(gapjunction)，通过交换小分子来实现代谢偶联或电偶联，从而实现功能调控。其中，分泌化学信号又可以分为内分泌、旁分泌、通过化学突触传递神经信号进行三种不同方式进行。
在本文中，术语“T细胞受体”，亦即TCR，是指T细胞上能够接收信号并跨细胞膜传送信号的分子或分子复合物。
在本文中，术语“抗原特异性”是指TCR能够特异性结合并识别其相应的抗原或抗原表位从而引发免疫响应的性质。
在本文中，术语“主要组织相容性复合物”或“MHC”，是指存在于大部分脊椎动物某条染色体(例如，对于小鼠，在第17号染色体上；对于人，在第6号染色体上)上的一组紧密连锁的基因群，其编码产物(主要组织相容性抗原)与特异性免疫应答、免疫调节和移植排斥等密切相关。小鼠中的MHC被称为H-2复合物，人中的MHC被称为人类白血球抗原(Human Leukocyte Antigen，HLA)。除了小鼠中的H-2以及人中的HLA外，其他动物中也存在相应的MHC，例如，兔白细胞抗原(RLA)、豚鼠白细胞抗原(GPLA)、家主白细胞抗原(SLA)、狗白细胞抗原(DLA)或猴白细胞抗原(RhLA)。MHC可分为三类基因群，即MHC-I类、MHC-II类和MHC-III类。对于人类而言，MHC-I类包括3个基因位点，即A、B、C，其编码产物为MHC-Ⅰ类分子或抗原；MHC-II类包括DP、DQ、DR三个亚区，其编码的经典产物为MHC-Ⅱ类分子或抗原，尚有与内源性抗原处理有关的LMP、TAP；MHC III类基因编码产物MHC-Ⅲ类分子或抗原。
在本文中，抗原递呈细胞(Antigen-Presenting Cells，APC)是机体内具有摄取、处理和传递抗原信息，诱发T、B细胞发生免疫应答作用的细胞。APC吞噬细菌、寄生虫等外部病源微生物，经细胞内溶酶体消化，形成多肽片段，和MHC-II类分子形成复合物，被呈递在细胞表面。APC主要包括巨噬细胞、树突状细胞、并指状细胞、郎罕细胞以及B细胞。
在一个实施方案中，所述TCR激活响应元件可以为活化T细胞核因子(NFAT)、AP1转录因子(AP1)或NF-κB。“核因子κB”或“NF-κB”是一个高度保守的多功能转录因子家族，包含五个成员，分别为p65(RelA)、RelB、c-Rel、NF-κB1和NF-κB2，它们会组成不同的同源或异源二聚体而发挥基因转录调节作用，进而调节许多重要的细胞行为，特别是炎症反应。
在本文中，术语“活化T细胞核因子”或“NFAT”(Nuclear Factor of Activated T cell)，是一类转录因子家族，在免疫反应中对诱导基因转录起重要作用。除T细胞外，这类蛋白质还可以在许多免疫细胞上进行表达，如B淋巴细胞、肥大细胞、嗜酸性粒细胞等，其活性受到钙离子依赖性的钙调蛋白磷酸酯酶C的调节。在本发明中，NFAT是优选的TCR激活响应元件。
在TCR激活相应元件为NFAT的情况下，如果TCR能够特异性地识别MHC-抗原肽复合物，则TCR胞内段结构会发生改变，并招募磷酸蛋白激酶K，其中包括ZAP70蛋白。在ZAP70蛋白下游，磷脂酰肌醇4,5-双磷酸酯的水解会产生三磷酸肌醇，结合内质网受体，引起钙离子释放，进而激活转录因子NFAT；当NFAT结合转染的NFAT-报告分子序列上游的NFAT结合区时，下游报告分子表达基因会被转录并翻译为报告分子(例如，在一个实施方案中为GFP蛋白)。在适当波长的激光激发下，报告分子会发出特定波长的光谱。使用光电倍增管(PMT)可以捕获报告分子的发射光，进而实现对抗原特异性TCR的表征和检测。
在本文中，CASB(Concanavalin A-based Sample Barcoding)策略用于单细胞测序，是一种借助刀豆蛋白的寡核苷酸序列标记技术，对细胞分别标记标签。CASB策略参见"CASB:a concanavalin A-based sample barcoding strategy for single-cell sequencing."Mol Syst Biol 17(4):e10060(2021)。
但是在测序时，受目前技术的限制，通常只能获得细胞的3’端序列信息，无法获得细胞所有序列信息或者5’端序列信息(TCR的序列信息主要在5’端)，因此不能从测序数据中直接获得TCR的激活情况及TCR序列。这里，发明人在3’端引入了报告分子，以辅助对T细胞是否被激活做出判断。在本文中，所谓“报告分子”是指能够指示TCR与MHC-抗原肽复合物之间的反应的物质。所述报告分子可以是一类可视化的报告基因编码蛋白或者说荧光蛋白，可以包括：绿色荧光蛋白(GFP)及其突变体如mGFP5蛋白、EGFP蛋白、D2EGFP蛋白等；红色荧光蛋白(RFP)，如DsRed蛋白、DsRed-express蛋白、mRFP1蛋白、mCherry蛋白、Kaede蛋白等；黄色荧光蛋白(YFP)；或者蓝色荧光蛋白(BFP)等。这类蛋白质在特定波长下可被激发出明亮荧光，具有易于检测、对细胞无毒害、荧光稳定、无物种特异性和无需底物等特点，因此可以广泛应用于动物、植物及真菌研究中。如果检测到某个液滴在特定波长的激光激发下能发出特定波长的荧光，则表明报告分子得到表达，所述TCR与所述MHC-抗原肽复合物之间发生特异性识别，所述第一细胞中包含抗原特异性TCR；如果检测到某个液滴在特定激光激发下不能发出特定波长的荧光，则表明所述报告分子没有被表达，所述TCR与所述MHC-抗原肽复合物之间没有发生特异性识别，所述第一细胞中不包含抗原特异性TCR。
因此，在一个实施方案中，所述报告分子为荧光蛋白，例如绿色荧光蛋白(GFP)、红色荧光蛋白(RFP)、或黄色荧光蛋白(YFP)、或者蓝色荧光蛋白(BFP)。
在本文中，T淋巴瘤细胞，例如Jurkat细胞、人来源T淋巴细胞，但不限于此。
在本文中，抗原呈递细胞能够表达MHC分子，例如T2细胞、K562细胞，但不限于此。
在本文中，术语“表达载体”是指包含重组多核苷酸的载体，该重组多核苷酸包含与待表达的核苷酸序列可操作地连接的表达调控序列。表达载体包含足以用于表达的顺式作用元件；用于表达的其他元件可由宿主细胞或在体外表达***中提供。在本发明中，表达载体可以包括本领域已知的所有表达载体，包括粘粒、质粒(例如，裸露的或包含在脂质体中)和掺有重组多核苷酸的病毒，例如慢病毒、逆转录病毒、腺病毒、单纯疱疹病毒和腺伴随病毒，但不限于此。
在一个实施方案中，所述TCR表达载体、所述MHC表达载体和所述表达所述TCR激活响应元件和所述报告分子的载体各自独立地为病毒表达载体，例如慢病毒表达载体、逆转录病毒载体、腺病毒载体、单纯疱疹病毒载体或腺伴随病毒载体。
在本文中，所谓“抗原肽”是指具有抗原表位并因此能被TCR识别的多肽。此外，在本发明中，术语“抗原”多指肿瘤抗原，即由肿瘤细胞或癌细胞单独或主要表达或过表达的任何分子，例如蛋白质、多肽、肽、脂质、碳水化合物等。肿瘤抗原也可以由正常细胞、非肿瘤细胞或非癌性细胞表达。然而，在此类情况下，正常细胞、非肿瘤细胞或非癌性细胞对肿瘤抗原的表达不如肿瘤细胞或癌细胞对肿瘤抗原的表达稳健。在这方面，与正常细胞、非肿瘤细胞或非癌性细胞表达抗原相比，肿瘤细胞或癌细胞可以过表达抗原或以显著更高的水平表达抗原。此外，肿瘤抗原可以由处于不同发育或成熟状态的细胞表达。例如，肿瘤抗原可以另外由胚胎期或胎儿期的细胞表达，所述细胞通常在成年宿主中不存在。可替代地，肿瘤抗原可以另外由干细胞或前体细胞表达，所述细胞通常在成年宿主中不存在。肿瘤抗原是本领域已知的，并且包括例如间皮素、CD19、CD22、CD276(B7H3)、gp100、MART-1、表皮生长因子受体变体III(EGFRVIII)、TRP-1、TRP-2、酪氨酸酶、突变的KRAS、NY-ESO-1(也称为CAG-3)、MAGE-1、MAGE-3等，但不限于此。此外，肿瘤细胞染色体不稳定，同时表观水平调控(如甲基化等)发生 异常，常常产生突变蛋白或融合蛋白，此类蛋白可以成为免疫治疗的靶点，被称为肿瘤新抗原(neoantigen)。
在一个实施方案中，所述抗原肽为肿瘤抗原肽。例如序列为SLLMWITQC(SEQ ID NO.1)的NY-ESO-1多肽、或者序列为LAGIGILTV(SEQ ID NO.2)的MART-1 (27-35)多肽，但不限于此。在一个实施方案中，本发明的方法可以包含五个步骤：1)细胞株构建；2)通过CASB策略标记细胞标签；3)利用液滴包裹细胞和细胞反应；4)液滴注射；5)单细胞测序；以及6)测序数据分析。具体步骤描述如图1所示。图1示出了根据本发明一个实施方案的方法示意图。以下对步骤分别进行描述：
(1)细胞株构建。首先构建两种细胞株，本领域技术人员可以理解，两种细胞株的构建没有先后次序：1)构建MHC慢病毒载体，并用所构建的MHC慢病毒载体转染APC细胞(如：T2细胞)，制备MHC稳定转染的APC细胞株，然后将抗原肽和所述稳定转染的APC细胞共同孵育，将多肽加载到APC细胞的MHC上；2)构建TCR慢病毒载体和增强型mCherry载体，并制备TCR文库，用所制备的TCR文库转染T细胞(如：Jurkat细胞)，制备稳定表达TCR和mCherry的T细胞株。其中，在mCherry上游加入激活T细胞核转录因子(NFAT)反应元件，用于检测TCR信号的激活。TCR文库可以包含多种不同的TCR，每个T细胞转染一种TCR。而抗原肽来自于：从肿瘤组织中分离出肿瘤细胞，通过质谱分析肿瘤细胞从而获得可能的肿瘤抗原，并根据抗原序列合成该抗原肽。
(2)为细胞添加标签。为不同类型的细胞(APC细胞和T细胞)添加不同的细胞标签ssDNA，以便于在后期单细胞测序数据分析时，拆分出液滴当中含有哪些细胞，哪些细胞之间发生了相互作用，从而实现液滴内信息的可视化。对于高通量检测，不同的细胞标签用于标记表达不同TCR的第一细胞(T细胞)，以及用于标记加载不同抗原肽的第二细胞(APC细胞)。因此，可以制备多种第一细胞，用于表达不同TCR，制备多种第二细胞，用于加载不同抗原肽；多种第一细胞分别用多种不同第一标签序列进行标记，多种第二细胞分别用多种不同第二标签序列进行标记。第一标签序列和第二标签序列可以均为DNA序列，长度为20-200bp，优选为100bp。第一标签序列与第二标签序列包含相同的共有序列，该共有序列可与磁珠上的捕获序列通过碱基互补配对进行结合，以实现细胞标签序列的捕获。
(3)液滴生成。使用液滴生成芯片(参见PCT/CN2019/108536，参见图1)，在芯片的两个加样孔内分别加入负载有抗原肽的APC细胞和磁珠，以及转染了mCherry报告分子的T细胞和磁珠；为了能够特异性地筛选TCR，调整细胞至合适浓度此浓度需要使大多数液滴中，每个液滴包裹有一个APC细胞，以及一个T细胞。具体设计为：按照泊松分布进行计算，使投入的细胞数符合λ＝1(即产生的液滴中单项细胞单胞的概率最高)，两种细胞都按照λ＝1投入，保证产生含有一个T细胞和1个APC细胞的目标液滴概率最高。使用着这种设计方案，尽可能地提升了APC细胞及T细胞的利用率。液滴生成后，将其置于37摄氏度、0.5％二氧化碳培养箱中，孵育4小时以实现细胞之间的相互作用，然后取出液滴。
(4)液滴注射。将裂解液注射进去以使细胞裂解，释放mRNA和细胞标签ssDNA。每个磁珠表面携带有独一无二的用于标记识别磁珠身份的唯一编码序列，与唯一编码序列相连接的是磁珠的捕获序列，捕获序列分为两种，一种是第一捕获序列，用于捕获样本靶标序列，例如使用TTTTTTTTTTTT(SEQ ID NO.3)序列作为第一捕获序列，它能够与样本mRNA的poly A尾互补，从而捕获细胞释放出来的mRNA；另一种是第二捕获序列，它是人为设计的固定序列，能够与细胞标签ssDNA上的共有序列(如GACGCTGCCGACGA(SEQ ID NO.4)序列)互补配对，从而捕获细胞标签ssDNA。正是由于存在唯一编码序列以及两种捕获序列，因此，利用磁珠的唯一编码序列可以识别出来自于同一个液滴的ssDNA和mRNA信息，进而完成mRNA信息和ssDNA信息的组装。
(5)mRNA反转录获得cDNA，细胞标签ssDNA进行PCR扩增，然后构建测序文库，并上机测序。
(6)数据处理与拆分。将带有相同的唯一编码序列的测序数据分为一组，并在组内进一步筛选出仅含有两种细胞标签信息(一种标签来自于APC、另一种标签来自于T细胞)的测序数据。然后对下机数据中T细胞的mRNA表达情况进行分析，重点关注T细胞激活相关的几个标志基因及mCherry基因的表达水平。由于细胞标签序列ssDNA是已知的，因此利用细胞标签序列即可分析出是哪两种细胞进行了互作。对于被激活的T细胞，通过数据中的ssDNA信息判断出是哪种抗原肽对它进行了刺激，也可根据ssDNA信息判断出是哪种第一细胞(T细胞)被激活，从而可以筛选出抗原肽，以及与该抗原肽具有配对关系的TCR序列。
实施例：两种抗原肽与其对应的TCR的配对关系的检测。
1、实验材料。
1)能识别DMF5并表达mCherry的Jurkat细胞，能识别IG4并表达mCherry的Jurkat细胞。上述两种细胞利用文献The Targeting Effect of Cetuximab Combined with PD-L1Blockade against EGFR-Expressing Tumors in a Tailored CD16-CAR T-Cell Reporter System(DOI:10.1080/07357907.2021.1894570)披露的方法进行构建，即通过慢病毒转染的方法将对应的激活T细胞核转录因子(NFAT)反应元件和mCherry序列导入到野生型Jurkat细胞(ATCC，TIB-152)内，从而获得这两种细胞。
2)表达MHC的T2细胞(Shanghai EK-Bioscience Biotechnology Co.,Ltd，CC-Y1650)、IG4多肽(Mart-1，LAGIGILTV，SEQ ID NO.2)、DMF5多肽(NY-ESO-1，SLLMWITQC，SEQ ID NO.1)。
3)CASB相关试剂：链霉亲和素(BBI C600432-0005)、伴刀豆球蛋白A(SIGMA L7647-100mg)、四种ssDNA(由北京六合合成)。
四种ssDNA序列分别为：
Figure PCTCN2022129973-appb-000001
下划线序列为细胞标签序列，在下机数据分析时，用于匹配拆分细胞。使用该序列在下机测序数据的读段中匹配，匹配上该序列的则认为该液滴中带有对应的ssDNA，从而判断出液滴中含有被标记了这种ssDNA的细胞；NNNNNNN区为测序的暗反应区，该部分序列无法通过测序获得，因而可以为随机序列；3’端GACGCTGCCGACGA(SEQ ID NO.9)为细胞标签的共有序列，该序列可以与磁珠上的人为设定的固定序列(即第二捕获序列)互补，用于后续磁珠的捕获。
4)液滴生成油(Bio-Rad，货号1863005)。
5)液滴生成装置(参见PCT/CN2019/108536，CN209144161U)。
6)液滴注射装置。
7)建库试剂盒：DNBelab C系列高通量单细胞RNA文库制备试剂盒(MGI，94-000047-00
2、实验步骤。
(1)多肽负载T2细胞的制备。
将T2细胞用IMDM完全培养基(IMDM完全培养基配方：89％IMDM培养基：GIBCO，12440053；10％FBS：HYCLONE SH30084.03HI；1％PS：HYCLONE，SV30010)调整到1*10 6个/ml的浓度，分别取1ml放置于24孔板的两个孔(一个孔内的细胞悬液标记为细胞悬液C，另外一个孔内的细胞悬液标记为细胞悬液D)，加3μL的10μg/μL的IG4多肽与细胞悬液C过夜共刺激，加入3μL的10μg/μL的DMF5多肽与细胞悬液D过夜共刺激。第二天离心弃上清，将负载了不同多肽的T2细胞分别重悬于1ml DPBS(GIBCO，C14190500BT)中。
(2)利用CASB策略为细胞添加标签。
将识别DMF5并表达mCherry的Jurkat细胞使用1640完全培养基(1640完全培养基配方：89％1640培养基；10％FBS：1％PS)调整到1*10 6个/ml的浓度(标记为细胞悬液A)。将识别IG4并表达mCherry的Jurkat细胞使用1640完全培养基调整到1*10 6个/ml的浓度(标记为细胞悬液B)。使用4种不同的ssDNA作为标签，分别构建4种CASB：在8μL ssDNA中加入2μL链霉亲和素，室温反应10分钟，再加入2μL伴刀豆球蛋白A，室温反应10分钟即构建好CASB。在4种CASB中，分别加入细胞悬液A、B、C和D，4℃反应10分钟，用DPBS清洗3遍，离心弃上清。CASB策略参见"CASB:a concanavalin A-based sample barcoding strategy for single-cell sequencing."Mol Syst Biol 17(4):e10060(2021)。每一种细胞悬液中的细胞被添加上对应的ssDNA标签。具体地，T2-IG4细胞标记ssDNA-1、T2-DMF5细胞标记ssDNA-2、Jurkat-IG4细胞标记ssDNA-3、Jurkat-DMF5细胞标记ssDNA-4。
(3)液滴生成。
按照PCT/CN2019/108536和CN209144161U披露的方法搭建液滴生成装置，并向液滴收集管中加入100μL液滴生成油作为缓冲液，使用液滴生成芯片，在一个加样 孔内加入步骤(2)获得的添加标签后的负载有IG4和DMF5的T2细胞混合液和磁珠，在另一个加样孔内加入步骤(2)获得的添加标签后的表达IG4和DMF5的Jurkat细胞的混合液和磁珠，并且细胞投入量为20000个/μL，磁珠投入量为10000个/μL。然后，将加样孔内的磁珠和细胞重悬于25μL工作液(工作液配方：5％FBS、2.5％HEP、1％PS、1％F68、30％Fical、60.5％纯培养基)中，在加油孔内加入400μL的液滴生成油，拉动液滴生成装置的注射器从刻度15至20以提供压力，共生成50μL液滴于收集管中。将收集管放在37℃温箱内孵育4小时。
(4)液滴注射。
搭建如图2所示的液滴注射装置。图2中A是注射装置实物图，其由“油相通道、液滴相通道和裂解液相通道”三个单元组成。图2中B是液滴注射装置的原理图。
从步骤(3)所获得的收集管中吸出液滴30μL至200μL枪头中，并置于注射装置的液滴相通道；用0.22μm无菌针头式过滤器(MILLIPORE，SLGP033RB)的过滤液滴生成油100μL，置于注射装置的油相通道；在裂解相通道中加入裂解液(DNBelab C系列高通量单细胞RNA文库制备试剂盒中配套的裂解液)100μL。调试机器电压(频率：15kHZ；电压：1VPP；电压放大器：200倍)，将液滴注射装置从刻度17.5拉到20(提供足够的压力)完成注射，注射后的液滴将放置于新的收集管中。
(5)建库。
使用华大智造的单细胞测序试剂盒——DNBelab C系列高通量单细胞RNA文库制备试剂盒进行。简而言之，将注射后的液滴在室温下孵育30分钟以使细胞裂解完全，然后进行破乳回收。
对样本依次进行逆转录反应、二链合成、cDNA产物扩增及扩增产物分选，获得第一次扩增产物。第一次扩增产物包含(1)磁珠捕获的细胞mRNA经逆转录获得的cDNA产物；(2)用于标记细胞的ssDNA，经扩增后获得的oligo产物。
对cDNA产物进行文库构建：cDNA打断、对打断产物进行片段筛选、末端修复、接头连接、产物纯化、cDNA产物二次扩增及纯化，目的是用于筛掉接头及其他不属于细胞mRNA逆转录出来的小分子片段。随即获得测序文库。
对oligo产物进行文库构建：oligo产物的二次扩增及片段筛选。由于此前获得的oligo产物中会含有很多磁珠上的接头，二次扩增的目的主要是为了去除接头等短的不属于用于细胞标记的ssDNA扩增出来的DNA片段。随即获得测序文库。
将上述两种文库进行高温变性、单链环化以及酶切等操作，使两种文库都变成符合单细胞测序要求的单链环化DNA文库。
(6)上机测序。
使用华大的单细胞测序平台DNBSEQ-T7，对步骤5获得的文库进行测序。
(7)数据处理分析。
按照唯一编码序列进行分组，将仅含有两种细胞标签信息(一种标签来自于T2、另一种标签来自于Jurkat细胞)的测序数据筛选出来。具体地，关注带有(ssDNA-1+ssDNA-3)、(ssDNA-1+ssDNA-4)、(ssDNA-2+ssDNA-3)或(ssDNA-2+ssDNA-4)这四种序列的液滴。对测序下机数据中的T细胞mRNA表达情况进行分析，重点关注T细胞激活相关的几个标志基因及mCherry基因的表达水平。由于细胞标签ssDNA序列是已知的，因此利用细胞标签序列即可分析出是哪两种细胞进行了相互作用。对于被激活的T细胞，通过数据中的ssDNA信息判断哪种抗原肽对它进行了刺激，以及哪种第一细胞(T细胞)被激活，从而筛选出抗原肽与TCR的对应关系。即筛选出携带有(ssDNA-1+ssDNA-3)或(ssDNA-2+ssDNA-4)这两种细胞标签的阳性液滴，并分析液滴中的细胞的mCherry基因及激活相关基因高表达情况。
此外，为了证明流程的可行性，还需要关注不配对的液滴是否符合Jurkat细胞未被激活的情况。具体地，关注带有(ssDNA-1+ssDNA-4)及(ssDNA-2+ssDNA-3)标签序列的阴性液滴中，细胞mCherry基因及激活相关基因是否低表达或不表达。
结果如图3所示。图3展示了三种液滴：positive 1为ssDNA-1+ssDNA-3的IG4阳性液滴；positive 2为ssDNA-2+ssDNA-4的DMF5阳性液滴，negative为(ssDNA-2+ssDNA-3)和(ssDNA-1+ssDNA-4)阴性液滴。从图3可以看到，T细胞激活的标志基因和mCherry基因表达情况完全符合预期情况：阳性组(positive)表达情况明显高于阴性组(negative)。即可判断阳性组液滴中的细胞间发生了互作。

Claims (26)

  1. 一种检测细胞间相互作用的方法，所述方法包括步骤：
    (1)制备表达TCR激活响应元件和报告分子的第一细胞，所述TCR激活响应元件位于所述报告分子上游，所述第一细胞表达TCR；
    (2)将抗原肽与表达MHC的第二细胞孵育，使抗原肽加载到所述第二细胞的MHC上，以形成抗原肽-MHC复合物，得到加载抗原肽的第二细胞；
    (3)使用第一标签序列对所述第一细胞进行标记，得到携带标签序列的第一细胞，以及使用第二标签序列对所述加载抗原肽的第二细胞进行标记，得到携带标签序列的第二细胞，其中所述第一标签序列与第二标签序列包含有相同的共有序列，并且所述第一标签序列与第二标签序列包含有彼此不相同的特异性序列；
    (4)使用油相包裹所述携带标签的第一细胞、所述携带标签的第二细胞和磁珠以制备多个液滴，其中，所述多个液滴中至少一部分的单个液滴中仅包含一个所述第一细胞、一个所述磁珠和至少一个所述第二细胞，并且所述磁珠表面携带有唯一编码序列、第一捕获序列和第二捕获序列，所述第一捕获序列用于捕获细胞靶标序列，所述第二捕获序列与细胞标签序列中的共有序列互补，用于捕获所述细胞标签序列，每个磁珠的唯一编码序列彼此不同，用于识别磁珠身份；
    (5)向单个液滴中注入细胞裂解液，将细胞裂解，释放并通过所述磁珠捕获细胞靶标序列和细胞标签序列，对捕获后获得的序列进行测序，得到测序数据；
    (6)对包含相同唯一编码序列的测序数据进行分析，确定单个液滴中第一细胞与第二细胞是否存在相互作用。
  2. 根据权利要求1所述的方法，在(5)中，对捕获后获得的序列进行扩增与文库构建，对获得的测序文库进行测序。
  3. 根据权利要求2所述的方法，在(5)中，捕获后获得的序列包含mRNA和DNA，对所述mRNA进行逆转录、二链合成、cDNA产物扩增，并对扩增的cDNA产物进行文库构建，获得第一测序文库；对所述DNA进行二次扩增及片段筛选，获得第二测序文库；将所述第一和第二测序文库合并进行测序。
  4. 根据权利要求1所述的方法，在(1)-(3)中，制备多种所述第一细胞，用于表达不同TCR，制备多种所述第二细胞，用于加载不同抗原肽；所述多种第一细胞分别用多种不同第一标签序列进行标记，所述多种第二细胞分别用多种不同第二标签序列进行标记。
  5. 根据权利要求1-4任一项所述的方法，在(1)中，所述第一细胞为T细胞，优选为Jurkat细胞或人来源T淋巴细胞。
  6. 根据权利要求1-4任一项所述的方法，在(1)中，构建TCR慢病毒载体和报告分子载体，与TCR序列共转染至第一细胞中，制备表达TCR和报告分子的第一细胞。
  7. 根据权利要求1-4任一项所述的方法，在(1)中，所述TCR激活响应元件为活化T细胞核因子、AP1转录因子或NF-κB。
  8. 根据权利要求1-4任一项所述的方法，在(1)中，所述报告分子为荧光蛋白，优选为绿色荧光蛋白、红色荧光蛋白、黄色荧光蛋白或者蓝色荧光蛋白。
  9. 根据权利要求1-4任一项所述的方法，在(2)中，所述第二细胞为抗原呈递细胞细胞，优选为T2细胞或K562细胞。
  10. 根据权利要求1-4任一项所述的方法，在(2)中，构建MHC慢病毒载体，转染至所述第二细胞中，制备表达MHC的第二细胞。
  11. 根据权利要求1-4任一项所述的方法，在(2)中，所述抗原肽为肿瘤特异性抗原肽，优选为序列为SLLMWITQC的NY-ESO-1多肽或者序列为LAGIGILTV的MART-1 (27-35)多肽。
  12. 根据权利要求1-4任一项所述的方法，在(3)中，利用CASB策略对第一细胞和第二细胞进行标记。
  13. 根据权利要求12所述的方法，所述第一标签序列和所述第二标签序列均为DNA序列。
  14. 根据权利要求13所述的方法，所述DNA序列长度为20-200bp，优选为100bp。
  15. 根据权利要求1-4任一项所述的方法，在(4)中，使用液滴微流控技术制备所述液滴。
  16. 根据权利要求15所述的方法，使用液滴生成芯片，在两个加样孔内分别加入携带标签的第一细胞和磁珠、以及携带标签的第二细胞和磁珠。
  17. 根据权利要求1-4任一项所述的方法，在(4)中，通过调整所述携带标签的第一细胞和所述携带标签的第二细胞的浓度，实现所述多个液滴中至少一部分的单个液滴中仅包含一个所述第一细胞和至少一个所述第二细胞。
  18. 根据权利要求17所述的方法，按液滴中单项细胞单胞的概率最高的浓度加入所述第一细胞和所述第二细胞。
  19. 根据权利要求1-4任一项所述的方法，在(4)液滴生成后包括孵育步骤，优选孵育时间为4-8小时。
  20. 根据权利要求1-4任一项所述的方法，在(5)中，使用液滴注射装置进行裂解液注射。
  21. 根据权利要求1-4任一项所述的方法，在(6)中，筛选出包含相同的唯一编码序列、且具有两种细胞标签序列的测序数据，根据测序数据中第一细胞的报告分子表达水平，确定第一细胞所表达的TCR与第二细胞的MHC-抗原肽复合物之间是否发生特异性识别，若发生特异性识别则判定单个液滴中第一细胞与第二细胞存在相互作用。
  22. 根据权利要求21所述的方法，在(6)中，判定第一细胞与第二细胞存在相互作用，进一步利用测序数据中的细胞标签序列得到对应的第一细胞与第二细胞。
  23. 根据权利要求1-4任一项所述的方法，细胞间相互作用包括细胞通过分泌化学信号进行细胞间的通讯、细胞间接触依赖性通讯和动物相邻细胞间形成间隙连接中的任意一种或多种。
  24. 权利要求1-23任一项的方法在高通量筛选抗原及其配对的T细胞受体中的应用，其中所述第一细胞是表达TCR的T细胞，所述第二细胞是加载抗原肽的APC细胞，所述T细胞有多种，用于表达不同TCR，所述APC细胞有多种，用于加载不同抗原肽；所述多种T细胞分别用多种不同第一标签序列进行标记，所述多种APC 细胞分别用多种不同第二标签序列进行标记。
  25. 根据权利要求24的应用，所述TCR为肿瘤特异性T细胞受体。
  26. 一种高通量筛选抗原及其配对的T细胞受体的方法，包括使用权利要求1-23任一项的方法筛选抗原及其配对的T细胞受体，其中所述第一细胞是表达TCR的T细胞，所述第二细胞是加载抗原肽的APC细胞，所述T细胞有多种，用于表达不同TCR，所述APC细胞有多种，用于加载不同抗原肽；所述多种T细胞分别用多种不同第一标签序列进行标记，所述多种APC细胞分别用多种不同第二标签序列进行标记。
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