WO2023019583A1 - 一种高通量检测和分选抗原特异性t细胞受体的方法 - Google Patents

一种高通量检测和分选抗原特异性t细胞受体的方法 Download PDF

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WO2023019583A1
WO2023019583A1 PCT/CN2021/113888 CN2021113888W WO2023019583A1 WO 2023019583 A1 WO2023019583 A1 WO 2023019583A1 CN 2021113888 W CN2021113888 W CN 2021113888W WO 2023019583 A1 WO2023019583 A1 WO 2023019583A1
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cell
cells
droplet
droplets
tcr
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PCT/CN2021/113888
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French (fr)
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王诗雨
李毅坚
高开
刘亚
董璇
刘杨
张隽语
陈琳喆
吕孟华
孙娜
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深圳华大生命科学研究院
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Priority to CN202180096479.1A priority Critical patent/CN117083516A/zh
Priority to PCT/CN2021/113888 priority patent/WO2023019583A1/zh
Publication of WO2023019583A1 publication Critical patent/WO2023019583A1/zh

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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • the invention belongs to the technical field of biological immune detection, in particular to a method for high-throughput detection and sorting of antigen-specific T cell receptors (TCR).
  • TCR antigen-specific T cell receptors
  • antigen-specific TCR In order to verify the antigen-specific TCR, researchers have developed over the past three decades including antigen-specific T cell expansion, major histocompatibility complex (MHC) tetramer labeling, flow cytometry sorting of antigen-reactive TCR These methods have been widely used in scientific research and clinical practice related to immunotherapy, but such technologies have major defects.
  • MHC major histocompatibility complex
  • Antigen-specific T cell expansion technology began to be applied in the 1990s, and is currently the mainstream method for expanding antigen-specific T cells from tumor patients.
  • This method needs to collect a large amount of peripheral blood from the patient, induce the monocytes in it to differentiate into antigen-presenting cells, load tumor antigens, and then co-culture with the patient's own T cells. After three generations of expansion, it takes 1-2 months Only then can antigen-specific T cells be obtained.
  • the advantage of this method is that it fully utilizes the first signal and the second signal (CD28-CD40L) required for T cell activation, and can prove that the corresponding TCR has physiological functions while verifying the antigen specificity of T cells.
  • MHC tetramer technology was developed by Mark Davis' group in the late 1990s. This technology overcomes the shortcomings of MHC monomer instability in vitro. Users can construct MHC tetramers, use ultraviolet light to load peptides of specified length (8-12 peptides) onto the tetramers, and add Labels, such as fluorescent molecules, use the principle of MHC-peptide-TCR specific recognition to mark cells expressing antigen-specific TCR, and then use flow cytometry for cell sorting and enrichment.
  • Labels such as fluorescent molecules
  • MHC-peptide-TCR specific recognition to mark cells expressing antigen-specific TCR
  • flow cytometry for cell sorting and enrichment.
  • the advantage of this technique is that the detection is fast, requires fewer cells than the amplification method, and has higher specificity. However, it also has many disadvantages, such as high cost of tetramer preparation and cumbersome experimental operations.
  • TCR-MHC due to the limited affinity of TCR-MHC, the fluorescent intensity of the label is limited, and the detection of trace antigen T cells fails.
  • T cell activation molecular markers CD154 and CD137, as markers of TCR activated cells, were used to sort antigen-specific T cells.
  • this method is very convenient to use, it is greatly affected by the bystander effect (activated T cells will activate its adjacent non-specific T cells), which leads to a high false positive rate of TCR screened by this method.
  • the MHC-expressing cells phagocytized the antigen and presented it to the TCR through the MHC.
  • the TCR signal will activate the intracellular luciferase, and after adding the enzyme substrate, the fluorescence released by the enzyme chemical reaction can be detected.
  • the advantages of the above-mentioned technology are: compared with MHC tetramers, the expression of enzymes is further utilized, the fluorescent signal is amplified, and traces of antigen-specific cells can be detected; it can prove that TCR intracellular signals have physiological functions.
  • the above methods rely on the presence of enzyme substrates and can only be done in well plates. At the same time, in order to identify the functioning TCR, only one TCR can be added to each well. Therefore, this method has obvious flux-limiting defects.
  • the present invention provides a method for high-throughput detection and sorting of antigen-specific T cell receptors (TCR), thereby solving the problems mentioned in the background technology section herein.
  • the present invention provides a method for detecting an antigen-specific T cell receptor (TCR), said method comprising the steps of:
  • MHC major histocompatibility complex
  • the first oil phase is used to wrap the first cell and the second cell loaded with the antigen polypeptide to prepare a droplet, wherein at least a part of the droplet is A single droplet contains only one of said first cell and at least one of said second cell;
  • said droplets are incubated prior to step d), preferably for a period of no more than 8 hours.
  • the first cell is transformed by co-incubating the source cell of the first cell, the TCR expression vector, the vector expressing the TCR activation response element and the reporter molecule, and optionally
  • the second cell is prepared by co-incubating the source cell of the second cell, the MHC expression vector and an optional transfection-promoting reagent.
  • 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, reverse transcription Viral vectors, adenoviral vectors, herpes simplex viral vectors or adeno-associated viral vectors.
  • a viral expression vector such as a lentiviral expression vector, reverse transcription Viral vectors, adenoviral vectors, herpes simplex viral vectors or adeno-associated viral vectors.
  • the transfection-promoting reagent is heidemine, Lipofectamine2000.
  • the source cell of the first cell is T cell lymphoma cell, preferably Jurkat cell or T lymphocyte derived from human; the source cell of the second cell is antigen presenting cell, preferably T2 cells or K562 cells.
  • the MHC is MHC class I, MHC class II or MHC class III, preferably MHC class I.
  • the TCR activation response element is 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
  • NF- ⁇ B 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 antigen polypeptide is a tumor antigen polypeptide, preferably NY-ESO-1 polypeptide with sequence SLLMWITQC or MART-1 (27-35) polypeptide with sequence LAGIGILTV.
  • the first cell comprises a plurality of cells, and at least some of the plurality of cells express different types of TCRs.
  • step c) at least a part of the individual droplets in the droplets only contain one of the first cell and at least one of the second cell by setting The diameter of the droplet, the multipack rate of the first cell and the proportion of empty droplets of the second cell are determined according to the Poisson distribution of the working concentration of the first cell and the working concentration of the second cell:
  • k is the number of cells in the droplet and is an integer > 0,
  • C is the cell suspension concentration (cell number/ ⁇ L)
  • ⁇ is the expected value
  • f(k; ⁇ ) is the probability of wrapping k cells in the droplet when k and ⁇ are determined
  • r is the droplet radius ( ⁇ m).
  • the diameter of the droplet is set to be 30 ⁇ m to 100 ⁇ m, preferably 30 ⁇ m to 80 ⁇ m, the multipack rate of the first cell is ⁇ 5% and the empty droplet ratio of the second cell is ⁇ 70%.
  • the working concentration of the first cells is 1 ⁇ 10 4 cells/mL to 4 ⁇ 10 6 cells/mL
  • the working concentration of the second cells is The working concentration is ⁇ 1.5 ⁇ 10 7 cells/mL.
  • the present invention provides a method of sorting antigen-specific T cell receptors (TCRs), said method comprising the steps of:
  • a single droplet contains only one of said first cell and at least one of said second cell;
  • the droplet When the droplet is detected to emit light, the light signal generated by the droplet is converted into a deflection force, and the deflection force is used to sort the containing
  • the droplets are incubated prior to step 2), preferably for a period of no more than 8 hours.
  • the droplets are passed individually through the laser irradiation site by dispersing the droplets in a second oil phase as a sheath fluid and flowing the second oil phase past the laser irradiation site. point.
  • the droplets are passed individually through the laser irradiation site by adjusting the concentration of the droplets in the second oil phase and adjusting the speed at which the second oil phase flows past the laser irradiation site.
  • said deflecting force is generated by converting said optical signal into an electrical signal.
  • said deflecting force is generated by dielectrophoresis or acoustic waves generated by said electrical signal.
  • the present invention provides a method for RNA sequencing, said method comprising the steps of:
  • the sequencing method is a high throughput sequencing method.
  • the method is a single cell RNA sequencing method.
  • step ii. of recovering cells in said positive droplets comprises the steps of:
  • the obtained liquid is subjected to low-speed centrifugation, preferably 30g-100g, more preferably 40g-70g, most preferably 50g, and the centrifugation lasts for 8-15 seconds, preferably 9-12 seconds, most preferably 10 seconds, thus the The liquid forms upper and lower layers with an interface in between;
  • the demulsifier is selected from the group consisting of tannin-based mixtures, ethoxylated or epoxidized PAGs, oxyalkylated poly(alkylene)poly(amines), poly(ether)poly( urethane) and octanols, preferably octanols, more preferably n-octanol, isooctyl alcohol, butanol, 1H, 1H, 8H-perfluoro-1-octanol or 1H, 1H, 2H, 2H - Perfluoro-1-octanol.
  • the positive droplet is located in the upper layer, and the droplet-forming oil is located in the lower layer, and it is preferable to remove part of the droplet-forming oil in the lower layer before adding the demulsifier.
  • the volume of the demulsifier added is 1 times the volume of the positive droplet.
  • the volume of demulsifier added again is less than the volume of demulsifier added in (1).
  • the present invention provides a use of the sequence information of the antigen-specific TCR obtained by the method of the third aspect of the present invention in cellular immunotherapy.
  • the cellular immunotherapy includes constructing TCR-T and/or CAR-T cells.
  • the present invention can screen a variety of TCRs with high throughput: the present invention can prepare millions of liquid droplets at one time by means of droplet microfluidic technology, and the proportion of living cells in the whole preparation process is not less than 70%. Living cells are suitable for subsequent single-cell RNA sequencing; and the detection of antigen specificity can be independently completed in the droplet. Compared with the detection of cell interaction in the well plate, the throughput of the present invention is significantly improved.
  • the present invention has better expansibility: compared with the prior art, the present invention can not only verify TCR specificity, but also be used for screening of antigen-specific TCR and recovery of TCR information. At the same time, the TCR data information obtained in the present invention can be used for subsequent construction of TCR-T/CAR-T cells, thereby providing greater convenience for the realization of TCR-T/CAR-T cell immunotherapy.
  • Figure 1 is a schematic diagram of one embodiment of a droplet generation device useful in carrying out the method of the present invention.
  • Figure 2 is a schematic diagram of one embodiment of a cell sorting chip useful for carrying out the methods of the present invention.
  • Fig. 3 shows a schematic diagram of the optical device in the sorting platform.
  • FIG. 4 shows a flow chart of optical signal-voltage signal processing.
  • Fig. 5 shows the change of GFP protein expression in T cells with pMHC stimulation time.
  • Figure 6 shows the statistical results of the viability of encapsulated cells as a function of incubation time.
  • Figure 7 shows the encapsulation rate statistics of cells in droplets.
  • Figure 8 shows the proportion of T cells expressing GFP stimulated by different concentrations of pMHC in the droplet.
  • Fig. 9 shows a statistical diagram of detecting the sorting effect of the method of the present invention based on flow cytometry.
  • Figure 10 shows the results of single-cell sequencing of sorted T cells.
  • Figure 11 shows the quality control results of single-cell sequencing.
  • the present invention aims to solve the technical problem of how to screen antigen-specific TCRs in high throughput.
  • the inventor constructed two kinds of cell lines: 1) construct MHC lentiviral vector, make it transfect T2 cell, prepare MHC stable transfection cell line thus; 2) construct TCR lentiviral vector and enhance Type GFP vector to prepare TCR library and transfect Jurkat cells to prepare cell lines stably expressing TCR and GFP; wherein, the nuclear factor of activated T cells (NFAT) response element was added upstream of GFP to detect the activation of TCR signal.
  • T2 cells were incubated with antigenic peptides, and then prepared into droplets together with Jurkat cells by droplet microfluidic technology.
  • the present invention provides a method for detecting an antigen-specific T cell receptor (TCR), said method comprising the steps of:
  • MHC major histocompatibility complex
  • the first oil phase is used to wrap the first cell and the second cell loaded with the antigen polypeptide to prepare a droplet, wherein at least a part of the droplet is A single droplet contains only one of said first cell and at least one of said second cell;
  • T cell receptor refers to a molecule or molecular complex on a T cell that is capable of receiving signals and transmitting them across the cell membrane.
  • the term “antigen specificity” refers to the property that TCR can specifically bind and recognize its corresponding antigen or antigen epitope, thereby triggering an immune response.
  • MHC major histocompatibility complex
  • H-2 complex MHC in mice
  • HLA human leukocyte antigen
  • corresponding MHCs also exist in other animals, for example, rabbit leukocyte antigen (RLA), guinea pig leukocyte antigen (GPLA), house owner leukocyte antigen (SLA), dog leukocyte antigen (DLA) or monkey leukocyte antigen (RhLA).
  • RLA rabbit leukocyte antigen
  • GPLA guinea pig leukocyte antigen
  • SLA house owner leukocyte antigen
  • DLA dog leukocyte antigen
  • RhLA monkey leukocyte antigen
  • MHC can be divided into three gene groups, namely MHC-I, MHC-II and MHC-III.
  • MHC-I class includes three gene loci, namely A, B, and C, and its encoded product is MHC-I class molecule or antigen;
  • MHC-II class includes three subregions of DP, DQ, and DR,
  • the classic products encoded by it are MHC-II molecules or antigens, as well as LMP and TAP related to endogenous antigen processing;
  • MHC class III genes encode MHC-III molecules or antigens.
  • All MHC class I molecules contain two unconnected polypeptide chains: one MHC-encoded ⁇ -chain or weighing chain, about 44 ⁇ 10 3 (for humans) or about 47 ⁇ 10 3 (for mice); One ⁇ -strand ( ⁇ 2-microglobulin) encoded by an independent chromosomal gene is 12 ⁇ 10 3 for both human and mouse.
  • the ⁇ chain is formed by a core polypeptide chain of about 40 ⁇ 10 3 , one (human) or two (mouse) oligosaccharides are connected to the N-terminus, and 3/4 of the ⁇ chain is outside the cell membrane, including the amino terminal and oligosaccharides.
  • the sugar group, the transmembrane region is a short hydrophobic segment, and the cytoplasmic region is the carboxyl terminal of 30 amino acid residues.
  • the ⁇ chain is non-covalently bound to the extracellular part of the heavy chain, and the ⁇ chain is not directly connected to the cell.
  • All MHC-II molecules are composed of two polypeptide chains connected by non-covalent bonds.
  • the overall structure of the two chains is similar.
  • the ⁇ chain (33 ⁇ 10 3 to 34 ⁇ 10 3 ), due to its extensive glycosylation , and thus slightly longer than the beta chain (29 ⁇ 10 3 to 32 ⁇ 10 3 ).
  • Both polypeptide chains contain oligosaccharides connected to the N-terminus, the extracellular is the amino-terminal, and the intracellular is the carboxyl-terminal. 2/3 of each chain is located outside the cell.
  • the two chains are encoded by different MHC genes and have multiple behavior (with few exceptions).
  • the molecular structure of these two types of glycoproteins has a common feature, that is, there is a groove on the surface.
  • the molecular groove of MHC-I is shallow and can accept peptide chains composed of 8-20 amino acids; the molecular groove of MHC-II is deep and can accept longer peptide chains.
  • MHC-I molecules mainly present intracellular antigens, which undertake the immune tolerance of normal cells and regulate the clearance of senescent cells; in tumor immunity, killer T cells recognize the abnormal antigens presented by MHC-I molecules on the surface of tumor cells, thereby Recognize tumor cells and complete the killing of tumor cells with the help of cytotoxic mechanisms such as perforin.
  • MHC-II molecules mainly present extracellular antigens, which are expressed on the surface of professional antigen-presenting cells (APCs) in humans.
  • APCs professional antigen-presenting cells
  • APC engulfs external pathogenic microorganisms such as bacteria and parasites, and is digested by intracellular lysosomes to form polypeptide fragments, which form complexes with MHC-II molecules and are presented on the cell surface.
  • MHC-II molecules The role of MHC-II molecules in tumor immunity is still unclear.
  • the preferred antigen-presenting molecule is MHC-I.
  • HLA-I MHC class I molecules
  • genes such as HLA-A, HLA-B, and HLA-C.
  • HLA-A gene was found to significantly affect the antigen affinity of TCR in tumor immunity. Therefore, in treatment, the priority of HLA-A gene Impact of differences on treatment.
  • the MHC may be MHC class I, MHC class II or MHC class III, preferably MHC class I.
  • the MHC is human leukocyte antigen I (HLA-I), such as HLA-A*0201, HLA-A*0101; for mice, the MHC is mouse histocompatibility system 2 (H-2).
  • HLA-I human leukocyte antigen I
  • H-2 mouse histocompatibility system 2
  • the TCR activation response element may 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
  • NF- ⁇ B NF- ⁇ B
  • nuclear factor of activated T cells or "NAFT” (nuclear factor of activated T cells) is a family of transcription factors that play an important role in inducing gene transcription in immune responses. In addition to T cells, this type of protein can also be expressed on many immune cells, such as B lymphocytes, mast cells, eosinophils, etc., and its activity is regulated by calcium ion-dependent calmodulin phosphatase C. In the present invention, NAFT is the preferred TCR activation responsive element.
  • the TCR can specifically recognize the MHC-antigen polypeptide complex, the structure of the intracellular segment of the TCR will change and recruit phosphoprotein kinase K, including the ZAP70 protein.
  • AP1 activator protein 1 transcription factor
  • AP1 is a transcription activator in cells, which is a heterodimer composed of c-Fos and c-Jun. This transcription factor can be induced by various stimuli such as hormones, growth factors, cytokines, nerve mediators, heat shock, electric shock, ultraviolet rays, oxygen stress, and overexpressed oncogenes, and is associated with cell differentiation, cell proliferation, and cell apoptosis. closely related to the regulation of cellular processes.
  • nuclear factor ⁇ B or "NF- ⁇ B” is a highly conserved family of multifunctional transcription factors comprising five members, namely p65(RelA), RelB, c-Rel, NF- ⁇ B1 and NF- ⁇ B1 - ⁇ B2, they will form different homologous or heterologous dimers to regulate gene transcription, and then regulate many important cell behaviors, especially inflammatory responses.
  • the first cell is transformed by co-incubating the source cell of the first cell, the TCR expression vector, the vector expressing the TCR activation response element and the reporter molecule, and optionally
  • the second cell is prepared by co-incubating the source cell of the second cell, the MHC expression vector and an optional transfection-promoting reagent.
  • the "source cell of the first cell” refers to a type of cell that can be transfected and express exogenous TCR molecules.
  • the source of the first cell may be T cell lymphoma cells, such as Jurkat cells, human T lymphocytes, but not limited thereto.
  • the "cell from which the second cell is derived” refers to a type of cell capable of expressing MHC molecules.
  • the source cell of the second cell may be an antigen-presenting cell, such as T2 cell, K562 cell, but not limited thereto.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed.
  • Expression vectors contain sufficient cis-acting elements for expression; other elements for expression can be provided by the 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 not limited thereto.
  • cosmids e.g., naked or contained in liposomes
  • viruses incorporating recombinant polynucleotides such as lentiviruses, Retroviruses, adenoviruses, herpes simplex viruses, and adeno-associated viruses, but 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, reverse transcription Viral vectors, adenoviral vectors, herpes simplex viral vectors or adeno-associated viral vectors.
  • a viral expression vector such as a lentiviral expression vector, reverse transcription Viral vectors, adenoviral vectors, herpes simplex viral vectors or adeno-associated viral vectors.
  • transfection-promoting reagent and “transfection-promoting agent” and “transfection reagent” are used interchangeably herein, and refer to reagents that facilitate the delivery of target genes/nucleic acids into cells.
  • transfection protocols and the simplification of transfection assays, it is necessary to select the appropriate transfection reagent in order to achieve the best transfection efficiency.
  • appropriate transfection reagents it is important to identify the cell type and culture conditions being assayed. Rare cell cultures, neurons, and primary cells are often difficult to transfect, and reagents that facilitate transfection are required, especially for such difficult-to-transfect cells. Additionally, reagent levels and cytotoxicity parameters should be considered before choosing an appropriate transfection agent. Ideal reagents should have low cytotoxicity and high transfection efficiency for the desired cell type.
  • the transfection-promoting agent may include, for example, hexadimethrine, Lipofectamine2000, but not limited thereto.
  • the method of the present invention is implemented b) step.
  • the so-called “antigenic polypeptide” refers to a polypeptide that has an antigenic epitope and thus can be recognized by TCR.
  • the term “antigen” mostly refers to tumor antigens, that is, any molecules expressed or overexpressed solely or mainly by tumor cells or cancer cells, such as proteins, polypeptides, peptides, lipids, carbohydrates, etc. Tumor antigens can also be expressed by normal cells, non-tumor cells, or non-cancerous cells. In such cases, however, expression of the tumor antigen by normal, non-tumor, or non-cancerous cells is less robust than expression of the tumor antigen by tumor cells or cancer cells.
  • tumor cells or cancer cells may overexpress the antigen or express the antigen at significantly higher levels than normal cells, non-tumor cells, or non-cancerous cells express the antigen.
  • tumor antigens can be expressed by cells in different states of development or maturation.
  • tumor antigens may additionally be expressed by embryonic or fetal cells that are not normally present in the adult host.
  • tumor antigens may additionally be expressed by stem or precursor cells that are not normally present in the adult host.
  • 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, mutated KRAS, NY-ESO-1 (also known as CAG-3), MAGE-1, MAGE-3, etc., but not limited thereto.
  • the chromosomes of tumor cells are unstable, and at the same time, the regulation of the apparent level (such as methylation, etc.) is abnormal, often producing mutant proteins or fusion proteins, which can become targets for immunotherapy, and are called neoantigens (neoantigens). ).
  • the antigenic polypeptide is a tumor antigenic polypeptide.
  • the NY-ESO-1 polypeptide whose sequence is SLLMWITQC, or the MART-1 (27-35) polypeptide whose sequence is LAGIGILTV, but not limited thereto.
  • Co-incubating may refer to incubating at 37°C and 5% carbon dioxide for a suitable period of time, such as 8-24 hours.
  • the droplet microfluidic technology is further used to wrap the first cell and the first oil phase with droplet generating oil, that is, the first oil phase.
  • the second cell loaded with the antigen polypeptide is used to prepare droplets, wherein at least a part of the droplets only contain one of the first cell and at least one of the second cell, i.e. Step c) of the process according to the invention is carried out.
  • Droplet microfluidic technology can adopt, for example, the microfluidic chip system disclosed in PCT application No.PCT/CN2019/108536 and Chinese utility model patent CN209144161U.
  • the disclosures in these two patents (applications) are incorporated herein by reference. Applying.
  • other devices capable of generating droplets are also contemplated by the present invention and may be used in the practice of the present invention.
  • FIG. 1 shows a schematic structural diagram of a microfluidic chip system 10 used in implementing the present invention.
  • the system 10 mainly includes: a droplet generating device 11, a droplet collection container 12 (in the invention, an Eppendorf tube is specifically used) and a power generating device 13 (in the present invention, a syringe is specifically used), wherein the liquid
  • the droplet generating device 11 and the power generating device 13 are respectively connected to the droplet collection container 12 through a first connecting pipe 14 and a second connecting pipe 15 .
  • the droplet generation device 11 includes a droplet generation chip body, on which a continuous phase (oil phase) inlet 111 is opened for introducing the first type of cells (such as Jurkat-TCR cells) ), a second dispersed phase inlet 113 for introducing a second type of cells (such as T2-MHC cells), and a continuous phase introduction channel 114 is opened inside the main body of the droplet generation chip, the second A dispersed phase introduction channel 115 , a second dispersed phase introduction channel 116 and a confluence channel 117 .
  • a continuous phase (oil phase) inlet 111 for introducing the first type of cells (such as Jurkat-TCR cells)
  • a second dispersed phase inlet 113 for introducing a second type of cells (such as T2-MHC cells)
  • a continuous phase introduction channel 114 is opened inside the main body of the droplet generation chip, the second A dispersed phase introduction channel 115 , a second dispersed phase introduction channel 116 and a conflu
  • the continuous phase inlet 111 communicates with the continuous phase introduction channel 114
  • the first dispersed phase inlet 112 communicates with the first dispersed phase introduction channel 115
  • the second dispersed phase inlet 113 communicates with the second dispersed phase inlet 113.
  • the phase introduction channel 116 communicates, and the continuous phase introduction channel 114, the first dispersed phase introduction channel 115 and the second dispersed phase introduction channel 116 intersect and communicate with one end of the confluence channel 117, and the confluence The other end of the channel 117 communicates with the first connecting pipe 14 .
  • the droplet generating oil is added to the chip body of the droplet generating device 11 through the continuous phase inlet 111, and the first type of cells is injected through the first dispersed phase inlet 112 and the second dispersed phase inlet 113.
  • the suspension of the cell and the suspension of the second cell are added into the chip body of the droplet generation device 11 .
  • start the power generating device 13 to form a pressure difference between the pressure in the second connecting pipe 15, the droplet collecting container 12, the first connecting pipe 14 and the chip main body and the atmospheric pressure, and the pressure difference promotes the cell suspension and the droplet generating oil flow together to form droplets, and the formed droplets are finally collected in the droplet collection container 12 .
  • the droplet is a water-in-oil structure formed by wrapping the suspension of the first cell and the suspension of the second cell with the droplet-forming oil, wherein the droplet-forming oil is the same as that described herein
  • first continuous phase or “first oily phase” mentioned elsewhere are used interchangeably, and the first cell suspension and the second cell suspension are the same as the terms used elsewhere herein.
  • Dispersed phase can be used interchangeably.
  • the selection of droplet generating oil is mainly based on the fluid mechanics of the droplet generating chip, without other special requirements.
  • the inventors have confirmed through experiments that the number of cells in the droplet in the technical solution of the present invention conforms to the Poisson distribution.
  • an empty droplet refers to a droplet that does not contain cells.
  • the proportion of empty droplets is also called the probability of empty droplets, which refers to the ratio of the number of empty droplets to the number of all generated droplets (f(0; ⁇ )).
  • the proportion of droplets enclosing a cell is also called the droplet probability enclosing a cell, which refers to the ratio of the number of droplets enclosing a cell to the number of all generated droplets (f(1; ⁇ )).
  • the proportion of droplets enclosing multiple cells is also called the droplet probability enclosing multiple cells, which refers to the ratio of the number of droplets containing multiple ( ⁇ 2) cells to the number of all generated droplets (1-f(0; ⁇ )-f(1; ⁇ )).
  • the multiplet rate refers to the ratio of the number of droplets containing multiple cells ( ⁇ 2) to the number of droplets containing at least one ( ⁇ 1) cell ((1-f(0; ⁇ )-f (1; ⁇ ))/(1-f(0; ⁇ )).
  • the required first cell can be determined according to the Poisson distribution formula (the following formula).
  • k is the number of cells in the droplet and is an integer > 0,
  • C is the cell suspension concentration (cell number/ ⁇ L), also known as cell concentration,
  • ⁇ is the expected value, which can be calculated from the cell concentration C and the droplet generation diameter by the above formula
  • f(k; ⁇ ) is the probability of wrapping k cells in the droplet when k and ⁇ are determined
  • r is the droplet radius ( ⁇ m).
  • the multipack ratio of the first type of cells is ⁇ 5%
  • the proportion of empty droplets of the second type of cells is ⁇ 70%
  • the first type of cell if it is expected that its multi-packet rate is ⁇ 0.05 ( ⁇ 5%), then (1-f(0; ⁇ )-f(1; ⁇ ))/(1-f(0; ⁇ )) ⁇ 0.05.
  • the second type of cells if the proportion of empty droplets is expected to be ⁇ 70%, then f(0; ⁇ ) ⁇ 0.7.
  • the proportion of empty droplets, the proportion of droplets enclosing one cell, the proportion of droplets enclosing multiple cells, and the multi-encapsulation rate can be calculated according to the above Poisson distribution formula.
  • an appropriate cell concentration that is, a cell working concentration, can be selected according to the desired multipack ratio of the first type of cells and the desired ratio of empty droplets of the second type of cells.
  • the droplet diameter may be set to be 30 ⁇ m to 100 ⁇ m, preferably 30 ⁇ m to 80 ⁇ m, most preferably 45 ⁇ m.
  • the working concentration of the first cell in order to make the multipackage rate of the first cell less than five percent (5%), can be selected as 1 ⁇ 10 4 cells/mL to 4 ⁇ 10 6 cells/mL.
  • the working concentration of the second cell in order to reduce the proportion of empty droplets of the second cell, for example, when the proportion of empty droplets of the second cell is ⁇ 70%, can be selected as ⁇ 1.5 ⁇ 10 7 cells/mL.
  • the droplet diameter is other sizes, those skilled in the art can predict the proportion of droplets in each state under different cell concentrations according to the Poisson distribution, and thus select the desired cell concentration.
  • the first cell comprises a plurality of cells, and at least a portion of the plurality of cells express different types of TCRs.
  • the inventors also tested the encapsulation rate of the first type of cells and the second type of cells by the droplet forming oil. Specifically, a cell tracer is added to the first cell and the second cell respectively. For example, one cell tracer is added to the first type of cells and another cell tracer is added to the second type of cells. After the droplets are generated, the encapsulation of cells in the droplets is detected with a fluorescence microscope at the corresponding wavelength. It was found that the encapsulation rate of the droplets formed by the method of the present invention containing only one of the first cell and at least one of the second cell is about 2%, which is close to the theoretical value of 3.29%.
  • the resulting droplets can be incubated.
  • the droplets can be incubated in an incubator placed at 37°C, 5% carbon dioxide, preferably for no more than 8 hours. Under this incubation condition, the proportion of viable cells can be ensured to be at least 70%, thereby meeting the requirements of single-cell sequencing.
  • said droplets are incubated prior to step d), preferably for a period of no more than 8 hours.
  • the purpose of incubating the droplets is to enable the TCR specific for the antigen polypeptide on the first cell to bind to the MHC-antigen polypeptide complex on the second cell.
  • step d) of the method of the present invention After incubating the droplets, irradiate the droplets one by one with a laser, and determine whether the droplets contain antigen-specific TCR according to whether the droplets emit light, that is, step d) of the method of the present invention is carried out.
  • reporter molecule refers to a substance capable of indicating the reaction between TCR and MHC-antigen polypeptide complex.
  • the reporter molecule can be a class of visualized reporter gene-encoded protein or fluorescent protein, which can include: green fluorescent protein (GFP) and mutants thereof 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 it is detected that a droplet can emit fluorescence of a specific wavelength under the excitation of a specific wavelength of laser light, it indicates that the reporter molecule is expressed, and specific recognition occurs between the TCR and the MHC-antigen polypeptide complex, and the first A cell contains an antigen-specific TCR; if it is detected that a droplet cannot emit fluorescence of a specific wavelength under specific laser excitation, it indicates that the reporter molecule is not expressed, and the TCR is complexed with the MHC-antigen polypeptide No specific recognition occurs between the cells, and the first cell does not contain 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
  • the method of the first aspect of the present invention can ensure that the proportion of living cells reaches at least 70%, and can achieve ideal encapsulation of two types of cells, thus meeting the requirements of single-cell sequencing, and can realize high-throughput detection of whether cells contain antigen-specific TCR .
  • the present invention provides a method of sorting antigen-specific T cell receptors (TCRs), said method comprising the steps of:
  • Liquid droplets are prepared by implementing steps a) to c) of the method of the first aspect of the present invention, at least a part of the single droplets contain only one of the first cell and at least one of the first cell. two kinds of cells;
  • the droplet luminescence is detected, the light signal generated by the droplet is converted into a deflection force, and the deflection force is used to sort positive droplets containing cells with antigen-specific TCR.
  • the droplets may be incubated, preferably not longer than 8 hours, before performing step 2) of the method of the invention.
  • step 2) of the method of the invention.
  • step 2) of the method of the present invention is performed.
  • the second oil phase can be used as the sheath liquid.
  • the second oil phase and the first oil phase can be the same or different, and are selected according to the fluid mechanics of the sorting equipment, without other special requirements.
  • the droplets are passed individually through the laser by dispersing the droplets in a second oil phase as a sheath fluid and flowing the second oil phase past the laser irradiation site. irradiation site.
  • the droplets are passed individually through the laser irradiation site by adjusting the concentration of the droplets in the second oil phase and adjusting the speed at which the second oil phase flows past the laser irradiation site.
  • the light signal generated by the droplet is converted into a deflection force, and the deflection force is used to sort positive droplets containing cells with antigen-specific TCR, that is, step 3) of the method of the present invention is performed.
  • droplet sorting is realized by converting detected optical signals into deflection forces. Specifically, the detected optical signal is first converted into an electrical signal, and then a dielectrophoresis or an acoustic wave is generated from the electrical signal, thereby generating a deflection force for sorting.
  • said deflecting force is generated by converting said optical signal into an electrical signal.
  • said deflecting force is generated by dielectrophoresis or acoustic waves generated by said electrical signal.
  • a droplet sorting device generally includes a droplet power generating device such as a droplet injection pump, a droplet sorting chip, an optical detection platform, and a droplet sorting component.
  • FIG. 2 schematically shows the structure of a droplet sorting chip 20 for implementing steps 2) and 3) of the method of the present invention.
  • the droplet sorting chip 20 includes: a sample injection part and a droplet sorting part.
  • the inventors designed the droplet inlet 201 and the second oil phase inlet 202 respectively, and used a droplet power generation device (not shown) to control the flow rate of the second oil phase and the droplet in the channel.
  • the purpose of controlling the droplet flow rate is to adjust the time interval between adjacent droplets, and the second oil phase acts as a sheath fluid, which can control the speed at which the droplets move within the chip.
  • the droplet sorting component includes at least a first channel 203, a second channel 204 (channel A in the figure) and a third channel 205 (channel B in the figure), and the first channel 203, the second channel 204 and all
  • the third channel 205 is arranged horizontally in a "Y" shape.
  • the inventors use the difference in flow rate ratio between the two sides of the "Y"-shaped pipe to make the droplet move to the second channel 204 (channel A shown in the figure) without external force.
  • an electrode is prepared using metal such as tin material.
  • a positive droplet that is, a droplet containing cells with a specific TCR
  • it will emit light under laser excitation, and the light signal generated by the droplet can generate a deflection force, which will make the liquid
  • the drop moves to the side of the third channel 205, so as to realize the sorting of the target.
  • the droplet sorting chip may also include a droplet collecting component for collecting the sorted droplets passing through each channel.
  • the optical detection platform may include: observation system, fluorescence excitation detection system and signal processing system.
  • an observation system and a fluorescence excitation detection system are constructed by adding optical components to a microscope (such as an Olympus IX83 microscope).
  • Fig. 3 shows a schematic structural diagram of an example of an observation system and a fluorescence excitation detection system that can be used in the method of the present invention.
  • This microscope provides two camera inlets, one of which is equipped with a high-speed camera (such as OlympusDP23 high-speed camera), and the other is equipped with a photomultiplier tube (such as a model H10770A-40 from Hamamatsu).
  • the excitation light path of the microscope is: the halogen lamp produces strong light, and the light of the target wavelength (for example, for GFP protein, the light with a wavelength of 488nm is filtered by the front color filter of the UIS2 lens group (such as the product of U-FBNA from Olympus) ). The filtered light is reflected by the central dichroic mirror of the mirror group, and irradiates the channel at the vertical electrode of the sorting chip through an objective lens (such as a product of UPLXAPO20X from Olympus).
  • the target wavelength for example, for GFP protein, the light with a wavelength of 488nm is filtered by the front color filter of the UIS2 lens group (such as the product of U-FBNA from Olympus) ).
  • the filtered light is reflected by the central dichroic mirror of the mirror group, and irradiates the channel at the vertical electrode of the sorting chip through an objective lens (such as a product of UPLXAPO20X from O
  • the reporter molecule When the droplet passes, if the cell in the droplet expresses a reporter molecule such as GFP protein, the reporter molecule will be excited, and then the emitted light generated by the reporter molecule will pass through the objective lens, the dichroic mirror, the bottom filter of the UIS2 lens group, and the microscope The self-contained mirror reaches a photomultiplier tube which then converts the optical signal into an electrical signal such as a voltage signal.
  • a reporter molecule such as GFP protein
  • the signal processing system can be composed of four independent devices, which are an electrical signal comparator, a single-chip microcomputer, a waveform generator and a voltage amplifier, see Figure 4.
  • the electrical signal generated by the photomultiplier tube is divided into two channels after being sent out, one of which is transmitted to the single-chip microcomputer (such as a product from Punctual Atom, model STM32F407ZGT6) through line 01, and the other signal is transmitted to the voltage comparator through line 02 (such as from Conway, model TLV3501).
  • the electrical signal comparator is triggered to generate a high level (such as a 4V high level).
  • the high level is transmitted to the microcontroller through the line 03.
  • the single-chip microcomputer is triggered, and starts to record the electrical signal transmitted through the 01 line at a certain frequency such as 100KHz.
  • the recording is stopped, and the maximum value of the electrical signals recorded this time is calculated.
  • the maximum value is greater than a preset threshold, it indicates that the droplet is a positive droplet.
  • the microcontroller emits a voltage for a period of time (eg 2V for 2 ms) and this voltage is passed on line 04 to a waveform generator (eg AFG2021 from Tektronix).
  • the waveform generator is set to the external trigger mode, and when it is triggered, it will generate a certain voltage with a certain frequency and a certain duration (for example, a 3V (3V-5V optional) voltage at 20kHz and a duration of 2ms).
  • This voltage is transmitted to a voltage amplifier (for example, from FLC, a product of model F10AD) through the connection 05, and after being amplified 200 times by the voltage amplifier, it is transmitted to the droplet sorting part through the line 06, where a deflection force is generated. This enables droplet deflection.
  • the deflection force may be generated by means of dielectrophoresis.
  • the electrodes are used to create an uneven electric field, and the droplets are induced to move to the direction of the high electric field, thereby realizing positive droplet sorting.
  • the deflection of the droplet can be realized at a lower voltage (600V).
  • the deflection force may be generated in the form of sound waves.
  • interdigitated electrodes are placed at the sorting part, and the characteristics of the electrode-generated acoustic wave propagation and attenuation on the chip surface are used to move the liquid droplets to the acoustic wave attenuation position, thereby realizing the deflection of the liquid droplets.
  • RNA sequencing comprising the following steps:
  • the method may be a high-throughput sequencing method; in a further embodiment, the method is a single-cell RNA sequencing method.
  • a chemical demulsifier can be used to recover the cells contained in the positive droplets according to the method disclosed in patent application 202110593330.6.
  • step ii. of recovering cells in said positive droplets comprises the steps of:
  • the obtained liquid is subjected to low-speed centrifugation, preferably 30g-100g, more preferably 40g-70g, most preferably 50g, and the centrifugation lasts for 8-15 seconds, preferably 9-12 seconds, most preferably 10 seconds, thus the The liquid forms upper and lower layers with an interface in between;
  • the demulsifier is selected from the group consisting of tannin-based mixtures, ethoxylated or epoxidized PAGs, oxyalkylated poly(alkylene)poly(amines), poly(ether)poly( urethane) and octanols, preferably octanols, more preferably n-octanol, iso-octanol, butano-octanol, 1H, 1H, 8H-perfluoro-1-octanol or 1H, 1H, 2H, 2H - Perfluoro-1-octanol.
  • the positive droplet is located in the upper layer, and the droplet-forming oil is located in the lower layer, and it is preferable to remove part of the droplet-forming oil in the lower layer before adding the demulsifier.
  • the volume of the demulsifier added is 1 times the volume of the positive droplet.
  • the volume of demulsifier added again in (3) is less than the volume of demulsifier added in (1).
  • step ii is performed on ice.
  • the recovered cells are sequenced to obtain the sequence information of the antigen-specific TCR.
  • the sequencing method may be single-cell RNA sequencing, which is a high-throughput sequencing method.
  • the use of the sequence information of the antigen-specific TCR obtained by the method of the third aspect of the present invention in cellular immunotherapy is provided.
  • TCR-T cells also known as T cell receptor chimeric T cells (TCR-modified T cells), CAR-T cells, also known as chimeric antigen receptor T cells (Chimeric antigen receptor T cells).
  • TCR-modified T cells T cell receptor chimeric T cells
  • CAR-T cells also known as chimeric antigen receptor T cells (Chimeric antigen receptor T cells).
  • Both CAR-T cell immunotherapy and TCR-T cell immunotherapy are immune cell therapy methods.
  • One of their common features is to improve the ability of T cell receptors to recognize and attack specific cancer cell antigens through genetic modification. However, there are some obvious differences between the two.
  • CAR-T cell immunotherapy works by recognizing membrane surface antigens (such as CD19, BCMA, etc.), and has a significant effect on hematological tumors, while TCR-T cells can recognize cell membrane surface Or tumor-specific antigens derived from intracellular sources, among which TCR-T cells targeting NY-ESO-1 have been used in clinical trials at home and abroad for the treatment of refractory recurrent melanoma, synovial sarcoma, multiple myeloma and lung cancer. It has demonstrated good safety and efficacy, and is currently the most likely T cell immunotherapy for solid tumor breakthroughs.
  • membrane surface antigens such as CD19, BCMA, etc.
  • the method of the present invention can provide greater possibilities for the realization of TCR-T and CAR-T cell immunotherapy.
  • the cellular immunotherapy includes constructing TCR-T and/or CAR-T cells.
  • TCR-T and/or CAR-T cells include constructing TCR-T and/or CAR-T cells.
  • Those of ordinary skill in the art know that after obtaining the antigen-specific TCR sequence information, conventional methods in the art can be used to construct TCR-T and CAR-T cells for subsequent immunotherapy.
  • the method for detecting, sorting and/or sequencing antigen-specific TCRs of the present invention has one or more of the following advantages:
  • the present invention can screen a variety of TCRs in a high-throughput manner: with the help of droplet microfluidic technology, millions of droplets can be prepared. Since the droplets move at high speed in the sorting chip, cells can Complete the detection of the specificity of multiple TCRs to antigens independently and simultaneously, and the throughput is significantly improved compared with the detection of cell interaction in the well plate;
  • a part of the technical solution of the present invention uses fluorescent protein as a reporter molecule, so it does not depend on the substrate; compared with other prior art, the signal of the present invention is stable, and the reporter molecule such as GFP protein can exist stably in the cell for 24 hours or even 48 hours, convenient for follow-up testing;
  • the present invention has better expansibility: Compared with the research published by Mark Davis team on Nature Biotechnology, the present invention can not only verify/detect TCR specificity, but also be used for screening of antigen-specific TCR;
  • Cell viability The proportion of living cells of the cells sorted by the method of the present invention can reach at least 70%, so it can meet the requirements of single-cell sequencing.
  • the inventors established T2-MHC cells and Jurkat DMF5 TCR cells respectively, and carried out droplet encapsulation, wherein the TCR on the surface of Jurkat cells recognized the MHC-antigen peptide complex on the surface of T2 cells.
  • the design of this experiment only when there are T2 cells and Jurkat cells in the droplet, the TCR on the surface of Jurkat cells can be activated, and the intracellular GFP fluorescent protein is expressed, which is then excited by the 488nm laser and sorted by the system.
  • T2 cells and Jurkat cells were labeled with different fluorescent molecules (T2 cells: CellTrace TM Violet; Jurkat cells: CellTracker TM Green CMFDA) before the experiment.
  • T2 cells CellTrace TM Violet
  • Jurkat cells CellTracker TM Green CMFDA
  • T2 cells CellTrace TM Violet
  • TM Green CMFDA Excited with a 488nm light source
  • CellTrace TM Violet excited with a 408nm light source
  • T2 cells American Culture Collection, No. CRL-1992
  • Retroviral concentrated liquid it comprises DMF5 TCR sequence (Creative Biolab, VP-TCR-C928)
  • Lentiviral concentrate which contains NFAT-GFP sequence (BPS Bioscience, 79922)
  • Droplet generation device the present invention adopts a droplet generation device similar to that disclosed in CN 209144161 U, wherein the droplet generation chip is replaced with the chip disclosed in the patent application PCT/CN2019/108536, and the syringe is replaced with a BD 50ml syringe (Cat. 309654).
  • a droplet sorting device which includes a sorting chip and an optical droplet sorting platform.
  • Working fluid containing the following ingredients:
  • This cell line was obtained from Jurkat cell transformation, and the NFAT-GFP gene and DMF5TCR gene were transfected into Jurkat cells by lentiviral vector, and then flow cytometry was used to obtain a cell line stably expressing TCR and GFP (Jurkat-TCR cell line ); wherein, the nuclear factor of activated T cell (NFAT) response element is a calcium ion-regulated transcription factor downstream of TCR in the cell.
  • NFAT nuclear factor of activated T cell
  • a TCR library can contain a variety of different TCRs, but only one TCR is transfected per cell.
  • the pro-infection agent hemelonium bromide (1:1000) was added to the cells, and the retrovirus concentrate (containing the DMF5TCR sequence) and the lentivirus concentrate (containing the NFAT- GFP sequence).
  • the pro-infection agent hemelonium bromide (1:1000) was added to the cells, and a lentivirus concentrate (containing the HLA-A*0201 sequence) was added in a volume of 10 microliters ( ⁇ L).
  • T2-MHC cells and 100 microliters of Jurkat-TCR cells were added to each well of a 96-well plate, and cultured in a 37° C., 5% CO 2 incubator for 0, 3, 6, 9, 12, and 24 hours, respectively.
  • the T2-MHC cell line and the Jurkat-TCR cell line are constructed, the T2-MHC cell line is incubated with the antigen polypeptide, thereby loading the antigen polypeptide onto the MHC of the T2-MHC cell line. Then, the T2-MHC cell line and the Jurkat-TCR cell line were encapsulated in the droplet by using the droplet generation chip and the droplet generation oil.
  • the concentration of the two cells it is necessary to properly adjust the concentration of the two cells so that most of the droplets are wrapped with T2-MHC cells (the ratio of empty droplets is ⁇ 70%), and at the same time, the multipack rate of Jurkat-TCR cells is ⁇ 5%. With this design, most Jurkat-TCR cells are exposed to the antigen.
  • CellTracker TM Green CMFDA dye 10 microliters ( ⁇ L) of dimethyl sulfoxide (DMSO) was used to dissolve the dye to prepare a stock solution of the dye with a final concentration of 10 mM.
  • DMSO dimethyl sulfoxide
  • CellTrace TM Violet dye 20 microliters ( ⁇ L) of dimethyl sulfoxide was used to dissolve the dye to prepare a stock solution of the dye with a final concentration of 5 mM.
  • Droplets are prepared using the microfluidic chip system 10 shown in FIG. 1 , which has been described in detail in the above detailed description. Specific steps are as follows:
  • the droplet prepared in part 2.3 of the experimental procedure was examined by a fluorescence microscope for the formation of oil-encapsulated cells from the droplet. Specifically, a 488nm light source was used to detect Jurkat-TCR cells labeled with CellTracker TM Green CMFDA dye, and a 405nm light source was used to detect T2-MHC cells labeled with CellTrace TM Violet dye, and then the encapsulation of each cell by droplets was counted.
  • the calculation method of the encapsulation rate of Jurkat-TCR cells is as follows: select 5 fields of view, and calculate the number of all droplets in the field of view in the bright field of the microscope, which is recorded as N1; turn on the laser light source of the microscope, adjust to a 488nm filter, and count a green fluorescent light wrapped in the field of view
  • N2 The number of droplets that label cells
  • N3 the number of droplets that enclose more than one green fluorescently labeled cell
  • the Jurkat-TCR multipack rate is N3/(N2+N3)*100%.
  • the calculation method of the encapsulation rate of T2-MHC cells is as follows: select 5 fields of view, and calculate the number of all droplets in the field of view in the microscope bright field, which is recorded as N1; turn on the laser light source of the microscope, adjust to a 488nm filter, and count one or one droplets in the field of view The number of droplets of the above blue fluorescently labeled cells is recorded as N2; the encapsulation rate of T2 cells is N2/N1*100%.
  • the droplets prepared in part 2.3 of the experimental procedure were placed in an incubator at 37°C and 5% carbon dioxide for a period of time, and then some of the droplets were taken out and demulsified with 1H, 1H, 2H, 2H-perfluoro-1-octanol, The cells were taken out, stained with acridine orange/propidium iodide (AO/PI), and the proportion of viable cells was counted.
  • AO/PI acridine orange/propidium iodide
  • the proportion of Jurkat-TCR cells and T2-MHC cells encapsulated in the droplets is about 2% ( Figure 7), which is close to the theoretical value of 3.29%.
  • Figure 6 shows the cell viability results of droplet-encapsulated T2-MHC and Jurkat-TCR cells. It can be seen from the figure that the proportion of living cells remained above 70% within 8 hours and below 60% after 12 hours. Therefore, the time for incubating the cells is selected, preferably no more than 8 hours, so as to ensure that the proportion of living cells can be above 70%, so as to ensure that the cells can be used in subsequent single-cell sequencing.
  • FIG 8 shows the results of T2-MHC activation of Jurkat-TCR cells within droplets.
  • the proportion of GFP positive cells also increased (left graph); in addition, in the case of antigen peptide loading concentration of 80 ⁇ g/ML, the control group (T2-MHC The proportion of cells expressing GFP in the unloaded DMF5 polypeptide was less than 1%, while the proportion of cells expressing GFP in the experimental group (T2-MHC loaded with 80 ⁇ g/ml DMF5 polypeptide) was close to 15% (right panel).
  • the droplets generated in part 2.3 of the experimental procedure were incubated in an incubator at 37°C and 5% carbon dioxide for 8 hours. Afterwards, some of the droplets were taken out, and the expression of GFP protein in the droplets was observed under a fluorescent microscope using a 488nm laser to excite. After the expression of GFP protein was observed under a fluorescence microscope, the droplets were used in subsequent sorting experiments.
  • Droplet sorting is performed in a droplet sorting device that uses dielectrophoretic forces to sort droplets. See above for a detailed description of the droplet sorting device used in the present invention.
  • the droplets are floating on the upper layer, and the lower layer is the continuous phase liquid (the droplet-forming oil is used as the sheath fluid in this experiment, and the continuous phase here is the droplet-forming oil).
  • RNA cDNA For the cells obtained by sorting and recovery, according to the instructions of DNBelab C4 scRNA-seq kit, the preparation of single-cell RNA cDNA was completed. At the same time, some Jurkat cells were taken as controls, and single-cell library sequencing was performed in parallel. The PE100 kit matched with the MGI T7 sequencer was used to complete the preparation and sequencing of the sequencing library.
  • PCA principal component analysis
  • the cell subset with high expression of CD2 molecule is Jurkat cell.
  • FIG. 10 shows the single-cell sequencing results of the sorted T cells
  • FIG. 11 shows the quality control results of the single-cell sequencing.
  • the cells can be divided into two large groups, the right cell subgroup expresses relatively high CD2 and CD3G, indicating that they are Jurkat cells, and the left cell subgroup highly expresses CD86, indicating that they are antigens presenting cells. Therefore, the above results prove that the present invention can maintain the characteristics of cells during operation, and is suitable for single-cell sequencing analysis process.
  • the number of genes detected in the 6 cell subpopulation cells is greater than 2000, indicating that the single-cell sequencing method can effectively capture the genes of the cells sorted by the method of the present invention; in all cells, mitochondria-related The genes are less than 10% of the total number of genes, further indicating that the state of the cells sorted by the method of the present invention meets the requirements of single-cell RNA sequencing.

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Abstract

一种检测和分选抗原特异性T细胞受体的方法，所述方法利用微流控技术来制备仅包含一个TCR表达细胞的液滴，并由此实现对不同TCR的高通量检测和分选。借助液滴微流控技术，可以一次性制备上百万个液滴，细胞可以在液滴内独立地完成抗原特异性的检测，相对在孔板内检测细胞相互作用，通量显著提高。

Description

一种高通量检测和分选抗原特异性T细胞受体的方法 技术领域
本发明属于生物免疫检测技术领域，具体涉及一种高通量检测和分选抗原特异性T细胞受体(TCR)的方法。
背景技术
为了验证抗原特异性TCR，近三十年来，研究者开发了包括抗原特异性T细胞扩增、主要组织相容性复合物(MHC)四聚体标记、流式细胞术分选抗原反应性T细胞等多种方法，这些方法目前已经在免疫治疗相关的科研和临床上广泛使用，但是此类技术存在着较大的缺陷。
抗原特异性T细胞扩增技术(以下简称扩增法)在20世纪90年代开始应用，是目前用来从肿瘤患者体内扩增抗原特异性T细胞的主流方法。此种方法需要采集患者大量外周血，诱导其中的单核细胞分化为抗原提呈细胞，负载肿瘤抗原后，再与患者自身来源的T细胞共同培养，经过三代扩增、历时1-2个月后，才能获得抗原特异性T细胞。这一方法的优点是充分利用了T细胞活化所需的第一信号和第二信号(CD28-CD40L)，能够在验证T细胞的抗原特异性的同时，证明相应的TCR有生理学功能。但是，这一方法存在诸多不足，例如样本量大，时间长，有T细胞培养失败的风险，扩增特异性有限。同时，随着TCR-T细胞技术(即将外源性TCR借助慢病毒转染患者自身细胞，进而制备用于免疫治疗所需的T细胞)和CAR-T技术的发展，此类技术逐渐被取代。
MHC四聚体技术于20世纪90年代末由Mark Davis团队开发。此技术克服了MHC单体在体外不稳定的缺点，使用者可以构建MHC四聚体，使用紫外线将指定长度(8-12肽)多肽负载到四聚体上，在四聚体尾端加上标签如荧光分子，借助MHC-多肽-TCR特异性识别原理，标记表达抗原特异性TCR细胞，再使用流式细胞术进行细胞分选、富集。此种技术的优势在于：检测快速，需要细胞较扩增法少，特异性较高。但是，其也有诸多缺陷，例如，四聚体制备成本高、实验操作繁琐，同时，由于TCR-MHC亲和力有限，导致标记的荧光强度有限，以及微量抗原T细胞检测失败。在2000年后，随着T细胞活化分子标记物的发现，CD154和CD137作为TCR活化细胞的标志物，被用于分选抗原特异性的T细胞。尽管此方法在使用上很便捷，但是，它受到bystander效应(活化T细胞会激活其临近非特异性T细胞)影响较大，进而导致此种方法筛选的TCR假阳性率较高。
2010年之后，随着分子生物学技术的发展，通过转染MHC至工具细胞，并负载多肽，用 于扩增抗原特异性T细胞的方法出现。此种方法通过共转染第二信号分子，使得抗原提呈细胞在易于获得的同时，能够激活T细胞的胞内信号，显著地改进治疗用细胞的扩增效率。在抗原特异性的验证中，2020年Mark Davis团队在Nature Biotechnology上发表研究，其中使用了光学报告系统，用于表征TCR的抗原特异性识别能力。这一系统包含两种工具细胞，其中一种工具细胞表达萤光酶(Luciferase)和特定TCR，另一种工具细胞表达特定的MHC。在实验前，令MHC表达细胞吞噬抗原，并通过MHC呈递给TCR。当这两种细胞特异性识别后，TCR信号会激活胞内荧光酶，在加入酶底物后，就可以检测酶化学反应释放的荧光。上述技术的优点是：相比MHC四聚体，进一步利用了酶的表达，放大了荧光信号，能够检测微量的抗原特异性细胞；能够证明TCR胞内信号有生理学功能。但是，上述方法依赖酶底物的存在，只能在孔板内完成。同时，为了识别发挥作用的TCR，每个孔只能加入一种TCR。因此，此种方法存在明显的通量限制缺陷。
因此，本领域亟需一种能够高通量检测和分选抗原特异性TCR的方案。
发明内容
有鉴于此，本发明提供了一种高通量检测和分选抗原特异性T细胞受体(TCR)的方法，由此解决了本文背景技术部分提及的问题。
因此，在第一方面，本发明提供了一种检测抗原特异性T细胞受体(TCR)的方法，所述方法包括以下步骤：
a)制备第一种细胞和第二种细胞，其中所述第一种细胞表达TCR、TCR激活响应元件和报告分子，所述第二种细胞表达主要组织相容性复合物(MHC)；
b)通过共同孵育所述第二种细胞与抗原多肽以使所述抗原多肽与所述MHC形成抗原多肽-MHC复合物，从而将所述抗原多肽加载于所述第二种细胞；
c)利用液滴微流控技术，用第一油相包裹所述第一种细胞和经所述抗原多肽加载后的第二种细胞来制备液滴，其中，所述液滴中至少一部分的单个液滴中仅包含一个所述第一种细胞和至少一个所述第二种细胞；
d)用激光照射液滴，并根据液滴是否发光确定该液滴是否包含抗原特异性TCR。
在一个实施方案中，在步骤d)之前孵育所述液滴，优选地孵育时间不超过8小时。
在一个实施方案中，所述第一种细胞通过共同孵育所述第一种细胞的来源细胞、TCR表达载体、表达所述TCR激活响应元件和所述报告分子的载体、和任选的促转染试剂来制备，所述第二种细胞通过共同孵育所述第二种细胞的来源细胞、MHC表达载体以及任选的促转染 试剂来制备。
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。
在一个实施方案中，所述促转染试剂为海地美铵、Lipofectamine2000。
在一个实施方案中，所述第一种细胞的来源细胞为T细胞的淋巴瘤细胞，优选为Jurkat细胞或人来源T淋巴细胞；所述第二种细胞的来源细胞为抗原呈递细胞，优选为T2细胞或K562细胞。
在一个实施方案中，所述MHC为MHC-I类、MHC-II类或MHC-III类，优选为MHC-I类。
在一个实施方案中，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，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，所述抗原多肽为肿瘤抗原多肽，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，所述第一种细胞含有多个细胞，且所述多个细胞中至少一部分细胞表达不同种类的TCR。
在一个实施方案中，在步骤c)中，通过以下方式来使所述液滴中至少一部分的单个液滴中仅包含一个所述第一种细胞和至少一个所述第二种细胞：设定液滴直径、第一种细胞的多包率和所述第二种细胞的空液滴比例，根据泊松分布来确定所述第一细胞的工作浓度以及所述第二细胞的工作浓度：
Figure PCTCN2021113888-appb-000001
λ＝C*πr 3/2
其中，
k是液滴内细胞数且为＞0的整数，
e是常数，
C是细胞悬液浓度(细胞数/μL)，
λ是期望值，
f(k；λ)是当k和λ确定时液滴内包裹k个细胞的概率，
r为液滴半径(μm)。
在一个实施方案中，设定所述液滴直径为30μm至100μm，优选为30μm至80μm，所述第一种细胞的多包率≤5％以及所述第二种细胞的空液滴比例≤70％。在一个实施方案中，在液滴直径为45μm的情况下，所述第一细胞的工作浓度为1×10 4个细胞/mL至4×10 6个细胞/mL，并且所述第二细胞的工作浓度为≥1.5×10 7个细胞/mL。
在第二方面，本发明提供了一种分选抗原特异性T细胞受体(TCR)的方法，所述方法包括以下步骤：
1)通过实施本发明第一方面的方法的步骤a)至c)来制备液滴，所述液滴中至少一部分
的单个液滴中仅包含一个所述第一种细胞和至少一个所述第二种细胞；
2)使所述液滴逐个地通过激光照射位点，并在此处检测液滴的发光情况；并且
3)在检测到液滴发光时，将液滴产生的光信号转化为偏转力，并利用该偏转力分选包含
具有抗原特异性TCR的细胞的阳性液滴。
在一个实施方案中，在步骤2)之前孵育所述液滴，优选地孵育时间不超过8小时。
在一个实施方案中，通过使所述液滴分散于作为鞘液的第二油相中并使所述第二油相流动通过激光照射位点，从而使所述液滴逐个地通过激光照射位点。
在一个实施方案中，通过调整液滴在第二油相中的浓度并调整第二油相流动通过激光照射位点的速度，从而使液滴逐个地通过激光照射位点。
在一个实施方案中，所述偏转力是通过将所述光信号转化为电信号产生的。
在一个实施方案中，所述偏转力是由所述电信号产生的介电泳或声波产生的。
在第三方面，本发明提供了一种RNA测序方法，所述方法包括以下步骤：
i.通过实施本发明第二方面的方法分选包含具有抗原特异性TCR的细胞的阳性液滴；
ii.回收所述阳性液滴中的细胞；并且
iii.对回收的细胞进行测序，由此获得所述细胞中包含的抗原特异性TCR的序列信息。
在一个实施方案中，所述测序方法为高通量测序方法。
在一个实施方案中，所述方法为单细胞RNA测序方法。
在一个实施方案中，回收所述阳性液滴中的细胞的步骤ii.包括以下步骤：
(1)向所述阳性液滴中加入破乳剂，然后静置，优选静置8-15分钟，更优选9-12 分钟，最优选10分钟；
(2)将静置后的液体最下层的油相移出；
(3)向所得的液体中再次加入所述破乳剂，然后静置，优选静置3-7分钟，更优选4-6分钟，最优选5分钟；
(4)将所得的液体进行低速离心，优选30g-100g，更优选40g-70g，最优选50g，所述离心持续8-15秒，优选9-12秒，最优选10秒，由此所述液体形成上下两层，中间有界面；
(5)收集所述界面之上包含所述内容物的液体，不触及所述中间的界面；
(6)任选地，一次或者多次地向所述界面上方的剩余液体中加入水性溶液优选细胞培养液，再次收集所述界面之上包含所述内容物的液体。
在一个实施方案中，所述破乳剂选自单宁基混合物、乙氧基化或环氧化的PAG、氧烷基化的聚(亚烷基)聚(胺)、聚(醚)聚(氨酯)和辛醇类，优选为辛醇类，更优选为正辛醇、异辛醇、丁辛醇、1H，1H，8H-全氟-1-辛醇或1H，1H，2H，2H-全氟-1-辛醇。
在一个实施方案中，在(1)中，所述阳性液滴位于上层，液滴生成油位于下层，优选在加入破乳剂之前除去部分的下层液滴生成油。
在一个实施方案中，在(1)中，加入破乳剂的体积是阳性液滴体积的1倍。
在一个实施方案中，在(3)中，再次加入的破乳剂的体积少于在(1)中加入的破乳剂的体积。
在第四方面，本发明提供了一种通过本发明第三方面的方法获得的抗原特异性TCR的序列信息在细胞免疫治疗中的用途。
在一个实施方案中，所述细胞免疫治疗包括构建TCR-T和/或CAR-T细胞。
本发明具有以下一个或者多个技术效果：
本发明可以高通量地筛选多种TCR：本发明借助液滴微流控技术，可以一次性制备上百万个液滴，在整个制备过程中活细胞比例不低于70％，该比例的活细胞适于后续的单细胞RNA测序；并且可以在液滴内独立地完成抗原特异性的检测，相对于在孔板内检测细胞相互作用，本发明通量显著提高。
本发明有较好的拓展性：与现有技术相比，本发明不仅可以验证TCR特异性，还可以用于抗原特异性TCR的筛选和TCR信息的回收。同时，本发明所获得的TCR数据信息可用于后续构建TCR-T/CAR-T细胞，从而为TCR-T/CAR-T细胞免疫疗法的实现提供更大的便利。
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是用于实施本发明方法的液滴生成装置的一个实施方案的示意图。
图2是用于实施本发明方法的细胞分选芯片的一个实施方案的示意图。
图3示出分选平台中光学装置示意图。
图4示出光学信号-电压信号处理流程图。
图5示出T细胞中的GFP蛋白表达随pMHC刺激时间的变化。
图6示出经包裹后的细胞的活力随孵育时间而变的统计结果。
图7示出细胞在液滴中的包裹率统计数据。
图8示出液滴内不同浓度pMHC刺激下表达GFP的T细胞比例。
图9示出基于流式细胞术检测本发明方法分选的效果的统计图。
图10示出分选出T细胞的单细胞测序结果。
图11示出单细胞测序质控结果。
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。
如上所述，本发明旨在解决如何高通量筛选抗原特异性TCR的技术问题。
为解决该技术问题，本发明人构建了两种细胞株：1)构建MHC慢病毒载体，使其转染T2细胞，由此制备MHC稳定转染细胞株；2)构建TCR慢病毒载体和增强型GFP载体，制备TCR文库，转染Jurkat细胞，由此制备稳定表达TCR和GFP的细胞株；其中，在GFP上游加入活化T细胞核因子(NFAT)反应元件，用于检测TCR信号的激活。接下来，使T2细胞与抗原多肽孵育，然后与Jurkat细胞一起通过液滴微流控技术制备成液滴。通过在细胞中加入不同的细胞示踪剂发现，当投入Jurkat细胞浓度为4*10 6/ml，T2细胞浓度为2*10 7/ml时，经试验发现约有2％的液滴同时包含Jurkat细胞和T2细胞，这 与理论值3.29％非常接近。在将这些液滴在液滴分选设备中进行分选时，成功地分选出包含抗原特异性TCR的液滴。由此，完成了本发明。
因此，在第一方面，本发明提供了一种检测抗原特异性T细胞受体(TCR)的方法，所述方法包括以下步骤：
a)制备第一种细胞和第二种细胞，其中所述第一种细胞表达TCR、TCR激活响应元件和报告分子，所述第二种细胞表达主要组织相容性复合物(MHC)；
b)通过共同孵育所述第二种细胞与抗原多肽以使所述抗原多肽与所述MHC形成抗原多肽-MHC复合物，从而将所述抗原多肽加载于所述第二种细胞；
c)利用液滴微流控技术，用第一油相包裹所述第一种细胞和经所述抗原多肽加载后的第二种细胞来制备液滴，其中，所述液滴中至少一部分的单个液滴中仅包含一个所述第一种细胞和至少一个所述第二种细胞；
d)用激光照射液滴，并根据液滴是否发光确定该液滴是否包含抗原特异性TCR。
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。
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。
所有MHC-I类分子都包含两条不相连的多肽链：一条为MHC编码的α链或称重链，约44×10 3(对于人类)或约47×10 3(对于小鼠)；另一条为独立染色体基因编码的β链(β2-微球蛋白)，对于人类和小鼠而言均为12×10 3。α链由一个约40×10 3的核心多肽链形成， N端连接有一个(人类)或两个(小鼠)寡糖，α链的3/4都在胞膜外，包括氨基端和寡糖群，跨膜区为一个疏水短片段，胞浆区为30个氨基酸残基的羧基末端。β链与重链的胞膜外部分以非共价键结合，β链不与细胞直接相连。
所有MHC-II类分子均由两条以非共价键相连的多肽链组成，两条链的整体结构相似，α链(33×10 3至34×10 3)，由于其广泛的糖基化，因而略长于β链(29×10 3至32×10 3)。两条多肽链均含有与N端相连的寡糖，胞外为氨基端，胞内为羧基端，每条链的2/3部分位于细胞外，两条链由不同的MHC基因编码，具有多态性(少数例外)。
这两类糖蛋白分子结构上具有共同特点，即表面都有一沟，MHC-I类分子沟较浅，可以接受8-20个氨基酸组成的肽链；MHC-II类分子沟较深，可接受较长的肽链。
MHC-I类和MHC-II类除了结构有异外，它们的主要区别是在免疫应答中激活机制和效果不同。MHC-I类分子主要呈递细胞内抗原，承担正常细胞的免疫耐受和调节衰老细胞的清除功能；在肿瘤免疫中，杀伤性T细胞通过识别肿瘤细胞表面MHC-I分子呈递的异常抗原，从而识别肿瘤细胞，并借助穿孔素等细胞毒机制，完成肿瘤细胞的杀伤。不同于MHC-I类分子，MHC-II类分子主要呈递细胞外抗原，在人体中此类分子表达于专职抗原提呈细胞(antigen-presenting cells，APC)表面。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。目前MHC-II类分子在肿瘤免疫中的作用尚不明确。而在肿瘤免疫治疗中，优选的抗原提呈分子为MHC-I。
在人类中，MHC-I类分子被命名为HLA-I。这类分子由HLA-A、HLA-B、HLA-C等基因编码，其中HLA-A基因被发现在肿瘤免疫中显著影响TCR的抗原亲和力，因此，在治疗中，优先考虑HLA-A基因的差异对治疗的影响。
因此，在一个实施方案中，所述MHC可以为MHC-I类、MHC-II类或MHC-III类，优选为MHC-I类。例如，对于人类而言，所述MHC为人白细胞抗原I(HLA-I)，例如HLA-A*0201、HLA-A*0101；对于小鼠而言，所述MHC为小鼠组织相容系统2(H-2)。
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。
在本文中，术语“活化T细胞核因子”或“NAFT”(nuclear factor of activated T cells)，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，NAFT是优选的TCR 激活响应元件。
在TCR激活相应元件为NAFT的情况下，如果TCR能够特异性地识别MHC-抗原多肽复合物，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，术语“AP1(activator protein 1)转录因子”或者“AP1”是细胞内的一个转录激活因子，为由c-Fos和c-Jun组成的异二聚体。该转录因子可以被激素、生长因子、细胞因子、神经介质、热休克、电休克、紫外线、氧应激以及过表达的癌基因等多种刺激所诱导，与细胞分化、细胞增殖和细胞凋亡等细胞进程的调控密切相关。
在本文中，术语“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。
在一个实施方案中，所述第一种细胞通过共同孵育所述第一种细胞的来源细胞、TCR表达载体、表达所述TCR激活响应元件和所述报告分子的载体、和任选的促转染试剂来制备，所述第二种细胞通过共同孵育所述第二种细胞的来源细胞、MHC表达载体以及任选的促转染试剂来制备。
在本文中，所述“第一种细胞的来源细胞”是指能够被转染并表达外源性TCR分子的一类细胞。
在一个实施方案中，所述第一种细胞的来源细胞可以是T细胞的淋巴瘤细胞，例如Jurkat细胞、人来源T淋巴细胞，但不限于此。
在本文中，所述“第二种细胞的来源细胞”是指能够表达MHC分子的一类细胞。
在一个实施方案中，所述第二种细胞的来源细胞可以是抗原呈递细胞，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系统中提供。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，术语“任选”是指该术语后面列出的元素/元件可以存在，也可以不存在，视情况而定。
术语“促转染试剂”与“转染促进剂”、“转染试剂”在本文中可以互换使用，指有助于将目标基因/核酸输送到细胞中的试剂。随着转染实验方案的发展和转染测定的日益简化，为了达到最佳转染效率，选择合适的转染试剂十分必要。在考虑合适的转染试剂时，重要的是鉴定测定的细胞类型和培养条件。稀有细胞培养物、神经元和原代细胞通常较难转染，因此需要能够促进转染的试剂，对这种难以转染的细胞更是如此。此外，在选择合适的转染剂之前，还应考虑试剂水平和细胞毒性参数。对于所需的细胞类型，理想的试剂应具有低细胞毒性和高转染效率。
在一个实施方案中，所述促转染剂可以包括，例如，海地美铵(hexadimethrine)、Lipofectamine2000，但不限于此。
在制备好所述第一种细胞和所述第二种细胞之后，通过共同孵育所述第二种细胞与抗原多肽以将所述抗原多肽加载于所述第二种细胞，即实施本发明方法的b)步骤。
在本文中，所谓“抗原多肽”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，所述抗原多肽为肿瘤抗原多肽。例如序列为SLLMWITQC的NY-ESO-1多肽、或者序列为LAGIGILTV的MART-1 (27-35)多肽，但不限于此。
共同孵育可以指在37℃、5％二氧化碳的条件下孵育合适长的一段时间，例如8-24小时。
在通过共同孵育所述抗原多肽与所述第二种细胞来进行抗原多肽加载之后，进一步利用液滴微流控技术，用液滴生成油亦即第一油相包裹所述第一种细胞和所述加载抗原多肽后的第二种细胞来制备液滴，其中，所述液滴中至少一部分的单个液滴中仅包含一个所述第一种细胞和至少一个所述第二种细胞，即进行本发明方法的步骤c)。
液滴微流控技术可以采用例如PCT申请No.PCT/CN2019/108536和中国实用新型专利CN209144161U中公开的微流控芯片系统，这两份专利(申请)中的公开内容通过援引的方式纳入本申请中。当然，可以理解，其他能够生成液滴的装置也在本发明的考虑之中，并且可以用于实施本发明。
图1示出了在实施本发明时使用的微流控芯片系统10的结构示意图。如图所示，该系统10主要包括：液滴生成装置11、液滴收集容器12(在发明中具体使用Eppendorf管)以及动力产生装置13(在本发明中具体使用注射器)，其中所述液滴生成装置11和所述动力产生装置13分别通过第一连接管14和第二连接管15与所述液滴收集容器12连接。进一步地，所述液滴生成装置11包括液滴生成芯片主体，在所述液滴生成芯片主体上开设有连续相(油相)入口111、用于引入第一种细胞(例如Jurkat-TCR细胞)的第一分散相入口112、用于引入第二种细胞(例如T2-MHC细胞)的第二分散相入口113，并且在所述液滴生成芯片主体内部开设有连续相引入通道114、第一分散相引入通道115、第二分散相引入通道116以及汇流通道117。所述连续相入口111与所述连续相引入通道114连通，所述第一分散相入口112与所述第一分散相引入通道115连通，所述第二分散相入口113与所述第二分散相引入通道116连通，并且所述连续相引入通道114、所述第一分散相引入通道115和所述第二分散相引入通道116相交并与所述汇流通道117的一端连通，而所述汇流通道117的另一端与所述第一连接管14连通。
使用时，经由所述连续相入口111将液滴生成油加入液滴生成装置11的芯片主体中，并经由所述第一分散相入口112和所述第二分散相入口113将第一种细胞的悬液和第二种 细胞的悬液加入液滴生成装置11的芯片主体中。然后，启动动力产生装置13，使第二连接管15、液滴收集容器12、第一连接管14和芯片主体内的压强与大气压强之间形成压强差，该压强差促使所述细胞悬液和所述液滴生成油汇流并形成液滴，并且形成的液滴最后收集于所述液滴收集容器12中。
在本发明中，液滴为液滴生成油包裹所述第一种细胞的悬液和所述第二种细胞的悬液而形成的油包水结构，其中，所述液滴生成油与本文他处提及的术语“第一连续相”或者“第一油相”可以互换使用，所述第一种细胞的悬液和所述第二种细胞的悬液与本文他处使用的术语“分散相”可以互换使用。在本发明中，对于液滴生成油的选择主要根据液滴生成芯片的流体力学，无其他特殊要求。
为了确保所述液滴中至少一部分的单个液滴中仅包含一个所述第一种细胞和至少一个所述第二种细胞，在液滴生成过程中，需要对诸如液滴直径、细胞浓度以及细胞悬浮状态等参数进行选择。
此外，本发明人经试验证实，本发明技术方案中的液滴内细胞数量符合泊松分布。
在本发明中，空液滴是指不包含细胞的液滴。空液滴比例也称为空液滴概率，是指空液滴数量占所有生成液滴数量的比值(f(0；λ))。包裹一个细胞的液滴比例也叫包裹一个细胞的液滴概率，是指包含一个细胞的液滴数量占所有生成液滴数量的比值(f(1；λ))。包裹多个细胞的液滴比例也叫包裹多个细胞的液滴概率，是指包含多个(≥2个)细胞的液滴数量占所有生成液滴数量的比值(1-f(0；λ)-f(1；λ))。多包率(multiplet rate)是指包含多个细胞(≥2个)的液滴数量占包含至少一个(≥1个)细胞的液滴数量的比值((1-f(0；λ)-f(1；λ))/(1-f(0；λ))。
因此，在设定液滴直径、所述第一种细胞的多包率、所述第二种细胞的空液滴比例下，可以根据泊松分布公式(下式)来确定所需的第一种细胞和第二种细胞的工作浓度：
Figure PCTCN2021113888-appb-000002
λ＝C*πr 3/2
其中，
k是液滴内细胞数且为＞0的整数，
e是常数，
C是细胞悬液浓度(细胞数/μL)，也称细胞浓度，
λ是期望值，可以通过上述公式由细胞浓度C和液滴生成直径计算，
f(k；λ)是当k和λ确定时液滴内包裹k个细胞的概率，
r为液滴半径(μm)。
在使用此公式时，需要在液滴直径确定的情况下，依据预先设定的条件，例如所述第一种细胞的多包率≤5％、所述第二种细胞的空液滴比例≤70％，才能确定并由此选择合适的细胞浓度。
对于第一种细胞，若期望其多包率≤0.05(≤5％)，则(1-f(0；λ)-f(1；λ))/(1-f(0；λ))≤0.05。对于第二种细胞，若期望其空液滴比例≤70％时，则f(0；λ)≤0.7。在液滴直径确定的情况下，根据上述泊松分布公式可以计算出空液滴比例、包裹一个细胞的液滴比例、包裹多个细胞的液滴比例和多包率。随后，可以根据第一种细胞期望的多包率和第二种细胞期望的空液滴比例，选择合适的细胞浓度，即细胞工作浓度。
在一个实施方案中，可以将液滴直径设定为30μm至100μm，优选为30μm至80μm，最优选为45μm。
在液滴直径为45μm的情况下，根据泊松分布预测的细胞浓度以及各状态液滴的比例情况如表1所示：
表1.根据泊松分布预测的细胞浓度以及各状态液滴的比例
Figure PCTCN2021113888-appb-000003
液滴直径＝45微米(μm)
由上表可知，在液滴直径为45μm的情况下，为了使所述第一种细胞的多包率≤百分之五(5％)，可以将第一种细胞的细胞工作浓度选择为1×10 4个细胞/mL至4×10 6个细胞/mL。同时，为了降低第二种细胞的空液滴比例，例如使所述第二种细胞的空液滴比例≤70％时， 可以将第二种细胞的细胞工作浓度选择为≥1.5×10 7个细胞/mL。在液滴直径为其他尺寸的情况下，本领域技术人员可以根据泊松分布预测不同细胞浓度下各状态液滴的比例，并由此选择所需的细胞浓度。
由此，可以获得包含仅一个所述第一种细胞和至少一个所述第二种细胞的液滴。借助这样的设计，多数所述第一种细胞均能接触抗原多肽，并且每个液滴中均只有一种TCR，由此能够避免非特异性TCR被富集。另外，所述第一种细胞中的各细胞可以表达相同或者不同种类的TCR。所述不同种类包括至少两个、三个、四个或更多个种类。在所述第一种细胞表达不同种类的TCR的情况下，可以实现对不同种类的抗原特异性TCR的高通量检测和分选。因此，在一个实施方案中，所述第一种细胞含有多个细胞，且所述多个细胞中至少一部分细胞表达不同种类的TCR。
另外，本发明人还对液滴生成油对所述第一种细胞和所述第二种细胞的包裹率进行了检测。具体地，分别在所述第一种细胞和所述第二种细胞中加入细胞示踪剂。例如，在所述第一种细胞中加入一种细胞示踪剂，在所述第二种细胞中加入另一种细胞示踪剂。在生成液滴之后，在相应的波长下用荧光显微镜检测液滴中的细胞包裹情况。结果发现，通过本发明方法形成的液滴仅包含一个所述第一种细胞和至少一个所述第二种细胞的包裹率大约为2％，该数值与理论值3.29％接近。
随后，可以对生成的液滴进行孵育。作为一个示例，可以将所述液滴在放置于37℃、5％二氧化碳的培养箱中孵育，优选地不超过8小时。在此孵育条件下可以确保活细胞比例为至少70％，由此能够满足单细胞测序的要求。因此，在一个实施方案中，在步骤d)之前孵育所述液滴，优选地孵育时间不超过8小时。
对液滴进行孵育是为了使所述第一种细胞上的所述抗原多肽特异的TCR能够与所述第二种细胞上的MHC-抗原多肽复合物结合。
在对液滴进行孵育之后，用激光逐个地照射液滴，并根据液滴是否发光确定该液滴是否包含抗原特异性TCR，即进行本发明方法的步骤d)。
可以理解，液滴是否发光与第二种细胞是否表达报告分子有关。
在本文中，所谓“报告分子”是指能够指示TCR与MHC-抗原多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-抗原多肽复合物之间发生特异性识别，所述第一种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-抗原多肽复合物之间没有发生特异性识别，所述第一种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)。
本发明第一方面的方法可以确保活细胞比例达到至少70％，可以实现对两种细胞的理想包裹，因此满足单细胞测序的要求，可以实现对细胞是否包含抗原特异性TCR的高通量检测。
在第二方面，本发明提供了一种分选抗原特异性T细胞受体(TCR)的方法，所述方法包括以下步骤：
1)通过实施本发明第一方面的方法的步骤a)至c)来制备液滴，所述液滴中至少一部分的单个液滴中仅包含一个所述第一种细胞和至少一个所述第二种细胞；
2)使所述液滴逐个地通过激光照射位点，并在此处检测液滴的发光情况；并且
3)在检测到液滴发光时，将液滴产生的光信号转化为偏转力，并利用该偏转力分选包含具有抗原特异性TCR的细胞的阳性液滴。
可以理解，上文针对本发明第一方面方法的步骤a)至c)的详细描述也适用于本发明这一方面的方法，故在此不再赘述。
在进行本发明方法的步骤2)前，可以孵育所述液滴，优选地不超过8小时。关于该操作更具体的描述，可以参见根据本发明第一方面的方法中的相关描述。
在孵育结束后，使所述液滴逐个地通过激光照射位点，并在此处检测液滴的发光情况，即进行本发明方法的步骤2)。
在实施本发明方法的步骤2)时，为了使所述液滴能够逐个地通过激光照射位点，可以使用第二油相作为鞘液。所述第二油相与所述第一油相可以相同，也可以不同，具体根据分选设备的流体力学进行选择，无其他特殊要求。因此，在一个实施方案中，通过使所述液滴分散于作为鞘液的第二油相中并使所述第二油相流动通过激光照射位点，从而使所述液滴逐个地 通过激光照射位点。
为了使液滴逐个地通过激光照射位点，需要控制液滴在分选芯片中的流速、以及液滴之间的距离。因此，在一个实施方案中，通过调整液滴在第二油相中的浓度并调整第二油相流动通过激光照射位点的速度，从而使液滴逐个地通过激光照射位点。
在检测到液滴发光时，将液滴产生的光信号转化为偏转力，并利用该偏转力分选包含具有抗原特异性TCR的细胞的阳性液滴，即进行本发明方法的步骤3)。
在本发明方法中，液滴分选是通过将检测到的光信号转化为偏转力来实现的。具体地，将检测到的光信号首先转化为电信号，再由该电信号产生介电泳或者声波，进而产生用于分选的偏转力。
因此，在一个实施方案中，所述偏转力是通过将所述光信号转化为电信号产生的。在另一个实施方案中，所述偏转力是由所述电信号产生的介电泳或声波产生的。
作为一个示例，本发明方法的步骤2)和3)可以在本领域的液滴分选设备中实施。液滴分选设备一般包括液滴动力产生装置例如液滴注射泵、液滴分选芯片、光学检测平台、以及液滴分选部件。
图2示意性地示出了用于实施本发明方法的步骤2)和3)的液滴分选芯片20的结构。如图所示，该液滴分选芯片20包括：样品进样部件和液滴分选部件。在样品进样部件，本发明人分别设计了液滴入口201和第二油相入口202，并采用液滴动力产生装置(未示出)控制第二油相和液滴在通道中的流速。控制液滴流速的目的是调节相邻液滴之间的时间间隔，并且第二油相作为鞘液，可以控制液滴在芯片内移动的速度。液滴分选部件至少包括第一通道203、第二通道204(图示通道A)和第三通道205(图示通道B)，并且所述第一通道203、所述第二通道204和所述第三通道205呈水平放置“Y”字形排布。在液滴和第二油相分别从液滴入口201和第二油相入口202进入液滴分选部件后，先流过所述第一通道203，随后根据检测结果流入第二通道204或第三通道205。
在本发明中，本发明人利用“Y”形管道两侧流速比的差异，使液滴在无外力作用的情况下，向第二通道204(图示通道A)移动。在第三通道205(图示通道B)一侧，使用锡材质等金属制备电极。当阳性液滴(即包含具有特异性TCR的细胞的液滴)通过第一通道203时，会在激光激发下发光，液滴由此产生的光信号可以产生偏转力，该偏转力会使液滴向第三通道205一侧移动，从而实现对目标的分选。
除了样品进样部件和液滴分选部件外，所述液滴分选芯片还可以包括液滴收集部件，用于收集分选通过各通道的液滴。
光学检测平台可以包括：观察系统、荧光激发检测系统和信号处理系统。
作为一个示例，观察系统和荧光激发检测系统是通过在显微镜(例如Olympus IX83显微镜)基础上加装光学部件而构成的。图3示出了本发明方法可以采用的观察系统和荧光激发检测系统的一个示例的结构示意图。此显微镜提供两个相机接入口，其中一个接入口加装高速相机(例如OlympusDP23高速相机)，另一接入口安装光电倍增管(例如来自Hamamatsu公司的型号为H10770A-40的产品)。
显微镜激发光路为：卤素灯产生强光，经UIS2镜组正面滤色片(例如来自Olympus的型号为U-FBNA的产品)过滤目标波长的光(例如，对于GFP蛋白，为波长为488nm的光)。经过滤得到的光经镜组中央二项色镜反射，并通过物镜(例如来自Olympus的型号为UPLXAPO20X的产品)照射分选芯片垂直电极处通道。当液滴通过时，如果液滴内细胞表达报告分子例如GFP蛋白，则报告分子会被激发，随后该报告分子产生的发射光经物镜、二项色镜、UIS2镜组底部滤镜、经显微镜自备反射镜抵达光电倍增管，该光电倍增管随后将光信号转化为电信号例如电压信号。
作为一个示例，该信号处理系统可以由4个独立设备构成，这四个设备分别为电信号比较器、单片机、波形发生器和电压放大器，参见图4。光电倍增管产生的电信号在传出后分成两路，其中一路信号经线路01传递至单片机(例如来自正点原子、型号为STM32F407ZGT6的产品)，另一路信号经线路02传递至电压比较器(例如来自康威、型号为TLV3501的产品)。当光电倍增管产生电信号，并且该电信号传导至电信号比较器时，该电信号比较器被触发，产生高电平(如4V高电平)。该高电平经过线路03传递至单片机。此时，单片机被触发，并以一定频率例如100KHz频率开始记录经01线路传递抵达的电信号。当高电平结束时，停止记录，并计算本次记录的电信号中的最大值。当最大值大于预先设定阈值时，表明液滴为阳性液滴。在这种情况下，单片机会发出持续一段时间的电压(例如持续2毫秒的2V电压)，并且该电压经线路04传递至波形发生器(例如来自Tektronix、型号为AFG2021的产品)。波形发生器设置为外部触发模式，当其被触发时会产生一定频率且持续一定时间的某个电压(例如20kHz且持续2ms的3V(3V-5V可选)电压)。该电压经连线05传递至电压放大器(例如来自FLC、型号为F10AD的产品)，并在经由该电压放大器放大200倍后，经过线路06传递至液滴分选部件，在此产生偏转力，从而实现液滴偏转。
作为一个示例，所述偏转力可以通过介电泳的形式产生。在这种情况下在分选部件处，利用物体在不均匀电场内受力不平衡的原理，用电极制造不均匀电场，诱导液滴向高 电场方向移动，从而实现阳性液滴分选。另外，通过优化电极和第三通道205(B通道)之间的距离，可以在较小的电压下(600V)，实现液滴的偏转。
作为另一个示例，所述偏转力可以通过声波的形式产生。在这种情况下，在分选部件处，放置叉指电极，利用电极产生声波在芯片表面传播衰减的特性，令液滴向声波衰减位置移动，从而实现液滴的偏转。
在本发明的第三方面，提供了一种RNA测序方法，所述方法包括以下步骤：
i.通过实施本发明第二方面的方法分选包含具有表达特异性TCR的细胞的阳性液滴；
ii.回收所述阳性液滴中的细胞；并且
iii.对回收的细胞进行测序，由此获得所述细胞中包含的抗原特异性TCR的序列信息。
在一个实施方案中，所述方法可以是高通量测序方法；在一个进一步的实施方案中，所述方法为单细胞RNA测序方法。
可以理解，在分选得到包含具有特异性TCR的细胞的阳性液滴之后，需要对该TCR的序列信息进行进一步的表征，以将其利用到有重大意义的CAR-T和/或TCR-T免疫疗法中。
因此，在分选得到阳性液滴之后，可以利用化学破乳剂，按照专利申请202110593330.6披露的方法从所述阳性液滴中回收其中包含的细胞。
在一个示例性实施方案中，回收所述阳性液滴中的细胞的步骤ii.包括以下步骤：
(1)向所述阳性液滴中加入破乳剂，然后静置，优选静置8-15分钟，更优选9-12分钟，最优选10分钟；
(2)将静置后的液体最下层的油相移出；
(3)向所得的液体中再次加入所述破乳剂，然后静置，优选静置3-7分钟，更优选4-6分钟，最优选5分钟；
(4)将所得的液体进行低速离心，优选30g-100g，更优选40g-70g，最优选50g，所述离心持续8-15秒，优选9-12秒，最优选10秒，由此所述液体形成上下两层，中间有界面；
(5)收集所述界面之上包含所述内容物的液体，不触及所述中间的界面；
(6)任选地，一次或者多次地向所述界面上方的剩余液体中加入水性溶液优选细胞培养液，再次收集所述界面之上包含所述内容物的液体。
在一个实施方案中，所述破乳剂选自单宁基混合物、乙氧基化或环氧化的PAG、氧烷基化的聚(亚烷基)聚(胺)、聚(醚)聚(氨酯)和辛醇类，优选为辛醇类，更优选为正辛醇、 异辛醇、丁辛醇、1H，1H，8H-全氟-1-辛醇或1H，1H，2H，2H-全氟-1-辛醇。
在一个实施方案中，在(1)中，所述阳性液滴位于上层，液滴生成油位于下层，优选在加入破乳剂之前除去部分的下层液滴生成油。
在一个实施方案中，在(1)中，加入破乳剂的体积是阳性液滴体积的1倍。
在一个实施方案中，在(3)中再次加入的破乳剂的体积少于在(1)中加入的破乳剂的体积。
在一个实施方案中，步骤ii在冰上进行操作。
然后，对回收得到的细胞进行测序，从而获得抗原特异性TCR的序列信息。在一个实施方案中，所述测序方法可以是单细胞RNA测序法，该方法是一种高通量测序方法。
在本发明的第四方面，提供了通过本发明第三方面的方法获得的抗原特异性TCR的序列信息在细胞免疫治疗中的用途。
TCR-T细胞，又称为T细胞受体嵌合T细胞(TCR-modified T cells)，CAR-T细胞又称为嵌合抗原受体T细胞(Chimeric antigen receptor T cells)。CAR-T细胞免疫疗法和TCR-T细胞免疫疗法都属于免疫细胞治疗方法，它们的一个共同点在于通过基因改造的手段提高T细胞受体对特异性癌症细胞抗原的识别能力和进攻能力。但是，两者又有一些明显差异，例如，CAR-T细胞免疫疗法是通过识别膜表面抗原(如CD19，BCMA等)而发挥作用，对血液肿瘤疗效显著，而TCR-T细胞可识别细胞膜表面或细胞内来源的肿瘤特异性抗原，其中靶向NY-ESO-1的TCR-T细胞在国内外治疗难治复发性黑色素瘤、滑膜肉瘤、多发性骨髓瘤和肺癌等临床试验研究中，已经展示了良好的安全性和有效性，是目前最有可能在实体瘤取得突破的T细胞免疫疗法。
由于TCR对于这两种细胞免疫疗法有着至关重要的作用，因此，本发明方法可以为TCR-T和CAR-T细胞免疫疗法的实现提供更大可能性。
在一个实施方案中，所述细胞免疫治疗包括构建TCR-T和/或CAR-T细胞。本领域普通技术人员可知，在获得抗原特异性TCR序列信息之后，可采用本领域常规方法构建TCR-T和CAR-T细胞用于后续的免疫治疗。
本发明的检测、分选和/或测序抗原特异性TCR的方法具有以下一个或者多个优势：
1)本发明可以高通量地筛选多种TCR：借助于液滴微流控技术，可以制备上百万个液滴，由于液滴在分选芯片内高速移动，因此细胞可以在液滴内独立地且同时地完成多种TCR对抗原的特异性的检测，相对在孔板内检测细胞相互作用，通量显著提高；
2)高准确性：本发明相比传统的细胞扩增法，可以显著降低bystander效应，显著降 低假阳性率；
3)操作快速：相比于细胞扩增法，本方法可以在短时间内筛选出抗原特异性的TCR；
4)灵敏度高：相比于MHC四聚体技术，本方法借助于报告分子例如GFP蛋白的累积作用，可以实现信号的放大，有更优的灵敏度；
5)信号稳定：本发明的一部分技术方案采用荧光蛋白作为报告分子，因而不依赖底物；相比其他现有技术，本发明的信号稳定，报告分子例如GFP蛋白可以在细胞稳定存在24小时甚至48小时，方便后续的检测；
6)本发明有较好的拓展性：与Mark Davis团队在Nature Biotechnology上发表的研究相比，本发明不仅可以验证/检测TCR特异性，还可以用于抗原特异性TCR的筛选；
7)细胞活力：通过本发明方法分选得到的细胞，其活细胞比例可以达到至少70％，因此能够满足单细胞测序的要求。
实施例
在本实施例中，本发明人分别建立了T2-MHC细胞和Jurkat DMF5 TCR细胞，并进行液滴包裹，其中Jurkat细胞表面TCR识别T2细胞表面的MHC-抗原肽复合物。依照本实验设计，只有当液滴中同时存在T2细胞和Jurkat细胞时，Jurkat细胞表面的TCR才能被激活，胞内GFP荧光蛋白表达，进而被488nm激光激发，被系统分选。依照本实验设计，约3.29％液滴中同时存在Jurkat细胞和T2细胞。为佐证结果的可靠性，在实验前对部分T2细胞和Jurkat细胞标记不同的荧光分子(T2细胞：CellTrace TM Violet；Jurkat细胞：CellTracker TM Green CMFDA)。在液滴生成后，检测液滴中CellTracker TM Green CMFDA(用488nm光源激发)和CellTrace TM Violet(用408nm光源激发)，分别统计CellTracker TM Green CMFDA和CellTrace TM Violet阳性液滴的比例，以证明细胞在液滴内分布接近理论值。随后，将负载抗原的T2细胞和Jurkat细胞包裹在液滴中，由于仅部分Jurkat细胞可以和T2共包裹，在完成孵育后，仅部分液滴内Jurkat细胞表达GFP蛋白，因此，液滴分选效果评估环节无需额外加入对照样本。随后进行液滴分选，使用流式细胞术检测分选所得细胞中表达GFP细胞所占比例，以评估液滴分选的效果。
实验材料
一、用于细胞报告系统构建的实验材料：
1.Jurkat细胞(美国菌种保藏中心，编号为TIB-152)
2.T2细胞(美国菌种保藏中心，编号为CRL-1992)
3.细胞培养液：
i.RPMI-1640培养基(GBICO，Cat.22400-089)
ii.10％胎牛血清(HYCLONE，SH30084.03HI)
4.逆转录病毒浓缩液，其包含DMF5 TCR序列(Creative Biolab，VP-TCR-C928)
5.慢病毒浓缩液，其包含NFAT-GFP序列(BPS Bioscience，79922)
6.二甲基亚砜(DMSO)(Sigma-Aldrich，D2650)
7.MART-1 (27-35)多肽LAGIGILTV(金斯瑞合成干粉，使用DMSO溶解)
8.促转染剂海美溴铵(Hexadimethrine bromide)(Sigma，H9268)
9. 12孔细胞培养板(JET BIOFIL，TCP011012)
二、用于抗原特异性TCR检测的实验材料：
1.液滴生成装置：本发明采用类似于CN 209144161 U中公开的液滴生成装置，其中，液滴生成芯片更换为专利中请PCT/CN2019/108536中公开的芯片，注射器更换为BD 50ml注射器(Cat.309654)。
2.液滴分选设备，其包括分选芯片和光学液滴分选平台。
3.液滴生成油(Bio-Rad，Cat.1864006)
4.CellTrace TMViolet染料(Invitrogen，Cat.C34557)
5.CellTracker TMGreen CMFDA染料(Invitrogen，Cat.C2925)
6.二甲基亚砜(DMSO)(Invitrogen，Cat.C34557)
7.DPBS(Invitrogen，Cat.C14190500BT)
8. 1640培养基(GBICO，Cat.22400-089)
9.工作液，包含以下成分：
·1640培养基(GBICO，Cat.22400-089)
·0.1％Pluronic F68(Invitrogen，24040-032)
·25mM HEPES pH 7.4(INVITROGEN，15630-106)
·5％超低IgG胎牛血清(THERMO，1921005PJ)
·1％Pen/Strep(INVITROGEN，15140122)
·6％Ficoll(GE，17-0300-10)
实验流程：
1.细胞株构建
首先构建了一种TCR信号报告细胞株：
本细胞株由Jurkat细胞改造获得的，通过慢病毒载体向Jurkat细胞中转染NFAT-GFP基因和DMF5TCR基因，随后利用流式细胞分选，获得稳定表达TCR和GFP的细胞株(Jurkat-TCR细胞)；其中，活化T细胞核因子(NFAT)反应元件是细胞内TCR下游钙离子调控转录因子，当TCR信号通路激活，NFAT被激活并结合NFAT-GFP上游NFAT结合区域，启动下游GFP蛋白的表达。TCR文库可以包含多种不同的TCR，但是每个细胞仅转染一种TCR。
1.1 Jurkat-TCR细胞株的构建：
1)取细胞状态良好的Jurkat细胞进行计数，然后加入12孔板中，每孔4×10 5个细胞，补液至1毫升(mL)。
2)将促感染剂海美溴铵(1∶1000)加入细胞中，并加入体积分别为10微升(μL)的逆转录病毒浓缩液(包含DMF5TCR序列)和慢病毒浓缩液(包含NFAT-GFP序列)。
3)将细胞混匀，封口膜封住，然后以800g离心30min。
4)离心后，撕去封口膜，再37℃培养过夜。
5)第二天进行细胞换液，用1000微升(μL)RPMI-1640+10％FBS培养。
1.2 T2-MHC细胞株的构建：
1)取细胞状态良好的T2细胞进行计数，然后加入12孔板中，每孔4×10 5个细胞，补液至1毫升(mL)。
2)将促感染剂海美溴铵(1∶1000)加入细胞中，并加入体积为10微升(μL)的慢病毒浓缩液(包含HLA-A*0201序列)。
3)将细胞混匀，封口膜封住，然后以800g离心30min。
4)离心后，撕去封口膜，再37℃培养过夜。
5)第二天进行细胞换液，用1000微升(μL)RPMI1640+10％FBS培养。
1.3细胞报告系统荧光表达能力的评估
1.3.1操作步骤
1)取10 6个T2-MHC细胞悬于1ml工作液，加入多肽MART-1 (27-35)LAGIGILTV，使其终浓度为80μg/mL。
2)孵育过夜后，以500g离心10分钟，弃上清，并加入工作液重悬细胞至终浓度 2×10 5/ml。
3)取1×10 6个Jurkat-TCR细胞加入15ml离心管，常温下以500g离心5分钟，弃上清，加入工作液重悬细胞至终浓度2×10 5/ml。
4)96孔板每孔加入100微升T2-MHC细胞和100微升Jurkat-TCR细胞，37℃、5％CO 2培养箱分别培养0、3、6、9、12、24小时。
5)在培养0、3、6、9、12、24小时后，取出细胞，进行流式细胞术以分析GFP蛋白表达。
1.3.2实验结果
如图5所示，随T2-MHC细胞刺激Jurkat-TCR细胞时间的增加，经激发的GFP蛋白荧光强度也随之增加，表明GFP蛋白的表达随着刺激时间的增加而增加。当刺激3小时后，Jurkat细胞内荧光信号和初始值(在0小时时)之间有显著的差异(Wilcox-ranked检验，P＜0.001)。
2.液滴制备
在构建好T2-MHC细胞株和Jurkat-TCR细胞株后，先使T2-MHC细胞株与抗原多肽共同孵育，由此将该抗原多肽加载到T2-MHC细胞株的MHC上。然后，利用液滴生成芯片并利用液滴生成油将所述T2-MHC细胞株和Jurkat-TCR细胞株包裹在液滴中。在进行液滴制备时，需要适当地调整两种细胞的浓度，以使多数液滴中包裹有T2-MHC细胞(空液滴比例≤70％)，同时使Jurkat-TCR细胞的多包率≤5％。借助这样的设计，多数Jurkat-TCR细胞均能够接触抗原。同时，由于每个液滴中只有一种TCR，因此可以避免非特异性TCR被富集。同时，为了评估液滴包裹和平台分选液滴的效率，我们对部分T2-MHC细胞和Jurkat-TCR细胞进行荧光染料标记，并借助荧光显微镜(Olympus IX83)进行了观察和统计。
2.1染料配制：
1.CellTracker TM Green CMFDA染料：使用10微升(μL)的二甲基亚砜(DMSO)溶解该染料，由此来配制该染料的储液，其终浓度为10mM。
2.CellTrace TM Violet染料：使用20微升(μL)的二甲基亚砜溶解该染料，由此来配制该染料的储液，其终浓度为5mM。
2.2标记细胞：
2.2.1 T2-MHC细胞标记
1)向1ml实验流程1.2部分中构建的T2-MHC细胞悬液中加入多肽MART-1 (27-35) LAGIGILTV，使其终浓度为80μg/mL。
2)过夜后，以500g离心10分钟，弃上清，并加入RPMI-1640培养基以重悬细胞。
3)取1mL DPBS，加入1μL CellTracker TM Green CMFDA染料的储液，混匀，由此制备获得该染料的工作液。
4)取4×10 6个T2-MHC细胞加入15mL离心管，常温下以500g离心5分钟，弃上清，加入1ml含有CellTracker TM Green CMFDA染料的工作液来重悬细胞。
5)37℃孵育细胞20分钟。
6)加入5mL含有10％FBS的RPMI-1640培养基，37℃孵育5分钟。
7)将细胞以500g离心5分钟，弃上清。
8)加入200微升(μL)工作液重悬细胞，由此制备得到经CellTracker TM Green CMFDA染料标记的T2-MHC细胞悬液，备用。
2.2.2.Jurkat-TCR细胞标记
1)取1mL DPBS，加入1μL CellTrace TM Violet染料的储液，混匀，由此制备获得该染料的工作液。
2)取8×10 5个Jurkat-TCR细胞加入15ml离心管，常温下以500g离心5分钟，弃上清，加入1mL含有CellTrace TM Violet染料的工作液来重悬细胞。
3)37℃孵育细胞20分钟。
4)加入5mL含有10％FBS的RPMI-1640培养基，37℃孵育5分钟。
5)将细胞以500g离心5分钟，弃上清。
6)加入200微升(μL)工作液来重悬细胞，由此制备得到经CellTrace TM Violet染料标记的Jurkat-TCR细胞悬液。
2.3液滴制备
使用图1所示的微流控芯片系统10来制备液滴，该微流控芯片系统已经在上面的具体实施方式部分进行了详细描述。具体步骤如下：
1)经由所述连续相入口111将120微升(μL)液滴生成油加入液滴生成装置11的芯片主体中，并经由所述第一分散相入口112和所述第二分散相入口113将各50微升(μL)的经荧光染料标记的Jurkat-TCR细胞悬液和T2-MHC细胞悬液加入液滴生成装置11的芯片主体中。
2)启动动力产生装置13，使第二连接管15、液滴收集容器12、第一连接管14和芯片主体内的压强与大气压强之间形成压强差，该压强差促使所述细胞悬液和所述液滴生成 油汇流并形成直径约为45μm的液滴。将形成的液滴最后收集于所述液滴收集容器12中。
2.4细胞包裹率检测
将实验流程2.3部分制备的液滴通过荧光显微镜检查液滴生成油包裹细胞的情况。具体地，采用488nm光源检测经CellTracker TM Green CMFDA染料标记的Jurkat-TCR细胞，采用405nm光源检测经CellTrace TM Violet染料标记的T2-MHC细胞，再统计液滴包裹各细胞的情况。
Jurkat-TCR细胞包裹率计算方法为：选取5个视野，显微镜明场计算视野内所有液滴的数量，记为N1；打开显微镜激光光源，调整至488nm滤光片，统计视野内包裹一个绿色荧光标记细胞的液滴数量，记为N2，包裹超过一个绿色荧光标记细胞的液滴数量，记为N3；则Jurkat-TCR多包率为N3/(N2+N3)*100％。
T2-MHC细胞包裹率计算方法为：选取5个视野，显微镜明场计算视野内所有液滴的数量，记为N1；打开显微镜激光光源，调整至488nm滤光片，统计视野内包裹一个或一个以上蓝色荧光标记细胞的液滴数量，记为N2；则T2细胞的包裹率为N2/N1*100％。
2.5细胞活力检测
将实验流程2.3部分制备的液滴放置在37℃、5％二氧化碳的培养箱中培养一段时间，然后取出部分液滴，用1H，1H，2H，2H-全氟-1-辛醇破乳，取出细胞，在使用吖啶橙/碘化丙啶(AO/PI)染色，统计活细胞所占比例。
2.6实验结果：
2.6.1细胞包裹率
理论值计算
1)Jurkat-TCR细胞液滴包裹率的统计结果：细胞浓度为4×10 6/ml，液滴直径约为45μm，依据泊松分布计算可知lamda(λ)＝0.095，则液滴中仅包裹一个细胞的概率是8.67％，包裹超过一个细胞的概率是0.43％。
2)T2-MHC细胞液滴包裹率的统计结果：细胞浓度为2×10 7/ml，液滴直径约为45μm，依据泊松分布计算可知lamda(λ)＝0.48，则液滴中包裹有至少一个T2-MHC细胞的概率是38％。
3)因此，液滴同时包裹有Jurkat-TCR细胞和T2-MHC细胞的比例是8.67％×38％＝3.29％。
实际测量值
根据实际测量，液滴中同时包裹有Jurkat-TCR细胞和T2-MHC细胞的比例约为2％(图 7)，接近理论值3.29％。
2.6.2液滴内T2-MHC和Jurkat-TCR细胞的细胞活力
图6示出了液滴包裹的T2-MHC和Jurkat-TCR细胞的细胞活力结果。从图中可以看到，在8小时内，活细胞比例保持在70％以上，12小时后低于60％。因此，选择孵育细胞的时间，优选地不超过8小时，由此保证活细胞比例能在70％以上，从而确保能将细胞用于后续的单细胞测序中。
2.6.4液滴内T2-MHC激活Jurkat-TCR细胞结果
图8示出了液滴内T2-MHC激活Jurkat-TCR细胞的结果。如图8所示，随着抗原肽浓度的增加，GFP阳性细胞的比例也随之上升(左图)；另外，在负载浓度为80μg/ML的抗原肽的情况下，对照组(T2-MHC未负载DMF5多肽)表达GFP的细胞比例低于1％，而实验组(T2-MHC负载80μg/ml DMF5多肽)表达GFP的细胞比例接近15％(右图)。以上结果表明，液滴中的T2-MHC细胞能够激活Jurkat-TCR细胞表达GFP蛋白。
3.液滴分选和细胞回收
在进行液滴分选之前，先将实验流程2.3部分中生成的液滴置于37℃、5％二氧化碳的培养箱中孵育8小时。之后，取出部分液滴，在荧光显微镜下，使用488nm激光激发并观察液滴中GFP蛋白的表达。在荧光显微镜下观察到GFP蛋白的表达后，将液滴用于后续的分选实验中。
液滴分选在液滴分选装置中进行，该液滴分选装置利用介电泳力来进行液滴分选。有关在本发明中使用的液滴分选装置的详细描述参见上文。
3.1步骤方法
按照以下方法步骤进行液滴分选和细胞回收：
1)将生成的液滴转移至1mL注射器，用导管连接注射器针头和分选芯片液滴入口201。
2)将注射器固定至注射泵，调节流速至50μL/小时。
3)取5mL注射器，吸取液滴生成油，固定于注射泵，调节流速至1200μL/小时，作为鞘液。
4)按图2所示，将分选芯片出口连接至1.5mL Eppendorf管。
5)打开注射泵，运行5分钟后，液路稳定。
6)打开488nm激光器，检测液滴，调节光电倍增管(PMT)增益至0.45，开始接收信号。
7)在将分选阈值设定为1V(实验组信号大于1的液滴比例为29％，对照为1％)时，可以有效地筛选阳性液滴。
8)设定信号发生器(即波形发生器)受外部信号触发，当存在外部信号时，所述信号发生器会被触发并由此发出持续2毫秒的20kHz方波，电压Vpp(峰-峰电压值)为4V，该电压再传递至电压放大器后在此被放大200倍，用于在分选芯片电极处形成介电泳，以偏转荧光信号触发阈值以上的液滴。
9)液滴分选结束后，从分选芯片内拔出导管，令导管直立，排出管内液滴。
10)此时液滴漂浮在上层，下层为连续相液体(液滴生成油作为本实验鞘液，此处连续相为液滴生成油)。
11)使用1mL注射器小心移除下层液滴生成油，由此获得阳性液滴。
12)向液滴收集管内缓慢滴加200μL 1H，1H，2H，2H-全氟-1-辛醇(英文缩写PFO，Sigma-Aldrich，货号370533-25G)，静置10分钟；此时样品收集管内有三层，下层是液滴生成油和PFO的油相混合物，中间是未破乳的液滴，上层是破乳释放的含细胞的水相。
13)使用1ml注射器移除液滴收集管下层油相，再向样品收集管内缓慢滴加100μL PFO，静置5分钟；
14)将所述样品收集管50×g离心10秒；此时样品收集管内液体分为两层，下层是液滴生成油和PFO的油相混合物，上层是破乳释放的含细胞的水相，两层之间有清晰的界面。
15)使用100μL量程移液器轻轻吹打上层细胞悬液，不触及界面。
16)向所述样品收集管中缓慢加入200μL的RPMI 1640完全培养基，并使用100μL移液器轻轻混匀上层细胞。
17)吸取上层细胞至新1.5mL EP管。
18)500×g离心该1.5mL EP管5分钟，使用100μL移液器丢弃上清，加入20μL含0.04％BSA的PBS溶液重悬细胞。
19)取部分细胞进行流式细胞术，检测GFP阳性细胞所占比例。
3.2实验结果
3.2.1液滴分选效果
1)操作时间：平台调整20分钟，分选用时4小时。
2)GFP阳性细胞比例接近70％(图9)，表明包含阳性细胞的液滴被富集。
4.目标液滴内细胞回收和单细胞测序
4.1实验步骤
4.1.1单细胞建库测序
对于分选和回收获得的细胞，按照DNBelab C4 scRNA-seq试剂盒说明书，完成单细胞RNA cDNA的制备。同时取部分Jurkat细胞作为对照，平行进行单细胞建库测序。使用MGI T7测序仪配套的PE100试剂盒完成测序文库的制备和测序。
4.1.2测序数据分析
1)使用PISA软件(https：//github.com/shiquan/PISA)处理测序获得的FASTQ文件，获得基因表达矩阵和细胞标签信息。
2)使用Seurat软件(v4.0.1，https：//github.com/satijalab/seurat)处理基因表达矩阵和细胞标签信息，删除仅在3个以下细胞中表达的基因，并删除检测基因数少于200的细胞。
3)使用principal component analysis(PCA)分析，选取对分群贡献度最高的10个成分，建立SNN网络，使用图聚类方法louvain algorithm鉴定细胞亚群，随后使用UMAP方法可视化聚类。
4)CD2分子高表达的细胞亚群为Jurkat细胞。
5)分析Jurkat细胞线粒体相关基因和细胞活化基因表达情况，确定本发明获取的细胞能否用于单细胞RNA测序。
4.2实验结果
本实验共获取了1500个细胞用于单细胞RNA数据分析。图10示出了分选出的T细胞的单细胞测序结果，图11示出了示出单细胞测序质控结果。
如图10所示，在UMAP图上，细胞可以分为两个大群，右侧细胞亚群相对高地表达CD2和CD3G，表明它们是Jurkat细胞，左侧细胞亚群高表达CD86，表明它们是抗原提呈细胞。因此，上述结果证明，本发明能够在操作过程中维持细胞的特征，适用于单细胞测序分析流程。
另外，如图11所示，在6个细胞亚群细胞中检测到的基因数均大于2000，表明单细胞测序方法能够有效捕获本发明方法分选的细胞的基因；在所有细胞中，线粒体相关基因均少于总基因数的10％，进一步表明经本发明方法分选获得的细胞的状态满足单细胞RNA测序的要求。

Claims (30)

  1. 一种检测抗原特异性T细胞受体(TCR)的方法，所述方法包括以下步骤：
    a)制备第一种细胞和第二种细胞，其中所述第一种细胞表达TCR、TCR激活响应元件和报告分子，所述第二种细胞表达主要组织相容性复合物(MHC)；
    b)通过共同孵育所述第二种细胞与抗原多肽以使所述抗原多肽与所述MHC形成抗原多肽-MHC复合物，从而将所述抗原多肽加载于所述第二种细胞；
    c)利用液滴微流控技术，用第一油相包裹所述第一种细胞和经所述抗原多肽加载后的第二种细胞来制备液滴，其中，所述液滴中至少一部分的单个液滴中仅包含一个所述第一种细胞和至少一个所述第二种细胞；
    d)用激光照射液滴，并根据液滴是否发光确定该液滴是否包含抗原特异性TCR。
  2. 根据权利要求1所述的方法，其中，在步骤d)之前孵育所述液滴，优选地孵育时间不超过8小时。
  3. 根据权利要求1或2所述的方法，其中，所述第一种细胞通过共同孵育所述第一种细胞的来源细胞、TCR表达载体、表达所述TCR激活响应元件和所述报告分子的载体、和任选的促转染试剂来制备，所述第二种细胞通过共同孵育所述第二种细胞的来源细胞、MHC表达载体以及任选的促转染试剂来制备。
  4. 根据权利要求1-3中任一项所述的方法，其中，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。
  5. 根据权利要求1-4中任一项所述的方法，其中，所述促转染试剂为海地美铵、Lipofectamine2000。
  6. 根据权利要求3-5中任一项所述的方法，其中，所述第一种细胞的来源细胞为T细胞的淋巴瘤细胞，优选为Jurkat细胞或人来源T淋巴细胞；所述第二种细胞的来源细胞为抗原呈递细胞，优选为T2细胞或K562细胞。
  7. 根据权利要求1-6中任一项所述的方法，其中，所述MHC为MHC-I类、MHC-II类或MHC-III类，优选为MHC-I类。
  8. 根据权利要求1-7中任一项所述的方法，其中，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。
  9. 根据权利要求1-8中任一项所述的方法，其中，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)。
  10. 根据权利要求1-9中任一项所述的方法，其中，所述抗原多肽为肿瘤抗原多肽，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。
  11. 根据权利要求1-10中任一项所述的方法，其中，所述第一种细胞含有多个细胞，且所述多个细胞中至少一部分细胞表达不同种类的TCR。
  12. 根据权利要求1-11中任一项所述的方法，其中，在步骤c)中，通过以下方式来使所述液滴中至少一部分的单个液滴中仅包含一个所述第一种细胞和至少一个所述第二种细胞：设定液滴直径、所述第一种细胞的多包率和所述第二种细胞的空液滴比例，根据泊松分布来确定所述第一细胞的工作浓度以及所述第二细胞的工作浓度：
    Figure PCTCN2021113888-appb-100001
    λ＝C*πr 3/2
    其中，
    k是液滴内细胞数且为＞0的整数，
    e是常数，
    C是细胞悬液浓度(细胞数/μL)，
    λ是期望值，
    f(k；λ)是当k和λ确定时液滴内包裹k个细胞的概率，
    r为液滴半径(μm)。
  13. 根据权利要求12所述的方法，其中，设定所述液滴直径为30μm至100μm、第一种细胞的多包率≤5％和第二种细胞的空液滴比例≤70％；优选地，设定所述液滴直径为30μm至80μm。
  14. 根据权利要求13所述的方法，其中，在液滴直径为45μm的情况下，所述第一细胞的工作浓度为1×10 4个细胞/mL至4×10 6个细胞/mL，并且所述第二细胞的工作浓度为≥1×10 7个细胞/mL。
  15. 一种分选抗原特异性T细胞受体(TCR)的方法，所述方法包括以下步骤：
    1)通过实施权利要求1-14中任一项所述的方法的步骤a)至c)来制备液滴，所述液滴中至少一部分的单个液滴中仅包含一个所述第一种细胞和至少一个所述第二种细胞；
    2)使所述液滴逐个地通过激光照射位点，并在此处检测液滴的发光情况；并且
    3)在检测到液滴发光时，将液滴产生的光信号转化为偏转力，并利用该偏转力分选包含具有抗原特异性TCR的细胞的阳性液滴。
  16. 根据权利要求所述的方法，其中，在步骤2)之前孵育所述液滴，优选地孵育时间不超过8小时。
  17. 根据权利要求15-16中任一项所述的方法，其中，通过使所述液滴分散于作为鞘液的第二油相中并使所述第二油相流动通过激光照射位点，从而使所述液滴逐个地通过激光照射位点。
  18. 根据权利要求15-17中任一项所述的方法，其中，通过调整液滴在第二油相中的浓度并调整第二油相流动通过激光照射位点的速度，从而使液滴逐个地通过激光照射位点。
  19. 根据权利要求15-18中任一项所述的方法，其中，所述偏转力是通过将所述光信号转化为电信号产生的。
  20. 根据权利要求19所述的方法，其中，所述偏转力是由所述电信号产生的介电泳或声波产生的。
  21. 一种RNA测序方法，所述方法包括以下步骤：
    i.通过实施权利要求15-20中任一项所述的方法分选包含具有抗原特异性TCR的细胞的阳性液滴；
    ii.回收所述阳性液滴中的细胞；并且
    iii.对回收的细胞进行测序，由此获得所述细胞中包含的抗原特异性TCR的序列信息。
  22. 根据权利要求21所述的方法，其中，所述方法为高通量测序方法。
  23. 根据权利要求21或22所述的方法，其中，所述方法为单细胞RNA测序方法。
  24. 根据权利要求21-23中任一项所述的方法，其中，回收所述阳性液滴中的细胞的步骤ii.包括以下步骤：
    (1)向所述阳性液滴中加入破乳剂，然后静置，优选静置8-15分钟，更优选9-12分钟，最优选10分钟；
    (2)将静置后的液体最下层的油相移出；
    (3)向所得的液体中再次加入所述破乳剂，然后静置，优选静置3-7分钟，更优选4-6分钟，最优选5分钟；
    (4)将所得的液体进行低速离心，优选30g-100g，更优选40g-70g，最优选50g，所述离心持续8-15秒，优选9-12秒，最优选10秒，由此所述液体形成上下两层，中间有界面；
    (5)收集所述界面之上包含所述内容物的液体，不触及所述中间的界面；
    (6)任选地，一次或者多次地向所述界面上方的剩余液体中加入水性溶液优选细胞培养液，再次收集所述界面之上包含所述内容物的液体。
  25. 根据权利要求24所述的方法，其中，所述破乳剂选自单宁基混合物、乙氧基化或环氧化的PAG、氧烷基化的聚(亚烷基)聚(胺)、聚(醚)聚(氨酯)和辛醇类，优选为辛醇类，更优选为正辛醇、异辛醇、丁辛醇、1H，1H，8H-全氟-1-辛醇或1H，1H，2H，2H-全氟-1-辛醇。
  26. 根据权利要求24-25中任一项所述的方法，在(1)中，所述阳性液滴位于上层，液滴生成油位于下层，优选在加入破乳剂之前除去部分的下层液滴生成油。
  27. 根据权利要求24-26中任一项所述的方法，其中，在(1)中，加入破乳剂的体积是阳性液滴体积的1倍。
  28. 根据权利要求24-27中任一项所述的方法，其中，在(3)中，再次加入的破乳剂的体积少于在(1)中加入的破乳剂的体积。
  29. 通过权利要求21-28中任一项所述的方法获得的抗原特异性TCR的序列信息在细胞免疫治疗中的用途。
  30. 根据权利要求29中任一项所述的方法，所述细胞免疫治疗包括构建TCR-T和/或CAR-T细胞。
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