MHC polymer compound coupled with barcode oligonucleotide as well as preparation method and application of MHC polymer compound

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses an MHC polymer compound coupled with barcode oligonucleotide as well as a preparation method and application of the MHC polymer compound. Wherein the kit comprises 1-4 MHC monomers, streptavidin, an oligonucleotide and a barcode oligonucleotide which is reversely complementary with the oligonucleotide sequence; and the oligonucleotide is combined with the streptavidin through a covalent bond. A double-strand oligonucleotide modification method is adopted, only one-time oligonucleotide coupling is needed, bar code oligonucleotide sequences can be varied according to needs, rapid production of multiple kinds of bar code oligonucleotide labeled streptavidin on a large scale is achieved, operation is easy, stability is high, and the coupling cost is reduced.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to an MHC multimer compound conjugated with barcode oligonucleotides, a preparation method thereof, and an application thereof. Background Art

[0002] Since the advent of MHC multimers, it has completely changed the way of analyzing antigen-specific T cells by indirect methods such as in vitro amplification culture in the past (Altman et al., 1996). MHC multimers can associate antigen peptides with their specific T cells. MHC monomer molecules loaded with antigen peptides can be affinity-linked to streptavidin through biotin modification sites. One streptavidin molecule can link four MHC molecules loaded with antigen peptides, thereby forming a multimer (MHC-tetramer).

[0003] Since the recognition of antigens by antigen-specific T cells occurs through MHC-restricted TCR, after TCR binds to the MHC multimer, antigen-specific T cells can be directly characterized. A common method is to conjugate a fluorescent dye to streptavidin, and then analyze or sort antigen-specific T cells by flow cytometry or other means. The combination with next-generation sequencing technology can further analyze antigen-specific T cells at the gene level or protein expression level.

[0004] However, due to the limited selectivity of streptavidin conjugated with fluorescence, it is impossible to perform mixed MHC multimer staining of multiple antigen epitopes and multiple samples. The challenge in single-cell library construction is that it is impossible to correspond antigen information to TCR sequences. Current studies have shown that barcode oligonucleotide labeling of streptavidin can achieve synchronous acquisition of antigen information during single-cell capture, which is used to correspond T cells recognized by MHC multimers to antigen epitopes, effectively breaking through the technical bottleneck of the antigen-TCR correspondence relationship in the high-throughput sequencing of large-scale samples and multiple antigen epitopes.

[0005] However, there are still the following problems: (1) The cost of streptavidin labeled with barcode oligonucleotides is relatively high, and different barcode oligonucleotides need to be conjugated for each antigen epitope, with complex operations, high technical difficulty, and high reagent costs; (2) The cell sorting operation steps in the library construction preparation process are cumbersome, the experimental period is long, the requirements for experimental equipment are high, and there are problems such as loss of T cell activity, which may lead to the loss of some antigen-specific T cells with low affinity, resulting in the loss of key information and incomplete data; (3) Interference by free oligonucleotides causes non-specific labeling, and it is impossible to simultaneously analyze the interaction and communication mechanisms between antigen-specific T cells and other cells in the sample, affecting data mining. Summary of the Invention

[0006] To solve the above problems, the present invention provides an MHC multimer compound conjugated with barcoded oligonucleotides, a preparation method thereof, and an application thereof.

[0007] The present invention adopts the following technical solutions: An MHC multimer compound conjugated with barcoded oligonucleotides, comprising:

[0008] (a) 1 to 4 MHC monomers;

[0009] (b) Streptavidin;

[0010] (c) An oligonucleotide;

[0011] (d) A barcoded oligonucleotide that is reverse complementary to the above oligonucleotide sequence;

[0012] The oligonucleotide is covalently bound to streptavidin, and a single fluorescent label is formed by attaching a fluorescent label to any one of the 5'-end of the oligonucleotide, the 5'-end of the barcoded oligonucleotide, and streptavidin.

[0013] An MHC multimer compound conjugated with barcoded oligonucleotides, comprising:

[0014] (a) 1 to 4 MHC monomers;

[0015] (b) Streptavidin;

[0016] (c) An oligonucleotide;

[0017] (d) A barcoded oligonucleotide that is reverse complementary to the above oligonucleotide sequence;

[0018] The oligonucleotide is covalently bound to streptavidin, and a double fluorescent label is formed by attaching a fluorescent label to the 5'-end of the barcoded oligonucleotide, streptavidin, or the 5'-end of the oligonucleotide.

[0019] Further, the barcoded oligonucleotide comprises a sequence complementary to the oligonucleotide described in (c), a barcode sequence, and a polyA sequence.

[0020] Further, the MHC monomer is MHC-I, MHC-II, wild-type loaded with CLIP, or pMHC loaded with a target antigen obtained by peptide exchange technology.

[0021] Further, the 3'-end of the oligonucleotide is covalently bound to streptavidin, and the covalent bond is a diarylhydrazone bond formed by 4-formylbenzamide and 6-hydrazinonicotinamide.

[0022] Further, the 3'-end of the oligonucleotide is subjected to amino modification or amino modification and iSpC18 spacer modification, and the oligonucleotide may or may not contain a spacer sequence.

[0023] Method for preparing MHC multimer compound conjugated with barcoded oligonucleotide, comprising the following steps:

[0024] (1) Conjugate oligonucleotide to streptavidin;

[0025] (2) Conjugate barcoded oligonucleotide to streptavidin: anneal the barcoded oligonucleotide to the oligonucleotide in a reverse complementary manner;

[0026] (3) Purify the barcoded conjugated streptavidin;

[0027] (4) Assemble the streptavidin conjugated with barcoded oligonucleotide and biotinylated MHC monomer into a multimer;

[0028] (5) Purify the MHC multimer compound conjugated with barcoded oligonucleotide.

[0029] Application of an MHC multimer compound conjugated with barcoded oligonucleotide in multi-epitope high-throughput single-cell sequencing.

[0030] Application of an MHC multimer compound conjugated with barcoded oligonucleotide in antigen-specific T cell flow cytometry analysis or sorting.

[0031] Application of an MHC multimer compound conjugated with barcoded oligonucleotide for preparing a kit for labeling antigen-specific T cells without sorting.

[0032] Furthermore, the kit comprises:

[0033] 1) Antigen-loaded MHC monomer for assembling the multimer;

[0034] 2) Oligonucleotide-conjugated streptavidin for assembling the multimer;

[0035] 3) Barcoded oligonucleotide with fluorescence modification;

[0036] 4) Sequencing primer for amplifying the above barcoded oligonucleotide.

[0037] Advantages of the present invention:

[0038] (1) The MHC multimer of the barcoded oligonucleotide provided by the present invention can recognize TCR on the surface of T cells, thereby characterizing MHC-restricted T cells. By using the double-stranded oligonucleotide modification method, only one oligonucleotide conjugation is required, and the barcoded oligonucleotide sequence can be varied in multiple ways as needed, realizing the rapid production of a large number of barcoded oligonucleotides of various types for labeling streptavidin, constructing a double-fluorescent-labeled pMHC multimer pool with barcoded information, with simple operation and high stability, and reducing the conjugation cost.

[0039] (2) The kit provided by this patent combines single-cell library construction with flow cytometry. After staining with MHC multimers conjugated with barcoded oligonucleotides, if antigen-specific T cells are identified in the sample by flow cytometry, the sample is directly subjected to single-cell library construction without sorting the antigen-specific T cells in the sample and then constructing the library. Finally, the antigen information loaded on MHC is corresponded with the TCR information through the barcodes on the MHC multimers. The obtained bioinformatics data is compared with the flow cytometry results, making the sequencing data more credible and effectively reducing cell damage and information loss during the sorting process of conventional MHC multimer-stained mixed samples.

[0040] (3) The dual purification strategy ensures that there are no free barcoded oligonucleotide chains in the pool of barcoded oligonucleotide-conjugated pMHC multimers, avoiding non-specific labeling, enabling whole-sample analysis of antigen-specific T cells and other cells such as non-antigen-specific T cells, facilitating the exploration of cell-cell interaction mechanisms, and revealing the cell-cell regulatory network. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is a schematic structural diagram of the MHC multimer compound conjugated with barcoded oligonucleotides of the present invention.

[0042] Figure 2 It is a schematic diagram of streptavidin conjugated with oligonucleotides of the present invention.

[0043] Figure 3 It is a schematic diagram of streptavidin conjugated with barcoded oligonucleotides of the present invention.

[0044] Figure 4 It is a flow chart for the preparation of the pool of MHC multimers conjugated with barcoded oligonucleotides of the present invention.

[0045] Figure 5 It is a schematic diagram of the covalent bond of streptavidin conjugated with oligonucleotides of the present invention.

[0046] Figure 6 It is a non-reducing SDS-PAGE electrophoresis result diagram of streptavidin conjugated with oligonucleotides of the present invention.

[0047] Figure 7 It is a dual-fluorescence localization result diagram of streptavidin and barcoded oligonucleotides in the present invention.

[0048] Figure 8 It is a single-cell application result diagram of barcoded streptavidin MHC multimers in the present invention.

[0049] Figure 9 It is a single-cell sequencing result diagram of Beta2M and CCR6 barcoded streptavidin MHC multimers in the present invention.

[0050] Figure 10 This is a statistical chart of the single-cell sequencing results of the Beta2M and CCR6 barcode streptavidin MHC multimers in the present invention. Detailed implementation manners

[0051] To enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0052] Example 1:

[0053] An MHC multimer compound (dual fluorescence) conjugated with a barcode oligonucleotide. On the basis of the MHC multimer compound, a segment of oligonucleotide sequence (the 3'-end of the oligonucleotide is connected to streptavidin) is conjugated by a chemical method, and then the oligonucleotide sequence is connected to its reverse complementary fluorescence-labeled barcode oligonucleotide. The structure is as Figure 1 shown, Figure 1 In which part A is the fluorescence-labeled streptavidin; part B is the oligonucleotide conjugated to streptavidin; part C is the barcode oligonucleotide reverse complementary to the oligonucleotide. Streptavidin has fluorescence labeling, and the 5'-end of the barcode oligonucleotide sequence is fluorescently modified.

[0054] As Figure 3 shown, the barcode oligonucleotide is first a sequence complementary to the oligonucleotide chain, followed by a 36-base-length barcode sequence, and finally a 25-base-length polyA sequence. The barcode sequence can be changed according to experimental needs, and the number of bases can also be increased or decreased. The polyA sequence can be changed according to experimental needs, and the number of bases can also be increased or decreased.

[0055] An MHC multimer compound (single fluorescence) conjugated with a barcode oligonucleotide. On the basis of the MHC multimer compound, a segment of oligonucleotide sequence (the 3'-end of the oligonucleotide is connected to streptavidin) is conjugated by a chemical method, and then the oligonucleotide sequence is connected to its reverse complementary fluorescence-labeled barcode oligonucleotide, in which only the 5'-end of the barcode oligonucleotide sequence is labeled with fluorescence.

[0056] An MHC multimer compound (single fluorescence) conjugated with a barcode oligonucleotide, with the same structure. The MHC monomer is 1, and only streptavidin is labeled with fluorescence, or only the 5'-end of the oligonucleotide sequence is labeled with fluorescence.

[0057] An MHC multimer compound (containing iSpC18 modification) conjugated with a barcode oligonucleotide, with the same structure. The 3'-end of the oligonucleotide is amino-modified and iSpC18 spacer-arm modified, as Figure 2The figure shows a schematic diagram of streptavidin conjugated with oligonucleotides. Part A is the fluorescent-labeled streptavidin conjugated with oligonucleotides; Part B is the sequence design of the oligonucleotides modified with an amino group at the 3'-end; Part C is the sequence design of the oligonucleotides with a shoulder-arm modification; Part D is the sequence design of the oligonucleotides modified with an amino group at the 3'-end with a spacer sequence (S); Part E is the sequence design of the oligonucleotides with a spacer sequence (S), a 3'-end amino modification, and a shoulder-arm modification.

[0058] The oligonucleotide sequence can be changed according to experimental needs.

[0059] Example 2: Preparation method, including the following steps:

[0060] (1) Conjugating oligonucleotides to streptavidin

[0061] Using the solulink kit, modify the oligonucleotides with 4FB (4-formylbenzamide) according to the kit requirements, and modify streptavidin with HyNic (6-hydrazinonicotinamide) at the same time. The 4FB group can form a stable covalent bond (biaryl hydrazone bond) with HyNic, as Figure 5 shown in the structure. It can withstand high temperatures of 92 °C and environments with pH values between 2.0 and 10.0.

[0062] The specific steps are as follows:

[0063] a) Desalting of oligonucleotides:

[0064] According to the kit requirements, order the required oligonucleotides from Genewiz (https: / / www.genewiz.com.cn / ) or IDT ( https: / / sg.idtdna.com / page / ) or other manufacturers; add a sufficient volume of the required reagent to the tube to form a 0.5 OD260 / μL solution, and desalt the oligonucleotides using a Zeba desalting column;

[0065] b) S-4FB modification of oligonucleotides

[0066] Measure the concentration and volume of the desalted product collected, and add an appropriate amount of S-4FB to the desalted product according to the instructions, and incubate at room temperature for 2 hours;

[0067] c) Determination of S-4FB modification efficiency

[0068] After the modification is completed, desalt the oligonucleotides modified with S-4FB again according to the previous steps to remove the excess S-4FB, measure the concentration and volume of the modified product collected, and calculate the molar substitution ratio (MSR) of the modified product according to the instructions;

[0069] d) Desalting of streptavidin:

[0070] Purchase fluorescently labeled or unlabeled streptavidin from any manufacturer (or synthesize it in the laboratory), with the requirement that the streptavidin concentration is ≥ 1 mg / mL and the volume is 50 - 130 μL; desalt the streptavidin using a Zeba desalting column.

[0071] e) S-HyNic modification of streptavidin

[0072] Measure the concentration and volume of the desalted streptavidin collected, and add an appropriate amount of S-HyNic to the desalted streptavidin according to the instructions, and incubate at room temperature for 2 hours.

[0073] f) Determination of S-HyNic modification efficiency

[0074] After the modification is completed, desalt the S-HyNic-modified streptavidin according to the previous steps, collect the desalted product and measure the protein concentration and volume collected. Calculate the molar substitution ratio (MSR) of HyNic-modified streptavidin according to the instructions.

[0075] g) Coupling of 4FB-modified oligonucleotide with HyNic-modified streptavidin

[0076] Take the required volume of HyNic-streptavidin and 4FB-oligonucleotide and add them to a centrifuge tube, gently mix, add the required volume of catalytic buffer according to the instructions, gently mix, and incubate overnight at 4 °C.

[0077] h) Purification and detection of the coupling product

[0078] After the coupling reaction is completed, excess oligonucleotides can be removed by methods known in the art such as molecular sieve or affinity chromatography. Determine the streptavidin concentration in the final product using methods known in the art, and verify the coupling of streptavidin and oligonucleotide by gel electrophoresis or SYBR dye.

[0079] As Figure 6 shown in the figure, h1 is the oligonucleotide; h2 is the oligonucleotide with shoulder arm modification; SA is streptavidin. The molecular weight of the SA protein is 52 kD, the molecular weight of h1 is 12.3 kD, and the molecular weight of h2 is 14.5 kD. SDS-PAGE migration indicates successful oligonucleotide coupling. Since the results of h1 and h2 are the same, for cost reduction, h1, that is, the oligonucleotide without shoulder arm modification, is used in subsequent experiments.

[0080] (2) Annealing barcode oligonucleotides

[0081] Take an appropriate volume of oligonucleotide-coupled streptavidin and barcode oligonucleotide to prepare an annealing reaction solution. React at 50 °C for 5 minutes, and then slowly cool to room temperature, about 45 - 60 minutes. Store on ice or at 4 °C until use.

[0082] The specific steps include:

[0083] a) Reverse-complement the barcode oligonucleotide to the oligonucleotide by annealing

[0084] Prepare the annealing solution and let it stand: 1 μL of Cutsmart buffer (10×), with a total volume of 9 μL for the oligonucleotide conjugated with streptavidin and the barcode oligonucleotide, ensuring that the molar concentration ratio of the barcode oligonucleotide to the oligonucleotide in the reaction system is at least 1:1; Set the annealing program on the PCR instrument: Step 1: 50 °C, 5 min, cycle 1; Step 2: -1 °C / min, cycle 25; Step 3: 4 °C, ∞. Put the prepared reaction solution into the PCR instrument;

[0085] b) Purify the streptavidin conjugated with the barcode oligonucleotide

[0086] Use an Ultra filter, with 1×PBS as the buffer, centrifuge at 14000 g for 5 minutes at 4 °C to wet the filter membrane; then add the sample and make up to a total volume of 500 μL with 1×PBS, and centrifuge again at 14000 g for 5 minutes at 4 °C; Repeat step 2 a total of 5 times; Invert the ultrafiltration device onto a new clean collection tube and centrifuge at 1000 g for 2 minutes at 4 °C; Transfer the collected samples to new brown centrifuge tubes respectively and measure the concentration of streptavidin;

[0087] c) Verify the annealing result

[0088] Verify the conjugation of streptavidin and the barcode oligonucleotide by SDS-PAGE and / or SYBR dye in gel electrophoresis.

[0089] (3) Purification of streptavidin conjugated with barcode

[0090] Remove the excess oligonucleotide according to the methods known in the art. For example, use a molecular sieve or an Ultra centrifugal ultrafiltration tube to ultrafilter the reaction solution of the second step, replace PBS 4 - 5 times according to the instructions, collect the purified product and measure the protein concentration.

[0091] (4) Assemble the streptavidin conjugated with the barcode oligonucleotide and the biotinylated MHC monomer into a multimer

[0092] Assemble streptavidin and the biotinylated MHC monomer together to form a multimer according to the methods known in the art. For example, use an ultraviolet-induced peptide exchange reaction to load the MHC with the target antigen peptide (i.e., pMHC monomer). The obtained pMHC monomer and the fluorescently labeled streptavidin form a multimer compound. Store the final product in a 4 °C amber (brown) polypropylene tube for future use.

[0093] Specific preparation steps:

[0094] a) UV-induced MHC monomer peptide exchange

[0095] Dissolve the target antigen peptide with sterile water or dimethyl sulfoxide to prepare a 10 mM stock solution. Note: The dimethyl sulfoxide in the final exchange reaction system should not exceed 10%. Dilute the antigen peptide stock solution with sterile water to 400 μM. Take the middle wells (such as C6, C7) of a clean 96-well U-bottom plate, add an equal volume of 400 μM antigen peptide dilution and MHC monomer, gently pipette and mix well, irradiate under an ultraviolet lamp for 30 minutes. Subsequently, seal the 96-well U-bottom plate with a sealing film and incubate at 37 °C in the dark for 30 minutes. Detect the peptide exchange efficiency by ELISA;

[0096] b) Assembly of barcode-conjugated streptavidin and peptide-loaded MHC monomers;

[0097] Take an appropriate volume of peptide-loaded MHC monomers and barcode-conjugated streptavidin in a brown centrifuge tube, ensure that the molar ratio of monomer: streptavidin is 5:1 - 6:1, gently pipette and mix well, then incubate on ice in the dark for 30 minutes. After incubation, block the vacant biotin-binding sites of streptavidin with D-biotin and incubate overnight at 4 °C, paying attention to keeping it in the dark. Store at 4 °C for future use.

[0098] Examples 3 to 5 are carried out using MHC multimer compounds (dual fluorescence) conjugated with barcode oligonucleotides.

[0099] Example 3: Application in antigen-specific T cell flow cytometry analysis or sorting

[0100] In the present invention, by constructing an MHC multimer pool, staining a mixed sample of PBMC from different sources, and using flow cytometry to discover and characterize antigen-specific T cells. After different subtypes or the same subtype of MHC monomers are loaded with different antigen peptides, they are combined with streptavidin with different barcode information, corresponding the antigen peptide with the fluorescence of the barcode and at the same time corresponding the MHC subtype with the fluorescence of streptavidin. According to the dual fluorescence labeling of streptavidin and barcode oligonucleotides, and in combination with conventional extracellular or intracellular flow antibody staining, such as CD3, CD4, CD8, CD45, CD19, TNF-α, IFN-γ antibodies, etc., analyze the phenotype and characteristics of antigen-specific cells, or use sorting technology to separately isolate the cells of interest for subsequent processing or continuous culture.

[0101] The steps are as follows:

[0102] (1) Preparation of MHC multimer compounds of barcode oligonucleotides. Load the target antigen peptides (P1, P2, P3) onto the corresponding MHC monomers (HLA-1, HLA-2). Select two different fluorescently labeled streptavidins (APC, PE) and bind them to three barcode oligonucleotides (BC1, BC2, BC3) respectively. The MHC monomers and the barcode oligonucleotide-conjugated streptavidins are assembled into multimers to obtain six multimer compounds as shown in Figure 4 Shown.

[0103] (2) Prepare a single-cell suspension of the target cells, measure the concentration and adjust the concentration of the single-cell suspension as needed. Remove dead cells, debris and aggregates by centrifugation or filtration.

[0104] (3) Incubate the cell sample with the multimer compound pool, set the incubation time and temperature according to the MHC subtype used under conditions known in the art, and pay attention to avoiding light throughout the process. After incubation, wash the cells with a suitable washing buffer.

[0105] (4) Collect samples according to the set flow cytometry gating strategy and collect data. Alternatively, select the sorting mode according to the experimental requirements, set the gating strategy to sort the target cell population, collect the sorted cells and perform viability detection. Note that aseptic operation should be ensured during the sorting process to avoid contamination.

[0106] The specific steps are as follows:

[0107] a) Prepare cells and multimers

[0108] Centrifuge the prepared multimers at 4 °C and 25,000 g for 5 minutes, and then store them on ice. Collect cells in good growth condition and count them. Take 2×10 6 cells into a round-bottom tube, add 4 mL of flow cytometry staining buffer (1×PBS containing 2% fetal bovine serum or bovine serum albumin), centrifuge at 4 °C and 500 g for 5 minutes, discard the supernatant to wash the cells, and repeat this step 2 times;

[0109] b) Multimer staining

[0110] Resuspend the cells with 200 μL of flow cytometry staining buffer, gently pipette to mix well, add 2 μL of each multimer, gently pipette to mix well, incubate on ice in the dark for 30 minutes, wash the cells twice with 4 mL of flow cytometry staining buffer, and then resuspend them in 400 μL of staining buffer;

[0111] c) Staining with other cell surface staining antibodies

[0112] Prepare a mixture of surface staining reagents and live / dead cell dyes. After the polybody incubation is completed, without washing, directly add the dye mixture to the sample, incubate in the dark on ice for 20 minutes, wash the cells twice with 4 mL of flow cytometry staining buffer, and then resuspend in 400 μL of staining buffer;

[0113] d) Flow sorting

[0114] As described above, prepare a single-cell suspension and stain it. Filter the cells through a 300-mesh cell sieve before loading. According to the gating strategy of flow cytometry analysis, select an appropriate sorting mode and collect the target cells.

[0115] As Figure 7 shown, it is the flow cytometry verification result of barcode streptavidin MHC tetramer. Select the antigen epitope (GILGFVFTL), assemble it into a tetramer with APC-streptavidin conjugated with Texas red-barcode oligonucleotide. The double-positive cell population of Texas red and APC proves that the tetramer is successfully prepared and has biological function.

[0116] Example 4: Application in multi-epitope high-throughput single-cell sequencing

[0117] The present invention can directly capture antigen-specific single T cells in a sample pool through the BD Rhapsody TM system without the sorting step in similar methods known in the art. In actual operation, use the BD Rhapsody platform to achieve single-cell separation. After each cell is lysed, a series of information of this cell will be captured by magnetic beads, including mRNA, TCR, BCR, sample tag (ST), and barcode oligonucleotide on the TCR-specific pMHC polybody. The oligonucleotide on the surface of the magnetic bead contains a universal sequence, cell label (CL), unique molecular identifier (UMI), etc. Subsequently, through reverse transcription reaction, the information of the magnetic bead is fused with the information of each cell. Carry out subsequent experiments in two programs: Program 1, obtain the supernatant after the magnetic bead is denatured, and use two-step nested PCR to amplify the sample tag and pMHC polybody barcode tag to construct a sequencing library. Program 2, perform PCR amplification on the cDNA, TCR, and BCR on the magnetic bead according to the program recommended in the instruction manual to construct a sequencing library. Finally, achieve high-throughput transcriptomic sequencing of antigen-specific T cells.

[0118] In single-cell sequencing, the target cell labeling can be completed in a single incubation, retaining all cell subsets (including low-frequency antigen-specific T cells), reducing the loss of cell subsets, cell state interference, and cell activity impairment caused by sorting, and avoiding selection bias.

[0119] This solution can synchronously obtain transcriptome, TCR sequences and phenotypic data to achieve multi-dimensional analysis. It directly associates TCR sequences with antigen information, clarifies the relationship between clonal expansion and effector function, and realizes the precise traceability of antigen-specific T cell clones. At the same time, it "in-situ captures" the information of antigen-specific T cells and other cells (such as non-antigen-specific T cells, etc.) in the whole sample, facilitating the exploration of the interaction mechanism between antigen-specific T cells and other cells and revealing the intercellular regulatory network.

[0120] By jointly applying single-cell library construction data and flow cytometry analysis results, the quality of single-cell data is verified. It not only shortens the experimental cycle and reduces the operation steps, but also ensures the authenticity and reliability of the data. It provides a solution for the functional research and clinical application of antigen-specific T cells.

[0121] The steps are as follows:

[0122] (1) Single-cell capture

[0123] Load the target cell sample onto the pre-pretreated BD Rhapsody TM HT chip, and then add enhanced cell capture magnetic beads.

[0124] (2) Cell labeling and mRNA capture

[0125] Perform cell lysis and magnetic bead recovery according to the BD instruction manual.

[0126] (3) cDNA amplification and library construction

[0127] The extracted sequences are reverse-transcribed into DNA, and then sequencing adapters are ligated to the cDNA ends. The library is amplified by PCR to obtain sufficient DNA for sequencing.

[0128] (4) Sequencing and data analysis

[0129] The constructed library is sequenced through a high-throughput sequencing platform (such as the Illumina platform), and the generated sequencing data undergoes analysis steps such as quality control, alignment, cell annotation, and quantification, and finally the gene expression profile of single cells is obtained.

[0130] The specific method steps are as follows:

[0131] a) Preparation of single-cell suspension.

[0132] The prepared single-cell suspension (with a clumping rate lower than 5%), after counting, take 5×10 5 ~1×10 6 cells into a 5 mL low-attachment centrifuge tube, centrifuge at 4°C and 500g for 5 minutes, discard the supernatant, and use BD Horizon TMBrilliant Stain Buffer (Cat. No. 563794 / 566349) Resuspend the cells; add 20 μL of Sample Tag and pipette gently to mix well, and record the tag number corresponding to the cell sample; incubate in the dark on ice for 30 minutes, gently flick the tube wall every 15 minutes during incubation to avoid cell precipitation; add 1 - 2 mL of BD Horizon TM Brilliant Stain Buffer, centrifuge at 500 g for 5 minutes at 4°C to wash the cells, and repeat the washing step 2 times;

[0133] b) Multimer Staining of Single - cell Mixture

[0134] Recount the single - cell suspension after labeling with the sample tag. Aliquot appropriate volumes of the single - cell suspension into 5 - mL low - attachment centrifuge tubes, with at least 1×10 6 cells, centrifuge at 500 g for 5 minutes at 4°C, discard the supernatant and resuspend the cells with 200 μL of hsDNA - FACS buffer (1×PBS containing 2% fetal bovine serum and 0.1 g / L herring sperm); add the above - mentioned barcode oligonucleotide - conjugated MHC multimer, incubate in the dark on ice for 30 minutes, gently flick the tube wall every 15 minutes during incubation to avoid cell precipitation; add 1 mL of BD Horizon TM Brilliant Stain Buffer and wash the cells 2 times; add 200 μL of BD Horizon TM Brilliant Stain Buffer to resuspend the cells, and then place them on ice for later use;

[0135] c) Quality Control of Single - cell Samples Using BD Rhapsody TM Scanner

[0136] Add live / dead dye to each tube, pipette gently to mix well, and incubate in the dark at 37°C for 5 minutes; then use the BD Rhapsody TM Scanner to count the cells; check the cell concentration, viability and quality control pictures. If the cell concentration of the sample is between 200 - 1000 cells / μL, subsequent operations can be carried out. Otherwise, it is recommended to dilute the cell suspension and retest the concentration;

[0137] d) Single - cell Isolation and Transcriptome Capture

[0138] Load the single - cell mixture onto the BD Rhapsody TM HT Xpress chip, and load the Enhanced Cell Capture Beads onto the BD Rhapsody TMOn the chip of HT Xpress, cells are lysed and the captured magnetic beads are recovered, and then immediately washed for Enhanced Cell Capture Beads and cDNA synthesis is carried out;

[0139] e) Construction of barcode and sample tag libraries

[0140] Prepare barcode-specific PCR primers on streptavidin, perform PCR1 reaction on barcodes and sample tags simultaneously, purify the PCR1 products with AMPure XP beads, perform PCR2 reaction on barcodes and sample tags separately, purify the PCR2 products with AMPure XP beads, quality control the barcode and sample tag PCR2 products, perform IndexPCR reaction on barcodes and sample tags separately, purify the Index PCR products with AMPure XP beads, and quality control the sequencing library;

[0141] f) Construction of whole transcriptome library (WTA), TCR and BCR libraries is carried out according to the instructions.

[0142] As Figure 8 shown, two TCR overexpressing Jurkat cell lines are mixed at 30% and 20% ratios respectively, with the wild-type Jurkat cell line as the negative control. Beta2M barcode oligonucleotide APC-MHC multimers specifically recognize approximately 90% of the CMV epitope (NLVPMVATV)-specific cells, and CCR6 barcode oligonucleotide PE-MHC multimers specifically recognize approximately 57% of the KRAS epitope (VVGAVGVGK)-specific BA12 cells.

[0143] By Figure 9 and 10 shown, the cells mainly labeled by Beta2M barcode oligonucleotides are CMV cells, accounting for 77% of the total CMV cells.

[0144] The cells mainly labeled by CCR6 barcode oligonucleotides are BA12 cells, accounting for 36% of the total BA12 cells.

[0145] Example 5: A kit for labeling antigen-specific T cells without sorting, comprising:

[0146] 1) pMHC multimer-oligonucleotide complex:

[0147] Customized antigen-loaded MHC monomers for assembling multimers;

[0148] Pre-coupled "streptavidin-oligonucleotide" complexes for assembling multimers;

[0149] Variable barcode oligonucleotides with "oligonucleotide" and "polyA" for assembling polymers (5'-complementary oligonucleotide sequence - antigen-specific barcode sequence - polyA - 3'), which can be customized with a fluorescent or non-fluorescent label at the 5'-end according to experimental requirements.

[0150] 2) Purification and Quality Control

[0151] Free oligonucleotide removal column: an ultrafiltration centrifugal column to remove unbound oligonucleotides;

[0152] Fluorescent quantitative detection reagent: SYBR Gold rapid fluorescent quantitative reagent for oligonucleotides.

[0153] 3) Cell Labeling and Verification

[0154] Cell labeling buffer: such as PBS containing a non-specific binding blocker, etc.;

[0155] Flow cytometry verification control: a T cell line with known antigen specificity (such as a CMV epitope-specific T cell) as a positive control.

[0156] 4) Compatibility with Single-Cell Library Construction

[0157] Sequencing primers for amplifying the above barcode oligonucleotides.

[0158] The above has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and the descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements fall within the scope of the present invention claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. An MHC multimer compound conjugated with a barcoded oligonucleotide, characterized in that: Comprising: (a) 1 to 4 MHC monomers; (b) streptavidin; (c) an oligonucleotide; (d) a barcode oligonucleotide that is reverse complementary to the above oligonucleotide sequence The oligonucleotide is covalently bound to streptavidin, and a single fluorescent label is formed by attaching a fluorescent label to any one of the 5'-end of the oligonucleotide, the 5'-end of the barcode oligonucleotide, and streptavidin.

2. An MHC multimer compound conjugated with a barcoded oligonucleotide, characterized in that: Comprising: (a) 1 to 4 MHC monomers; (b) streptavidin; (c) an oligonucleotide; (d) a barcode oligonucleotide that is reverse complementary to the above oligonucleotide sequence; The oligonucleotide is covalently bound to streptavidin, and a double fluorescent label is formed by attaching a fluorescent label to the 5'-end of the barcode oligonucleotide, streptavidin, or the 5'-end of the oligonucleotide.

3. The MHC multimer compound conjugated with barcoded oligonucleotides according to claim 1 or 2, characterized in that: The barcode oligonucleotide contains a sequence complementary to the oligonucleotide described in (c), a barcode sequence, and a polyA sequence.

4. The MHC multimer compound conjugated with a barcoded oligonucleotide according to claim 1 or 2, characterized in that: The MHC monomer is MHC-I, MHC-II, wild type loaded with CLIP, or pMHC loaded with a target antigen obtained by peptide exchange technology.

5. The MHC multimer compound conjugated with a barcoded oligonucleotide according to claim 1 or 2, characterized in that: The 3'-end of the oligonucleotide is covalently bound to streptavidin, and the covalent bond is a diarylhydrazone bond formed by 4-formylbenzamide and 6-hydrazinonicotinamide.

6. The MHC multimer compound conjugated with a barcoded oligonucleotide according to claim 5, wherein: The 3'-end of the oligonucleotide is subjected to amino modification or amino modification and iSpC18 spacer modification, and the oligonucleotide may or may not contain a spacer sequence.

7. The method for preparing an MHC multimer compound conjugated with a barcoded oligonucleotide according to claim 1 or 2, characterized in that: Comprising the following steps: (1) Coupling an oligonucleotide to streptavidin; (2) Coupling the barcode oligonucleotide to streptavidin: annealing the barcode oligonucleotide to be reverse complementary to the oligonucleotide; (3) Purifying the barcode-coupled streptavidin; (4) Assembling a multimer of streptavidin linked to the barcode oligonucleotide and a biotinylated MHC monomer; (5) Purifying the MHC multimer compound coupled with the barcode oligonucleotide.

8. Use of an MHC multimer compound coupled with a barcode oligonucleotide in multi-epitope high-throughput single-cell sequencing.

9. Use of an MHC multimer compound coupled with a barcode oligonucleotide in antigen-specific T cell flow cytometry analysis or sorting.

10. Use of an MHC multimer compound coupled with a barcode oligonucleotide for preparing a kit for labeling antigen-specific T cells without sorting.

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