A method for detecting multiple HLA-specific T cell killing effects

Abstract

This invention discloses a method for detecting the killing effect of multiple HLA-specific T cells, belonging to the field of biotechnology. This invention enables T2 cells to express various HLA-I molecular subtypes. After the receptor is expressed, antigen fragments loaded with various HLA-I molecules can be captured in the culture supernatant for in vitro evaluation of the killing effect of HLA-I-adapted antigen-specific T cells, thus expanding the application of T2 cells in in vitro killing effect evaluation. Simultaneously, it avoids the enormous workload and high cost of using the K562 cell line to construct a cell line for each HLA subtype-specific antigen fragment during the HLA subtype construction process.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method for detecting multiple HLA-specific T cell killing effects. Background Technology

[0002] Antigen-specific T cells specifically kill tumor cells expressing the antigen by recognizing antigen fragments presented by HLA-I molecules on the surface of tumor cells via their TCRs. In practice, the TCR receptors of T cells need to be compatible with the HLA on the surface of tumor cells for specific killing assays. Therefore, when evaluating the in vitro killing effect of a particular T cell, tumor cells with the same HLA subtype as that T cell and expressing the specific antigen are required. However, currently commercially available tumor cell lines have unknown, unalterable, and do not necessarily express a specific antigen.

[0003] Tumor antigens can be hydrolyzed into polypeptides of 9-11 amino acids after enzymatic digestion within cells. Under the action of antigen-processing peptide transporters (TAPs), these polypeptides form polypeptide-HLA-I complexes with newly synthesized HLA-I molecules in the endoplasmic reticulum. After transport, they are co-expressed on the cell surface. After cross-antigen presentation between the complex and dendritic cells (DCs), the antigen peptide is transferred to the HLA-I molecules of the DCs. The HLA-I molecules of the DCs bind to the TCRs of T cells, thereby presenting antigens to specifically activate T lymphocytes. When T cells activated by the antigen encounter tumor cells, they recognize the tumor cells loaded with the antigen on the HLA-I molecules through the TCRs and can specifically kill the tumor cells by secreting active cytokines such as perforin and IFN-γ. Therefore, when it is necessary to evaluate the in vitro tumor-killing effect of a certain specific T cell, the selected human tumor cell line must meet the following conditions: (1) The HLA-I molecules of the tumor cells are consistent with the HLA-I typing of the T cells. (2) The antigen fragments presented by the HLA-I molecules of the tumor cells are consistent with the antigen fragments of the activated T cells. However, currently commercially available human tumor cell lines not only have unknown HLA subtypes, but also express fixed HLA-I molecules, meaning they are identical to the cell host. Furthermore, even if the HLA subtype of the tumor cell line is detected through sequencing technology, it is still necessary to determine whether the tumor cell line expresses the required antigen fragment in practical applications. In summary, if a single tumor cell line could be used to detect the in vitro killing effect of T cells recognizing various HLA subtypes and specific antigen fragments, it would simplify the detection of antigen-specific T cell killing and accelerate its clinical application. Summary of the Invention

[0004] This invention provides a method for detecting multiple HLA-specific T cell killing effects.

[0005] The present invention solves its technical problem by adopting the following technical solution:

[0006] A method for detecting multiple HLA-specific T cell killing effects includes the following steps:

[0007] (1) Construct an in vitro transcription mRNA vector for HLA-I molecules to obtain a pmRNA-HLA-I molecule expression vector;

[0008] (2) After linearization by enzyme digestion and purification, the pmRNA-HLA-I molecular expression vector was transcribed in vitro using T7 RNA polymerase and then purified by lithium chloride precipitation to obtain HLA-I molecular mRNA.

[0009] (3) Prepare an electroporation suspension by mixing HLA-I molecule mRNA with T2 cells, perform electroporation using an electroporator, and then transfer the cells to a culture medium to obtain T2 cells expressing the target HLA-I molecule;

[0010] (4) Add the target antigen fragment to the culture medium of T2 cells expressing the target HLA-I molecule, incubate to load the T2 cells with antigen, and then co-incubate with the HLA-specific T cells to be tested. The killing effect of T cells is evaluated by apoptosis detection.

[0011] This invention enables T2 cells to express other HLA-I subtypes via electroporation of mRNA, and can be used for T cell-specific killing detection of the same HLA-I subtype. Compared with existing T2 cell application technologies, this invention constructs expression plasmids for HLA-I subtypes based on T2 cells, transcribes mRNA in vitro, and then uses electroporation of mRNA to enable T2 cells to express other HLA-I subtypes. It can also load other HLA-I subtype-specific antigen fragments for killing evaluation, expanding the application of T2 cells in specific T cell killing evaluation. Compared with the K526 technique for constructing HLA-I subtype cell lines, this invention does not require inserting the antigen fragment sequence to be detected into the HLA expression plasmid for co-expression when constructing the HLA-I expression plasmid. Antigen loading is performed by subsequent addition to the cell culture medium, thus constructing only one HLA-I subtype expression plasmid, but allowing for specific T cell killing detection of multiple HLA-I subtype restriction antigen fragments. For example, when it is necessary to detect 10 restriction antigen fragments of HLA-A*11:01, this application only requires the construction of one HLA-A*11:01 expression plasmid, while the K562 cell line requires the construction of 10 HLA-A*11:01 expression plasmids containing antigen fragments. Furthermore, when constructing HLA-I subtype expression plasmids, this application only requires replacing the HLA-A*11:01 sequence with other subtype sequences to achieve the construction of T2 cell lines for other HLA-I subtypes, such as HLA-A*24:02, HLA-A*02:07, HLA-A*33:03, HLA-C*01:02, HLA-C*07:02, HLA-C*03:04, HLA-C*06:02, HLA-C*08:01, etc.

[0012] In this invention application, T2 cells are made to express other HLA-I subtypes by electroporation of mRNA. If the cell line is constructed by lentivirus, its design principle and subsequent application are consistent with this invention application.

[0013] As an embodiment of this application, the HLA-I molecule includes at least one of HLA-A*24:02, HLA-A*02:07, HLA-A*33:03, HLA-C*01:02, HLA-C*07:02, HLA-C*03:04, HLA-C*06:02, and HLA-C*08:01.

[0014] As an implementation of this application, the in vitro transcription mRNA vector for constructing the HLA-I molecule is specifically constructed as follows: starting with the T7 promoter, the HLA-I molecule sequence is inserted between the 5'UTR and 3'UTR sequences, and a PolyA sequence is ligated after the 3'UTR. The vector is then transformed into DH5α competent cells for cloning and amplification to obtain the pmRNA-HLA-I molecule expression vector.

[0015] As an embodiment of this application, the enzyme digestion linearization specifically involves reacting 1-3 μL of restriction endonuclease, 15-20 μL of buffer, 4-6 μg of pmRNA-HLA-I molecular expression vector, and water at 36-38℃ for 1-4 h, and then taking 4-6 μL of the digestion product for agarose gel electrophoresis.

[0016] As an embodiment of this application, the restriction endonuclease is Hind III.

[0017] As an implementation scheme of this application, the lithium chloride precipitation method specifically comprises:

[0018] S1. Prepare a 5-10M LiCl and 40-60M EDTA solution, filter and autoclave;

[0019] S2. After transcription is complete, add 0.5-2 μL of RNase-free DNase I and incubate at 36-38℃ for 30-50 min to remove the DNA template;

[0020] S3. Add RNase-free H2O and lithium chloride solution to make the final lithium chloride concentration 2~3M. Cool, centrifuge, collect the precipitate, precipitate twice with ethanol, centrifuge, add 40~60μL RNase-free H2O and dissolve.

[0021] As an embodiment of this application, the electroporation buffer is Opti-MEM; before electroporation, T2 cells are washed twice with Opti-MEM medium, each time 5 × 10⁶ cells / mL. 6 30 μg mRNA was added to each cell to prepare an electroporation suspension.

[0022] As an implementation scheme of this application, the electrical transfer conditions are: voltage 200V, Pulse Duration 2000μs, Pulse No. 4, Interval 1000ms.

[0023] As an implementation scheme of this application, after the T cells to be tested were co-incubated with T2 cells loaded with antigen overnight, apoptosis was detected using APC-Annexin V and PI. The target of the test was a CFSE-negative T2 cell population.

[0024] As an embodiment of this application, the final concentration of the added antigen fragment is 10~30μM, and the incubation time is 1~5h.

[0025] The beneficial effects of this invention are:

[0026] (1) This invention enables T2 cells to express various HLA-I molecular subtypes through technical means. After the receptor is expressed, antigen fragments loaded with various HLA-I molecules can be captured in the culture supernatant for evaluation of the in vitro killing effect of HLA-I-adapted antigen-specific T cells, thus expanding the application of T2 cells in evaluating the in vitro killing effect. At the same time, it avoids the huge workload and high cost of using the K562 cell line to construct a cell line for each HLA subtype-specific antigen fragment.

[0027] (2) This invention enables T2 cells to express other specific HLA-I subtypes on top of the original HLA-A*02:01 mRNA via electroporation, and can be used for the detection of specific T cell killing of other HLA-I subtypes besides HLA-A*02:01. Simultaneously, this application avoids the construction of multiple plasmids for the same HLA-I subtype, simplifying the antigen loading method. This invention utilizes the lack of TAP in T2 cells, avoiding the need for K526 cell lines to construct co-expression plasmids for HLA-I subtypes and the target antigen fragment, reducing the workload of detecting multiple antigen fragments for the same HLA-I subtype. Furthermore, the antigen loading method for T2 cells is simple; it only requires adding the antigen fragment to the culture medium for co-incubation. Attached Figure Description

[0028] Figure 1 This is a gel electrophoresis image of the linearized pmRNA-HLA-A*11:01 plasmid.

[0029] Figure 2 This is a gel electrophoresis image of HLA-A*11:01 mRNA.

[0030] Figure 3 This is a diagram showing the expression of HLA-A*11:01 in T2 cells.

[0031] Figure 4 This is a comparison of the killing effects of T2 cells and T2 cells (HLA-A*11:01 mRNA). Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0034] In this invention, numerical ranges are involved. Unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe features or characteristics, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0035] In this invention, there are no particular limitations on the specific dispersion and stirring methods.

[0036] Unless otherwise specified, all reagents or instruments used in this invention are commercially available conventional products. Unless otherwise specified, the raw materials used in each comparative example and the parallel experiments of each embodiment are the same commercially available products.

[0037] Example 1 A method for detecting multiple HLA-specific T cell killing effects includes the following steps:

[0038] (1) Constructing an in vitro transcription vector for HLA-I molecules:

[0039] The technical solution is detailed using HLA-A*11:01 as an example. This expression vector starts with a T7 promoter, which binds to T7 RNA polymerase in vitro, leading to the synthesis of single-stranded mRNA under enzymatic catalysis. The HLA-A*11:01 sequence was constructed using an integrated ultra-rapid cloning kit (catalog number: C115-01, Novizan) between the 5'UTR and 3'UTR sequences, with a PolyA sequence following the 3'UTR. A 3xFlag fragment is appended to the signal peptide of the HLA-A*11:01 sequence; this fragment is used for HLA-A*11:01 expression analysis. After plasmid construction, the cells were transformed into DH5α competent cells for cloning and amplification. The cells were then plated on kanamycin plates for PCR screening and sequencing to obtain the pmRNA-HLA-A*11:01 expression vector. The specific sequence of HLA-A*11:01 is shown in Table 1.

[0040]

[0041] (2) In vitro transcription of mRNA from HLA-A*11:01 expression vector

[0042] S1. The in vitro transcription kit used was the T7 RNAi reverse transcription kit (TR102-01, Novizan), the plasmid linearization enzyme was Hind III (ER0502, Thermo Scientific), the RNase-free DNase I (M0303S, New England Biolabs), and the agarose gel DNA recovery kit (DP219, TIANGEN).

[0043] Plasmid (pmRNA-HLA-A*11:01) was linearized by enzyme digestion. The plasmid linearization system was prepared according to the conditions in Table 2 and reacted at 37℃ for 2 h. After the reaction, 5 μL of the digestion product was run on a 1% agarose gel electrophoresis. If a single bright band was observed and the band was >5000 bp, the digestion product could be recovered using an agarose gel DNA recovery kit.

[0044] Gel electrophoresis, such as Figure 1 As shown, the total size of the pmRNA-HLA-A*11:01 plasmid is 4088 bp. After linearization, the gel electrophoresis bands (left lane) are shown between the 3000 bp (second band from top to bottom) and 5000 bp (first band from top to bottom) indicator bands (right lane), and the plasmid has been confirmed by base sequencing.

[0045]

[0046] S2. In vitro transcription of linearized plasmids. Prepare the in vitro transcription system of linearized plasmids according to the conditions in Table 3, mix well, and incubate at 42°C overnight.

[0047]

[0048] S3. Lithium chloride precipitation method for purifying mRNA

[0049] 1. Prepare a 7.5M LiCl, 50 mM pH=8.0 EDTA solution using RNAse-free water, filter it through a 0.22 μm filter and autoclave to remove RNAse.

[0050] 2. After transcription is complete, add 1 μL of RNase-free DNase I, mix well, and incubate at 37°C for 45 min to remove the DNA template.

[0051] 3. After incubation, add 80 μL of RNase-free H2O, mix gently, then add 50 μL of lithium chloride (final concentration 2.5M), mix gently, and then cool the resulting solution at -20℃ for 3 h.

[0052] 4. Centrifuge at 15,000 rpm and 4℃ for 15 minutes. A clear white precipitate will be visible. Discard the supernatant as much as possible and wash the precipitate twice with 70% ethanol in a -20℃ ice bath to remove residual salts.

[0053] 5. Centrifuge at 4 ℃ and maximum speed for 1 min to precipitate RNA, discard the supernatant as much as possible, and then leave the centrifuge tube open for 5 min to allow the ethanol to evaporate.

[0054] 6. Add 50 μL of RNase-free H2O to the RNA precipitate and dissolve at 70℃ for 5 min until no flocculent precipitate remains. You can gently tap the bottom of the test tube during this process to promote dissolution.

[0055] 7. Use a NanDrop one spectrophotometer to detect RNA concentration and run 1% agarose gel electrophoresis. If the bands are intact and bright, they can be used for downstream experiments.

[0056] Among them, the gel electrophoresis image is as follows Figure 2 As shown, the molecular weight of the HLA-A*11:01 sequence after transcription into mRNA is around 1000 bp (left lane), which is close to the brightest band (1000 bp) in the indicator band (right lane).

[0057] (3) Electroporation of HLA-A*11:01 mRNA in T2 cells

[0058] The electroporation buffer used was Opti-MEM (31985062, Thermo Scientific), and the electroporator used was X-Porator (EBXP-H1, Yida Biotechnology).

[0059] 1. Preparation before electroporation. Remove the Opti-MEM 30 minutes in advance and allow it to return to room temperature. Pre-fill a certain volume of culture medium (3 mL per well) into a 6-well cell culture plate and place it in a 37℃, 5% CO2 incubator for immediate transfer of cells after electroporation for continued culture.

[0060] 2. Take the required number of cells and place them in a new centrifuge tube. Centrifuge at 300 g for 5 min.

[0061] 3. After centrifugation, discard the supernatant, add 2.5 mL of Opti-MEM medium (5*106 cells), centrifuge at 300g for 5 min.

[0062] 4. After centrifugation, discard the supernatant, add 1 mL of Opti-MEM medium (5*10⁶ cells), resuspend, and centrifuge at 300g for 5 min. Aspirate the supernatant as thoroughly as possible to obtain the desired cells. Add the required amount of Opti-MEM (100 μL of medium per 5*10⁶ cells) and gently pipette to mix.

[0063] 5. Add the required amount of mRNA for the corresponding cell volume (per 5*10) 6 Add 30 μg mRNA to the cells, mix thoroughly by pipetting about 5 times with a pipette tip, and prepare an electroporation suspension.

[0064] 6. Add the mixed cell suspension along the wall into the H1 electroporation cup, insert the electroporation cup into the base, and select the correct electroporation conditions (voltage: 200V; Pulse Duration: 2000 μs; Pulse No.: 4; Interval: 1000ms).

[0065] 7. After electroporation, add 2 mL of preheated culture medium to the electroporation cup. Gently pipette 4-5 times, then transfer the medium to a plate and incubate in an incubator.

[0066] (4) Expression and functional verification of HLA-A*11:01 mRNA in T2 cells after electroporation

[0067] 1. Select PBMCs derived from HLA-A*11:01 subtype, taking 2×10⁻⁶ samples. 7PBMCs were suspended in AIM-V medium (containing 10% plasma) and cultured in 10 mm culture dishes for 3 h for adherence. After 3 h, adherent monocytes were collected, while non-adherent suspension cells were collected and cryopreserved. Adherent monocytes were then re-cultured in AIM-V medium (containing 10% plasma) containing 100 ng / mL recombinant human GM-CSF and 50 ng / mL IL-4 for induction, designated day 1. On day 3, the medium was partially replaced with AIM-V medium containing the same concentrations of recombinant human GM-CSF and IL-4. On day 5, immature dendritic cells (DCs) were collected using a cell scraper.

[0068] 2. Immature DC cells were seeded into 12-well plates, with each well containing 1×10⁶ cells. 5 One dendritic cell (DC) cell and 1 mL of AIM-V (containing 10% plasma) medium were prepared. Immunoepitopes KRAS-G12V-7 (VVVGAVGVG) and KRAS-G12D-7 (VVVGADGVG) were added to the DC cells at a concentration of 40 μM, and incubated for 24 h. Recombinant human GM-CSF (final concentration 50 ng / mL) and IL-4 (final concentration 25 ng / mL) were added simultaneously. After incubation, LPS (10 ng / mL) and IFN-γ (10 ng / mL) were added, and the DC cells were incubated for another 24 h to mature. The medium was removed by centrifugation, and the mature DC cells were collected. These mature DC cells were then co-incubated with the non-adherent suspension cells from the PBMCs in step 1 in fresh AIM-V (containing 10% plasma) medium at a cell ratio of DC:T = 1:20, and this was designated as day 1. On day 3 of co-incubation, recombinant human IL-2 (1250 IU / mL) was added. On day 5 of co-incubation, the suspended T cells were transferred to another batch of matured DC cells loaded with the same candidate immune epitopes and co-incubated for 5 days. IL-2 (1250 IU / mL) was added on days 1 and 3 of the second co-incubation.

[0069] 3. Collect T2 cells that have been electroporated with HLA-A*11:01 mRNA and wash them twice with PBS buffer. After washing, resuspend the collected T2 cells in RPMI-1640 basal medium (serum-free) and seed them into 12-well plates (non-adherent) at a density of 1 × 10⁶ cells per well. 54 × 10⁴ cells, 1 mL RPMI-1640 basal medium. KRAS-G12V-7 and KRAS-G12D-7 epitopes were added to T2 cells electroporated with HLA-A*11:01 mRNA to a final concentration of 20 μM, and incubated for 4 h to load the T2 cells with the antigen; another group was treated with a solvent containing no candidate immunoepitaxes as a control. T cells were collected by centrifugation after two pulses of KRAS-G12V-7 and KRAS-G12D-7 epitope stimulation, 4 × 10⁴ cells. 5 T cells were suspended in 400 μL of PBS and stained with CFSE fluorescent dye at a final concentration of 0.5 μM / mL. After staining, the cells were centrifuged, collected, and resuspended in 400 μL of fresh AIM-V medium. This resuspended medium was then evenly added to T2 cells treated with KRAS-G12V-7 and KRAS-G12D-7 epitopes and untreated cells electroporated with HLA-A*11:01 mRNA. Cells were incubated overnight. After incubation, all cells were collected, and apoptosis was detected using APC-Annexin V and PI. The detection target was the CFSE-negative T2 cell population, and the apoptosis status of T2 cells was compared.

[0070] like Figure 3 As shown, the Flag fragment co-expressed with HLA-A*11:01 was detected by flow cytometry antibody detection. The results showed that the Flag tag could be detected in T2 cells after electroporation of HLA-A*11:01 mRNA.

[0071] like Figure 4 As shown, Figure 4 -A represents untreated T2 cells (expressing only HLA-A*02:01). KRAS-G12D-7 and KRAS-G12V-7 fragments are restriction peptides of HLA-A*11:01. Using HLA-A*11:01-restricted T cells containing KRAS-G12D-7 and KRAS-G12V-7 to kill native T2 cells showed no difference in killing power between the two groups, regardless of whether the corresponding KRAS-G12D-7 and KRAS-G12V-7 antigen fragments were added to the T2 cells. However, the killing power of NY-ESO-1-restricted HLA-A*02:01-restricted T cells against native T2 cells differed with and without the addition of the NY-ESO-1 peptide. Figure 4The T2 cells used in -B were electroporated with HLA-A*11:01 mRNA and expressed HLA-A*11:01. These T2 cells were killed using HLA-A*11:01-restricted T cells containing KRAS-G12D-7 and KRAS-G12V-7 antigen fragments. The results showed that the T cells had a stronger killing effect on T2 cells (HLA-A*11:01) with added KRAS-G12D-7 and KRAS-G12V-7 antigen fragments. Therefore, T2 cells with surface electroporated HLA-A*11:01 mRNA can be used for in vitro killing assays of HLA-A*11:01-specific T cells.

[0072] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the technical spirit of the present invention. The technical scope of the present invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A method for detecting multiple HLA-specific T cell killing effects, characterized in that, Includes the following steps: (1) Construct an in vitro transcription mRNA vector for HLA-I molecules to obtain a pmRNA-HLA-I molecule expression vector; (2) After linearization by enzyme digestion and purification, the pmRNA-HLA-I molecular expression vector was transcribed in vitro using T7 RNA polymerase and then purified by lithium chloride precipitation to obtain HLA-I molecular mRNA. (3) Prepare an electroporation suspension by mixing HLA-I molecule mRNA with T2 cells, perform electroporation using an electroporator, and then transfer the cells to a culture medium to obtain T2 cells expressing the target HLA-I molecule; (4) Add the target antigen fragment to the culture medium of T2 cells expressing the target HLA-I molecule, incubate to load the T2 cells with antigen, and then co-incubate with the HLA-specific T cells to be tested. The killing effect of T cells is evaluated by apoptosis detection.

2. The method for detecting multiple HLA-specific T cell killing according to claim 1, characterized in that, The HLA-I molecule includes at least one of HLA-A*24:02, HLA-A*02:07, HLA-A*33:03, HLA-C*01:02, HLA-C*07:02, HLA-C*03:04, HLA-C*06:02, and HLA-C*08:

01.

3. The method for detecting multiple HLA-specific T cell killing according to claim 1, characterized in that, The in vitro transcription mRNA vector for constructing the HLA-I molecule is specifically constructed as follows: starting with the T7 promoter, the HLA-I molecule sequence is inserted between the 5'UTR and 3'UTR sequences, and a PolyA sequence is ligated after the 3'UTR. The vector is then transformed into DH5-α competent cells for cloning and amplification to obtain the pmRNA-HLA-I molecule expression vector.

4. The method for detecting multiple HLA-specific T cell killing according to claim 3, characterized in that, The specific process for linearization by enzyme digestion is as follows: 1-3 μL of restriction endonuclease, 15-20 μL of buffer, 4-6 μg of pmRNA-HLA-I molecular expression vector and water are reacted at 36-38℃ for 1-4 h, and 4-6 μL of the enzyme digestion product is taken for agarose gel electrophoresis.

5. The method for detecting multiple HLA-specific T cell killing according to claim 4, characterized in that, The restriction endonuclease is Hind III.

6. The method for detecting multiple HLA-specific T cell killing according to claim 1, characterized in that, The lithium chloride precipitation method is specifically as follows: S1. Prepare a 5-10M LiCl and 40-60M EDTA solution, filter and autoclave; S2. After transcription is complete, add 0.5-2 μL of RNase-free DNase I and incubate at 36-38℃ for 30-50 min to remove the DNA template; S3. Add RNase-free H2O and lithium chloride solution to make the final lithium chloride concentration 2~3M. Cool, centrifuge, collect the precipitate, precipitate twice with ethanol, centrifuge, add 40~60μL RNase-free H2O and dissolve.

7. The method for detecting multiple HLA-specific T cell killing according to claim 1, characterized in that, The electroporation buffer was Opti-MEM; T2 cells were washed twice with Opti-MEM medium before electroporation, each time 5 × 10⁶ cells / mL. 6 30 μg mRNA was added to each cell to prepare an electroporation suspension.

8. The method for detecting multiple HLA-specific T cell killing according to claim 7, characterized in that, The electrical discharge conditions are: voltage 200V, Pulse Duration 2000μs, Pulse No.4, Interval 1000ms.

9. The method for detecting multiple HLA-specific T cell killing according to claim 1, characterized in that, After co-incubating T cells and antigen-loaded T2 cells overnight, apoptosis was detected using APC-Annexin V and PI. The target population was CFSE-negative T2 cells.

10. The method for detecting multiple HLA-specific T cell killing according to claim 1, characterized in that, The final concentration of the added antigen fragment is 10-30 μM, and the incubation time is 1-5 h.

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