阅读说明：本技术 一种筛选肿瘤特异性t细胞及tcr的方法 (Method for screening tumor specific T cells and TCR ) 是由 罗微 毛晓帆 余思菲 张贝莹 林凯容 金亚彬 于 2019-09-17 设计创作，主要内容包括：本公开提供了一种筛选肿瘤特异性T细胞及TCR的方法,具体步骤为：(1)首先将肿瘤组织进行浸润单细胞消化分离；(2)然后从分离的单细胞中流式分选出CD3<Sup>+</Sup>T细胞；(3)利用免疫磁珠去除流式分选出CD3<Sup>+</Sup>T细胞中的死细胞；(4)将去除死细胞后的CD3<Sup>+</Sup>T细胞进行单细胞转录组建库、VDJ建库及测序；(5)将测序后的数据进行分析,然后筛选肿瘤特异CD8<Sup>+</Sup>T细胞及TCR。所述方法针对每个未知抗原的样本,能够在其肿瘤抗原未知的情况下,筛选出其肿瘤特异性T细胞及TCR。(The present disclosure provides a method for screening tumor specific T cells and TCRs, comprising the steps of: (1) firstly, carrying out infiltration unicellular digestion and separation on tumor tissues; (2) the separated single cells were then flow sorted for CD3 + A T cell; (3) flow sorting of CD3 using immunomagnetic bead removal + Dead cells in T cells; (4) removing CD3 after dead cells + Performing single cell transcriptome bank building, VDJ bank building and sequencing on the T cells; (5) analyzing the sequenced data, and then screening the tumor specific CD8 + T cells and TCR. The method can be used for each sample of unknown antigenCan screen out the tumor specific T cells and TCR under the condition that the tumor antigen is unknown.)

1. A method for screening tumor specific T cells and TCR, which comprises the following steps:

(1) firstly, carrying out infiltration unicellular digestion and separation on tumor tissues;

(2) the separated single cells were then flow sorted for CD3⁺A T cell;

(3) flow sorting of CD3 using immunomagnetic bead removal⁺Dead cells in T cells;

(4) removing CD3 after dead cells⁺Performing single cell transcriptome bank building, VDJ bank building and sequencing on the T cells;

(5) analyzing the sequenced data, and then screening the tumor specific CD8⁺T cells and TCR.

2. The method of screening for tumor specific T cells and TCRs as claimed in claim 1, wherein the specific steps of step (1) are:

(a) firstly, in vitro surgical resection of tumor tissue is placed in a sterile plate, the tumor tissue is repeatedly washed by sterile PBS or Hank's solution, connective tissue and adipose tissue are subtracted, and then the tumor tissue is cut into pieces;

(b) adding tissue digestive juice into the cut tumor tissue, mixing uniformly, and digesting for 1-2h at 37 ℃ by a shaking table;

(c) adding HBSS solution into the digested tumor tissue to terminate digestion, transferring the tissue fluid to a 70-micron screen by using a pipette, filtering the tissue fluid into a centrifuge tube, centrifuging the tissue fluid at 1800r/min for 8 min;

(d) and removing supernatant of the centrifuged tissue fluid, washing the tissue fluid by using Hank's solution, centrifuging the tissue fluid to remove the supernatant, adding culture fluid to resuspend cells, and counting the cells.

3. The method of screening for tumor specific T cells and TCRs as claimed in claim 1, wherein the step (2) comprises the following steps:

① will count as 10-50X 10⁶Infiltrating single nuclear cells into the tissue, centrifuging, removing supernatant, cleaning with purified buffer solution, and centrifuging at 1800rpm/min at 4 ℃ for 8 min;

② discarding the supernatant after centrifugation, resuspending the precipitated cells with 200 μ L of purification buffer, adding fluorescent antibody, incubating at 4 deg.C in dark for 30min, washing with purification buffer, centrifuging at 1800rpm/min at 4 deg.C for 8min, discarding the supernatant, and resuspending the precipitated cells with 500 μ L of purification buffer;

③ filtering the cell resuspension through a 40 μm mesh to remove adherent cell masses, then subjecting the filtered cell resuspension to surface molecular staining with 7-AAD dye, and collecting 7-AAD-CD45-V450 in single cells using BD Aria II⁺CD3-PE-CF594⁺A population of cells.

4. The method of screening for tumor specific T cells and TCRs as claimed in claim 1, wherein the step (3) is specifically performed by: sorting streaming CD3⁺Centrifuging T cells, washing with sterile PBS, re-suspending the cells with 45-55 μ L sterile PBS, mixing the same amount of cell suspension with placenta blue, and counting cells and cell survival rate by Life Count cell counter<80% of the cells are killed by using magnetic beads which are whirly and beautiful.

5. The method of screening for tumor specific T cells and TCRs as claimed in claim 1, wherein the step (4) is specifically performed by: labeling single cells obtained after dead cells are removed by using a magnetic bead labeling technology, labeling each RNA of the same cell with the same label, performing transcriptome amplification and VDJ amplification after reverse transcription and enzyme digestion, and finally sequencing the amplified transcriptome and VDJ.

6. The method of screening for tumor specific T cells and TCRs of claim 1, wherein step (5) is specifically performed by:

(I) converting the data obtained after sequencing into an expression matrix of cell gene and obtaining the TCR of each cell by using a single cell sequencing data processing method;

(II) reading in an expression matrix by utilizing Seurat software, removing the data of the cells with too low or too high UMI, and performing interception on the front and the back 3 percent;

(III) integrating the removed data by using a CCA method in Seurat software, dividing the cells into 30-35 groups by using a cell clustering method, and calculating a mean expression profile of each cell group;

(IV) reading in the mean expression profile of the cell population using SingleR software, initially defining the cell population according to the ENCODE and BLUEPRINT databases, and filtering non-T cells in the data;

(V) in the CD8 positive cell population, looking at the expression quantity of ITGAE genes, selecting an ITGAE high expression cell population, and observing the expression quantity of KLRC2 and ENTPD1 in the high expression cell population, wherein the cell population with high expression quantity of KLRC2 is IEL, the cell population with high expression quantity of ENTPD1 is CD8 Trm Ex, and the other cell population is CD8 Trm;

(VI) using SingleR software to match the ITGAE gene low expressing cell population with GSE107011 data to obtain definition and annotation of ITGAE gene low expressing cell population which can be defined as: CD8EM, CD8EM Ex promoter, CD8

(VII) the CD8 cells with tumor specific TCR are the cells which are highly expanded in the CD8Tem and CD8 Trm Ex cells in the tumor tissues, and the cells with highest TCR frequency are searched to be specific T cells in the CD8Tem and CD8 Trm Ex cells respectively, and the sequences of the cells are the tissue tumor specific TCR sequences.

7. A method of screening for tumor specific T cells and TCRs as claimed in claim 6 wherein the single Cell sequencing data processing method is Cell Ranger's method, STAR method or BD Rhapbody system processing method.

8. Use of a tumor-reactive TCR obtainable by a method of screening tumor-specific T cells and TCRs according to any of claims 1-7 for the screening of tumor antigens, vaccine preparation, TCR-T therapy.

Technical Field

The disclosure relates to the field of biotechnology, and in particular relates to a method for screening tumor specific T cells and TCR.

Background

At present, the traditional operation, radiotherapy and chemotherapy can not thoroughly eliminate tumor cells and can not solve the problem of tumor metastasis and recurrence. The immune system performs the functions of monitoring, eliminating, regulating and immunological memory of mutant cells and tumor cells, activates the patient's own immune system through immunotherapy, and kills the tumor cells by means of the immune function of the patient, thereby preventing the recurrence and metastasis of the tumor. With the rapid and rapid research on the aspect of tumor immunotherapy in recent years, people have come to step with the dream of cancer, 90% of leukemia can be cured by Chimeric Antigen Receptor T cell therapy (CAR-T), the PD-1/CTLA-4 antibody normalizes immunity by regulating T cell function, the remission rate and 5-year survival rate of various solid tumors are greatly improved, and the Nuobell biomedical prize of 2018 is obtained. Thus, more and more scholars insist that immunotherapy will be the ultimate weapon for humans to combat tumors.

The recognition and elimination of tumor cells by an immune system mainly depend on T cell mediated cellular immunity, the limitations of the traditional treatment technology are overcome by the Adoptive Cell Therapy (ACT) which enhances the immune response of anti-tumor cells and restores the immune monitoring, defense and regulation functions of an organism, and through the development of most centuries, the adoptive cell therapy has been developed from the basic theory research to the clinical application stage, and a new way is provided for middle and late stage patients to realize long-term tumor survival by outstanding safety and effectiveness.

ACT therapies include Tumor Infiltrating T cell (TIL) therapy, CAR-T cell therapy, and T Cell Receptor (TCR) therapy. TIL adoptive reinfusion therapy showed good clinical efficacy in various malignant tumors represented by melanoma, but sufficient amount of TI was obtained due to in vitro amplificationL is very difficult and its clinical application is greatly limited. CAR-T cell therapy and TCR therapy are two latest technologies in the field of ACT at present, T cells in peripheral blood of patients are modified through genetic engineering to express CAR or new TCR capable of recognizing cancer cells, so that ordinary T cells are endowed with the capability of targeting tumor recognition, and specific function-enhanced T cells can be obtained based on genetic modification technology, so that the CAR-T cell therapy and the TCR therapy are widely concerned and become research hotspots. Unlike TCRs, CARs are chimeric single chain antibodies (scFv) that recognize tumor-associated antigens with T cell activation sequences, so their use is limited to tumors with known target antigens, such as CD19⁺The B cell lymphoma is easy to cause 'cytokine storm' and 'off-target effect', generates stronger side reaction, and the prior clinical research finds that the CAR-T has far lower treatment effect on solid tumor than TCR-T.

TCR is a receptor molecule on the surface of T cells that specifically recognizes the antigen peptide-MHC complex on antigen presenting cells, thereby triggering a T cell immune response. Since TCR molecules determine the antigen recognition specificity of T cells, if TCR specific to tumor antigens is transferred into common T cells, the T cells can be endowed with the recognition capability of the tumor antigens, and the T cells can be transferred into patients after being activated and proliferated in vitro, so that the anti-tumor effect can be exerted. Therefore, a large number of T cells for identifying specific antigens can be conveniently obtained by utilizing a TCR gene introduction method, the T cells modified by the TCR gene are called TCR-T, and the TCR-T becomes a research hotspot in tumor immunotherapy in recent years and shows good treatment effect in clinical experiments.

The TCR molecule mainly consists of two chains of alpha and beta, and encoding genes V, (D) J and C of the TCR molecule have MHC recognition restriction through germline rearrangement in the process of T cell development and through positive selection and negative selection processes in thymus. The TCR α β produced by the mature T cells of the body constitutes a Repertoire of antigen recognition receptors (reportire) capable of binding tens of millions of antigens, and theoretically, the TCR α β receptor Repertoire has a capacity of more than 1015.

The successful acquisition of tumor antigen specific TCR is an important prerequisite for the treatment of tumors by TCR-T cells, and the screening of tumor specific TCR genes at present mainly comprises the steps of acquiring T cells identified by tumor antigen specificity and then cloning the TCR genes thereof. The method mainly comprises the following steps: 1. (ii) a multicellular RT-PCR based amplification technique; MHC-peptide Pentamer (Pentamer) flow cytometry is a sensitive method for detecting epitope specificity, and after fluorescent labeled MHC-peptide Pentamer is incubated with T cells, the T cells specifically recognizing the MHC-peptide Pentamer can be excited to fluoresce and sorted by flow, so that single T cell clones with antigen specificity can be separated. The general process flow is that mononuclear cells are separated from peripheral blood of a tumor patient, wherein adherent cells are stimulated by colony stimulating factors and IL-4 to obtain Dendritic Cells (DCs), and then synthesized tumor antigen peptides are incubated with the DCs to obtain antigen-loaded mature DCs; the non-adherent T cells are stimulated multiple times by antigen-loaded DC to induce Cytotoxic T Lymphocytes (CTL); according to the HLA type of a tumor patient, preparing a corresponding tumor antigen peptide-MHC pentamer, and screening out single specific CTL by using a flow cytometry technology. Finally, a certain number of T cells are obtained by amplifying single specific CTL in vitro, and then the specific TCR gene is obtained by RT-PCR amplification. 2. The single cell RT-PCR amplification technology is utilized, the single cell RT-PCR amplification technology is approximately based, after the tumor antigen specific T cell monoclonal is obtained through separation, reverse transcription is carried out on the single cell clone, and a target sequence is specifically amplified through PCR cloning according to the difference of the used primers (based on 5' RACE amplification or PCR amplification based on multiple primers).

However, in the multi-cell RT-PCR amplification technology, T cells specifically recognizing tumor antigens need to be screened first, and undergo long-term in vitro cloning culture, which is time-consuming and has high cost and technical requirements, and it is not easy to obtain sufficient number of cells for PCR amplification, and only a few laboratories have succeeded in obtaining tumor-specific TCR genes by this method at present.

Although the amplification technology of single cell RT-PCR does not need complicated and complicated in vitro culture and proliferation, the method can not obtain the attributes of the cells expressing the TCR, including the state and the subgroup of the cells and which effector is expressed, and the tumor reactivity of the cells can not be known, so that the method is not beneficial to the effectiveness evaluation of the TCR sequence applied to TCR-T.

More importantly, single-cell or multi-cell RT-PCR techniques require the prior acquisition of T-cells with known tumor antigen reactivity, but in practice, the vast majority of tumor antigens are currently unknown, undefined and variable. The bottleneck of the prior art is as follows: specific T cells or TCRs targeting tumors of unknown antigen cannot be obtained.

Disclosure of Invention

The present disclosure provides a method for screening tumor-specific T cells and TCRs to obtain specific T cells and TCRs targeting unknown tumors.

In order to realize the purpose, the technical scheme is as follows:

a method for screening tumor specific T cells and TCR, the method comprises the following steps:

(1) firstly, carrying out infiltration unicellular digestion and separation on tumor tissues;

(2) the separated single cells were then flow sorted for CD3⁺A T cell;

(3) flow sorting of CD3 using immunomagnetic bead removal⁺Dead cells in T cells;

(4) removing CD3 after dead cells⁺Performing single cell transcriptome bank building, VDJ bank building and sequencing on the T cells;

(5) analyzing the sequenced data, and then screening the tumor specific CD8⁺T cells and TCR.

The specific operation steps of the step (1) are as follows:

(a) firstly, placing tumor tissues removed in a fresh operation in a sterile plate, repeatedly washing the tumor tissues by using sterile PBS or Hank's solution, subtracting connective tissues and adipose tissues, and then shearing the tumor tissues into pieces;

(b) adding tissue digestive juice into the cut tumor tissue, mixing uniformly, and digesting for 1-2h at 37 ℃ by a shaking table;

(c) adding HBSS solution into the digested tumor tissue to terminate digestion, transferring the tissue fluid to a 70-micron screen by using a pipette, filtering the tissue fluid into a centrifuge tube, centrifuging the tissue fluid at 1800r/min for 8 min;

(d) and removing supernatant of the centrifuged tissue fluid, washing the tissue fluid by using Hank's solution, centrifuging the tissue fluid to remove the supernatant, adding culture fluid to resuspend cells, and counting the cells.

The specific operation steps of the step (2) are as follows:

① the above separation is counted as 10-50X 10⁶Infiltrating single nuclear cells into the tissue, centrifuging, removing supernatant, cleaning with purified buffer solution, and centrifuging at 1800rpm/min at 4 ℃ for 8 min;

② discarding the supernatant after centrifugation, resuspending the precipitated cells with 200 μ L of purification buffer, adding fluorescent antibody, incubating at 4 deg.C in dark for 30min, washing with purification buffer, centrifuging at 1800rpm/min at 4 deg.C for 8min, discarding the supernatant, and resuspending the precipitated cells with 500 μ L of purification buffer;

③ filtering the cell resuspension through a 40 μm mesh to remove adherent cell masses, then subjecting the filtered cell resuspension to surface molecular staining with 7-AAD dye, and collecting 7-AAD-CD45-V450 in single cells using BD Aria II⁺CD3-PE-CF594⁺A population of cells.

The specific operation of the step (3) is as follows: sorting the above-described flow-sorted CD3⁺Centrifuging T cells, washing with sterile PBS, re-suspending the cells with 45-55 μ L sterile PBS, mixing the same amount of cell suspension with placenta blue, and counting cells and cell survival rate by Life Count cell counter<80% of the cells are killed by using magnetic beads which are whirly and beautiful.

The step (4) comprises the following specific steps: and (3) marking the obtained single cells by using a magnetic bead label technology, marking the same label on each RNA of the same cell, performing reverse transcription and enzyme digestion, respectively performing transcriptome and VDJ amplification, and finally sequencing the amplified transcriptome and VDJ.

The specific operation steps of the step (5) are as follows:

(I) converting the data obtained after sequencing into an expression matrix of cell gene and obtaining the TCR of each cell by using a single cell sequencing data processing method;

(II) reading in an expression matrix by utilizing Seurat software, removing the data of the cells with too low or too high UMI, and performing interception on the front and the back 3 percent;

(III) integrating the removed data by using a CCA method in Seurat software, dividing the cells into 30-35 groups by using a cell clustering method, and calculating a mean expression profile of each cell group;

(IV) reading in the mean expression profile of the cell population using SingleR software, initially defining the cell population according to the ENCODE and BLUEPRINT databases, and filtering non-T cells in the data;

(V) in the CD8 positive cell population, looking at the expression quantity of ITGAE genes, selecting an ITGAE high expression cell population, and observing the expression quantity of KLRC2 and ENTPD1 in the high expression cell population, wherein the cell population with high expression quantity of KLRC2 is IEL, the cell population with high expression quantity of ENTPD1 is CD8 Trm Ex, and the other cell population is CD8 Trm;

(VI) using SingleR software to match the ITGAE gene low expressing cell population with GSE107011 data to obtain definition and annotation of ITGAE gene low expressing cell population which can be defined as: CD8EM, CD8EM Ex promoter, CD8

CD8 Tcm、CD8 TE、MAIT、CD4

CD4 Tcm, Th1, Th17, th1.th17, Th2, TFH, Treg or CD8 Tem;

(VII) the CD8 cells with tumor specific TCR are the cells which are highly expanded in the CD8Tem and CD8 Trm Ex cells in the tumor tissues, and the cells with highest TCR frequency are searched to be specific T cells in the CD8Tem and CD8 Trm Ex cells respectively, and the sequences of the cells are the tissue tumor specific TCR sequences.

The single Cell sequencing data processing method is a Cell Ranger method, a STAR method or a BD Rhapbody system processing method.

The tumor reactive TCR obtained by the method for screening the tumor specific T cell and the TCR is applied to the aspects of tumor antigen screening, vaccine preparation and TCR-T treatment.

The beneficial effects of this disclosure are: a method for screening tumor specific T cells and TCR is provided, which can screen tumor specific T cells and TCR of each patient under the condition of unknown tumor antigen, can determine the activity state of the T cells, whether to express or express inhibitory receptors, express chemokines and immunoactive factors, belong to cell subgroups, evaluate the tumor reactivity, and obtain the information to be beneficial to immunotherapy based on specific tumor specific T cells or TCR-T therapy based on specific TCR sequences.

Drawings

FIG. 1 is a graph showing the results of the first step of grouping cells in example 2 using the Seurat CCA method.

FIG. 2 is a preliminary definition of cell subpopulations according to the ENCODE and BLUEPRINT databases using SingleR software in example 2.

FIG. 3 is a graph showing the result of defined clustering of cells based on the expression level of ITGAE gene and GSE107011 data in example 2.

Detailed Description

The following steps are only used for illustrating the technical scheme of the disclosure and are not limited; although the present disclosure has been described in detail with reference to the foregoing steps, those of ordinary skill in the art will understand that: the technical solutions recorded in the foregoing steps may still be modified, or some or all of the technical features may be equivalently replaced; and such modifications or substitutions do not depart from the scope of the respective technical solutions of the steps of the present disclosure.