Tumor reactive inexhaustible T cell subtype and separation and identification method thereof

Abstract

The invention relates to the field of cell biology, in particular to a tumor reactive inexhaustible T cell subtype and a separation and identification method thereof. The applicant of the invention firstly discovers a novel tumor reactive Non-exposed T-cell (TNT) named as CD8HLA-DRA + Tem in research, and provides a separation and identification method of the cell subtype, in a multicolor immunohistochemical experiment, the subtype can be positioned through CD8 +, HLA-DR + and TIM-3-, and through cell function analysis of the subtype, it is found that the tumor reactive Non-exposed T-cell (TNT) which is named as CD8HLA-DRA + Tem and the TNT can be separated and identified through the CD8HLA-DRA + Tem. The subtype has an anti-tumor function.

Description

Technical Field

[0001] The present invention relates to the field of cell biology, and in particular, to a tumor-reactive non-exhausted T cell subtype and a method for separating and identifying the subtype. Background Art

[0002] As the executor of cellular immunity, T cells have great differences in functional status. T cells can be distinguished from other immune cells based on the expression of CD3 surface protein. Classical T cells include naive T cells, effector T cells (effector T cells, Teff, also known as cytotoxic T cells, Cytotoxic T cells, CTL) and memory T cells (memory T cells, Tm), among which naive T cells can differentiate into the latter two after encountering antigen stimulation. Tm can be divided into central memory T cells (central memory T cell, Tcm), effector memory T cells (effector memory T cell, Tem) and tissue resident memory T cells (tissue resident memory T cell, Trm), etc.

[0003] T cells recognize MHC-antigen complexes through T cell receptors (TCR) on the cell surface. T cells with TCRs that can specifically recognize tumor antigens are called tumor-reactive T cells, and T cells that cannot specifically recognize tumor antigens are called bystander T cells. In the tumor microenvironment, due to the long-term stimulation of TCR by tumor antigens, the cytotoxicity of tumor-reactive T cells usually decreases, and this process is called T cell exhaustion. As T cells are exhausted, their cell proliferation ability gradually decreases, their effector function gradually decreases, and they are accompanied by increased expression of co-inhibitory receptors, such as PD-1 (PDCD1), LAG-3 (LAG3), CTLA4, TIM-3 (HAVCR2), etc.

[0004] We previously analyzed gastric cancer-related single-cell transcriptome data and found a special T cell subtype. We used the shared TCR with exhausted T cells to indicate tumor antigen specificity and found that some tumor-reactive T cells not only have the characteristics of exhausted T cells responding to tumor antigens, but also do not have exhaustion characteristics. Public data confirmed that this cell subtype exists in a variety of tumor tissues, but has not been individually identified, separated, and characterized before. Summary of the invention

[0005] In the process of studying T cells, the applicant unexpectedly discovered a new tumor-reactive non-exhausted T cell subtype, which is the first time this subtype has been discovered. Based on this, the present invention provides the following technical solutions:

[0006] First, in a first aspect, the present invention provides a method for isolating a tumor-reactive non-exhausted T (TNT) cell subtype, the method comprising sorting CD8+HLA-DR+TIM-3- cells by flow cytometry.

[0007] In one embodiment, the method comprises the steps of:

[0008] 1. Collect cells into sample tubes and adjust the cell concentration to 1×10^6 cells / 100ul. Wash the cells with 2ml PBS and centrifuge at 500g for 5 minutes. Discard the supernatant and repeat twice. Resuspend the cells with 100ul flow cytometry staining buffer and add 5-10ul 7-AAD dye to the cell suspension. Mix evenly and incubate at 4℃ in dark for 30 minutes. Detect 7-AAD staining with flow cytometry and remove dead cells.

[0009] 3. Add anti-Fc antibody blocking solution to the washed cell suspension for blocking incubation;

[0010] 4 Repeat step 1 above to stain with 5 antibodies, including CD45, CD3, CD8, HLA-DR, and TIM-3. After incubation for 1 hour, wash the cells twice and resuspend them into a single cell suspension.

[0011] 5 The flow cytometry instrument starts cell sorting. According to the set parameters such as fluorescence intensity and cell gate, CD8+HLA-DR+TIM-3- cells are successfully sorted out as TNT cells.

[0012] The second aspect of the present invention provides a method for identifying tumor-reactive non-exhausted T cell subtypes, the method comprising performing multicolor immunohistochemical staining of three proteins, CD8, HLA-DR, and TIM-3, on the sample, and CD8+HLA-DR+TIM-3- cells are tumor-reactive non-exhausted T cell subtypes.

[0013] The third aspect of the present invention provides the use of tumor-reactive non-exhausted T cell subtypes in the preparation of anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0015] Figure 1: Comparison of markers expressed by TNT cells and exhausted T cells;

[0016] Figure 2 : Comparison of the expression of T cell exhaustion markers with different TCR amplification degrees;

[0017] Figure 3 :The proportion of TNT cells in T cells in different tissues;

[0018] Figure 4 :The proportion of TNT cells in T cells in primary lesions of gastric cancer;

[0019] Figure 5 : Public database data show that TNT cells are more commonly distributed in primary tumor lesions;

[0020] Figure 6 : Trajectory inference results;

[0021] Figure 7 : Comparison of pathway enrichment between TNT cells and exhausted T cells;

[0022] Figure 8 :Analysis and prediction of the interaction between TNT cells and other cells;

[0023] Fig. 9 : Public data indicate that TNT cells are abundant in other malignancies;

[0024] Fig.10 :Multiple immunohistochemical staining confirmed the existence of TNT cells. DETAILED DESCRIPTION

[0025] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0026] Example 1 Isolation and identification of tumor-reactive non-exhausted T cell subtypes

[0027] 1. Generate expression matrix from sequencing data: Single-cell transcriptome sequencing was performed on primary lesions, metastases, negative lymph nodes, peripheral blood, and adjacent samples of gastric cancer patients (from Peking University Cancer Hospital, all of whom had signed informed consent). The single-cell sequencing data generated by 10xGenomics was initially presented in fastq format and was initially processed by the official Cellranger (version 6.1.2) to obtain the original single-cell gene expression matrix. The single-cell transcriptome data used in this study used the 5' end sequencing method to simultaneously obtain T cell receptor (TCR) and B cell receptor (BCR) information. This sequencing method can provide complete TCR chain information, including the sequences of the V (Variable), D (Diversity), and J (Joining) segments. In order to simultaneously analyze the gene expression matrix and TCR and BCR information, the cellranger multi function was first used to simultaneously input all sequencing data of the same sample, and GRCh38 was used as the reference genome to obtain the gene expression profile corresponding to each cell and the TCR and BCR sequence information.

[0028] 2. Quality control: Then, the gene expression matrix was read in using the CreateSeuratObject function in the Seurat package (version 4.2.0). Quality control was performed according to the following criteria: cells were screened based on the number of genes detected between 300 and 6000 and the percentage of mitochondrial genes was less than 20%, genes were screened based on expression in at least three cells, and potential double cells were removed using the Scrublet package with default parameters. Next, for the single-cell gene expression matrix that passed quality control, the sum of the gene expression of each cell was first normalized to 10,000 and further processed by log1p.

[0029] 3. Identify major cell categories: Use the FindVariableFeatures function in the Seurat package to find the top 2,000 HVGs, and manually remove more than 1,000 genes related to the cell cycle, mitochondria, ribosomes, heat shock proteins, and dissociation. Use PCA to reduce the dimensionality of the data, extract the main dimensional information, and select the appropriate number of PCs for each group of data. In the analysis of all cells, this study selected the top 20 PCs. The UMAP method was then used to visualize the reduced-dimensional data to show the relationship and distribution between cells. The Louvain algorithm was used to perform unsupervised clustering of the reduced-dimensional data to identify cell groups. Use the FindAllMarkers function to obtain the highly expressed genes of each Cluster based on the Wilcox rank sum test, and combine the expression of classic marker genes to identify the cell type to which each Cluster belongs.

[0030] 4. T cell subtype identification: further select cells identified as T cells in the general classification for separate analysis, and then further identify the subtype. The RPCA (Robust Principal Component Analysis) method is used to correct the batch effect of the data to ensure the consistency of the data between different batches. Then the data is integrated, and the subtypes are identified using the same dimensionality reduction, clustering, and cell type identification methods. First, according to the clustering results, T cells are divided into two groups: CD4+T cells and CD8+T cells. Then the above-mentioned batch effect removal, dimensionality reduction, clustering and other steps are repeated for the two groups of T cells, and subtypes are identified according to gene expression. Among them, the CD8_HLA-DRA+Tem subtype was obtained.

[0031] 5. TCR sequence analysis: The contig file of TCR generated by Cell Ranger is output and processed by the scRepertoire software package. Use the combineTCR function to extract TCR clonotypes. Different degrees of expansion can be defined according to the number of cells in which the clonotype is located, such as single, small, medium, large, hyperexpanded, etc. The TCR clonotype carried by the cell is associated with the cell type according to the cell barcode. Use the clonalOverlap function to compare the clonotype similarity between different samples or different T cell subtypes in the same sample. The specific overlap value is the number of cells carrying the intersecting TCR clonotypes in the two cell populations divided by the relatively smaller total number of cells in the two populations. This proportion is defined as the overlap degree of the two cell populations. Use the quantContig function to calculate the proportion of the number of clonotypes in the TCR carried by each cell population after deduplication in the total number of cells. The lower the value, the higher the degree of TCR clonal expansion of the cell population.

[0032] 6. TNT discovery: Each T cell has a TCR, and the protein sequences corresponding to the TCR are completely consistent and are considered to be the same clone. If the number of cells belonging to the same clone between two cell subtypes is relatively large, it is considered that the two have a high degree of clonal sharing. Therefore, by having a relatively high degree of clonal sharing with the TCR of exhausted T cells, its potential tumor-responsive T cell subtype can be located, that is, the CD8_HLA-DRA+Tem subtype named in this article. By examining the gene expression of these T cell subtypes, it was found that the CD8_HLA-DRA+Tem T cell subtype expresses genes related to cytotoxic function, such as granzymes and perforins ( Figure 1 ). However, unlike exhausted T cells, the target subtype barely expresses exhaustion markers ( Figure 1 ), including cells with highly expanded TCRs ( Figure 2 ), so we named them tumor-reactive non-exhausted T cells (TNT cells).

[0033] 7. Verification of the existence of TNT cells: We confirmed the existence of this subtype by performing multicolor immunohistochemistry (mIHC) of three proteins, CD8, HLA-DR, and TIM-3, on paraffin sections of the samples (detailed steps are as follows) Fig.10 ), based on the gene expression of this subtype, flow cytometry confirmed that this subtype could be sorted by CD8+, HLA-DR+ and TIM-3-, and confirmed the existence of the TNT subtype in primary gastric cancer lesions.

[0034] The steps of mIHC are:

[0035] 1. Dewax the paraffin sections to water: Dewax in environmentally friendly dewaxing agent (1), environmentally friendly dewaxing agent (2), and environmentally friendly dewaxing agent (3) for 10 minutes respectively, then dewax in anhydrous ethanol, 95% ethanol, and 75% ethanol for 5 minutes each. Wash with distilled water 3 times, 3 minutes each time, and then soak.

[0036] 2. Antigen repair: High temperature and high pressure repair (EDTA, pH 9.0): Add EDTA (pH 9.0) antigen repair solution to the pressure cooker. The volume of the repair solution should be enough to immerse the slices. Cover the lid lightly and heat it on the induction cooker until it boils (the induction cooker is 2100 watts). Put the dewaxed and hydrated tissue slices into the boiling buffer solution, cover the lid tightly, wait for a while until the air nozzle is lifted, add the pressure limiting valve (adjust the power of the induction cooker to 1200 watts), and strictly count for 1 minute and 30 seconds after the pressure cooker sprays, then stop the fire. Rinse the pressure cooker with cold water, open the lid after the air pressure drops, and wait for the repair solution to cool naturally to room temperature (25-30°C, about 1 hour). Then take the slices out of the repair solution and immerse them in TBST 3 times, 5 minutes each time.

[0037] 3. Blocking: Place the slices in 3% H2O2 for blocking at room temperature for 10 minutes, then soak in distilled water for 3 minutes.

[0038] 4. Draw circles: After drawing circles with a tissue pen, place the slices in TBST.

[0039] 5. Blocking: Add 10% goat serum and incubate at 37°C for 30 minutes.

[0040] 6. Incubate with primary antibody: shake off the serum, take CD8 antibody stock solution, add 50-100 μl primary antibody working solution to each section, and incubate at 4°C overnight.

[0041] 7. Incubate with secondary antibody: Take the slices out of the refrigerator the next day, place them at room temperature for 15 minutes (rewarm), rinse with TBST 3 times, and then immerse 3 times, 3 minutes each time. Take goat anti-rabbit IgG H&L (HRP) secondary antigen solution, add it to TBST, prepare a secondary antibody working solution with a concentration of 1:4000, mix it with a vortex mixer, add 50-100μl (depending on the size of the tissue) of secondary antibody working solution to each slice, and incubate at 37℃ for 45 minutes. Rinse 3 times with TBST, and then immerse 3 times, 3 minutes each time.

[0042] 8. TSA staining: remove TBST, take 488tyramide stock solution was added to 0.003% H2O2 to prepare a concentration of 1:300 488tyramide working solution, add 50-100 μl to each slice 488tyramide working solution, incubate at room temperature for 10 min. After incubation, wash with TBST three times.

[0043] 9. Elution: Add elution solution to the repair box. The elution solution should cover the slices. Place the repair box in a 42℃ water bath to preheat until it reaches the temperature, then place the slices in the repair box and elute for 20 minutes. Take the slices out of the elution solution and soak them in TBST for 3 minutes.

[0044] 10. Blocking: Add 10% goat serum and incubate at 37°C for 30 minutes.

[0045] 11. Incubate with primary antibody: shake off the serum, take the HLA-DRB5 antibody stock solution and add it to TBST to prepare a primary antibody working solution with a concentration of 1:200. Mix it with a vortex mixer, add 50-100 μl of primary antibody working solution to each section, and incubate overnight at 4°C.

[0046] 12. Incubate the secondary antibody: Take the slices out of the refrigerator the next day, place them at room temperature for 15 minutes (rewarm), rinse them 3 times with TBST, and then immerse them 3 times, 3 minutes each time. Take the goat anti-rabbit IgG H&L (HRP) secondary antigen solution, add it to TBST, prepare a secondary antibody working solution with a concentration of 1:4000, mix it with a vortex mixer, add 50-100μl (depending on the size of the tissue) of the secondary antibody working solution to each slice, and incubate at 37℃ for 45 minutes. Rinse 3 times with TBST, and then immerse 3 times, 3 minutes each time.

[0047] 13. TSA staining: Remove TBST, take CY3tyramide stock solution and add it to 0.003% H2O2 to prepare CY3tyramide working solution with a concentration of 1:600, add 50-100μl CY3tyramide working solution to each slice, incubate at room temperature for 10 minutes, and then wash with TBST for 3 times.

[0048] 14. Elution: Add elution solution to the repair box. The elution solution should cover the slices. Place the repair box in a 42℃ water bath to preheat until it reaches the temperature, then place the slices in the repair box and elute for 20 minutes. Take the slices out of the elution solution and soak them in TBST for 3 minutes.

[0049] 15. Blocking: Add 10% goat serum and incubate at 37°C for 30 minutes.

[0050] 16. Incubate with primary antibody: shake off the serum, take the TIM3 antibody stock solution and add it to TBST to prepare a primary antibody working solution with a concentration of 1:400. Mix it with a vortex mixer, add 50-100 μl of primary antibody working solution to each section, and incubate overnight at 4°C.

[0051] 17. Incubate with secondary antibody: Take the slices out of the refrigerator the next day, place them at room temperature for 15 minutes (rewarm), rinse with TBST 3 times, and then immerse 3 times, 3 minutes each time. Take goat anti-rabbit IgG H&L (HRP) secondary antigen solution, add it to TBST, prepare a secondary antibody working solution with a concentration of 1:4000, mix it with a vortex mixer, add 50-100μl (depending on the size of the tissue) of secondary antibody working solution to each slice, and incubate at 37℃ for 45 minutes. Rinse 3 times with TBST, and then immerse 3 times, 3 minutes each time.

[0052] 18. TSA staining: Remove TBST, take CY5tyramide stock solution and add it to 0.003% H2O2 to prepare CY5tyramide working solution with a concentration of 1:400. Add 50-100μl CY5tyramide working solution to each slice and incubate at room temperature for 10 minutes. After incubation, wash with TBST for 3 times.

[0053] 19. Nuclear staining: Remove TBST, add 50-100 μl (depending on the size of the tissue) DAPI working solution (prepared with DAPI stock solution at a dilution of 1:500) to each section, stain the nucleus for 5 minutes in the dark, and then rinse with TBST.

[0054] 20. Seal the slides: Seal the slides with fluorescent mounting medium and store at 4°C away from light.

[0055] 21. Microscopic examination: examination under a microscope, image acquisition and analysis.

[0056] Use flow cytometry to sort TNT cells: 1. Sample preparation: prepare single cell suspension, fluorescent labeled antibodies, adjust the cell concentration to 1-10×10^6 cells / ml, use a filter to filter the cells to avoid clogging, then transfer the cells to a sample tube, press them into the flow chamber under high pressure on ice away from light. Adjust the computer sorting parameters and settings.

[0057] 2. Collect cells into a sample tube and adjust the cell concentration to 1×10^6 cells / 100ul. Wash the cells with 2ml PBS and centrifuge at 500g for 5 minutes. Discard the supernatant and repeat twice. Then resuspend the cells with 100ul of flow cytometry staining buffer, add 5-10ul 7-AAD dye to the cell suspension, mix evenly and incubate at 4℃ in the dark for 30 minutes. Use flow cytometry to detect 7-AAD staining and remove dead cells.

[0058] 3. Add anti-Fc antibody blocking solution to the washed cell suspension for blocking incubation.

[0059] 4 Repeat step 2 above to stain and label 5 antibodies (CD45, CD3, CD8, HLA-DR, TIM-3). Among them, CD45 expression can be used to exclude epithelial cells and fibroblasts, CD3 expression can be used to screen T cells, and CD8, HLA-DR but not TIM-3 expression can be used to screen TNT cells. After 1 hour of labeling and incubation, wash the cells twice, then resuspend them into a single cell suspension and prepare for sorting.

[0060] 5 The flow cytometry instrument started cell sorting and successfully sorted out TNT cells based on the set parameters such as fluorescence intensity and cell gate.

[0061] Example 2 Functional verification of tumor-reactive non-exhausted T cell subtypes

[0062] TNT cells are more abundant in primary gastric cancer lesions, less abundant in metastatic lymph nodes, and least abundant in negative lymph nodes ( Figure 3 The proportion of gastric cancer primaries in patients with and without lymph node metastasis was also significantly different, with the proportion being higher in patients without lymph node metastasis ( Figure 4 ). Analysis of hundreds of samples also showed that this subtype was more abundant in tumor samples compared with lymph nodes and PBMCs ( Figure 5 ). These results suggest that TNT cells may be associated with anti-cancer and anti-metastasis. Trajectory inference shows that TNT cells are in a terminal state rather than a state of impending exhaustion ( Figure 6 ). Since tumor-reactive T cells are usually exhausted under long-term antigen stimulation, we further analyzed the differences between this CD8_HLA-DRA+Tem subtype and exhausted CD8+T (Tex) cells. TNT cells are enriched for pathways that positively regulate T cell activation, while Tex cells are enriched for pathways that negatively regulate ( Figure 7When analyzing cell-cell interactions in gastric cancer primaries in patients without lymph node metastasis, it was found that TNT cells received different signals from Tex cells. Tex cells received many exhaustion signals, such as PDCD1 and CTLA4, while TNT cells mainly received signals from co-stimulatory receptors and experienced lower levels of immunosuppression than exhausted T cells ( Figure 8 ). Examination of signals from epithelial cells revealed that TNT cells were recipients of CDH1 signals in Pri.GC from patients without metastasis ( Figure 8 ). This suggests that in patients without metastasis, there is an adhesion relationship between epithelial cells and TNT cells in Pri.GC, and this subtype has anti-tumor function and is therefore associated with the absence of metastasis. Taken together, these results indicate that TNT cells are a tumor-reactive subtype with low exhaustion and high cytotoxicity. Public data analysis shows that they are abundant in ovarian cancer, head and neck cancer, gastrointestinal cancer, lung cancer, esophageal cancer, etc. ( Fig. 9 ).

[0063] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for isolating a tumor-reactive non-exhausted T (TNT) cell subtype, characterized in that: The method includes sorting CD8+HLA-DR+TIM-3- cells by flow cytometry.

2. The separation method according to claim 1, characterized in that The method comprises the following steps: (1) Collect cells into a sample tube and adjust the cell concentration to 1×10^6 cells / 100ul. Wash the cells with 2ml PBS and centrifuge at 500g for 5 minutes. Discard the supernatant and repeat twice. Resuspend the cells with 100ul flow cytometry staining buffer and add 5-10ul 7-AAD dye to the cell suspension. Mix well and incubate at 4℃ in the dark for 30 minutes. Detect 7-AAD staining with flow cytometry and remove dead cells. (2) adding anti-Fc antibody blocking solution to the washed cell suspension for blocking incubation; (3) Repeat the above step (1) to stain with five antibodies, including CD45, CD3, CD8, HLA-DR, and TIM-3. After incubation for 1 hour, wash the cells twice and resuspend them into a single cell suspension. (4) The flow cytometry instrument starts to sort cells. Based on the set parameters such as fluorescence intensity and cell gate, CD8+HLA-DR+TIM-3- cells are successfully sorted as TNT cells.

3. A method for identifying tumor-reactive non-exhausted T cell subtypes, characterized in that: The method includes performing multicolor immunohistochemical staining of three proteins, CD8, HLA-DR, and TIM-3, on the sample, and CD8+HLA-DR+TIM-3- cells are tumor-reactive non-exhausted T cell subtypes.

4. Use of the tumor-reactive non-exhausted T cell subtype obtained by the separation method according to claim 1 in the preparation of anti-tumor drugs.

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