Rapid detection method for cytotoxic T cells in peripheral blood

Abstract

The invention provides a rapid detection method for cytotoxic T cells in peripheral blood. Specifically, the method provided by the invention is used for rapidly identifying the cytotoxic T cells in the peripheral blood by detecting the expression of cell surface proteins CX3CR1 and GPR56. The method provided by the invention is quick to operate, does not involve cell fixation and membrane rupture, and does not affect cell activity.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine and in particular to a method for rapid detection of cytotoxic T cells in peripheral blood. Background Art

[0002] Cytotoxic T cells are the main force of anti-tumor immunity, and the biological field often needs to detect cytotoxic T cells. The existing technical solution is to identify cytotoxic T cells by detecting the ability of cells to produce cytotoxic molecules such as granzymes and perforins. The specific method is as follows: Use T cell stimulators PMA, ionomycin, etc. to stimulate T cells to promote the expression of cytotoxic molecules, and add Brefeldin A (BFA) to inhibit protein transport so that the cytotoxic molecules produced by them remain in the cells. Subsequently, the cells are fixed and membrane-permeated, and stained with fluorescent antibodies targeting cytotoxic molecules such as granzyme B and perforin, and flow cytometry is used to detect the expression of cytotoxic molecules.

[0003] However, the detection methods of the prior art have the following disadvantages: (1) It is time-consuming: the entire detection takes about 8 hours; (2) T cells need to be subjected to TCR stimulation, fixation, and membrane disruption. After fixation and membrane disruption, the cells will die, and it is impossible to obtain active cytotoxic T cells for further culture.

[0004] Therefore, there is a need in the art to develop a method that is time-saving and capable of identifying cytotoxic T cells while the cells are alive. Summary of the invention

[0005] The object of the present invention is to provide a method which is time-saving and capable of identifying cytotoxic T cells in a viable state.

[0006] In a first aspect of the present invention, a method for identifying cytotoxic T cells is provided, the method comprising the steps of:

[0007] (1) Providing T cells to be tested;

[0008] (2) detecting the expression of the T cell surface marker protein to identify whether the T cell is a cytotoxic T cell;

[0009] Wherein, the marker protein is CX3CR1, or CX3CR1 and GPR56.

[0010] In another preferred embodiment, if the T cells are detected to be CX3CR1-positive, or CX3CR1- and GPR56-positive T cells, the T cells are cytotoxic T cells.

[0011] In another preferred embodiment, the CX3CR1 positivity refers to that the ratio (E1 / E0) of the expression level of CX3CR1 on the surface of the T cells (E1) to the expression level of CX3CR1 on the surface of non-cytotoxic T cells (E0) is ≥1.5, preferably ≥1.7, and more preferably ≥2.0.

[0012] In another preferred embodiment, the GPR56 positivity means that the ratio (E1 / E0) of the expression level of GPR56 on the surface of the T cells (E1) to the expression level of CX3CR1 on the surface of non-cytotoxic T cells (E0) is ≥1.5, preferably ≥1.7, and more preferably ≥2.0.

[0013] In another preferred embodiment, the T cells to be detected are derived from humans or non-human mammals, such as humans, mice, rats, rabbits, and monkeys.

[0014] In another preferred embodiment, the T cells to be detected are derived from human peripheral blood mononuclear cells.

[0015] In another preferred embodiment, the detection is an in vitro detection.

[0016] In another preferred embodiment, the detection is non-diagnostic and non-therapeutic.

[0017] In another preferred embodiment, the detection is flow cytometry detection.

[0018] In another preferred embodiment, the flow cytometry detection comprises the steps of:

[0019] (1) Use flow cytometry antibodies against CX3CR1 and GPR56 to stain the T cells to be detected;

[0020] (2) washing the T cells to remove excess antibodies;

[0021] (3) Flow cytometry was used to detect cells expressing CX3CR1, CX3CR1 and GPR56, which were cytotoxic T cells.

[0022] In another preferred embodiment, the cytotoxic T cells are selected from the following group: CD4 + Cytotoxic T cells, CD8 + cytotoxic T cells, or a combination thereof.

[0023] In another preferred embodiment, the method does not include the steps of cell fixation and / or cell membrane disruption.

[0024] In another preferred embodiment, the method does not include the step of detecting intracellular proteins.

[0025] In a second aspect of the present invention, a method for enriching cytotoxic T cells is provided, comprising the steps of:

[0026] (1) Providing a cell population to be enriched;

[0027] (2) Screening the cell population for cells that highly express the cell surface marker protein CX3CR1, or CX3CR1 and GPR56, thereby enriching cytotoxic T cells.

[0028] In another preferred embodiment, the cell population to be enriched is derived from humans or non-human mammals, such as humans, mice, rats, rabbits, and monkeys.

[0029] In another preferred embodiment, the cell population to be enriched is a human peripheral blood mononuclear cell population.

[0030] In another preferred embodiment, the screening is performed by flow cytometry.

[0031] In another preferred embodiment, the flow cytometry comprises the steps of:

[0032] (1) Use flow cytometry antibodies against CX3CR1 and GPR56 to stain the enriched cell population;

[0033] (2) washing the T cells to remove excess antibodies;

[0034] (3) Cells expressing CX3CR1, CX3CR1 and GPR56 are sorted out using a flow cytometer, and the cells are collected to obtain enriched cytotoxic T cells.

[0035] In another preferred embodiment, the cytotoxic T cells are selected from the following group: CD4 + Cytotoxic T cells, CD8 + cytotoxic T cells, or a combination thereof.

[0036] In another preferred embodiment, the enriched cytotoxic T cells are active.

[0037] In the third aspect of the present invention, a cytotoxic T cell is provided. The cytotoxic T cell is enriched by the method according to the second aspect of the present invention.

[0038] In another preferred embodiment, the cytotoxic T cells are enriched from human peripheral blood mononuclear cell population.

[0039] In another preferred embodiment, the cytotoxic T cells are selected from the following group: CD4 + Cytotoxic T cells, CD8 + cytotoxic T cells, or a combination thereof.

[0040] In another preferred embodiment, the enriched cytotoxic T cells are active.

[0041] In a fourth aspect of the present invention, a pharmaceutical composition is provided, comprising the cytotoxic T cells according to claim 3, and a pharmaceutically acceptable carrier.

[0042] In the fifth aspect of the present invention, there is provided use of the cytotoxic T cells as described in the third aspect of the present invention or the pharmaceutical composition as described in the fourth aspect of the present invention in the preparation of a drug for treating a disease.

[0043] In another preferred embodiment, the disease is a tumor, an autoimmune disease, an inflammatory disease, or a combination thereof.

[0044] In a sixth aspect of the present invention, a reagent combination for identifying or enriching cytotoxic T cells is provided, wherein the reagent combination comprises reagents for detecting CX3CR1 and GPR56 proteins.

[0045] In another preferred embodiment, the reagent is an antibody, such as a flow cytometry detection antibody.

[0046] In a seventh aspect of the present invention, a device for detecting cytotoxic T cells is provided, characterized in that the device comprises:

[0047] 1) an input module, the input module being used to receive T cells to be detected;

[0048] 2) a detection module, wherein the detection module is used to detect a reagent for detecting the T cell surface marker protein, wherein the marker protein is CX3CR1, or CX3CR1 and GPR56;

[0049] 3) An output module, which is used to output the detection results.

[0050] In another preferred example, if the detection module detects that the T cell is CX3CR1-positive, or CX3CR1- and GPR56-positive T cell, the output module outputs: the T cell is a cytotoxic T cell.

[0051] In another preferred embodiment, the CX3CR1 positivity refers to that the ratio (E1 / E0) of the expression level of CX3CR1 on the surface of the T cells (E1) to the expression level of CX3CR1 on the surface of non-cytotoxic T cells (E0) is ≥1.5, preferably ≥1.7, and more preferably ≥2.0.

[0052] In another preferred embodiment, the GPR56 positivity means that the ratio (E1 / E0) of the expression level of GPR56 on the surface of the T cells (E1) to the expression level of CX3CR1 on the surface of non-cytotoxic T cells (E0) is ≥1.5, preferably ≥1.7, and more preferably ≥2.0.

[0053] In another preferred embodiment, the T cells to be detected are derived from humans or non-human mammals, such as humans, mice, rats, rabbits, and monkeys.

[0054] In another preferred embodiment, the T cells to be detected are derived from human peripheral blood mononuclear cells.

[0055] In another preferred embodiment, the detection is flow cytometry detection.

[0056] In another preferred embodiment, the detection module comprises a reagent for detecting CX3CR1, or CX3CR1 and GPR56.

[0057] In another preferred embodiment, the detection does not include the steps of cell fixation and / or cell membrane disruption.

[0058] In another preferred embodiment, the detection does not include the step of detecting intracellular proteins.

[0059] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The following drawings are used to illustrate specific embodiments of the present invention and are not used to limit the scope of the present invention defined by the claims.

[0061] Figure 1 The correlation between CX3CR1 and ADGRG1 (GPR56 encoding gene) and the RNA expression of cytotoxicity molecule-related genes granzyme B (GZMB) and perforin (PRF1) was shown.

[0062] Figure 2 The correlation between CX3CR1 and GPR56 and the expression of cytotoxic molecules granzyme B and perforin protein is shown. (A) The expression heat map of CX3CR1, GPR56, granzyme B and perforin shows that the four are significantly co-expressed; (B-C) The expression levels of cytotoxic molecules granzyme B and perforin in CX3CR1+GPR56+ T cells and CX3CR1-GRP56- T cells.

[0063] Figure 3 The correlation of CX3CR1 with the expression of the cytotoxic molecules granzyme B and perforin protein in mice is shown. DETAILED DESCRIPTION

[0064] After extensive and in-depth research, the inventors have developed a rapid detection method for cytotoxic T cells in peripheral blood for the first time. The present invention discovered two cell surface proteins CX3CR1 and GPR56 that are specific to cytotoxic T cells. Based on this, the present invention provides a method for quickly identifying cytotoxic T cells in peripheral blood using only surface staining CX3CR1 and GPR56. The method of the present invention is fast to operate, does not require fixation and membrane permeabilization, and does not destroy cell activity. On this basis, the present invention is completed.

[0065] the term

[0066] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0067] As used herein, the terms "comprise", "include", and "contain" are used interchangeably and include not only closed definitions, but also semi-closed and open definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0068] As used herein, the term "flow cytometry (FCM)" is a detection method for quantitative analysis and sorting of single cells or other biological particles at the functional level. It can analyze tens of thousands of cells at high speed and can simultaneously measure multiple parameters from one cell. The present invention detects the expression of T cell surface protein CX3CR1, or CX3CR1 and GPR56 by flow cytometry, thereby identifying cytotoxic T cells.

[0069] As used herein, the term "cytotoxic T cells" refers to T cells that express cytotoxic molecules (such as granzyme B, perforin).

[0070] As used herein, the term "CX3CR1" refers to C-X3-C Motif Chemokine Receptor 1. Accession number: HGNC:2558, NCBI Gene:1524, Ensembl:ENSG00000168329, 601470,UniProtKB / Swiss-Prot:P49238.

[0071] As used herein, the term "GPR56" refers to Adhesion G Protein-Coupled Receptor G1, accession number: HGNC:4512 NCBI Gene:9289 Ensembl:ENSG00000205336 604110UniProtKB / Swiss-Prot:Q9Y653.

[0072] The main advantages of the present invention include:

[0073] (1) The method of the present invention significantly shortens the detection time of cytotoxic T cells from 8 hours in the prior art to 30 minutes;

[0074] (2) The method of the present invention does not require the use of stimulants to stimulate T cells, nor does it require the use of BFA blocking and fixation permeabilization reagents, thereby reducing the detection cost;

[0075] (3) This method does not require fixation and membrane permeabilization, and can obtain active cytotoxic T cells: the identified cytotoxic T cells can be flow sorted or immunomagnetic bead sorted, which is conducive to the further use and in-depth research of cytotoxic T cells.

[0076] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0077] Example 1 Screening of cytotoxic T cell-specific surface molecules

[0078] The results of single-cell RNA sequencing of human peripheral blood T cells were analyzed, and the differentially expressed genes highly expressed in CD8_Cytotoxic and CD4_Cytotoxic T cells were screened according to the expression change ratio (avg_log2FC), and the genes CX3CR1 and ADGRG1 were screened. Figure 1 As shown, the expression of T cell CX3CR1 and ADGRG1 (the encoding gene of GPR56) is highly correlated with the expression of GZMB and perforin PRF1. The redder the color, the higher the gene expression level.

[0079] Example 2 Cytotoxicity detection of CX3CR1+GPR56+T cells

[0080] To further verify whether CX3CR1 and GPR56 are specific indicators of cytotoxic T cells, human peripheral blood T cells were stimulated and blocked with PMA, ionomycin and BFA, and then the surface proteins CX3CR1 and GPR56 and the intracellular cytokines granzyme B and perforin were stained and evaluated by flow cytometry.

[0081] like Figure 2 As shown, CX3CR1+GPR56+T cells in human peripheral blood highly expressed the cytotoxic molecules granzyme B and perforin, indicating that they are cytotoxic T cells.

[0082] Example 3 Identification of mouse cytotoxic T cells

[0083] Mouse peripheral blood T cells were stimulated and blocked with PMA, ionomycin, and BFA, and then stained for surface protein CX3CR1 and intracellular cytokines granzyme B and perforin, and evaluated by flow cytometry. Figure 3 As shown, CX3CR1+ T cells expressed higher levels of the cytotoxic molecules granzyme B and perforin compared to CX3CR1- T cells.

[0084] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A device for detecting cytotoxic T cells, characterized in that The device comprises: 1) an input module, the input module being used to receive T cells to be detected; 2) a detection module, wherein the detection module is used to detect a reagent for detecting the T cell surface marker protein, wherein the marker protein is CX3CR1, or CX3CR1 and GPR56; 3) An output module, which is used to output the detection results.

2. The device according to claim 1, characterized in that If the detection module detects that the T cell is CX3CR1-positive, or CX3CR1- and GPR56-positive T cell, the output module outputs: the T cell is a cytotoxic T cell.

3. A method for identifying cytotoxic T cells, characterized in that: The method comprises the steps of: (1) Providing T cells to be tested; (2) detecting the expression of the T cell surface marker protein to identify whether the T cell is a cytotoxic T cell; Wherein, the marker protein is CX3CR1, or CX3CR1 and GPR56.

4. The method according to claim 3, characterized in that If the T cells are detected to be CX3CR1-positive, or CX3CR1- and GPR56-positive T cells, the T cells are cytotoxic T cells.

5. The method according to claim 3, characterized in that The T cells to be detected are derived from humans or non-human mammals.

6. The method according to claim 3, characterized in that The T cells to be detected are derived from human peripheral blood mononuclear cells.

7. The method according to claim 3, characterized in that The detection is flow cytometry detection.

8. The method according to claim 3, characterized in that The method does not include cell fixation and / or cell permeabilization steps.

9. A method for enriching cytotoxic T cells, characterized in that: Includes steps: (1) Providing a cell population to be enriched; (2) Screening the cell population for cells that highly express the cell surface marker protein CX3CR1, or CX3CR1 and GPR56, thereby enriching cytotoxic T cells.

10. The method according to claim 9, characterized in that The cell population to be enriched is a human peripheral blood mononuclear cell population.

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