Rapid detection method for cytotoxic t cell in peripheral blood
By detecting the expression levels of T cell surface marker proteins CX3CR1 and GPR56, flow cytometry can be used to rapidly identify cytotoxic T cells, solving the problems of long processing time and cell death in existing technologies, and achieving rapid and low-cost detection of cytotoxic T cells and acquisition of viable cells.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- SHANGHAI MAAGI MEDICAL TECH CO LTD
- Filing Date
- 2025-10-23
- Publication Date
- 2026-05-07
AI Technical Summary
Existing technologies are time-consuming and cannot identify cytotoxic T cells while the cells are alive, resulting in low detection efficiency and cell death.
By detecting the expression levels of T cell surface marker proteins CX3CR1 and GPR56, cytotoxic T cells can be rapidly identified using flow cytometry, avoiding cell fixation and membrane perforation.
It enables rapid identification of cytotoxic T cells within 30 minutes, maintains cell viability, reduces detection costs, and provides viable cells for further research.
Smart Images
Figure PCTCN2025129583-FTAPPB-I100001 
Figure PCTCN2025129583-FTAPPB-I100002 
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Abstract
Description
A rapid detection method for cytotoxic T cells in peripheral blood Technical Field
[0001] This invention relates to the field of biomedicine. Specifically, this invention relates to a rapid detection method for cytotoxic T cells in peripheral blood. Background Technology
[0002] Cytotoxic T cells are the main force in anti-tumor immunity, and their detection is frequently required in the biological field. Current techniques identify cytotoxic T cells by detecting their ability to produce cytotoxic molecules such as granzyme B and perforin. The specific method is as follows: T cells are stimulated with T cell stimulants such as PMA and iomycin to promote the expression of cytotoxic molecules. Brefeldin A (BFA) is then added to inhibit protein transport, ensuring that the produced cytotoxic molecules remain within the cells. Subsequently, the cells are fixed and permeabilized. Fluorescent antibodies targeting cytotoxic molecules such as granzyme B and perforin are used for staining, and flow cytometry is used to detect the expression of these cytotoxic molecules.
[0003] However, the existing detection methods have the following disadvantages: (1) They are time-consuming: the entire detection process takes about 8 hours; (2) They require TCR stimulation, fixation and membrane rupture of T cells. After fixation and membrane rupture, 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 field to develop a method that is time-efficient and can identify cytotoxic T cells while the cells are still alive. Summary of the Invention
[0005] The purpose of this invention is to provide a method that is time-efficient and can identify cytotoxic T cells while the cells are still alive.
[0006] In a first aspect of the invention, a method for identifying cytotoxic T cells is provided, the method comprising the steps of:
[0007] (1) Provide the T cells to be tested;
[0008] (2) Detect the expression of the marker proteins on the surface of the T cells to identify whether the T cells are cytotoxic T cells;
[0009] 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, then the T cells are cytotoxic T cells.
[0011] In another preferred embodiment, CX3CR1 positivity means that the ratio (E1 / E0) of the expression level of CX3CR1 on the surface of T cells (E1) to the expression level of CX3CR1 on the surface of non-cytotoxic T cells 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 the non-cytotoxic T cells 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 a flow cytometry detection.
[0018] In another preferred embodiment, the flow cytometry detection includes the steps of:
[0019] (1) Flow cytometry antibodies CX3CR1 and GPR56 were used to stain the T cells to be tested;
[0020] (2) Wash the T cells to remove excess antibodies;
[0021] (3) Flow cytometry was used to detect cells expressing CX3CR1, CX3CR1 and GPR56, which are cytotoxic T cells.
[0022] In another preferred embodiment, the cytotoxic T cells are selected from the group consisting of: CD4+ + Cytotoxic T cells, CD8 + Cytotoxic T cells, or a combination thereof.
[0023] In another preferred embodiment, the method does not include cell fixation and / or cell permeabilization steps.
[0024] In another preferred embodiment, the method does not include the step of detecting intracellular proteins.
[0025] In a second aspect of the invention, a method for enriching cytotoxic T cells is provided, comprising the steps of:
[0026] (1) Provide the cell population to be enriched;
[0027] (2) Screen cells that highly express cell surface marker proteins CX3CR1 or CX3CR1 and GPR56 in the cell population to enrich cytotoxic T cells.
[0028] In another preferred embodiment, the cell population to be enriched is derived from human 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 includes the steps of:
[0032] (1) The cell population to be enriched was stained using flow cytometry antibodies against CX3CR1 and GPR56.
[0033] (2) Wash the T cells to remove excess antibodies;
[0034] (3) Cells expressing CX3CR1, CX3CR1 and GPR56 were sorted using flow cytometry and the cells were collected to obtain enriched cytotoxic T cells.
[0035] In another preferred embodiment, the cytotoxic T cells are selected from the group consisting of: 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 a third aspect of the invention, a cytotoxic T cell is provided, which is obtained by enrichment as described in the second aspect of the invention.
[0038] In another preferred embodiment, the cytotoxic T cells are enriched from human peripheral blood mononuclear cell populations.
[0039] In another preferred embodiment, the cytotoxic T cells are selected from the group consisting of: 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 invention, a pharmaceutical composition is provided comprising the cytotoxic T cells as described in claim 3 and a pharmaceutically acceptable carrier.
[0042] In a fifth aspect of the invention, the use of cytotoxic T cells as described in the third aspect of the invention or pharmaceutical compositions as described in the fourth aspect of the invention in the preparation of medicaments for treating diseases is provided.
[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 invention, a reagent combination for identifying or enriching cytotoxic T cells is provided, the reagent combination comprising reagents for detecting CX3CR1 and GPR56 proteins.
[0045] In another preferred embodiment, the reagent is an antibody, such as a flow cytometry antibody.
[0046] In a seventh aspect of the invention, an apparatus for detecting cytotoxic T cells is provided, characterized in that the apparatus comprises:
[0047] 1) Input module, the input module being used to receive T cells to be detected;
[0048] 2) Detection module, wherein the detection module is used to detect the reagent of the T cell surface marker protein, wherein the marker protein is CX3CR1, or CX3CR1 and GPR56;
[0049] 3) Output module, which is used to output the detection results.
[0050] In another preferred embodiment, if the detection module detects that the T cells are CX3CR1 positive, or CX3CR1 and GPR56 positive T cells, the output module outputs: the T cells are cytotoxic T cells.
[0051] In another preferred embodiment, CX3CR1 positivity means that the ratio (E1 / E0) of the expression level of CX3CR1 on the surface of T cells (E1) to the expression level of CX3CR1 on the surface of non-cytotoxic T cells 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 the non-cytotoxic T cells 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 a flow cytometry detection.
[0056] In another preferred embodiment, the detection module includes reagents for detecting CX3CR1, or CX3CR1 and GPR56.
[0057] In another preferred embodiment, the detection does not include cell fixation and / or cell permeabilization steps.
[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 this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description
[0060] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.
[0061] Figure 1 shows the correlation between CX3CR1 and ADGRG1 (GPR56 encoding genes) and the RNA expression of cytotoxic molecule-related genes granzyme B (GZMB) and perforin (PRF1).
[0062] Figure 2 shows the correlation between CX3CR1 and GPR56 and the expression of cytotoxic molecules granzyme B and perforin. (A) The heatmap of expression of CX3CR1, GPR56, granzyme B and perforin shows that the four are significantly co-expressed; (BC) Expression levels of cytotoxic molecules granzyme B and perforin in CX3CR1+GPR56+ T cells and CX3CR1-GRP56- T cells.
[0063] Figure 3 shows the correlation between CX3CR1 and the expression of cytotoxic molecules granzyme B and perforin in mice. Detailed Implementation
[0064] Through extensive and in-depth research, the inventors have developed, for the first time, a rapid detection method for cytotoxic T cells in peripheral blood. This invention identifies two cytotoxic T cell-specific cell surface proteins, CX3CR1 and GPR56. Based on this, the invention provides a method for rapidly identifying cytotoxic T cells in peripheral blood using only surface staining of CX3CR1 and GPR56. The method is rapid and does not require fixation or cell disruption, thus preserving cell viability. Based on this, the invention was completed.
[0065] the term
[0066] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.
[0067] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting 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 simultaneously measure multiple parameters from a single cell. This invention uses flow cytometry to detect the expression of the T cell surface proteins CX3CR1, or CX3CR1 and GPR56, thereby identifying cytotoxic T cells.
[0069] As used in this article, the term "cytotoxic T cell" refers to a T cell that expresses cytotoxic molecules such as granzyme B and 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 in this article, the term "GPR56" refers to Adhesion G Protein-Coupled Receptor G1, accession number: HGNC:4512 NCBI Gene:9289 Ensembl:ENSG00000205336 604110 UniProtKB / Swiss-Prot:Q9Y653.
[0072] The main advantages of this invention include:
[0073] (1) The method of the present invention significantly reduces 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 membrane-breaking reagents, thus reducing the detection cost;
[0075] (3) This method does not require fixation and membrane rupture treatment, and can obtain active cytotoxic T cells: the identified cytotoxic T cells can be sorted by flow cytometry or immunomagnetic beads, which is helpful for the further utilization and in-depth research of cytotoxic T cells.
[0076] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally 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 as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.
[0077] Example 1: Screening of cytotoxic T cell-specific surface molecules
[0078] Analysis of single-cell RNA sequencing results from human peripheral blood T cells was performed. Differentially expressed genes highly expressed in CD8_Cytotoxic and CD4_Cytotoxic T cells were screened based on fold change in expression (avg_log2FC), identifying CX3CR1 and ADGRG1 as genes. As shown in Figure 1, the expression of CX3CR1 and ADGRG1 (encoding genes of GPR56) in T cells was 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 blocked by stimulation with PMA, iomycin and BFA, and the surface proteins CX3CR1 and GPR56 and the intracellular cytokines granzyme B and perforin were stained and evaluated by flow cytometry.
[0081] As shown in Figure 2, CX3CR1+GPR56+ T cells in human peripheral blood highly express 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 blocked after stimulation with PMA, iomycin, and BFA. The surface protein CX3CR1 and intracellular cytokines granzyme B and perforin were stained, and the results were evaluated by flow cytometry. As shown in Figure 3, CX3CR1+ T cells expressed higher levels of the cytotoxic molecules granzyme B and perforin compared to CX3CR1-T cells.
[0084] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.
Claims
1. A method for identifying cytotoxic T cells, the method comprising the steps of: (1) Provide the T cells to be tested; (2) Detect the expression of the marker proteins on the surface of the T cells to identify whether the T cells are cytotoxic T cells; in, The marker protein is CX3CR1, or CX3CR1 and GPR56.
2. The method as described in claim 1, characterized in that, If the T cells are detected to be CX3CR1 positive, or CX3CR1 and GPR56 positive T cells, then the T cells are cytotoxic T cells.
3. The method as described in claim 2, characterized in that, The CX3CR1 positivity refers to the ratio (E1 / E0) of the expression level of CX3CR1 on the surface of T cells (E1) to the expression level of CX3CR1 on the surface of non-cytotoxic T cells (E0) being ≥1.5, preferably ≥1.7, and more preferably ≥2.
0.
4. The method as described in claim 2, characterized in that, The GPR56 positivity refers to the ratio (E1 / E0) of the expression level of GPR56 on the surface of T cells (E1) to the expression level of CX3CR1 on the surface of non-cytotoxic T cells (E0) being ≥1.5, preferably ≥1.7, and more preferably ≥2.
0.
5. The method as described in claim 1, characterized in that, The T cells to be detected are derived from humans or non-human mammals, such as humans, mice, rats, rabbits, and monkeys.
6. The method as described in claim 1, characterized in that, The T cells to be tested were derived from human peripheral blood mononuclear cells.
7. The method as described in claim 1, characterized in that, The aforementioned detection is an in vitro detection.
8. The method as described in claim 1, characterized in that, The aforementioned detection is non-diagnostic and non-therapeutic.
9. The method as described in claim 1, characterized in that, The cytotoxic T cells mentioned are selected from the following group: CD4 + Cytotoxic T cells, CD8 + Cytotoxic T cells, or a combination thereof.
10. The method as described in claim 1, characterized in that, The method does not include cell fixation and / or cell permeabilization steps.
11. The method as described in claim 1, characterized in that, The method does not include the step of detecting intracellular proteins.
12. A method for enriching cytotoxic T cells, comprising the steps of: (1) Provide the cell population to be enriched; (2) Screen cells that highly express cell surface marker proteins CX3CR1 or CX3CR1 and GPR56 in the cell population to enrich cytotoxic T cells.
13. The method as described in claim 12, characterized in that, The cell population to be enriched is derived from humans or non-human mammals, such as humans, mice, rats, rabbits, and monkeys.
14. The method as described in claim 12, characterized in that, The cell population to be enriched is human peripheral blood mononuclear cells.
15. The method as described in claim 12, characterized in that, The cytotoxic T cells mentioned are selected from the following group: CD4 + Cytotoxic T cells, CD8 + Cytotoxic T cells, or a combination thereof.
16. The method as described in claim 12, characterized in that, The enriched cytotoxic T cells are active.
17. A cytotoxic T cell, said cytotoxic T cell being enriched by the method of any one of claims 12-16.
18. A pharmaceutical composition comprising the cytotoxic T cells as described in claim 17 and a pharmaceutically acceptable carrier.
19. Use of the cytotoxic T cells of claim 17 or the pharmaceutical composition of claim 18 in the preparation of a medicament for treating a disease.
20. The use as described in claim 19, characterized in that, The diseases mentioned are tumors, autoimmune diseases, inflammatory diseases, or combinations thereof.
21. A reagent combination for identifying or enriching cytotoxic T cells, said reagent combination comprising reagents for detecting CX3CR1 and GPR56 proteins.
22. A device for detecting cytotoxic T cells, characterized in that, The device includes: 1) Input module, the input module being used to receive T cells to be detected; 2) Detection module, wherein the detection module is used to detect the reagent of the T cell surface marker protein, wherein the marker protein is CX3CR1, or CX3CR1 and GPR56; 3) Output module, which is used to output the detection results.
23. The device as claimed in claim 22, characterized in that, If the detection module detects that the T cells are CX3CR1 positive, or CX3CR1 and GPR56 positive T cells, the output module outputs: the T cells are cytotoxic T cells.
24. The device as claimed in claim 23, characterized in that, The CX3CR1 positivity refers to the ratio (E1 / E0) of the expression level of CX3CR1 on the surface of T cells (E1) to the expression level of CX3CR1 on the surface of non-cytotoxic T cells (E0) being ≥1.5, preferably ≥1.7, and more preferably ≥2.
0.
25. The device as claimed in claim 23, characterized in that, The GPR56 positivity refers to the ratio (E1 / E0) of the expression level of GPR56 on the surface of T cells (E1) to the expression level of CX3CR1 on the surface of non-cytotoxic T cells (E0) being ≥1.5, preferably ≥1.7, and more preferably ≥2.
0.
26. The device as claimed in claim 22, characterized in that, The T cells to be detected are derived from humans or non-human mammals, such as humans, mice, rats, rabbits, and monkeys.
27. The device as claimed in claim 22, characterized in that, The detection module includes reagents for detecting CX3CR1, or CX3CR1 and GPR56.