KR20240083054A - The Method of Analyzing Antigen-Specific Polyfunctional T Lymphocytes - Google Patents

Abstract

The method for analyzing antigen-specific multifunctional T lymphocytes according to the present invention relates to a method of measuring cellular immune response to a specific antigen using antigen-specific multifunctional T lymphocytes, or a method of providing information to determine whether acquired immunity to a specific antigen has been acquired. As a result, it is possible to more accurately determine whether acquired immunity to a specific antigen has been acquired, so it can be used effectively in the treatment of diseases including infectious diseases and in the drug development process, and can also be used as a material for medicines, etc.

Description

{The Method of Analyzing Antigen-Specific Polyfunctional T Lymphocytes}

The present invention relates to a method for analyzing antigen-specific multifunctional T lymphocytes, and specifically, a method for measuring cellular immune responses to a specific antigen using antigen-specific multifunctional T lymphocytes, or information for determining whether acquired immunity to a specific antigen has been acquired. It's about how to provide it.

The human body's immune system is divided into innate immunity and acquired immunity, and acquired immunity is further divided into humoral immunity by B lymphocytes and cellular immunity by T lymphocytes. Acquired immunity is an immune system that acts against external pathogens that have already been experienced and remembered. Compared to innate immunity, acquired immunity is known to respond to external pathogens faster, stronger, and more accurately, allowing more efficient removal of pathogens from the body. In addition, in acquired immunity, cellular immunity by T lymphocytes refers to antigen-specific immunity by T lymphocytes, which means that T lymphocytes are directly involved in the immune response.

Currently, it is known that acquisition of immunity to pathogenic infectious agents such as viruses or cancer cells is mainly measured by measuring humoral immune responses such as the ability to form neutralizing antibodies or the amount of neutralizing antibodies.

However, concerns have been raised that neutralizing antibodies will not play a significant role in evaluating vaccine efficacy against viral mutations, and in order to accurately measure vaccination or infection history through a vaccine against a specific antigen, it is better to use humoral immunity related to antibodies than humoral immunity related to antibodies. It is desirable to measure the acquisition of acquired immunity by measuring cellular immunity by T lymphocytes specific to infectious agents, but research on this is lacking.

Republic of Korea Patent No. 10-2405649

The purpose of the present invention is to provide a method for measuring cellular immune response to a specific antigen using T lymphocytes, which includes the step of detecting antigen-specific multifunctional T lymphocytes in a sample stimulated with an antigen.

Another object of the present invention is to provide a method of providing information for confirming whether acquired immunity to a specific antigen has been acquired, which includes the step of detecting antigen-specific multifunctional T lymphocytes in a sample stimulated with an antigen.

This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Additionally, the scope of the present application cannot be considered limited by the specific description described below.

In one aspect for achieving the above object, the present invention provides a method for measuring cellular immune response to a specific antigen using T lymphocytes, including the step of detecting antigen-specific multifunctional T lymphocytes in a sample stimulated with an antigen.

In the present invention, the method for measuring the cellular immune response to a specific antigen may be to obtain a sample stimulated with the antigen by contacting a separated sample with an antigen-derived peptide and culturing the sample.

In the present invention, the method for measuring the cellular immune response to a specific antigen may be to determine whether immunity to the antigen has been acquired by detecting antigen-specific multifunctional T lymphocytes in the sample.

In the present invention, the antigen-specific multifunctional T lymphocyte may be a memory T lymphocyte, preferably a late memory T lymphocyte, but is not limited thereto.

In the present invention, the term 'T lymphocyte (T cell, T lymphocyte)' is one of the lymphocytes that control acquired immunity specific to a specific antigen, and matures in the thymus. Depending on its function, T lymphocytes are called naïve T lymphocytes, effector T lymphocytes, etc. They are classified into lymphocytes and memory T lymphocytes, and memory T lymphocytes are further classified into early, intermediate, and late T lymphocytes depending on the time of differentiation, and each produces various cytokines and chemokines depending on the time of differentiation or external and internal stimuli. and/or express cell surface markers.

In the present invention, antigen-specific multifunctional T lymphocytes mean secreting two or more cytokines, but are not limited thereto.

In the present invention, the term 'antigen-specific multifunctional T lymphocyte' refers to a T lymphocyte that specifically reacts to a specific antigen and has the ability to perform various functions, such as various cytokines, chemokines, cell surface markers, or cytotoxic granules. refers to T lymphocytes that secrete.

In the present invention, the term 'cytokine' refers to a type of protein secreted by cells, which plays an important role in cell signaling and is secreted by immune cells. Cytokines are secreted from cells and can affect other cells or the secreting cell itself, and types include interleukins (IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, etc. ), CSF (GM-CSF, G-CSF, etc.), tumor necrosis factor (TNFα, β), interferon (IFNα, β, γ), MIP1a (Macrophage inflammatory protein 1a), or MIP1b.

In the present invention, the term 'chemokine' refers to a highly conserved small cytokine secreted by white blood cells as well as other cells that induce directional movement, and types include CCL2, CCL1, CCL22, or CCL17.

In the present invention, the term 'cell surface marker' is a special protein or sugar expressed on the cell surface, and is often used as a marker for a specific cell type, including CD3, CD4, CD8, CD25, CD27, and CD45RA. or CD45RO.

In the present invention, antigen-specific multifunctional T lymphocytes mean secreting two or more cytokines, but are not limited thereto.

In the present invention, antigen-specific multifunctional T lymphocytes contain interleukin (IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, etc.), CSF (GM-CSF, G-CSF, etc.) ), tumor necrosis factor (TNFα, β), interferon (IFNα, β, γ), MIP1a or MIP1b, and may secrete two or more cytokines, preferably consisting of IL-2, IFNγ, TNFα and MIP1b. It may secrete two or more cytokines selected from the group, but is not limited thereto.

In the present invention, the sample may be human or animal blood, tears, sweat, saliva, nasopharyngeal fluid, urine or feces, or may be separated from human or animal blood, tears, sweat, urine or feces, Specifically, it may be isolated from human blood, but is not limited thereto.

In the present invention, the sample may be a cell, specifically peripheral blood mononuclear cell (PBMC), but is not limited thereto.

In the present invention, the antigen may be an environmental allergen such as bacteria, virus, fungus, cancer cells, food, or pollen, an autoantigen, or an antigen derived from a vaccine, preferably a virus, but is not limited thereto.

In the present invention, the virus may be Adenovirus, Coronavirus, Norovirus, Epstein-Barr virus, Retrovirus, or Herpesvirus, etc. Preferably, it may be a coronavirus, and more preferably, it may be SARS-CoV2, but is not limited thereto.

In the present invention, the term 'coronavirus' is a general term for RNA viruses belonging to the Coronavirinae subfamily of the Coronaviridae family , and is a virus that causes respiratory and digestive system infections in animals, including humans, and causes infections in humans. It is known to be the cause of a significant portion of common colds, and in particular, several strains of human coronavirus are known, specifically SARS-CoV, SARS-CoV2, HCoV-229E, HCoV-OC43, HCoV-NL63, MERS-CoV, etc. There is.

In the present invention, the term 'SARS-CoV2 (Severe acute respiratory syndrome coronavirus 2)' is a type of coronavirus and has strong genetic similarity to the Bat SARS-like coronavirus. It is believed to have originated from this, and refers to the virus known to be the cause of the coronavirus infection-19 epidemic that occurred in Wuhan, China in 2019 and developed into an international public health emergency.

In the present invention, the term 'autoantigen' refers to a tissue component that exerts immunogenicity within one's own body and stimulates the production of autoantibodies within one's own tissue.

In the present invention, the step of measuring the antigen-specific multifunctional T lymphocytes may be analysis using Cytometry by time of flight (CyTOF) or flow cytometry, preferably analysis using CyTOF, but is not limited thereto. no.

In the present invention, when detecting antigen-specific multifunctional T lymphocytes using CyTOF, there is an advantage in that immunological expression can be measured at a lower frequency than when using a flow cytometer.

In the present invention, the term 'flow cytometry' refers to a laser-based electrical detection technology used in protein engineering to count or classify cells and detect biomarkers by stopping or sending flowing cells. It means technology.

In the present invention, the term 'mass cytometry' refers to a technology that determines cell characteristics by counting cells or detecting biomarkers by analyzing the mass of rare elements labeled on antibodies, and CyTOF is representative. The CyTOF is an application device for mass cytometry used to quantify targets displayed on the surface and inside of single cells, and CyTOF can quantify multiple cellular components simultaneously.

According to the method for determining whether acquired immunity to a specific antigen has been acquired according to the present invention, whether acquired immunity to a specific antigen can be measured more accurately than the method of measuring acquisition of acquired immunity to a specific antigen.

In the present invention, the term 'acquired immunity' refers to immunity obtained by already having a disease due to infection with viruses, pathogens, etc. or the production of cancer cells, etc., or through vaccination, etc., and reacts to external pathogens faster, stronger, and more accurately than innate immunity. This means that it is known to be able to remove pathogens from the body more efficiently.

In the present invention, the term 'acquired immunity' may be acquired through contact with an antigen included in a bacterium, virus, fungus, cancer cell, food, pollen, autoantigen, or vaccine, but is not limited thereto.

In the present invention, the term 'comprising' is intended to designate the presence of steps, components, operations, numbers, or a combination thereof described in the specification, but one or more other steps, components, actions, numbers, or these. It should be understood that it does not exclude in advance the existence or addition possibility of combinations of.

In another aspect for achieving the above object, the present invention provides a method of providing information for confirming whether acquired immunity to a specific antigen has been acquired, including the step of detecting antigen-specific multifunctional T lymphocytes in a sample stimulated with an antigen.

In the present invention, a method of providing information to confirm whether acquired immunity to a specific antigen has been acquired may be to obtain a sample stimulated with the antigen by contacting a separated sample with an antigen-derived peptide and culturing the sample.

In the present invention, a method of providing information for confirming whether acquired immunity to a specific antigen has been acquired may be to confirm whether immunity to the antigen has been acquired by detecting antigen-specific multifunctional T lymphocytes in a sample.

In the present invention, antigen-specific multifunctional T lymphocytes contain interleukin (IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, etc.), CSF (GM-CSF, G-CSF, etc.) ), tumor necrosis factor (TNFα, β), interferon (IFNα, β, γ), MIP1a or MIP1b, and may secrete two or more cytokines, preferably consisting of IL-2, IFNγ, TNFα and MIP1b. It may secrete two or more cytokines selected from the group, but is not limited thereto.

In the present invention, the sample may be human or animal blood, tears, sweat, saliva, nasopharyngeal fluid, urine or feces, or may be separated from human or animal blood, tears, sweat, urine or feces, Specifically, it may be isolated from human blood, but is not limited thereto.

In the present invention, the sample may be a cell, specifically peripheral blood mononuclear cells, but is not limited thereto.

In the present invention, the antigen may be an environmental allergen such as bacteria, virus, fungus, cancer cells, food, or pollen, an autoantigen, or an antigen derived from a vaccine, preferably a virus, but is not limited thereto.

In the present invention, the virus may be adenovirus, coronavirus, norovirus, Epstein-Barr virus, retrovirus, or herpes virus, preferably a coronavirus, and more preferably SARS-CoV2. It is not limited.

In the present invention, the step of detecting antigen-specific multifunctional T lymphocytes may be measured using CyTOF or flow cytometry, preferably using CyTOF, but is not limited thereto.

In the present invention, when detecting antigen-specific multifunctional T lymphocytes using CyTOF, there is an advantage in that immunological expression can be measured at a lower frequency than when using a flow cytometer.

The above ‘antigen’, ‘antigen-specific multifunctional T lymphocyte’, ‘cytokine’, ‘chemokine’, ‘cell surface marker’, ‘sample’, ‘antigen’, ‘virus’, ‘flow cytometry’, ‘mass cytometry’ ', 'acquired immunity', 'acquired immunity', 'coronavirus' and 'including' are as described above.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention should not be construed as limited by these examples.

A method for confirming whether acquired immunity to a specific antigen has been acquired, comprising the steps of contacting an antigen with a sample according to the present invention and analyzing antigen-specific multifunctional T lymphocytes contained in the sample contacted with the antigen. It can more accurately determine whether acquired immunity has been acquired, so it can be used effectively in the treatment of diseases including infectious diseases and in the drug development process, and can also be used as a material for medicines, etc.

Figure 1 is a schematic diagram schematically showing the method of analyzing antigen-specific multifunctional T lymphocytes of the present invention.
Figure 2 shows the results of CyTOF analysis of the expression of CD45RO, CD27, IL-2, IFNγ, TNFα, and MIP1b in antigen-specific multifunctional T lymphocytes.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1. Antigen-specific multifunctional T lymphocyte analysis

According to the schematic method shown in Figure 1, antigen-specific multifunctional T lymphocytes were analyzed. Samples of human peripheral blood mononuclear cells (huPBMC) were collected from healthy donors previously exposed to the SARS-CoV2 virus and cryopreserved in liquid nitrogen. Afterwards, liquid nitrogen was removed from the cryopreserved sample and it was quickly thawed in a water bath at 37°C. Afterwards, the sample was washed with RPMI and incubated at room temperature for 20 minutes in RPMI/10% FBS medium supplemented with 1 Cultured cells were incubated with PMA/ionomycin (positive control, Sigma Aldrich, #P1585, #I9657), Pan-SARS-CoV2 peptide mixture (PM-Pan-SARS2select01- 1, JPT company), SARS-CoV2 BA1.1/Omicron peptide mixture (PM-SARS2-SMUT08-1, JPT Company), or SARS-CoV2 Omicron variant (PepTivator ^® SARS-CoV2 Prot_S B.1.1.529/BA .2 WT Reference Pool, manufactured by Mitenyi, etc. were stimulated. The mutation information of the SARS-CoV2 Omicron variant compared to the SARS-CoV2 spike Wuhan wild type is shown in Table 1 below. Thereafter, cells stimulated with each antigen were washed with 1% BSA (Thermo Fisher Scientific) and PBS without calcium or magnesium (Gibco, Thermo Fisher Scientific). Then, to stain antigens on the cell surface, the cells were stained with 50 μL PBS containing an antibody mixture (Standard Biotools, Maxpar® Human T-Cell Phenotyping Panel Kit, 16 Marker-25 Tests, Product Number 201305) and 1% BSA. The stained cells were washed with PBS and 1% BSA and then fixed with 1.6% paraformaldehyde (Electron Microscopy Sciences).

SARS-COV-2 spike protein Wuhan wild type Mutations in the B.1.1.529 lineage Sequence position Amino acid BA.1 BA.2 19 T19 - T19I 24 L24 - L24deletion 25 P25 - P25deletion 26 P26 - P26deletion 27 A27 - A27S 67 A67 A67V - 69 H69 H69deletion 70 V70 V70deletion - 95 T95 T95I - 142 G142 G142D G142D 143 V143 V143deletion - 144 Y144 Y144deletion - 145 Y145 Y145deletion - 211 N211 N211deletion - 212 L212 L212I - 213 V213 - V213G 214 - Insertion214EPE - 339 G339 G339D G339D 371 S371 S371L S371L 373 S373 S373P S373P 375 S375 S375F S375F 376 T376 - T376A 405 D405 - D405N 408 R408 - R408S 417 K417 K417N K417N 440 N440 N440K N440K 446 G446 G446S - 477 S477 S477N S477N 478 T478 T478K T478K 484 E484 E484A E484A 493 Q493 Q493R Q493R 496 G496 G496S - 498 Q498 Q498R Q498R 501 N501 N501Y N501Y 505 Y505 Y505H Y505H 547 T547 T547K - 614 D614 D614G D614G 655 H655 H655Y H655Y 679 N679 N679K N679K 681 P681 P681H P681H 764 N764 N764K N764K 796 D796 D796Y D796Y 856 N856 N856K - 954 Q954 Q954H Q954H 969 N969 N969K N969K 981 L981 L981F -

Afterwards, the cells were washed once with PBS, then resuspended in cold methanol (ThermoFisher Scientific) to increase cell permeability and permeabilize the cells. The permeabilized cells were cultured at -20°C overnight, and then intracellularly To stain the antigen, staining was performed with 50 μL PBS containing the antibody cocktail and 1% BSA. Afterwards, the cells were washed with PBS and 1% BSA, then washed once more with PBS, and stained with 1.6% paraformaldehyde using an iridium DNA intercalator (Standard BioTools) at room temperature for 20 minutes. Afterwards, the cells were washed with PBS and diH2O, resuspended in 1Х EQ Four Element Calibration Beads (Fluidigm), and collected in a Helios mass cytometer (Standard BioTools), a type of CyTOF. Afterwards, further analysis and data visualization were performed with Cytobank.

As a result, as shown in Figure 2, when stimulated with PMA/ionomycin (positive control), IL-2, IFNγ, TNFα, and MIP1b were strongly produced, but when stimulated with SARS-CoV2 peptide, only one As observed in clusters, IFNγ, TNFα, and MIP1b were produced. In other words, it was confirmed that SARS-CoV2-specific T lymphocytes belong to the late memory T lymphocyte subgroup that produces IFNγ, TNFα, and MIP1b. In addition, it was confirmed that the functional activation of antigen-specific T lymphocytes can be evaluated using CyTOF.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

Claims (16)

A method of measuring cellular immune response to a specific antigen using T lymphocytes, comprising the step of detecting antigen-specific multifunctional T lymphocytes in a sample stimulated with an antigen. According to paragraph 1,
A method of measuring cellular immune response to a specific antigen using T lymphocytes, wherein a sample stimulated with an antigen is obtained by contacting and culturing a separated sample with an antigen-derived peptide. According to paragraph 1,
The method is to determine whether immunity to an antigen has been acquired by detecting antigen-specific multifunctional T lymphocytes in a sample. According to paragraph 1,
A method of measuring cellular immune response to a specific antigen using T lymphocytes, wherein the antigen-specific multifunctional T lymphocytes secrete two or more cytokines selected from the group consisting of IL-2, IFNγ, TNFα, and MIP1b. According to paragraph 1,
A method of measuring cellular immune response to a specific antigen using T lymphocytes, wherein the sample is blood or peripheral blood mononuclear cells isolated from blood. According to paragraph 1,
A method of measuring cellular immune response to a specific antigen using T lymphocytes, wherein the antigen is derived from one or more selected from the group consisting of bacteria, viruses, fungi, cancer cells, food, pollen, autoantigens, and vaccines. According to clause 6,
A method of measuring cellular immune response to a specific antigen using T lymphocytes, wherein the virus is a coronavirus. According to paragraph 1,
A method of measuring cellular immune response to a specific antigen using T lymphocytes, wherein the step of detecting the antigen-specific multifunctional T lymphocytes is measured using CyTOF or flow cytometry. A method of providing information for confirming whether acquired immunity to a specific antigen has been acquired, comprising the step of detecting antigen-specific multifunctional T lymphocytes in a sample stimulated with an antigen. According to clause 9,
A method of providing information to confirm whether acquired immunity to a specific antigen is obtained by obtaining a sample stimulated with an antigen by contacting and culturing a separated sample with an antigen-derived peptide. According to clause 9,
The method is a method of providing information for confirming whether acquired immunity to a specific antigen has been acquired by detecting antigen-specific multifunctional T lymphocytes in the sample. According to clause 9,
In the step of detecting the antigen-specific multifunctional T lymphocytes,
When the antigen-specific multifunctional T lymphocyte secretes two or more cytokines selected from the group consisting of IL-2, IFNγ, TNFα, and MIP1b, it is confirmed that acquired immunity to the specific antigen has been acquired. How to provide information to determine whether acquired immunity has been acquired. According to clause 9,
A method of providing information for determining whether acquired immunity to a specific antigen is obtained, wherein the sample is blood or peripheral blood mononuclear cells isolated from blood. According to clause 9,
A method of providing information for determining whether acquired immunity to a specific antigen is obtained, wherein the antigen is derived from one or more selected from the group consisting of bacteria, viruses, fungi, cancer cells, food, pollen, autoantigens, and vaccines. . According to clause 14,
A method of providing information for determining whether acquired immunity to a specific antigen is obtained, wherein the virus is a coronavirus. According to clause 9,
A method of providing information for confirming whether acquired immunity to a specific antigen has been acquired, wherein the step of detecting the antigen-specific multifunctional T lymphocytes is analyzed by CyTOF or flow cytometry.