Novel CD8 < + > original T cell subgroup and application thereof

Abstract

The invention relates to a novel CD8 < + > original T cell subgroup and application thereof. The invention discloses a brand new separated cell population, namely an immune cell population which takes whether CCL5 is expressed or not as a marker, namely a CCL5 + CD8 + # imgabs0 # T cell population or a CCL5-CD8 + # imgabs1 # T cell population (# imgabs2 # (TSN) subpopulation). Wherein the CCL5 + CD8 + # imgabs3 # T cell population can be used as an analysis target and is used for analyzing functions of immune cells. The invention also provides a method for sorting the cells and the like. The invention provides a new way for application research and clinical regulation of immune cells.

Description

Technical Field

[0001] The present invention relates to the field of medical diagnosis; more specifically, the present invention relates to a CD8 + New primitive T cell subsets and their application in detecting human CD8 + Application of T cell immune function. Background Art

[0002] CD8 + T cells are a key component of the adaptive immune system and play an important role in defending against viral, bacterial and tumor cell infections. + After being activated by antigens, T cells proliferate and differentiate into cytotoxic effector cells. They kill target cells by releasing cytotoxic granules and inducing apoptosis of target cells, thus exerting antiviral and anti-tumor effects. + Primitive T cells are a subset of T cells that develop from the thymus without antigen stimulation. Most of them express αβ-type T cell receptors (TCRs) and differentiate into cytotoxic effector CD8 T cells after being activated by antigen stimulation. + T cells can be further subdivided according to the type of cytokines they secrete. For example, Tc1 cells that secrete large amounts of IFN-γ, TNF-α, etc. play an antiviral and anti-tumor role, Tc2 cells that secrete IL-4, IL-5, etc. participate in immune defense against parasitic infections, and Tc17 cells secrete cytokines such as IL-17, which play a role in resisting bacterial and fungal infections. Therefore, the ability of cells to activate, proliferate, differentiate, and secrete effector factors is an important factor in evaluating CD8 + An important indicator of T cell function.

[0003] TCR plays a key role in T cell activation and immune response. It activates T cells by recognizing antigen peptides presented on MHC molecules and initiates a series of signal transduction pathways to induce T cell activation, proliferation, differentiation and subsequent effects. The diversity of TCR is the basis of adaptive immune response. + Naive T cells have a wide range of TCR diversity and circulate in the periphery, waiting to be activated by antigens. During the aging process of the human body, due to the degeneration of hematopoiesis and thymus, peripheral blood CD8 + The decrease in naive T cells and the increase in the proportion of memory cells, as well as the reduction in T cell receptor polymorphism, lead to CD8 + The decline in T cell function is specifically manifested in the decline in the ability to recognize antigens, activate proliferation, and secrete cytokines. These changes will reduce the body's defense against new infections and tumors. Impairment of the body's immune surveillance function will accelerate the accumulation of senescent cells and further accelerate the aging process. Therefore, monitoring and maintaining the function of the immune system is of great significance to delaying aging. Summary of the invention

[0004] The present invention aims to provide a CD8 + A new subset of primitive T cells, its isolation method and its application. Its application includes CCL5 + CD8 + T cells and CCL5 - CD8 + T cells.

[0005] In a first aspect of the present invention, a cell population is provided for use as an analysis target and for preparing a reagent for analyzing immune cell function; wherein the cell population is selected from: an immune cell population expressing CCL5; or an immune cell population not expressing (including low expression) CCL5.

[0006] In one or more embodiments, the immune cells are CD8 + T cells.

[0007] In one or more embodiments, the immune cell population expressing CCL5 is CCL5 + CD8 + T cell populations.

[0008] In one or more embodiments, the immune cell population that does not express CCL5 is CCL5 - CD8 + T cell populations ( (T SN ) subgroup).

[0009] In one or more embodiments, the analyzing immune cell function comprises: analyzing CD8 + T cell function; if the test subject's CCL5 + CD8 + T cells account for the total CD8 + A significantly high proportion of T cells indicates that CD8 + T cell function was significantly low.

[0010] In one or more embodiments, the analysis includes prognosis, for CCL5 + CD8 + T cells account for the total CD8 + In subjects with a significantly high proportion of T cells, CCL5-non-expressing CD8 + T cell therapy options.

[0011] In one or more embodiments, the CCL5 + CD8 + T cells account for the total CD8 + The proportion of T cells is significantly high, which means that the proportion of total CD8 + The proportion of T cells is higher than 10% or higher; preferably higher than 12% or higher; more preferably higher than 15% or higher; more preferably higher than 18% or higher; and further preferably higher than 20% or higher.

[0012] In one or more embodiments, in subjects of relatively high age, the population of immune cells expressing CCL5 accounts for a proportion of the total CD8 + The proportion of T cells was significantly high.

[0013] In one or more embodiments, the relatively old subject is a human, aged above 50 years (e.g., 50-100 years), above 55 years, above 60 years, above 65 years, above 70 years, above 75 years, above 80 years, above 85 years, above 90 years, or above 95 years.

[0014] In one or more embodiments, a 50-year-old human is equivalent to a 10-month-old mouse.

[0015] In one or more embodiments, analyzing the immune cell function of the test subject includes: analyzing the anti-tumor ability of the test subject, analyzing the anti-pathogen (including virus) ability of the test subject, or analyzing the diversity of the T cell receptor (TCR) of the test subject.

[0016] In one or more embodiments, the immune cell population expressing CCL5 accounts for the total CD8 + A significantly high proportion of T cells indicates that the anti-tumor ability of the test subject is relatively low (significantly lower than that of ordinary patients, or significantly lower than that of patients who do not present "CCL5 + CD8 + T cells account for the total CD8 + T cells were significantly high in the patients).

[0017] In one or more embodiments, the immune cell population expressing CCL5 accounts for the total CD8 + A significantly high proportion of T cells indicates that the test subject's ability to resist pathogens is relatively low (significantly lower than that of ordinary patients, or significantly lower than that of patients who do not present "CCL5 + CD8 + T cells account for the total CD8 + T cells were significantly high in the patients).

[0018] In one or more embodiments, the immune cell population expressing CCL5 accounts for the total CD8 + A significantly high proportion of T cells indicates that the diversity of the T cell receptor (TCR) of the test subject is relatively low (significantly lower than that of ordinary patients, or significantly lower than that of patients who do not present "CCL5 + CD8 + T cells account for the total CD8 + T cells were significantly high in the patients).

[0019] In one or more embodiments, the CCL5 + CD8 + T cell populations are activated by specific binding or capture of CCL5 + and CD8 + Binding molecules (such as antibodies) or capture molecules from T cells ( T cells) are sorted in samples; the CCL5-CD8 + T cell populations ( (T SN ) subset) is CD8 + Subtraction (reduction) of CCL5 in T cell population + CD8 + T cells are obtained after the cell population.

[0020] In one or more embodiments, the T cell-containing sample is peripheral blood; more preferably, the CCL5 + CD8 + T cell population or CCL5-CD8 + T cell populations are sorted and / or the ratio is determined; more preferably, methods for sorting cells include (but are not limited to): flow cytometry sorting, immunomagnetic bead sorting, microfluidic cell sorting, and adhesion method.

[0021] In another aspect of the present invention, a method for isolating or enriching a cell population is provided, wherein the cell population is CCL5 + CD8 + T cell population or CCL5-CD8 + The method comprises: using a binding molecule (such as an antibody) or a capture molecule that specifically binds to or captures CCL5 to express negative and positive CD8 + T cells are sorted and / or the ratio is determined; preferably, the method of sorting cells includes (but is not limited to): flow cytometry sorting, immunomagnetic bead sorting, microfluidic cell sorting, and adhesion method.

[0022] In another aspect of the present invention, a separated cell population is provided; the cell population is a CD8 + T immune cell population; preferably, the immune cell population expressing CCL5 is CCL5 + CD8 + T cell population, the immune cell population not expressing CCL5 is CCL5 - CD8 + T cell populations.

[0023] In one or more embodiments, the method of isolating or enriching cells is a method that is not directly aimed at disease diagnosis or treatment (non-diagnostic or non-therapeutic).

[0024] In another aspect of the present invention, a method for specifically detecting and separating (sorting) CCL5 + The use of a reagent for the preparation of a reagent for analyzing the function of immune cells; preferably, the specific detection and separation (sorting) of CCL5 + Cell-specific reagents include: + Binding molecules (such as antibodies) or capture molecules (including antibodies that define surface markers of the cells or antibodies conjugated to fluorescence).

[0025] In another aspect of the present invention, there is provided an immune cell population (CCL5 - CD8 + T cell population, or (T SN ) subset) in the preparation of a pharmaceutical composition for improving immune cell function (enhancing immunity) and / or inhibiting tumors; preferably, the immune cell population that does not express CCL5 is deposited in the China Center for Type Culture Collection with the accession number CCTCC NO: C2024243.

[0026] In another aspect of the present invention, a method for screening a substance (potential substance) for improving immune cell function (enhancing immunity) is provided, the method comprising: (1) treating separated CD8 + T cell population (e.g., isolated from peripheral blood cells); and (2) detecting the CD8 + CCL5 in T cell population + CD8 + The proportion of T cell population, if CCL5 + CD8 + The proportion of T cell populations was statistically reduced (e.g., CCL5 + CD8 + If the amount of the T cell population is reduced by more than 5%, more than 10%, more than 20%, more than 50%, more than 80%, etc.), the candidate substance is a substance (potential substance) that improves the function of immune cells (enhances immunity).

[0027] In one or more embodiments, step (1) comprises: in the test group, adding the candidate substance to the isolated CD8 + T cell population; step (2) comprises: detecting the CD8 + CCL5 in T cell population + CD8 + and compared with a control group, wherein the control group is a CD8 T cell population without adding the candidate substance. + T cell population; if CCL5 + CD8 + If the proportion of the T cell population is statistically reduced, the candidate substance is a substance that improves the function of immune cells (enhances immunity).

[0028] In one or more embodiments, the improving the function of immune cells includes: improving the ability to resist infection by pathogens (including viruses).

[0029] In one or more embodiments, the improving the function of immune cells includes: improving anti-tumor ability.

[0030] In one or more embodiments, the method further comprises: conducting further cell experiments and / or animal experiments on the obtained potential substances to further select and determine substances useful for improving immune cell function (enhancing immunity) from the candidate substances.

[0031] In one or more embodiments, the candidate substance includes a small molecule compound or a biological molecule designed for T cells.

[0032] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 .CCL5 marks CD8 + New subsets of naive T cells;

[0034] A.CD8 + Heterogeneity of naive T cell subsets in young and old individuals, highlighting the dominance of novel subsets in older individuals;

[0035] B.CD8 + Characteristic gene expression analysis of a new subset of naive T cells, characterized by high expression of CCL5;

[0036] C.CD8 + Gene pathway enrichment analysis of the new primitive T cell subsets suggests that this subset plays a negative regulatory role in immune effects;

[0037] D.CD8 + The new subset of naive T cells is closely related to cell state aging;

[0038] E.CD8 + The proportion of new primitive T cell subsets increases as people age and becomes dominant.

[0039] Figure 2 .CCL5-labeled CD8 + The new subset of naive T cells is determined by CD8 + T cell diversity subsets;

[0040] A. Combined analysis of TCR sequencing and single-cell sequencing showed that the reduction of TCR diversity was concentrated in CD8 + T cells;

[0041] B. CD8 characterized by CCL5 expression + The new subset of naive T cells is the decisive subset in the reduction of TCR diversity;

[0042] C. CD8 in different age groups + The TCR diversity of new subsets of naive T cells gradually decreases.

[0043] Figure 3 .CD8 + New primitive T cell subsets indicate CD8 + T cell immune efficacy;

[0044] A. Detection of mouse CD8 after flow cytometry sorting + Gene pathways are enriched in the new subset of naive T cells, with high expression of cellular pathways related to immune effects and cell proliferation;

[0045] B. In vitro experiments were performed to detect the difference in differentiation efficiency between the sorted new subpopulation and the control cells into Tc1 cells. The upper figure shows the new subpopulation and the lower figure shows the control.

[0046] C. In vitro experiments detected the difference in proliferation ability between the new subpopulations after sorting and the control cells. Green represents the new subpopulation and gray represents the control cells.

[0047] Figure 4 .CD8 + The role of new naive T cell subsets in viral infection;

[0048] A. Detection of CD8 in patients infected with SARS-CoV-2 + Differences in the proportion of new subsets of naive T cells, CD8 + The new subset of primitive T cells was significantly higher than that of patients with mild symptoms;

[0049] B. For samples from the mild and severe groups of the new coronavirus, CCL5 was detected by flow cytometry + CD8 + T cell ratio.

[0050] C. Use a high proportion of CD8 + When mice with the new subset of naive T cells were compared with a control group and subjected to influenza virus infection experiments, a high proportion of mice developed severe disease symptoms and experienced dramatic weight loss after infection;

[0051] D. Measurement of CD8 in mice infected with influenza virus + T cell effector capacity, high proportion of CD8 + New subsets of naive T cells emerge during infection in mice CD8 + The effector capacity of T cells to secrete cytokines was significantly weaker than that of normal mice at a low rate.

[0052] Figure 5 .CCL5 + CD8 + ROC curve and test parameters corresponding to low antiviral ability of T cell population.

[0053] Figure 6 .CCL5 in peripheral blood + CD8 + T cells and their proportions; 10mo means 10 months of age.

[0054] Figure 7 .CD8 + The role of new naive T cell subsets in fighting tumors;

[0055] A. Comparison of tumor size at the end point of the model after mice were inoculated with B16 melanoma and treated with cells or without treatment;

[0056] B. The melanoma growth curve obtained by measuring and recording the tumor growth of mice with a vernier caliper every three days from the beginning of modeling. The arrow indicates the time when cell therapy started;

[0057] C. The weight of melanoma was measured after tumor removal at the end point of modeling. DETAILED DESCRIPTION

[0058] The inventors are committed to the study of immune cell function. After in-depth research, they have revealed a new isolated cell population, which is defined by whether it expresses CCL5, preferably CCL5 - CD8 + T cell populations ( (T SN ) subpopulation) and CCL5 + CD8 + T cell population. CCL5 + CD8 + T cells can be used as analysis targets and for analyzing immune cell functions. The present invention also provides methods for sorting the cells, etc. The present invention provides a new approach for the study and clinical regulation of immune cells.

[0059] the term

[0060] As used herein, "isolated" means that a substance is separated from its original environment (e.g., a natural organism or natural environment). For example, a living cell is not isolated and purified when it exists in the body, but the same cell is isolated or purified when it is separated from other substances present in the natural state / original environment.

[0061] As used in the present invention, "enriched" cells (groups) refer to increasing the content of desired target cells / target cells contained in a mixed cell population, generally referring to increasing the frequency of "target cells" in total cells. Therefore, an enriched cell population refers to a cell population with a higher frequency of target target cells produced by an enrichment step.

[0062] As used in the present invention, unless otherwise specified, the "target cell" is a cell of interest in the present invention that has the intended function; more specifically, the "target cell" refers to a CD8 T cell defined by whether it expresses CCL5. + T cell populations.

[0063] As used in the present invention, the "CCL5+ " or "CCL5 positive" means that cells express CCL5 and can be bound or captured by their specific binding molecules (such as antibodies) or capture molecules, thereby separating and enriching the positive cells (populations).

[0064] As used in the present invention, "cell population" includes pre-enrichment and post-enrichment cell populations. After the enrichment step, the cell population can be enriched. Each cell population can be used directly in the next step, can be partially or completely frozen for long-term or short-term storage and used in subsequent steps. At the same time, the cells of the cell population can be suspended individually to obtain a single cell suspension.

[0065] As used in the present invention, "marker" and "marker" can be used interchangeably. In the present invention, unless otherwise specified, the "marker" is a molecule of interest (such as a protein surface molecule) used to determine a specific cell (group) or its characteristics. For example, in the present invention, CCL5 is used as a marker.

[0066] New cells (populations) and their isolation

[0067] Based on the novel findings of the present inventors, an isolated cell (group) or cell culture is provided, wherein the function of the cell is an immune cell group defined by the expression of CCL5.

[0068] The CCL5 is a protein. The amino acid sequence of the human CCL5 protein can be found in Gene ID: 6352 in NCBI; the amino acid sequence of the mouse CCL5 protein can be found in Gene ID: 20304 in NCBI.

[0069] In a preferred embodiment of the present invention, the cells of the present invention are separated from peripheral blood cells, and may also be passaged cells.

[0070] According to the new discovery of the present invention, a method for isolating or enriching cells is also provided, wherein the cells are an immune cell population defined by the expression of CCL5, preferably CCL5 + CD8 + T cell populations and CCL5 - CD8 + T cell populations.

[0071] Methods for separating or enriching cells with specific identifiable signals (such as CCL5) are known to those skilled in the art, and a variety of methods for separating or enriching cells can be used in the present invention. In the embodiments of the present invention, specific cell sorting methods are provided, but it should be understood that after the cell surface molecules of interest are known in the present invention, cell sorting is not limited to those specifically listed in the embodiments, and other methods are also available. Some commonly used cell sorting methods in the art include but are not limited to the following:

[0072] (a) Flow cell sorting, which is a method for separating heterogeneous cell groups by using a flow cytometer and a fluorescent probe; when a single cell suspension that has been fluorescently stained or labeled is placed in a sample tube, it is pressed into a flow chamber by high pressure; the flow chamber is filled with sheath fluid, and under the wrapping and pushing of the sheath fluid, the cells are arranged in a single row and ejected from the flow chamber nozzle at a certain speed; an ultra-high frequency piezoelectric crystal is provided on the nozzle of the flow chamber, which vibrates after being charged, so that the ejected liquid flow breaks into uniform droplets, and the cells to be tested are dispersed in these droplets; these droplets are charged with different positive and negative charges, and when the droplet flow passes through a deflection plate with several thousand volts, it is deflected under the action of a high voltage electric field and falls into its respective collection container, and the uncharged droplets fall into the waste liquid container in the middle, thereby achieving cell separation; it is particularly suitable for sorting single cells, sorting based on intracellular markers, sorting based on the expression level of cell surface markers, and sorting when complex cell types are separated by multiple markers and the purity requirement is high.

[0073] (b) Immunomagnetic cell sorting is a technique for separating target cells from a cell population using magnetic beads. First, the magnetic beads bind to specific cell surface proteins on the target cells through antibodies, biotin or avidin. Then the sample is placed in a magnetic field, and the cells labeled with the magnetic beads are adsorbed under the action of the magnetic field. Unlabeled cells are retained in the supernatant, thereby physically separating the target cells and non-target cells in the sample. Because it is fast and easy to operate, immunomagnetic cell sorting is one of the commonly used methods for researchers to isolate high-purity specific cell subsets.

[0074] (c) Microfluidic cell sorting, which separates single cells by manipulating fluids at the microscopic level. Microfluidics is often built on microchips, often referred to as "chip labs". Microfluidics has great advantages when the volume of samples and reagents used is very small. In addition, chip labs are portable and can be used almost anywhere, making them particularly suitable as tools for on-site diagnosis. Several different microfluidic cell sorting methods include: ultrasonic sorting, two-phase aqueous system, biomimetic microfluidic sorting, affinity sorting, deterministic lateral deviation, electrophoresis sorting, field flow classification, gravity and sedimentation, magnetophoresis, microfiltration, and optical sorting.

[0075] (d) Adhesion method, which can separate target cells from heterogeneous cell populations based on the unique adhesion ability of different types of cells; by selecting appropriate growth factors and cell culture plates to selectively promote or inhibit the adhesion ability of cells, the adhered cells can be separated from the suspension. The effective use of this method also depends on the observation of the adhesion characteristics of the cell population separated in the present invention.

[0076] Different types of identifiable signals can be selected according to different sorting methods. For example, a cell population is enriched by flow cytometry sorting to form an enriched cell population containing at least one target cell, and the identifiable signal used is preferably a fluorescent dye, which is interpreted according to the specific excitation light wavelength or emission light wavelength of the fluorescent dye.

[0077] The binding molecules or capture molecules used to separate or enrich specific cells are easily prepared or obtained by those skilled in the art. Commonly used binding molecules include, but are not limited to, antibodies and ligands, which may be free or fixed on a solid phase carrier.

[0078] The cells obtained by the method of the present invention can be frozen, revived, passaged, and maintained in culture for a long time.

[0079] application

[0080] In the current diagnosis field, hospitals often use flow cytometry to detect the T cell status of the body under disease (such as tumor patients) or to detect the number ratio of various common immune cell subsets in peripheral blood, such as CD4 + The ratio of T cells, NK cells, macrophages, etc. can be used to determine whether the immune system is active. However, no method has yet focused on the importance of TCR diversity in T cell function, which is an indicator that determines CD8 + T cells are key factors in recognizing foreign antigens and maintaining autoimmune stability. Monitoring them can help to screen disease risks early and accurately evaluate CD8 + The immune function of T cells.

[0081] Therefore, in order to more accurately evaluate the functional + T cell function, the present invention innovatively provides + A new subset of T cells that affects TCR diversity and cell function, namely CD8 marked by CCL5 expression + Naive T cell subsets that participate in CD8 + Negative regulation of the immune effect of T cells. By detecting the TCR diversity, proliferation and effector function of this new subset, a comprehensive evaluation of the CD8 + The immune function of T cells.

[0082] The technical solution of the present invention can detect CD8 + The proportion of new functional subsets of T cells, determination of TCR diversity, determination of CD8 + As a preferred embodiment, the CD8 + Methods for determining T cell immune function include:

[0083] S1, obtain CD8 + Proportion of new subsets of naive T cells;

[0084] S2, obtain the CD8 + An indicator of TCR diversity in new subsets of naive T cells;

[0085] S3, based on CD8 + The diversity and immune function of new primitive T cell subsets TCR to quantify the CD8 + The immune function of new T cell subsets.

[0086] The human CD8 + The method for determining T cell immune efficacy is simple, rapid and accurate, which helps in early screening and intervention of disease risks among people of all ages, especially the middle-aged and elderly people, and is suitable for clinical promotion and application.

[0087] After knowing the specific properties of CCL5-labeled cells and their close correlation with immune cell function, substances that improve immune cell function (enhance immunity) can be screened based on this feature. Drugs that are truly useful for improving immune cell function can be found from the substances.

[0088] Therefore, the present invention provides a method for screening a substance (potential substance) that improves the function of immune cells (enhances immunity), the method comprising: (1) treating the separated CD8 + T cell population (e.g., isolated from peripheral blood cells); and (2) detecting the CD8 + CCL5 in T cell population + CD8 + The proportion of T cell population, if CCL5 + CD8 + The proportion of T cell populations was statistically reduced (e.g., CCL5 + CD8 + If the amount of the T cell population is reduced by more than 5%, more than 10%, more than 20%, more than 50%, more than 80%, etc.), the candidate substance is a substance (potential substance) that improves the function of immune cells (enhances immunity).

[0089] In a preferred embodiment of the present invention, in order to more easily observe CCL5 + CD8 + The amount of T cells can be changed, and a control group (Control) can be set up. The control group can be an isolate without adding the candidate substance.

[0090] As a preferred embodiment of the present invention, the method further comprises: conducting further cell experiments and / or animal experiments and / or clinical experiments on the obtained potential substances to further select and determine substances that are truly useful for improving immune cell function.

[0091] The potential substances for improving the function of immune cells obtained by the screening method of the present invention can be used to form a screening library, so that people can eventually further screen drugs therefrom.

[0092] Based on the new discovery of the inventors, the present invention also provides a method for separating, enriching or detecting the immune cell population expressing CCL5 of the present invention, preferably CCL5 + CD8 + A kit for a T cell population, comprising: a cell binding molecule or a capture molecule associated with CCL5 expression.

[0093] In an optional mode of the present invention, the kit may further include reagents for hydrolyzing / decomposing cells, such as collagenase, decomposing enzymes; reagents for cell suspension; culture medium for cell culture, etc.

[0094] In order to facilitate the use by those skilled in the art, the kit of the present invention also includes instructions for use that explain how to separate or enrich the cells (groups) of the present invention. For example, the aforementioned method of the present invention can be recorded in the instructions for use.

[0095] Based on the new discovery of the inventors, the present invention also provides an immune cell population (T SN ) in the preparation of a pharmaceutical composition for improving immune cell function and / or inhibiting tumors. The immune cell population that does not express CCL5 can be derived from animal or human cells, in which CCL5 is reduced or removed. Preferably, the immune cell population that does not express CCL5 has a preservation number of CCTCC NO: C2024243 in the China Center for Type Culture Collection.

[0096] The present invention also provides a composition, which contains an effective amount of the cell population that does not express (including low expression) CCL5, and a pharmaceutically acceptable carrier.

[0097] The cells can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably, the pH is about 6-8. Pharmaceutically acceptable carriers may include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should match the mode of administration, and the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount.

[0098] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention 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 carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, Science Press, or according to the conditions recommended by the manufacturer.

[0099] Example 1: CCL5 Marking CD8 + New subsets of naive T cells

[0100] By analyzing the single-cell transcriptome sequencing data of bone marrow immune cells from 3-91 years old (recorded in the GSA for Human database of the National Center for Bioinformatics, BioProject No.: PRJCA023058), CD8 + After T cell population extraction, further analysis was performed to compare CD8 + Transcriptomic information of T cell populations, in which the new subpopulation highlighted in yellow-green (arrow area) is the subpopulation that gradually occupies the main part in the elderly cells (CD8 + T cell population)( Figure 1 -A).

[0101] Further comparative analysis of the gene expression characteristics of the subgroups that gradually occupied the majority of the aged cells revealed that the CCL5 gene was highly expressed ( Figure 1 -B).

[0102] The enriched characteristic expression genes were analyzed for biological function pathways, showing that the new subpopulation with high expression of CCL5 (enriched in the elderly immune system) was closely related to the negative regulation of immunity ( Figure 1 -C).

[0103] And the GSEA analysis method also suggests that the new subpopulation with high expression of CCL5 is associated with the overall aging state of cells ( Figure 1 -D).

[0104] Use the data in the database to expand single-cell data analysis to all age groups:

[0105] Kid group: 3-12 years old;

[0106] Young group: 20-30 years old;

[0107] Middle group: 40-50 years old;

[0108] Pre-Old group: 55-65 years old;

[0109] Old group: 70-95 years old.

[0110] The results showed that the proportion of the new subpopulation with high expression of CCL5 increased with age, gradually occupying the CD8 + The dominant position of T cell population ( Figure 1 -E).

[0111] In summary, the analysis of human single-cell transcriptome sequencing showed that the new subgroup with high expression of CCL5 dominated the elderly individuals; and gene pathway analysis showed that the gene group with high expression of CCL5 was closely related to aging characteristics and negative regulation of T cell function. + T cell population (T SN ) subpopulation, CD8 + T cell population is a new cell subset associated with immune aging.

[0112] These results suggest that targeting new subpopulations with high CCL5 expression for intervention may affect T cell function in elderly individuals.

[0113] Example 2, CCL5-labeled CD8 + The new subset of naive T cells is determined by CD8 + T cell diversity subsets

[0114] In addition to the single-cell transcriptome sequencing data of immune cells in the bone marrow of 3-91 years old, the inventors conducted a targeted sequencing data analysis of the T cell receptor (TCR) that affects T cell function.

[0115] The results of the analysis using the scRepertoire R package showed that the polyclonal T cells were mainly CD8 + T cells, that is, the reduction of TCR repertoire diversity (a key functional change point associated with aging of T cells) is concentrated in CD8 + T cells instead of CD4 + T cells ( Figure 2 -A).

[0116] CD8 + T cells were extracted separately for a more detailed analysis, comparing the CD8 + TCR data of T cells, showing that the purple subgroup with the most reduced TCR diversity is the CD8 with high expression of CCL5 identified in the previous section. + T cell populations ( Figure 2 -B).

[0117] The data of all age groups were also statistically analyzed and it was found that CD8 + The TCR diversity of T cell populations decreases with age ( Figure 2 -C).

[0118] The above two parts of data show that CD8 + The T cell population is a key new subpopulation that affects T cell immunosenescence in terms of gene expression, number and TCR diversity (T cell function).

[0119] TCR is a key factor in the function of T cells. The diversity of its TCR library directly affects the CD8 + In order to effectively recognize antigens and then amplify them to exert their effects in fighting infections, tumors and other diseases, the inventors tested the changes in the TCR repertoire in people of different ages. The results showed that the diversity of the TCR repertoire varied greatly in CD8 + The greatest changes were within the T cell population (rather than CD4 + T cells).

[0120] Furthermore, after further in-depth analysis of cell subsets, it was found that the CD8 + This T cell population is CD8 + The new T cell subset is the main subgroup with the largest reduction in the diversity of the human TCR library, which fully suggests that this subgroup affects CD8 + T cells have distinct characteristics in terms of function.

[0121] Example 3, CD8 + New primitive T cell subsets indicate CD8 + T cell immune function

[0122] In addition to the above sequencing data, the inventors sorted a sufficient number of CD8 T cells from the spleen of mice (CCL5-EYFP mice, obtained from Saiye Bio) by flow cytometry (FACS) (using antibody staining of cell surface markers and CCL5-coupled EYFP fluorescence to sort negative and positive cell populations). + T cells (including CCL5 - CD8 + T cells (T SN cells), CCL5 + CD8 + T cells, >2*10 6 For more in-depth sequencing and characteristic gene pathway enrichment analysis, we compared T SN The results of cell comparison showed that CCL5 + CD8 + T cell populations are functionally related to the effector and proliferation of immune cells ( Figure 3 -A).

[0123] The sorted cells were resuspended in 1640 complete medium and the cells were cultured at a rate of 1*10 5 The cells were seeded at a density of 1000 cells / well onto a 96-well U-bottom plate pre-coated with anti-human CD3 (6μg / ml) and anti-human CD28 (8μg / ml) antibodies, and cytokines IL-2 (10ng / ml) and IL-12 (10ng / ml) were added for culture. After 72 hours, the cells were collected and cultured in a 5% CO2, 37°C incubator with 1640 complete medium supplemented with 1000ng / mlionomycin and 50ng / ml phorbolmyristate acetate (PMA) and BD's GolgiPlug for 4 to 6 hours. The cells were resuspended and washed once with FACS and then analyzed for CD8 + Surface staining of T cells was performed at 4°C for 30 min. The cells were permeabilized with BD's permeabilization solution at 4°C for 20 min, and intracellular staining of IFN-γ was performed and flow cytometry was performed ( Figure 3 -B). The results showed that CCL5 + CD8 + T cell subsets ( Figure 3 -B upper part) The effector ability of secreting IFN-γ is significantly weaker than that of T SN cell( Figure 3 -B lower part).

[0124] At the same time, the sorted cells were resuspended in PBS and then added with CellTrace TM Violet staining reagent was used to stain the cells for CTV proliferation. The cells were incubated in a 37°C incubator in the dark for 20 minutes. 1640 complete medium was added and the cells were incubated in a 37°C incubator in the dark for 5 minutes to terminate the staining. 5 The cells were seeded at a density of 100 cells / well on a 96-well U-bottom plate pre-coated with anti-human CD3 (6 μg / ml) and anti-human CD28 (8 μg / ml) antibodies. The cells were cultured in a 5% CO2, 37°C incubator for 72 hours and then detected by flow cytometry to observe cell proliferation ( Figure 3 -C). The results showed that CCL5 + CD8 + The proliferation peak of the new T cell subset was weaker than that of the control, indicating that its proliferation ability was weakened.

[0125] Therefore, by detecting the transcriptome gene pathways, proliferation and effector functions of the new subgroup, it is shown that this new subgroup does have functional differences, which can indicate that CD8 + T cell function.

[0126] Example 4, CD8 + The role of new naive T cell subsets in viral infection

[0127] 1. Patient analysis

[0128] In order to further explore and verify the function of the new subpopulation, we first selected elderly patients (65-95 years old) with mild (outpatient visit) and severe (ICU admission) infection with the new coronavirus (SARS-CoV-2) and obtained their peripheral blood samples (based on the hospital's admission information and sample delivery) to detect CCL5 in their immune cells. + CD8 + The proportion of T cell subsets.

[0129] The results showed that CCL5 + CD8 + T cell subsets in severe patients were significantly higher than those in mild control patients ( Figure 4 -A), indicating a key role for this novel subset in immune senescence, frailty, and increased inflammation.

[0130] For samples from the mild and severe groups, CCL5 was detected by flow cytometry. + CD8 + T cell ratio data, such as Figure 4 -B.

[0131] In the case data, CCL5 + CD8 + ROC curves and test parameters of T cell populations corresponding to low antiviral ability Figure 5 , indicating that this cell population has good sensitivity and specificity for isolating populations with low antiviral ability.

[0132] 2. Animal analysis

[0133] To analyze the immune cell function of animals, C57BL6 / J mice of the same age were used. Blood was collected from the eyelids (50 μl / mouse) and flow cytometry was performed to determine the CCL5 + CD8 + The proportion of T cells in peripheral blood was divided into the following groups:

[0134] Control: mice not infected by intranasal drip;

[0135] Normal mice: CCL5 + CD8 + Total CD8 T cells in peripheral blood + The proportion in T cells is less than 5%.

[0136] CCL5 + CD8 + T cell high ratio mice: CCL5 + CD8 + Total CD8 T cells in peripheral blood + The proportion in T cells is greater than 15%.

[0137] The inventors used influenza virus (Influenza A) to infect mice for an infectious modeling experiment. The mice were infected with influenza virus (dose 40 PFU) via nasal drops on the first day, and the weight of the mice was observed and recorded every day thereafter.

[0138] Observations show that CCL5+ CD8 + The symptoms of mice with a high proportion of T cells after infection were significantly more severe than those of normal mice, and their body weight decreased significantly ( Figure 4 -C).

[0139] At the end of infection, mice in each group were euthanized and spleens were removed. TM Mouse CD8 + T Cell Isolation Kit (STEMCELL Technologies, Catalog #19853) was used to isolate mouse CD8 + T cells were stimulated to produce effector factors by adding 1000 ng / ml ionomycin and 50 ng / ml phorbolmyristate acetate (PMA) and BD's GolgiPlug, and the cells were cultured in a 5% CO2, 37°C incubator for 4 to 6 hours. The cells were resuspended and washed once by FACS and then analyzed for CD8 + Surface staining of T cells, staining at 4℃ for 30min. Use BD's membrane permeabilization solution to permeabilize cells at 4℃ for 20min, stain for IFN-γ and perform flow cytometry ( Figure 4 -D). The results showed that CCL5 + CD8 + Mice with a high proportion of T cells CD8 + The effector capacity of T cells to secrete cytokines was significantly weaker than that of normal mice at a low rate.

[0140] Through viral infection, a key in vivo experiment, it was shown that the proportion of this subset in vivo affects CD8 + T cells function.

[0141] Example 5, CD8 + New subsets of naive T cells (T SN The role of cell subsets in fighting tumors

[0142] Select CCL5 in 10-month-old + CD8 + Female / male C57BL / 6 mice with a high proportion of T cells. Flow cytometry analysis showed that the mice in this group had: CCL5 + CD8 + The proportion of T cells in total CD8 + T cells greater than 15%, such as Figure 6 .

[0143] The animals were divided into groups of 6 each. After anesthesia, the hair on the lower right side of the back was removed. 2*10 5 B16F10 cells (skin melanoma cells cultured in vitro). Observe the size of the tumor at the injection site. If there is a rice-sized tumor visible to the naked eye on the 4th day, it means that the tumor is successfully injected. At the same time, the tumor growth of the mice is measured and recorded every 3 days using a vernier caliper ( Figure 7 -B).

[0144] On the 3rd and 8th day after tumor transplantation, mouse CD4 antibody (1 mg / ml) was injected intraperitoneally, 200 μl per mouse each time, to exclude CD4 + T cells' intervention in tumors.

[0145] On day 10 after tumor inoculation, 4-week-old mice (CD8 + T cell populations with low expression of CCL5) CD8 in the spleen + T cell population (T SN The T cells sorted above were injected into the tumor SN The cell subsets were injected into the treated mice, with 3*10 6 The model was completed on the 18th day after tumor inoculation. The mice were euthanized and the tumors were removed, weighed, and photographed. Figure 7 -A, C).

[0146] Experimental data showed that compared with the control group, the SN Mice with cell subsets supplemented with non-T SN The tumors in the mice treated with cells were significantly suppressed, and CD8 + The killing effect of T cells is good.

[0147] Biomaterial Deposit

[0148] The cells isolated and obtained in the present invention (human umbilical cord blood T cells T SN Homo Sapiens), has been deposited in the China Center for Type Culture Collection (CCTCC) (Wuhan, China), with the deposit number being CCTCC NO:C2024243 and the deposit date being August 14, 2024.

[0149] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. Application of cell populations as analytical targets and preparation of reagents for analyzing immune cell functions; wherein: The cell population is selected from: an immune cell population expressing CCL5; or an immune cell population not expressing CCL5.

2. The use according to claim 1, characterized in that The immune cell population expressing CCL5 is CCL5 + CD8 + T cell population; or The immune cell population that does not express CCL5 is CCL5 - CD8 + T cell populations.

3. The use according to claim 2, characterized in that The analysis of immune cell function includes: analyzing CD8 + T cell function; if the test subject's CCL5 + CD8 + T cells account for the total CD8 + A significantly high proportion of T cells indicates that CD8 + T cell function is significantly low; Preferably, the analysis includes prognosis, for CCL5 + CD8 + T cells account for the total CD8 + In subjects with a significantly high proportion of T cells, CCL5-non-expressing CD8 + T cell therapy options.

4. The use according to any one of claims 1 to 3, characterized in that: Analyzing the immune cell function of the test subject includes: analyzing the anti-tumor ability of the test subject, analyzing the anti-pathogen ability of the test subject, or analyzing the diversity of the T cell receptors of the test subject.

5. The use according to claim 4, characterized in that The CCL5-expressing immune cell population accounts for the total CD8 + A significantly high proportion of T cells indicates that the test subject has relatively low anti-tumor ability; or The CCL5-expressing immune cell population accounts for the total CD8 + A significantly high proportion of T cells indicates that the test subject has a relatively low ability to resist pathogens; or The CCL5-expressing immune cell population accounts for the total CD8 + A significantly high proportion of T cells indicates that the diversity of T cell receptors in the test subject is relatively low.

6. The use according to any one of claims 2 to 3, characterized in that: The CCL5 + CD8 + T cell populations are activated by specific binding or capture of CCL5 + and CD8 + The binding molecules or capture molecules are used to sort out T cells from a sample containing T cells; the CCL5-CD8 + T cell population is CD8 + Subtraction of CCL5 from T cell population + CD8 + Cell populations obtained after T cells; Preferably, the sample containing T cells is peripheral blood; more preferably, the CCL5 + CD8 + T cell population or CCL5 - CD8 + Sorting and / or determining the proportion of T cell populations; More preferably, the method of sorting cells includes: flow cytometry sorting, immunomagnetic bead sorting, microfluidic cell sorting, and adhesion method.

7. A method for isolating or enriching a cell population, wherein the cell population is CCL5 + CD8 + T cell population or CCL5 - CD8 + T cell population; the method comprising: Using a binding molecule or a capture molecule that specifically binds to or captures CCL5, the CCL5 expression negative and positive CD8 + T cells are sorted and / or the proportion is determined; preferably, the method of sorting cells includes: flow cytometry sorting, immunomagnetic bead sorting, microfluidic cell sorting, and adhesion method.

8. Specific Detection and Isolation of CCL5 + The use of a reagent for the specific detection and separation of CCL5 + Reagents for cells include: Specific binding or capture of CCL5 + Binding molecules or capture molecules.

9. Use of an immune cell population that does not express CCL5 in the preparation of a pharmaceutical composition for improving immune cell function and / or inhibiting tumors; preferably, the collection number of the immune cell population that does not express CCL5 in the China Center for Type Culture Collection is CCTCC NO: C2024243.

10. A method for screening a substance that improves immune cell function, the method comprising: (1) Treating isolated CD8 + T cell population; and (2) Detection of the CD8 + CCL5 in T cell population + CD8 + The proportion of T cell population, if CCL5 + CD8 + If the proportion of the T cell population is statistically reduced, the candidate substance is a substance that improves the function of immune cells.

Images