Method for determining the relative amount of a specific cell subgroup in a CD4 + t

Abstract

The present invention provides a method for more efficiently measuring the relative amount of a specific cell subgroup in a CD4 + T cell group, and a kit for use in the method. The present invention provides a method for determining the relative amount of CCR4-CCR6 + cell subgroups in a CD4 + T cell group in a sample derived from a subject, the method comprising the steps of: selecting a CXCR3-cell subgroup in the CD4 + T cell group; setting a boundary between the CCR4 <-> cell subgroup and the CCR4 < + > cell subgroup in the CXCR3 <-> cell subgroup; and determining the relative amount of CCR4-CCR6 + cell subgroups in the CD4 + T cell group using the boundary described above.

Description

Technical Field

[0001] This invention relates to CD4 + The determination of the relative amounts of medically important cell subgroups within the T-cell group. Background Technology

[0002] Given CD4 + The T cell group contains some medically important cell groups. For example, CD4 + CD62L low Cell subgroups, CCR4 - CCR6 + The relative amounts of cellular subgroups are medically important cellular subgroups that are particularly useful for responding to cancer treatment (Patent Document 1, Patent Document 2).

[0003] These cell subgroups are typically separated as follows: more than 8 ml of capillary blood is collected, and laboratory technicians use centrifuges or similar equipment at the blood collection site to separate PBMCs (capillary mononuclear cells), freeze them, transport the frozen cells to the assay facility, preserve them as needed after transport, and then culture and stain them for FACS (Fluorescence-Activated Cell Sorting) assay.

[0004] Existing technical documents Patent documents Patent Document 1: International Publication No. 2018 / 147291 Patent Document 2: International Publication No. 2022 / 054796 Summary of the Invention The technical problem that the invention aims to solve The objective of this invention is to provide a more efficient method for measuring CD4. + A method for determining the relative amounts of specific cell subgroups within a T cell population, and a kit for using this method.

[0005] Solution for solving the problem The inventors discovered that firstly, CXCR3 was isolated. + / - Cell populations are beneficial for measuring CD4. + CCR4 in the T cell group - CCR6 + The relative amounts of cellular subgroups.

[0006] Furthermore, the inventors of this invention have discovered a method for successfully measuring CD4 through whole blood samples. + Preferred sample preservation conditions for the relative amounts of specific cell subgroups within the T cell group.

[0007] This invention provides, for example, the following items.

[0008] (Project 1) CD4 from a sample of the test subject + CCR4 in T cell group - CCR6 + A method for determining the relative amounts of cell subgroups, comprising the following steps: Select the CD4 above. + CXCR3 in the T cell group - Cellular subgroups; In the aforementioned CXCR3 - In the cell subgroup, CCR4 was set. - Cellular subgroups and CCR4 + The boundaries of cellular subgroups; and Using the above boundaries, CD4 was measured. + CCR4 in the T cell group - CCR6 + The relative amounts of cellular subgroups.

[0009] (Project 2) According to the method described in Project 1, the above-mentioned sample is a whole blood sample or a peripheral blood mononuclear cell (PBMC) sample from the subject.

[0010] (Project 3) According to the method described in Project 2, the above-mentioned sample is a whole blood sample.

[0011] (Project 4) According to the method described in Project 3, the above-mentioned sample is a sample that has been collected from the above-mentioned subject and stored for more than one day.

[0012] (Project 5) According to the method described in Project 4, the above-mentioned samples are samples taken from the above-mentioned subjects and stored for 1 to 6 days.

[0013] (Project 6) According to the method described in Project 4, the above-mentioned samples are samples taken from the above-mentioned subjects and stored for 1 to 3 days.

[0014] (Project 7) According to the method described in Project 4, the above-mentioned sample is a sample stored at approximately 10°C to approximately 40°C.

[0015] (Project 8) According to the method described in Project 4, the above-mentioned sample is a sample stored at approximately 12°C to approximately 25°C.

[0016] (Project 9) According to the method described in Project 3, the sample is approximately 10 μl to approximately 500 μl.

[0017] (Project 10) CD4 from a sample of the test subject + CCR4 in T cell group - CCR6 + A method for determining the relative amounts of cell subgroups, comprising the following steps: Prepare whole blood samples from the above-mentioned subjects; and Measure CD4 in the above whole blood samples + CCR4 in the T cell group - CCR6 + The relative amounts of cellular subgroups.

[0018] (Project 11) According to the method described in Project 10, the above-mentioned sample is a sample that has been collected from the above-mentioned subject and stored for more than one day.

[0019] (Project 12) According to the method described in Project 11, the above-mentioned samples are samples taken from the above-mentioned subjects and stored for 1 to 6 days.

[0020] (Project 13) According to the method described in Project 11, the above-mentioned samples are samples taken from the above-mentioned subjects and stored for 1 to 3 days.

[0021] (Project 14) According to the method described in Project 11, the above-mentioned sample is a sample stored at about 10°C to about 40°C.

[0022] (Project 15) According to the method described in Project 11, the above-mentioned sample is a sample stored at approximately 12°C to approximately 25°C.

[0023] (Project 16) According to the method described in Project 10, the sample is approximately 10 μl to approximately 500 μl.

[0024] (Project 17) A kit for use in any one of items 1 to 16, wherein the kit comprises a detection reagent for CD4, a detection reagent for CCR4, and a detection reagent for CCR6.

[0025] (Project 18) The kit described in Project 17 further includes a detection reagent for CXCR3.

[0026] (Project 19) According to the kit described in Project 16, the detection agent is an antibody.

[0027] Invention Effects According to the present invention, it is possible to more accurately determine medically important cell subgroups (e.g., CCR4) within the CD4+ T cell population. - CCR6 + Cell subgroup or CD62L low (Cellular subgroups).

[0028] In addition, it solves the problems of limited blood collection sites (having centrifuges, etc., and professional technicians) and the large gap between personnel / institutions skilled in PBMC separation and those who are not skilled, enabling sample collection with no processing at the blood collection site and minimal changes between equipment. Attached Figure Description

[0029] Figure 1 This is a bar chart of CXCR3 and a scatter plot of CCR4 vs CCR6 for whole blood and PBMCs.

[0030] Figure 2 These are bar charts of CXCR3 and scatter plots of CCR4 vs CCR6 when the storage time of whole blood is changed.

[0031] Figure 3 This is a scatter plot of CD4 vs CD8 when the storage time and temperature are changed.

[0032] Figure 4 When storing data by changing the temperature and number of days, it is relative to CD4. + A bar chart of CD62L in cells.

[0033] Figure 5 When changing the temperature and number of days for storage, it is relative to CD8. + A bar chart of CD62L in cells.

[0034] Figure 6 It is the Th7R used when changing the temperature and number of days for storage.

[0035] Figure 7 When changing the temperature and number of days for storage, it relates to CD4. + Scattering diagram of CCR7 and CD45RA in cells.

[0036] Figure 8 When changing the temperature and number of days for storage, it relates to CD8. + Scattering diagram of CCR7 and CD45RA in cells.

[0037] Figure 9This is a comparison of the results obtained from the separation and cryopreservation of PBMCs from whole blood and preserved whole blood.

[0038] Figure 10 A comparison of each preservation method on CD4 + A diagram showing the expression of CCR4, CCR6, and CXCR3 in cells.

[0039] Figure 11A It's about CD4 + Scatter plot of CCR6 vs CCR4 in cells, gates not shown.

[0040] Figure 11B It's about CD4 + CXCR3 - Scatter plot of CCR6 vs CCR4 in cells, gates not shown.

[0041] Figure 11C It's about CD4 + The scatter plot of CCR6 vs CCR4 in cells also shows the gate.

[0042] Figure 11D It's about CD4 + CXCR3 - The scatter plot of CCR6 vs CCR4 in cells also shows the gate.

[0043] Figure 12 This is the result of WB panel 3 staining of a small amount of blood sample.

[0044] Figure 13 This is a schematic diagram representing the Th differentiation of human CD4 T cells.

[0045] Figure 14 This indicates peripheral blood CD4 + A schematic diagram of T cell clusters and the chemokine receptors expressed in each region. Detailed Implementation

[0046] The invention will now be described with reference to its preferred form. It should be understood that throughout this specification, unless otherwise specified, the singular form includes the concept of its plural form. Therefore, it should be understood that unless otherwise specified, articles in the singular form (e.g., in the case of English, “a,” “an,” “the,” etc.) also include the concept of their plural forms. Furthermore, it should be understood that unless otherwise specified, the terms used in this specification are used in the sense commonly understood in the art. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention pertains. In cases of conflict, this specification (including definitions) takes precedence.

[0047] The following provides appropriate definitions of terms and / or basic technical content used in this specification.

[0048] In this specification, “about” means ±10% of the following value.

[0049] In this specification, "biomarker" means a characteristic that can be objectively measured and evaluated as an indicator of a normal biological process, pathological process, or pharmacological response to a therapeutic intervention.

[0050] In this specification, the terms "cancer" and "tumor" are used interchangeably, referring to malignant tumors that exhibit strong atypia, proliferate faster than normal cells, and are capable of destructively infiltrating surrounding tissues or metastasizing, or the presence of such malignant tumors. In this invention, cancer includes solid tumors and hematopoietic organ tumors, such as non-small cell lung cancer, small cell lung cancer, kidney cancer, Hodgkin's disease, head and neck cancer, breast cancer, gastric cancer, malignant melanoma, colorectal cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, gastrointestinal stromal tumors, pancreatic neuroendocrine tumors, skin cancer, etc., but is not limited to these.

[0051] In this specification, "cancer immunotherapy" or "cancer immunotherapy" refers to a method of treating cancer by using biological defense mechanisms such as the immune mechanisms possessed by an organism.

[0052] In this specification, "anti-tumor immune response" refers to any immune response against tumors in the body.

[0053] In this specification, "correlation" means that two events are statistically significantly correlated. For example, "the relative quantity of B related to A" means that, given the occurrence of event A, the relative quantity of B is statistically significantly affected (e.g., increased or decreased).

[0054] In this specification, "cell subgroup" refers to any collection of cells within a cell group that contains cells with diverse characteristics and shares certain common features. This term may be used to refer to a specific cell subgroup by those who are familiar with specific names in the art, or by describing any property (e.g., the expression of cell surface markers) to refer to a specific cell subgroup.

[0055] In this specification, the cell-related term "relative amount" is used interchangeably with "ratio." Typically, the terms "relative amount" and "ratio" refer to: relative to the formation of a specific cell cluster (e.g., CD4). + The number of T cell groups and the cell subgroups required for their formation (e.g., CCR4) - CCR6 + The number of cells in a cell subgroup.

[0056] In this specification, "relative value" refers to a value calculated by comparing other values ​​with respect to a given value.

[0057] In the context of this specification, the term "detector" is used broadly to refer to all factors capable of detecting a target substance (such as cell surface markers).

[0058] In this specification, the “amount” of a cell subgroup includes the absolute number of cells and the relative amount of the proportion of cells in the cell group.

[0059] In this specification, "flow cytometry analysis" refers to a technique for measuring the number of cells, individuals, and other biological particles suspended in a liquid, as well as their respective physical, chemical, and biological characteristics. The results of flow cytometry analysis are typically presented as a dot plot with FSC as the X-axis and SSC as the Y-axis. Each cell is represented by a dot in the plot, and their position is determined by the relative values ​​of FSC and SSC. Relatively small lymphocytes with simple internal structures are located in the lower left, while larger granulocytes with internal granules are located in the upper right. Additionally, larger monocytes with simple internal structures are located between lymphocytes and granulocytes, forming separate clusters. Furthermore, the results of flow cytometry analysis can be represented using bar charts, dot plots, etc.

[0060] In this specification, the "subject" can be any animal, typically a mammal. There is no limitation on the term "mammal," which includes livestock (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans, and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In a particular embodiment, the subject is a human.

[0061] In this specification, “cancer treatment” means any treatment administered to a cancer patient to remove or shrink a tumor, including, but not limited to, cancer immunotherapy, radiation therapy, molecularly targeted therapy, surgery, cell transplantation, or any combination of these treatments.

[0062] (Th7R) In this specification, "effective cells" refer to immune cells that differentiate from unstimulated naïve cells and acquire actual immune-related functions, especially human helper T cells, which are known to mainly include three types: Th1, Th2, and Th17. These cells release characteristic cytokines such as interferon-γ (IFNγ) and interleukin-17 (IL-17). In addition, each Th subset contains characteristic transcription factors, such as Th1 expressing T-bet and Th2 expressing GATA-3. However, cells that simultaneously express both T-bet and GATA-3 also exist; these cells are referred to as "Th1 / Th2" or "Th1 / 2". In this specification, "Th1 / Th17" or "Th1 / 17" expresses the characteristic Th1 T-bet and the characteristic Th17 RORγt.

[0063] In addition, effector cells such as Th1, Th2, and Th17 can also be classified according to the types of chemokine receptors (CCRs) they express. For example, Th1 cells express CXCR3, Th2 cells express CCR4, and Th17 cells express both CCR4 and CCR6. The classification of Th subsets is as follows: Figure 13 (Annual Review of Immunology, Vol.34:317-334 Heterogeneity of Human CD4 + As shown in TCells Against Microbes (Fig. 1). In this specification, "Th1 / Th17" or "Th1 / 17" expresses CCR6, one of the two CCRs characteristic of Th1 (CXCR3) and Th17 (CXCR3). Additionally, in this specification, CCR6SP (Single-positive) is a previously unclassified type in the CCR-based Th type classification, representing a Th subgroup that expresses only CCR6 from CCR4, CCR6, and CXCR3. Furthermore, in this specification, TP (Triple-positive) is a previously unclassified type in the CCR-based Th type classification, representing a Th subgroup that expresses all three CCRs (CCR4, CCR6, and CXCR3).

[0064] As disclosed in International Publication No. 2022 / 054796, Th1 / 17 and CCR6 SP CD4 in the Th cluster... + T cell clusters showed a high correlation with progression-free survival. The chemokine receptors expressed in each cluster were summarized in a graph, becoming... Figure 14 (Corresponding to Figure 17 of International Publication No. 2022 / 054796). As shown in the figure, the part enclosed by the dashed line is CD62L. lowThe cell clusters contained four clusters (C, D, E, and H) with different expressions of chemokine receptors. Among them, block D was expressed using CXCR3. + CCR4 - CCR6 + The representation is Th1 / 17, and block H is represented by CXCR3. - CCR4 - CCR6 + The CCR6 SP is represented.

[0065] In this specification, "Th7R" refers to the Th1 / 17 cell subgroup (CD4+). + CXCR3 + CCR4 - CCR6 + ) and CCR6SP cell subgroup (CD4) + CXCR3 - CCR4 - CCR6 + The Th7R cell subgroup is composed of cells that are part of the immune system. The Th7R cell subgroup in the blood is considered responsible for systemic immunity. For example, it can be used to predict the efficacy of cancer immunotherapy, especially immune checkpoint inhibitors (anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, etc.), and to predict responses to cancer treatments such as chemotherapy, radiation therapy, or molecularly targeted drugs (e.g., epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors). Furthermore, it is found in patients with hepatitis B, mouse models of chlamydial pneumonia, patients with opportunistic infectious diseases, dengue fever, and patients with acetylcholine-positive myasthenia gravis, making it an important cell subgroup in medicine.

[0066] Thus, the Th7R includes CXCR3. + Cellular subgroups and CXCR3 - The concepts of cellular subgroups and CD4 subgroups are therefore relevant in determining CD4. + When determining the ratio of Th7R cells in a cell subgroup, it is generally not necessary to measure CXCR3 expression. However, as disclosed in this specification, by measuring CXCR3... - Gating CCR4 in cell subgroups is more effective than gating CD4 without confirming CXCR3 expression. + When CCR4 is gated as a whole cell, gateding can be performed more precisely (see Example 5). Therefore, according to the present invention, the following series of steps are preferred when measuring Th7R cell subgroups: 1. Select CD4 + CXCR3 in the T cell group - Cellular subgroups; 2. In CXCR3 - In the cell subgroup, CCR4 was set. - Cellular subgroups and CCR4 + Boundaries of cell subgroups (defined by phyla); 3. Using the established boundary (gate), determine CD4. + CCR4 in the T cell group - CCR6 + The relative amounts of cellular subgroups.

[0067] (cancer) In this invention, examples of cancers targeted include: malignant melanoma, non-small cell lung cancer, renal cell carcinoma, malignant lymphoma (Hodgkin lymphoma or non-Hodgkin lymphoma), head and neck tumors, urinary system cancers (bladder cancer, urothelial carcinoma, prostate cancer), small cell lung cancer, thymic carcinoma, gastric cancer, esophageal cancer, esophagogastric junction cancer, liver cancer (hepatocellular carcinoma, intrahepatic cholangiocarcinoma), primary brain tumors (glioblastoma, primary central nervous system lymphoma), malignant pleural mesothelioma, gynecological cancers (ovarian cancer, cervical cancer, endometrial cancer), soft tissue sarcoma, biliary tract cancer, multiple myeloma, breast cancer, colorectal cancer, etc., but are not limited to these.

[0068] (whole blood sample) For samples used to differentiate and isolate T cells, appropriate samples can be taken from the subject using standard methods. For example, samples can be taken from peripheral blood, bone marrow, tumor tissue, hematopoietic tissue, spleen, normal tissue, or lymph. Taking samples from peripheral blood is particularly advantageous due to its non-invasiveness and ease of operation.

[0069] In this invention, whole blood samples derived from capillary blood or PBMC samples prepared therefrom can be used, with whole blood samples being preferred. Whole blood samples can be collected using blood collection tubes containing an anticoagulant. In this invention, any anticoagulant known in the art can be used, preferably heparin (sodium heparin, lithium heparin), EDTA, sodium citrate, ACD-A, etc., more preferably heparin. As shown in the examples described later, when using EDTA, the proportion of cells changing with increasing storage days in the blood collection tube is greater, but for Th7R (CD4) cells… + CCR6 + CCR4 - It has the characteristic that even when using EDTA, the rate of change is relatively small.

[0070] As can be seen from the examples in this specification, the preferred storage conditions for whole blood samples can vary depending on the cellular markers of the target organism. For Th7R, the optimal storage conditions are suitable over a wide temperature range, and even with long-term storage, the variation in measurement results is minimal. Therefore, when measuring CD4... + In the case of Th7R in cells, whole blood samples can be stored at approximately 10°C to approximately 40°C, more preferably at approximately 12°C to approximately 25°C. Furthermore, when measuring Th7R, storage at approximately 12°C to approximately 25°C allows for adequate measurement of whole blood samples within one week of blood collection (preferably within six days, more preferably within three days), which is unexpected compared to other markers described below. Additionally, since the ability to perform measurements immediately after blood collection depends on the collection location and facilities, the ability to perform measurements stably even after one day or more, rather than immediately after blood collection, is an advantage. Therefore, in this invention, the storage time for whole blood samples used to measure Th7R can be, for example, 1 day to 1 week, 1 day to 6 days, 1 day to 3 days, etc.

[0071] It can be seen that in the measurement of CD4 + CD62L in cells low In this case, a higher temperature range than that for Th7R is preferred. Specifically, in the determination of CD62L low In such cases, whole blood samples can be stored at approximately 15°C to approximately 40°C, more preferably at approximately 18°C ​​to approximately 37°C. Additionally, in the determination of CD62L... low Under suitable conditions, when stored at approximately 18°C ​​to 37°C, whole blood samples can be adequately tested within one week of collection (preferably within three days). Furthermore, since whether testing can be performed immediately after collection depends on the collection location and equipment, the ability to perform stable testing even after one day rather than immediately after collection is an advantage. Therefore, this method is suitable for measuring CD4. + CD62L in cells low Whole blood samples can be stored for, for example, 1 day to 1 week, 1 day to 6 days, or 1 day to 3 days.

[0072] In a specific implementation, Th7R was measured. CD62L low The ratio of cell subpopulations is advantageous. In this case, by storing at approximately 18°C ​​to approximately 25°C, sufficient assays can be performed within one week of blood collection (preferably within three days). Therefore, this method is suitable for measuring Th7R. CD62L low Whole blood samples from cellular subgroups can be stored for, for example, 1 day to 1 week, 1 day to 6 days, or 1 day to 3 days.

[0073] On the other hand, for CD4 + Regarding CCR7 in cells, if more than 2 days have passed since blood collection, the results show significant changes compared to samples collected immediately after collection. Therefore, it can be concluded that when measuring CD4... + In the case of CCR7 in cells, whole blood samples should not be stored for more than 3 days. If stored for up to 2 days, a storage temperature of 18℃~25℃ is more stable.

[0074] Regarding CD8 + The fluorescence intensity of CD8 cells remained stable for 2 days. During this short period (within 2 days), the temperature should ideally be between 18°C ​​and 25°C.

[0075] Thus, depending on the type of cell and the type of marker used for testing, the storage temperature and storage time of whole blood samples vary. For Th7R, it is unexpected that it can be stored for a long time (within 1 week, preferably within 6 days, more preferably within 3 days after blood collection, for example, 1 day to 1 week, 1 day to 6 days, 1 day to 3 days, etc.) within a wide temperature range (about 10°C to about 40°C, more preferably about 12°C to about 25°C).

[0076] The amount of whole blood sample collected can be the amount commonly used in this field, typically about 16 ml or less. However, as shown in Example 6 of this specification, even a trace amount of about 100 μl can stably achieve the determination of Th7R. This amount allows the subject to collect the blood themselves, reducing the burden on the subject, which is advantageous. Therefore, in a preferred embodiment of the present invention, the whole blood sample volume is about 10 μl or more, about 50 μl or more, more preferably about 100 μl or more, and about 500 μl or less, more preferably about 200 μl or less, and preferably about 100 μl to 150 μl. Examples of whole blood sample collection kits that can be used in the present invention include: MBS capillary tubes (lithium heparin, 100 μl to 150 μl collection line) (Micro Blood Science Co., Ltd.), micro-collection tubes (lithium heparin, without separator) 250 μL "Micro health Tube" (Health Wave Japan Co., Ltd.), CAPIJECT II (Terumo), and kits with BD Microguard. TM Capped BD Microtainer (registered trademark) micro-volume blood collection tubes, etc., but not limited to these.

[0077] To date, for Th7R-specific testing, medical personnel collect blood, immediately isolate PBMCs, cryopreserve them as needed, and transport the samples to the testing facility. In this case, the blood collection facility requires biosafety cabinets, cooling centrifuges, and cryogenic freezers, while the testing facility requires specialized equipment such as biosafety cabinets, cooling centrifuges, CO2 incubators, cryogenic freezers, and liquid nitrogen tanks. Even when testing is performed at the blood collection facility, biosafety cabinets and cooling centrifuges are still required, and if residues are to be preserved for testing, cryogenic freezers and liquid nitrogen tanks are also necessary. However, in cases where whole blood samples can be used, as in this invention, testing can be performed solely at the blood collection facility, requiring only a microcentrifuge. If whole blood samples are used and subsequently transported to the testing facility, no equipment is needed at the blood collection facility. This approach offers significant advantages over conventional methods.

[0078] As shown in the comparative example, there are problems with the detection of CD4 and CXCR3 when a stabilizer is added to anticoagulated blood to fix the cells. Therefore, the efficient assay of Th7R found in whole blood samples in this specification is unexpectedly advantageous.

[0079] In a preferred embodiment, whole blood samples are not subjected to hemolysis, centrifugation, or both before being analyzed using a flow cytometer or similar instrument. Data leakage due to the large number of red blood cells present in the analysis of white blood cells (T cells in this invention) in whole blood samples can be problematic. In most cases, hemolysis and centrifugation are performed to remove red blood cells, but these procedures are cumbersome and can cause cell damage, loss, and agglutination. In this invention, a simple method for measuring blood without hemolysis and washing (No-Wash, No-Lyse method) can also be used. In the No-Wash, No-Lyse method, whole blood is aliquoted (approximately 5 μl to 10 μl), subjected to antibody reaction, diluted with buffer, and then analyzed. During the assay, white blood cells can be separated using a purple laser or by Trigger staining.

[0080] For example, white blood cells can be separated from whole blood samples by using side-scattered light (SSC) from both blue and violet lasers. White blood cells and red blood cells have different scattering characteristics under violet laser light; red blood cells absorb violet laser light, thus their light intensity is lower compared to white blood cells.

[0081] Alternatively, white blood cells can be isolated from whole blood samples by staining to distinguish them from other cells. Red blood cells and other cells can be gated out using CD45 antibodies (a white blood cell marker) and specific markers for each cell population. Furthermore, since red blood cells and platelets lack nuclei, cell-penetrating pigments (such as invitrogen) that specifically stain the nuclei of living cells can be used. TM Vybrant TM DyeCycle TM Pigments, etc., can specifically stain only white blood cells.

[0082] (PBMC) When measuring th7R and other cell subpopulations, if a long period (e.g., more than one week) is required between blood collection and cell differentiation and measurement, PBMCs can be isolated and frozen. They can then be thawed for cell differentiation and measurement and cultured as needed. However, as shown in Example 5 of this specification, separating PBMCs after a period of time following whole blood collection is not preferable for th7R measurement. Therefore, in this invention, when measuring th7R and other cell markers by isolated PBMCs, it is preferable to isolate PBMCs within 3 hours, more preferably within 2 hours, after blood collection and freeze them.

[0083] Individual isolation and cryopreservation of PBMCs can be performed according to methods known in the field, such as those using Vacutainer Spitz. TM Mononuclear cells were isolated and cryopreserved using liquid nitrogen (see Example 1).

[0084] (Cell differentiation and separation) For CXCR3 + CCR4 - CCR6 + T cell subgroups, or CXCR3 - CCR4 - CCR6 + When counting the number of cells in each cell subgroup, such as T cells, the number can be determined beforehand by experimentally removing cells outside the respective subgroup from the whole cell line. For example, human CD4... + Effector memory T cell isolation kits (Militenyi Biotech) and similar products can isolate cells corresponding to target T cell subpopulations from peripheral blood without the use of specific antibodies. For example, if human CD4 is used... + Effector memory T cell isolation kit (Militenyi Biotech) allows for the isolation of CD4-like cells from peripheral blood without the use of CD4 and CD62L antibodies.+ CD62L low T cell subsets. The total number of viable cells can be pre-counted and recorded, and the number of cells obtained using this kit can also be counted and recorded. Human CD4 cells can also be selected using the same kit. + CD25 + CD127dim / - Regulatory T Cell Isolation Kit (Militenyi Biotech), Human CD25 + CD49d - Regulation T cell isolation kit (Militenyi Biotech). In one embodiment of the invention, any kit can be used as long as the cell number of each target T cell subgroup can be counted.

[0085] Alternatively, antibodies can be used without them. Antibodies specifically recognize and bind to molecules expressed by individual cells, allowing for color detection when the antibody binds to molecules expressed on the cell surface or inside the cell, thus counting the number of cells that show color. Here, since these molecules expressed on the cell surface or inside the cell are proteins, the mRNA encoding them is also formed inside the cell when the protein is expressed. That is, it is sufficient to investigate the mRNA inside each cell and whether there is mRNA encoding the protein molecule of interest. This idea can be realized through single-cell gene expression analysis, i.e., single-cell level mRNA analysis. Examples of single-cell gene expression analysis include: 1) next-generation sequencing using Quartz-Seq; 2) using the Fluidigm C1 System or ICELL8 Single-Cell System to isolate cells and then constructing a gene library using SMART-Seq v4; 3) using a cell sorter to isolate cells and then using the Ambion Single Cell-to-CT kit for quantitative PCR; 4) CyTOF SYSTEM (Standard Biotools), etc.

[0086] That is, blood is obtained, the number of viable cells is counted, and cells are separated using a cell sorting instrument. For each separated cell, the expression level of a specific gene can be measured using a quantitative PCR device, for example, with the Ambion Single Cell-to-CT kit. Based on the results, it can also be determined whether each cell belongs to the CXCR3 group. + CCR4 - CCR6 + T cell subgroups, CXCR3 - CCR4 - CCR6 + Which subgroup of T cells is it? Count the number of cells belonging to each subgroup.

[0087] The determination of the ratio of cell subgroups in this invention, or the comparison with reference values ​​or thresholds, can be performed using standard samples with specified signals. The signal of a standard (e.g., particles coated with fluorescent dye) prepared in a manner that generates a fluorescence signal corresponding to a specified cell subgroup is compared with that of a sample containing cell subgroups. By comparing with the standard, the amount or ratio of cell subgroups in the sample is determined. Furthermore, the signal of a standard (e.g., particles coated with fluorescent dye) prepared in a manner that generates a fluorescence signal corresponding to a specified reference value or threshold is compared with that of a sample containing cell subgroups. By comparing with the standard, the presence or amount of the marker of this invention in the T cell composition of the sample is determined.

[0088] In this invention, regarding specific biomarkers, when determining whether an expression is high (high expression) or low (low expression), those skilled in the art can use classification criteria for expression intensity commonly used in this technical field. For example, for CD62L, the signal intensity corresponding to the 10E2 signal when using the PE biomarker anti-human CD62L antibody can be used as a boundary to clearly classify CD62L. low and CD62L high (WO2018 / 147291).

[0089] In one embodiment of the invention, those skilled in the art can appropriately identify the surface markers of the cells shown to distinguish or count the cells.

[0090] In this specification, "flow cytometry" refers to the technique of measuring the number of cells, individuals, and other biological particles suspended in a liquid, and their respective physical / chemical / biological characteristics. Flow cytometry allows for the analysis of various cell types. In particular, it enables the determination of blood cell differentiation. This differentiation determination is used not only for research but is also beginning to be applied to diagnosis. Advantages of flow cytometry include: easy determination of the percentage of blast cells; high specificity and sensitivity; high reproducibility; the ability to analyze multiple cells; and shorter processing time.

[0091] The device using this technology is called a "flow cytometer." A flow cytometer is an instrument for measuring the optical properties of suspended matter (cells) in a homogeneous suspension of cells. Cells flow through the focal point of a laser beam as the liquid flows through them. During this flow, for each cell at a rate of 500 to 4000 cells per second, the optical properties of forward-scattered light, side-scattered light, and fluorescence at more than one different wavelength can be measured simultaneously. Furthermore, the size, internal structure, and biological characteristics of these cells, such as the amount of various antigens or nucleic acids present in the cell membrane, cytoplasm, and nucleus, can be rapidly and accurately determined.

[0092] Scattered light refers to the light scattered around a cell when a laser beam hits it. Forward scatter (FSC) light is detected in front of the laser axis, and its intensity is proportional to the cell's surface area. That is, a larger FSC value generally indicates a larger cell, and a smaller FSC value generally indicates a smaller cell. Side scatter (SSC) light is detected at a 90-degree angle (right angle) relative to the laser axis, and its intensity is proportional to the cell's granules and the state of its internal structure. That is, a larger SSC value generally indicates a more complex internal cell structure, and a smaller SSC value generally indicates a simpler internal cell structure.

[0093] The results of flow cytometry analysis are typically represented as a dot plot with FSC as the X-axis and SSC as the Y-axis. Each cell is represented by a dot in the plot, and their position is determined by the relative values ​​of FSC and SSC. Cells within a cluster are separated as follows: relatively small lymphocytes with simple internal structures are located in the lower left; larger granulocytes with internal granules are located in the upper right; and larger monocytes with simple internal structures are located between the lymphocytes and granulocytes.

[0094] Fluorescence refers to the light produced when fluorescent pigments labeled on cells are excited by a laser and release energy. Typically, flow cytometers (e.g., Becton & Dickinson FACSCalibur) irradiate cells with a single wavelength laser at 488 nm and a single wavelength laser at 635 nm. Although cells themselves possess the property of emitting weak fluorescence (autofluorescence), in practical applications requiring the specific detection of molecules present in cells using fluorescence, the cells or their molecules must be pre-labeled with fluorescent pigments in some form. For example, FITC (fluorescein isothiocyanate) absorbs excitation light at 488 nm and primarily emits fluorescence (green) at 530 nm. If FITC is pre-labeled with antibodies, the amount of antibody bound varies depending on the amount of antigen present on the cell surface, resulting in different fluorescence intensities of FITC, thus allowing estimation of the amount of antigen present on the cell surface. The FACSCalibur example used in this study is equipped with four fluorescence detectors capable of detecting different fluorescence wavelength regions. If multiple fluorescent pigments emitting different wavelengths of light are pre-prepared, up to four different antigens can be detected simultaneously. As fluorescent pigments other than FITC excited by a single-wavelength 488nm laser, PE (phycoerythrin) mainly emits fluorescence at 585nm, while PerCP (polydinoflavin-chlorophyll-protein complex) and PE-Cy5 (carbonylcyanin-5) mainly emit fluorescence at 670nm. APC (allophycocyanin), a fluorescent pigment excited by a single-wavelength 635nm laser, mainly emits fluorescence at 670nm. Combining these fluorescent pigments with various antibodies can be used for double and triple staining of cells. For molecules such as CD4, CD8, CD62L, CD25, CCR4, CCR6, and CXCR3 expressed on the surface of T lymphocytes, monoclonal antibodies that specifically react with them can be used for detection.

[0095] In the method of this invention, the expression pattern of cell markers can be determined by any method used to analyze the expression distribution of marker proteins, such as flow cytometry, gene analysis, or CyTOF. Typically, the determination of the expression pattern of cell markers in the method of this invention can be performed on cell distribution maps representing the results of flow cytometry (also known as flow cytometry analysis), such as bar charts or dot plots. Particularly for CXCR3... - In the cell distribution map of cell subgroups, CCR4 is defined. - Cellular subgroups and CCR4 + The boundaries of cell subgroups (gates) are used to determine CD4. + CCR4 in the T cell group - CCR6 + The relative amounts of cellular subgroups.

[0096] Strictly speaking, there are two types of flow cytometers: those that analyze only cells and those that can sort (sort) the analyzed cells; the latter is called "FACS". In this specification, "FACS" is an abbreviation for fluorescence-activated cell sorter, which refers to a device that can be used in methods such as using a laser beam to analyze surface antigens on free cells such as lymphocytes, or to sort specific cells based on the presence or absence of surface antigens.

[0097] Regarding the results of flow cytometry analysis, based on the detected optical information, for one or more assays, the cells can be presented as a distribution map of light originating from particles, such as a bar chart or a dot plot (scatter plot), for each assay. In this manual, a "bar chart" refers to a graph plotted using the intensity of the light signal for each parameter as the X-axis and the cell count as the Y-axis during fluorescence assays using a flow cytometer. This format allows for the counting of more than 10,000 cells. In this manual, a "dot plot" refers to a graph plotted using the fluorescence intensity of two fluorescent pigments as the X-axis and Y-axis. In the case of double and triple staining, the analysis can be performed using the following representation: each cell corresponds to a point on a two-dimensional graph with its respective fluorescence intensity as the X-axis or Y-axis. In this manual, "gating," "setting boundaries," and "setting gates" refer to selecting a specific distribution area in the distribution map corresponding to the assay for each assay, in order to perform appropriate measurements.

[0098] For example, after collecting peripheral blood or bone marrow fluid, red blood cells are removed using hemolysis or hydrocentrifugation. The sample is then reacted with fluorescent marker antibodies (antibodies against the target antigen and their control antibodies). After thorough washing, flow cytometry analysis can be used for observation. The detected scattered light and fluorescence are converted into electrical signals, which are then analyzed by a computer. The results show that the intensity of the FSC (freezing cell superposition) indicates cell size, and the intensity of the SSC (saturated cell superposition) indicates intracellular structure, thus allowing differentiation between lymphocytes, monocytes, and granulocytes. Subsequently, gating can be performed on target cell groups as needed to study the expression patterns of cell markers in these cells.

[0099] In the implementation of the method of the present invention, those skilled in the art can appropriately identify the surface markers of the cells shown to distinguish or count the cells.

[0100] Regarding the No Wash-No Lyse method, as described above, since the purpose of this invention is to determine the ratio of specific cell subgroups in T cells using whole blood or PBMCs, data analysis can be facilitated by adding CD45 antibodies. Because CD45 antibodies react with lymphocytes containing T cells but not with erythrocytes and platelets, the target cell is limited to CD45. bright Cell subgroups are used to exclude unwanted cells such as red blood cells and platelets from the analysis.

[0101] (Reagent test kit) In one embodiment of the invention, the kit may include a detection agent for CD4, CCR4, and CCR6 used in samples prepared according to the method of the invention. In another embodiment, the kit may further include a detection agent for CXCR3 and CD62L. In one embodiment, the detection agent is an antibody. Preferably, the antibody is appropriately labeled to facilitate the detection of the biomarker.

[0102] (General Technology) The molecular biology, biochemical, and microbiological methods used in this manual are well-known and commonly used methods in the field, as described in the following references: Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene... Pub. Associates; Ausubel, FM (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, MA et al. (1995). PCR Strategies, Academic Press; Ausubel, FM (1999).Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press; Experimental Medicine Volume: Gene Introduction & Expression Analysis Experimental Methods, Yangtu Publishing House, 1997, etc. Relevant sections (which may be in their entirety) are cited in this manual for reference.

[0103] In this specification, "or" is used when there are "at least one" of the items listed in the text. The same applies to "or". In this specification, when explicitly stated as a "range" of "two values", that range also includes the two values ​​themselves.

[0104] All scientific literature, patents, patent applications, and other references cited in this specification are cited in this specification for reference to the same extent as the specific content described therein.

[0105] The preferred embodiments have been described above to facilitate understanding of the present invention. The present invention will now be described based on embodiments, but the above description and the following embodiments are merely illustrative and not intended to limit the scope of the invention. Therefore, the scope of the present invention is defined only by the claims, and not by the embodiments or examples specifically described in this specification.

[0106] [Example] (Panel) In this embodiment, the following sets (WB set 2 or WB set 3) are used as shown in Table 1.

[0107] [Table 1]

[0108] (Example 1: Comparison of whole blood method and method for separating PBMCs) Blood collected using blood collection tubes containing an anticoagulant (sodium heparin) was used as whole blood and compared with whole blood that was immediately separated into PBMCs and frozen. The sample analyzed was an 8 ml sample of blood collected from the same subject (H-34) that had been heparinized and then divided into two equal parts: one part was used as heparinized whole blood, and the other part was used for PBMC separation.

[0109] For whole blood, staining antibody was added to 100 μl of whole blood, and staining was performed at room temperature after shielding from light for 15 minutes. Then, 2 ml of hemolysis reagent (Becton Dickinson, Cat. 349202) was added, and the mixture was shielded from light for 10 minutes and placed at room temperature to hemolyze the red blood cells. The cells were then centrifuged at 500 × g for 5 minutes at room temperature, and the supernatant was discarded. Next, 2 ml of 2% FBS / PBS was added for resuscitation, and centrifugation was performed under the same conditions. This resuscitation and centrifugation process was repeated, and after removing the supernatant, the cells were resuspended in 250 μl of 2% FBS / PBS for analysis.

[0110] On the other hand, to obtain PBMCs, the remaining whole blood was transferred to a 15 ml conical tube, and twice the volume of physiological saline was added and mixed. This mixture was aliquoted into two Leucosep lymphocyte separation tubes (Greiner, Cat. 163288-013) and centrifuged at 800 × g for 15 minutes at room temperature. The plasma and cell layer at the top was stirred by pipetting, and the volume equivalent to two Leucosep lymphocyte separation tubes was recovered into a 50 ml conical tube. 15 ml of 5% FBS / PBS was added to the recovered cell solution, and the mixture was centrifuged at 500 × g for 5 minutes at 4°C. After removing the supernatant, 10 ml of 5% FBS / PBS was added to resuspend the cells. The cell count was then performed, and the cell solution was aliquoted for the day of analysis and for freezing.

[0111] For the same day's assay, add 10 ml of 5% FBS / PBS and centrifuge again. Remove the supernatant, then add 50 μl of PBS to resuspend the sample, transfer it to an FACS tube, immediately add the staining antibody, protect from light for 20 minutes, and stain at room temperature. Afterward, add 1 ml of 2% FBS / PBS to resuspend the sample, centrifuge at 500×g at 4°C for 5 minutes, remove the supernatant, add 250 μl of 2% FBS / PBS to resuspend the sample, and perform the assay (this is the PBMC staining step). These are considered fresh PBMCs.

[0112] For the frozen fraction, centrifuge at 500×g at 4°C for 5 minutes, remove the supernatant, and add 1 ml of CELLBANKER2 to resuspend it. Aliquot the entire fraction into cryovials (Cryo Tubes), place them in BICELLs cooled to 4°C, and then quickly transfer them to a deepfreezer at -80°C. The next day, transfer them to a liquid nitrogen tank for cryopreservation. After 5 days, thaw them in a 37°C water bath, resuspend them in 12 ml of 10% FBS / RPMI 1640 medium (NacalaiTesque, Cat. 30264-85), and centrifuge at 500×g at 4°C for 5 minutes, removing the supernatant. Add another 10 ml of 10% FBS / RPMI 1640 medium to resuspend them, count the cells, and then halve the cell suspension. One sample was centrifuged at 500×g at 4°C for 5 minutes. After removing the supernatant, 100 μl of PBS was added to resuspend the cells, and PBMC staining was performed for analysis. This was designated as the frozen group. Another sample was aliquoted into 24-well plates and cultured at 37°C in the presence of 5% CO2 for 48 hours. Subsequently, the entire volume of culture medium containing cells was recovered into 15 ml conical tubes, diluted to 10 ml with PBS, and the cell count was performed. The cells were then centrifuged at 500×g at 4°C for 5 minutes. After removing the supernatant, 100 μl of PBS was added to resuspend the cells, and PBMC staining was performed for analysis. This was designated as the PBMC culture group. The following steps were the same.

[0113] Staining was performed using antibodies against CD45, CD4, CCR4, CCR6, and CXCR3 (WB set 2 in Table 1), and assays were performed using a BDFortessa X-20. FlowJo... TM (Becton Dickinson) analyzed the measurement results and plotted them.

[0114] The graphs of whole blood and fresh PBMC are similar. Figure 1 That is, from top to bottom, the scatter plots of CCR4 vs CCR6 for the entire CD4+ cell line, and the scatter plots of CCR4 vs CCR6 for CD4+ cells. + Cells were used to plot a bar chart of CXCR3 and the CXCR3 values ​​in the bar chart were collected. - Similarly, scatter plots of CCR4 vs CCR6 were drawn, and CXCR3 was collected. + When plotting the CCR4 vs CCR6 scatter plots, the patterns in the whole blood and fresh groups are similar in both plots. However, the frozen PBMC group differs from both the whole blood and fresh PBMC groups in any of the plots. For the cultured PBMC group, the distribution of CCR4 vs CCR6 is significantly different. + CXCR3 - CXCR3+ In the scatter plot of CCR4 vs CCR6, CCR6 shows a pattern somewhere between the fresh and frozen groups of PBMCs, but CCR4... + Compared to the fresh group of PBMC, the frozen group showed a decrease. Furthermore, the bar chart for CXCR3 was similar to that of the frozen group of PBMC.

[0115] In summary, the following points have been clarified.

[0116] • Whole blood is similar to the fresh group of PBMC.

[0117] • After PBMCs are frozen, they differ from those in whole blood and fresh blood groups, and they also differ from those in fresh blood groups after culturing.

[0118] • The isolation of CXCR3 deteriorated after cryopreservation and could not be recovered even after culturing.

[0119] CCR4 + The number of cells did not decrease even after cryopreservation, but decreased after incubation.

[0120] CCR6 + The number of blood cells decreased after cryopreservation, recovered slightly after culturing, but was still less than immediately after blood collection.

[0121] (Example 2: Study on the effect of storage time) In this embodiment, the storage time of whole blood was varied and the effect of storage time was studied.

[0122] The following whole blood samples were prepared: freshly collected, stored at room temperature (20℃~22℃) for 3 days, and stored at room temperature for 6 days. Hemolysis, staining, and analysis were performed immediately after collection or after storage. Experiments were conducted using whole blood samples prepared in Example 1 (H-30) and whole blood samples with different collection dates (H-34). The Western blot kit 2 (Table 1) was used for the analysis. No significant differences were observed between freshly collected and stored blood samples. Figure 2 ).

[0123] Furthermore, by increasing the sample size, counting the number of Th7R cells, and comparing the number of days whole blood was stored, the experiment was conducted.

[0124] Whole blood samples prepared in Example 1 (H-30) and samples with different collection dates (H-34) were used. The results are shown in Table 2. H-34 in this table is displayed numerically. Figure 2 The result.

[0125] [Table 2]

[0126] Table 2 also compares whole blood and PBMCs (Table 2, bottom section "2. Whole Blood Method Day 0, Data for Fresh Group, Frozen Group, and Culture Group after PBMC Separation from Whole Blood"). The PBMC processing method is as described in Example 1. H-34 at the bottom of Table 2 shows the numerical values ​​for Example 1 (…). Figure 1 The results were obtained. As a testing kit, WB kit 2 was used. Preparations equivalent to [prepared kit] were made for each sample. Figure 1 Bar charts and scatter plots were generated. FlowJo was used for plotting to obtain cell numbers. CXCR3 was obtained from the CXCR3 bar chart. - and CXCR3 + The number of cells.

[0127] In the upper part of Table 2 (1. Whole blood was stored at room temperature for 0, 3, and 6 days), a total of more than 10,000 CD4 were obtained. + Then, CD4 will be used in subsequent tabulation. + The total number has been adjusted to 10,000.

[0128] The lower part of Table 2 (2. Data from the whole blood method on day 0, and the fresh, frozen, and cultured groups after PBMC isolation from whole blood) shows that approximately 10,000 CD4+ cells were obtained using FlowJo. + The value obtained under the given circumstances.

[0129] Th1 / 17 is about CXCR3 + CCR4 vs CCR6 scatter plot CCR4 - CCR6 + The number of cells in the region. Shown relative to CXCR3. + The ratio.

[0130] CCR6 SP (single positive) is related to CXCR3. - CCR4 vs CCR6 scatter plot CCR4 - CCR6 + The number of cells in the region. Shown relative to CXCR3. - The ratio.

[0131] Th7R represents the total number of Th1 / 17 and CCR6 SPs.

[0132] Th1 is about CXCR3 + CCR4 vs CCR6 scatter plot CCR4 - CCR6 - Cells in the region. Shown relative to CXCR3. + The ratio.

[0133] CCR4 + CCR6- It's about the CX-CR3. + CCR4 vs CCR6 scatter plot CCR4 + CCR6 - Cells in the region. Shown relative to CXCR3. + The ratio.

[0134] TP (triple positive) is related to CXCR3 + CCR4 vs CCR6 scatter plot CCR4 + CCR6 + Cells in the region. Shown relative to CXCR3. + The ratio.

[0135] TN (triple negative) is about CXCR3 - CCR4 vs CCR6 scatter plot CCR4 - CCR6 - Cells in the region. Shown relative to CXCR3. - The ratio.

[0136] Th2 is about CXCR3 - CCR4 vs CCR6 scatter plot CCR4 + CCR6 - Cells in the region. Shown relative to CXCR3. - The ratio.

[0137] Th17 is about CXCR3 - CCR4 vs CCR6 scatter plot CCR4 + CCR6 + Cells in the region. Shown relative to CXCR3. - The ratio.

[0138] (Inspection) 1. Differences caused by the number of days whole blood is stored. Whole blood samples were measured on the day of collection, after 3 days of storage at room temperature, and after 6 days of storage at room temperature. The results were compared, and the results showed that CXCR3 decreased with increasing storage time. + Reduced, but Th1 / 17 in CXCR3 + The proportion of CXCR3 shows a slight increasing trend (Table 2). - Increase, CCR6 SP in CXCR3 - The proportion of Th7R also increased. Additionally, Th7R, which is the sum of Th1 / 17 and CCR6SP, increased slightly. That is, CXCR3... + Reduce CXCR3 - Added, as Th7R CCR4- CCR6 + The total increase. Of which, by Figure 2 As can be seen, the changes were all minor, and as will be discussed later, the data were similar to those of the fresh PBMC group compared to the frozen and cultured groups. That is, it can be concluded that whole blood undergoes relatively small changes during preservation compared to other treatment methods.

[0139] Th1 in CXCR3 + The proportion of TN in CXCR3 - The proportion of CCR4 - CCR6 - The ratio increases slightly. Therefore, CCR4 - CCR6 - The ratio increased.

[0140] CXCR3 + CCR4 in + CCR6 - The ratio and CXCR3 - The proportion of Th2 in CCR4 decreases. + CCR6 - The ratio decreased.

[0141] TP (CCR4) + CCR6 + In CXCR3 + The proportion of Th17 in CXCR3 - The proportions of all of them decreased.

[0142] Combining the above Th1 / 17 and CCR6 SP results, we can observe the ratio in CCR4 vs CCR6, regardless of whether it is CXCR3. - Or CXCR3 + The ratios tend to increase or decrease in a consistent manner, CCR4 - CCR6 - and CCR4 - CCR6 + Increase, CCR4 + CCR6 - and CCR4 + CCR6 + Reduce. That is, CCR4. + Reduce CCR4 - Increased. However, there is no difference to the extent seen when comparing with PBMC.

[0143] 2. Comparison of whole blood and PBMC Compared with the fresh group of whole blood PBMC, CXCR3 + More, CXCR3- Fewer (Table 2). In addition, Th1 / 17 and CCR6 SP are slightly more, and Th7R is also slightly more. But the difference is small.

[0144] Furthermore, considering the cell ratios in the regions defined by CCR4 vs CCR6, the results showed that Th1, TP, and TN were essentially the same in whole blood compared to the fresh PBMC group, while CXCR3 was... + CCR4 + CCR6 - Less, Th2 slightly less, Th17 slightly less. However, the difference is smaller compared to the frozen and cultured groups of PBMC.

[0145] Subsequently, the frozen and cultured groups of PBMCs were included in the comparison. Regarding CXCR3... + Compared to the fresh group, the frozen PBMC group showed variations depending on the specimen, while the variations were significantly reduced for the cultured group. Conversely, CXCR3... - The frozen group was similar to the fresh group, while the cultured group had more cells than the fresh group. Regarding Th1 / 17 relative to CXCR3... + In terms of the ratio, the frozen group of PBMCs had significantly fewer cells than the fresh group, while the culture group had more cells than the frozen group but still fewer than the fresh group. The ratio of CCR6 SPs was similar to that of Th1 / 17, with a significant decrease in the frozen group and a slight decrease in the culture group compared to the fresh group. Regarding the number of Th7Rs, the frozen group had significantly fewer cells than the fresh group, while the culture group had more cells than the frozen group but still fewer than the fresh group.

[0146] That is, due to CXCR3 + The number of Th1 / 17 cells decreased significantly after culture, resulting in a substantial decrease in their number after cryopreservation. A slight increase followed by cryopreservation did not restore them to their initial isolation levels. Conversely, the ratio of CCR6 SP cells decreased significantly due to cryopreservation. Some specimens showed an increase in the CCR6 SP ratio after culture, returning to pre-cryopreservation levels, while others showed only a slight increase. Furthermore, when CD4+ was... + When the cell number is fixed and measurements are performed, compared to immediately after isolation, the CXCR3 level after culture is significantly higher. - The number of Th1 / 17 and CCR6SP increases, while the number of CCR6SP varies depending on the specimen; it can increase or decrease. Therefore, Th7R, which is the sum of Th1 / 17 and CCR6SP, decreases after cryopreservation and increases after culture, but cannot return to the level immediately after isolation. That is, when using PBMC to measure Th7R, it is desirable to perform the measurement immediately after isolation and before cryopreservation.

[0147] Subsequently, the frozen and cultured groups of PBMCs were included in the comparison, focusing on CCR4 vs CCR6. - CCR6 +Comparison with cells existing in regions other than CXCR3. Th1 relative to CXCR3. + The ratio of [CCR4] was slightly higher in the frozen group than in the fresh group, while the cultured group, although slightly lower than the frozen group, was still higher than the fresh group. Regarding CCR4... + CCR6 - The ratio of TP increased in the frozen group and decreased significantly in the cultured group. Regarding the TP ratio, it decreased significantly in the frozen group, and although it increased in the cultured group, it was much lower than in the fresh group. Regarding CXCR3... - The expression patterns of TN, Th2, Th17, CCR4, and CCR6 exist and correspond to CXCR3. + Th1 and CCR4 at different times + CCR6 - The increase and decrease of TP show the same trend. This is also consistent with the relationship between Th1 / 17 and CCR6 SP. That is, the effects of cryopreservation and culture are unrelated to the presence or absence of CXCR3. Overall, CCR4 + While the cells are unaffected by cryopreservation, their numbers decrease if cultured. CCR6 + The number of CCR4 cells decreases when cryopreserved, but recovers when cultured. However, CCR4... + CCR6 + CCR4 exists + Cells, CCR6 + The effects of both cell reduction and cryopreservation are reduced, but not restored after culturing.

[0148] (Example 3: The effect of storage time (2)) The data from Example 2 were used to compare the number of Th7R cells.

[0149] [Table 3]

[0150] In Table 3, the measurements on the left (whole blood) are the same as those in the upper part of Table 2 (1. Whole blood stored at room temperature for 0, 3, and 6 days), and the measurements on the right (whole blood and PBMCs) are the same as those in the lower part of Table 2 (2. Data on day 0 of the whole blood method, and data on the fresh, frozen, and cultured groups after PBMC separation from whole blood).

[0151] The WB kit 2 (Table 1) was used in the determination.

[0152] Here, we will not perform CXCR3-based separation, but will focus on CD4. + Draw a scatter plot of CCR4 vs CCR6. Then plot the scatter plot of CCR4 vs CCR6. Figure 1 and Figure 2 . Figure 1 and Figure 2In addition to the diagram shown, the same graph was also plotted using FlowJo to count the cell number. Because CXCR3 was not isolated... - / CXCR3 + Therefore, only the number of Th7R cells is shown (Table 3).

[0153] Regarding whole blood, no significant differences were observed between immediately after collection and after storage. That is, no problems were found even when testing was performed 6 days after collection.

[0154] The Th7R in the fresh PBMC group was slightly less than that in whole blood.

[0155] The number of Th7Rs in the frozen PBMC group was significantly lower than that in the whole blood and fresh groups. The number of culture groups subsequently obtained did not recover to the level of the whole blood and fresh groups.

[0156] That is, when PBMCs are separated, the Th7R assay must be performed immediately after separation. When PBMCs were separated after whole blood was preserved, the desired assay results were not obtained (see Example 5). Therefore, it is desirable to separate PBMCs and measure them immediately after blood collection. On the other hand, if whole blood is used, results of the same quality as immediately after blood collection can be obtained even after 6 days. Therefore, it is possible to perform the assay within 6 days after transportation at room temperature.

[0157] (Example 4: The effect of storage temperature (3)) In this embodiment, the optimal temperature range for whole blood preservation was investigated. Two experiments were conducted. In the first experiment, measurements were taken after 1, 2, 3, and 6 days at 10°C, 15°C, 20°C, 25°C, 30°C, and 37°C. In the second experiment, measurements were taken after 1, 3, and 6 days at 12°C, 15°C, and 18°C. Heparinized blood was aliquoted into CRYO tubes at 250 μl and stored in water baths or incubators set to their respective temperatures. WB kits 2 and 3 (Table 1) were used for the measurements.

[0158] In the first experiment, when stored at 15°C, water droplets adhered to the inside of the cap, suggesting that the blood might have been concentrated. Therefore, in the second experiment, efforts were made to maintain the temperature effectively, preventing water droplets from adhering to the cap.

[0159] 1. Regarding the expression of CD4 and CD8. The scattering diagram of CD4 vs CD8 when saving with different temperatures and number of days is shown in the figure. Figure 3 The horizontal axis represents the distribution of CD4, and the vertical axis represents the distribution of CD8.

[0160] Will Figure 3 The conditions within the box are evaluated as stable. Regarding CD4 staining, specifically CD4...+ Regarding the frequency and staining pattern, it remained stable for 3 days between 12°C and 25°C. Furthermore, regarding CD8, regardless of the temperature, the fluorescence intensity of CD8 decreased after 2 days, indicating a reduction in expression. It should be noted that in the following analysis, the decrease in fluorescence intensity corresponds to the decrease in CD8 expression. + The door can be moved within its designated area.

[0161] 2. Regarding the expression of CD62L in CD4 Targeting CD4 + Cells, presented as a bar chart showing the staining pattern of CD62L ( Figure 4 The horizontal axis represents the distribution of CD62, and the vertical axis represents the count. Figure 4 The conditions within the box are evaluated as stable. Regarding CD62L... low This indicates that separation is good after several days at high temperatures. Furthermore, at temperatures close to 37°C, staining remains stable even with increased storage time.

[0162] 3. Regarding the expression of CD62L in CD8. For CD8 + Cells, presented as a bar chart showing the staining pattern of CD62L ( Figure 5 The horizontal axis represents the distribution of CD62, and the vertical axis represents the count.

[0163] Will Figure 5 The conditions within the box are evaluated as stable. Regarding CD8... + Cells, in CD8 + When the position is lowered, the corresponding range of movement is used to select CD8. + Cells. With CD4 + The same applies to cells. If the temperature is close to 37°C, the staining remains stable even with increased storage days. However, if the storage time exceeds 2 days, the overall fluorescence intensity weakens.

[0164] 4. Regarding the expression of Th7R in CD4 Targeting CD4 + Cells, focusing on those belonging to CCR4 - CCR6 + A bar chart of fractionated Th7R cells ( Figure 6 The horizontal axis represents the distribution of CCR6, and the vertical axis represents the distribution of CCR4.

[0165] Will Figure 6 The conditions within the box are evaluated as stable. Th7R exhibits good stability over a relatively long storage period, including a storage period of 3 days, with a wide storage temperature range.

[0166] 5. Regarding the expression of CCR7 and CD45RA in CD4. Targeting CD4 + Cells, scatter plots of CCR7 and CD45RA were created ( Figure 7 That is, observe central memory cells, effector memory cells, and naïve cells. The horizontal axis represents the distribution of CCR7, and the vertical axis represents the distribution of CD45RA.

[0167] Will Figure 7 The conditions within the box were assessed as stable. However, the separation of CCR7 staining deteriorated daily with increasing storage days. Therefore, effector memory cells (CCR7)... - CD45RA - ), effector cells (CCR7) － CD45RA + The expression of CD45RA tends to decrease at low temperatures, but remains stable at around 20°C. Therefore, the expression of naïve cells (CCR7) increases. + CD45RA + It works well at around 20℃, and the frequency decreases when the temperature is below or above this.

[0168] 6. Regarding the expression of CCR7 and CD45RA in CD8. For CD8 + Cells, scatter plots of CCR7 and CD45RA were created ( Figure 8 That is, observe central memory cells, effector memory cells, naïve cells, and EMRA cells. The horizontal axis represents the distribution of CCR7, and the vertical axis represents the distribution of CD45RA.

[0169] Will Figure 8 The conditions within the box were evaluated as stable. Up to day 2 of the storage period, the expression of CCR7 and CD45RA was good, basically the same as on day 1. However, after day 2, the expression of CCR7 decreased significantly, and CD45RA also gradually decreased.

[0170] 7. Summary Regarding CD8 + For cell assays, the fluorescence intensity of CD8 remains stable for 2 days. During short periods (less than 2 days), the temperature should ideally be between 18°C ​​and 25°C.

[0171] Regarding CD4, it is stable for 3 days between 12℃ and 25℃, but CD4 + Individual variations exist in the stability of various markers within cells.

[0172] Regarding CD62L, at high temperatures, separation is good after several days, and at around 37°C, it remains stable even with increased storage time.

[0173] The separation of CCR7 staining deteriorated daily with increasing storage time. It was stable at temperatures ranging from 18°C ​​to 25°C.

[0174] Regarding Th7R, it can be well measured over a relatively long storage period, including a storage period of 3 days, with a wide storage temperature range (12℃~25℃).

[0175] (Example 5: PBMC separation from self-preserved whole blood) The usability of residual samples after whole blood testing was investigated. As mentioned above, if whole blood is stored directly, some cellular markers gradually deteriorate and become undetectable. Therefore, this study investigated the separation and preservation of PBMCs after storage.

[0176] Blood was drawn from two healthy individuals using heparinized blood collection tubes. The following four items were prepared:

[0177] #1: The test was performed immediately after blood was collected.

[0178] #2: After storing #1 at room temperature (20.4℃~21.0℃) for 3 days, the results were measured.

[0179] #3: PBMCs were separated from #2 and frozen for 23 days. They were then thawed and analyzed.

[0180] #4: After culturing #3 for 48 hours, the results were measured.

[0181] The WB kit 2 (Table 1) was used for the measurement.

[0182] The results are shown in Figure 9 .

[0183] Table 4 below shows the cell frequencies obtained by each preservation method. It should be noted that the items in Table 4 are as follows.

[0184] CD45: The ratio of CD45 to single cells Lymphocytes: The ratio of lymphocytes to CD45 CD4: The ratio of CD4 to lymphocytes CXCR3 + CXCR3 + Ratio relative to CD4 Th1 / 17: The ratio of Th1 / 17 to CD4 CXCR3 - CXCR3 - Ratio relative to CD4 CCR6 SP: The ratio of CCR6 SP to CD4 [Table 4]

[0185] CD4 under different saving methods + The expression of CCR4, CCR6, and CXCR3 in cells was compared, and the results are shown in... Figure 10 .

[0186] Figure 9 and Figure 10 The samples were identical. Table 4 shows the cell frequencies of the two samples. According to... Figure 9 CXCR3 in PBMCs isolated from self-preserved whole blood, as shown in Table 4, #3 + The number of cells was significantly reduced.

[0187] The culture of #4 yielded a slight increase, but it was still less than that of whole blood from #1 or #2.

[0188] As CXCR3 + CCR4 - CCR6 + The presence of Th1 / 17 with CXCR3 + The same trend was observed. However, the ratio after culture was close to that before culture.

[0189] Additionally, CCR6 SP (CXCR3) - CCR4 - CCR6 + Although it is also similar to the CX-CR3 - It is unrelated, but exists in relation to CXCR3. + The same tendency.

[0190] Therefore, it is shown Figure 10 This was used to identify which biomarkers were affected by the sample processing conditions. Immediate blood samples from #1 were compared with those from #2 to #4. According to the graph, after 3 days of storage, #2 whole blood showed no change in any biomarkers compared to #1. Furthermore, regarding CCR4, #3 and #4 showed no change compared to #1. However, regarding CCR6… + and CXCR3 + CCR6 in #3 and #4 is significantly lower than that in #1. Compared to #3, CCR6 in #4 is lower in #3. + and CXCR3 + It increased slightly, but less than #1 and #2.

[0191] In summary, PBMC separation occurred 3 days later, and CXCR3... + and CCR6 +The reduced number of cells suggests that PBMCs may not be optimal for CXCR3 and CCR6 assays. Therefore, PBMCs isolated from whole blood are not suitable for Th7R assays. Ideally, the blood should be divided into two portions immediately after collection. One portion should be tested within 2 days if tested as a whole blood sample including CD8, or within 3 days if tested as a whole blood sample without CD8. The remaining blood should be immediately separated into PBMCs and frozen.

[0192] (Example 6: The Importance of CXCR3 Measurement) Th7R is defined as CCR4 - CCR6 + CD4 + including CXCR3 + Also includes CX-CR3 - Therefore, in this embodiment, it is also assumed that CXCR3 does not need to be measured when measuring Th7R, and the significance of measuring CXCR3 is investigated.

[0193] Blood was collected from 6 healthy individuals using heparinized blood collection tubes, and measurements were performed immediately using Western blotting kit 2 (Table 1). Data analysis and graphing were performed using FlowJo. The results are presented below. Figures 11A-11D Figure 11 is about CD4. + Or CXCR3 - Scatter plot of CCR6 vs CCR4 in cells.

[0194] Figure 11A CD4 + T cells. Phylogenetic level not shown.

[0195] Figure 11B CXCR3 - CD4 + T cells. Phylogenetic level not shown.

[0196] Figure 11C :and Figure 11A Same. Displayed on CXCR3 - The door constructed in the middle.

[0197] Figure 11D :and Figure 11B Same. Displayed on CXCR3 - The door constructed in the middle.

[0198] according to Figure 11B Depending on the sample, CXCR3 can be clearly observed. - CCR4 high CCR6 - Cells form a cluster, with CXCR3 - CCR4med CCR6 - The cells are separated into groups. On the other hand, according to Figure 11A In the case of CD4 + In the overall scatter plot, CCR4 high CCR6 - Cells and CCR4 med CCR6 - The boundaries between the two cell clusters are difficult to distinguish. Therefore, in Figure 11B In the middle, it is easy to determine CCR4 high The group's gate, as determined in the diagram, is... Figure 11D , Figure 11C The text also records this gate.

[0199] Therefore, it is easier to measure CXCR3. - Perform CCR4 - CCR6 + The gate is set. Therefore, it can be known that in order to perform CCR4 - CCR6 + For specific groups, whole blood samples are ideal for CXCR3 measurement. Compared to PBMCs, this tendency is more pronounced when using whole blood samples, especially after 3 days, as whole blood samples are more likely to show CXCR3 levels compared to PBMCs. - Perform CCR4 - CCR6 + The door is set up.

[0200] (Example 7: Determination of self-collected blood samples using micro-blood collection equipment) The above experiments demonstrate that whole blood can be measured, and only a small amount of whole blood is needed. Therefore, this embodiment investigates the possibility of using a micro-blood collection device. Using a micro-blood collection device as described in this embodiment, the subject can collect blood themselves, which is relatively simple.

[0201] Blood was collected as follows: a BD safety lancet (Becton Dickinson) was used to puncture the fingertip, and blood was collected using an MBS capillary tube (lithium heparin, 100μl–150μl suture) (Micro Blood Science, Inc.). Immediately after collection, blood was stained and analyzed using Western blotting kit 3 (Table 1). The results are presented below. Figure 12 Even extremely small blood samples of 100μl to 150μl can be tested without any problems.

[0202] A sample of 150 μl of blood (approximately 3 drops) can be collected and tested. This method allows for immediate testing in a laboratory setting, even without the presence of a medical professional. Previously, the PBMC method required culture kits, making it unsuitable for urgent control needs. Furthermore, preparing controls required frequent blood collection of approximately 16 ml from healthy individuals each day. However, with this method, only 3 drops of blood are needed for an urgent control.

[0203] (Comparative example: cell immobilization) Paraformaldehyde (PFA) or BD Cell FIX (a solution containing 10.0% formaldehyde, 3.55% methanol, and 0.93% sodium azide) was added to the anticoagulated blood. After being stored at the specified temperature for 3 days, PBMC treatment was performed, followed by staining and measurement.

[0204] (1) Paraformaldehyde Blood was collected from four 4ml blood collection tubes (Nipro, Cat. 31-764) from each of two subjects (H-15 and H-34) and treated with heparin sodium. The blood was then aliquoted into 1ml portions into 10 separate tubes. Two tubes were prepared with PBS added to 4% paraformaldehyde-phosphate buffer, resulting in paraformaldehyde concentrations of 0.5%, 1%, 1.5%, and 2% respectively; the other two tubes contained no additives. Each of the 0% to 2% concentration tubes was incubated at 4°C or room temperature for 3 days.

[0205] For tubes containing 0 to 2% paraformaldehyde, twice the volume of blood in physiological saline was added after the tubes were brought to room temperature. These were then added to Leucosep lymphocyte separation tubes (Greiner, Cat. 163288-013).

[0206] Centrifuge the tubes at 800×g for 15 minutes at room temperature. Stir the plasma and cell layer at the top using a pipette and collect it into a 15ml conical tube. Add an equal volume or more (or 5ml) of 5% FBS / PBS to the collected suspension, centrifuge at 500×g for 5 minutes at 4°C, remove the supernatant, add 10ml of 5% FBS / PBS to resuspend the cells, and count the cells.

[0207] In addition, on the same day, two 8ml blood samples were collected from each BD Vacutainer (blood collection tubes containing anticoagulant and separating agent (BD, Cat. 362753)) and treated with heparin sodium. The samples were centrifuged at 2200×g, 20°C for 20 minutes, and the plasma and cell layer at the top was stirred with a pipette and collected into a 50ml conical tube. 15ml of 5% FBS / PBS was added to the collected suspension, and the samples were centrifuged at 500×g, 4°C for 5 minutes. After removing the supernatant, 10ml of 5% FBS / PBS was added to resuspend the blood, and the cell count was performed. For the blood samples collected using the 8ml Vacutainer, the suspension was allocated to the same-day testing portion and the frozen portion.

[0208] For the portion stored for 3 days and the portion measured on the same day using 8 ml of BD Vacutainer, after counting, centrifugation was performed. After removing the supernatant, 50 μl of PBS was added to suspend the sample, which was then transferred to FACS tubes. Immediately, staining antibody was added, and the sample was stained at 4°C in the dark for 20 minutes. Subsequently, 1 ml of 2% FBS / PBS was added to suspend the sample, and centrifugation was performed at 500×g and 4°C for 5 minutes. After removing the supernatant, 250 μl of 2% FBS / PBS was added to suspend the sample, and the measurement was performed.

[0209] For the 8ml frozen portion of the BD Vacutainer, centrifuge at 500×g and 4℃ for 5 minutes, remove the supernatant, and then divide into approximately 5.0×10 frozen tubes per tube. 6 Cellbanker 2 was added to a concentration of cells / ml to suspend the cells. 1 ml of the solution was aliquoted into cryovials, placed in BICELL containers cooled to 4°C, and then immediately stored at -80°C. The next day, the cells were transferred to liquid nitrogen for cryopreservation. Two days later, the cells were thawed in a 37°C water bath and resuspended in 12 ml of 10% FBS / RPMI 1640 medium (Nacalai Tesque, Cat. 30264-85). The cells were centrifuged at 500×g for 5 minutes at 4°C, and the supernatant was removed. Another 10 ml of 10% FBS / RPMI 1640 medium was added to suspend the cells. After cell count, the cell culture was aliquoted into 24-well plates and cultured at 37°C in the presence of 5% CO2 for 48 hours. Subsequently, the entire volume of culture medium containing the cells was recovered into 15 ml conical tubes, and the cell count was re-established. Subsequently, the samples were centrifuged at 500×g and 4℃ for 5 minutes. After removing the supernatant, 100μL of PBS was added to resuspend the samples, and PBMC staining and analysis were performed. This was designated as the PBMC culture group. The same procedure was followed below. A sample overview is shown below.

[0210] No. Specimen name, contents after 3 days, or other processing. 1. H-15 PBMCs were isolated, frozen, and then cultured for 2 days after thawing (PBMC culture group). 2. H-15 PBMCs were not preserved after isolation (fresh PBMC group) 3. H-152% paraformaldehyde, 4℃ 4. H-15 1.5% paraformaldehyde, 4℃ 5. H-151% paraformaldehyde, 4℃ 6. H-150.5% paraformaldehyde, 4℃ 7. H-150% paraformaldehyde, 4℃ 8. H-152% paraformaldehyde, room temperature 9. H-15 1.5% paraformaldehyde, room temperature 10. H-15 1% paraformaldehyde, room temperature 11. H-15 0.5% paraformaldehyde, room temperature 12. H-15 0% paraformaldehyde, room temperature 13. H-34 PBMCs were isolated, cryopreserved, and cultured for 2 days after thawing (PBMC culture group). 14. H-34 PBMCs were not preserved after isolation (fresh PBMC group) 15. H-34 2% paraformaldehyde, 4℃ 16. H-34 1.5% paraformaldehyde, 4℃ 17. H-34 1% paraformaldehyde, 4℃ 18. H-34 0.5% paraformaldehyde, 4℃ 19. H-34 0% paraformaldehyde, 4℃ 20. H-34 2% paraformaldehyde, room temperature 21. H-34 1.5% paraformaldehyde, room temperature 22. H-34 1% paraformaldehyde, room temperature 23. H-34 0.5% paraformaldehyde, room temperature 24. H-34 0% Paraformaldehyde, room temperature The results are shown below.

[0211] <CD4> Room temperature: CD4 expression could not be confirmed in subjects with a final concentration of 2-1% PFA.

[0212] Even for those who added 1%, only weaker expressions were confirmed.

[0213] 4℃: CD4 expression could not be confirmed in subjects with a final concentration of 2-1% PFA.

[0214] Even with the addition of 0.5%, only a weak expression was confirmed.

[0215] <CXCR3> Room temperature: Since CD4 was not expressed, the clear bimodality of CXCR3 could not be confirmed.

[0216] 4℃: Although it exhibits a bimodal distribution, expression is unstable, with CXCR3 showing a gradual decrease as the final PFA concentration decreases from 2% to 0.5%. + The ratio tends to gradually increase and peak at 1%.

[0217] <CCR6> In the samples where expression was confirmed, CCR6 without added PFA was observed. + The expression is 10 3 Left and right, in contrast, add those with PFA to confirm up to 10. 4 The above expressions are quite high, but the expression is unstable.

[0218] <CCR4> Among the samples where expression was confirmed, those with added PFA had a narrower expression range and poorer separation compared to those without added PFA.

[0219] (2) BD Cell FIX Blood was collected from two subjects (H-15 and H-34) in 4ml tubes and treated with heparin sodium. 1ml of each tube was then aliquoted into four separate tubes. A solution obtained by diluting BD Cell FIX (BD, Cat. 340181: discontinued, currently sold as a 3X stabilizer concentrate) with 2% FBS / PBS was added to each tube, resulting in final dilution ratios of ×4, ×10, and ×20. No additives were added to any of the tubes. The tubes were then incubated at 4°C for 3 days.

[0220] The following procedure is the same as for paraformaldehyde. That is, to separate PBMCs. A sample overview is shown below.

[0221] No. Specimen Name Processing 25. H-15 without Cell Fix, 4℃, 3 days 26. H-15×4 Cell FIX, 4℃, 3 days 27. H-15×10 Cell FIX, 4℃, 3 days 28. H-15×20 Cell FIX, 4℃, 3 days 29. H-34 without Cell Fix, 4°C, 3 days 30. H-34×4 Cell FIX, 4℃, 3 days 31. H-34×10 Cell FIX, 4℃, 3 days 32. H-34×20 Cell FIX, 4℃, 3 days The results are shown below.

[0222] 4-fold dilution: No lymphocytes were observed.

[0223] 10-fold dilution: Lymphocytes are barely visible.

[0224] 20-fold dilution: Although CD4 expression is still visible, it is below the usual expression position, and the CXCR3 cutoff is also shifted to the left and unstable.

[0225] As mentioned above, there are problems with the detection of CD4 or CXCR3, especially when stabilizers are added to anticoagulated blood to fix cells. Therefore, an efficient assay for Th7R found in whole blood samples is advantageous.

[0226] (Example 8: Comparison of anticoagulants) Purpose In most cases of blood collection at the medical site, blood collection tubes containing EDTA as an anticoagulant are used. Therefore, heparin sodium, EDTA, sodium citrate, and sodium fluoride are compared as anticoagulants.

[0227] method Blood was collected from two healthy individuals. Two 2mL blood collection tubes (1.8mL for sodium citrate only) were used, each containing one of the following coagulants: sodium heparin (Nipro, NEO tube NP-HE0405), EDTA 2kJ (Sekisui Medical Co., Ltd., INSEPACKII SMD520EK-Purple-ST), 3.8% sodium citrate (Nipro, NEO tube OP-CB0165-12), or sodium fluoride + disodium EDTA (Nipro, NEO tube OP-FN0205). After collection, the blood was thoroughly mixed. One tube was kept unopened and stored horizontally at room temperature. The other tube was opened, and 100μL was aliquoted into a FACS tube, which was also stored horizontally, similarly to the unopened tube. Antibody WB kit 3 was added to this FACS tube, and the tube was protected from light for 15 minutes before staining at room temperature. Next, 2 mL of hemolysis reagent (solution solution, BD 349202) was added, the mixture was shielded from light for 10 minutes, and placed at room temperature for hemolysis. Then, the mixture was centrifuged at 500×g at room temperature for 5 minutes, and the supernatant was discarded. 2 mL of 2% FBS / PBS was added to suspend the mixture, and centrifugation was performed under the same conditions. This suspension and centrifugation process was repeated, and after removing the supernatant, the mixture was resuspended in 250 μL of 2% FBS / PBS for analysis using FACSymphony. For opened blood collection tubes, 100 μL samples were taken after 1 and 2 days for hemolysis, staining, and analysis. For unopened blood collection tubes, they were opened after 3 days, and 100 μL samples were subjected to hemolysis, staining, and analysis using the same method.

[0228] Results and Discussion Using the day of blood collection with the most stable heparin sodium as the baseline, the ratio of cell clusters was compared for samples collected with different coagulants and for different storage days. The results are shown in Tables 5-1 to 5-4. Underlined numbers indicate increases or decreases of more than 20% compared to the immediate blood collection with heparin sodium.

[0229] [Table 5-1]

[0230] [Table 5-2]

[0231] [Table 5-3]

[0232] [Table 5-4]

[0233] In the case of EDTA, as the number of days of storage increases, CD4...+ CD62L low The ratio increases, CD4 + CD62L high The ratio decreased (Table 5). Additionally, the expression intensity of CXCR3 decreased, and CXCR3... - The ratio of CXCR3 increased. + The ratio decreased. Among them, Th7R (CD4) + CCR6 + CCR4 - The changes in the ratio of [specific cells] were relatively small. Results were consistent with those obtained with heparin sodium in measurements taken on the day of blood collection and Day 1. However, after preservation, changes in cell populations were observed. The following cells (preservation days) showed increases or decreases of more than 20% compared to the day of heparin sodium collection. Differences in the increase or decrease also existed between different samples.

[0234] Add: CD4 + CD62L low (Day 3), CXCR3 - CCR6 - CCR4 - (Day 3) Subtract: CCR6 - CCR4 + (Day 2, Day 3), CCR6 + CCR4 + (Day 1, Day 2, Day 3), CXCR3 + (Day 3), CXCR3 + CCR6 - CCR4 + (Day 1, Day 2, Day 3), CXCR3 + CCR6 + CCR4 + (Day 1, Day 2, Day 3), CXCR3 + CCR6 + CCR4 - (Day 2, Day 3), CXCR3 - CCR6 - CCR4 + (Day 3), CXCR3 - CCR6 + CCR4 + (Day 2, Day 3), CD62L high CD4 + CD25+Treg (Day 2, Day 3) Increases and decreases were observed for all cell markers in the presence of sodium citrate and sodium fluoride (not illustrated). The following can be gleaned from the above results.

[0235] The best-performing anticoagulant was heparin sodium.

[0236] EDTA can also be used for the determination of Th7R.

[0237] (Note) As illustrated above, the present invention has been described using preferred embodiments, but it should be understood that the scope of the invention should be interpreted solely by the claims. The contents of patents, patent applications, and other documents referenced in this specification are themselves equivalent to the contents specifically described herein and should be understood as being cited as references to this specification. This application claims priority to Japanese Patent Application No. 2023-72026, filed with the Japan Patent Office on April 26, 2023, the contents of which are incorporated herein by reference as constituting the entirety of this application.

[0238] Industrial availability This invention can be used to measure CD4 + The relative amounts of medically important cell subgroups within the T cell group.

Claims

1. A CD4 sample derived from a subject + CCR4 in T cell group - CCR6 + A method for determining the relative amounts of cell subgroups, comprising the following steps: Select the CD4 + CXCR3 in the T cell group - Cellular subgroups; In the CXCR3 - In the cell subgroup, CCR4 was set. - Cellular subgroups and CCR4 + The boundaries of cellular subgroups; and Using the aforementioned boundary, CD4 was determined. + CCR4 in the T cell group - CCR6 + The relative amounts of cellular subgroups.

2. The method according to claim 1, wherein, The sample is a whole blood sample or a peripheral blood mononuclear cell (PBMC) sample from the subject.

3. The method according to claim 2, wherein, The sample was a whole blood sample.

4. The method according to claim 3, wherein, The sample is a sample taken from the subject and stored for more than one day.

5. The method according to claim 4, wherein, The samples were collected from the subjects and stored for 1 to 6 days.

6. The method according to claim 4, wherein, The samples were collected from the subjects and stored for 1 to 3 days.

7. The method according to claim 4, wherein, The samples were stored at approximately 10°C to approximately 40°C.

8. The method according to claim 4, wherein, The samples were stored at approximately 12°C to approximately 25°C.

9. The method according to claim 3, wherein, The sample size is approximately 10 μl to approximately 500 μl.

10. A CD4 sample derived from a subject + CCR4 in T cell group - CCR6 + A method for determining the relative amounts of cell subgroups, comprising the following steps: Prepare whole blood samples from the subjects; and The CD4 count of the whole blood sample was measured. + CCR4 in the T cell group - CCR6 + The relative amounts of cellular subgroups.

11. The method according to claim 10, wherein, The sample is a sample taken from the subject and stored for more than one day.

12. The method according to claim 11, wherein, The samples were collected from the subjects and stored for 1 to 6 days.

13. The method according to claim 11, wherein, The samples were collected from the subjects and stored for 1 to 3 days.

14. The method according to claim 11, wherein, The samples were stored at approximately 10°C to approximately 40°C.

15. The method according to claim 11, wherein, The samples were stored at approximately 12°C to approximately 25°C.

16. The method of claim 10, wherein, The sample size is approximately 10 μl to approximately 500 μl.

17. A kit for use in the method of any one of claims 1 to 16, wherein, It includes detection reagents for CD4, CCR4, and CCR6.

18. The kit according to claim 17, wherein, It further includes a detection reagent for CXCR3.

19. The kit according to claim 16, wherein, The detection reagent is an antibody.

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