Method for measuring cellular immune response activity and kit therefor

Abstract

Provided is a method for measuring a subject's cellular immune response activity, the method comprising: (1) incubating an aqueous medium comprising peripheral blood mononuclear cells (PBMCs) collected from a subject, an antigen, and human serum or plasma; and (2) subsequent to (1), measuring immune effector molecules within the PBMCs.

Description

Method for measuring cellular immune response activity and kit therefor

[0001] The present invention relates to a method for measuring cellular immune response activity and a kit therefor.

[0002] Cellular immunity is one of the immune responses of the body that eliminates foreign substances that invade from the outside. Cellular immunity recognizes foreign substances such as pathogens that have invaded the body and reacts with CD4 through antigen-presenting cells. ＋ T cells and / or CD8 ＋ It is an immune response system that activates T cells to eliminate foreign substances (or cells containing foreign substances, such as infected cells) (Non-Patent Document 1). By measuring the activity of the cellular immune response, the level of the immune response of the living body can be determined.

[0003] The activity of the cellular immune response can be measured by adding an antigen derived from a pathogen or the like to a sample containing peripheral blood mononuclear cells (PBMCs) derived from a subject to stimulate leukocytes such as T cells contained in the PBMCs, and then detecting effector molecules such as cytokines (e.g., interferon-γ) produced by the stimulated leukocytes, detecting gene expression of effector molecules in leukocytes, or detecting leukocytes expressing effector molecules. As examples of methods for measuring the activity of a subject's cellular immune response against a pathogen, methods for measuring the activity of a subject's cellular immune response against SARS-CoV-2 have been reported, including a method using ELISpot technology (Non-Patent Document 2), an ELISA method (Non-Patent Document 3), a reverse transcription polymerase chain reaction (RT-PCR) method (Patent Document 1), and a flow cytometry technology (Non-Patent Document 4).

[0004] Samples containing leukocytes such as T cells used in methods for measuring cellular immune response activity include whole blood, which is collected as is, and PBMCs separated from collected blood. From the viewpoint of long-term storage and transportation of the sample, it is preferable that the sample can be cryopreserved. As a preservation solution for stably cryopreserving cells, cell cryopreservation solutions such as CELLBANKER (registered trademark) (manufactured by Nippon Zenyaku Kogyo Co., Ltd.) are known.

[0005] The method of measuring cellular immune response activity using whole blood does not require a step of separating PBMCs from whole blood and is simpler than the method of measuring cellular immune response activity using PBMCs. However, it has the disadvantage that whole blood (fresh blood) must be used immediately after collection because it cannot be cryopreserved using the above-mentioned cell cryopreservation solution.

[0006] On the other hand, PBMCs can be stored in a frozen state for a long period of time by using the cell cryopreservation solution, and are therefore more suitable than whole blood in terms of long-term storage.

[0007] WO 2023 / 167250

[0008] Current Opinion in Immunology Vol3 pp471-475, 1991International Journal of Infectious Diseases, Vol 113. pp 155-162, 2021International Journal of Infectious Diseases, Vol 106. pp 338-347, 2021med Rxiv 2020 Nov3;2020.10.30.20223099.

[0009] For the above reasons, PBMCs are often used as samples for measuring cellular immune response activity. However, when the present inventors compared the activity signal obtained when measuring cellular immune response activity using PBMCs as a subject's sample with the activity signal obtained when measuring cellular immune response activity using whole blood as a subject's sample, they found that the activity signal obtained when measuring cellular immune response activity using PBMCs as a subject's sample was lower.

[0010] The present invention aims to provide a method for measuring cellular immune response activity with sufficient sensitivity using PBMCs as a subject's sample, and a measurement kit for measuring cellular immune response activity with sufficient sensitivity using PBMCs as a subject's sample.

[0011] As a result of extensive research, the present inventors have found that in a method for measuring cellular immune response activity using PBMCs as a subject's sample, by performing a reaction in which PBMCs are stimulated with an antigen in the presence of human serum or plasma, it is possible to measure cellular immune response activity with sufficient sensitivity, and have thus completed the present invention.

[0012] The present invention includes, for example, the following aspects: [1] A method for measuring the cellular immune response activity of a subject, comprising: (1) a step of incubating an aqueous medium containing peripheral blood mononuclear cells (PBMCs) collected from the subject, an antigen, and human serum or plasma; and (2) a step of measuring immune effector molecules in the PBMCs after step (1). [2] The method of [1], wherein the human serum or plasma is derived from the subject. [3] The method of [1] or [2], wherein the content of human serum or plasma in the aqueous medium in step (1) is 20% or more and less than 100% based on the total mass of the aqueous medium. [4] In step (1), the mass of human serum or plasma in the aqueous medium before incubation is 0.3 x 10 ６

[0014] The method of any one of [1] to [3], wherein the amount of the aqueous medium is 0.05 mL to 1.0 mL per PBMC of cells. [5] The method of any one of [1] to [4], wherein the PBMCs have been frozen and thawed after collection from the subject. [6] The method of any one of [1] to [5], further comprising, before step (1), a step of separating and / or isolating PBMCs from whole blood collected from the subject. [7] The method of any one of [1] to [6], wherein the aqueous medium does not contain a liquid culture medium. [8] The method of any one of [1] to [7], wherein measuring immune effector molecules is measuring the expression level of mRNA of the immune effector molecules. [9] A kit for measuring cellular immune response activity of a subject by the method of any one of [1] to [8].

[10] A kit for measuring a subject's cellular immune response activity using peripheral blood mononuclear cells (PBMCs) incubated with an antigen in human serum or plasma, comprising: (1) an antigen; and (2) a reagent for measuring immune effector molecules.

[11] The kit according to

[10] , further comprising: (3) a PBMC separation tube or reagent for separating and / or isolating PBMCs; (4) a reagent for purifying RNA from PBMCs; and (5) a reagent for synthesizing cDNA from mRNA, wherein reagent (2) is a reagent for measuring the mRNA expression level of an immune effector molecule.

[12] The kit according to any of [9] to

[11] , further comprising an instruction manual or label describing how to use the kit for measuring cellular immune response activity.

[13] The kit according to any of [9] to

[12] , further comprising a density gradient medium for density gradient centrifugation.

[14] The kit according to any of [9] to

[13] , further comprising a blood collection tube.

[15] The method according to any one of [1] to [8], further comprising the step of collecting peripheral blood mononuclear cells (PBMCs) from the subject.

[0013] According to the present invention, it is possible to measure cellular immune response activity with sufficient sensitivity by using PBMCs, which can be stored in a frozen state for a long period of time, as a specimen from a subject.

[0014] 1 is a graph showing the results of measuring IFN-γ (IFNG) gene expression using fresh blood.

[0033] FIG. 1 is a graph showing the results of measuring IFN-γ (IFNG) gene expression using PBMC.

[0034] FIG. 1 is a graph showing the results of measuring IFN-γ (IFNG) gene expression after antigen stimulation of PBMC in plasma from the same subject.

[0035] FIG. 1 is a graph showing the results of a Student's t-test performed on the cellular immune activity when PBMCs were antigen-stimulated in plasma from the same subject and the cellular immune activity when PBMCs were antigen-stimulated in RPMI medium containing no human serum or human plasma.

[0036] FIG. 1 is a graph showing the average values ​​of six cases of the cellular immune activity when PBMCs were antigen-stimulated in plasma from the same subject and the cellular immune activity when PBMCs were antigen-stimulated in RPMI medium containing no human serum or human plasma relative to the measured value of fresh blood (relative values ​​when the measured value of fresh blood is set to 100).

[0015] Preferred embodiments of the present invention will be described in detail below, but the present invention is not limited to the following embodiments.

[0016] The method for measuring the cellular immune response activity of a subject in this embodiment includes: (1) a step of incubating an aqueous medium containing peripheral blood mononuclear cells (PBMCs) collected from the subject, an antigen, and human serum or plasma; and (2) a step of measuring immune effector molecules in the PBMCs after step (1).

[0017] The incubation step (step (1)) in this embodiment is a step of stimulating PBMCs by adding an antigen. This causes the stimulated PBMCs to produce immune effector molecules. By stimulating PBMCs in the presence of human serum or plasma, cellular immune response activity can be measured with sufficient sensitivity using PBMCs.

[0018] By incubating PBMCs with antigens, antigen-presenting cells take up the antigens, process them as needed, and present them on the cell surface together with major histocompatibility complex (MHC) molecules. When T cell receptors (TCRs) recognize the MHC-antigen complex, T cells are activated and produce immune effector molecules.

[0019] In this embodiment, the subject may be a human or a non-human animal. The non-human animal is preferably a mammal. The subject may be an individual infected with a specific pathogen, an individual suspected of being infected, an uninfected individual, or an individual vaccinated against the specific pathogen. For example, if the specific pathogen is SARS-CoV-2, the subject may be an individual infected with SARS-CoV-2, an individual suspected of being infected with SARS-CoV-2, an individual uninfected with SARS-CoV-2, or an individual vaccinated against SARS-CoV-2.

[0020] Whole blood is usually collected from a vein and stored in a blood collection tube containing an anticoagulant. Examples of blood collection tubes include EDTA blood collection tubes and sodium heparin blood collection tubes. Because EDTA may affect lymphocyte activity, sodium heparin blood collection tubes are more preferred.

[0021] Peripheral blood mononuclear cells (PBMCs) are obtained by removing plasma components, red blood cells, platelets, and granulocytes from whole blood and separating them. They contain monocytes and T cells (CD4 ＋ Cell / CD8 ＋ The term "peripheral blood mononuclear cells (PBMCs) collected from a subject" refers to PBMCs derived from a subject and obtained by "separation" from whole blood. Therefore, in step (1), "peripheral blood mononuclear cells (PBMCs) collected from a subject," "antigen," and "human serum or plasma" are, in principle, prepared separately. However, when the PBMCs and human serum or plasma are derived from the same subject, the PBMCs and human serum or plasma can be simultaneously separated from whole blood and used.

[0022] The method of this embodiment may include, prior to step (1), a step of preparing PBMCs from a subject. The step of preparing PBMCs from a subject may be or may include a step of separating and / or isolating PBMCs from whole blood (a specimen) collected from the subject. Separation of PBMCs from whole blood may be performed by density gradient centrifugation, which may be performed using a density gradient medium such as Ficoll or LymphoPrep. For example, whole blood may be stratified in a centrifuge tube containing a density gradient medium and centrifuged, whereby PBMCs gather at the interface between the density gradient medium and plasma. PBMCs can be obtained by collecting the white layer (PBMC layer) formed at this interface.

[0023] PBMCs can be cryopreserved and can maintain their cellular function for a long period of time. They can be cryopreserved using a preservation solution that stably freezes and preserves cells. Examples of such preservation solutions include cell cryopreservation solutions such as CELLBANKER (registered trademark) (manufactured by Nippon Zenyaku Kogyo Co., Ltd.). PBMCs used in the method of this embodiment may also be frozen and thawed after collection from a subject. Even when frozen and thawed PBMCs are used, the method of this embodiment allows for the measurement of cellular immune response activity with sufficient sensitivity.

[0024] Human serum or plasma can be obtained by centrifuging collected blood and recovering the liquid portion. Serum is obtained by allowing collected blood to clot naturally and then centrifuging. Meanwhile, plasma is obtained by centrifuging in the presence of an anticoagulant. Serum does not contain coagulation factors such as fibrinogen. Human serum or plasma may be derived from a single donor, for example, from the subject in the method of this embodiment. When human serum or plasma is derived from a subject, whole blood collected from the subject can be separated and used as PBMCs and human serum or plasma.

[0025] The antigen used in the method of this embodiment is not particularly limited, and examples thereof include proteins derived from pathogens such as viruses, bacteria, and parasites; proteins derived from cells recognized as non-self in vivo, such as tumor cells, infected cells, and inflammatory cells; and peptides containing the amino acid sequences constituting the above proteins. Two or more types of antigens may be combined.

[0026] Examples of viruses from which antigens can be derived include SARS-CoV-2, human immunodeficiency virus (HIV), influenza virus, cytomegalovirus (CMV), hepatitis B virus, measles virus, rubella virus, poliovirus, and human papillomavirus (HPV). Examples of proteins derived from viruses include the spike protein (S protein) of SARS-CoV-2, HIV Gag protein, HIV envelope protein (gp120), influenza virus M1 protein, influenza virus hemagglutinin (HA), CMV pp65 protein, hepatitis B virus surface antigen (HBsAg), and HPV E7 protein.

[0027] Examples of bacteria from which antigens can be derived include Mycobacterium tuberculosis, Listeria monocytogenes, Salmonella enterica, Staphylococcus aureus, and Escherichia coli. Examples of proteins derived from bacteria include ESAT-6 from Mycobacterium tuberculosis and listeriolysin O (LLO) from Listeria monocytogenes.

[0028] Examples of tumor cells from which antigens are derived include neoantigen-expressing cells, gp100, MART-1, PSA, CEA, AFP, MUC-1, CA125, CA19-9, HER2, Survivin, and WT-1 positive cells. Examples of proteins derived from tumor cells include tumor-specific proteins expressed on the cell surface of tumor cells.

[0029] Infected cells from which antigens are derived include cells infected with the above-mentioned viruses, etc. Proteins derived from infected cells include, for example, viral proteins displayed on the cell surface of infected cells.

[0030] Inflammatory cells from which antigens are derived include cells involved in autoimmune responses that occur in autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, and type 1 diabetes. Proteins derived from inflammatory cells include, for example, proteins expressed on the cell surface of inflammatory cells.

[0031] It is well established in the art that antigen presentation and antigen recognition can be carried out in an aqueous medium in vitro or ex vivo, and those skilled in the art can select or prepare an aqueous medium suitable for that purpose based on their ordinary knowledge.

[0032] In this embodiment, the aqueous medium contains PBMCs, an antigen, and human serum or plasma. The content of human serum or plasma in the aqueous medium during the incubation step may be 20% or more but less than 100% based on the total mass of the aqueous medium. For example, when adding PBMCs and antigen, the medium other than the PBMCs and antigen (e.g., a storage buffer) can be replaced with human serum or plasma. When the PBMCs and antigen prepared in this manner are added to human serum or plasma, the aqueous medium excluding the PBMCs and antigen becomes approximately 100% human serum or plasma. The content of human serum or plasma in the aqueous medium in the incubation step may be 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 99.9% or more, based on the total mass of the aqueous medium. The aqueous medium may contain, for example, water and / or a liquid medium for cell culture in addition to PBMCs, antigen, and human serum or plasma, or may not contain a liquid medium.

[0033] In the incubation step, the mass of human serum or plasma in the aqueous medium before incubation is not particularly limited as long as the effects of the present invention are achieved. ６ For PBMC cells, the amount may be, for example, 0.05 mL to 1.0 mL, 0.05 mL to 0.5 mL, 0.1 mL to 0.3 mL, or 0.1 mL to 0.2 mL.

[0034] The conditions for incubating an aqueous medium containing PBMCs, an antigen, and human serum or plasma are not particularly limited and can be appropriately determined by a person skilled in the art, but can be carried out, for example, at a temperature in the range of 30° C. to 40° C., preferably 36.5° C. to 37.5° C. Furthermore, incubation can be carried out for, for example, 1 hour or more, 1 hour to 72 hours, 1 hour to 48 hours, 1 hour to 24 hours, 1 hour to 2 hours, 2 hours to 3 hours, 3 hours to 4 hours, 4 hours to 5 hours, 5 hours to 6 hours, 6 hours to 7 hours, 6 hours to 8 hours, 8 hours to 24 hours, 1 hour to 4 hours, or 2 hours to 4 hours.

[0035] In the incubation step, the content of the antigen in the aqueous medium before incubation is not particularly limited and can be appropriately determined by a person skilled in the art, and may be, for example, 0.1 to 20 μg / mL, 0.2 to 10 μg / mL, or 0.5 to 5 μg / mL.

[0036] Furthermore, the number of PBMC cells in the aqueous medium before incubation in the incubation step is not particularly limited and can be appropriately determined by those skilled in the art. For example, 0.2 × 10 ６ cells~0.3×10 ６ It may be cells / mL.

[0037] The measuring step (step (2)) in this embodiment is a step of measuring immune effector molecules in PBMCs after stimulating the PBMCs with an antigen in the incubation step. The term "measuring" used in the context of cellular immune response activity may include not only quantifying the cellular immune response activity, but also determining or testing for the presence or absence of significant cellular immune response activity based on the quantification result (e.g., in comparison with a negative control and / or a positive control).

[0038] White blood cells include lymphocytes, neutrophils, eosinophils, basophils, and monocytes. Monocytes differentiate into macrophages and dendritic cells, so they are also considered to be part of white blood cells. Examples of lymphocytes include T cells, B cells, and natural killer cells. Furthermore, T cells contain CD4 ＋ T cells and CD8 ＋ PBMCs are white blood cells excluding granulocytes (neutrophils, eosinophils, basophils, and mast cells).

[0039] The activity of the cellular immune response can be measured by measuring immune effector molecules in PBMCs after stimulating the PBMCs with an antigen in an incubation step.

[0040] Measurement of immune effector molecules can be performed by detecting the immune effector molecules, detecting gene expression of the immune effector molecules in PBMCs, detecting PBMCs expressing the immune effector molecules, etc. Measuring immune effector molecules may involve measuring the expression level of mRNA of the immune effector molecules, or may involve detecting and / or quantifying proteins that are immune effector molecules.

[0041] Methods for measuring the expression level of immune effector molecule mRNA include, for example, hybridization-based methods such as Northern blot and microarray, RNase protection, and RNA-Seq (sequencing). However, from the viewpoints of speed, simplicity, reliability, etc., reverse transcription polymerase chain reaction (RT-PCR) is preferred, and real-time RT-PCR is particularly preferred due to its excellent quantitative properties. Examples of real-time RT-PCR include the SYBR (registered trademark) Green method and the TaqMan (registered trademark) method. In addition, examples of methods that can be used simply and quickly in clinical settings include the loop mediated isothermal amplification (LAMP) method, which is an isothermal nucleic acid amplification method, the helicase dependent amplification method, the rolling circle amplification method, the recombinase polymerase amplification method, the invader assay method, etc. In addition, examples of more quantitative methods include the digital droplet PCR method and the digital droplet LAMP method.

[0042] To measure the expression level of immune effector molecule mRNA, RNA may be extracted from PBMCs, and specific cell subsets (e.g., CD4 ＋ T cells and CD8 ＋ Alternatively, RNA may be extracted after isolating cells (e.g., T cells) from the target cells. RNA extraction can be performed by a total RNA extraction method using commonly available commercially available magnetic beads, centrifugal columns, or the like. Examples of RNA include mRNA, RNA fractions containing mRNA, and total RNA. When measuring the expression level of mRNA of an immune effector molecule, the mRNA to be measured may be mature mRNA that has undergone various processes such as splicing, or RNA that has not yet been processed to become mature mRNA (pre-mRNA).

[0043] When using reverse transcription polymerase chain reaction (RT-PCR), the extracted RNA is then converted to cDNA using reverse transcriptase, and quantitative PCR (qPCR) can be performed using the cDNA as a template and primers specific to the immune effector molecule. The qPCR master mix used for quantitative PCR can be the above-mentioned SYBR® Green, TaqMan®, or the like. The conditions for the qPCR reaction can be appropriately set by those skilled in the art. The mRNA expression level of the immune effector molecule can be measured by normalizing the obtained relative mRNA expression level to an internal control gene. In the case of real-time RT-PCR, fluorescent signals can be collected for each cycle of the qPCR reaction, allowing the amplification of the PCR product to be monitored in real time.

[0044] The step of measuring immune effector molecules in PBMCs may be, for example, an RT-PCR method comprising the following steps (a) to (b) or (a) to (c): (a) lysing PBMCs to obtain a lysate containing mRNA, (b) capturing mRNA from the lysate, and (c) measuring the mRNA level.

[0045] Step (a) may involve, for example, lysing cells using a cell lysis buffer. Cell lysis elutes mRNA contained in PBMCs, yielding a lysate containing mRNA. Any known cell lysis buffer can be used.

[0046] Step (b) may involve, for example, transferring the lysate obtained in step (a) to a substrate for capturing mRNA (e.g., an oligo(dT)-immobilized substrate) and incubating at 4°C for 1 to 24 hours to capture mRNA on the substrate.

[0047] Step (c) may involve, for example, adding a reverse transcription buffer containing a primer and a reverse transcriptase to the mRNA isolated in step (b) to generate complementary DNA (cDNA), and contacting the cDNA with sense and antisense primers specific to the nucleic acid sequence of the immune effector molecule gene and a DNA polymerase to obtain a DNA amplification product, which allows measurement of the expression level of the immune effector molecule mRNA.

[0048] After the PCR reaction is completed or while the PCR reaction is in progress (in the case of real-time RT-PCR), the expression level of the immune effector molecule mRNA is quantified. Quantification may be calculated by comparing the amount of mRNA encoding one or more markers with a reference value. Examples of reference values ​​that can be used include the expression level of a gene whose expression is neither induced nor suppressed by a stimulant (e.g., an antigen peptide), and a specific example is the expression level of a known housekeeping gene (e.g., β-actin). The mRNA expression level can be evaluated, for example, based on the cycle threshold (Ct value) obtained using real-time RT-PCR. Normalizing the Ct value of the immune effector molecule with the Ct value of the housekeeping gene allows for easy comparison of the expression levels of the immune effector molecule between samples. In real-time RT-PCR, the detected amounts of one or more immune effector molecules and housekeeping genes are measured, the immune effector molecule is normalized using the Ct value of the housekeeping gene, and the normalized Ct value is converted to gene expression level, allowing for an approximate estimation of the amount of mRNA whose expression has changed (e.g., increased) due to antigen stimulation.

[0049] Furthermore, for example, changes in the expression level of one or more immune effector molecules induced by stimulation with an antigen can be evaluated by comparing the expression level of the same immune effector molecules from a non-induction-treated (solvent-only control) sample. The degree of induction can be estimated from the fold increase (F.I.) value, which is the value obtained by comparing the gene expression level of the induced immune effector molecule with the gene expression level of the immune effector molecule that has not been subjected to induction treatment.

[0050] Methods for detecting and / or quantifying immune effector molecule proteins include, for example, methods using ELISpot technology, ELISA, and flow cytometry. The ELISpot technology is a method for detecting and / or quantifying molecules secreted by individual cells. The secreted immune effector molecules are captured by antibodies and visualized as spots, allowing the number of individual cells secreting the immune effector molecules to be quantified. The ELISA method is a method for detecting and / or quantifying the concentration of a specific protein. The immune effector molecules secreted by stimulating PBMCs are captured using specific antibodies, and the concentration can be quantified using chemiluminescence or fluorescence. The flow cytometry technology detects immune effector molecules on the surface or inside individual cells using fluorescently labeled antibodies, and measures the fluorescence intensity of each cell, allowing the analysis of cellular characteristics and molecular expression.

[0051] Common immune effector molecules that can be used as indicators for measuring the activity of cellular immune responses include, for example, Th1 cytokines. Th1 cytokines are cytokines secreted by Type 1 T helper (Th1) cells. Specific examples of Th1 cytokines include known T cell function-related markers such as interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), interleukin 2 (IL2), and interleukin 10 (IL10). These markers are induced to express in stimulated PBMCs as part of cellular immunity. Note that although the above markers are listed by their human names, the same markers may be referred to by different names depending on the animal species or context.

[0052] The method of this embodiment allows the activity of cellular immune responses to be measured ex vivo.

[0053] The kit of this embodiment is for measuring the cellular immune response activity of a subject, and the measurement of the cellular immune response activity of a subject may be carried out by the method described above.

[0054] The kit of this embodiment may be for measuring the cellular immune response activity of a subject using peripheral blood mononuclear cells (PBMCs) incubated with an antigen in the presence of human serum or plasma.

[0055] The kit of this embodiment may include a reagent for measuring an antigen and / or an immune effector molecule. The antigen, immune effector molecule, and the method for measuring them are as described above.

[0056] The reagent for measuring immune effector molecules may be a reagent for measuring the expression level of mRNA of immune effector molecules. Examples of the reagent for measuring the expression level of mRNA of immune effector molecules include a total RNA extraction reagent, a reverse transcriptase, a random primer or an oligo-dT primer, and a qPCR master mix (typically containing DNA polymerase, dNTPs, MgCl ２The kit may include, for example, SsoAdvanced Universal SYBR Green Supermix (manufactured by Bio-Rad), a primer specific to the gene of the target immune effector molecule, a primer for an internal standard gene (e.g., a housekeeping gene), or a combination thereof, including buffers, fluorescent dyes, etc. Furthermore, as a reagent for measuring the mRNA expression level of the immune effector molecule, the kit may include a reagent for purifying RNA from PBMCs and / or a reagent for synthesizing cDNA from mRNA. Furthermore, the kit of this embodiment may optionally include an RNase inhibitor, a PCR reaction plate, and / or a reagent for isolating mRNA from total RNA.

[0057] The reagent for measuring an immune effector molecule may be a reagent for detecting and / or quantifying a protein that is an immune effector molecule. Examples of the reagent for detecting and / or quantifying a protein that is an immune effector molecule include a reagent selected from a capture antibody specific to the immune effector molecule, a secondary antibody that binds to the captured immune effector molecule, a substrate solution for an enzyme reaction, a stop solution for an enzyme reaction, a cell culture buffer, and combinations thereof, as well as a reagent selected from a fluorescently labeled antibody that detects an immune effector molecule or a cell surface marker, a cell fixation / permeabilization reagent, a cell staining buffer, a dead cell staining reagent, and combinations thereof.

[0058] The kit of this embodiment may further include one or more selected from a blood collection tube (e.g., a heparin sodium blood collection tube); a PBMC separation tube (e.g., SepMate-50 (manufactured by STEMCELL TECHNOLOGIES)) or a reagent (e.g., a density gradient medium such as Ficoll (registered trademark) or Lymhoprep (registered trademark)) for separating and / or isolating PBMCs from whole blood; a cell preservation solution (e.g., CultureSure DMSO (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), CELLBANKER (registered trademark) (manufactured by Nippon Zenyaku Kogyo Co., Ltd.)); a wash buffer; a detergent; a surfactant; a cell culture vessel (e.g., a cell culture plate); and a cell culture medium (e.g., RPMI medium). The blood collection tube may contain an anticoagulant. Common anticoagulants include, for example, ethylenediaminetetraacetic acid (EDTA), heparin, and sodium citrate.

[0059] The above-mentioned components that may be contained in a reagent for measuring immune effector molecules can be combined in any manner and used in the above-mentioned method for measuring cellular immune response activity. For example, the components may be combined to prepare one or more mixed solutions, which can then be used in the above-mentioned method for measuring cellular immune response activity.

[0060] The kit of this embodiment may include one or more containers containing the above-mentioned reagents, etc. The reagents, etc. may be contained in separate containers for each type, or may be contained in any combination in a container.

[0061] The kit of this embodiment may include instructions for using the kit and / or a label indicating how to use the kit. A label includes any written or recorded material provided on or with the kit, or otherwise associated with the kit. The instructions and label may be directly written on the instructions and label, or may be provided as a link (e.g., a URL or QR code) to a site that describes the instructions.

[0062] As specific aspects of the kit of this embodiment, the aspects described above in the method for measuring cellular immune response activity can be applied without limitation.

[0063] Hereinafter, the embodiments of the present disclosure will be described in more detail with reference to examples. However, it should be understood that these examples are merely illustrative of representative experimental results, and that the embodiments of the present invention are not limited to these details. In the following examples, reagents from the following manufacturers were used.

[0064] [Reagents] SARS-CoV-2 Spike Glycoprotein (PepGen Sf) (manufactured by Genscript); CultureSure DMSO (hereinafter referred to as DMSO), PBS(-) (hereinafter referred to as PBS buffer), polyoxyethylene (20) sorbitan monolaurate (hereinafter referred to as Tween 20) (all manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.); M-MLV Reverse Transcriptase, M-MLV (manufactured by Promega); RNasin™ Ribonuclease Inhibitor (hereinafter referred to as RNasin) (manufactured by Promega); Venoinject II vacuum blood collection tube with heparin sodium (hereinafter referred to as heparin sodium blood collection tube) (manufactured by Terumo Corporation); 96-well transparent round-bottom cell culture surface-treated microplate (hereinafter referred to as cell culture plate), Costar [registered trademark] 6-well transparent cell culture-treated multiwell plate (hereinafter referred to as 6-well plate), Falcon [registered trademark] conical tube (50 mL) (hereinafter referred to as 50 mL Falcon tube) (all manufactured by Corning); MicroAmp (registered trademark) Fast Optical 96-Well Reaction Plate, 0.1 mL (hereinafter referred to as 96-well PCR plate) (manufactured by Applied Biosystems); SsoAdvanced Universal SYBR Green Supermix (manufactured by Bio-Rad); SepMate-50, Lymphoprep (all manufactured by STEMCELL TECHNOLOGIES); RPMI Medium 1640 (hereinafter referred to as RPMI medium), FBS (all manufactured by ThermoFisher); CELLBANKER (registered trademark) 1 (manufactured by Nippon Zenyaku Kogyo Co., Ltd.); human AB type male donor defibrinated serum (hereinafter referred to as human serum) (manufactured by GRIFOLS); Turks solution (hereinafter referred to as Turk's solution) (manufactured by Sigma-Aldrich).

[0065] Test Example 1: Method for measuring cellular immune response activity (1) Sample preparation: Samples described as "fresh blood" refer to samples collected in heparin sodium collection tubes, mixed by inversion, and then used for antigen stimulation within 24 hours. After collection, the number of cells in the fresh blood was counted using Turk's solution. Samples described as "frozen PBMC" refer to samples in which PBMCs were separated from blood collected in heparin sodium collection tubes and stored frozen. The PBMC separation process is shown below. The blood collected in the heparin sodium collection tubes was dispensed into 50 mL Falcon tubes and centrifuged at 300 g and 25°C for 10 minutes to separate it into plasma and blood cell components. The plasma was dispensed into another 50 mL Falcon tube, frozen in a -80°C freezer, and stored. The blood cells remaining in the 50 mL Falcon tube were mixed with an equal volume of "PBS buffer containing 2% FBS (referred to as PBS-2%FBS)" as the plasma dispensed into another 50 mL Falcon tube. An equal volume of PBS-2% FBS as the blood volume collected was then added and mixed. The blood cell suspension prepared in PBS-2% FBS as described above was dispensed into a SepMate-50 tube containing Lymphoprep and centrifuged at 1200 g and 25°C for 10 minutes to separate PBMCs. The PBMCs were transferred to another 50 mL Falcon tube, and 10 mL of PBS-2% FBS was added to the suspension and the suspension was suspended. The suspension was then centrifuged at 300 g and 25°C for 8 minutes, after which the supernatant was removed. The same procedure was repeated to wash the PBMCs. The PBMCs were then suspended in 1 mL of Cell Banker and frozen in a -80°C freezer for storage.

[0066] (2) Peptide Pool for Stimulation: PepGen Sf, an overlapping peptide pool product based on the amino acid sequence of the spike protein constituting the wild-type Wuhan-Hu-1 strain of SARS-CoV-2, was prepared. These peptide pools consisted of multiple peptides corresponding to 15-amino acid fragments of the spike protein amino acid sequence, with adjacent peptides overlapping by 11 amino acids.

[0067] (3) Preparation and storage of peptide plates The peptide pool was dissolved in antigen diluent (PBS buffer containing 20% ​​DMSO) to a concentration of 20 μg / mL. 5 μL of each peptide pool solution was dispensed into each well of a cell culture plate. In addition to each peptide pool solution, 5 μL of the antigen diluent was also dispensed into other wells of the cell culture plate as a negative control. The cell culture plates into which each solution was dispensed (hereinafter referred to as peptide plates) were frozen and stored in a -80°C freezer.

[0068] (4) Peptide stimulation of fresh blood and PBMCs. Frozen PBMCs were kept at 25°C and thawed. The PBMCs were dispensed into a 6-well plate, and 5 mL of RPMI medium containing 5% human serum (RPMI-5% human serum) was added. Recovery culture was performed at 37°C for 16 hours. After 16 hours of culture, 5 mL of RPMI medium containing the PBMCs was transferred to a 50 mL Falcon tube. Subsequently, 5 mL of RPMI medium containing no human serum (hereinafter referred to as human serum-free RPMI medium) was added to the empty well where the PBMCs had been present, and the RPMI medium was washed. The RPMI medium was then transferred to the 50 mL Falcon tube (at this point, 10 mL of RPMI medium containing PBMCs was present in the 50 mL Falcon tube). The RPMI medium containing the PBMCs was centrifuged at 300 g for 10 minutes at 25°C, and the supernatant was removed. The PBMCs were washed again with human serum-free RPMI medium in the same manner as above, and then suspended in human serum-free RPMI medium. After that, the cells were counted and adjusted to a cell concentration similar to that of fresh blood.

[0069] The frozen peptide plate was kept at 25°C, and the peptide pool solution was thawed. Fresh blood or PBMCs with an adjusted cell concentration were added in 120 μL portions to each well of the peptide plate and mixed by stirring. After stirring, the mixture was incubated at 37°C for 4 hours to stimulate the leukocytes contained in the fresh blood or PBMCs (frozen and stored). After incubation, the peptide plate containing the peptide-stimulated leukocytes or PBMCs was frozen and stored at -80°C.

[0070] (5) Capture of leukocytes contained in fresh blood after antigen stimulation. The peptide plate frozen in (4) was kept at 37°C for 15 minutes, and the sample in each well was thawed. 150 μL of filter wash solution (PBS buffer containing 0.05% Tween 20) was added to each well of a multi-well filter plate, and the plate was centrifuged at 2500 g and 4°C for 1 minute. 100 μL of the whole blood sample was then added to each well, and the plate was left to stand at 4°C for 10 minutes, followed by centrifugation at 2500 g and 4°C for 2 minutes to capture leukocytes on the filter. The PBMCs cryopreserved in (4) were also subjected to the same procedure as for fresh blood, and the PBMCs were captured on the filter. In the context of this example, "centrifugation" refers to rotating the plate in a centrifuge and applying centrifugal force toward the bottom of the plate.

[0071] (6) Elution and Capture of mRNA. Cell lysis buffer (60 μL) was added to the filter of each well of the filter plate and incubated at 37°C for 10 minutes to lyse the leukocytes or PBMCs on the filter. Another multiwell plate (hereinafter referred to as the mRNA capture plate) with oligo(dT) immobilized in each well was placed below the filter plate, with the wells of both plates stacked vertically. The plate was then centrifuged at 2500 g and 4°C for 5 minutes. The filter plate was removed, and the mRNA capture plate containing the cell lysate in its wells was incubated at 4°C for 1 to 24 hours. After incubation, each well was washed with wash buffer to remove any non-oligo(dT)-binding substances contained in the cell lysate.

[0072] (7) Synthesis of cDNA 30 μL of a reverse transcription buffer containing reverse transcriptase was added to each well of the mRNA capture plate, followed by incubation at 37° C. for 2 hours. By this procedure, complementary DNA (cDNA) of the mRNA with a polyA tail that had been captured on the mRNA capture plate was synthesized.

[0073] (8) qPCR Primer pairs (sequences shown in Table 1) for each gene (actin beta: ACTB, interferon gamma: IFNG) were mixed with SsoAdvanced Universal SYBR Green Supermix and dispensed into wells of a 96-well PCR plate at 6 μL each. 4 μL of the cDNA solution obtained in (6) above was added to each well, and real-time PCR was performed using a 7500 Fast Real-Time PCR (ThermoFisher Scientific) according to the system's instructions, with the following cycle: Stage 1: 95°C 10:00, Stage 2: 40 cycles of 95°C 00:30 and 65°C 1:00, Stage 3: Melting curve

[0074]

[0075] (9) Analysis of measurement results The measured values ​​of each gene obtained by real-time PCR were expressed as Ct values, which are the cycle numbers that meet the reaction threshold. In addition, the Ct values ​​of the target gene (IFNG) were normalized using the Ct value of the housekeeping gene ACTB gene, and the normalized Ct values ​​were converted to gene expression levels to estimate the amount of mRNA expression increased by stimulation. That is, the estimated mRNA expression level was compared with the mRNA expression level of an unstimulated sample to determine the mRNA expression level specific to peptide stimulation, and the results were expressed as log ２ Expressed in F.I. For example, log ２ When the F.I. is 0, it means that the mRNA expression level of the unstimulated sample is the same as that of the stimulated sample, and the log ２ When the F.I. is 1, it means that the mRNA expression level in the stimulated sample is twice that of the unstimulated sample. ２ A larger FI means that the amount of mRNA expression induced by peptide stimulation is increased.

[0076] [Test Example 2] [When PBMCs were stimulated with an antigen in human serum-free RPMI medium] Blood was collected in sodium heparin blood collection tubes from six subjects who had received the COVID-19 mRNA vaccine, and fresh blood and frozen PBMCs from the same subjects were used to compare the cellular immune activity when fresh blood was used with that when frozen PBMCs were used, according to the method described in Test Example 1. In the measurement of cellular immune activity using PBMCs, the step of antigen stimulation of PBMCs was carried out in RPMI medium that did not contain human serum or human plasma.

[0077] The results of measuring the IFN-γ (IFNG) gene as an index of cellular immune activity in fresh blood are shown in Figure 1, and the results in PBMC are shown in Figure 2. When fresh blood was used, the error range of gene expression fluctuations (-1 < LOG ２ While the expression increased above the error margin when PBMCs were stimulated with antigen in RPMI medium containing no human serum or plasma, no increase in expression was observed beyond the error margin.

[0078] Example 1 Antigen-Stimulated PBMCs in Human Plasma Blood was collected in sodium heparin blood collection tubes from six subjects who had received the COVID-19 mRNA vaccine, and fresh blood and frozen PBMCs from the same subjects were used to compare the cellular immune activity measured using fresh blood with that measured using frozen PBMCs according to the method described in Test Example 1. Note that in the measurement of cellular immune activity using PBMCs in this Example 1, the antigen stimulation step was performed in the plasma of the same subject, rather than in RPMI medium containing no human serum or human plasma as in Test Example 2. Furthermore, for Subjects 1, 2, and 5, the PBMC cell count was 0.3 x 10 ６ For subjects 3, 4, and 6, 0.1 mL of plasma was added to each sample, and the PBMC cell count was 0.2 × 10 ６ 0.1 mL of plasma was added to each sample.

[0079] PBMCs were stimulated with an antigen in the plasma of the same subject, and the IFN-γ (IFNG) gene was measured as an index of cellular immune activity. The results are shown in Table 2 and Figure 3. When PBMCs were stimulated with an antigen in the plasma of the same subject, the error range of gene expression fluctuations (-1 < LOG２ The expression of PBMCs increased beyond the F.I. <1. The average values ​​of the results for six cases are shown in Figure 4 . A Student's t-test was performed on the cellular immune activity of PBMCs stimulated with antigen in the plasma of the same subject compared to the cellular immune activity of PBMCs stimulated with antigen in RPMI medium without human serum or plasma. The cellular immune activity of PBMCs stimulated with antigen in the plasma of the same subject was significantly higher (p<0.0001). The relative values ​​(relative to the fresh blood value set as 100) of the cellular immune activity of PBMCs stimulated with antigen in the plasma of the same subject compared to the cellular immune activity of PBMCs stimulated with antigen in RPMI medium without human serum or plasma are shown in Figure 5 for six cases. The cellular immune activity of PBMCs stimulated with antigen in the plasma of the same subject was close to 100%.

[0080]

Claims

1. A method for measuring the cellular immune response activity of a subject, comprising: (1) incubating an aqueous medium containing peripheral blood mononuclear cells (PBMCs) collected from the subject, an antigen, and human serum or plasma; and (2) measuring immune effector molecules in the PBMCs after step (1).

2. The method of claim 1, wherein human serum or plasma is derived from the subject.

3. The method according to claim 1, wherein the content of human serum or plasma in the aqueous medium in step (1) is 20% or more and less than 100% based on the total mass of the aqueous medium.

4. In step (1), the mass of human serum or plasma in the aqueous medium before incubation is 0.3 × 10 ６ The method according to any one of claims 1 to 3, wherein the amount of the solution is 0.05 mL to 1.0 mL per PBMC of cells.

5. The method according to any one of claims 1 to 3, wherein the PBMCs are frozen and thawed after collection from the subject.

6. The method according to any one of claims 1 to 3, further comprising, prior to step (1), a step of separating and / or isolating PBMCs from whole blood collected from the subject.

7. The method according to any one of claims 1 to 3, wherein the aqueous medium does not comprise a liquid culture medium.

8. The method according to any one of claims 1 to 3, wherein measuring the immune effector molecule comprises measuring the expression level of mRNA of the immune effector molecule.

9. A kit for measuring the cellular immune response activity of a subject by the method according to any one of claims 1 to 3.

10. A kit for measuring the cellular immune response activity of a subject using peripheral blood mononuclear cells (PBMCs) incubated with the antigen in human serum or plasma, comprising: (1) an antigen; and (2) a reagent for measuring immune effector molecules.

11. The kit according to claim 10, further comprising: (3) a PBMC separation tube or reagent for separating and / or isolating PBMCs; (4) a reagent for purifying RNA from PBMCs; and (5) a reagent for synthesizing cDNA from mRNA, wherein reagent (2) is a reagent for measuring the expression level of mRNA of an immune effector molecule.

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