Method for measuring natural killer cell activity

Abstract

To provide a simple and highly safe method of measuring NK cell activity.SOLUTION: NK cell activity is measured by measuring the level of expression of a natural killer (NK) cell activating receptor in a blood sample collected from a subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for measuring natural killer (NK) cell activity. [Background technology]

[0002] There are a variety of immune cells in the body. Among them, natural killer (NK) cells are a type of lymphocyte responsible for innate immunity, and are important cells with strong killing power and the ability to recognize, attack, and eliminate virus-infected cells, stress cells, and malignant cells.

[0003] The immune function of human NK cells is regulated via activating and inhibitory receptors. The natural cytotoxicity receptors (NCRs) CD337 (NKp30), CD336 (NKp44), and CD335 (NKp46) and the C-type lectin family NKG2D act as activating receptors, while killer cell immunoglobulin-like receptors (KIRs) and CD94 / NKG2A act as inhibitory receptors. NK cells activated through signal transduction via these receptors release cytotoxic granules (e.g., perforin and granzymes) and produce cytokines such as IFN-γ and TNF-α, which are known to affect dendritic cell maturation and T cell differentiation (Non-Patent Document 1).

[0004] It has been reported that NK cell activity declines with age and is significantly affected by various environmental factors such as stress, diet, and sleep (Non-Patent Documents 1 and 2). Therefore, it is expected that NK cell activity can be used as an indicator of immune strength to monitor the immune status of an individual living organism.

[0005] The cytotoxic activity of NK cells can be measured using the following methods: 51 The Cr-free method is widely used. 51 The Cr release method typically involves separating lymphocytes, including NK cells, from the patient's peripheral blood by specific gravity centrifugation, culturing them, and then 51Cr-labeled leukemia cells (K562) were co-cultured, and K562 cells were killed by NK cells, and released into the culture supernatant. 51 The cytotoxicity rate (%) obtained by measuring the radioactivity of Cr using a gamma ray counter is taken as NK cell activity. 51 The Cr release method requires a relatively large amount of blood sample (approximately 5-10 mL), takes several days to complete the test (approximately 2-5 days), and requires skilled techniques such as peripheral blood mononuclear cell isolation and cell preparation. 51 The Cr release method has the problem that the results are easily affected by the number of lymphocytes used, the ratio of target cells K562, and the treatment and culture conditions of those cells before the assay, making it difficult to obtain stable data. 51 The Cr release method uses radioisotopes, which not only requires dedicated facilities and dedicated measuring equipment, but also poses the risk of radiation exposure to the experimenter (Patent Document 1, Non-Patent Document 3). Therefore, there is a need for the development of a simpler method for measuring NK cell activity that can provide stable test results.

[0006] Progress is being made in the development of methods for measuring NK cell activity without using radioisotopes. For example, a method is known in which K562 cells and NK cells are co-cultured, and the CD107a protein, which is specifically expressed on the cell membrane surface during NK cell degranulation, is stained with a fluorescently labeled antibody and quantified by flow cytometry. Alternatively, a method has been developed in which NK cells are stimulated using compounds such as PMA (phorbol-12-myristate-13-acetate) instead of K562 cells (Non-Patent Documents 3 and 4). A method has also been reported in which NK cells are cultured with an anti-NK cell activating receptor antibody and the amount of cytokines produced is measured (Patent Document 1). In addition, a reagent kit has been developed that uses an enzyme substrate to measure the activity of the cytoplasmic enzyme LDH (lactate dehydrogenase), which is released from K562 cells damaged by NK cells, using a colorimeter (Non-Patent Document 5). However, this method is 51 It is known to be less sensitive than the Cr-free method. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 2019-045215 [Non-patent literature]

[0008] [Non-Patent Document 1] Aging Res. Rev., 12(4): 1069-1078 (2013) [Non-patent document 2] Prev. Med., 44(2): 117-23 (2007) [Non-patent document 3] Indian J. Exp. Biol., 52(10): 983-8 (2014) [Non-patent document 4] J. Immunol. Res., 2016:3769590 (2016) [Non-patent document 5] J. Immunol. Methods, 64:313-320 (1983) Summary of the Invention [Problem to be solved by the invention]

[0009] As described above, conventional methods for measuring NK cell activity use radioisotopes, leaving safety and other issues unresolved. The methods described in Non-Patent Documents 3 to 5 and Patent Document 1 all require cell culture equipment such as a CO2 incubator. Furthermore, all of these methods require time for stimulation and culture of blood or cells separated from blood, making it difficult to analyze many samples at once.

[0010] An object of the present invention is to provide a simpler and safer method for measuring NK cell activity. [Means for solving the problem]

[0011] As a result of extensive research, the inventors discovered that the expression level of NK cell-activating receptors in a certain amount of whole blood sample from human peripheral blood shows a high correlation with NK cell activity measured by conventional methods, leading to the completion of the present invention.

[0012] That is, the present invention provides the following. (1) A method for measuring natural killer (NK) cell activity, comprising a step of measuring the expression level of NK cell activating receptors in a blood sample collected from a subject. (2) The method described in (1), wherein the NK cell activating receptor is NKp30, NKp46, or NKG2D. (3) The method according to (1) or (2), wherein the expression level is measured by quantifying the NK cell activating receptor protein in a blood sample per unit volume. (4) The method according to (3), wherein the protein is quantified by flow cytometry, immunoassay, Western blotting, or mass spectrometry. (5) The method according to (4), wherein the protein is quantified by flow cytometry. (6) The method according to (1) or (2), wherein the expression level is measured by quantifying the NK cell activating receptor mRNA in a blood sample per unit volume. (7) A reagent for measuring NK cell activity, comprising the following a) or b): a) an antibody that specifically binds to an NK cell activating receptor; b) A nucleic acid having a sequence complementary to at least a portion of an NK cell activating receptor mRNA. (8) The reagent according to (7), wherein the NK cell activating receptor is NKp30, NKp46, or NKG2D. (9) The reagent according to (7) or (8), comprising an antibody that specifically binds to an NK cell activating receptor. [Effects of the Invention]

[0013] According to the present invention, it is possible to provide a method for measuring NK cell activity that is simpler and safer than known methods. [Brief explanation of the drawings]

[0014] [Figure 1] The results of flow cytometry analysis of an example whole blood sample are shown below. WBCs were analyzed using FSC and SSC (A), and the proportion of lymphocytes in total WBCs was measured by gating on the small lymphocyte fraction in both FSC and SSC (B). The lymphocyte fraction was expanded by CD56 and CD3 expression intensity, and the CD56-positive / CD3-negative NK cell fraction was gated and analyzed for the expression levels of NKp30 (C), NKp46 (D), and NKG2D (E). [Figure 2] Scatter plots showing the correlation between the mean fluorescence intensity (MFI) of various receptors per NK cell and the 51Cr release assay. (A) Correlation between the MFI of NKp30 and the 51Cr release assay. (B) Correlation between the MFI of NKp46 and the 51Cr release assay. (C) Correlation between the MFI of NKG2D and the 51Cr release assay. [Figure 3] Scatter plots showing the correlation between the total fluorescence intensity (Total FI) of various receptors and the measurement results of the 51Cr release method. (A) Correlation between Total FI of NKp30 and the 51Cr release method. (B) Correlation between Total FI of NKp46 and the 51Cr release method. (C) Correlation between Total FI of NKG2D and the 51Cr release method. [Figure 4] Scatter plots showing the corrected correlation between the measurement results of total FI and 51Cr release method for various receptors. (A) Correlation between total FI and 51Cr release method for NKp30. (B) Correlation between total FI and 51Cr release method for NKp46. (C) Correlation between total FI and 51Cr release method for NKG2D. DETAILED DESCRIPTION OF THE INVENTION

[0015] 1. Method for measuring natural killer (NK) cell activity The method for measuring NK cell activity of the present invention (hereinafter also referred to as "the method of the present invention") is characterized by including a step of measuring the expression level of an NK cell activating receptor in a blood sample collected from a subject.

[0016] In the present invention, a "blood sample" collected from a subject is used as the specimen. In the present invention, a "blood sample" must be blood containing at least NK cells. An example of such a blood sample is whole blood (whole blood sample). In the present invention, "whole blood" or a "whole blood sample" refers to a blood sample that, unlike serum or plasma, has not undergone separation or removal of blood components. Alternatively, the blood sample may be a sample from which blood cell components other than lymphocytes have been removed. Such a sample can be obtained, for example, by hemolyzing red blood cells in a whole blood sample and washing away all but the cells.

[0017] In the present invention, blood samples also include those to which anticoagulants have been added to avoid difficulties in measurement due to blood coagulation. Anticoagulants include, but are not limited to, heparin (heparin sodium, heparin lithium, etc.), EDTA (EDTA-2Na, EDTA-2K, EDTA-3K, etc.), sodium fluoride, sodium citrate, sodium oxalate, etc.

[0018] A blood sample can be obtained by drawing blood from a subject. The blood sample is typically, but not limited to, peripheral blood. The subject can be any animal, but is preferably a mammal, such as primates (e.g., humans, gorillas, chimpanzees, orangutans, Japanese macaques), rodents (e.g., mice, rats, hamsters, guinea pigs), and livestock and pets (e.g., cows, horses, sheep, goats, donkeys, llamas, camels, rabbits, dogs, cats, ferrets). Humans are more preferred.

[0019] In the present invention, "NK cells" are lymphocytes that are CD3 - / CD56 + The number of NK cells in whole blood is usually counted by flow cytometry using anti-CD3 and anti-CD56 antibodies.

[0020] In the present invention, the term "NK cell activating receptor" refers to an NK receptor responsible for transmitting activation signals to NK cells. It is preferable that the NK cell activating receptor is not present on the surface of blood cells other than NK cells, and more preferably, it is present specifically on the surface of NK cells. Examples of such NK cell activating receptors include NKp30, NKp46 (see Pende D., et al., J. Exp. Med., 190 (10): 1505-1516 (1999); NCBI Gene IDs: 259197 and 9437), and NKG2D (see Houchins JP, et al., J. Exp. Med., 173: 1017-1020 (1991); NCBI Gene ID: 22914).

[0021] In a first embodiment of the method of the present invention, the expression level of an NK cell-activating receptor can be measured by quantifying the amount of NK cell-activating receptor protein in a blood sample per unit volume. Any known protein quantification method can be used to quantify the NK cell-activating receptor protein, including, for example, flow cytometry, immunoassay, Western blotting, and mass spectrometry. Flow cytometry is particularly preferred because it is simple and enables accurate measurement.

[0022] Specifically, flow cytometry involves reacting an anti-NK cell activating receptor antibody fluorescently labeled with FITC, PE, or the like with the cell surface in a blood sample and measuring the fluorescence intensity of the label bound to the cell surface for each cell. Here, the expression level of NK cell activating receptor must be determined not as the mean fluorescence intensity (MFI) of the cells, but as the integrated value of the fluorescence intensity of each cell in a unit volume of blood sample (total fluorescence intensity: Total FI). Total FI can be calculated from the area under the peak (AUC) of the NK cell activating receptor in flow cytometry of a unit volume of blood sample. Alternatively, it can be calculated as the product of MFI and cell number.

[0023] In flow cytometry, in addition to anti-NK cell activating receptor antibodies, anti-CD3 antibodies and anti-CD56 antibodies labeled with different fluorescent dyes can be used. In this case, CD3 - / CD56 + By gating only on cells, i.e., NK cells, the total FI in NK cells can be determined. Because NK cell activating receptors are specifically expressed on NK cells, theoretically there is no significant difference in fluorescence intensity compared to when measuring whole cells, but the S / N ratio can be increased by avoiding the influence of nonspecific reactions.

[0024] Any known immunoassay method can be used. The method used may be any of the direct competitive method, the indirect competitive method, the sandwich method, the colloidal metal method, etc. Furthermore, the immunoassay method may be any of the chemiluminescent enzyme immunoassay (CLEIA), the chemiluminescent immunoassay (CLIA), the turbidimetric immunoassay (TIA), the enzyme immunoassay (EIA) (e.g., direct competitive ELISA, indirect competitive ELISA, and sandwich ELISA), the radioimmunoassay (RIA), the latex agglutination method, the fluorescent immunoassay (FIA), and the immunochromatography method. It is particularly preferable to use a method that does not use radioisotopes.

[0025] Specifically, immunoassays involve reacting anti-NK cell-activating receptor antibodies with a blood sample to measure NK cell-activating receptor protein in the sample. The blood sample may be a hemolyzed sample, or may be a sample from which red blood cell components have been removed after hemolysis. Furthermore, the sample may be one in which blood cell components have been lysed using a cell lysing agent containing an ionic or non-ionic surfactant. Particularly when using an optical detection system, removing red blood cell components and lysing other blood cell components reduces the influence of sample-derived pigments, enabling more accurate values ​​to be obtained.

[0026] Immunoassay methods allow for simple and direct quantification of anti-NK cell-activating receptor protein in a unit blood sample.

[0027] In the flow cytometry and immunoassay methods, the antibody used is an anti-NK cell activating receptor antibody, preferably an anti-NKp30 antibody, an anti-NKp46 antibody, or an anti-NKG2D antibody. An anti-NKp30 antibody is more preferred. The anti-NK cell activating receptor antibody is particularly preferably an antibody that specifically binds to a human NK cell activating receptor. The anti-NK cell activating receptor antibody may be of any immunoglobulin class, preferably IgG, IgD, or IgE, more preferably IgG. The anti-NK cell activating receptor antibody may be of any subclass, for example, IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3, or IgG4. Various anti-NK cell activating receptor antibodies are commercially available, and any of them can be used.

[0028] In mass spectrometry, blood samples, either directly diluted or after hemolysis or lysis, are subjected to a mass spectrometer to measure the intensity of signals specific to the NK cell-activating receptor protein, allowing for quantification of NK cell-activating receptor protein. Mass spectrometers, such as MALDI-TOF-MS, can be used. While this method is simple and highly sensitive, depending on the conditions, it is possible that the sample may contain substances whose ionization behavior and mass spectral pattern are similar to those of the target protein, making it difficult to distinguish the target protein. Therefore, sample pretreatment and measurement conditions must be optimized in advance.

[0029] In a second embodiment of the method of the present invention, the expression level of an NK cell-activating receptor can be measured by quantifying the NK cell-activating receptor mRNA in a blood sample per unit volume. Any known nucleic acid quantification method can be used to quantify the NK cell-activating receptor mRNA, including RT-PCR, real-time RT-PCR, microarray analysis, Northern blotting, dot blot analysis, and RNase protection assay. Real-time RT-PCR is particularly preferred. Any known real-time RT-PCR method, such as SYBR® Green or TaqMan® PCR, can be used.

[0030] When using real-time RT-PCR, the primer sequences used to quantify various NK cell-activating receptor mRNAs are not limited as long as they are sequences that can reverse transcribe and amplify the target mRNA. For example, the sequences shown in Table 1 can be used.

[0031] [Table 1]

[0032] All of the above-mentioned methods are methods for measuring the expression level of NK cell-activating receptors, and the present inventors have found that the expression level of NK cell-activating receptors measured by these methods shows a high correlation with NK cell activity measured by known methods. Therefore, the method of the present invention corresponds to a method for measuring NK cell activity. The method of the present invention does not use radioisotopes or require a cell culture step, and therefore can measure NK cell activity safely and simply.

[0033] 2. Reagents for measuring NK cell activity The reagent for measuring NK cell activity of the present invention (hereinafter also referred to as "the reagent of the present invention") is characterized by comprising the following a) or b): a) Antibodies that specifically bind to NK cell activating receptors b) A nucleic acid having a sequence complementary to at least a portion of an NK cell activating receptor mRNA.

[0034] The reagent of the present invention is a reagent for measuring NK cell activity by measuring the expression level of an NK cell-activating receptor in a sample. The "sample" referred to here refers to a "blood sample" collected from a subject.

[0035] A first embodiment of the reagent of the present invention comprises an antibody that specifically binds to an NK cell-activating receptor, i.e., an anti-NK cell-activating receptor antibody, for quantifying an NK cell-activating receptor protein in a sample. The NK cell-activating receptor to be quantified is preferably NKp30, NKp46, or NKG2D. NKp30 is particularly preferred.

[0036] The first embodiment is preferably a form suitable for a protein quantification method using an antigen-antibody reaction. The protein quantification method is not particularly limited as long as it uses an antibody, and flow cytometry and immunoassay methods can be used. The immunoassay method may be, for example, a direct competitive method, an indirect competitive method, a sandwich method, a metal colloid method, or a Western blot method. Furthermore, the immunoassay method may be any of CLEIA, CLIA, TIA, EIA, ELISA, RIA, latex agglutination reaction, FIA, and immunochromatography.

[0037] The anti-NK cell activating receptor antibody may be of any immunoglobulin class, preferably IgG, IgD, or IgE, and more preferably IgG. The anti-NK cell activating receptor antibody may be of any subclass, for example, IgG1, IgG2, IgG2a, IgG2b, IgG2c, IgG3, or IgG4. Various anti-NK cell activating receptor antibodies are commercially available, and any of them can be used.

[0038] The anti-NK cell activating receptor antibody may be labeled with a fluorescent dye, enzyme, biotin, radioisotope, or the like, depending on the protein measurement method. In this case, it is preferable that the antibody be labeled with a material other than a radioisotope. The anti-NK cell activating receptor antibody may be contained in an aqueous solution, or may be immobilized on magnetic beads, latex particles, a microwell plate, a membrane filter, or the like, depending on the protein quantification method.

[0039] In addition to the antibody, the first embodiment may contain stabilizers (BSA, sugars, etc.), pH buffers (Tris, MES, etc.), antioxidants, preservatives, etc., as needed.

[0040] A second embodiment of the reagent of the present invention comprises a nucleic acid having a sequence complementary to at least a portion of an NK cell-activating receptor mRNA. More specifically, the reagent comprises a nucleic acid having a sequence complementary to all or a portion of a gene encoding NKp30 (e.g., NCBI Gene ID: 259197), a gene encoding NKp46 (e.g., NCBI Gene ID: 9437), or a gene encoding NKG2D (e.g., NCBI Gene ID: 22914). The nucleic acid referred to here is preferably DNA. When the nucleic acid has a sequence complementary to a portion of an NK cell-activating receptor mRNA, it preferably has a sequence complementary to a contiguous nucleotide sequence of 16 or more bases, particularly 20 or more bases, of the nucleotide sequence of the gene encoding NKp30, the gene encoding NKp46, or the gene encoding NKG2D.

[0041] The second embodiment is preferably a form suitable for a nucleic acid quantification technique. The nucleic acid quantification technique is not particularly limited as long as it uses nucleic acid hybridization, and examples thereof include RT-PCR, real-time RT-PCR, microarray, Northern blot, dot blot, and RNase protection assay. In particular, any of the real-time RT-PCR methods may be used.

[0042] In the second embodiment, the nucleic acid may be a primer for reverse transcribing mRNA to generate cDNA and further amplifying the cDNA. Alternatively, the nucleic acid may be a probe for capturing and detecting RNA or cDNA. When a primer is used, its sequence is not limited as long as it is a sequence that can reverse transcribe and amplify the target mRNA, and for example, the sequences shown in Table 1 above can be used.

[0043] In the second embodiment, the nucleic acid may be labeled with a fluorescent dye, an enzyme, biotin, a radioisotope, or the like. In this case, it is preferable that the nucleic acid is labeled with a substance other than a radioisotope. The nucleic acid may be contained in an aqueous solution, or, depending on the mRNA quantification method, may be immobilized on a DNA chip, magnetic beads, a microwell plate, a membrane filter, or the like.

[0044] In the second embodiment, in addition to the nucleic acid, the nucleic acid may contain, as necessary, metal salts, pH buffers, etc. In particular, when PCR is used, the nucleic acid may contain divalent metal salts, polymerase (such as Tth polymerase), dNTPs, etc.

[0045] The reagent of the present invention can be used in the method of the present invention described in "1. Method for measuring natural killer (NK) cell activity." In the reagent of the present invention, the definitions of terms and detailed conditions for measurement are as described in "1. Method for measuring natural killer (NK) cell activity," unless otherwise inconsistent. [Example]

[0046] Example: Correlation test between NK activating receptor expression in human whole blood and NK cell activity A total of 15 male and female volunteers aged 20 to under 60 years who agreed to the study plan were given 11 mL of blood each (2 mL for peripheral blood general testing (EDTA-2K blood collection tube), 2 mL for NK cell receptor expression analysis (heparin sodium blood collection tube), 51 Venous blood was collected for the Cr release method (including 5 mL of dedicated blood collection tubes (PNK)). 51NK cell activity testing using the Cr release method was performed by SRL Co., Ltd. in the usual manner. Table 2 shows the volunteers' gender, medical history, and other background information, as well as their total white blood cell count (WBC). Since the WBC counts of all 15 patients were within the normal range, it was confirmed that there was no suspicion of an infectious disease. 51 The values ​​of NK cell activity measured by the Cr release method are shown in Table 4.

[0047] [Table 2]

[0048] 30 μL of FcR blocker (422302 / BioLegend) solution was added to 2 mL of collected whole blood and incubated at room temperature for 15 minutes. Then, 200 μL of each solution was dispensed into four tubes. Each labeled antibody listed in Table 3 was added at 4 μL per tube and incubated at 4°C for 45 minutes. After that, 2 mL of hemolysis buffer (349202 / Becton Dickinson (BD)) was added and incubated at room temperature for 15 minutes to lyse red blood cells. After centrifugation at 1500 rpm for 5 minutes, the supernatant was removed and resuspended in 0.5 mL of FACS buffer (PBS(-) with 2% FBS). After washing the cells twice by centrifugation at 1500 rpm for 5 minutes and resuspension, three-color fluorescence analysis was performed using a flow cytometer (FACS Calibur (BD)).

[0049] [Table 3]

[0050] Using flow cytometry, white blood cells were analyzed by forward scatter (FSC) and side scatter (SSC), and the proportion of lymphocytes in total WBCs was measured by gating on lymphocyte fractions with low FSC and SSC. The lymphocyte fraction was then analyzed by CD56 and CD3 expression intensity, and the CD56-positive / CD3-negative NK cell fraction was gated and the expression levels of various receptors (NKp30, NKp46, NKG2D) were analyzed (Figure 1). Detailed information such as the proportion of positive cells and the fluorescence intensity of expression was analyzed using the analysis software FlowJo ver. 8.6.

[0051] The analysis results of the number of NK cells in the blood and the mean fluorescence intensity (MFI) of various receptors per NK cell are shown in Table 4. In addition, the MFI of various receptors per NK cell and 51 The results of the analysis of the correlation with the Cr release method are shown in Figure 2. Figure 2A shows the correlation between the MFI of NKp30 and 51 Figure 2B shows the correlation between the MFI of NKp46 and the Cr release assay. 51 Figure 2C shows the correlation between the MFI of NKG2D and the Cr release assay. 51 The correlation with the Cr release assay is shown. Regarding various receptors, NKp30 and NKp46 had a weak correlation with correlation coefficients of 0.289 and 0.268, respectively, while NKG2D had a correlation coefficient of 0.061, indicating no correlation. Regarding correlation, a correlation coefficient of 0-0.2 was evaluated as no correlation, 0.2-0.4 as a weak correlation, 0.4-0.7 as a moderate correlation, and 0.7-1 as a strong correlation.

[0052] The total expression level (Total FI) of various receptors in whole blood samples is shown in Table 4, and the total FI of various receptors is shown in Table 5. 51 Figure 3 shows the results of an analysis of the correlation with the measurement results of the Cr release method. Here, Total FI is the integrated value of the fluorescence intensity (FI) of each receptor on cells in the blood. Since each receptor is specific to NK cells, the value of Total FI is approximately equal to the product of the number of NK cells in the blood and the MFI of the receptor on the NK cells. Figure 3A shows the relationship between the Total FI of NKp30 and 51 Figure 3B shows the correlation between the total FI of NKp46 and the Cr release method. 51 Figure 3C shows the correlation between the total FI of NKG2D and the Cr release method. 51 The correlation with the Cr release method is shown. Compared with MFI, Total FI and 51 The correlations with the Cr release assay results improved, with NKp30 and NKG2D showing correlation coefficients of 0.525 and 0.575, respectively. 51 It was found that there was a moderate correlation with the Cr-free method.

[0053] In Figure 3, the Total FI of NKp30 and NKp46 from only one volunteer (No. 8) showed a significantly high value, deviating from the correlation curve. This whole blood sample had a significantly higher NK cell count than the other samples, suggesting that the subject was in an immune-enhanced state due to factors such as having recently contracted some kind of infectious disease. With the exception of the data for No. 8, the Total FI of various receptors and 51 The correlation with the Cr release assay results was analyzed again, and the results are shown in Figure 4. Figure 4A shows the relationship between the total FI of NKp30 and 51 Figure 4B shows the correlation between the total FI of NKp46 and the Cr release method. 51 Figure 4C shows the correlation between the total FI of NKG2D and the Cr release method. 51 The correlation with the Cr release method is shown. All correlations improved, with correlation coefficients of NKp30, NKp46, and NKG2D reaching 0.917, 0.740, and 0.587, respectively, with NKp30 showing a particularly high correlation.

[0054] [Table 4-1]

[0055] [Table 4-2]

Claims

1. measuring the expression level of a natural killer (NK) cell activating receptor in a blood sample collected from a subject; does not include a step of separating peripheral blood mononuclear cells or NK cells from a blood sample; the NK cell activating receptor is NKp30, NKp46, or NKG2D; Method for measuring NK cell activity.

2. The method according to claim 1, wherein the expression level is measured by quantifying the amount of NK cell-activating receptor protein in a blood sample per unit volume.

3. The method of claim 2, wherein the protein is quantified by flow cytometry, immunoassay, Western blotting, or mass spectrometry.

4. The method according to claim 3, wherein the protein is quantified by flow cytometry.

5. The method according to claim 1, wherein the expression level is measured by quantifying NK cell-activating receptor mRNA in a blood sample per unit volume.

6. A reagent for measuring NK cell activity to be used in the method according to any one of claims 1 to 5, comprising the following a) or b): a) an antibody that specifically binds to an NK cell activating receptor; b) A nucleic acid having a sequence complementary to at least a portion of an NK cell activating receptor mRNA.

7. The reagent according to claim 6, wherein the NK cell activating receptor is NKp30, NKp46, or NKG2D.

8. The reagent according to claim 6 or 7, comprising an antibody that specifically binds to an NK cell activating receptor.

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