JP2010022339A - Method for evaluating activation activity of substance toward natural killer cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for screening a component activating NK cells, capable of performing simply and in a large scale. <P>SOLUTION: This method for evaluating the activation activity of a test substance toward the NK cells is characterized by comprising an activation process of giving the test substance to cell-lined natural killer cells, and an evaluating process for evaluating the cell-injuring activity of the natural killer cells after the activation process. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for simply evaluating the activation activity of a candidate substance expected to have functionality on natural killer cells.

  Natural killer (NK) cells are responsible for eliminating tumor cells and virus-infected cells in vivo. Therefore, if screening of components that activate NK cells (ie, increase the cytotoxic activity of NK cells) can be performed easily and on a large scale, it is extremely useful for searching for new functional food materials. It is.

Conventionally, in order to evaluate the NK cell activation activity of a candidate substance, it has been common to cause the candidate substance to act on human peripheral blood mononuclear cells (PBMC) or mouse spleen cells obtained by collecting blood from healthy individuals. there were. Since these cells are difficult to prepare in large quantities, it has heretofore been difficult to perform large-scale screening. In addition, since multiple cells coexist in PBMC, it was not possible to measure purely the action of candidate substances on NK cells.
JP 2007-297291 A

  An object of the present invention is to provide a method that enables simple and large-scale screening of components that activate NK cells.

The present inventors have surprisingly found that activation of NK cells can be measured even when a human NK cell line is used instead of PBMC, and the present invention has been completed. The present invention includes the following inventions.
(1) A method for evaluating the activation activity of a test substance on natural killer cells,
An activation step of providing a test substance to the established natural killer cells;
An evaluation step of evaluating the cytotoxic activity of the natural killer cells after the activation step.
(2) The method according to (1), wherein the established natural killer cells are derived from human.
(3) The human-derived cell is any one of KHYG-1 cells, NK-92 cells, YT cells, NKL cells, SNT-8 cells, HANK-1 cells, and NK-YS cells (2) the method of.
(4) In the evaluation step, the natural killer cell and the target cell after the activation step are added to the medium to cause the natural killer cell to act on the target cell, and then L-lactate dehydrogenase (LDH in the medium) ) The method according to any one of (1) to (3), which is a step of measuring the amount and evaluating the cytotoxic activity of natural killer cells based on the measured LDH amount.
(5) In the evaluation step, the natural killer cell after the activation step and the target cell labeled with chromium are added to the medium to cause the natural killer cell to act on the target cell, and then released into the medium. The method according to any one of (1) to (3), wherein the radioactivity of chromium is measured with a gamma counter and the cytotoxic activity of natural killer cells is evaluated based on the measured radioactivity.
(6) The evaluation step measures the gene expression level of γ-type interferon (INF-γ) or CD69 in the natural killer cells after the activation step, and based on the measured gene expression level, The method according to any one of (1) to (3), which is a step of evaluating cytotoxic activity.
(7) The step in which the evaluation step measures the protein expression level of INF-γ in the natural killer cells after the activation step, and evaluates the cytotoxic activity of the natural killer cells based on the measured protein expression level The method according to any one of (1) to (3).

  Since the established NK cells can be easily proliferated in large quantities, the NK cell activator can be screened on a large scale according to the method of the present invention. According to the method of the present invention, an NK cell activator can be screened purely based only on the action on NK cells, so that highly accurate screening is possible.

1. Natural Killer Cells NK cells used in the present invention are established NK cells, that is, a stable NK cell line that has acquired unlimited proliferation in culture. NK cells are preferably derived from mammals, particularly humans.

  Specific NK cell lines include KHYG-1 cells, NK-92 cells, YT cells, NKL cells, SNT-8 cells, HANK-1 cells, and NK-YS cells.

  KHYG-1 cells (JCRB0156) is a cell line derived from a malignant NK leukemia patient having a point mutation at p53, and was established by Yagita M. et al. (Leukemia. 2000; 14: 922-930). The cells are available from the JCRB cell bank. NK-92 cells (CRL-2407) is an IL-2-dependent NK cell line derived from peripheral blood mononuclear cells of non-Hodgkin lymphoma patients and was established by Gong JH et al. (Leukemia. 1994; 8: 652- 658). The cells are available from ATCC. YT cells (ACC434) were established by Yodoi J. et al. (J. Immunol. 1985; 134: 1623-1630) from patients with acute lymphocytic leukemia. The cells are available at the DSMZ-German Collection of Microorganisms and Cell Cultures. NKL cells were established by Robertson M.J. et al. (Exp. Hematol. 1996; 24: 406-415) from the peripheral blood of patients with large granular lymphocyte leukemia. SNT-8 cells are an EB virus-positive NKT cell line derived from a patient with nasal lymphoma and were established by Nagata H. et al. (Blood. 2001; 97: 708-713). HANK-1 cells were established by Kagami Y. et al. (Br. J. Haematol. 1998; 103: 669-677) from a patient with retroperitoneal CD56 + NK / T cell lymphoma. NK-YS cells were established by Tsuchiyama J. et al. (Blood. 1998; 92: 1374-1383) from a patient with primary malignant lymphoma of the nose.

The KHYG-1 cells used in the present invention are preferably cultured under the following conditions. KHYG-1 cells are cultured in RPMI1640 medium containing 1-10% fetal calf serum (FCS) and 5-40 unit / ml recombinant human IL-2 (rIL-2, PeproTech EC Ltd, London, UK). For subculture, inoculate a 60 mm dish at a cell density of 1.0 × 10 ⁵ cells / ml and repeat subculture 48 hours later.

2. Activation Process In the present invention, the activation process is a process in which a test substance is given to the established NK cells and the test substance is allowed to act on the established NK cells for a certain period of time. At this time, inoculate the established NK cells in a medium at a cell density of 8.0 × 10 ^{4 to} 3.0 × 10 ⁵ cells / ml, add the test substance at a final concentration of 1 nM to 2 mM, and act for a certain period of time. Is preferred. The action time is preferably 1 to 48 hours.

3. Evaluation Step In the present invention, the evaluation step refers to a step of directly or indirectly evaluating the cytotoxic activity of the established NK cells after the activation step.

As a method for directly evaluating the cytotoxic activity, the established NK cell and the target cell after the activation step are added to the medium so that the established NK cell acts on the target cell, and then the target cell is damaged. A method for measuring the change can be mentioned. Such changes include the release of L-lactate dehydrogenase (LDH) by target cells and ⁵¹ Cr release. Evaluation of cytotoxic activity by the amount of LDH is a well-known technique as an LDH assay. ^{In the 51} Cr release method, target cells are labeled with chromium (Na ₂ ⁵¹ CrO ₄ ) in advance, mixed with NK cells for 4 hours, and the radioactivity of free chromium released into the culture supernatant is measured using a gamma counter. This is a highly reliable technology.

  Examples of target cells to be used include K562 cells, HL-60 cells, and Daudi cells that are widely used for screening, but are not limited to these cells. Target cells can be appropriately selected according to the purpose of screening. K562 cells (RCB0027) are human leukemia cells and were established by Lozzio CB. Et al. (Blood. 1975; 45: 321-334). Because it is sensitive to human natural killer cells, it is frequently used for screening. The cells are available at RIKEN CELL BANK. HL-60 cells (CCL-240) were established by Gallagher R. et al. (Blood. 1979; 54: 713-733) from patients with acute promyelocytic leukemia. The cells are available at ATCC. Daudi cells (RCB1640) were established in human Burkitt lymphoma by Klein E. et al. (Cancer Res. 1968; 28: 1300-1310). The cells are available at RIKEN CELL BANK.

For example, K562 cells can be cultured by the following method. K562 cells are cultured in RPMI1640 medium containing 10% FCS. For subculture, inoculate a 60 mm dish at a cell density of 1.0 × 10 ⁵ cells / ml, and perform subculture 48 hours later.

  The ratio of the established NK cells to the target cells (E: T ratio) is preferably 3: 1 to 40: 1. The mixed culture is preferably performed at 37 ° C. The mixed culture time is preferably 2 to 8 hours.

  As a method of indirectly evaluating the cytotoxic activity, the step of allowing the established NK cell to act on the target cell is not performed, but instead, the increase / decrease depends on the cytotoxic activity in the established NK cell after the activation step. A method of measuring the gene expression level or protein expression level of the protein to be processed (γ-type interferon (INF-γ), CD69, etc.). The present inventors surprisingly found that the gene expression level and the protein expression level of γ-type interferon (INF-γ) and CD69 in the established NK cell are strong and weak in cytotoxic activity against the target cell by the established NK cell. We found that it increased or decreased correspondingly. The stronger the cytotoxic activity of the established NK cell against the target cell, the greater the gene expression level and protein expression level of INF-γ and CD69 in the established NK cell. The gene expression level can be measured, for example, by reverse transcription PCR (RT-PCR) using appropriate primers. The amount of protein expression can be measured by an immunochemical measurement method such as ELISA.

4). Test substance Any substance can be used as the test substance in the present invention. Typically, a substance that is a candidate for an active ingredient of a functional food can be mentioned.

5. Preferred embodiments The most preferred embodiments of the present invention include, but are not limited to, the following examples.
5.1. NK activity measurement method by LDH assay ( ⁵¹ Cr release method is also acceptable)
KHYG-1 cells are seeded in a 35 mm dish at 1.0 × 10 ⁶ cells / ml, and the test substance is allowed to act for 24-36. KHYG-1 cells are collected, washed with PBS, and resuspended in 1% BSA / RPMI1640 phenol red-free medium (SIGMA) to a cell density of 1.0 × 10 ⁶ cells / ml. Separately, K562 cells are prepared in 1% BSA / RPMI1640 medium to a cell density of 1.0 × 10 ⁵ cells / ml. Next, the medium and cell suspension shown in Table 1 are added to the 96-well plate in this order. Under this condition, the E: T ratio is 10: 1.

After the cells are prepared, the 96-well plate is incubated at 37 ° C. in a CO ₂ incubator equilibrated with 5.0% CO ₂ gas for 4 hours. Subsequently, centrifugation is performed at 1,200 rpm × 10 minutes, and the amount of released LDH is measured with 100 μl of the supernatant using an LDH assay kit (Roche). After calculating the cytotoxic activity by the following formula, the NK activity activation activity of the test compound is calculated as the relative activity with respect to control.
Cytotoxic activity (%) = 100 x (Test-Effector control-Low control) / (High control-Low control)

5.2. RT-PCR method (can be implemented in real-time PCR as well)
KHYG-1 cells are seeded in a 60 mm dish at a cell density of 1.5 × 10 ⁵ cells / ml, a test substance is added, and the cells are collected 24 hours later. The collected cells are washed twice with PBS, and RNA is extracted as usual using TRIzoL Reagent (Invitrogen).

  For reverse transcription, ReverTra Ace- (TOYOBO) is used to synthesize cDNA according to the product manual. Next, the following primers and TaKaRa ExTaq (TaKaRa) are used for PCR reaction using cDNA as a template according to the product manual.

CD69 (NM_001781, product size: 451bp)
5'-CCTTCCAAGTTCCTGTCC-3 '(sense),
5'-CATTCCATGCTGCTGACCTC-3 '(anti-sense).
IFN-γ (NM_000619, product size: 393bp) is
5'-GCATCGTTTTGGGTTCTCTTGGCTGTTACTGC-3 '(sense),
5'-CTCCTTTTTCGCTTCCCTGTTTTAGCTGCTGG-3 '(anti-sense).
β-actin (NM_001101, product size: 661bp) is
5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3 '(sense),
Use 5'-CTAGAAGCATTGCGGTGGACGATGGAGGG-3 '(anti-sense).

  The PCR reaction product is subjected to agarose electrophoresis, and the band intensity is analyzed by a conventional method. Divide the band intensity of IFN-γ and CD69 by the band intensity of β-actin to obtain the expression level of each gene. Furthermore, the relative value with respect to the control is obtained and used as the NK activity activation activity of the test compound.

5.3. ELISA method
KHYG-1 cells are seeded in a 60 mm dish at a cell density of 1.5 × 10 ⁵ cells / ml, a test compound is added, and 100 μl of the culture supernatant is recovered 24 hours later. Using a Human IFN-γ ELISA development kit (PEPROTECH), the amount of IFN-γ in the culture supernatant is quantified, the relative value to the control is determined, and the NK activity activation activity of the test compound is obtained.

(1a) Experimental procedure
1a-1 Separation density gradient centrifugation of PBMC (peripheral blood mononuclear cell) was performed. Ficoll-paque plus (Amersham) or LSM (Organon Teknika) was used as the specific gravity solution. Blood was collected from a healthy person, 1.6 mg of ethylenediaminetetraacetic acid (EDTA) was added to 1 ml of blood, and diluted twice with phosphate-buffered saline (PBS). Next, 3 ml of Ficoll-paque plus or LSM was added to a centrifuge tube having a capacity of 15 ml, and 4 ml of the previously diluted blood was overlaid, followed by centrifugation at 400 × g for 25 minutes. Of the four separated layers, the second lymphocyte layer from the top and half of the reagent layer below it were transferred to another centrifuge tube and washed twice with PBS. Subsequently, the cells were resuspended in RPMI1640 medium, and the cell density and viability were measured.

1a-2 Cell culture method
KHYG-1 cells (JCRB0156) were obtained from the Japanese Collection of Research Bioresources (JCRB) cell bank and cultured in RPMI1640 medium containing 10% FCS, 20 U / ml recombinant human IL-2. K562 cells and PBMC were cultured in RPMI1640 medium containing 10% FCS.

1a-3 Cytotoxic activity assay (LDH release assay)
Cytotoxic activity was measured by LDH release assay.
When using PBMC, inoculate in a 35 mm dish at 1.0 × 10 ⁶ cells / ml. When using cell lines such as KHYG-1 cells, inoculate in a 35 mm dish at 1.5 × 10 ⁵ cells / ml. Test compounds such as food ingredients were added to the cells and allowed to act for 1-36 hours. After the action time of the food components, etc., the PBMC or the established cells are collected and washed with PBS, and the cell density is 1.0 × 10 ^{6 in} 1% BSA / RPMI1640 phenol red-free medium (SIGMA) containing no food components. Resuspended in cells / ml. Separately, K562 cells were prepared in 1% BSA / RPMI1640 medium to a cell density of 1.0 × 10 ⁵ cells / ml. Next, the medium and cell suspension shown in Table 2 were sequentially added to the 96-well plate (the experiment was performed in triplicate). The ET ratio is 10: 1.

After the cells were prepared, the 96-well plate was incubated for 4 hours in a CO ₂ incubator equilibrated with 5.0% CO ₂ gas at 37 ° C. Subsequently, centrifugation was performed at 250 × g for 10 minutes, and the amount of released LDH was measured with 100 μl of the supernatant using an LDH assay kit (Roche). After calculating the cytotoxic activity by the following formula, the relative cytotoxic activity of each treatment group was calculated as the relative activity with respect to the control. Statistical analysis used t-test.
Cytotoxic activity (%) = 100 x (Test-Effector control-Low control) / (High control-Low control)

1a-4 IFN-γ and CD69 gene expression level measurement (RT-PCR)
KHYG-1 cells were seeded in a 60 mm dish at a cell density of 1.5 × 10 ⁵ cells / ml, a test compound was added, and the cells were collected 24 hours later. The collected cells were washed twice with PBS, and RNA was extracted as usual using TRIzoL Reagent (Invitrogen). That is, 1 ml of TRIzoL Reagent was added to 5 to 10 × 10 ⁶ cells of KHYG-1 cells. Pipetting several times, transferred to a 1.5 ml microtube and left at room temperature for 5 minutes. Next, 0.2 ml of chloroform was added, and the mixture was stirred with a vortex mixer for 15 seconds and allowed to stand at room temperature for 5 minutes. After centrifugation at 12,000 × g for 15 minutes at 4 ° C., 0.25 ml was recovered from the upper layer portion (RNA layer) and transferred to a new 1.5 ml capacity microtube. Then, 0.25 ml of chloroform was added and re-extraction was performed. Thereafter, in order to precipitate RNA, 0.5 ml isopropyl alcohol was added and stirred. After standing at room temperature for 15 minutes, centrifugation at 12,000 × g was performed at 4 ° C. for 15 minutes. Next, the supernatant was removed, 1 ml of cold 75% ethanol was added, the mixture was stirred with a vortex mixer, and centrifuged at 4,500 × g at 4 ° C. for 5 minutes. After removing the supernatant and air-drying for 5 to 10 minutes, the RNA pellet was redissolved with 20 μl of DEPC (Diethyl Pyrocarbonate) -treated water. Then, RNA concentration and RNA purity were measured using ND-1000 (NanoDrop). Gloves were worn during the experimental operation.

For reverse transcription, ReverTra Ace (TOYOBO) was used, and cDNA was synthesized according to the product manual. That is, it was prepared with DEPC-treated water so that the extracted RNA was 1 μg / μl. Next, add RNAse free H ₂ O (10 μl), 5 × RT buffer (4 μl), dNTP mixture (20 μl), RNAse inhibitor (1.0 μl), Primer oligo (dT) 20 (1.0 μl), RNA in a 0.2 ml PCR tube (1.0 μl) and ReverTra Ace (1.0 μl) were added. This tube was set in a thermal cycler (Bio-Rad) and operated at 42 ° C. (20 min) → 99 ° C. (5 min) → 4 ° C. to perform a reverse transcription reaction.

  Subsequently, PCR reaction using cDNA as a template was performed using TaKaRa ExTaq (TaKaRa). That is, the composition of the reaction solution is TaKaRa ExTaq (0.1 μl), 10 × ExTaq buffer (2 μl), dNTP mixture (1.6 μl), Templete (1.0 μl), 10 pmol / μl Primer-sense (1.0 μl), 10 pmol / μl Primer-antisense (1.0 μl) and DEPC treated water (13.3 μl). Each primer is as follows.

CD69 (NM_001781, product size: 451bp)
5 ^' -CCTTCCAAGTTCCTGTCC-3 ^' (sense),
5 ^' -CATTCCATGCTGCTGACCTC-3 ^' (anti-sense).
IFN-γ (NM_000619, product size: 393bp) is
5 ^' -GCATCGTTTTGGGTTCTCTTGGCTGTTACTGC-3 ^' (sense),
5 ^' -CTCCTTTTTCGCTTCCCTGTTTTAGCTGCTGG-3 ^' (anti-sense).
β-actin (NM_001101, product size: 661bp) is
5 ^' -TGACGGGGTCACCCACACTGTGCCCATCTA-3 ^' (sense),
5 ^' -CTAGAAGCATTGCGGTGGACGATGGAGGG-3 ^' (anti-sense)
It was used.

  Using a thermal cycler, the reaction is a program of 94 ° C (3 min) → {94 ° C (30 sec) → 55 ° C (30 sec) → 72 ° C (1 min)} → 72 ° C (5 min) → 4 ° C (Repetition in {}. IFN-γ 30 times, β-actin 21 times, CD69 27 times). For CD69, a program of 94 ° C (3 min) → {94 ° C (1 min) → 52 ° C (1 min) → 72 ° C (1 min)} → 72 ° C (7 min) → 4 ° C ({} The inside is repeated 25 times.)

  The PCR reaction product was subjected to agarose electrophoresis, and the band intensity was analyzed by a conventional method. The band intensity of IFN-γ and CD69 was divided by the band intensity of β-actin to obtain the expression level of each gene. Furthermore, the relative value with respect to control was calculated | required and it was set as the NK activity activation activity of the test compound.

1a-5 Measurement of IFN-γ production (ELISA)
KHYG-1 cells were seeded in a 60 mm dish at a cell density of 1.5 × 10 ⁵ cells / ml, a test compound was added, and 100 μl of the culture supernatant was collected 24 hours later. Using a human IFN-γ ELISA development kit (PEPROTECH), the amount of IFN-γ in the culture supernatant was quantified according to the product instructions, the relative value to the control was determined, and the NK activity activation activity of the test compound was determined.

(1b) Explanation of results
1b-1 Differences in PBMC and KHYG-1 cell reactivity (LDH assay) to food components
Add food ingredients (genistein, resveratrol, curcumin, beta-carotene, vitamin E, DHA) that are already known to affect NK activity as test compounds to both PBMC and KHYG-1 It was measured. As a result, when 60 μM DHA was added, a significant increase in activity was observed in both cells as shown in FIG. 1a. In addition, when 0.1 μM genistein was added, a trend of increased activity was observed in PBMC, but a significant increase in activity was observed in KHYG-1 cells (FIG. 2a). On the other hand, when 14nM curcumin (Fig. 2b) or 0.1µM beta-carotene (Fig. 1b) was added, a significant increase in activity was observed in PBMC, but there was a tendency for an increase in activity in KHYG-1 cells. Observed. When Vitamin E was added, almost no effect was observed in both cells (FIG. 2c). With the above components, the measurement results with the same or the same tendency were obtained for both PBMC and KHYG-1 cells. In contrast, when 0.6 or 1.3 μM resveratrol was added, a significant increase in activity was observed in PBMC at an action time of 24 hours as shown in FIG. 1c, but no effect was observed in KHYG-1 cells. . When the Resveratrol concentration was 1.3 μM, a significant increase in activity was observed by extending the action time of the components to 36 hours as shown in FIG. 1d. Some food ingredients have been found to require longer exposure times to cells, but with these conditions, PBMC and KHYG-1 cells have the same reactivity with respect to food ingredients for cytotoxic activity. It was.

  In summary, it was found that KHYG-1 cells can be used as substitute cells for PBMC when measuring the NK activity (cytotoxic activity) activation effect of food ingredients.

1b-2 Difference in gene expression level and cytotoxic activity of KHYG-1 cells for food components Since the measurement of cytotoxic activity requires the preparation of target cells (K562 cells) in addition to NK cells, large-scale experiments Not suitable for. Therefore, the difference between the gene expression level of IFN-γ and CD69 in the NK cell line and the cytotoxic activity was examined (Table 3). As a result, regarding genistein, curcumin, and DHA, both IFN-γ and CD69 showed values of 1.0 or more, which was similar to the results of the LDH assay. In beta-carotene, there was a commonality between IFN-γ and LDH assay. For Vitamin E, it was determined that there was almost no effect of the components in both measurement methods. In resveratrol, both IFN-γ and CD69 increased in expression, but no change in cytotoxicity was observed when the exposure time (action time) of the components was 24 hours. Since the cytotoxic activity increased after 36 hours, it was considered that the increase in the gene expression level in a short time was a reasonable phenomenon.

  In summary, in KHYG-1 cells, there is a correlation between cytotoxic activity by LDH assay and IFN-γ gene expression level and CD69 gene expression level measured by RT-PCR, especially IFN-γ gene It was found that the evaluation method of cytotoxic activity based on the expression level could be an alternative measurement method of LDH assay.

1b-3 Difference in IFN-γ protein expression and cytotoxic activity in KHYG-1 cells
An attempt was made to measure NK activity by ELISA, which is simpler than RT-PCR (real-time PCR). Regarding the NK activity activation action of Genistein, curcumin, beta-carotene on KHYG-1 cells, the relative expression level of IFN-γ (protein) measured by ELISA was compared with the relative cytotoxic activity by LDH assay (Table 4). As a result, it was determined by both methods that any compound increased NK activity. From this, it was found that NK activity can be easily measured by measuring IFN-γ produced by cell lines such as KHYG-1.

It is a figure which shows the measurement result of the cytotoxic activity of PMBC and KHYG-1. It is a figure which shows the measurement result of the cytotoxic activity of PMBC and KHYG-1.

Claims (7)

A method for evaluating the activation activity of a test substance on natural killer cells, comprising:
An activation step of providing a test substance to the established natural killer cells;
An evaluation step of evaluating the cytotoxic activity of the natural killer cells after the activation step.   The method according to claim 1, wherein the established natural killer cell is derived from a human.   The method according to claim 2, wherein the human-derived cell is any one of KHYG-1 cell, NK-92 cell, YT cell, NKL cell, SNT-8 cell, HANK-1 cell, and NK-YS cell.   In the evaluation step, the natural killer cells and target cells after the activation step are added to the medium to cause the natural killer cells to act on the target cells, and then the amount of L-lactate dehydrogenase (LDH) in the medium is determined. The method according to any one of claims 1 to 3, which is a step of measuring and evaluating the cytotoxic activity of natural killer cells based on the measured LDH amount.   In the evaluation step, the natural killer cells after the activation step and the target cells labeled with chromium are added to the medium to cause the natural killer cells to act on the target cells, and then the chromium released in the medium The method according to any one of claims 1 to 3, which is a step of measuring the radioactivity with a gamma counter and evaluating the cytotoxic activity of natural killer cells based on the measured radioactivity.   The evaluation step measures the gene expression level of γ-type interferon (INF-γ) or CD69 in natural killer cells after the activation step, and the cytotoxic activity of natural killer cells based on the measured gene expression level The method according to any one of claims 1 to 3, wherein the method is a step of assessing.   The evaluation step is a step of measuring the protein expression level of INF-γ in the natural killer cells after the activation step, and evaluating the cytotoxic activity of the natural killer cells based on the measured protein expression level. The method according to claim 1.

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