Method for evaluating immunostimulatory capacity of target substance

Abstract

Disclosed is a method for evaluating the immunostimulatory capacity of a target substance, the method including a contacting step in which a human plasmacytoid dendritic cell line is cultured in the presence of the target substance, wherein the human plasmacytoid dendritic cell line is the CAL-1 cell line (accession number FERM BP-10914), and the CAL-1 cells are in a starvation state during at least part of the contacting step.

Description

Method for evaluating the immunostimulatory activity of a target substance

[0001] The present disclosure relates to a method for evaluating the immunostimulatory activity of a substance of interest.

[0002] Plasmacytoid dendritic cells (pDCs), also known as plasmacytoid dendritic cells, are a type of dendritic cell that constitutes the innate immune system. pDCs are the main producers of the cytokine type I interferon in the body and play an extremely important role in biodefense. Type I interferons exhibit growth inhibitory activity against viruses and other pathogens. Representative type I interferons are interferon α (hereinafter also referred to as IFN-α) and interferon β (hereinafter also referred to as IFN-β), which are thought to have immunostimulatory properties. Therefore, substances that activate pDCs and promote the production of type I interferons may be useful as immunostimulatory substances. For example, Patent Document 1 discloses an immunostimulatory food composition containing lactic acid bacteria that activate pDCs and induce IFN-α production.

[0003] Evaluation of the immunostimulatory activity of a substance and search for substances with immunostimulatory activity can be performed using as an indicator whether the production of type I interferon is promoted in pDCs exposed to the substance. Mouse bone marrow-derived dendritic cells (BM-DCs) are widely used for such evaluation and search. BM-DCs are a cell population consisting of multiple types of dendritic cells, including mouse-derived pDCs.

[0004] CAL-1 cells are a human pDC cancer cell line with the deposit number FERM BP-10914. Because CAL-1 cells are a human-derived pDC cell line, they are expected to be useful in the search for substances involved in human immune responses or drugs that regulate the antigen-presenting function of dendritic cells (Patent Document 2).

[0005] JP 2017-201984 A JP 2007-044008 A

[0006] Because mouse-derived BM-DCs are derived from mice, not humans, the results of evaluation and exploration in tests using them may not match the immunostimulatory activity of human cells. Furthermore, because BM-DCs are a cell population consisting of multiple types of dendritic cells including pDCs, it may not be possible to determine whether the results of evaluation and exploration in tests using BM-DCs are due to pDCs alone or dendritic cells other than pDCs. In addition, tests using human primary pDCs and BM-DCs are expensive.

[0007] An object of the present disclosure is to provide a method for evaluating the immunostimulatory activity of a target substance using human-derived pDCs.

[0008] The present inventors focused on CAL-1 cells, a cell line that can be used inexpensively as a human-derived pDC. However, the present inventors found that, under commonly used media and culture conditions, CAL-1 cells express only a small amount of type I interferon, making it difficult to evaluate the immunostimulatory activity of CAL-1 cells using the expression level of type I interferon as an index.

[0009] As a result of further intensive research, the present inventors discovered conditions under which the IFN-α production ability of CAL-1 cells can be enhanced in an evaluation of immunostimulatory activity using CAL-1 cells, thereby completing the present invention.

[0010] The present disclosure relates to, for example, the following: [1] A method for evaluating the immunostimulatory activity of a target substance, comprising a contacting step of culturing a human plasmacytoid dendritic cell line in the presence of the target substance, wherein the human plasmacytoid dendritic cell line is CAL-1 cells (deposit number FERM BP-10914), and the CAL-1 cells are in a starved state during at least a part of the contacting step. [2] The method according to [1], wherein the starved state is a state in which the cells are cultured in a medium having a serum content of less than 1.0% by volume based on the total volume of the medium. [3] The method according to [1] or [2], wherein the contacting step satisfies at least one of the following conditions selected from the group consisting of A) and B): A) the CAL-1 cells are cultured under conditions that starve the CAL-1 cells during a part or the entire contacting step; B) the CAL-1 cells are in a starved state at the start of the contacting step. [4] The method according to any one of [1] to [3], further comprising a pretreatment step of starving CAL-1 cells before the contact step. [5] The method according to [4], wherein the culture time in the pretreatment step is 1 hour or more and 72 hours or less. [6] The concentration of CAL-1 cells in the medium during culture in the contact step is 0.1 x 10 ４ cells / mL or more 5.0×10 ８[7] The method according to any one of [1] to [6], wherein the culture time in the contacting step is 1 hour or more and 168 hours or less. [8] The method according to any one of [1] to [7], wherein the target substance is a bacterium. [9] The method according to [8], wherein the bacterium is a lactic acid bacterium and / or an acetic acid bacterium.

[10] The method according to any one of [1] to [9], wherein the target substance is a substance that is phagocytosed by the CAL-1 cells.

[11] The method according to any one of [1] to

[10] , wherein the concentration of the target substance in the medium in the culture in the contacting step is 1.0 μg / mL or more and 1000 μg / mL or less.

[12] The method according to any one of [1] to

[11] , wherein the immunostimulatory ability is a dendritic cell activation ability.

[13] The method according to

[12] , wherein the dendritic cell activation ability is evaluated using the expression level of interferon α by the CAL-1 cells as an index.

[14] The method according to

[13] , wherein the expression level of interferon α is the amount of interferon α secreted extracellularly and / or the amount of interferon α in the CAL-1 cells, or the amount of mRNA encoding interferon α. ​​

[15] The method according to

[13] or

[14] , further comprising a comparison step of comparing the measured value of the expression level of interferon α in the CAL-1 cells contacted with the substance of interest in the contacting step with the measured value of the expression level of interferon α in the CAL-1 cells not contacted with the substance of interest.

[16] The method according to any one of

[13] to

[15] , wherein the measurement of the expression level of interferon α is carried out at a time point of 15 to 72 hours after the start of the contacting step.

[17] The method according to

[12] , wherein the dendritic cell activation ability is evaluated using the proportion of interferon α-positive cells among the CAL-1 cells as an index.

[18] The method according to any one of [1] to

[17] , comprising, before the contacting step, a preparation step of preparing the CAL-1 cells to be used in the contacting step, wherein the preparation step comprises a step of preparing CAL-1 cells that are susceptible to activation by single-cell cloning of the CAL-1 cells.

[19] The method according to

[18] , wherein the preparing step comprises: isolating at least one single cell from a population of CAL-1 cells; culturing each of the at least one single cell to obtain at least one colony with guaranteed monoclonality; and selecting a colony containing CAL-1 cells that are easily activated from the at least one colony.

[20] The CAL-1 cells are cultured at a density of 5.0 × 10 in RPMI-1640 medium containing no serum components. ５

[21] The method according to any one of [1] to

[19] , wherein the CAL-1 cells are CAL-1 cells, which are contacted with 10 μg / mL of Lactococcus lactis subsp. lactis JCM 5805 at an initial concentration of 5.0 × 10 cells / mL for 24 hours, and the interferon α concentration in the medium is 100.0 pg / mL or more. ５CAL-1 cells (deposit number FERM BP-10914), in which the interferon α concentration in the medium after 24 hours of contact with 10 μg / mL of Lactococcus lactis subsp. lactis JCM 5805 at an initial concentration of 100.0 pg / mL.

[22] A method for screening for a substance having immunopotentiating ability, using the method described in any one of [1] to

[20] .

[23] A kit for use in the method described in any one of [1] to

[20] .

[24] Use of a human plasmacytoid dendritic cell line in evaluating the immunostimulatory activity of a substance of interest, the use comprising a contacting step of culturing the human plasmacytoid dendritic cell line in the presence of the substance of interest, the human plasmacytoid dendritic cell line being CAL-1 cells (deposit number FERM BP-10914), and the CAL-1 cells being in a starved state during at least a portion of the contacting step.

[25] Use of a human plasmacytoid dendritic cell line in screening for substances having immunostimulatory activity, the use comprising a contacting step of culturing the human plasmacytoid dendritic cell line in the presence of a test substance, the human plasmacytoid dendritic cell line being CAL-1 cells (deposit number FERM BP-10914), and the CAL-1 cells being in a starved state during at least a portion of the contacting step.

[0011] According to the present disclosure, a method for evaluating the immunostimulatory activity of a target substance using CAL-1 cells can be provided. For example, by using CAL-1 cells in a low-serum medium and / or by using CAL-1 cells in a starved state, the immunostimulatory activity of the target substance can be evaluated using the expression level of interferon α by CAL-1 cells as an indicator. For example, the evaluation method of the present disclosure uses an inexpensive cell line, which reduces costs compared to methods using mouse-derived BM-DCs or human-derived primary pDCs, and allows for the evaluation and screening of a larger number of substances. Furthermore, because the evaluation method of the present disclosure uses a human-derived cell line, it is possible to screen substances with immunostimulatory activity in humans with a higher probability.

[0012] 1 is a diagram showing the results of measuring the IFN-α concentration in the culture supernatant for each serum concentration in the medium in Example 1. FIG. 2 is a diagram showing the results of measuring the IFN-α concentration in the culture supernatant for each culture time in the pretreatment step in Example 2. FIG. 3 is a diagram showing the results of measuring the IFN-α concentration in the culture supernatant for each concentration of CAL-1 cells in Example 3. FIG. 4 is a diagram showing the results of measuring the IFN-α concentration in the culture supernatant when a 48-well microplate and a 96-well microplate are used in Example 4. FIG. 5 is a diagram showing the change over time in the fluorescence detection area for each concentration of Lactococcus lactis subsp. lactis JCM5805 (LC-Plasma) in Example 5. FIG. 6 is a diagram showing the results of measuring the IFN-α concentration in the culture supernatant for each concentration of Lactococcus lactis subsp. lactis JCM5805 (LC-Plasma) in Example 5. 1 is a diagram showing the fluorescence detection area 24 hours after the addition of Lactococcus lactis subsp. lactis JCM5805 (LC-Plasma) in a test using a serum-free medium (FBS 0%) and an FBS 10% medium in Example 5. 2 is a diagram showing the measurement results of the IFN-α concentration in the culture supernatant recovered 24 hours after the addition of Lactococcus lactis subsp. lactis JCM5805 (LC-Plasma) in a test using a serum-free medium (FBS 0%) and an FBS 10% medium in Example 5. 3 is a diagram showing the fluorescence detection area 24 hours after the addition of bacteria for the 10 types of bacteria tested in Example 6. 4 is a diagram showing the measurement results of the IFN-α concentration in the culture supernatant recovered 24 hours after the addition of bacteria for the 10 types of bacteria tested in Example 6. 1 is a diagram showing the IFN-α concentration in the supernatant when CAL-1 cells treated with a serum-free medium (0% FBS) or a 10% FBS medium (10% FBS) in the pretreatment step and the contact step were contacted with LC-Plasma in Example 7. FIG. 1 is a diagram showing the percentage of IFN-α-positive cells among CAL-1 cells contacted with PBS or Lactococcus lactis subsp. lactis JCM5805 (LC-Plasma).This figure shows the IFN-α concentration in the culture supernatant when the original CAL-1 cells and CAL-1 cell lines A to M newly obtained by single cell cloning were contacted with Lactococcus lactis subsp. lactis JCM5805 (LC-Plasma).

[0013] Hereinafter, an embodiment for carrying out the present invention will be described, but the present invention is not limited to the following embodiment.

[0014] A first embodiment of the present invention is a method for evaluating the immunostimulatory activity of a target substance, the method comprising a contacting step of culturing a human plasmacytoid dendritic cell line in the presence of the target substance, wherein the human plasmacytoid dendritic cell line is CAL-1 cells (deposit number FERM BP-10914), and the CAL-1 cells are in a starved state during at least a part of the contacting step (hereinafter also referred to as the "evaluation method of the first embodiment").

[0015] CAL-1 cells are a human plasmacytoid dendritic cell (pDC) cancer cell line established from tumor cells in a patient's peripheral blood (Patent Document 2), and are a cell line deposited at the National Institute of Technology and Evaluation, Biotechnology Center, Patent Microorganisms Depositary (NPMD, Japan) under deposit number FERM BP-10914.

[0016] In the evaluation method of the first embodiment, it is sufficient that CAL-1 cells are in a starved state during at least a portion of the contacting step. The CAL-1 cells may be in a starved state throughout the entire contacting step (total culture time), or during a portion of the contacting step (a portion of the culture time). Here, the contacting step begins when the target substance is contacted with the CAL-1 cells (starting time) and ends when the target substance is removed (ending time). If a medium change is performed during the contacting step, the target substance may be temporarily removed (within approximately one hour) by the medium change, and this time is counted as part of the contacting time. The term "starved state" in which CAL-1 cells are in a starved state during at least a portion of the contacting step includes cases where the CAL-1 cells are in a starved state only at the start of the contacting step, and also includes cases where the CAL-1 cells are not in a starved state at the start of the contacting step but become starved by the end of the contacting step depending on the culture conditions. The contacting step may be, for example, either A) culturing the CAL-1 cells so that they are in a starved state during the contacting step, or B) the CAL-1 cells are in a starved state at the start of the contacting step. A) above means, in other words, culturing the CAL-1 cells under conditions that put the CAL-1 cells in a starved state during part or the entire contacting step. B) above can be achieved, for example, by including a pretreatment step described below in the evaluation method of the first embodiment, or by using CAL-1 cells that have been stored or distributed in a starved state in the contacting step. It is sufficient for the contacting step to satisfy at least one condition selected from the group consisting of A) and B), and it is preferable that A) be satisfied, and it is more preferable that both A) and B) be satisfied. The present inventors have found that when the contact step satisfies at least one selected from the group consisting of A) and B), CAL-1 cells are more likely to be activated, making it easier to evaluate the immunostimulatory ability of the target substance; and that when the contact step satisfies both A) and B), CAL-1 cells are even more likely to be activated, making it even easier to evaluate the immunostimulatory ability of the target substance.Furthermore, in the contacting step, the CAL-1 cells are preferably in a starved state for 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% of the total culture time.

[0017] In the present disclosure, "starvation state" refers to a state in which components necessary for survival and / or proliferation are lacking. In the present disclosure, for example, the starvation state of CAL-1 cells can be confirmed using the method described in Example 5 of the present specification. More specifically, when the CAL-1 cells are exposed to Lactococcus lactis subsp. lactis JCM 5805 (hereinafter also referred to as "LC-Plasma") stained with pHrodo Red SE (Invitrogen, P36600) and the fluorescence detection area is measured, the starvation state can be confirmed by the fluorescence detection area being 1.5 times or more, preferably 2 times or more, 5 times or more, more preferably 10 times or more, and even more preferably 50 times or more, larger than the fluorescence detection area when CAL-1 cells cultured in a medium containing 10% by volume of fetal bovine serum (FBS) are exposed. Instead of using LC-Plasma stained with pHrodo Red SE (Invitrogen, P36600), it is also possible to use, without particular limitation, latex beads stained with fluorescently labeled rabbit IgG (Phagocytosis Assay Kit, IgG FITC, Funakoshi, 500290), latex beads stained with pHrodo Red SE (Invitrogen, P36600) (Aliphatic Amine Latex Beads, 2% w / v, 1.0 μm, Invitrogen, A37362), or LC-Plasma stained with another fluorescent substance.

[0018] In the present disclosure, the starvation state may refer to a state in which cells are cultured in a medium (low-serum medium) containing less than 1.0% by volume of serum based on the total volume of the medium. The serum content of the low-serum medium may be, for example, 0%, 0.01%, 0.02%, or 0.05% by volume at its lower limit, and 1.0%, 0.80%, 0.50%, 0.30%, 0.25%, 0.20%, 0.10%, 0.05%, 0.02%, 0.01%, or 0% by volume at its upper limit, based on the total volume of the medium. These upper and lower limits can be combined in any desired manner. For example, the serum content in a low serum medium can be 0% by volume or more and 1.0% by volume or less, 0% by volume or more and 0.80% by volume or less, 0% by volume or more and 0.50% by volume or less, 0% by volume or more and 0.30% by volume or less, 0% by volume or more and 0.25% by volume or less, 0% by volume or more and 0.20% by volume or more, 0% by volume or more and 0.10% by volume or more, 0% by volume or more and 0. 0.05% by volume or less, 0% by volume or more and 0.02% by volume or less, 0% by volume or more and 0.01% by volume or less, 0.01% by volume or more and 1.0% by volume or less, 0.01% by volume or more and 0.80% by volume or less, 0.01% by volume or more and 0.50% by volume or less, 0.01% by volume or more and 0.30% by volume or less, 0.01% by volume or more and 0.25% by volume or less, 0.01% by volume or more and 0.20% by volume or less, 0.01% by volume or more and 0.10% by volume or less, 0 0.01% by volume or more and 0.05% by volume or less, 0.01% by volume or more and 0.02% by volume or less, 0.02% by volume or more and 1.0% by volume or less, 0.02% by volume or more and 0.80% by volume or less, 0.02% by volume or more and 0.50% by volume or less, 0.02% by volume or more and 0.30% by volume or less, 0.02% by volume or more and 0.25% by volume or less, 0.02% by volume or more and 0.20% by volume or less, 0.02% by volume or more and 0.10% by volume or less, 0.0 The concentration may be 2% by volume or more and 0.05% by volume or less, 0.05% by volume or more and 1.0% by volume or less, 0.05% by volume or more and 0.80% by volume or less, 0.05% by volume or more and 0.50% by volume or less, 0.05% by volume or more and 0.30% by volume or less, 0.05% by volume or more and 0.25% by volume or less, 0.05% by volume or more and 0.20% by volume or less, or 0.05% by volume or more and 0.10% by volume or less, or may be 0% by volume (serum-free).When the serum content in the low-serum medium is within the above range, the production of IFN-α is likely to be promoted when the target substance is brought into contact with CAL-1 cells, and the amount of IFN-α expressed by CAL-1 cells is likely to be improved, making it possible to suitably evaluate the immunostimulatory activity of the target substance.

[0019] In a preferred embodiment, the low-serum medium may be a medium having a serum content of 0.10% by volume or less based on the total volume of the medium. In a more preferred embodiment, the low-serum medium may be a medium having a serum content of 0% by volume based on the total volume of the medium, i.e., a serum-free medium. When the low-serum medium contains multiple types of serum, the serum content (volume %) refers to the total volume % of those multiple types of serum. Furthermore, when the low-serum medium contains a serum substitute (a component that is not serum but promotes cell growth similar to serum), it is preferable that the total serum content (volume %) and serum substitute content (volume %) in the low-serum medium be within the above range.

[0020] In the present specification, serum generally refers to serum derived from humans or other animals that is used for culturing animal cells, including humans, and includes, for example, serum derived from humans, bovine serum, and equine serum, and more specifically, fetal bovine serum (FBS).

[0021] The medium can be the same as the medium (e.g., basal medium) commonly used by those skilled in the art for cell culture. For example, the medium may be a medium containing water as a solvent, sugars such as D-glucose, and amino acids such as L-glutamine, or may further contain serum in the above-mentioned amounts. The medium may further contain additives. The additives may be those commonly used by those skilled in the art for cell culture, such as at least one selected from the group consisting of antibiotics (penicillin, streptomycin, etc.), vitamins, inorganic salts, and buffers. In one aspect, the lower limit of the D-glucose content of the medium can be 500 mg / L or 1200 mg / L, and the upper limit of the D-glucose content of the medium can be 6000 mg / L or 3000 mg / L. These upper and lower limits can be combined in any desired manner, and for example, the D-glucose content of the medium can be 500 mg / L to 6000 mg / L, 500 mg / L to 3000 mg / L, 1200 mg / L to 6000 mg / L, or 1200 mg / L to 3000 mg / L. In one aspect, the lower limit of the L-glutamine content of the medium can be 100 mg / L or 250 mg / L, and the upper limit of the L-glutamine content of the medium can be 1000 mg / L or 600 mg / L. These upper and lower limits can be combined in any desired manner. For example, the L-glutamine content of the medium may be 100 mg / L to 1000 mg / L, 100 mg / L to 600 mg / L, 250 mg / L to 1000 mg / L, or 250 mg / L to 600 mg / L.

[0022] The medium can be prepared by a method commonly used by those skilled in the art. For example, it can be prepared by adding sugars, amino acids, and other components to water. For example, the medium can also be prepared by adding serum and other missing components at the desired concentration to a basal medium that can be purchased or easily prepared by those skilled in the art, such as RPMI-1640 medium (Roswell Park Memorial Institute-1640 medium) and DMEM medium (Dulbecco's Modified Eagle Medium). The prepared medium may be sterilized using an autoclave or the like before use in the contact step.

[0023] In the contacting step of the present invention, a target substance is contacted with CAL-1 cells in a medium to culture the CAL-1 cells. The medium used in the contacting step is not particularly limited, and a medium prepared by a method commonly used by those skilled in the art can be used, although a low-serum medium is preferable. Note that, in the case where the evaluation method of the first embodiment includes culturing CAL-1 cells under conditions that starve the CAL-1 cells in part or all of the above-mentioned A) contacting step, the state in which the CAL-1 cells are cultured in a medium containing less than 1.0% by volume of serum based on the total volume of the medium can also be said to be a low-serum medium in the contacting step.

[0024] In the contacting step, contacting the target substance with CAL-1 cells may be performed by a method commonly used by those skilled in the art. For example, contacting the target substance with CAL-1 cells may be performed by adding a suspension of CAL-1 cells to the target substance or a solution or suspension thereof, and culturing the cells in a medium for a predetermined period of time. In addition, contacting the target substance with CAL-1 cells may be performed by adding a solution or suspension of the target substance to CAL-1 cells, and culturing the cells in a medium for a predetermined period of time. In addition, contacting the target substance with CAL-1 cells may be performed by adding the target substance or a solution or suspension thereof to a suspension of CAL-1 cells, and culturing the cells in a medium for a predetermined period of time. The culturing in the contacting step may be performed under an environment commonly used by those skilled in the art for cell culturing, for example, at 37°C and 5% CO ２ The process may be carried out in air at 85% humidity.

[0025] CAL-1 cells are usually suspension cells, but in a starvation state, they exhibit an adherent cell-like behavior, and therefore the contacting step is preferably carried out in a state in which the CAL-1 cells are adhered to a substrate. That is, the contacting step is preferably carried out using a culture vessel to which CAL-1 cells can adhere. Examples of culture vessels to which CAL-1 cells can adhere include cell-adhesive well plates (e.g., 96-well microplates, 48-well microplates) and glass-bottom dishes.

[0026] The concentration of CAL-1 cells in the medium in the contact step is not particularly limited as long as it is a cell concentration that allows evaluation of the expression level of IFN-α by CAL-1 cells contacted with the substance of interest. In one embodiment, the concentration of CAL-1 cells in the medium in the contact step is 0.1 × 10 as the lower limit of the concentration before adhesion of CAL-1 cells. ４ cells / mL, 0.1×10 ５ cells / mL, 0.1×10 ６ cells / mL, or 0.2 x 10 ６ The upper limit of the concentration before CAL-1 cells adhere may be 5.0 × 10 ８ cells / mL, 5.0×10 ７ cells / mL, 5.0×10 ６ cells / mL, or 2.0 x 10 ６ These upper and lower limits can be arbitrarily combined, and for example, the concentration of CAL-1 cells in the medium in the contact step can be 0.1 × 10 cells / mL as the concentration before the CAL-1 cells adhere. ４ cells / mL or more 5.0×10 ８ cells / mL or less, 0.1×10 ４ cells / mL or more 5.0×10 ８ cells / mL or less, 0.1×10 ４ cells / mL or more 5.0×10 ８ cells / mL or less, 0.1×10 ４ cells / mL or more 5.0×10 ８ cells / mL or less, 0.1×10 ５cells / mL or more 5.0×10 ７ cells / mL or less, 0.1×10 ５ cells / mL or more 5.0×10 ７ cells / mL or less, 0.1×10 ５ cells / mL or more 5.0×10 ７ cells / mL or less, 0.1×10 ５ cells / mL or more 5.0×10 ７ cells / mL or less, 0.1×10 ６ cells / mL or more 5.0×10 ６ cells / mL or less, 0.1×10 ６ cells / mL or more 5.0×10 ６ cells / mL or less, 0.1×10 ６ cells / mL or more 5.0×10 ６ cells / mL or less, 0.1×10 ６ cells / mL or more 5.0×10 ６ cells / mL or less, 0.2×10 ６ cells / mL or more 2.0×10 ６ cells / mL or less, 0.2×10 ６ cells / mL or more 2.0×10 ６ cells / mL or less, 0.2×10 ６ cells / mL or more 2.0×10 ６ Cells / mL or less, and 0.2×10 ６ cells / mL or more 2.0×10 ６cells / mL or less. When the concentration of CAL-1 cells is within the above range, for example, it becomes easier to evaluate the degree of increase in the expression level of IFN-α in CAL-1 cells depending on the immunostimulatory activity of the target substance, and therefore the immunostimulatory activity of the target substance can be suitably evaluated. The concentration before CAL-1 cells adhere is the concentration before CAL-1 cells adhere to a culture vessel to which all of the components involved in the contacting step have been added. In other words, the concentration before CAL-1 cells adhere is the concentration when it is assumed that all cells are in a non-adherent state. The number of CAL-1 cells in the medium in the contacting step is not particularly limited as long as it is a cell number that allows evaluation of the expression level of IFN-α by CAL-1 cells contacted with the target substance. For example, in order to make it easier to evaluate the degree of increase in the expression level of IFN-α, the lower limit of the number of CAL-1 cells in the medium in the contacting step is 1.0 × 10 ４ cells or more, 0.4 x 10 ５ cells or more, 1.0 x 10 ５ cells or more, or 1.0 x 10 ６ The upper limit is 1.0 × 10 ８ cells, 5.0×10 ７ cells, or 1.0 x 10 ７ These upper and lower limits can be arbitrarily combined. In order to facilitate evaluation of the degree of increase in the expression level of IFN-α, the number of CAL-1 cells in the medium in the contact step can be set to 1.0 × 10 ４ cells or more 1.0 x 10 ８ Below cells, 1.0×10 ４ cells or more 5.0 x 10 ７ Below cells, 1.0×10 ４ cells or more 1.0 x 10 ７ Cells or less, 0.4 x 10 ５ cells or more 1.0 x 10 ８ Cells or less, 0.4 x 10 ５ cells or more 5.0 x 10 ７ Cells or less, 0.4 x 10 ５ cells or more 1.0 x 10 ７ Below cells, 1.0×10５ cells or more 1.0 x 10 ８ Below cells, 1.0×10 ５ cells or more 5.0 x 10 ７ Below cells, 1.0×10 ５ cells or more 1.0 x 10 ７ Below cells, 1.0×10 ６ cells or more 1.0 x 10 ８ Below cells, 1.0×10 ６ cells or more 5.0 x 10 ７ cells or less, or 1.0 x 10 ６ cells or more 1.0 x 10 ７ It may be less than cells.

[0027] The culture time (contact time) in the contacting step is not particularly limited, as long as it is a time that allows evaluation of the expression level of a cytokine such as IFN-α by CAL-1 cells contacted with the substance of interest. In one aspect, the lower limit of the culture time in the contacting step can be 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 15 hours, 18 hours, 20 hours, or 24 hours, or can be 168 hours, 72 hours, 48 ​​hours, 36 hours, 24 hours, 16 hours, 12 hours, 10 hours, or 8 hours.These upper and lower limits can be combined arbitrarily. For example, the culture time in the contact step can be set to 1 hour or more and 8 hours or less, 1 hour or more and 10 hours or less, 1 hour or more and 12 hours or less, 1 hour or more and 16 hours or less, 1 hour or more and 24 hours or less, 1 hour or more and 36 hours or less, 1 hour or more and 48 hours or less, 1 hour or more and 72 hours or less, 1 hour or more and 168 hours or less, 2 hours or more and 8 hours or less, 2 hours or more and 10 hours or less, 2 hours or more and 12 hours or less, 2 hours or more and 16 hours or less, 2 hours or more and ... more than 24 hours, more than 2 hours and less than 36 hours, more than 2 hours and less than 48 hours, more than 2 hours and less than 72 hours, more than 2 hours and less than 168 hours, more than 4 hours and less than 8 hours, more than 4 hours and less than 10 hours, more than 4 hours and less than 12 hours, more than 16 hours 4 to 24 hours, 4 to 36 hours, 4 to 48 hours, 4 to 72 hours, 4 to 168 hours, 8 to 10 hours, 8 to 12 hours, 8 to 16 hours, 8 hours more than 24 hours, more than 8 hours and less than 36 hours, more than 8 hours and less than 48 hours, more than 8 hours and less than 72 hours, more than 8 hours and less than 168 hours, more than 12 hours and less than 24 hours, more than 12 hours and less than 36 hours, more than 12 hours 48 hours or less, 12 hours to 72 hours, 12 hours to 168 hours, 15 hours to 16 hours, 15 hours to 24 hours, 15 hours to 36 hours, 15 hours to 48 hours, 15 hours to 72 hours, 3 p.m. The incubation time may be from 1 hour to 168 hours, from 18 hours to 24 hours, from 18 hours to 36 hours, from 18 hours to 48 hours, from 18 hours to 72 hours, or from 18 hours to 168 hours, from 20 hours to 24 hours, from 20 hours to 36 hours, from 20 hours to 48 hours, from 20 hours to 72 hours, from 20 hours to 168 hours, from 24 hours to 36 hours, from 24 hours to 48 hours, from 24 hours to 72 hours, or from 24 hours to 168 hours. When the incubation time is equal to or greater than the lower limit, the expression level of IFN-α in CAL-1 cells increases, making its measurement easier, and the immunostimulatory activity of the target substance can be suitably evaluated.

[0028] The evaluation method of the first embodiment may further include a pretreatment step of starving CAL-1 cells prior to the contacting step. When the evaluation method of the first embodiment includes the pretreatment step, the above-described condition B) that CAL-1 cells are in a starved state at the start of the contacting step is satisfied. The pretreatment medium used in the pretreatment step can be the same as that described for the medium used in the contacting step. For example, the pretreatment medium may have a serum content of 0 vol%, 0.01 vol%, 0.02 vol%, or 0.05 vol%, based on the total volume of the medium, with the lower limit being 0 vol%, 0.01 vol%, 0.02 vol%, or 0.05 vol%, and the upper limit being 0.80 vol%, 0.50 vol%, 0.30 vol%, 0.25 vol%, 0.20 vol%, 0.10 vol%, 0.05 vol%, 0.02 vol%, 0.01 vol%, or 0 vol% (serum-free). These upper and lower limits can be combined arbitrarily. For example, the serum content in the pretreatment medium can be 0% by volume or more and 0.80% by volume or less, 0% by volume or more and 0.50% by volume or less, 0% by volume or more and 0.30% by volume or less, 0% by volume or more and 0.25% by volume or less, 0% by volume or more and 0.20% by volume or less, 0% by volume or more and 0.10% by volume or less, 0% by volume or more and 0.05% by volume or less, 0% by volume or more and 0.02% by volume or less, 0% by volume or more and 0.01% by volume or less, 0.01% by volume or more and 0.80% by volume or less, 0.01% by volume or more and 0.50% by volume or less, 0.01% by volume or more and 0.30% by volume or less, 0.01% by volume or more and 0.25% by volume or less, 0.01% by volume or more and 0.20% by volume or less, 0.01% by volume or more and 0.10% by volume or less , 0.01% by volume or more and 0.05% by volume or less, 0.01% by volume or more and 0.02% by volume or less, 0.02% by volume or more and 0.80% by volume or less, 0.02% by volume or more and 0.50% by volume or less, 0.02% by volume or more and 0.30% by volume or less, 0.02% by volume or more and 0.25% by volume or less, 0.02% by volume or more and 0.20% by volume or less, 0.02% by volume or more and 0.10% by volume or less, 0.02% by volume or more and 0.05% by volume or less, 0.05% by volume or more and 0.80% by volume or less, 0.05% by volume or more and 0.50% by volume or less, 0.05% by volume or more and 0.30% by volume or less, 0.05% by volume or more and 0.25% by volume or less, 0.05% by volume or more and 0.20% by volume or less, or 0.05% by volume or more and 0.10% by volume or less, or it may be 0% by volume (serum-free).In this case, the starvation state of CAL-1 cells is more likely to be maintained in the contact step, and the enhanced IFN-α production activity of the starved CAL-1 cells is more likely to be maintained, allowing for favorable evaluation of the immunostimulatory activity of the target substance.

[0029] The pretreatment medium is not particularly limited and can be a medium prepared by a method commonly used by those skilled in the art. It may be a medium having the same composition as the medium used in the contacting step, or a medium having a different composition from the medium used in the contacting step. When the composition of the pretreatment medium is the same as that of the medium used in the contacting step, the contacting step may be performed by adding the target substance to the culture supernatant (medium) used in the pretreatment step and then culturing the culture medium. For example, when the composition of the pretreatment medium and the composition of the medium used in the contacting step are different, the CAL-1 cells used in the contacting step may be CAL-1 cells separated from the pretreatment medium after the pretreatment step by removing the culture supernatant or by centrifugation or the like.

[0030] In the pretreatment step, CAL-1 cells are cultured in a pretreatment medium. The culture in the pretreatment step may be performed under an environment in which a person skilled in the art would normally culture cells, for example, at 37°C and 5% CO ２ The pretreatment step may be carried out in air with a humidity of 100%. CAL-1 cells are usually suspension cells, but since the pretreatment step causes them to take on an adherent cell-like appearance, the pretreatment step is preferably carried out using a culture vessel to which CAL-1 cells can adhere. Examples of culture vessels to which CAL-1 cells can adhere include cell-adhesive well plates (e.g., 96-well microplates) and glass-bottom dishes. The concentration of CAL-1 cells in the pretreatment step is not particularly limited, and for example, the concentration before the CAL-1 cells adhere can be 0.1 x 10 ６ cells / mL or more 10.0×10 ６ The concentration may be 1000 cells / mL or less.

[0031] The culture time in the pretreatment step is not particularly limited as long as it is a time that allows evaluation of the expression level of IFN-α by CAL-1 cells contacted with the substance of interest in the subsequent contact step. In one aspect, the lower limit of the culture time in the pretreatment step can be 1 hour, 2 hours, 4 hours, 6 hours, or 8 hours, and the upper limit can be 72 hours, 48 ​​hours, 36 hours, 24 hours, 16 hours, 12 hours, 10 hours, or 8 hours. These upper and lower limits can be combined arbitrarily. For example, the culture time in the pretreatment step can be 1 hour or more and 72 hours or less, 1 hour or more and 48 hours or less, 1 hour or more and 36 hours or less, 1 hour or more and 24 hours or less, 1 hour or more and 16 hours or less, 1 hour or more and 12 hours or less, 1 hour or more and 10 hours or less, 1 hour or more and 8 hours or less, 2 hours or more and 72 hours or less, 2 hours or more and 48 hours or less, 2 hours or more and 36 hours or less, 2 hours or more and 24 hours or less, 2 hours or more and 16 hours or less, 2 hours or more and 12 hours or less, 2 hours or more and 10 hours or less, 2 hours or more and 8 hours or less, 4 hours or more and 72 hours or less, 4 hours or more and 48 hours or less. The incubation time in the pretreatment step may be, for example, 4 hours to 36 hours, 4 hours to 24 hours, 4 hours to 16 hours, 4 hours to 12 hours, 4 hours to 10 hours, 4 hours to 8 hours, 6 hours to 72 hours, 6 hours to 48 hours, 6 hours to 36 hours, 6 hours to 24 hours, 6 hours to 16 hours, 6 hours to 12 hours, 6 hours to 10 hours, 6 hours to 8 hours, 8 hours to 72 hours, 8 hours to 48 hours, 8 hours to 36 hours, 8 hours to 24 hours, 8 hours to 16 hours, 8 hours to 12 hours, or 8 hours to 10 hours. When the incubation time in the pretreatment step is within the above range, the expression level of IFN-α can be increased while suppressing a decrease in cell viability in the subsequent contact step, allowing the immunostimulatory ability of the target substance to be suitably evaluated.

[0032] The target substance in the evaluation method of the first embodiment may be any substance that can be brought into contact with cells. The target substance may be, for example, a cell, a virus, a protein, an amino acid, a nucleic acid, a sugar, or a low molecular weight compound, or a complex thereof, or may be a cell, a virus, a protein, an amino acid, a nucleic acid, or a sugar, or may be a cell or a virus, or may be a cell. The cell may be a prokaryotic cell and / or a eukaryotic cell, or may be a prokaryotic cell. The prokaryotic cell may be a bacterium. The low molecular weight compound may be, for example, a compound with a molecular weight (g / mol) of 1000 or less.

[0033] In one embodiment, the target substance may be a bacterium, and in particular a bacterium that is useful to the human body. When the target substance is a bacterium, it tends to be phagocytosed by CAL-1 cells when the target substance is brought into contact with the CAL-1 cells. The phagocytosed bacterium is decomposed within the CAL-1 cells and can exert its physiological action. By using bacteria as the target substance, the immunostimulatory activity of bacteria having immunostimulatory activity can be evaluated in a simple and low-cost manner. Suitable bacteria may be gram-positive bacteria or gram-negative bacteria. For example, the bacterium may be a lactic acid bacterium or an acetic acid bacterium, and preferably a lactic acid bacterium.

[0034] Lactic acid bacteria are bacteria that produce lactic acid as a metabolite. Examples of lactic acid bacteria include bacteria of the genus Oenococcus, Bifidobacterium, Weissella, Tetragenococcus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus, Enterococcus, Lactobacillus, acetic acid bacteria, and Bacillus.

[0035] In this specification, the term "Lactobacillus" includes bacteria that were classified into the genus Lactobacillus before the reclassification of the genus Lactobacillus.For example, with the reclassification of the genus Lactobacillus, the genus Acetilactobacillus, the genus Agrilactobacillus, the genus Amylolactobacillus, the genus Apilactobacillus, the genus Bombilactobacillus, the genus Companilactobacillus, the genus Dellaglioa, the genus Fructilactobacillus, the genus Furfurilactobacillus, the genus ), Holzapfelia genus, Lacticaseibacillus genus, Lactiplantibacillus genus, Lapidilactobacillus genus, Latilactobacillus genus, Lentilactobacillus genus, Levilactobacillus genus, Ligilactobacillus genus, Limosilactobacillus This includes bacteria classified into the genera Liquorilactobacillus, Loigolactobacillus, Paralactobacillus, Paucilactobacillus, Schleiferilactobacillus, Secundilactobacillus, etc.

[0036] Among the above, bacteria of the genus Oenococcus, Bifidobacterium, Lentilactobacillus, Weissella, Tetragenococcus, Lactococcus, Leuconostoc, Pediococcus, Enterococcus, Lactobacillus, and Lactiplantibacillus are preferred.

[0037] Examples of the Oenococcus include Oenococcus oeni, etc. Specific examples of the Oenococcus include Oenococcus oeni JCM6125, etc.

[0038] Examples of Bifidobacterium bacteria include Bifidobacterium animalis subsp. lactis, Bifidobacterium pseudolongum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum subsp. longum, and Bifidobacterium longum subsp. infantis. Specific examples of Bifidobacterium bacteria include Bifidobacterium animalis subsp. lactis JCM 10602, Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium longum subsp. infantis JCM 1222, Bifidobacterium longum subsp. infantis M-63, and Bifidobacterium longum subsp. longum. BB536, Bifidobacterium longum subsp. longum N61, Bifidobacterium bifidum OLB6378, Bifidobacterium breve M-16V, Bifidobacterium breve MCC1274, and Bifidobacterium pseudolongum JCM1205.

[0039] Examples of Weissella include Weissella paramesenteroides and Weissella viridescens. Specific examples of Weissella include Weissella paramesenteroides JCM 9890 and Weissella viridescens JCM 1174.

[0040] Examples of bacteria of the genus Tetragenococcus include Tetragenococcus halophilus, etc. Specific examples of bacteria of the genus Tetragenococcus include Tetragenococcus halophilus NRIC 0098 and Tetragenococcus halophilus No. 1.

[0041] Examples of Lactococcus bacteria include Lactococcus lactis, Lactococcus lactis subsp. lactis, Lactococcus garvieae, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. hordniae, and Lactococcus plantarum.

[0042] Specific examples of Lactococcus bacteria include Lactococcus lactis subsp. lactis JCM 5805, Lactococcus lactis subsp. lactis NBRC 12007, Lactococcus lactis subsp. lactis NRIC 1150, Lactococcus lactis subsp. lactis JCM 20101, Lactococcus lactis subsp. lactis JCM 7638, Lactococcus lactis subsp. lactis ATCC 7963, Lactococcus lactis subsp. lactis ATCC 7962, Lactococcus lactis subsp. lactis ATCC 29146, and Lactococcus lactis subsp. lactis ATCC lactis ATCC 15577, Lactococcus lactis subsp. lactis ATCC 15346, Lactococcus lactis subsp. lactis ATCC 13675, Lactococcus lactis subsp. lactis ATCC 12929, Lactococcus lactis subsp. lactis ATCC 11955, Lactococcus lactis subsp. lactis ATCC 11454, Lactococcus lactis subsp. lactis ATCC 11007, Lactococcus garvieae NBRC 100934, Lactococcus lactis subsp. cremoris JCM 16167, Lactococcus lactis subsp. cremoris NBRC 100676, Lactococcus lactis subsp. holdoniae JCM 1180, Lactococcus lactis subsp. holdoniae JCM 11040, and Lactococcus plantarum JCM 11056.

[0043] Examples of bacteria of the genus Leuconostoc include Leuconostoc carnosum and Leuconostoc lactis. Specific examples of bacteria of the genus Leuconostoc include Leuconostoc carnosum JCM 9695 and Leuconostoc lactis NBRC 12455.

[0044] Examples of bacteria of the genus Pediococcus include Pediococcus acidilactici, Pediococcus pentosaceus, Pediococcus cellicola, Pediococcus claussenii, Pediococcus damnosus, Pediococcus ethanolidurans, Pediococcus inopinatus, Pediococcus parvulus, and Pediococcus stilesii. Specific examples of the genus Pediococcus include Pediococcus acidilactici JCM 8797, Pediococcus acidilactici K15, and Pediococcus damnosus JCM 5886.

[0045] Examples of bacteria of the genus Streptococcus include Streptococcus thermophilus, etc. Specific examples of bacteria of the genus Pediococcus include Streptococcus thermophilus SBC 8781, etc.

[0046] Examples of bacteria of the genus Enterococcus include Enterococcus alcedinis, Enterococcus faecalis, etc. Specific examples of bacteria of the genus Enterococcus include Enterococcus faecalis EC-12, etc.

[0047] Examples of Lactobacillus bacteria include Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fructivorans, Lactobacillus hilgardii, Lactobacillus rhamnosus, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus bulgaricus, and Lactobacillus parakefii. parakefiri), Lactobacillus plantarum, and Lactobacillus pentosus.

[0048] Specific examples of Lactobacillus bacteria include Lactobacillus paracasei KW3110, Lactobacillus paracasei MCC1849, Lactobacillus paracasei K71, Lactobacillus paracasei K-2, Lactobacillus rhamnosus GG, Lactobacillus rhamnosus CRL1505, Lactobacillus gasseri SBT2055, Lactobacillus gasseri OLL2716, Lactobacillus Lactobacillus gasseri PA-3, Lactobacillus acidophilus L-92, Lactobacillus casei subsp. casei 327, Lactobacillus (newly classified as Lacticaseibacillus) casei Shirota, Lactobacillus bulgaricus OLL1073R-1, Lactobacillus bulgaricus 2038, Lactobacillus parakeefili (newly classified as Lentilactobacillus parakeefili) JCM 8573, Lactobacillus plantarum (newly classified as Lactipranchibacillus plantarum) L-137, Lactobacillus pentosus (newly classified as Lactipranchibacillus pentosus) ONRICb0240, and the like.

[0049] The acetic acid bacteria are not particularly limited, but examples thereof include bacteria of the genus Gluconacetobacter, Acetobacter, and Gluconobacter, preferably bacteria of the genus Gluconacetobacter, more preferably Gluconacetobacter hansenii, and even more preferably Gluconacetobacter hansenii GK-1.

[0050] The Bacillus bacteria are not particularly limited, but include, for example, Bacillus coagulans, etc. Specific examples of Bacillus bacteria include Bacillus coagulans SANK 70258 strain, etc.

[0051] Among the above, the target substance is preferably selected from the group consisting of bacteria of the genus Lactobacillus, Lactococcus, and Gluconacetobacter in terms of flavor for consumption.

[0052] In addition to the above, the bacteria that are the target substance in one embodiment may be bacteria of the genus Akkermansia, Bacteroides, Blautia, Clostridium, Collinsella, Faecalibacterium, Faecalicatena, Lacrimispora, Paeniclostridium, Parabacteroides, or Roseburia.

[0053] Examples of the Akkermansia genus include Akkermansia muciniphila JCM30893.

[0054] Examples of Bacteroides include Bacteroides caccae JCM9498T, Bacteroides fragilis JCM 11019T, Bacteroides fragilis JCM 11017, Bacteroides fragilis JCM 17586, Bacteroides fragilis JCM 17587, Bacteroides ovatus JCM 5824T, Bacteroides setaiotaomicron ATCC 29148T, Bacteroides setaiotaomicron ATCC 29741, Bacteroides setaiotaomicron ATCC 12290, Bacteroides uniformis JCM 5828T, Bacteroides uniformis JCM 13286, Bacteroides uniformis JCM 13287, and Bacteroides uniformis JCM 13288.

[0055] Examples of Blautia include Blautia acetigens JCM 34803T, Blautia ammoniilytica JCM 34802T, Blautia algi JCM 31394T, Blautia caekimuris JCM 34498T, Blautia coccoides JCM 1395T, Blautia faeces JCM 17205T, Blautia glucellacea JCM 17039T, Blautia hansenii JCM 14655, Blautia hansenii JCM 35484, Blautia hominis JCM 32276T, Blautia hydrogenotrophica JCM 31266, and Blautia liquorice JCM 34498T. 34225T, Blautia luti JCM 17040T, Blautia obeum JCM 31340, Blautia producta JCM 1471T, Blautia pseudococcoides JCM 35243T, Blautia cinquii JCM 14657T, Blautia wechslerae JCM 31267, and Blautia wechslerae JCM 35486.

[0056] Examples of bacteria of the genus Clostridium include Clostridium butyricum JCM NT, Clostridium neccille JCM 31500T, and Clostridium symbiosum JCM 1297T.

[0057] Examples of Collinsella bacteria include Collinsella aerofaciens JCM 10188T, Collinsella intestinalis JCM 10643T, Collinsella stercoris JCM 10641T, and Collinsella tanakaei JCM 16071T.

[0058] Examples of the genus Faecalibacterium include Faecalibacterium hattorii JCM 39210, Faecalibacterium longum JCM 39208, Faecalibacterium prausnitzii JCM 31915, Faecalibacterium prausnitzii JCM 39207, and Faecalibacterium prausnitzii JCM 39209.

[0059] Examples of the genus Faecalicatena include Faecalicatena oroticum JCM 1429T.

[0060] Examples of the fungi of the genus Lacrimispora include Lacrimispora celerecrescens JCM 15734T, Lacrimispora sphenoides JCM 1415T, and Lacrimispora xylanolytica JCM 15735T.

[0061] Examples of the genus Paeniclostridium include Paeniclostridium sordellii JCM 3814T.

[0062] Examples of the Parabacteroides genus include Parabacteroides meldae JCM 9497T.

[0063] Examples of the genus Roseburia include Roseburia hominis JCM 17582, Roseburia intestinalis JCM 17583, and Roseburia inulinovorans JCM 17584.

[0064] When the target substance is bacteria, the target substance may be a single type of bacteria or a mixture of two or more types of bacteria, for example, a mixture of lactic acid bacteria and acetic acid bacteria.

[0065] In one embodiment, the target substance may be a substance that is phagocytosed by CAL-1 cells. Phagocytosis is a phenomenon that occurs when cells ingest relatively large objects, such as solid particles, other cells, or bacteria. During phagocytosis, a cell extends its cell membrane to engulf the object and ingest it intracellularly. After phagocytosis, a vesicle (phagosome) containing the ingested object fuses with a lysosome to degrade the contents. The present inventors have found that contacting CAL-1 cells with a target substance in a low-serum medium (described below) enhances the phagocytic activity of the target substance by CAL-1 cells compared to contacting the cells in a medium containing a high serum concentration (e.g., 10% by volume), as commonly used for cell culture. Furthermore, the present inventors have found a strong positive correlation between the amount of phagocytosis for each target substance and its immunostimulatory activity. Therefore, if the target substance is a substance that is phagocytosed by CAL-1 cells, the immunostimulatory activity of the target substance can be more reliably evaluated.

[0066] Whether a target substance is a substance that is phagocytosed by CAL-1 cells can be determined, for example, by the method described in the Examples. Specifically, this determination can be based on whether fluorescence is detected from CAL-1 cells when a target substance labeled with a fluorescent dye that becomes highly fluorescent in an acidic environment (e.g., pHrodo Red SE, Invitrogen, P36600) is contacted with CAL-1 cells in serum-free medium for a sufficient period of time (e.g., 24 hours). Furthermore, this determination can be based on whether the fluorescence intensity of CAL-1 cells when a target substance labeled with a fluorescent dye (e.g., an amine-reactive dye is preferred, and an amine-reactive dye pH-sensitive (preferably a fluorescent dye that becomes highly fluorescent in an acidic environment, such as within an endosome) dye is more preferred) is contacted with CAL-1 cells in serum-free medium for a sufficient period of time (e.g., 24 hours) is greater (e.g., three times or more) than the fluorescence intensity when a similar test is performed in a medium further containing a component that inhibits phagocytosis, such as cytochalasin D.

[0067] The concentration of the target substance in the medium in the contacting step is not particularly limited, as long as it is for a period of time that allows evaluation of the expression level of IFN-α by CAL-1 cells contacted with the target substance. In one aspect, the lower limit of the concentration of the target substance in the medium in the contacting step can be, for example, 0.1 μg / mL, 1.0 μg / mL, 3.0 μg / mL, 5.0 μg / mL, or 10.0 μg / mL, and the upper limit can be, for example, 10,000 μg / mL, 5,000 μg / mL, 1,000 μg / mL, 300 μg / mL, 200 μg / mL, or 100 μg / mL. These upper and lower limits can be combined arbitrarily. For example, the concentration of the target substance in the medium in the contact step can be set to 0.1 μg / mL or more and 10,000 μg / mL or less, 0.1 μg / mL or more and 5,000 μg / mL or less, 0.1 μg / mL or more and 1,000 μg / mL or less, 0.1 μg / mL or more and 300 μg / mL or less, 0.1 μg / mL or more and 200 μg / mL or less, 0.1 μg / mL or more and 100 μg / mL or less. g / mL or less, 1.0 μg / mL or more and 10000 μg / mL or less, 1.0 μg / mL or more and 5000 μg / mL or less, 1.0 μg / mL or more and 1000 μg / mL or less, 1.0 μg / mL or more and 300 μg / mL mL or less, 1.0 μg / mL or more and 200 μg / mL or less, 1.0 μg / mL or more and 100 μg / mL or less, 3.0 μg / mL or more and 10,000 μg / mL or less, 3.0 μg / mL or more and 5,000 μg / mL or less Bottom, 3.0 μg / mL or more and 1000 μg / mL or less, 3.0 μg / mL or more and 300 μg / mL or less, 3.0 μg / mL or more and 200 μg / mL or less, 3.0 μg / mL or more and 100 μg / mL or less, 5.0 μg / mL to 10000 μg / mL, 5.0 μg / mL to 5000 μg / mL, 5.0 μg / mL to 1000 μg / mL, 5.0 μg / mL to 300 μg / mL, 5.0 μg / mL or more and 200 μg / mL or less, 5.0 μg / mL or more and 100 μg / mL or less, 10.0 μg / mL or more and 10,000 μg / mL or more, 10.0 μg / mL or more and 5,000 μg / mL or less, 10.0 μg / mL or more and 1,000 μg / mL or more, 10.0 μg / mL or more and 300 μg / mL or less, 10.0 μg / mL or more and 200 μg / mL or less, or 10.0 μg / mL or more and 100 μg / mL or less.When the concentration of the target substance is within the above range, it becomes easier to evaluate the degree of increase in the expression level of IFN-α in CAL-1 cells in accordance with the immunostimulatory ability of the target substance, and therefore the immunostimulatory ability of the target substance can be suitably evaluated.

[0068] In the present invention, the immunopotentiating ability (immune activation ability) refers to the ability of a target substance to activate (activate) the innate immune system in cells contacted with the substance or in an animal to which the substance is administered. In one aspect, the immunopotentiating ability may be a dendritic cell activation ability that activates the innate immune system of dendritic cells, and more specifically, may be a plasmacytoid dendritic cell activation ability (pDC activation ability). In one aspect, the immunopotentiating ability may be an increase in the expression level of a cytokine or cell surface marker secreted from dendritic cells, preferably an increase in the expression level of IFN-α, or an increase in the expression level of IFN-α in pDC, and more preferably an increase in the expression level of IFN-α in CAL-1 cells. In vivo, pDC is the main producer of IFN-α, and IFN-α is a type of cytokine responsible for the host defense mechanism via the innate immune system. Therefore, a substance capable of promoting IFN-α expression by pDC can be said to be a substance having dendritic cell activation ability and pDC activation ability, or a substance having immunopotentiating ability. In other words, a substance that activates CAL-1 cells and has the ability to promote IFN-α expression by CAL-1 cells can also be said to have the ability to activate dendritic cells and pDCs, or to have immunostimulatory activity.

[0069] Activation of CAL-1 cells may refer to, for example, the promotion of cytokine production by CAL-1 cells. In this case, CAL-1 cells that are easily activated refer to CAL-1 cells that are easily stimulated to produce cytokines. As a more specific example, they refer to CAL-1 cells that are likely to produce increased amounts of cytokines when contacted with a substance known to have the effect of activating CAL-1 cells. In these cases, the cytokine is preferably IFN-α. In these cases, the immunostimulatory ability of CAL-1 cells can be measured using as an indicator the amount of IFN-α produced and / or the proportion of IFN-α-positive cells when CAL-1 cells are contacted with a substance known to have the effect of activating CAL-1 cells. As a more specific example, the measurement can also be performed by contacting a substance known to have the effect of activating CAL-1 cells as a target substance according to an evaluation method according to one aspect of the first embodiment of the present disclosure. When easily activated CAL-1 cells are contacted with a certain target substance, the degree of activation tends to be higher compared to when less easily activated CAL-1 cells are contacted with the same target substance, specifically, the amount of IFN-α secreted extracellularly and / or the amount of IFN-α in the cells, the amount of mRNA encoding IFN-α, and the proportion of interferon-α-positive cells in the CAL-1 cells are higher. Such CAL-1 cells can be said to be easily activated. In this way, the fact that CAL-1 cells are easily activated can also be rephrased as the fact that CAL-1 cells are highly active or that CAL-1 cells are easily activated.

[0070] In the evaluation method of the first embodiment, the immunostimulatory ability of a target substance can be evaluated using as indicators the amount of IFN-α expressed by CAL-1 cells contacted with the target substance in the contacting step and / or the proportion of interferon-α-positive cells in the CAL-1 cells. The evaluation method of the first embodiment may include a measuring step of measuring the amount of IFN-α expressed by CAL-1 cells after contact with the target substance and / or the proportion of interferon-α-positive cells in CAL-1 cells after contact with the target substance.

[0071] In one aspect of the evaluation method of the first embodiment, the immunostimulatory ability of a target substance may be evaluated using the expression level of IFN-α by CAL-1 cells contacted with the target substance in the contacting step as an index. When the immunostimulatory ability of a target substance is evaluated using the expression level of IFN-α by CAL-1 cells contacted with the target substance in the contacting step as an index, the measured value measured in the measuring step is the amount of a biomolecule that can serve as an evaluation index for the amount of IFN-α expressed by CAL-1 cells upon contact with the target test substance (hereinafter also referred to as a contact measurement value). In one aspect, the expression level of IFN-α may be the amount of IFN-α secreted extracellularly and / or the amount of IFN-α in CAL-1 cells, or the amount of mRNA encoding IFN-α. In a preferred aspect, the expression level of IFN-α may be the amount of IFN-α secreted extracellularly.

[0072] The amount of extracellularly secreted IFN-α can be evaluated, for example, by the ELISPOT method, in which extracellularly secreted IFN-α is adsorbed onto a cell culture well and measured, or by measuring the amount of extracellularly secreted IFN-α present in the culture supernatant. In this case, the culture supernatant is the medium obtained after contacting the target substance with CAL-1 cells in the contacting step. The amount of IFN-α in the culture supernatant can be measured after collecting the culture supernatant using a method commonly used by those skilled in the art to quantify a specific protein, for example, by ELISA or Western blotting.

[0073] The amount of IFN-α in CAL-1 cells can be measured by methods commonly used by those skilled in the art, such as intracellular cytokine staining using a flow cytometer, or by preparing a CAL-1 cell lysate and then measuring the amount of IFN-α in the lysate using a method commonly used by those skilled in the art to quantify a specific protein. In these cases, the amount of IFN-α can be measured by, for example, ELISA or Western blotting, or by ELISA. For example, the Human IFN-Alpha Multi-Subtype ELISA Kit (PBL Assay Science) can be used to measure the amount of IFN-α by ELISA.

[0074] Alternatively, the amount of mRNA encoding IFN-α in CAL-1 cells can be measured by preparing a CAL-1 cell lysate by a method commonly used by those skilled in the art, and then measuring the amount of mRNA encoding IFN-α in the lysate by a method commonly used by those skilled in the art for quantifying mRNA. Quantification of mRNA may be performed, for example, by quantitative PCR (q-PCR) such as real-time PCR, direct digital counting (e.g., nCounter®), or a next-generation sequencer (NGS).

[0075] In the evaluation method of this embodiment, the immunostimulatory ability of a target substance may be evaluated using the expression level of IFN-α measured in the measurement step, i.e., the contact measurement value, as an index. In this case, a higher expression level of IFN-α by CAL-1 cells indicates that the target substance has immunostimulatory ability. For example, the presence or absence, and the level of immunostimulatory ability of the target substance may be evaluated based on whether or not the contact measurement value exceeds the expression level of IFN-α (hereinafter also referred to as a reference substance contact measurement value) obtained when a similar test is performed on CAL-1 cells that have not been contacted with the target substance or on CAL-1 cells that have been contacted with a substance that does not have immunostimulatory ability (reference substance), and the extent to which this exceeds the expression level. Furthermore, the presence or absence, and the level of immunostimulatory ability of the target substance may be evaluated based on whether or not the expression level of IFN-α by CAL-1 cells measured in the measurement step (contact measurement value) exceeds the expression level of IFN-α (hereinafter also referred to as a non-contact measurement value) by CAL-1 cells that have not been subjected to the contact step (i.e., not contacted with the target substance), and the extent to which this exceeds the expression level. In these cases, the measurement value (contact measurement value) measured in the measurement step exceeding the comparison target measurement value (e.g., a reference substance contact measurement value or a non-contact measurement value) may mean, for example, that the average value of the measurement value measured in the measurement step is greater than the average value of the comparison target measurement value. Furthermore, for example, the measurement value measured in the measurement step exceeding the comparison target measurement value may mean that the measurement value measured in the measurement step is statistically significantly greater than the comparison target measurement value (e.g., a p-value of less than 0.05 or 0.01 in a Student's t-test). In one aspect, if the measurement value measured in the measurement step exceeds the comparison target measurement value, the target substance may be determined to have immunostimulatory activity. Thus, in one aspect, the evaluation method of the first embodiment may further include, in addition to the measurement step, a comparison step of comparing the contact measurement value with the reference substance contact measurement value or non-contact measurement value, and in one aspect, the comparison step may include a determination step of determining that the target substance has immunostimulatory activity when the contact measurement value exceeds the reference substance contact measurement value or non-contact measurement value.In addition, in one aspect, the evaluation method of the first embodiment may further include, in addition to the measurement step, a comparison step of comparing the contact measurement value with the non-contact measurement value, and in one aspect, the comparison step may include a determination step of determining that the target substance has immunostimulatory activity when the contact measurement value exceeds the non-contact measurement value.

[0076] In one aspect of the evaluation method of the first embodiment, the immunostimulatory activity of a target substance may be evaluated using as an index the proportion of interferon-α-positive cells among CAL-1 cells contacted with the target substance in the contacting step. The proportion of IFN-α-positive cells among CAL-1 cells can be measured as the proportion of cells in a population of CAL-1 cells from which a significant signal corresponding to the IFN-α labeling is detected, relative to the number of cells contained in the population of CAL-1 cells from which the dead cells have been removed, after labeling intracellular IFN-α and dead cells in the population of CAL-1 cells. The dead cell label is preferably one whose signal does not weaken even after the fixation step performed in intracellular cytokine labeling. For example, the label may be a fluorescent label with an amine-reactive fluorescent dye that is impermeable to live cells but permeable only to dead cells and can be fixed. Dead CAL-1 cells can be labeled, for example, by contacting CAL-1 cells with a fixable amine-reactive fluorescent dye (e.g., Zombie Aqua® Fixable Viability Kit (Biolegend, 423101)) before fixation and permeabilization. IFN-α in CAL-1 cells can be labeled with any label that can provide an index of the amount of IFN-α in CAL-1 cells, and may be, for example, a fluorescent label. IFN-α in CAL-1 cells can be labeled, for example, by contacting fixed and permeabilized CAL-1 cells with a labeled anti-IFN-α antibody (e.g., BD Pharmingen (registered trademark) PE Mouse anti-Human IFN-α[2b] (BD Pharmingen (registered trademark), 560097)), or by genetically fusing a labeled protein to IFN-α in CAL-1 cells, or by binding a reporter gene to the promoter region of interferon regulatory factor 7 (IRF7), a transcription factor that regulates IFN-α gene expression. The method for detecting a signal corresponding to the labeled IFN-α may be any method capable of detecting an intracellular signal, and may be, for example, flow cytometry or fluorescence observation. Significant detection of a signal corresponding to the IFN-α label from CAL-1 cells may mean, for example, that the signal is greater than a predetermined lower limit.The predetermined lower limit may be determined, for example, as the signal magnitude that corresponds to a threshold value of a predetermined upper percentage (e.g., 0.01%, 0.1%, or 1.0%) when a signal is measured in CAL-1 cells contacted with a labeled antibody that does not bind to IFN-α at the same concentration instead of the labeled anti-IFN-α antibody. The predetermined lower limit may also be determined, for example, as the signal magnitude that corresponds to a threshold value of a predetermined upper percentage (e.g., 0.01%, 0.1%, or 1.0%) when a signal is measured in CAL-1 cells that have not been subjected to the contacting step.

[0077] In the evaluation method of this embodiment, the immunostimulatory activity of a target substance may be evaluated using as an index the proportion of IFN-α-positive cells among CAL-1 cells measured in the measurement step (hereinafter also referred to as the contact positive proportion). In this case, a higher proportion of IFN-α-positive cells indicates that the target substance has immunostimulatory activity. For example, the presence or absence, and the level of immunostimulatory activity, of a target substance may be evaluated based on whether or not the contact positive proportion exceeds the proportion of IFN-α-positive cells (hereinafter also referred to as the reference substance contact positive proportion) obtained when a similar test is performed on CAL-1 cells that have not been contacted with the target substance or CAL-1 cells that have been contacted with a substance that does not have immunostimulatory activity (reference substance), and the extent of this difference. Furthermore, for example, the presence or absence and level of the immunostimulatory activity of a target substance may be evaluated based on whether or not the proportion of IFN-α-positive cells measured in the measurement step (contact positive proportion) exceeds the proportion of IFN-α-positive cells among CAL-1 cells that have not been subjected to the contact step (i.e., not contacted with the target substance) (hereinafter also referred to as the non-contact positive proportion), and the extent to which this occurs. In these cases, the positive proportion (contact positive proportion) measured in the measurement step exceeding the positive proportion of a comparison target (e.g., the reference substance contact positive proportion or non-contact positive proportion) may mean, for example, that the average positive proportion measured in the measurement step is greater than the average positive proportion of the comparison target. Furthermore, for example, the positive proportion measured in the measurement step exceeding the positive proportion of a comparison target may mean that the positive proportion measured in the measurement step is statistically significantly greater than the positive proportion of the comparison target (e.g., a p-value of less than 0.05 or less than 0.01 in Student's t-test). In one aspect, if the positive rate measured in the measuring step exceeds the positive rate of the comparison target, the target substance may be determined to have immunostimulatory activity. Thus, in one aspect, the evaluation method of the first embodiment may further include, in addition to the measuring step, a comparison step of comparing the contact positive rate with the reference substance contact positive rate or non-contact positive rate, and in one aspect, the comparison step may include a determination step of determining that the target substance has immunostimulatory activity when the contact positive rate exceeds the reference substance contact positive rate or non-contact positive rate.In addition, in one aspect, the evaluation method of the first embodiment may further include, in addition to the measurement step, a comparison step of comparing the contact positive rate with the non-contact positive rate, and in one aspect, the comparison step may include a determination step of determining that the target substance has immunostimulatory activity when the contact positive rate exceeds the non-contact positive rate.

[0078] The measuring step may be carried out after the contacting step has been completed. Furthermore, when the amount of IFN-α in the culture supernatant is used as the expression level of IFN-α, the measuring step may be carried out at each time point by collecting the culture supernatant at multiple time points during the contacting step. That is, the measuring step may be carried out after the contacting step and / or simultaneously with the contacting step. When the measuring step is carried out at each time point by collecting the culture supernatant at multiple time points during the contacting step, immune activation in CAL-1 cells contacted with the substance of interest can be evaluated over time.

[0079] In one aspect, the measurement of the expression level of IFN-α by CAL-1 cells and / or the proportion of IFN-α-positive cells in the measurement step may be performed at a time point 6 hours or more, 15 hours or more, 18 hours or more, or 24 hours or more after the start of contact of the target substance with CAL-1 cells in the contact step, or at a time point 168 hours or less, 72 hours or less, or 36 hours or less. In another aspect, the measurement of the expression level of IFN-α by CAL-1 cells and / or the proportion of IFN-α-positive cells in the measurement step may be performed at a time point of 6 to 168 hours, 6 to 72 hours, 6 to 36 hours, 15 to 168 hours, 15 to 72 hours, 15 to 36 hours, 18 to 168 hours, 18 to 72 hours, 18 to 36 hours, 24 to 168 hours, 24 to 72 hours, or 24 to 36 hours after the start of contact between the target substance and CAL-1 cells in the contacting step. If a time within the above range has elapsed since the start of contact, there is no accelerated increase in the expression level of IFN-α by CAL-1 cells and / or the proportion of IFN-α-positive cells compared to immediately after the start of contact, and therefore, a measurement value of the expression level of IFN-α and / or the proportion of IFN-α-positive cells that is highly reliable as an index of immunostimulatory activity can be obtained.

[0080] The evaluation method of the first embodiment may include a preparation step of preparing CAL-1 cells to be used in the contacting step, prior to the contacting step. The preparation step may be a step of preparing CAL-1 cells that are easily activated by single-cell cloning of CAL-1 cells. In the present disclosure, single-cell cloning of cells means doubling the number of cells from a single cell to obtain a cell population. For example, the preparation step may include three steps: a step of isolating at least one single cell from a population of CAL-1 cells (separation step), a step of culturing each of the at least one single cell to obtain at least one colony with guaranteed monoclonality (colony formation step), and a step of selecting a colony containing easily activated CAL-1 cells from the at least one colony (selection step). When the evaluation method of the first embodiment includes a preparation step, easily activated CAL-1 cells can be used as the CAL-1 cells to be used in the contacting step, making it easier to evaluate the immunostimulatory activity of a target substance. The preparation step typically includes a separation step, a colony formation step, and a selection step, in this order. The evaluation method of the first embodiment may include multiple cycles of the preparation step, in which case it is possible to obtain CAL-1 cells that are more easily activated.

[0081] In the separation step, single CAL-1 cells are separated from a population of CAL-1 cells. In the above, a population of CAL-1 cells refers to a collection of multiple CAL-1 cells contained in the same system, and may be, for example, a collection of multiple (e.g., 100 or more) CAL-1 cells contained in the same cell suspension. Single cells can be separated using techniques commonly used by those skilled in the art in single-cell cloning of cells, for example, by diluting a cell suspension containing CAL-1 cells to a concentration such that the number of cells stochastically contained in a volume corresponding to one well of a microwell plate is one or less, and then seeding the cells on the microwell plate.

[0082] In the colony formation step, the single cells obtained in the separation step are cultured to obtain a collection of cells (colonies) with guaranteed monoclonality. Such colonies are collections of cells derived from a single cell, obtained by repeated doubling of the single cell. Therefore, the individual CAL-1 cells contained in such colonies have the same or nearly the same properties as the single cell from which they were derived, ensuring monoclonality. The culture in the colony formation step may be performed under culture conditions typically used for culturing CAL-1 cells, for example, in a serum-containing medium. In the colony formation step, each colony formed by culturing the single cells is recovered. The CAL-1 cells forming the colonies recovered in the colony formation step may be used in the subsequent selection step, or the CAL-1 cells obtained by further expansion culture of the recovered colonies may be used in the subsequent selection step.

[0083] In the selection step, colonies containing easily activated CAL-1 cells are selected from the colonies obtained in the colony formation step. Colonies containing easily activated CAL-1 cells can be selected, for example, as colonies containing CAL-1 cells with higher immunostimulatory activity among the obtained colonies by measuring the immunostimulatory activity of the CAL-1 cells forming each colony or cells obtained by expanding the same, or, for example, as colonies containing CAL-1 cells with higher immunostimulatory activity than CAL-1 cells that have not been subjected to single cell cloning.

[0084] The CAL-1 cells obtained according to the preparation steps described above are easily activated. For example, the CAL-1 cells obtained according to the preparation steps described above can be cultured at a concentration of 5.0 × 10 in a serum-free RPMI-1640 medium. ５In other words, in one aspect of the first embodiment of the present disclosure, the CAL-1 cells used are cultured at an initial concentration of 5.0 × 10 cells / mL in a serum-free RPMI-1640 medium, and the interferon α concentration in the medium after 24 hours of contact with 10 μg / mL of Lactococcus lactis subsp. lactis JCM 5805 is 100.0 pg / mL or more. ５ The method may also be an evaluation method in which the concentration of interferon α in the medium is 100.0 pg / mL or more after 24 hours of contact with 10 μg / mL of Lactococcus lactis subsp. lactis JCM 5805 at an initial concentration of 100.0 pg / mL. More specifically, the susceptibility of CAL-1 cells to activation may be measured according to the method described in the Examples of the present application.

[0085] Furthermore, such easily activated CAL-1 cells have not been known until now. That is, another aspect of the present disclosure is to culture CAL-1 cells at a concentration of 5.0 × 10 in a serum-free RPMI-1640 medium. ５ The cells may also be CAL-1 cells (deposit number FERM BP-10914) in which the interferon α concentration in the medium is 100.0 pg / mL or higher after 24 hours of contact with 10 μg / mL of Lactococcus lactis subsp. lactis JCM 5805 at an initial concentration of 100.0 pg / mL. The susceptibility of CAL-1 cells to activation may be measured in more detail according to the method described in the Examples of the present application. Such CAL-1 cells can be suitably used to evaluate the immunostimulatory activity of a target substance.

[0086] According to the evaluation method of the first embodiment, the immunostimulatory ability of a target substance can be evaluated using CAL-1 cells. For example, according to the evaluation method of the first embodiment, by using CAL-1 cells in a low-serum medium, the immunostimulatory ability of a target substance can be evaluated using the expression level of interferon α by CAL-1 cells as an index.

[0087] In the evaluation method of the first embodiment, CAL-1 cells, a cell line of human-derived pDC, are used as pDCs, and thus the immunostimulatory activity of a target substance in human cells can be evaluated without concerns about the validity of evaluation results based on species differences, as occurs when mouse-derived BM-DCs are used.

[0088] In the evaluation method of the first embodiment, the immunostimulatory ability of a target substance can be evaluated under conditions in which CAL-1 cells are contained as the sole pDC (i.e., conditions in which the contacting step is performed in an environment that does not contain pDCs other than CAL-1 cells). Thus, unlike when a cell population consisting of multiple types of dendritic cells including pDCs, such as BM-DCs, is used, the immunostimulatory ability of a target substance can be evaluated under conditions that ensure that the results obtained are attributable to pDCs.

[0089] In the evaluation method of the first embodiment, CAL-1 cells, an established cancer cell line, are used as pDCs, and therefore the number of pDCs can be increased by culturing. Therefore, the immunostimulatory activity of a target substance can be evaluated more inexpensively than when primary cultured cells collected from an animal, such as BM-DCs, are used as they are.

[0090] Here, among the target substances according to one embodiment of the present invention described above, a target substance having immunostimulatory ability can be contained in a composition, and the composition can be used as an immunostimulatory composition (preferably an immunostimulatory food composition). In this case, the immunostimulatory ability of the target substance is a measured value when the expression level of IFN-α measured in the measurement step of the evaluation method according to one aspect of the first embodiment of the present invention is the amount of IFN-α secreted extracellularly in the culture supernatant, and is 0.1 pg / mL (preferably, 1 pg / mL, 10 pg / mL, 20 pg / mL, 30 pg / mL, 50 pg / mL, more preferably 60 pg / mL, more preferably 70 pg / mL, 80 pg / mL). , 90 pg / mL, 100 pg / mL, 150 pg / mL, more preferably 200 pg / mL, 210 pg / mL, 220 pg / mL, 230 pg / mL, 240 pg / mL, 250 pg / mL, even more preferably 300 pg / mL, 400 pg / mL, 500 pg / mL, 600 pg / mL, 700 pg / mL, and particularly preferably 800 pg / mL) or more, the substance can be considered to have immunostimulatory activity.

[0091] Thus, one embodiment of the present invention may be an immunostimulating composition containing a bacterium as an active ingredient, wherein the bacterium has an IFN-α expression level equal to or higher than the lower limit as measured in the measurement step of the evaluation method according to one aspect of the present invention. Another embodiment of the present invention may be an immunostimulating composition containing a bacterium as an active ingredient, wherein the bacterium comprises Lactococcus lactis subsp. lactis ATCC 7963, and wherein the IFN-α expression level measured in the measurement step of the evaluation method according to one aspect of the present invention is 200 pg / mL or higher. Another embodiment of the present invention may be an immunostimulating composition containing a bacterium as an active ingredient, wherein the bacterium comprises Lactococcus lactis subsp. lactis ATCC 7962, and wherein the IFN-α expression level measured in the measurement step of the evaluation method according to one aspect of the present invention is 300 pg / mL or higher. One embodiment of the present invention may be an immunostimulating composition containing a bacterium as an active ingredient, wherein the bacterium comprises Lactococcus lactis subsp. lactis ATCC 15346, and wherein the expression level of IFN-α measured in the measurement step of the evaluation method according to one aspect of the present invention is 10 pg / mL or more. Another embodiment of the present invention may be an immunostimulating composition containing a bacterium as an active ingredient, wherein the bacterium comprises Lactococcus lactis subsp. lactis ATCC 13675, and wherein the expression level of IFN-α measured in the measurement step of the evaluation method according to one aspect of the present invention is 400 pg / mL or more. Another embodiment of the present invention may be an immunostimulating composition containing a bacterium as an active ingredient, wherein the bacterium includes Lactococcus lactis subsp. lactis ATCC 12929, and the expression level of IFN-α measured in the measurement step of the evaluation method according to one aspect of the present invention is 400 pg / mL or more.One embodiment of the present invention may be an immunostimulating composition containing a bacterium as an active ingredient, wherein the bacterium comprises Lactococcus lactis subsp. lactis ATCC 11955, and wherein the expression level of IFN-α measured in the measurement step of the evaluation method according to one aspect of the present invention is 200 pg / mL or more. Another embodiment of the present invention may be an immunostimulating composition containing a bacterium as an active ingredient, wherein the bacterium comprises Lactococcus lactis subsp. lactis ATCC 11454, and wherein the expression level of IFN-α measured in the measurement step of the evaluation method according to one aspect of the present invention is 600 pg / mL or more. Another embodiment of the present invention may be an immunostimulating composition containing a bacterium as an active ingredient, wherein the bacterium comprises Lactococcus lactis subsp. lactis ATCC 11007, and the expression level of IFN-α measured in the measurement step of the evaluation method according to one aspect of the present invention is 30 pg / mL or more. In these cases, the expression level of IFN-α may be a value measured according to the method of Example 6 of the present specification.

[0092] Furthermore, the immunostimulating composition of the above-mentioned one embodiment may be preferably an immunostimulating food composition. Furthermore, the bacterium in the immunostimulating composition of the above-mentioned one embodiment may be a single bacterium or a mixture of two or more types of bacteria. The bacterium in the immunostimulating composition of the above-mentioned one embodiment may be, for example, a mixture of lactic acid bacteria and acetic acid bacteria.

[0093] The immunostimulating composition of one embodiment described above is preferably used orally in order to reduce the burden of ingestion. When used orally, the lactic acid bacteria or a composition or agent containing the lactic acid bacteria preferably has high resistance to gastric juice, intestinal juice, etc., and for example, strong acid resistance. The lactic acid bacteria are not particularly limited, and either live or killed bacteria can be used, but killed bacteria are preferred in terms of immunostimulating effect, stability, production efficiency, etc., and heat-killed bacteria are more preferred.

[0094] The immunostimulatory composition of one embodiment described above can be provided in the form of, for example, food, medicine, quasi-drug, feed (including pet food), additive, or the like, and can be implemented as described below.

[0095] The immunostimulating composition of one embodiment described above can be orally ingested by humans and non-human mammals, and a typical intake form is food (e.g., a food composition). When the immunostimulating composition of one embodiment described above is provided as a food, it may be provided as a food as is, or may be incorporated into a food. The provided food contains an effective amount of the immunostimulating composition of one embodiment described above. Here, "containing an effective amount" refers to a content such that the active ingredient of the present invention is ingested to an extent that the effect of immunostimulation, etc. is exerted when ingested in an amount normally consumed in a particular food. Furthermore, the term "food" is used to include health foods, functional foods, nutritional supplements, health-promoting foods (e.g., foods for specified health uses, nutritionally functional foods, foods with functional claims), foods for special dietary uses (e.g., foods for infants, foods for pregnant women, foods for the sick), and supplements. It goes without saying that when the active ingredient of the present invention is ingested by mammals other than humans, the food referred to in the present invention is used as feed.

[0096] The immunostimulating composition of one embodiment described above has immunostimulatory effects and can therefore be provided by being contained in foods that are consumed daily. In this case, the immunostimulating composition of one embodiment described above can be provided in a unit-packaged form in which the amount to be ingested per meal is predetermined. Examples of unit-packaged forms per meal include forms in which a fixed amount is specified in a pack, package, can, bottle, or the like. In order to better exert the various effects of the immunostimulating composition of one embodiment described above, the intake amount per meal can be determined according to the daily intake amount of the active ingredient of the present invention, which will be described later. The food of the present invention may be provided with instructions regarding the intake amount displayed on the packaging, or may be provided together with a document or the like containing the instructions.

[0097] The predetermined intake amount per meal in the unit package form may be an effective daily intake amount, or an intake amount obtained by dividing the effective daily intake amount into two or more (preferably 2 to 6) doses. Therefore, the unit package form of the immunostimulating composition of one embodiment described above can contain the active ingredient of the present invention at the daily intake amount described below, or can contain the active ingredient of the present invention in an amount that is one-half to one-sixth of the daily intake amount described below. For convenience of intake, the immunostimulating composition of one embodiment described above is preferably provided in a unit package form per meal (i.e., a unit package form per day) in which the intake amount per meal is the effective daily intake amount.

[0098] The form of the "food" in the immunostimulating composition of one embodiment described above is not particularly limited, and may be, for example, a beverage, a semi-liquid or gel form, or a solid or powder form. Examples of "supplements" include tablets prepared by kneading the active ingredient of the immunostimulating composition of one embodiment described above with excipients, binders, etc., followed by tableting; granules prepared by granulating the active ingredient of the present invention with excipients, binders, etc., orally disintegrating tablets; and capsules in which the active ingredient of the present invention is encapsulated in a capsule or the like. When provided as a supplement, in addition to the above-described unit packaging form per meal or per day, it is also suitable to provide it in a unit packaging form per week, per two weeks, per month, or per two months. It is preferable that the latter unit packaging form, for example, display the intake amount per meal or per day, so that the consumer can ingest an effective amount of the active ingredient of the present invention by following the labeling.

[0099] The food provided as the immunostimulating composition of one embodiment described above is not particularly limited as long as it contains the active ingredient of the present invention, and examples thereof include soft drinks, carbonated drinks, fruit juice drinks, vegetable juice drinks, fruit and vegetable juice drinks, livestock milk such as cow's milk, soy milk, dairy drinks, drink-type yogurt, drink-type or stick-type jelly, coffee, cocoa, tea drinks, nutritional drinks, energy drinks, sports drinks, mineral water (including both sparkling and non-sparkling water), near-water, non-alcoholic beer-flavored drinks and other non-alcoholic drinks; carbohydrate-containing foods and drinks such as rice, noodles, bread, or pasta; cheeses, hard or soft yogurt, livestock milk and other raw materials made from fats and oils; dairy products such as cream and ice cream; Western sweets such as cookies, cakes and chocolates, Japanese sweets such as manju or yokan, tablet sweets (refreshing sweets) such as ramune, candies, chewing gum, gummy candies, frozen desserts and frozen confections such as jelly or pudding, snacks, and other sweets; alcoholic beverages such as whiskey, bourbon, spirits, liqueur, wine, fruit wine, sake, Chinese alcohol, shochu, beer, non-alcoholic beer with an alcohol content of 1% or less, happoshu, other miscellaneous alcohol, and chuhai; processed foods such as processed products using eggs, processed seafood or meat (including liver and other offal) products (including delicacies), soups such as miso soup, condiments such as miso, soy sauce, furikake, and other seasonings, and liquid foods such as concentrated liquid foods.

[0100] Tea beverages include all types of fermented tea, semi-fermented tea, and non-fermented tea, such as black tea, green tea, barley tea, brown rice tea, sencha, gyokuro tea, roasted green tea, oolong tea, turmeric tea, pu-erh tea, rooibos tea, rose tea, chrysanthemum tea, ginkgo leaf tea, and herbal tea (e.g., mint tea, jasmine tea).

[0101] Examples of fruits used in fruit juice beverages and fruit and vegetable juice beverages include apples, mandarin oranges, grapes, bananas, pears, peaches, mangoes, acai, blueberries, and plums. Examples of vegetables used in vegetable juice beverages and fruit and vegetable juice beverages include tomatoes, carrots, celery, pumpkins, cucumbers, and watermelons.

[0102] When the immunostimulatory composition of one aspect described above is provided as feed, it can be provided in accordance with the description regarding food products above.

[0103] When the immunostimulating composition according to one embodiment of the present invention is provided as a drug, quasi-drug, or pharmaceutical composition, it can be formulated into an oral or parenteral formulation. Oral formulations include granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, liquids, jellies, emulsions, and suspensions. Parenteral formulations include injections suitable for local administration (including intradermal, subcutaneous, intramuscular, and intravenous injections), inhalants (e.g., inhalation aerosols, inhalation powders, and inhalation liquids), nasal drops (e.g., nasal powders and nasal liquids), ointments, creams, gels, suppositories, patches, and poultices. These formulations can be formulated using pharmaceutically acceptable carriers according to techniques commonly used in the art. Pharmaceutically acceptable carriers include excipients, binders, diluents, additives, flavorings, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, and preservatives.

[0104] When the immunostimulating composition of one embodiment described above is used as a drug, quasi-drug, or pharmaceutical composition, examples of the target diseases include cancers already known to be suitable for Type I IFN, including renal cancer, multiple myeloma, chronic myeloid leukemia, hairy cell leukemia, glioblastoma, medulloblastoma, astrocytoma, malignant melanoma, mycosis fungoides, and adult T-cell leukemia; viral infections including subacute sclerosing panencephalitis, HTLV-1 myelopathy, hepatitis B, and hepatitis C; bacterial infections such as chlamydia (sexually transmitted disease), mycobacterium (tuberculosis), listeria (sepsis, etc.), staphylococcus (food poisoning), and helicobacter (gastritis); and autoimmune diseases including multiple sclerosis. The immunostimulating composition of one embodiment described above can be used for the prevention or treatment of the above diseases. Furthermore, the immunostimulating composition of one embodiment described above can also be used for the prevention or treatment of osteoporosis and the like, since Type I IFN is known to have the function of inhibiting differentiation from osteoblasts to osteoclasts.

[0105] The immunostimulatory composition of one embodiment described above can be used as a vaccine by expressing an antigen corresponding to a specific disease in or on the surface of the lactic acid bacteria, which are the active ingredients, or by secreting it outside the lactic acid bacteria using genetic engineering techniques. In particular, since the cell wall of lactic acid bacteria has the function of protecting antigens from gastric acid, heterologous antigen-expressing strains in which antigens are expressed in or on the surface of lactic acid bacteria are suitable as hosts for oral vaccines.

[0106] When the immunostimulating composition of the above-mentioned embodiment is provided as an additive, it can be carried out according to the description regarding the food, feed, drug, quasi-drug, or pharmaceutical composition. When the immunostimulating composition of the above-mentioned embodiment is provided as a food additive, the immunostimulating composition of the above-mentioned embodiment can be used as a functional ingredient in a food with functional claims having an immunostimulating effect.

[0107] The amount of the immunostimulating composition of the above-mentioned embodiment can be determined depending on the recipient's sex, age, and weight, symptoms, time of ingestion, dosage form, ingestion route, and materials or drugs to be combined, etc. The daily intake amount of the immunostimulating composition of the above-mentioned embodiment for an adult can be determined, for example, by the bacterial count of the lactic acid bacteria that are the active ingredient, and the lower limit thereof is 1 x 10 ８ pieces, 1×10 ９ pcs or 1 x 10 １０ The upper limit is 1 × 10 １４ pieces, 1×10 １３ pieces, 1×10 １２ These upper and lower limits can be arbitrarily combined, and the intake range can be, for example, 1 × 10 ８ ~1 x 10 １４ pieces, 1×10 ９ ~1 x 10 １３ pcs or 1 x 10 １０ ~1 x 10 １２ The number of lactic acid bacteria can be measured using a known microscope, flow cytometer, or non-culture rapid microorganism testing device (for example, ELESTA PixeeMo (AFI Technology Co., Ltd.)), but measurement using a microscope is preferred from the viewpoint of high versatility.

[0108] The daily intake amount of the immunostimulating composition of one embodiment for an adult can also be determined by the dry cell mass of the lactic acid bacteria that is the active ingredient, and the lower limit is 2.5 × 10 －２ mg, 2.5 x 10 －１ mg or 2.5 mg, with the upper limit being 2.5×10 ４ mg, 2.5 x 10 ３ mg, 2.5 x 10 ２ These upper and lower limits can be arbitrarily combined, and the intake range can be, for example, 2.5 × 10 －２ mg to 2.5 x 10 ４ mg, 2.5 x 10 －１ mg to 2.5 x 10 ３ mg, 2.5 mg ~ 2.5 x 10 ２ It can be expressed as mg.

[0109] The intake amount of the immunostimulating composition of one embodiment described above and the intake timing and intake period described below are applicable whether the immunostimulating composition of one embodiment described above is used for non-therapeutic purposes or therapeutic purposes, and in the case of therapeutic purposes, "intake" can be read as "administration."

[0110] It is preferable to continue taking the immunostimulating composition of one embodiment described above for the period during which the immunostimulating effect is expected. From the viewpoint of better exerting the immunostimulating effect, the intake period of the active ingredient of the present invention can be, for example, one week or more, two weeks or more, three weeks or more, preferably one month or more (four weeks or more) at the above daily dose. The intake interval of the active ingredient of the present invention can be once every three days, once every two days, or once a day at the above daily dose, and is preferably once a day.

[0111] Intake of the immunostimulating composition of one embodiment described above may also be initiated before an event or period when an immunostimulating effect is expected. Examples of events when an immunostimulating effect is expected include behaviors that may result in viral infection (e.g., participating in an event with a high risk of viral infection, traveling to an endemic area), and examples of periods when an immunostimulating effect is expected include a viral infection epidemic period. Examples of intake times before an event when an immunostimulating effect is expected include at least one day, at least three days, at least one week, at least two weeks, at least three weeks, at least one month (at least four weeks), or at least two months (at least eight weeks). Furthermore, although not particularly limited, the composition may be taken continuously, with intervals between intakes, from the start of intake until the event when an immunostimulating effect is expected. Intake of the active ingredient of the present invention may also be initiated after an event or period when an immunostimulating effect is expected. Examples of intake times after an event when an immunostimulating effect is expected include at least one day, at least three days, at least one week, or at least two weeks later. Furthermore, although not particularly limited, when the active ingredient of the present invention is taken after an event in which an immunostimulatory effect is expected, it may be taken continuously with intervals in between ingestion. In the present invention, it is particularly preferred to start taking the active ingredient of the present invention before an event in which an immunostimulatory effect is expected, and continue taking the active ingredient until after the event.

[0112] Furthermore, the evaluation method of the first embodiment of the present invention can be used as a second embodiment, a screening method for substances having immunopotentiating ability (hereinafter also referred to as the "screening method of the second embodiment"). The screening method of the second embodiment aims to screen for substances having immunopotentiating ability, and can be carried out in the same manner as the evaluation method of the first embodiment, except that a test substance is used instead of a target substance in the contacting step. The medium and CAL-1 cells used in the contacting step are the same as those described for the evaluation method of the first embodiment. Furthermore, the method may further include a pretreatment step, a measurement step, a comparison step, and / or an evaluation step, and the detailed methods for these steps are also the same as those described for the evaluation method of the first embodiment.

[0113] The test substance in the screening method of the second embodiment can be the same as that described with respect to the target substance in the evaluation method of the first embodiment. The test substance may be a substance known to have or have the potential to have immunostimulatory activity (e.g., the ability to promote IFN-α production), a substance known to have some effect on the immune system other than immunostimulatory activity, or a substance known to have no effect on the immune system. The conditions and method for contacting the test substance with CAL-1 cells may be the same as those described with respect to contacting the target substance with CAL-1 cells in the evaluation method of the first embodiment.

[0114] According to the screening method of the second embodiment, CAL-1 cells can be used to screen (select) substances having immunopotentiating ability. For example, according to the screening method of the second embodiment, by using CAL-1 cells in a culture medium, substances having immunopotentiating ability can be screened (selected) using the expression level of interferon α by CAL-1 cells as an index. For example, a substance that increases the expression level of interferon α by CAL-1 cells upon contact can be selected as a substance having immunopotentiating ability. Detailed measurement of the expression level of interferon α and evaluation of the immunopotentiating ability of a candidate substance based on the measurement can be performed in the same manner as described for the evaluation method of the first embodiment.

[0115] A third embodiment of the present invention is a kit for use in the evaluation method of the first embodiment and / or the screening method of the second embodiment (hereinafter also referred to as a "kit of the third embodiment"). The kit of the third embodiment may include, for example, as reagents, an anti-interferon α antibody and / or primers that can be used to amplify mRNA encoding interferon α, and may include an anti-interferon α antibody. The kit of the third embodiment may further include fetal bovine serum. The kit of the third embodiment may further include instructions that describe performing evaluation according to the method described in the evaluation method of the first embodiment and / or performing screening according to the screening method of the second embodiment, and the instructions may be electronic.

[0116] Furthermore, the kit of the third embodiment may include CAL-1 cells or a composition containing them, and preferably may include starved CAL-1 cells or a composition containing them. More specifically, for example, the kit of the third embodiment may be obtained by separating a liquid component from a solid component of a cell suspension obtained by detaching cultured CAL-1 cells in the same manner as described in the pretreatment step of the evaluation method of the first embodiment, and may include CAL-1 cells or a composition containing them as the recovered solid component, or may include CAL-1 cells or a composition containing them as the pellet obtained by centrifuging a medium in which the CAL-1 cells are suspended.

[0117] When the kit of the third embodiment includes a composition containing CAL-1 cells, the content of CAL-1 cells (preferably CAL-1 cells in a starved state) in the composition, based on the total amount of the composition, can have a lower limit of 10 mass%, 40 mass%, 70 mass%, 85 mass%, 90 mass%, 95 mass%, 98 mass%, or 99 mass%, and an upper limit of 99 mass%, 98 mass%, 90 mass%, 80 mass%, 50 mass%, or 30 mass%. These upper and lower limits can be combined in any desired manner. For example, when the kit of the third embodiment includes a composition containing CAL-1 cells, the content of CAL-1 cells (preferably CAL-1 cells in a starved state) in the composition is, based on the total amount of the composition, 10% by mass or more and 30% by mass or less, 10% by mass or more and 50% by mass or less, 10% by mass or more and 80% by mass or less, 10% by mass or more and 90% by mass or less, 10% by mass or more and 98% by mass or less, 10% by mass or more and 99% by mass or less, 40% by mass or more and 50% by mass or less, 40% by mass or more and 80% by mass or less, The content may be 40% by mass or more and 90% by mass or less, 40% by mass or more and 98% by mass or less, or 40% by mass or more and 99% by mass or less, 70% by mass or more and 80% by mass or less, 70% by mass or more and 90% by mass or less, 70% by mass or more and 98% by mass or less, 70% by mass or more and 99% by mass or less, 85% by mass or more and 90% by mass or less, 85% by mass or more and 98% by mass or less, 85% by mass or more and 99% by mass or less, 90% by mass or more and 98% by mass or less, 90% by mass or more and 99% by mass or less, 95% by mass or more and 98% by mass or less, or 95% by mass or more and 99% by mass or less. In one aspect, the content of CAL-1 cells (preferably starved CAL-1 cells) in the composition included in the kit of the third embodiment may be 100% by mass based on the total amount of the composition.

[0118] The present invention will be described in more detail below using examples, but the present invention is not limited to the following examples.

[0119] In the following examples, CAL-1 cells provided by Nagasaki University (National University Corporation) were used as a sample (hereinafter referred to as the "sample"). (All rights to the sample belong to Nagasaki University, and permission to use the sample has been obtained from Nagasaki University.) The cells were cultured at 37°C and 5% CO. ２ The results are shown as the mean ± standard deviation.

[0120] In the following examples, bacteria (including LC-plasma) were used as bacterial bulk powder prepared as follows. Using a 50 ml Falcon tube, bacteria were cultured in MRS medium (MRS BROTH, CODE: CM0359, Oxoid). The culture solution was centrifuged (5000 rpm) and the supernatant was removed to obtain a bacterial solution. The resulting bacterial solution was then mixed with phosphate-buffered saline (PBS) (Takara Bio Inc.) at a ratio of 1:19 and washed twice by centrifugation (5000 rpm). The washed bacterial solution was heat-treated to kill the lactic acid bacteria in the bacterial solution. The heat treatment involved heating the solution from room temperature to 80°C over 30 minutes, maintaining it at 80°C for 30 minutes, and then cooling it to room temperature over 30 minutes. The cooled bacterial solution was then freeze-dried to obtain a bacterial bulk powder.

[0121] Example 1: IFN-α production by CAL-1 cells 1 The effect of serum concentration in the medium on the amount of IFN-α produced by CAL-1 cells was examined. The basal medium used was RPMI-1640 medium (Sigma, R8758) supplemented with penicillin / streptomycin (Gibco, 15140-12) at a final concentration of 1.0% by volume (hereinafter also referred to as "serum-free medium"). Fetal bovine serum (FBS, Sigma-Aldrich, 173012) was added to the serum-free medium to a concentration of 0%, 0.10%, 1.0%, or 10% by volume to prepare media. CAL-1 cells were cultured at 1.0 x 10 ６ A cell suspension containing 5.0 × 10 cells / mL was prepared, and the cell suspension was seeded at 10 mL / well in a 10 cm diameter culture dish and cultured for 16 hours (pretreatment step). CAL-1 cells were recovered by pipetting. The recovered CAL-1 cells were then resuspended in the same medium as used in the pretreatment step at a concentration of 5.0 × 10 cells.５ The cells were resuspended at a concentration of 100 μg / mL and seeded at 500 μL / well into each well of a 48-well microplate. To each well, LC-Plasma (Kirin Holdings Co., Ltd.), CpG ODN2216 (Invivogen, tlrl-2216) at a final concentration of 0.5 μM, or R848 (Invivogen, tlrl-r848) at a final concentration of 1 μg / mL were added as IFN-α production-promoting substances. These were designated the LC-Plasma group, CpG group, and R848 group, respectively. Negative groups were also prepared without the addition of these IFN-α production-promoting substances. CAL-1 cells in the wells were cultured for 24 hours (contact step), and the culture supernatant was then collected. The IFN-α concentration in the culture supernatant was measured by ELISA using a Human IFN-Alpha Multi-Subtype ELISA Kit (PBL Assay Science).

[0122] Fig. 1 shows the results of measuring the IFN-α concentration in the culture supernatant for each serum concentration in the medium. In Fig. 1, 0%, 0.1%, 1.0%, and 10% represent the volume percentage of FBS in the medium.

[0123] LC-Plasma is a bacterium that promotes the production of IFN-α (Patent Document 1). According to the results shown in Figure 1, significant IFN-α production was observed in CAL-1 cells exposed to LC-Plasma in a medium containing 0% or 0.10% FBS by volume. This demonstrates that contact with an IFN-α production-promoting substance can promote IFN-α production by CAL-1 cells when cultured in a low-serum medium. Therefore, it has become clear that the immunostimulatory activity of a target substance can be evaluated by an evaluation method including a step of contacting the target substance with CAL-1 cells in a low-serum medium, i.e., a contact step of culturing CAL-1 cells in a starvation state.

[0124] Furthermore, the results in Figure 1 unexpectedly showed that the expression level of IFN-α by CAL-1 cells in low-serum medium was significantly higher when exposed to LC-Plasma than when exposed to CpG ODN2216 or R848 (selective activating ligands for human TLR7 and TLR8), which also promote IFN-α production. Thus, it was revealed that an evaluation method including a step of contacting a target substance with CAL-1 cells in low-serum medium can suitably evaluate the immunostimulatory activity of a target substance that is a bacterium or a substance derived from a bacterium.

[0125] Example 2: IFN-α production by CAL-1 cells 2 The effect of the culture time in the pretreatment step on the expression level of IFN-α in CAL-1 cells was evaluated. CAL-1 cells were cultured in the serum-free medium at 1.0 × 10 ６ A cell suspension containing 100 ng / mL of IFN-α was prepared, and 10 mL of the cell suspension was seeded into a 10 cm diameter culture dish and cultured for 0, 4, 8, 16, or 24 hours (pretreatment step). After culture, LC-Plasma was added to the serum-free medium at a final concentration of 10 μg / mL or 20 μg / mL, and the cells were cultured for an additional 24 hours (contact step). A negative group, to which LC-Plasma was not added, was also prepared and cultured for 24 hours in the same manner. After culture, the culture supernatant was collected, and the IFN-α concentration in the culture supernatant was measured using the same method as in Example 1.

[0126] FIG. 2 shows the results of measuring the IFN-α concentration in the culture supernatant for each incubation time in the pretreatment step. The results in FIG. 2 show that the amount of IFN-α produced by CAL-1 cells upon exposure to LC-Plasma was increased in the groups with pretreatment times of 4, 8, 16, and 24 hours, compared to the group with a pretreatment time of 0 hour (i.e., the group without the pretreatment step). This demonstrates that an evaluation method including a pretreatment step can suitably evaluate immunostimulatory activity. Furthermore, the results in FIG. 2 show that the amount of IFN-α produced by CAL-1 cells upon exposure to LC-Plasma was greater when the incubation time in the pretreatment step was 4, 8, 16, or 24 hours, and was particularly high when the incubation time was 8 or 16 hours. On the other hand, because prolonged incubation in serum-free medium reduces cell viability, the amount of IFN-α expressed by CAL-1 cells decreased when the pretreatment step using serum-free medium was prolonged. This indicates that immunostimulatory activity can be evaluated by using CAL-1 cells that have undergone a pretreatment step, i.e., CAL-1 cells in a starved state, and furthermore, the optimal culture time for the pretreatment step was identified.

[0127] Example 3: IFN-α production by CAL-1 cells 3 The effect of CAL-1 cell concentration on the expression level of IFN-α in CAL-1 cells was evaluated. CAL-1 cells were grown at a concentration of 1.0 × 10 in a serum-free medium. ６ A cell suspension containing 0.5 × 10 cells / mL was prepared, and 10 mL of the cell suspension was seeded in a 10 cm diameter culture dish and cultured for 16 hours (pretreatment step). The pretreated CAL-1 cells were collected by pipetting and added to serum-free medium. ６ cells / mL, 1.0×10 ６ cells / mL or 2.0 x 10 ６The cells were resuspended at a concentration of 1000 cells / mL and seeded at 500 μL / well into each well of a 48-well microplate. LC-Plasma was added to a final concentration of 50 μg / mL or 100 μg / mL, or CpG ODN2216 (Invivogen, tlrl-2216) was added to a final concentration of 0.1 μM. A negative group was also prepared without these treatments. CAL-1 cells in the wells were cultured for 24 hours (contact step), and the culture supernatant was collected. The IFN-α concentration in the culture supernatant was measured using the same method as in Example 1.

[0128] Fig. 3 shows the results of measuring the IFN-α concentration in the culture supernatant for each concentration of CAL-1 cells. The results in Fig. 3 show that the IFN-α concentration in the culture supernatant increases in a CAL-1 cell concentration-dependent manner. This further demonstrates that the evaluation method according to the present invention can measure IFN-α produced by CAL-1 cells.

[0129] Also, according to the results of Figure 3, 0.5 × 10 ６ cells / mL, 1.0×10 ６ cells / mL and 2.0 x 10 ６ Regardless of the cell suspension concentration, IFN-α concentration in the culture supernatant increased with the addition of LC-Plasma, and the increase in IFN-α concentration was cell concentration dependent.

[0130] Example 4: IFN-α production test 4 by CAL-1 cells Whether the amount of IFN-α produced by CAL-1 cells increases in a cell number-dependent manner was investigated. Pretreatment and recovery of CAL-1 cells were carried out in the same manner as in Example 3. The recovered CAL-1 cells were placed in serum-free medium at a concentration of 0.2 × 10 ６ The resulting cell suspension was resuspended at a concentration of 0.1 × 10 cells / mL. 500 μL (0.1 × 10 cells / mL) of the resulting cell suspension was placed in a 48-well microplate. ６ cells) / well or 200 μL (0.4 × 10 ５CAL-1 cells were seeded into each well at a concentration of 10 μg / mL. LC-Plasma was added to each well at a final concentration of 10 μg / mL. A negative group to which LC-Plasma was not added was also prepared. CAL-1 cells in the wells were cultured for 24 hours (contact step), and then the culture supernatant was collected. The IFN-α concentration in the culture supernatant was measured in the same manner as in Example 1.

[0131] Figure 4 shows the results of measuring the IFN-α concentration in the culture supernatant when a 48-well microplate and a 96-well microplate were used. As shown in Figure 4, at the same cell concentration, a sufficient increase in the IFN-α concentration in the culture supernatant was observed with the addition of LC-Plasma in both the 96-well and 48-well microplates, but the IFN-α concentration in the 96-well microplate (0.4 × 10 ５ The number of cells in a 48-well microplate (0.1 × 10 cells / well) was higher than that in a 48-well microplate (0.1 × 10 cells / well). ６ A higher increase in IFN-α concentration was observed in the 96-well microplate (100 cells / well), demonstrating that the increase in IFN-α concentration is cell number dependent. On the other hand, a sufficient increase in IFN-α concentration was also observed in the 96-well microplate, demonstrating that the 96-well microplate can also be suitably used in the evaluation method of the present invention.

[0132] Example 5: Correlation between phagocytosis amount and IFN-α production CAL-1 cells were placed in serum-free medium at 0.5 × 10 ６ A cell suspension containing 0.4 × 10 cells / mL was prepared. 10 mL of the cell suspension was seeded in a 10 cm diameter culture dish and cultured for 16 hours (pretreatment step). The pretreated CAL-1 cells were recovered by pipetting and 200 μL (0.4 × 10 cells) was added to a 96-well microplate. ５Lactic acid bacteria were seeded into each well at a concentration of 10 μg / mL, 50 μg / mL, or 100 μg / mL. LC-Plasma stained with pHrodo Red SE (Invitrogen, P36600) was added to each well to a final concentration of 10 μg / mL, 50 μg / mL, or 100 μg / mL. The staining method using pHrodo Red SE (Invitrogen, P36600) was as described below in <Staining of lactic acid bacteria with pHrodo Red SE 1>. A negative group to which LC-Plasma was not added was also prepared. The fluorescence of pHrodo Red SE in each well was then measured over time using an Incucyte® SX5 Live-Cell Analysis System (Sartorius). After further culturing for 21 hours, the culture supernatant from each well was collected, and the IFN-α concentration in the culture supernatant was measured using the same method as in Example 1. pHrodo Red SE is a staining reagent whose fluorescence intensity is enhanced in a low pH environment. When bacteria stained with pHrodo Red SE are phagocytosed by CAL-1 cells, the bacteria enter endosomes, creating a low pH environment, which increases the fluorescence intensity of pHrodo Red SE. Therefore, the amount of bacteria phagocytosed by CAL-1 cells can be quantified using the total area in which pHrodo Red SE fluorescence is detected (fluorescence detection area) within the entire field of view as an index. The fluorescence intensity analysis conditions for the Incucyte (registered trademark) SX5 Live-Cell Analysis System (Sartorius) were as described below in <Incucyte Fluorescence Intensity Analysis Conditions 1>.

[0133] <Staining Lactic Acid Bacteria with pHrodo Red SE 1> pHrodo Red SE (Invitrogen, P36600) was dissolved in DMSO to prepare a 10.2 mM solution. The bacterial bulk powder was weighed into a 2 mL Eppendorf tube, and 0.1 M sodium bicarbonate adjusted to pH 9.0 was added to prepare a 20 mg / mL bacterial solution. 95 μL was then transferred to a new 2 mL Eppendorf tube, and 5 μL of the prepared 10.2 mM pHrodo Red SE was added. After dispersion by vortexing, the mixture was shielded from light with aluminum foil and incubated at room temperature for 60 minutes. Then, 750 μL of PBS (Takara Bio Inc.) was added, vortexed, and centrifuged at 20,000 g for 2 minutes at room temperature. After discarding 800 μL of the supernatant, 1.5 mL of PBS was added and the precipitate was completely suspended using a vortex mix. After another centrifugation at 20,000 g for 2 minutes at room temperature, 1.5 mL of the supernatant was discarded. Finally, 140 μL of PBS was added and suspended to prepare a 10 mg / mL stained lactic acid bacteria solution. The stained bacterial solution was protected from light by aluminum foil and stored at 4°C until use. Immediately before use, the solution was diluted 10-fold with PBS and used as a 1 mg / mL lactic acid bacteria solution. The stained bacterial solution was used within 24 hours of preparation.

[0134] <Incucyte Fluorescence Intensity Analysis Condition 1> In the incucyte fluorescence intensity analysis, two channels, a "Phase channel" and an "Orange channel," were set. In the Phase channel, CAL-1 cells were detected based on phase difference. In the Orange channel, pHrodo Red SE fluorescence was detected. Detailed detection conditions are as shown in the Scan Settings and Analysis Settings below. Note that in the Scan Settings and Analysis Settings below, the points indicated as "(Conditions in Figure 5)" or "(Conditions in Figure 7)" indicate points where the measurement conditions differ between the results in Figure 5 and the results in Figure 7. Scan Settings Scan Type: Standard (with lock) (Conditions in Figure 5) / Non-Adherent Cell-by-Cell (Conditions in Figure 7) Vessel Type: 96-well Sartorius Imagelock (Conditions in Figure 5) / 48-well Iwaki (Conditions in Figure 7) Image Channels: Phase, Orange (Acquisition Time: 400 ms) Objective: 20× Images per Well: 3 Analysis Settings Analysis Type: Basic Analyzer Phase Channel Segmentation: AI Confluence Cleanup: - Hole Fill (μm 2 ): 0.000 - Adjust Size (pixels): 0 Filters: - Area (μm 2): 60.000 or more - Eccentricity: No setting Orange Channel Segmentation: Surface Fit - Threshold (OCU): 5.000 - Edge Split: On - Edge Sensitivity: 0 Cleanup: - Hole Fill (μm 2 ): 0.000 - Adjust Size (pixels): 0 Filters: - Area (μm 2 ): 20,000 or more (conditions in Figure 5) / Not set (conditions in Figure 7) - Eccentricity: Not set - Mean Intensity: Not set - Mean Intensity: Not set

[0135] FIG. 5 shows the change in fluorescence detection area over time for each LC-Plasma concentration. FIG. 6 shows the results of measuring the IFN-α concentration in the culture supernatant for each LC-Plasma concentration. As shown in FIG. 5, in the group to which LC-Plasma stained with pHrodo Red SE was added, an increase in the fluorescence detection area over time was observed, and the rate of increase was dependent on the LC-Plasma concentration. This indicates that LC-Plasma was phagocytosed by CAL-1 cells over time and in a concentration-dependent manner. Furthermore, as shown in FIG. 6, the amount of IFN-α expressed by CAL-1 cells also showed a concentration-dependence on LC-Plasma. From FIGS. 5 and 6, it was found that there is a correlation between the amount of LC-Plasma phagocytosed and the amount of IFN-α expressed in CAL-1 cells. These results suggest that LC-Plasma was phagocytosed by CAL-1 cells, thereby promoting the expression of IFN-α by CAL-1 cells.

[0136] To further investigate the correlation between the amount of LC-Plasma phagocytosis and the expression level of IFN-α, a comparative test was conducted in the contact step using two types of media: a serum-free medium and a serum-free medium supplemented with 10% by volume of FBS (hereinafter also referred to as "FBS 10% medium"), with the final concentration of LC-Plasma being 10 μg / mL.

[0137] FIG. 7 shows the fluorescence detection area 24 hours after the addition of LC-Plasma in tests using serum-free medium (0% FBS) or 10% FBS medium. FIG. 8 shows the results of measuring the IFN-α concentration in the culture supernatant collected 24 hours after the addition of LC-Plasma in tests using serum-free medium (0% FBS) or 10% FBS medium. The results in FIG. 7 indicate that the fluorescence detection area was significantly smaller in the test using 10% FBS medium, demonstrating that the amount of LC-Plasma phagocytosed by CAL-1 cells was significantly smaller. Therefore, it was inferred that the low amount of IFN-α produced in the test using 10% FBS medium, as shown in FIG. 8, was due to a decrease in the amount of LC-Plasma phagocytosed by CAL-1 cells.

[0138] 5 to 8 suggest that the enhanced IFN-α production ability of CAL-1 cells in low-serum medium is due to increased phagocytic activity. In other words, it was suggested that the evaluation method according to the present invention can suitably evaluate the immunostimulatory ability of a substance to be evaluated (e.g., bacteria) that is taken up by CAL-1 cells by phagocytosis.

[0139] Example 6: Immunostimulatory Activity Evaluation Test Using CAL-1 Cells in Low-Serum Medium A pretreatment step was performed in the same manner as in the test using serum-free medium in Example 5. After culturing, 5 μL of the various lactic acid bacteria shown below, stained with pHrodo Red SE (Invitrogen, P36600) according to the method described above in <Staining of Lactic Acid Bacteria with pHrodo Red SE 1>, was added to each well to a final concentration of 10 μg / mL. A control group was also prepared to which the same amount of PBS was added. Subsequently, the fluorescence of pHrodo Red SE was measured over time, and the concentration of IFN-α in the culture supernatant was measured using the same method as in Example 5, except for the fluorescence intensity analysis conditions of the Incucyte® SX5 Live-Cell Analysis System (Sartorius). The conditions for fluorescence intensity analysis using the Incucyte (registered trademark) SX5 Live-Cell Analysis System (Sartorius) were as described below in <Incucyte fluorescence intensity analysis 2>.

[0140] <Incucyte Fluorescence Intensity Analysis Condition 2> For incucyte fluorescence intensity analysis, two channels, "Phase channel" and "Orange channel," were set. CAL-1 cells were detected in the Phase channel based on phase contrast. pHrodo Red SE fluorescence was detected in the Orange channel. Detailed detection conditions are as shown in the Scan Settings and Analysis Settings below. Scan Settings Scan Type: Non-Adherent Cell-by-Cell Vessel Type: 96-well Corning Image Channels: Phase, Orange (Acquisition Time: 400 ms) Objective: 20× Images per Well: 9 Analysis Settings Analysis Type: Basic Analyzer Phase Channel Segmentation: AI Confluence Cleanup: - Hole Fill (μm 2): 0.000 - Adjust Size (pixels): 0 Filters: - Area (μm 2 ): 60.000 or more - Eccentricity: No setting Orange Channel Segmentation: Surface Fit - Threshold (OCU): 5.000 - Edge Split: On - Edge Sensitivity: 0 Cleanup: - Hole Fill (μm 2 ): 0.000 - Adjust Size (pixels): 0 Filters: - Area (μm 2 ): Not set - Eccentricity: Not set - Mean Intensity: Not set - Mean Intensity: Not set

[0141] Lactic acid bacteria: LC-Plasma, Lactococcus lactis subsp. lactis ATCC 15577 (referred to as "155" in Figure 9 or Figure 10), Lactococcus lactis subsp. lactis ATCC 7962 (referred to as "7962" in Figure 9 or Figure 10), Lactococcus lactis subsp. lactis ATCC 7963 (referred to as "7963" in Figure 9 or Figure 10), Lactococcus lactis subsp. lactis ATCC 11007 (referred to as "11007" in Figure 9 or Figure 10), Lactococcus lactis subsp. lactis ATCC 11454 (referred to as "11454" in Figure 9 or Figure 10), Lactococcus lactis subsp. lactis ATCC 11955 (referred to as "11955" in Figure 9 or 10), Lactococcus lactis subsp. lactis ATCC 12929 (referred to as "12929" in Figure 9 or 10), Lactococcus lactis subsp. lactis ATCC 13675 (referred to as "13675" in Figure 9 or 10), and Lactococcus lactis subsp. lactis ATCC 15346 (referred to as "15346" in Figure 9 or 10).

[0142] Figure 9 shows the fluorescence detection area 24 hours after the addition of the 10 types of bacteria tested, and Figure 10 shows the measurement results of the IFN-α concentration in the culture supernatant collected 24 hours after the addition of the bacteria, for the 10 types of bacteria tested. According to the results in Figures 9 and 10, various phagocytic behaviors and immunostimulatory abilities were observed for each bacterium, including LC-Plasma, Lactococcus lactis subsp. lactis ATCC 7962, Lactococcus lactis subsp. lactis ATCC 7963, Lactococcus lactis subsp. lactis ATCC 11007, Lactococcus lactis subsp. lactis ATCC 11454, Lactococcus lactis subsp. lactis ATCC 11955, Lactococcus lactis subsp. lactis ATCC 12929, Lactococcus lactis subsp. lactis ATCC 13675, and Lactococcus lactis subsp. lactis ATCC 14006. 9 and 10, there is a strong positive correlation (R ２ = 0.87). This further suggests that the enhanced IFN-α production capacity of CAL-1 cells in low-serum medium is due to increased phagocytic activity. Furthermore, to evaluate the validity of these results, the activity of LC-Plasma and Lactococcus lactis subsp. lactis ATCC15577 was evaluated using primary pDCs obtained from humans, and results similar to those obtained using CAL-1 cells were observed. Therefore, it has become clear that the evaluation of immunostimulatory activity using CAL-1 cells in low-serum medium is also useful as a screening system for substances with immunostimulatory activity.

[0143] Example 7: Necessity of serum-free medium in the pretreatment step and contact step The following test was carried out to examine the effect of serum on IFN-α production in the pretreatment step and contact step. CAL-1 cells were cultured at a concentration of 0.5 × 10 in a 10% FBS medium or a serum-free medium. ６ A cell suspension containing 0.4 × 10 cells / mL was prepared. 10 mL of the cell suspension was seeded in a 10 cm diameter culture dish and cultured for 16 hours (pretreatment step). The pretreated CAL-1 cells were recovered by pipetting and resuspended in 10% FBS medium or serum-free medium, and 200 μL (0.4 × 10 cells) was placed in a 96-well microplate. ５ Cells were seeded into each well at 100 μg / well. LC-Plasma was then added to each well to a final concentration of 10 μg / mL, and the cells were cultured for 21 hours (contact step). Thus, a total of four groups were obtained, each using 10% FBS medium or serum-free medium in the pretreatment step and contact step. After culture, the culture supernatant was collected from each well, and the IFN-α concentration in the culture supernatant was measured in the same manner as in Example 1.

[0144] 11 shows the IFN-α concentration in the supernatant of CAL-1 cells treated with serum-free medium (0% FBS) or 10% FBS medium (10% FBS) in the pretreatment and contact steps, and then contacted with LC-Plasma. As shown in FIG. 11, CAL-1 cells treated with serum-free medium in both the pretreatment and contact steps (0% FBS → 0% FBS) produced the highest level of IFN-α. Furthermore, as in Example 1, no IFN-α was detected in CAL-1 cells treated with 10% FBS medium in both the pretreatment and contact steps (10% FBS → 10% FBS). However, in CAL-1 cells treated with serum-free medium in only either the pretreatment step or the contact step (FBS 0% → FBS 10%, (FBS 10% → FBS 0%)), a sufficiently detectable level of IFN-α was confirmed, although the level was about one-tenth that of CAL-1 cells treated with serum-free medium in both the pretreatment step and the contact step. These results indicate that IFN-α production can be evaluated by culturing using serum-free medium in at least either the pretreatment step or the contact step. CAL-1 cells treated with serum-free medium in both the pretreatment step and the contact step are more likely to have a lower cell viability than CAL-1 cells treated with serum-free medium in only one of the steps. Therefore, when conducting long-term tests, it may be more preferable to use CAL-1 cells treated with serum-free medium in only one of the steps.

[0145] Example 8: Evaluation based on the percentage of intracellular IFN-α positive cells The following test was carried out to examine whether IFN-α production by CAL-1 cells can also be evaluated by detecting intracellular cytokines. A pretreatment step was carried out in the same manner as in the test using serum-free medium in Example 5. The pretreated CAL-1 cells were recovered by pipetting. The recovered CAL-1 cells were diluted in serum-free medium to 5 × 10 ５The cell suspension was prepared by suspending the cells in 1000 μL of PBS / mL and seeded in a 96-well microplate at 200 μL per well. Then, 5 μL of LC-Plasma was added to each well to a final concentration of 25 μg / mL. A control group was also prepared to which 5 μL of PBS was added. After gently shaking the plate, the cells were incubated in a CO ２The cells were incubated in an incubator for 6 hours. 5 μL of Brefeldin A (Biolegend, 420601) was added to each well to a final concentration of 5 μg / mL. After further incubation for 16 hours, the cell suspension was collected by pipetting. The collected cell suspension was centrifuged at 400 g for 3 minutes at 4°C, the supernatant was discarded, and the remaining cells were washed with Cell staining buffer (Biolegend, 420201). After repeating the same washing once more, 200 μL of Intracellular Staining Fixation Buffer (Biolegend, 420801) was added to the remaining cells and allowed to stand for 20 minutes at room temperature in the dark (fixation treatment). The mixture containing the cells after standing was centrifuged at 400 g for 3 minutes at 4 °C, the supernatant was discarded, and the remaining cells were washed with 200 μL of cell staining buffer. The mixture containing the washed cells was centrifuged again at 400 g for 3 minutes at 4 °C, and the supernatant was discarded. 200 μL of 1 × Intracellular Staining Perm Wash Buffer, prepared by diluting 10 × Intracellular Staining Perm Wash Buffer (BioLegend Cat. No. 421002) with pure water, was added to the remaining cells, and the resulting cell suspension was centrifuged at 400 g for 3 minutes at 4 °C, and the supernatant was discarded (membrane permeabilization). After repeating this process twice more, 100 μL of 1× Intracellular Staining Perm Wash Buffer was added to the remaining cells. To stain intracellular IFN-α, 20 μL of BD Pharmingen® PE Mouse anti-Human IFN-α[2b] (BD Pharmingen®, 560097) or its isotype control, BD Pharmingen® PE Mouse IgG1,κ Isotype Control (BD Pharmingen®, 555749), was added to the resulting cell suspension, and the cells were incubated for 20 minutes at room temperature in the dark. After incubation, the suspension was centrifuged at 400 g for 3 minutes at 4° C., and the supernatant was discarded.To the remaining cells, 200 μL of 1× Intracellular Staining Perm Wash Buffer was added, and the resulting cell suspension was centrifuged again at 400 g for 3 minutes at 4°C, after which the supernatant was discarded. The same washing procedure was repeated once more. To the remaining cells, 200 μL of cell staining buffer was added, and the percentage of IFN-α-positive cells among the CAL-1 cells was calculated by FACS (fluorescence-activated cell sorting) using a flow cytometer.

[0146] The FACS results were analyzed as follows. First, a population containing CAL-1 cells was gated using FSC-A / SSC-A. Next, doublets (data points containing two or more cells) were removed using FSC-A / FSC-H and SSC-A / SSC-H, and only the singlet population (a population of data points containing only one cell) was extracted. This singlet population was then gated using wavelengths corresponding to SSC / PE, and the proportion of the singlet population that fluoresced at the wavelength corresponding to PE (PE+ population) was calculated. In this study, IFN-α was stained with a PE-labeled antibody, so the proportion of this PE+ population is synonymous with the proportion of IFN-α-positive cells. The threshold for the PE+ population was set so that the PE+ population was 0.1% or less in groups to which isotype control antibodies at the same concentration as the respective IFN-α antibodies were added.

[0147] FIG. 12 is a diagram showing the percentage of IFN-α-positive cells among CAL-1 cells contacted with PBS or LC-Plasma. As shown in FIG. 12, the percentage of IFN-α-positive cells was significantly increased in the group contacted with LC-Plasma compared to the group to which PBS was added (control group). This result is consistent with the result shown in FIG. 10 of Example 6, in which the IFN-α concentration in the supernatant was significantly increased in the LC-Plasma group compared to the group to which PBS was added (control group). This demonstrates that the IFN-α-producing activity of CAL-1 cells can be evaluated not only by ELISA but also by the percentage of intracellular IFN-α-positive cells measured using FACS.

[0148] Example 9: Obtaining highly active CAL-1 cells by single cell cloning 37°C, 5% CO ２ CAL-1 cells were maintained in 10% FBS medium under the above conditions, and cells that entered the proliferation phase were collected. After medium replacement, the cells were counted and serially diluted with 10% FBS medium to prepare a cell suspension at 1 cell / 100 μL. The obtained cell suspension was seeded in 100 μL aliquots (i.e., stochastically 1 cell per well) into a 96-well plate, and then incubated at 37°C, 5% CO in 10% FBS medium. ２ The cells were maintained under this environment for approximately 1-2 weeks, and only wells in which single colonies had formed were collected. The strains obtained from each single colony were expanded under the same conditions until the cell number had increased sufficiently. Thereafter, the original CAL-1 cells and 13 cell lines newly obtained by single cell cloning (CAL-1 cell lines A to M) were subjected to the same pretreatment process as in the test using serum-free medium in Example 5, and 200 μL (0.4 × 10 ５ The cells were seeded into each well at a concentration of 10 μg / mL. LC-Plasma was then added to each well to a final concentration of 10 μg / mL. After culturing for 21 hours, the culture supernatant was collected from each well, and the IFN-α concentration in the culture supernatant was measured in the same manner as in Example 1.

[0149] Figure 13 shows the IFN-α concentration in the culture supernatant when the original CAL-1 cells and CAL-1 cell lines A to M newly obtained by single cell cloning were contacted with LC-Plasma. As shown in Figure 13, line C exhibited IFN-α production activity 10 times or more higher than that of the original line. These results demonstrate that a more active CAL-1 cell line can be obtained by single cell cloning. Furthermore, it was demonstrated that the immunostimulatory activity evaluation system using the highly active CAL-1 cell line obtained by single cell cloning is a system that makes it easier to evaluate immunostimulatory activity.

Claims

1. A method for evaluating the immunostimulatory activity of a target substance, comprising a contacting step of culturing a human plasmacytoid dendritic cell line in the presence of the target substance, wherein the human plasmacytoid dendritic cell line is CAL-1 cells (deposit number FERM BP-10914), and the CAL-1 cells are in a starved state during at least a part of the contacting step.

2. The method according to claim 1, wherein the starvation state is a state in which the cells are cultured in a medium containing less than 1.0% by volume of serum based on the total volume of the medium.

3. The method according to claim 1, wherein the contacting step satisfies at least one of the following conditions selected from the group consisting of A) and B): A) the CAL-1 cells are cultured under conditions that starve the CAL-1 cells during part or all of the contacting step; B) the CAL-1 cells are in a starved state at the start of the contacting step.

4. The method of claim 1, further comprising a pretreatment step of starving CAL-1 cells prior to said contacting step.

5. The method according to claim 4, wherein the culture time in the pretreatment step is from 1 hour to 72 hours.

6. The concentration of CAL-1 cells in the medium during the culture in the contact step is 0.1 x 10 ４ cells / mL or more 5.0×10 ８ The method of claim 1, wherein the concentration is less than 1000 cells / mL.

7. The method according to claim 1, wherein the culture time in the contact step is from 1 hour to 168 hours.

8. The method according to any one of claims 1 to 7, wherein the target substance is a bacterium.

9. The method according to claim 8, wherein the bacteria are lactic acid bacteria and / or acetic acid bacteria.

10. A method according to any one of claims 1 to 7, wherein the target substance is a substance that is phagocytosed by the CAL-1 cells.

11. The method according to any one of claims 1 to 7, wherein the concentration of the target substance in the medium during the culture in the contact step is 1.0 μg / mL or more and 1000 μg / mL or less.

12. The method according to any one of claims 1 to 7, wherein the immunostimulatory activity is the ability to activate dendritic cells.

13. The method according to claim 12, wherein the dendritic cell activation ability is evaluated using the expression level of interferon α by the CAL-1 cells as an index.

14. The method according to claim 13, wherein the expression level of interferon α is the amount of interferon α secreted extracellularly and / or the amount of interferon α in the CAL-1 cells, or the amount of mRNA encoding interferon α.

15. The method according to claim 14, further comprising a comparison step of comparing the measured value of the expression level of interferon α in the CAL-1 cells contacted with the target substance in the contact step with the measured value of the expression level of interferon α in the CAL-1 cells not contacted with the target substance.

16. The method according to claim 13, wherein the expression level of interferon α is measured at a time point between 15 hours and 72 hours after the start of the contacting step.

17. The method according to claim 12, wherein the dendritic cell activation ability is evaluated using as an index the proportion of interferon α-positive cells among the CAL-1 cells.

18. The method according to any one of claims 1 to 7, further comprising, prior to the contacting step, a preparation step of preparing the CAL-1 cells to be used in the contacting step, wherein the preparation step comprises a step of preparing CAL-1 cells that are susceptible to activation by single-cell cloning of the CAL-1 cells.

19. The CAL-1 cells were cultured at a density of 5.0 × 10 in serum-free RPMI-1640 medium. ５ The method according to any one of claims 1 to 7, wherein the CAL-1 cells are those which, after contact with 10 µg / mL of Lactococcus lactis subsp. lactis JCM 5805 for 24 hours at an initial concentration of 100.0 pg / mL, have an interferon α concentration in the medium of 100.0 pg / mL or more.

20. 5.0 x 10 cells were cultured in serum-free RPMI-1640 medium. ５ CAL-1 cells (deposit number FERM BP-10914), in which the concentration of interferon α in the medium is 100.0 pg / mL or more after 24 hours of contact with 10 μg / mL of Lactococcus lactis subsp. lactis JCM 5805 at an initial concentration of 100.0 pg / mL.

21. A method for screening for a substance having immunopotentiating properties, using the method according to any one of claims 1 to 7.

22. A kit for use in the method of any one of claims 1 to 7.

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