Method for detecting respiratory sensitization potential of a test substance, method for screening sensitization inhibitors, and method for producing a composition for bioadministration.

Abstract

This invention provides a novel method for determining the respiratory sensitization potential of a test substance without using a living organism. [Solution] The present invention provides a method for determining respiratory sensitization, comprising the steps of: preparing a three-dimensional co-culture system of a dendritic cell layer and a bronchial epithelial cell layer; adding a test substance to the bronchial epithelial cell layer of the three-dimensional co-culture system and incubating it; detecting mRNA expression of a gene marker in the dendritic cell layer of the three-dimensional co-culture system; and determining the respiratory sensitization of the test substance based on the mRNA expression level. The dendritic cell layer is a layer of immature dendritic cells derived from a monocyte cell line or a monocyte-like cell line. The determination step involves comparing the detected mRNA expression level with a determination criterion for mRNA expression level corresponding to respiratory sensitization. The gene marker is at least one selected from the group consisting of TNFSF4, a subunit of the TSLP receptor, a subunit of the IL-25 receptor, and a subunit of the IL-33 receptor, or a combination thereof.

Description

[Technical Field]

[0001] The present invention relates to a method for detecting respiratory sensitization, a method for screening sensitization inhibitors, and a method for producing a composition for biological administration. [Background technology]

[0002] Allergies are broadly classified into two types: skin allergies and respiratory allergies. Respiratory allergies, in particular, often lead to serious conditions such as asthma, requiring a high level of risk management. Therefore, it is crucial to determine whether various substances, including chemicals, have respiratory sensitizing properties that could trigger respiratory allergies.

[0003] On the other hand, in the medical field, confirming the properties of various substances has generally involved animal testing, but in recent years, from the perspective of animal welfare, there has been a demand for alternative methods that do not use animals. Research is also being conducted on alternative methods to animal testing for confirming respiratory sensitization, but none have yet been established, and their development is urgently needed (Non-Patent Literature 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Arts, J. (2020). How to assess respiratory sensitization of low molecular weight chemicals? Int J Hyg Environ Health 225, 113469. [Overview of the project] [Problems that the invention aims to solve]

[0005] Therefore, the present invention aims to provide a novel method for determining the respiratory sensitization potential of a test substance without using a living organism. [Means for solving the problem]

[0006] To achieve the above objective, the respiratory sensitization determination method of the present invention is: The process of preparing a three-dimensional co-culture system of dendritic cell layer and bronchial epithelial cell layer, A step of adding the test substance to the bronchial epithelial cell layer of the three-dimensional co-culture system and incubating it. A step of detecting mRNA expression of a gene marker in the dendritic cell layer of the three-dimensional co-culture system, and The process includes determining the respiratory sensitization potential of the test substance based on the mRNA expression level of the gene marker; In the preparation step described above, the dendritic cell layer is a layer of immature dendritic cells derived from a monocyte cell line or a monocyte-like cell line. The aforementioned determination step is a step of determining the mRNA expression level by comparing the detected mRNA expression level with the criteria for determining mRNA expression levels according to respiratory sensitization. The aforementioned gene marker is at least one selected from the group consisting of TNFSF4, a subunit of the TSLP receptor, a subunit of the IL-25 receptor, and a subunit of the IL-33 receptor, or a combination thereof. It is characterized by the following: [Effects of the Invention]

[0007] According to the present invention, as described above, the respiratory sensitization potential of a test substance can be determined by using a three-dimensional co-culture system of a dendritic cell layer derived from the monocyte cell line or the monocyte-like cell line and a bronchial epithelial cell layer, and by detecting the mRNA expression of a specific gene marker in the dendritic cell layer.

[0008] Furthermore, since the mechanisms of action of skin sensitization and respiratory sensitization are thought to be similar, it is difficult to distinguish whether the immune system's response is due to skin sensitization or respiratory sensitization by a substance. In contrast, according to the present invention, it is possible to distinguish whether the sensitization of the test substance is respiratory sensitization rather than skin sensitization by the method described above. [Brief explanation of the drawing]

[0009] [Figure 1] Fig. 1 is a cross-sectional view schematically showing a DC co-culture system. [Figure 2A] Fig. 2A is a graph showing the relative expression levels of mRNA of a specific gene for the skin sensitizing substance group and the respiratory sensitizing substance group in Example 1, wherein the left graph shows the relative expression level of CD80 mRNA, and the right graph shows the relative expression level of CD86 mRNA. [Figure 2B] Fig. 2B is a graph showing the relative expression levels of mRNA of a specific gene for the skin sensitizing substance group and the respiratory sensitizing substance group in Example 1, wherein the left graph shows the relative expression level of TNFSF4 (OX40 ligand) mRNA, the middle graph shows the ratio of the relative expression level of TNFSF4 mRNA to the relative expression level of CD80 mRNA, and the right graph shows the ratio of the relative expression level of TNFSF4 mRNA to the relative expression level of CD86 mRNA. [Figure 2C] Fig. 2C is a graph showing the relative expression levels of mRNA of a specific gene for the skin sensitizing substance group and the respiratory sensitizing substance group in Example 1, wherein the left graph shows the relative expression level of CRLF2 mRNA, and the right graph shows the ratio of the relative expression level of CRLF2 mRNA to the relative expression level of CD80 mRNA. [Figure 2D] Fig. 2D is a graph showing the relative expression levels of mRNA of a specific gene for the skin sensitizing substance group and the respiratory sensitizing substance group in Example 1, wherein the left graph shows the relative expression level of IL-7Rα mRNA, the middle graph shows the ratio of the relative expression level of IL-7Rα mRNA to the relative expression level of CD80 mRNA, and the right graph shows the ratio of the relative expression level of IL-7Rα mRNA to the relative expression level of CD86 mRNA. [Figure 2E]Fig. 2E is a graph showing the relative expression of mRNA of a specific gene for the skin sensitizer group and the respiratory sensitizer group in Example 1, the left is a graph showing the relative expression of IL-17RB mRNA, and the right is a graph showing the ratio of the relative expression of IL-17RB mRNA to the relative expression of CD80 mRNA. [Figure 2F] Fig. 2F is a graph showing the relative expression of mRNA of a specific gene for the skin sensitizer group and the respiratory sensitizer group in Example 1, the left is a graph showing the relative expression of ST2 mRNA, the middle is a graph showing the ratio of the relative expression of ST2 mRNA to the relative expression of CD80 mRNA, and the right is a graph showing the ratio of the relative expression of ST2 mRNA to the relative expression of CD88 mRNA. [Figure 3A] Fig. 3A shows the result of comparing the skin sensitizer NBB and the respiratory sensitizer PZA in Example 2, and is a graph of cell viability. [Figure 3B] Fig. 3B shows the result of comparing the skin sensitizer NBB and the respiratory sensitizer PZA in Example 2, and from the top, it is a graph of cytokine production amount, a graph of cytokine production amount corrected by cell viability, and a graph showing the relative value when the cytokine production amount corrected by the cell viability of a control (10% DMSO) is set as 1. [Figure 3C] Fig. 3C is a graph showing the ratio between the highest production relative values for two types of cytokines produced by stimulation with the same sensitizer in Example 2. [Figure 4A] Fig. 4A shows the result of comparing the skin sensitizer DNCB and the respiratory sensitizer TMA in Example 2, and is a graph of cell viability. [Figure 4B] Fig. 4B shows the result of comparing the skin sensitizer DNCB and the respiratory sensitizer TMA in Example 2, and from the top, it is a graph of cytokine production amount, a graph of cytokine production amount corrected by cell viability, and a graph showing the relative value when the cytokine production amount corrected by the cell viability of a control (10% DMSO) is set as 1. [Figure 4C] Figure 4C is a graph showing the ratio between the highest relative production values ​​for two types of cytokines produced by the same sensitizing substance in Example 2. [Figure 5A] Figure 5B is a graph showing the ratio of the production of specific cytokines relative to the control for the skin sensitizing substance group and the respiratory sensitizing substance group in Example 2. [Figure 5B] Figure 5B is a graph showing the ratio of the production of specific cytokines to the control group for the skin sensitizing substance group and the respiratory sensitizing substance group. [Figure 6] Figure 6 shows the flow cytometry results of alloantigen-specific Th2 cell lines in Example 3. [Modes for carrying out the invention]

[0010] [1] A step to prepare a three-dimensional co-culture system of dendritic cell layer and bronchial epithelial cell layer, A step of adding the test substance to the bronchial epithelial cell layer of the three-dimensional co-culture system and incubating it. A step of detecting mRNA expression of a gene marker in the dendritic cell layer of the three-dimensional co-culture system, and The process includes determining the respiratory sensitization potential of the test substance based on the mRNA expression level of the gene marker; In the preparation step described above, the dendritic cell layer is a layer of immature dendritic cells derived from a monocyte cell line or a monocyte-like cell line. The aforementioned determination step is a step of determining the mRNA expression level by comparing the detected mRNA expression level with the criteria for determining mRNA expression levels according to respiratory sensitization. The aforementioned gene marker is at least one selected from the group consisting of TNFSF4, a subunit of the TSLP receptor, a subunit of the IL-25 receptor, and a subunit of the IL-33 receptor, or a combination thereof. A method for determining respiratory sensitization, characterized by the following features. [2] The TSLP receptor subunit is CRLF2 or IL-7Rα, The IL-25 receptor subunit is either IL-17RA or IL-17RB. The determination method according to [1], wherein the subunit of the IL-33 receptor is ST2 or IL-1RAP. [3] The monocyte cell line is A cell line into which the c-MYC gene, BMI1 gene, and BCL-2 gene have been introduced into monocyte cells, or The determination method described in [1] or [2], wherein the cell line is a cell line into which the c-MYC gene, the BMI1 gene, and the BCL-2 gene have been introduced into monocyte cells induced from induced pluripotent stem cells. [4] The method for determining that the monocyte-like cell line is an acute monocytic leukemia cell line, as described in [1] or [2]. [5] The determination method according to any one of [1] to [4], wherein the monocyte cell line is a human monocyte cell line and the monocyte-like cell line is a human monocyte-like cell line. [6] The determination method according to any one of claims [1] to [5], wherein in the three-dimensional co-culture system, the lower layer is the dendritic cell layer, the upper layer is the bronchial epithelial cell layer, and the test substance is added to the bronchial epithelial cell layer of the upper layer. [7] The above criteria are the criteria for determining respiratory nonsensitization, The criteria for determining respiratory nonsensitization is the mRNA expression level of the gene marker when a respiratory nonsensitizing substance is used. In the determination process described above, If the detected mRNA expression level is greater than the criteria for determining respiratory non-sensitization, the test substance is determined to be respiratory sensitizing. The determination method according to any one of [1] to [6], wherein the detected mRNA expression level is equivalent to or lower than the criteria for determining respiratory non-sensitization, and the test substance is determined to be non-respiratory sensitizing. [8] The above criteria are the criteria for respiratory sensitization, The criterion for determining sensitization is the mRNA expression level of the gene marker when a respiratory sensitizing agent is used. The determination method according to any one of [1] to [7], wherein, in the determination step, if the detected mRNA expression level is equal to or greater than the criteria for determining respiratory sensitization, the test substance is determined to be respiratory sensitizing. [9] In the step of preparing the three-dimensional co-culture system, Immature dendritic cells are prepared by stimulating the monocyte cell line or monocyte-like cell line with interleukin-4 using a culture medium containing GM-CSF and M-CFS. The determination method according to any one of [1] to [8], wherein an immature dendritic cell layer is prepared by culturing the immature dendritic cells.

[10] Furthermore, After the incubation step, a step of collecting the dendritic cell layer from the three-dimensional co-culture system, The step of adding antigen-specific T cells to the dendritic cell layer and incubating it in the presence of antigen-specific T cells, A process for detecting cytokine markers in cell supernatant, and The process includes determining the respiratory sensitization potential of the test substance based on the amount of the cytokine marker; The aforementioned antigen-specific T cells are protein antigen-specific T cells, The cytokine marker is at least one selected from the group consisting of IL-4, IFN-γ, and IL-2, or a combination of multiple markers. The determination method according to any one of [1] to [9], wherein the determination step is to determine by comparing the detected cytokine marker amount with a determination criterion for the amount of cytokine marker according to respiratory sensitization.

[11] The determination method according to

[10] , wherein the subset of T cells is at least one selected from the group consisting of Th0 cells, Th1 cells, and Th2 cells.

[12] The determination method according to

[10] or

[11] , wherein the protein antigen for the protein antigen-specific T cell is an alloantigen.

[13] The determination method according to any one of

[10] to

[12] , wherein the protein antigen for the protein antigen-specific T cells is at least one selected from the group consisting of pollen, dust mites, house dust, serum albumin, antibody immunoglobulin, and keratin.

[14] The determination method according to any one of

[10] to

[13] , wherein the detection of the cytokine marker amount is the detection of a protein.

[0011] <1. Method for detecting respiratory sensitization> The present invention's method for determining respiratory sensitization involves the steps of preparing a three-dimensional co-culture system of dendritic cell layer and bronchial epithelial cell layer, as described above. A step of adding the test substance to the bronchial epithelial cell layer of the three-dimensional co-culture system and incubating it. A step of detecting mRNA expression of a gene marker in the dendritic cell layer of the three-dimensional co-culture system, and The process includes determining the respiratory sensitization potential of the test substance based on the mRNA expression level of the gene marker; In the preparation step described above, the dendritic cell layer is a layer of immature dendritic cells derived from a monocyte cell line or a monocyte-like cell line. The aforementioned determination step is a step of determining the mRNA expression level by comparing the detected mRNA expression level with the criteria for determining mRNA expression levels according to respiratory sensitization. The gene marker is characterized by being at least one selected from the group consisting of TNFSF4, a subunit of the TSLP receptor, a subunit of the IL-25 receptor, and a subunit of the IL-33 receptor, or a combination thereof.

[0012] In the present invention, the test substance used to determine respiratory sensitization is also referred to as the sensitizing test substance.

[0013] If the sensitizing test substance has respiratory sensitizing properties, when the sensitizing test substance is added to the bronchial epithelial cell layer, the interaction between the bronchial epithelial cells of the bronchial epithelial cell layer and the sensitizing test substance causes the immature dendritic cells in the dendritic cell layer to differentiate into mature dendritic cells. The inventors have found that the expression of a specific group of genes during this differentiation process is associated with respiratory sensitization and exhibits different behavior from skin sensitization. Therefore, by detecting the specific group of genes as a genetic marker, the present invention makes it possible to easily determine whether the sensitizing test substance exhibits respiratory sensitizing properties, or more specifically, whether it exhibits respiratory sensitization rather than skin sensitization.

[0014] (Embodiment 1) As an example of the method for determining respiratory sensitization according to the present invention, Embodiment 1 is a method for determining the respiratory sensitization of a sensitizing test substance using the three-dimensional co-culture system of the dendritic cell layer and the bronchial epithelial cell layer, as described above, and specifically includes the steps of preparing the three-dimensional co-culture system, adding the sensitizing test substance and incubating it, detecting the mRNA expression of the gene marker, and determining the respiratory sensitization of the sensitizing test substance. The method of Embodiment 1 will also be referred to below as the DC co-culture system determination method.

[0015] The preparation step described above is a step of preparing a three-dimensional co-culture system of a dendritic cell layer and a bronchial epithelial cell layer. The dendritic cell layer is a layer of immature dendritic cells derived from a monocyte cell line or a monocyte-like cell line, and the bronchial epithelial cell layer is a layer of bronchial epithelial cells. The cells of the dendritic cell layer and the cells of the bronchial epithelial cell layer may be cells from the same species or cells from different species, but it is preferable that they are both of human origin. The species is preferably, for example, the species whose respiratory sensitization properties of the sensitizing test substance are to be determined. The species is, for example, a human or a non-human animal, but it is preferably a human. Examples of non-human animals include mice, rats, rabbits, dogs, cats, monkeys, cattle, pigs, sheep, and horses. The following are specific examples of human-derived cells, but are not limited thereto.

[0016] The monocyte cell line is, for example, a cell line obtained by introducing transcription factor genes into monocyte cells. The transcription factor genes are, for example, the c-MYC gene, the BMI1 gene, and / or the BCL-2 gene, and it is preferable to introduce all three of these genes and establish a cell line. The monocyte cells into which the genes are introduced may be, for example, monocyte cells isolated from the aforementioned species (preferably human), monocyte cells differentiated from induced pluripotent stem cells (iPS), or cell lines established from these. The monocyte cell line is hereinafter referred to as the monocyte cell line (c-MYC / BMI1 / BCL-2).

[0017] Monocyte cells or monocyte cell lines before the introduction of the aforementioned genes will, for convenience, be referred to below as monocyte cells (-) or monocyte cell lines (-). The monocyte cells (-) or monocyte cell lines (-) are, for example, monocyte cells or monocyte cell lines isolated from the peripheral blood of a normal human, for better or for worse.

[0018] The aforementioned monocyte cell line (c-MYC / BMI1 / BCL-2) can be prepared as a cell line (cell line) by introducing the various genes mentioned above into human monocyte cells, for example, based on the following paper. “Generation of Large Numbers of Antigen-Expressing Human Dendritic Cells Using CD14-ML Technology” Haruta et al., PLoS One 2016 The monocyte cell line (c-MYC / BMI1 / BCL-2) into which each of the aforementioned genes has been introduced can be a commercially available product, for example, aMylc (MiCAN Corporation). The human monocyte cell line (c-MYC / BMI1 / BCL-2), aMylc, is also known as CD14-ML, for example.

[0019] Furthermore, the aforementioned monocyte cell line (c-MYC / BMI1 / BCL-2) can be prepared as a cell line by introducing the various genes into monocyte cells induced from induced pluripotent stem cells (iPS cells), for example, based on the following paper. Such cells are called iPS-ML cells, and the following paper can be referenced. Commercially available iPS-ML cells can be used, for example. “Generation of dendritic cells and macrophages from human induced pluripotent stem cells aiming at cell therapy.” Senju, S. et al., “Gene Ther 2011, 18, 874-883.

[0020] The aforementioned monocyte-like cell line refers to a cell line in which cells expressing CD14, a cell surface marker for human monocytes, can be induced to differentiate into immature dendritic cells by stimulation such as cytokines. Examples of such monocyte-like cell lines include cells that have spontaneously formed from cells derived from cancer patients, and a specific example is the monocyte-like leukemia cell line derived from acute monocytic leukemia patients, i.e., THP-1 cells. THP-1 cells can be obtained, for example, from ATCC.

[0021] Hereinafter, the monocyte cell line (c-MYC / BMI1 / BCL-2) and the monocyte-like cell line used for the preparation of immature dendritic cells will also be referred to as "raw material cell lines." Unless otherwise specified, the following descriptions of the monocyte cell line (c-MYC / BMI1 / BCL-2) and the monocyte-like cell line are interchangeable.

[0022] The monocyte cell line (c-MYC / BMI1 / BCL-2) can be subcultured using a medium containing, for example, granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). The medium can be, for example, a basic medium for animal cells, such as αMEM medium. The culture conditions are not particularly limited, and the culture temperature is, for example, 36-37°C, preferably 37°C. The monocyte-like cell line can be subcultured using a medium containing, for example, fetal bovine serum (FBS), with a FBS concentration of, for example, 10-20%.

[0023] Immature dendritic cells derived from the aforementioned raw material cell line (the monocyte cell line (c-MYC / BMI1 / BCL-2) or the monocyte-like cell line) can be obtained from the raw material cell line, for example, by a general method for differentiating monocyte cells into immature dendritic cells. The differentiation method may include, for example, cytokine stimulation, which can be carried out by culturing in a cytokine-containing medium.

[0024] The aforementioned bronchial epithelial cell layer is a layer of bronchial epithelial cells. The bronchial epithelial cells may be, for example, cells isolated from the surface epithelium of the bronchi, or a cell line may be used. Examples of bronchial epithelial cells that can be used include BEAS-2B (trade name CRL-3588, ATCC) and NHBE (CC-2540S, LONZA).

[0025] The three-dimensional co-culture system preferably comprises the dendritic cell layer and the bronchial epithelial cell layer, and is a cell stack of the dendritic cell layer and the bronchial epithelial cell layer. Preferably, the cell stack has the dendritic cells as the lower layer and the bronchial epithelial cell layer as the upper layer above it. The cell stack can be prepared, for example, by separately forming the dendritic cell layer and the bronchial epithelial cell layer and stacking them.

[0026] The number of cells in the three-dimensional co-culture system is, for example, 4 × 10 cells in the dendritic cell layer. 5 In the cells, the number of cells in the bronchial epithelial cell layer is 2.5 × 106 The cells are, and the ratio of the number of cells in the dendritic cell layer (D) to the bronchial epithelial cell layer (B) (D:B) is, for example, about 1:6±2 or about 1:6.

[0027] An example of a method for preparing the aforementioned cell stack will be described. However, the present invention is not limited to these examples.

[0028] The raw material cell line is cultured under cytokine stimulation as described above to differentiate it into immature dendritic cells. For the culture, for example, the basic medium for animal cells described above can be used as the culture medium. The cytokine added to the medium is, for example, interleukin, hematopoietic factor, etc. A specific example of interleukin is interleukin-4 (IL-4). Specific examples of hematopoietic factor are granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), etc., preferably both. The culture conditions for differentiation are, for example, a culture temperature of 37°C and a culture time of 2 days.

[0029] Next, a cell scaffold is placed inside the culture vessel, and the suspension of immature dendritic cells is seeded onto the scaffold to adhere the immature dendritic cells to the scaffold. The liquid solvent of the suspension is not particularly limited; for example, a culture medium can be used, and specifically, the basic culture medium for animal cells described above can be used. Then, the immature dendritic cells seeded in the culture vessel are cultured to form a layer of immature dendritic cells (dendritic cell layer). For the culture, for example, the basic culture medium for animal cells described above can be used as the culture medium. The cytokines added to the culture medium are, for example, interleukins, hematopoietic factors, etc. A specific example of the interleukin is interleukin-4 (IL-4). Specific examples of the hematopoietic factors are granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), etc., and preferably both. The culture conditions for differentiation are, for example, a culture temperature of 37°C and a culture time of 2 days.

[0030] Meanwhile, a cell scaffold is placed inside another culture vessel, and the suspension of bronchial epithelial cells is seeded onto the scaffold to adhere the bronchial epithelial cells to the scaffold. The liquid solvent of the suspension is not particularly limited; for example, a culture medium can be used, and specifically, the basic culture medium for animal cells described above can be used. Then, the bronchial epithelial cells seeded in the culture vessel are cultured to form a layer of bronchial epithelial cells (bronchial epithelial cell layer). For the culture, for example, the basic culture medium for animal cells described above can be used as the culture medium. The culture conditions are, for example, a culture temperature of 37°C and a culture time of 2 days.

[0031] Then, the dendritic cell layer and the bronchial epithelial cell layer are detached from each culture vessel along with their respective scaffolds, and the bronchial epithelial cells are stacked on top of the dendritic cell layer. This cell stack is placed in a new culture vessel, and culture is performed by adding culture medium. In this way, the three-dimensional co-culture system can be prepared. As the culture medium, for example, the basic culture medium for animal cells can be used. Additives may also be added to the basic culture medium. The cytokines added to the culture medium are, for example, interleukins and hematopoietic factors. A specific example of the interleukin is interleukin-4 (IL-4). Specific examples of the hematopoietic factors are granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), and preferably both. The culture conditions for differentiation are, for example, a culture temperature of 37°C and a culture time of 1 day.

[0032] Next, the incubation step involves adding a sensitizing test substance to the bronchial epithelial cell layer of the three-dimensional co-culture system and incubating it. The three-dimensional co-culture system is preferably a laminate in which the upper layer is the bronchial epithelial cell layer and the lower layer is the dendritic cell layer, and it is thought that by adding the sensitizing test substance to the bronchial epithelial cell layer, the sensitizing test substance and its stimuli move from the upper layer (bronchial epithelial cell layer) to the lower layer (dendritic cell layer).

[0033] The sensitizing test substance is not particularly limited, and any substance whose respiratory sensitization potential you wish to assess can be used. Examples of the sensitizing test substance include low molecular weight compounds, high molecular weight compounds, nucleic acids, proteins, peptides, and the like.

[0034] The sensitizing test substance is added to the bronchial epithelial cell layer, for example, as a mixture with a liquid solvent. The type of liquid solvent is not particularly limited, and it is preferable to use one that does not exhibit sensitizing properties such as respiratory sensitization on its own. Specific examples of the liquid solvent include aqueous solvents, organic solvents, oily solvents, and mixtures thereof. Examples of aqueous solvents include water, physiological saline, buffer solutions, and culture media. Examples of organic solvents include alcohols such as ethanol, acetone, and dimethyl sulfoxide (DMSO). Examples of oily solvents include soybean oil and olive oil. The mixture may be a suspension, a solvent, or any other form. If the sensitizing test substance is poorly soluble in an aqueous solvent, for example, it is preferable to first dissolve it in the organic solvent and then dilute it with the aqueous solvent to prepare the mixture. Since the mixture is added to the bronchial epithelial cell layer, the final concentration of the organic solvent in the mixture is preferably, for example, 0.1-20% (v / v), 1-15% (v / v), 5-15% (v / v), 10±1% (v / v), or 10% (v / v).

[0035] The sensitizing test substance may be added to the bronchial epithelial cell layer in the cell stack after culturing the cell stack as described above, after removing the culture medium, and then a new culture medium may be added. For example, a basic culture medium for animal cells as described above can be used as the culture medium. The incubation conditions are not particularly limited, and the incubation temperature is, for example, 37°C. An example of incubation is given below, but the present invention is not limited to this specific example.

[0036] As described above, it is preferable to add the sensitizing test substance as a mixture to at least one, preferably multiple, locations on the bronchial epithelial cell layer. Surface area of ​​the bronchial epithelial cell layer: 19 mm² 2 (For example, the number of cells on the surface is 2.5 × 10⁻⁶) 6For each cell, the number of locations where the mixture is added is, for example, 1 to 5 locations, preferably 2 to 5 locations. The amount of the mixture added per location is, for example, 1 to 50 μL, 5 to 50 μL, 5 to 25 μL, or 5 to 10 μL. A specific example of incubation conditions is a surface area of ​​19 mm² of the bronchial epithelial cell layer. 2 Add 25 μL of the sensitizing test substance (for example, 5 μL each at 5 locations) to the sample, incubate at room temperature for 20 minutes, then add 2.5 mL of culture medium and incubate for a further 8 hours.

[0037] Next, the detection step is to detect the mRNA expression of a gene marker in the dendritic cell layer of the three-dimensional co-culture system. If the sensitizing test substance has respiratory sensitizing properties, when inflammatory cytokines such as damage-associated molecular patterns (DAMPs), TSLP, IL-25, and IL-33 produced from the bronchial epithelial cell layer stimulated by the sensitizing test substance move to the dendritic cell layer, the immature dendritic cells in the dendritic cell layer mature (become activated).

[0038] The mRNA expression can be detected, for example, by separating the dendritic cell layer from the three-dimensional co-culture system, extracting RNA from the dendritic cell layer, and performing the detection of the obtained RNA sample. The method for detecting mRNA expression is not particularly limited and can include, for example, RT-qPCR. The mRNA expression level may be, for example, the actual amount of mRNA, the corresponding signal intensity (e.g., fluorescence intensity), or their relative values.

[0039] The aforementioned gene marker is at least one selected from the group consisting of TNFSF4, TSLP receptor, its subunits, IL-25 receptor, its subunits, and IL-33 receptor, its subunits. These gene markers are also hereinafter referred to, for example, as "respiratory sensitization gene markers." The aforementioned gene markers are preferably human gene markers.

[0040] TNFSF4 is tumor necrosis factor (TNF) Superfamily Member 4, also known as the OX40 ligand. The TSLP receptor is the thymic stromal lymphopoietin (TSLP) receptor.

[0041] Examples of subunits of the TSLP receptor (TSLP-R) include CRLF2 and IL-7Rα. Examples of subunits of the IL-25 receptor (IL-25-R) include IL-17RA and IL-17RB. Examples of subunits of the IL-33 receptor (IL-33-R) include ST2 (also called IL-1R1) and IL-1 receptor accessory protein (also called IL-1RAP).

[0042] In the detection step described above, the number of gene markers detected for a single RNA sample may be, for example, one or a combination of two or more. The gene marker is preferably TNFSF4 (OX40 ligand). A combination of two or more gene markers is also preferred, and a specific example is the combination of TNFSF4 (OX40 ligand) and ST2 (IL-1R1).

[0043] The determination step is a step of determining the respiratory sensitization potential of the sensitizing test substance based on the mRNA expression level of the gene marker. The determination can be made by comparing the detected mRNA expression level with a determination criterion for mRNA expression level corresponding to respiratory sensitization. The determination criterion can be set in advance, for example.

[0044] The aforementioned determination criterion is, for example, a determination criterion for respiratory non-sensitization, and can be set to the mRNA expression level of the gene marker when a respiratory non-sensitizing substance is used. In this case, for example, if the detected mRNA expression level is greater than the determination criterion for respiratory non-sensitization, the sensitizing test substance is determined to be respiratory sensitizing, and if the detected mRNA expression level is equal to or less than the determination criterion for respiratory non-sensitization, the sensitizing test substance is determined to be non-respiratory sensitizing.

[0045] Furthermore, the judgment criterion can be, for example, a judgment criterion for respiratory sensitization, and can be set to the mRNA expression level of the gene marker when a respiratory sensitizing substance is used. In this case, in the judgment step, if the detected mRNA expression level is equivalent to or greater than the judgment criterion for respiratory sensitization, the sensitizing test substance can be determined to be respiratory sensitizing.

[0046] (Extreme Variation 1-1) For co-culture using the aforementioned three-dimensional co-culture system, for example, culture tools for three-dimensional cell culture can be used. As such culture tools, a co-culture set combining an outer container, an inner container, and a cell scaffold can be used.

[0047] The outer container, for example, can accommodate the inner container and serves as a culture vessel (also called a culture tank) for the cells in the inner container. The outer container can be, for example, placed in the through-holes of a plate having multiple through-holes to form a so-called 6-well plate or a 12-well plate. On the other hand, the inner container has a structure in which its sides are partially cut out and its bottom is open, allowing the scaffold to be placed at the bottom.

[0048] As described above, the cell stack can be prepared by stacking the dendritic cell layer containing the scaffold and the bronchial epithelial cell layer containing the scaffold. Therefore, the cell stack can be placed in the inner container by inserting the cell stack containing the scaffold into the inner container. Then, the culture medium is placed in the outer container, and the inner container with the cell stack placed in it is inserted thereafter. Since the inner container has partially missing sides and an open bottom, the cell stack comes into contact with the culture medium in the outer container in those areas. Therefore, the cell stack can be cultured in contact with the culture medium in the outer container.

[0049] As the aforementioned co-culture set, commercially available products such as the Alvetex® Scaffold 12-well insert and 24-well plate (Reinnervate Ltd.) can be used. Furthermore, the scaffold is not particularly limited, and for example, a porous material can be used, such as one made of polystyrene or other resin. As a commercially available product of the scaffold, for example, the Alvetex® Scaffold membrane can be used.

[0050] (Variations 1-2) Further specific examples of the criteria and methods for determination are given below. For example, the following mRNA expression levels can be set as the criteria. (1) mRNA expression level when the sensitizing test substance is not added to the three-dimensional co-culture system. (2) mRNA expression level when a respiratory non-sensitizing substance that does not cause respiratory sensitization is added to the three-dimensional co-culture system. (3) mRNA expression level when a sensitizing substance exhibiting respiratory sensitization is added to the three-dimensional co-culture system

[0051] The aforementioned gene marker (e.g., TNFSF4) can be used to determine, for example, that the sensitizing test substance may have respiratory sensitizing properties if the detected mRNA expression level is greater than the determination criterion (1) (preferably by a statistically significant difference).

[0052] Furthermore, the increase in production of the gene marker (e.g., TNFSF4) due to respiratory sensitizing substances is higher than that due to skin sensitizing substances (the increase in production due to skin sensitizing substances is lower than that due to respiratory sensitizing substances). From this, if the detected mRNA expression level is greater than that of (2) above (preferably significantly greater), it can be determined that the sensitizing test substance may have respiratory sensitization properties rather than skin sensitization properties. In the determination criterion (2), respiratory non-sensitizing substances that do not show respiratory sensitization are, for example, skin sensitizing substances. If the detected mRNA expression level is greater than that of determination criterion (1) above and about the same as that of determination criterion (2) above (e.g., no significant difference), it can also be determined that the sensitizing test substance may have skin sensitization properties rather than respiratory sensitization properties.

[0053] The aforementioned gene marker (e.g., TNFSF4) can be used to determine if the sensitizing test substance may have respiratory sensitizing properties if, for example, the detected mRNA expression level is at the same level as (e.g., no significant difference) or greater than the determination criterion (3). Furthermore, it can also be determined that the sensitizing test substance may have respiratory sensitizing properties rather than cutaneous sensitizing properties. Additionally, if the detected mRNA expression level is greater than the determination criterion (1) and less than the determination criterion (3), it can also be determined that the sensitizing test substance may have cutaneous sensitizing properties rather than respiratory sensitizing properties.

[0054] The aforementioned criteria are preferably set based on the mRNA expression level detected by a method under the same conditions except for the use of the sensitizing test substance.

[0055] (Variations 1-3) The mRNA expression level of the gene marker may be, for example, a measured value of the actual amount of mRNA, an equivalent measured value, or a corrected value thereof.

[0056] Examples of measured values ​​corresponding to the actual quantity include luminescence intensity and relative values ​​of luminescence intensity obtained by analytical methods such as real-time RT-PCR.

[0057] The correction value may be, for example, a correction value for the actual amount, or a correction value for a measured value corresponding to the actual amount. The correction method is not particularly limited, and for example, the measured value of mRNA of an endogenous control gene in a cell can be used. The correction value can be expressed, for example, as the ratio of the measured value of mRNA of the gene marker (M) to the measured value of mRNA of the control gene (C), specifically as the ratio of the measured value of mRNA of the gene marker (M) to the measured value of mRNA of the control gene (M) (M / C). The endogenous control gene is not particularly limited, and for example, it may be a housekeeping gene, and specific examples include the HPRT (hypoxanthine phosphoribosyltransferase) gene, β-actin, GAPDH (glyceraldehyde 3-phosphate dehydrogenase), rRNA (18S ribosomal RNA), etc.

[0058] Furthermore, as a correction method, for example, the mRNA measurement value of a cell surface molecule of a dendritic cell, which is an antigen-presenting cell, can be used. The correction value can be expressed, for example, as the ratio of the mRNA measurement value of the gene marker (M) to the mRNA measurement value of the cell surface molecule (S), specifically as the ratio of the mRNA measurement value of the gene marker (M) to the mRNA measurement value of the cell surface molecule (S) (M / S). The mRNA measurement value of the gene marker (M) and the mRNA measurement value of the cell surface molecule (S) may be the correction values ​​(M / C and S / C) based on the endogenous control gene, respectively. The cell surface molecule is not particularly limited and is, for example, CD80, CD86, or CD11c, preferably CD80 or CD86.

[0059] Preferably, the mRNA expression level in the judgment criteria and the mRNA expression level when using the test substance are of the same type as the measured value or the corrected value.

[0060] (Variations 1-3) In the modified example 1-2 described above, the mRNA expression level in the judgment criterion (2) may be, for example, the mRNA expression level set from the results of adding one type of respiratory non-sensitizing substance, but it is preferable that it be the mRNA expression level set from the results of using two or more types of respiratory non-sensitizing substances. Specifically, it is preferable that the mRNA expression level be set from the average of the results of adding two or more types of respiratory non-sensitizing substances. (2) mRNA expression level when a respiratory non-sensitizing substance that does not cause respiratory sensitization is added to the three-dimensional co-culture system.

[0061] The aforementioned non-respiratory sensitizing substance may include, for example, the skin sensitizing substances listed in the examples, and may be one or more of them.

[0062] In the modified examples 1-3 described above, the mRNA expression level in the judgment criterion (3) may be, for example, the mRNA expression level set from the results of adding one type of respiratory sensitizing agent, but it is preferable that it be the mRNA expression level set from the results of using two or more types of respiratory sensitizing agents. Specifically, it is preferable that the mRNA expression level be set from the average of the results of adding two or more types of respiratory sensitizing agents. (3) mRNA expression level when a sensitizing substance exhibiting respiratory sensitization is added to the three-dimensional co-culture system

[0063] The aforementioned respiratory sensitizing substance may be, for example, one of the respiratory sensitizing substances listed in the examples, or two or more of them.

[0064] (Embodiment 2) As an example of a method for determining respiratory sensitization according to the present invention, Embodiment 2 is a method for determining the respiratory sensitization of a sensitizing test substance using the dendritic cell layer after the incubation step of Embodiment 1 and newly added antigen-specific T cells. Specifically, the method of Embodiment 2 includes, after the preparation step and the incubation step (hereinafter referred to as the first incubation step) of Embodiment 1, a step of collecting the dendritic cell layer from the three-dimensional co-culture system, a step of adding antigen-specific T cells to the dendritic cell layer and incubating it in the presence of antigen-specific T cells (hereinafter referred to as the second incubation step), a step of detecting cytokine markers in the cell supernatant, and a step of determining the respiratory sensitization of the sensitizing test substance based on the amount of cytokine markers.

[0065] The method of Embodiment 2 may, for example, perform a further cytokine marker detection step (hereinafter referred to as the second detection step) and a determination step (hereinafter referred to as the second determination step) after the detection step (hereinafter referred to as the first detection step) and determination step (hereinafter referred to as the first determination step) in the method of Embodiment 1, or it may omit the first detection step and the first determination step in the method of Embodiment 1 and perform only the second detection step and the second determination step. Since the method of Embodiment 2 involves further steps after the method of Embodiment 1, it will hereinafter also be referred to as the two-step DC / T cell co-culture system determination method.

[0066] The aforementioned collection step is a step of collecting the dendritic cell layer from the three-dimensional co-culture system after the first incubation of Embodiment 1. Since the three-dimensional co-culture system is a cell stack in which the bronchial epithelial cell layer is stacked on top of the dendritic cell layer, for example, the dendritic cell layer may be collected by peeling off the bronchial epithelial cell layer from the cell stack, or the dendritic cell layer may be collected by peeling off the dendritic cell layer.

[0067] Next, the second incubation step involves adding antigen-specific T cells to the dendritic cell layer and incubating it in the presence of antigen-specific T cells. In living organisms, when immature dendritic cells capture an antigen, they are activated (matured) and become mature dendritic cells. These mature dendritic cells, with their enhanced antigen-presenting ability, migrate to lymph nodes and efficiently exhibit antigen-presenting functions to T cells. Therefore, in this embodiment, by isolating the dendritic cell layer from the three-dimensional co-culture system to which the test substance has been added, and further adding antigen-specific T cells, a model can be created that spatiotemporally reproduces the phenomenon occurring in living organisms.

[0068] It is preferable to place the dendritic cell layer in a culture vessel such as a well plate and incubate it with the added antigen-specific T cells in the presence of a culture medium.

[0069] The aforementioned protein antigen-specific T cells are, for example, T cells that specifically react to a certain protein antigen, that is, helper CD4 + This refers to T cells that induce a T(Th) immune response. In the present invention, any T cell that recognizes some kind of protein antigen and thereby induces a Th immune response is acceptable, and the type of protein antigen is not particularly limited, and examples include pollen such as cedar pollen, dust mites, house dust, serum albumin, antibody immunoglobulins, and keratin.

[0070] Specific examples of the protein antigen include keyhole limpet hemocyanin (KLH), cedar pollen (Cryj1, Cryj2), etc. However, the protein antigen is not limited to these and may also be, for example, a protein that is abundant in the body (e.g., serum albumin, antibody immunoglobulin, etc.) or keratin present in epithelial cells. The protein antigen may also be, for example, an alloantigen. A T cell specific to the alloantigen refers to, for example, a T cell that recognizes allogeneic HLA. An example of the alloantigen is an HLA antigen.

[0071] The type (subset) of said antigen-specific T cells is, for example, Th0 cell, Th1 cell or Th2 cell, with Th2 cell being preferred. The antigen-specific T cells may be any one type, or a mixture of two or more types.

[0072] It is preferable that said antigen-specific T cells are added to said dendritic cell layer, for example, in the form of a cell suspension, and it is also preferable that said antigen-specific T cells are added to the upper surface of said dendritic cell layer. The addition amount of said antigen-specific T cells relative to said dendritic cell layer is not particularly limited, for example, per 4×10 5 cells of said dendritic cells, the amount of said antigen-specific T cells is 4×10 5 cells (2×10 5 to 1×10 6 cells), and per 2 cm 2 of area on the upper surface of said dendritic cell layer, the amount of said antigen-specific T cells is 4×10 5 cells (2×10 5 to 1×10 6 cells). The liquid solvent of said cell suspension is not particularly limited, and for example, a medium can be used. Said medium is not particularly limited, and examples thereof include the basal media for animal cells described above.

[0073] The medium used during incubation is not particularly limited, and for example, the basal medium for animal cells as described above can be used. Incubation conditions are not particularly limited, for example, the incubation temperature is 37°C, and the incubation time is, for example, 24 hours.

[0074] Next, said second detection step is a step of detecting a cytokine marker in cell supernatant. That is, it is a step of detecting a cytokine marker contained in the supernatant (cell supernatant) of a culture after incubating said dendritic cell layer in the presence of said antigen-specific T cells.

[0075] The cytokine marker can be detected, for example, by separating the culture supernatant from the culture in the culture vessel and using a sample of the obtained culture supernatant. The method for detecting the cytokine marker is not particularly limited and may include immunological measurement methods using antibodies such as ELISA. The amount of the cytokine marker may be, for example, the actual amount of protein, the corresponding signal intensity (e.g., fluorescence intensity), or their relative values.

[0076] The cytokine marker is, for example, at least one selected from the group consisting of IL-4, IFN-γ, and IL-2. In the second detection step, the number of cytokine markers detected for a single sample may be, for example, one or a combination of two or more. The cytokine marker is preferably IL-4, and also preferably a combination of IL-4 and IL-2, or a combination of IL-4 and IFN-γ.

[0077] Next, the second determination step is to determine the respiratory sensitization potential of the sensitizing test substance based on the amount of cytokine markers. This determination can be made by comparing the detected amount of cytokine markers with a determination criterion for the amount of cytokine markers corresponding to respiratory sensitization. The determination criterion can be set in advance, for example.

[0078] The aforementioned determination criterion is, for example, a determination criterion for respiratory non-sensitization, and can be set to the amount of cytokine marker when a respiratory non-sensitizing substance is used. In this case, for example, if the detected amount of cytokine marker is greater than the determination criterion for respiratory non-sensitization, the sensitizing test substance is determined to be respiratory sensitizing, and if the detected amount of cytokine marker is equal to or less than the determination criterion for respiratory non-sensitization, the sensitizing test substance is determined to be non-respiratory sensitizing.

[0079] Furthermore, the judgment criterion can be, for example, a judgment criterion for respiratory sensitization, and can be set to the amount of cytokine marker when a respiratory sensitizing substance is used. In this case, in the judgment step, if the detected amount of cytokine marker is equal to or greater than the judgment criterion for respiratory sensitization, the sensitizing test substance can be determined to be respiratory sensitizing.

[0080] (Variation 2-1) Further specific examples of the criteria and methods for evaluation are given below. For example, the following cytokine marker levels can be set as the criteria. (1) The amount of cytokine markers in the three-dimensional co-culture system when the sensitizing test substance is not added in Embodiment 1. (2) The amount of cytokine markers when a respiratory non-sensitizing substance that does not exhibit respiratory sensitization is added to the three-dimensional co-culture system in the above embodiment 1. (3) The amount of cytokine markers when a sensitizing substance exhibiting respiratory sensitization is added to the three-dimensional co-culture system in the above embodiment 1.

[0081] Examples of cytokine markers include IL-4 alone, or a combination of IL-4 and IL-2. In this case, the cytokine marker amount can be determined by, for example, the ratio of IL-4 amount to IL-2 amount. Specifically, the ratio of IL-4 amount to IL-2 amount, i.e., IL-4 amount / IL-2 amount (hereinafter referred to as IL-4 / IL-2), can be set as the cytokine marker amount.

[0082] When IL-4 / IL-2 is used as the cytokine marker level, for example, if the detected IL-4 / IL-2 is greater than the IL-4 / IL-2 of the judgment criterion (1) (preferably significantly greater), it can be determined that the sensitizing test substance may have respiratory sensitizing properties.

[0083] For example, the increase in production of IL-4 / IL-2 due to respiratory sensitizing substances is higher than that due to skin sensitizing substances (the increase in production due to skin sensitizing substances is lower than that due to respiratory sensitizing substances). Therefore, when using IL-4 / IL-2 as a cytokine marker, for example, if the detected IL-4 / IL-2 is greater than the IL-4 / IL-2 in the judgment criterion (2) (preferably significantly greater), it can be determined that the sensitizing test substance may have respiratory sensitization rather than skin sensitization. In the judgment criterion (2), a respiratory non-sensitizing substance that does not show respiratory sensitization is, for example, a skin sensitizing substance. Furthermore, if the detected IL-4 / IL-2 is greater than the IL-4 / IL-2 of criterion (1) and is similar to the IL-4 / IL-2 of criterion (2) (for example, there is no significant difference), it can be determined that the sensitizing test substance may have skin sensitization rather than respiratory sensitization.

[0084] Furthermore, when IL-4 / IL-2 is used as the cytokine marker level, for example, if the detected IL-4 / IL-2 is at the same level as (e.g., no significant difference) or greater than the IL-4 / IL-2 in criterion (3), it can be determined that the sensitizing test substance may have respiratory sensitizing properties. Also, for example, it can be determined that the sensitizing test substance may have respiratory sensitizing properties rather than skin sensitizing properties. Moreover, if the detected IL-4 / IL-2 is greater than the IL-4 / IL-2 in criterion (1) and less than the IL-4 / IL-2 in criterion (3), it can be determined that the sensitizing test substance may have skin sensitizing properties rather than respiratory sensitizing properties.

[0085] Furthermore, examples of cytokine markers include a combination of IL-4 and IFN-γ. In this case, the amount of cytokine marker can be determined by, for example, the ratio of IL-4 amount to IFN-γ amount. Specifically, the ratio of IL-4 amount to IFN-γ amount, i.e., IL-4 amount / IFN-γ amount (hereinafter referred to as IL-4 / IFN-γ), can be set as the amount of cytokine marker.

[0086] When IL-4 / IFN-γ is used as the cytokine marker, for example, if the detected IL-4 / IFN-γ is greater than the IL-4 / IFN-γ of the judgment criterion (1) (preferably significantly greater), it can be determined that the sensitizing test substance may have respiratory sensitizing properties.

[0087] For example, the increase in production of IL-4 / IFN-γ due to respiratory sensitizing substances is higher than that due to skin sensitizing substances (the increase in production due to skin sensitizing substances is lower than that due to respiratory sensitizing substances). Therefore, when IL-4 / IFN-γ is used as a cytokine marker, for example, if the detected IL-4 / IFN-γ is greater than the IL-4 / IFN-γ of the judgment criterion (2) (preferably significantly greater), it can be determined that the sensitizing test substance may have respiratory sensitizing properties. Alternatively, for example, it can be determined that the sensitizing test substance may have respiratory sensitizing properties rather than skin sensitizing properties. In the judgment criterion (2), a respiratory non-sensitizing substance that does not show respiratory sensitizing properties is, for example, a skin sensitizing substance. Furthermore, if the detected IL-4 / IFN-γ is greater than the IL-4 / INF-γ of criterion (1) and is similar to the IL-4 / INF-γ of criterion (2) (for example, there is no significant difference), it can be determined that the sensitizing test substance may have skin sensitization rather than respiratory sensitization.

[0088] Furthermore, when IL-4 / IFN-γ is used as the cytokine marker, for example, if the detected IL-4 / IFN-γ is at the same level as (e.g., no significant difference) or greater than the IL-4 / IFN-γ of criterion (3), it can be determined that the sensitizing test substance may have respiratory sensitizing properties. Also, for example, it can be determined that the sensitizing test substance may have respiratory sensitizing properties rather than skin sensitizing properties. Moreover, if the detected IL-4 / INF-γ is greater than the IL-4 / INF-γ of criterion (1) and less than the IL-4 / INF-γ of criterion (3), it can be determined that the sensitizing test substance may have skin sensitizing properties rather than respiratory sensitizing properties.

[0089] The aforementioned criteria are preferably set based on the amount of cytokine markers detected by a method under the same conditions except for the use of the sensitizing test substance.

[0090] (Variation 2-2) The protein amount of the cytokine marker may be, for example, a measured value of the actual amount of protein, an equivalent measured value, or a corrected value thereof.

[0091] The measured value corresponding to the actual amount may be, for example, the luminescence intensity obtained by an analytical method such as ELISA, or the relative value of the luminescence intensity. The correction value may be, for example, a correction value for the actual amount, or a correction value for the measured value corresponding to the actual amount.

[0092] Preferably, the protein amount in the judgment criteria and the protein amount when the test substance is used are the same type of measured value or corrected value.

[0093] (Variations 2-3) The cytokine marker amount in the aforementioned judgment criterion (2) may be, for example, the cytokine marker amount determined from the results of adding one type of respiratory non-sensitizing substance, but it is preferable that it be the cytokine marker amount determined from the results of using two or more types of respiratory non-sensitizing substances. Specifically, it is preferable to use two or more types of respiratory non-sensitizing substances and determine the cytokine marker amount from the average of the results of adding each of them. (2) Cytokine marker levels when a respiratory non-sensitizing substance that does not cause respiratory sensitization is added to the three-dimensional co-culture system.

[0094] The cytokine marker amount in the judgment criterion (3) above may be, for example, the cytokine marker amount determined from the results of adding one type of respiratory sensitizing agent, but it is preferable that it be the cytokine marker amount determined from the results of using two or more types of respiratory sensitizing agents. Specifically, it is preferable that the cytokine marker amount is determined from the average of the results of adding two or more types of respiratory sensitizing agents. (3) Cytokine marker levels when a sensitizing substance exhibiting respiratory sensitization is added to the three-dimensional co-culture system.

[0095] <Screening methods for respiratory sensitization inhibitors> The present invention provides a screening method for respiratory sensitization inhibitors, which involves using a respiratory sensitization-inducing substance involved in inducing sensitization and an inhibitory test substance that is the target for determining its inhibitory effect on respiratory sensitization. Specifically, the screening method of the present invention involves adding the respiratory sensitization-inducing substance and the inhibitory test substance to the bronchial epithelial cell layer instead of the sensitizing test substance, performing the respiratory sensitization detection method of the present invention, and evaluating the respiratory sensitization inhibitory ability of the inhibitory test substance by comparing the expression level of the gene marker in the absence of the inhibitory test substance with the expression level of the gene marker in the presence of the inhibitory test substance. The screening method for respiratory sensitization inhibitors of the present invention can also be described, for example, as a method for evaluating respiratory sensitization inhibitors.

[0096] The screening method of the present invention can be carried out in the same manner as, for example, the respiratory sensitization detection method of the present invention, except that the respiratory sensitization-inducing substance and the inhibitory test substance are added to the bronchial epithelial cell layer, and the description of the respiratory sensitization detection method of the present invention can be incorporated.

[0097] The screening method of the present invention includes, for example, the steps of adding the respiratory sensitization-inducing substance and the inhibitory test substance to the bronchial epithelial cell layer of the three-dimensional co-culture system and incubating them; detecting the mRNA expression of a gene marker in the dendritic cell layer of the three-dimensional co-culture system; and determining the inhibitory ability of the inhibitory test substance on respiratory sensitization based on the mRNA expression level of the gene marker.

[0098] The determination step can be performed, for example, by comparing the mRNA expression level when the respiratory sensitization-inducing substance and the inhibitory test substance are added with the mRNA expression level when the respiratory sensitization-inducing substance is not added. That is, if the mRNA expression level when the respiratory sensitization-inducing substance is not added is smaller than the mRNA expression level when the respiratory sensitization-inducing substance and the inhibitory test substance are added, it can be determined that the inhibitory test substance has inhibitory ability against sensitization.

[0099] The screening method of the present invention further includes, for example, After the incubation step, a step of collecting the dendritic cell layer from the three-dimensional co-culture system, The step of adding antigen-specific T cells to the dendritic cell layer and incubating it in the presence of antigen-specific T cells, A process for detecting cytokine markers in cell supernatant, The process includes a step of determining the inhibitory ability of the inhibitory test substance to suppress respiratory sensitization based on the amount of the cytokine marker.

[0100] The determination step can be performed, for example, by comparing the amount of cytokine markers when the respiratory sensitization-inducing substance and the inhibitory test substance are added with the amount of cytokine markers when the respiratory sensitization-inducing substance is not added. That is, if the amount of cytokine markers when the respiratory sensitization-inducing substance is not added is smaller than the amount of cytokine markers when the respiratory sensitization-inducing substance and the inhibitory test substance are added, it can be determined that the inhibitory test substance has inhibitory ability against sensitization.

[0101] <Method for producing the composition> The present invention provides a method for producing a bioadministered composition, characterized in that the respiratory sensitization properties of a test substance are confirmed using the respiratory sensitization detection method of the present invention, and the test substance that does not induce sensitization is used as a component of the bioadministered composition for production.

[0102] The present invention is characterized by confirming the safety of the components constituting a composition for biological administration by the respiratory sensitization detection method of the present invention, and no other conditions or steps are limited.

[0103] In the present invention, the test substance may be, for example, a candidate active ingredient that exhibits the desired effect of the bioadministered composition, or it may be any other additive component.

[0104] The aforementioned compositions for biological administration are not limited in any way and include, for example, pharmaceuticals, quasi-drugs, food and beverages, and cosmetics. [Examples]

[0105] Next, examples of the present invention will be described. However, the present invention is not limited to the following examples. Commercial reagents were used according to their respective protocols unless otherwise specified. [Examples]

[0106] A DC co-culture system was constructed, and sensitizing substances were evaluated.

[0107] (1)Cell culture The human bronchial epithelial cell line BEAS-2B (CRL-9609) was purchased from the American Type Culture Collection. For culturing BEAS-2B, Eagle's Minimum Essential Medium MEM (Gibco) containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin (Invitrogen) was used (hereinafter referred to as the BEAS-2B medium). Culture conditions were 5% CO2, 95% air, and 37°C. BEAS-2B cells were detached from plates with trypsin and subcultured twice weekly.

[0108] As the aforementioned monocyte cell line (c-MYC / BMI1 / BCL-2), the CD14-ML cell line, constructed based on the aforementioned paper (Haruta et al., PLoS One 2016), was obtained from Mycan Technologies. For culturing CD14-ML, αMEM medium (Thermo Fisher Scientific) containing 20% ​​FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin was used as the CD14-ML basic medium. CD14-ML was subcultured twice a week using the aforementioned basic medium.

[0109] For culturing immature dendritic cells (hereinafter referred to as immature DCs) derived from CD14-ML, the CD14 basic medium described above was used. Furthermore, for the co-culture system described later, the CD14-ML basic medium (αMEM medium) containing 50 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF), 50 ng / mL macrophage colony-stimulating factor (M-CSF), and 100 ng / mL interleukin-4 (IL-4) was used. This medium is hereinafter referred to as GM-CSF / M-CSF / IL-4 medium (50 / 50 / 100). Unless otherwise specified, the final concentrations of the components in each medium are as described above; if different, they are indicated separately.

[0110] Mycoplasma testing was performed using a commercially available kit (e-MycoTMVALiD Mycoplasma PCR Detection Kit, iNtRON Biotechnology, Inc.).

[0111] (2) Reagents Human recombinant GM-CSF, M-CSF, and IL-4 were commercially available (BioLegend). The substances evaluated included 11 skin-sensitizing substances and 9 respiratory-sensitizing substances, as well as 5 non-sensitizing substances, as listed below. (Skin sensitizer group) 2,4-Dinitrochlorobenzene (DNCB) 1,4-Benzoquinone (BQ) 4-Nitrobenzylbromide (NBB) 2-Mercaptobenzothiazole (MBT) Isoeugenol (IEU) Eugenol (EU) Phenyl benzoate (PB) Ethylene glycol dimethacrylate (EGDM) Butyl glycidyl ether (BGE) Oxazolone (OXA) Formaldehyde (FA) (Respiratory sensitizer group) Trimellitic anhydride (TMA) Hexamethylene diisocyanate (HDI) Chloramine T (Ch-T) Piperazine (PZA) Hexahydrophthalic anhydride (HHPA) Toluene diisocyanate (TDI) 8 Propylene glycol Glutaraldehyde (GA) Methyltetrahydrophthalic anhydride (MTHPA) Orthophthaldialdehyde (OPA) (Non-sensitizing substance group) 4-Aminobenzoic acid (ABB) a-Isomethylionone (AIM) Benzyl benzoate (BB) n-Hexane (HX) Isopropanol (IP)

[0112] (3) Preparation of DC co-culture system For the DC co-culture system, commercially available three-dimensional culture kits were used, including a standard bottomed well plate (6-well plate or 12-well plate) with the bottoms of the wells sealed, a 12-well insert with a scaffold membrane at the bottom (Alvetex® Scaffold 12-well insert, Reprocell), and a 24-well plate with a scaffold membrane at the bottom (Alvetex® Scaffold 24-well plate, Reprocell). The 12-well insert containing the scaffold membrane, or the 24-well plate containing the scaffold membrane, were washed in the following order: ethanol, phosphate-buffered saline, and culture medium (the basic medium according to the cell type).

[0113] The 12-well insert containing the scaffold membrane after washing was placed in the bottomed 6-well plate. Then, BEAS-2B was suspended in the BEAS-2B culture medium, and 100 μL (2.5 × 10) of the suspension was added. 6 Cells were seeded in the center of the scaffold membrane within the insert and left for 4-6 hours to allow the cells to adhere to the scaffold membrane. After this period, 5 mL of fresh BEAS-2B culture medium was added to the insert placed in the bottomed 6-well plate, and the cells were cultured for 2 days.

[0114] CD14-ML cultured in the αMEM medium (Thermo Fisher Scientific) further containing 100 ng / mL GM-CSF and 100 ng / mL M-CSF was suspended in the GM-CSF / M-CSF / IL-4 medium (50 / 50 / 100) to prepare a suspension. 100 μL of the suspension (4 × 10⁶) was placed in the center of the scaffold membrane in a freshly washed 24-well plate. 5 The cells were seeded and left for 4-6 hours to adhere to the scaffold membrane. After this period, 0.3 mL of fresh GM-CSF / M-CSF / IL-4 medium (50 / 50 / 100) was added to the scaffold membrane containing the cells in the 24-well plate, and the cells were cultured for 2 days to prepare immature dendritic cells (hereinafter referred to as immature DCs) derived from CD14-ML.

[0115] After culturing, the BEAS-2B layer, along with the scaffold membrane, was peeled off from one insert, and the immature DC layer, along with the scaffold membrane, was peeled off from the other insert. The scaffold membrane of the immature DC layer and the scaffold membrane of the BEAS-2B layer were then stacked to form a cell stack. In the cell stack, the lower layer was the immature DC layer including the scaffold membrane, and the upper layer was the BEAS-2B layer including the scaffold membrane. The cell stack was then set up as shown in Figure 1. Figure 1 is a cross-sectional view showing a schematic of the DC co-culture system. As shown in Figure 1, the cell stack 10 of the lower layer 11 and the upper layer 12 was placed at the bottom of a new insert 20. The insert 20 was then placed in each well 30 of a bottomed 12-well plate. Medium 40 was added to each well of the bottomed 12-well plate, and the cells were cultured overnight. The culture medium used was the αMEM medium (Thermo Fisher Scientific) further containing GM-CSF (final concentration 10 ng / mL), M-CSF (final concentration 10 ng / mL), and IL-4 (final concentration 20 ng / mL). This medium was hereafter referred to as GM-CSF / M-CSF / IL-4 medium (10 / 10 / 20). After culturing, the medium was removed from the bottomed 12-well plate. These cell stacks were used as a DC co-culture system for the following evaluations.

[0116] (4) Stimulation of DC co-culture systems with sensitizing agents Eleven evaluation samples were prepared from the group of skin-sensitizing substances, nine evaluation samples from the group of respiratory-sensitizing substances, and five evaluation samples from the group of non-sensitizing substances. The evaluation samples were prepared by one of the following methods, depending on the chemical properties of the evaluation substances, which are the sensitizing and non-sensitizing substances. Specifically, the evaluation substance was dissolved in DMSO, and then MEM containing 10% FBS was added so that the final concentration of DMSO was 10% (v / v) to prepare the evaluation sample. MEM containing 10% FBS, which contained only 10% (v / v) DMSO, was used as a control.

[0117] Stimulation was performed in the DC co-culture system as follows: Specifically, the evaluation sample was added to five locations on the upper surface of the cell stack in each insert of the bottomed well plate. 5 μL of the same evaluation sample was added to each of the five locations of the cell stack. After addition, it was left to stand for 20 minutes, and then 2.5 mL of αMEM medium (Thermo Fisher Scientific) was added to each well of the bottomed well plate, and stimulation with the evaluation sample was performed for 8 hours. For the control, instead of the evaluation sample containing the sensitizing substance, MEM containing 10% FBS with a final concentration of 10% (v / v) DMSO was added in the same manner, and it was left to stand for 8 hours. The concentrations of the evaluation substance in the evaluation sample are shown in Table 1 below. The concentration of each sensitizing substance among the evaluated substances was set to the optimal concentration determined in advance for each, and each non-sensitizing substance was basically added to a maximum concentration of 2%. If the HPRT expression level fell to one-tenth or less of that when MEM (control) containing 10% FBS and 10% (v / v) DMSO was added, the addition concentration was reduced to less than 2%.

[0118] After 8 hours of stimulation, the lower layer (including the scaffold membrane) was detached from the cell stack and collected. In the lower layer of the cell stack, immature dendritic cells differentiate into mature cells upon stimulation with the evaluation sample; therefore, the lower layer after stimulation is hereinafter referred to as the mature DC layer. To remove the evaluation sample (the sensitizing substance), the mature DC layer, along with the scaffold membrane, was washed three times with 2 mL of phosphate-buffered saline (PBS). After washing, the mature DC layer, along with the scaffold membrane, was immersed in a commercially available cell lysis reagent (product names Buffer RLT, Qiagen) to extract total RNA and obtain a total RNA sample.

[0119] (5) Real-time RT-PCR cDNA was prepared from the aforementioned total RNA samples, and real-time RT-PCR of target genes was performed on these cDNA samples using commercially available reagents (product name SYBR Premix Ex Taq II, Takara Bio Inc.) and equipment (product name Thermal Cycler Dice Real Time System, Takara Bio Inc.). To standardize mRNA, the HPRT (hypoxanthine phosphoribosyltransferase) gene was also analyzed as a housekeeping gene. The relative expression of PCR products was determined by the ΔΔCt method to compare the expression of target genes with the expression of HPRT mRNA. RNA samples with HPRT mRNA expression levels of 1 / 10 or less compared to the control without the evaluation sample were not used in the statistical analysis in the next section.

[0120] (6) Statistical analysis Relative expression results are shown as mean ± standard error (SE). Statistical analysis used the receiver-operating characteristic (ROC) curve and the area under the curve to evaluate the cutoff level between the two groups. P < 0.05 was considered statistically significant.

[0121] (7) Enhancement of gene marker expression These results are shown in Figure 2. These graphs show the relative expression of mRNA of specific genes for the skin sensitizing substance group and the respiratory sensitizing substance group. In each graph in Figure 2, the vertical axis represents the relative expression of the specific gene when the expression level in the control without the evaluation sample is set to 1, and shows (expression level of mRNA of the specific gene) / (expression level of HPRT mRNA, a housekeeping gene).

[0122] Figure 2A shows the relative expression of CD80 mRNA on the left and the relative expression of CD86 mRNA on the right. In Figure 2A, the horizontal line in the middle of the plot represents the mean value. As shown in Figure 2A, both C80 mRNA and CD86 mRNA showed relative expression levels greater than 1, confirming that stimulation occurred.

[0123] Figure 2B shows, on the left, the relative expression of TNFSF4 (OX40 ligand) mRNA; in the middle, the ratio of the relative expression of TNFSF4 mRNA to the relative expression of CD80 mRNA shown in Figure 2A; and on the right, the ratio of the relative expression of TNFSF4 mRNA to the relative expression of CD86 mRNA shown in Figure 2A. In Figure 2B, the horizontal line in the middle of the plot represents the mean value, and the dotted line represents the threshold value, which will be discussed later. In Figure 2B, * indicates P < 0.05 (the same applies to other figures). As shown in Figure 2B, in all graphs, the relative expression of TNFSF4 mRNA was significantly increased when a respiratory sensitizer was added compared to when a skin sensitizer was added. These results confirm that TNSF4 mRNA can serve as a genetic marker for respiratory sensitizers even when used alone. Furthermore, in each graph in Figure 2B, the values ​​shown by the dotted lines can be set as thresholds (cutoff values) for distinguishing between skin-sensitizing substances and respiratory-sensitizing substances. If the value is greater than this cutoff value, it is highly likely to be a respiratory-sensitizing substance, and if it is below this cutoff value, it is highly likely to be a skin-sensitizing substance rather than a respiratory-sensitizing substance.

[0124] Figure 2C shows, on the left, a graph of the relative expression of CRLF-2 mRNA, and on the right, a graph showing the ratio of the relative expression of CRLF-2 mRNA to the relative expression of CD80 mRNA shown in Figure 2A. In Figure 2C, the horizontal line in the middle of the plot represents the mean value. CRLF-2 is one of the subunits of TSLP-R. As shown in Figure 2C, in both graphs, the relative expression of CRLF-2 mRNA tended to increase when respiratory sensitizers were added compared to when skin sensitizers were added. These results suggest that CRLF-2 mRNA alone, or in combination with other genetic markers such as TNFSF4, may serve as genetic markers for respiratory sensitizers.

[0125] Figure 2D shows the relative expression of IL-7Rα mRNA on the left, the ratio of the relative expression of IL-7Rα mRNA to the relative expression of CD80 mRNA shown in Figure 2A in the middle, and the ratio of the relative expression of IL-7Rα mRNA to the relative expression of CD86 mRNA shown in Figure 2A on the right. In Figure 2D, the horizontal line in the middle of the plot represents the mean value. IL-7Rα is one of the TSLP-R subunits. As shown in Figure 2D, in all graphs, the relative expression of IL-7Rα mRNA tended to increase when respiratory sensitizers were added compared to when skin sensitizers were added. These results suggest that IL-7Rα mRNA alone, or in combination with other genetic markers such as TNFSF4, may serve as genetic markers for respiratory sensitizers.

[0126] Figure 2E shows a graph on the left illustrating the relative expression of IL-17RB mRNA, and a graph on the right illustrating the ratio of the relative expression of IL-17RB mRNA to the relative expression of CD80 mRNA shown in Figure 2A. In Figure 2E, the horizontal line in the middle of the plot represents the mean value. IL-17RB is one of the subunits of IL-25-R. As shown in Figure 2E, in both graphs, the relative expression of IL-17RB mRNA tended to increase when respiratory sensitizers were added compared to when skin sensitizers were added. These results suggest that IL-17RB mRNA alone, or in combination with other genetic markers such as TNFSF4, may serve as genetic markers for respiratory sensitizers.

[0127] Figure 2F shows the relative expression of ST2 mRNA on the left, the ratio of the relative expression of ST2 mRNA to the relative expression of CD80 mRNA shown in Figure 2A in the middle, and the ratio of the relative expression of ST2 mRNA to the relative expression of CD86 mRNA shown in Figure 2A on the right. In Figure 2F, the horizontal line in the middle of the plot represents the mean value. ST2 is one of the subunits of IL-33-R. As shown in Figure 2F, in all graphs, the relative expression of ST2 mRNA tended to increase when respiratory sensitizers were added compared to when skin sensitizers were added. These results suggest that ST2 mRNA alone, or in combination with other genetic markers such as TNFSF4, may serve as a genetic marker for respiratory sensitizers.

[0128] (8) Evaluation of respiratory sensitization based on the expression behavior of gene markers Using the three relative expression cutoff values ​​shown in Figure 2B below as criteria, sensitization was determined for the aforementioned 11 skin-sensitizing substances, 9 respiratory-sensitizing substances, and 5 non-sensitizing substances. These results are shown in Tables 1 to 4 below. In Tables 1 to 4 below, "N, Negative" indicates that the relative expression is below the cutoff value, and "P, Positive" indicates that the relative expression is above the cutoff value.

[0129] In Table 4, Sensitivity, Specificity, Accuracy, and Balanced Accuracy were calculated as follows. Sensitivity = [Rp / (Rn+Rp)] × 100 Rn: Number of TNFSF4-negative substances in respiratory sensitizers Rp: Number of TNFSF4-positive substances in respiratory sensitizers Specificity=[Sn / (Sn+Sp)]×100 Sn: Number of TNFSF4 negative substances in skin sensitizing agents Sp: Number of TNFSF4-positive substances in skin sensitizing agents Accuracy=[(Sn+Rp) / A]×100 Sn: Number of TNFSF4 negative substances in skin sensitizing agents Rp: Number of TNFSF4-positive substances in respiratory sensitizers A: Total number of sensitizing substances Balanced Accuracy = (Sensitivity + Specificity) / 2

[0130] [Table 1]

[0131] [Table 2]

[0132] [Table 3]

[0133] [Table 4]

[0134] As shown in Tables 1 to 4 above, the evaluation of various chemical substances using the evaluation criteria set in Figure 2B revealed that respiratory sensitizers and skin sensitizers could be distinguished with extremely high accuracy. Furthermore, the correlation between sensitivity and specificity was found to be P = 0.0167. [Examples]

[0135] A two-step DC / T cell co-culture system was prepared, and sensitizing substances were evaluated.

[0136] (1) Preparation of alloantigen-specific Th2 cell lines The cell line prepared in Example 3, described later, was used.

[0137] (2) Stimulation by sensitizing agents in DC co-culture systems A DC co-culture system was prepared using the same method as in Example 1, and stimulation with a sensitizing substance was performed. Specifically, the DC co-culture system was prepared in the same manner as in Example 1(4), and the evaluation sample was added to five locations on the upper surface of the cell stack in each insert of the bottomed 12-well plate. 5 μL of the same evaluation sample was added to each of the five locations of the cell stack, and it was left to stand for 20 minutes after addition. Then, 2.5 mL of αMEM medium (Thermo Fisher Scientific) was added to each well of the bottomed 12-well plate, and it was left to stand for a further 12 to 18 hours to stimulate with the evaluation sample. For the control (10% DMSO), MEM containing 10% FBS, which contained only 10% (v / v) DMSO at a final concentration, was added in the same manner, and it was left to stand for 12 to 18 hours.

[0138] (3) Preparation of a 2-step DC / T co-culture system After standing for 12-18 hours as described above, the cell stack in the insert was washed three times with 2 mL of phosphate-buffered saline (PBS). The BEAS-2B layer, which includes the scaffold membrane and is the upper layer, was peeled off and removed from the cell stack in the insert. Then, the insert, which still contained the mature DC layer stimulated with the evaluation sample, was transferred to a new bottomed 24-well plate. On top of this mature DC layer, 100 μL (4 × 10) of the suspension of the alloantigen-specific Th2 cell line prepared in (1) was placed. 5 Cells were added and allowed to stand for 24 hours. The suspension was prepared using RPMI medium containing 10% FBS.

[0139] The control (mature DC(-)) was prepared as follows: The cell-free scaffold membrane placed on the bottom of the washed 24-well plate was transferred to the wells of a new bottomed 24-well plate. Then, 100 μL (4 × 10) of the suspension of the alloantigen-specific Th2 cell line prepared in (1) was placed on top of this scaffold. 5 Cells were added and left to stand for 24 hours.

[0140] (4) ELISA After the aforementioned 24-hour incubation period, cytokine detection by ELISA and cell viability were measured as follows.

[0141] First, cytokine detection was performed by ELISA after the 24-hour incubation period by collecting the supernatant from the insert.

[0142] Next, cell viability was measured using WST-8 as follows: After collecting the supernatant, 0.5 mL of RPMI medium containing 10% FBS was added to the insert containing the remaining cells, and then 25 μL of WST-8 reagent (product name Cell Counting Kit-8, Dojin Chemical Research Institute) was added and allowed to stand for 6 hours. Next, 50 μL of the supernatant was taken from the insert, transferred to a 96-well plate, and the absorbance at 450 nm was measured using a plate reader. The absorbance of the control (10% DMSO) was then set as 100% cell viability, and the cell viability was calculated as a relative value.

[0143] (5) Enhancement of cytokine production Some of these results are shown in Figures 3 and 4. Figure 3 shows the results of comparing the skin sensitizer NBB with the respiratory sensitizer PZA, and Figure 4 shows the results of comparing the skin sensitizer DNCB with the respiratory sensitizer TMA. The bars in each figure, from left to right, show the results for control (mature DC(-)), control (10% DMSO), skin sensitizer (four or three concentrations), and respiratory sensitizer (four or three concentrations).

[0144] Specifically, Figures 3A and 4A are graphs of cell viability. Figures 3B and 4B are graphs of cytokine production, a graph showing cytokine production corrected for cell viability, and a graph showing the relative value when the cytokine production corrected for the control (10% DMSO) is set to 1. In Figures 3B and 4B, for the sensitizing substance at different concentrations, the highest relative value (IL-4) is shown. high , IFN-γ high IL-2 high A star was placed next to it.

[0145] Figures 3C and 4C are graphs showing the ratio of the highest relative production values ​​for two types of cytokines produced by the same sensitizing agent. Specifically, the highest relative production value for IL-4 is shown. high ) and the highest relative production value of IL-2 (IL-2 high ) ratio (IL-4 high / IL-2 high ) a graph showing the highest relative production value of IL-4 (IL-4 high ) and the highest relative production value of IFN-γ (IFN-γ high ) ratio (IL-4 high / IFN-γ high This graph shows the relationship between IL-2 production and T cell activation. Since IL-2 production correlates with the degree of T cell activation, the production of each cytokine was divided by IL-2 production to correct for variations in the degree of T cell activation. IL-4 high / IFN-γ high The ratio indicates the proportion of Th2 reactions to Th1 reactions.

[0146] As shown in Figures 3C and 4C, when a respiratory sensitizer was added compared to when a skin sensitizer was added, the ratio of cytokine production, IL-4 / IL-2, increased significantly, and IL-4 / IFN-γ also showed an increasing trend. These results suggest that skin sensitizers and respiratory sensitizers can be distinguished by the ratio of cytokine production, IL-4 / IL-2 and IL-4 / IFN-γ.

[0147] (6) Assessment of respiratory sensitization based on cytokine production ratios IL-4 / IL-2 and IL-4 / IFN-γ Figure 5 shows the cytokine production results of the two-step DC / T co-culture system using the following five types (n=5) of skin sensitizing substances and six types (n=6) of respiratory sensitizing substances. Figure 5A is a graph showing the ratio of the production of specific cytokines to the control for the skin sensitizing substance group and the respiratory sensitizing substance group, with the vertical axis representing the ratio of cytokine production corrected for cell viability. The correction formula was as follows. The cytokines were IL-4, IFN-γ, or IL-2. The ratio of cytokine production corrected for cell viability = (cytokine production / cell viability) / (control cytokine production / control cell viability)

[0148] Furthermore, Figure 5B is a graph showing the ratio of the production of specific cytokines relative to the control for the skin sensitizing substance group and the respiratory sensitizing substance group, with the vertical axis showing the result of dividing the ratio of IL-4 production, corrected for cell viability, by the ratio of IL-2 production or IFN-γ production.

[0149] As shown in Figures 5A and 5B, compared to the case where a skin sensitizer was added, IL-4 / IL-2 levels significantly increased when a respiratory sensitizer was added, and IL-4 / IFN-γ also showed an increasing trend. These results suggest that IL-4 / IL-2 and IL-4 / IFN-γ can serve as cytokine markers for respiratory sensitizers.

[0150] Regarding IL-4 / IL-2, in the graph of Figure 5B, the value of 1.56 shown by the dotted line can be set as a threshold (cutoff value) for distinguishing between skin-sensitizing substances and respiratory-sensitizing substances. If the value is greater than this cutoff value, it is more likely to be a respiratory-sensitizing substance, and if it is below this cutoff value, it is more likely to be a skin-sensitizing substance rather than a respiratory-sensitizing substance.

[0151] Using the cutoff values ​​in Figure 5B as the criteria, the five skin-sensitizing substances and six respiratory-sensitizing substances mentioned above were evaluated. These results are shown in Tables 5 to 7 below. In Tables 5 to 7 below, "N, Negative" indicates that the relative expression is below the cutoff value, and "P, Positive" indicates that the relative expression is above the cutoff value.

[0152] In Table 6, Sensitivity, Specificity, Accuracy, and Balanced Accuracy were calculated as follows. Sensitivity = [Rp / (Rn+Rp)] × 100 Rn: Number of IL-4 / IL-2 negative substances in respiratory sensitizers Rp: Number of IL-4 / IL-2 positive substances in respiratory sensitizers Specificity=[Sn / (Sn+Sp)]×100 Sn: Number of IL-4 / IL-2 negative substances in skin sensitizing agents Sp: Number of IL-4 / IL-2 positive substances in skin sensitizing agents Accuracy=[(Sn+Rp) / A]×100 Sn: Number of IL-4 / IL-2 negative substances in skin sensitizing agents Sp: Number of IL-4 / IL-2 positive substances in respiratory sensitizers A: Total number of sensitizing substances Balanced Accuracy = (Sensitivity + Specificity) / 2

[0153] [Table 5]

[0154] [Table 6]

[0155] [Table 7]

[0156] As shown in Tables 5 to 7 above, the evaluation of various chemical substances using the evaluation criteria set in Figure 5B revealed that respiratory sensitizers and skin sensitizers can be distinguished with extremely high accuracy. [Examples]

[0157] The alloantigen-specific Th2 cell line in Example 2 was prepared as follows.

[0158] CD14-ML cells were first cultured overnight in a low-adsorption 24-well plate (Sumitomo Bakelite) using the GM-CSF / M-CSF / IL-4 medium (50 / 50 / 100). Then, 0.05 mL of LPS was added to achieve a final concentration of 500 ng / mL, and the cells were cultured overnight to prepare mature DC cells. The cultured cells were then collected in a 15 mL tube, and the tube was irradiated with 15 Gy of X-rays. These were designated as X-ray-treated mature DC cells. This series of steps is referred to as the preparation of X-ray-treated mature DC cells.

[0159] Meanwhile, human blood was collected, and peripheral blood-derived mononuclear cells were isolated using a reagent (product name Lympholyte-H, CEDARLANE). Then, antibody beads from the Naive CD4-Positive T Cell Isolation Kit II (product name Milteny Biotech) were reacted with the peripheral blood mononuclear cells, and naive CD4-positive T cells were isolated and purified using an isolation device (product name AutoMACS® pro, Milteny Biotech). The isolated naive CD4-positive T cells were added to the aforementioned X-ray-treated mature DC cells and cultured for two weeks. The culture medium used was RPMI medium containing 10% FBS with 50 ng / mL IL-4, 50 unit / mL IL-2, 1 μg / mL anti-IL-12 antibody, and 1 μg / mL IFN-γ antibody (Th2 differentiation medium containing IL-4 / IL-2 / anti-IL-12 antibody / anti-IFN-γ antibody), and half of the volume was replaced with fresh medium every 3 to 4 days.

[0160] At the end of the two-week culture period for the naive CD4-positive T cells, new X-ray-treated mature DC cells were prepared using the aforementioned X-ray-treated mature DC cell preparation process. The naive CD4-positive T cells, which had been cultured for two weeks, were again stimulated with these X-ray-treated mature DC cells and cultured for two weeks in the new Th2 differentiation medium. This process of preparing new X-ray-treated mature cells, stimulating the naive CD4-positive T cells with them, and culturing for two weeks was repeated to maintain an alloantigen-specific Th2 cell line.

[0161] Then, the alloantigen-specific Th2 cell line was limitably diluted and seeded into a 96-well plate. As described above, the X-ray-treated mature DC cells were added and stimulated every two weeks, and the cells were maintained in the Th2 differentiation medium containing IL-4 / IL-2 / anti-IL-12 antibody / anti-IFN-γ antibody. Then, cells from each well of the well plate in which cell proliferation was confirmed were cultured as alloantigen-specific Th2 clone cells.

[0162] The alloantigen-specific Th2 cell lines and alloantigen-specific Th2 clones prepared in this manner were stimulated with 50 ng / mL PMA, 500 ng / mL ionomycin, and 5 μg / mL Brefeldin A for 4 hours, and then stained with staining reagents (Pcific Blue-conjugated anti-CD3C antibody and APC-conjugated anti-CD4 antibody) for 15 minutes. After staining, the cells were fixed with fixation reagent (4% paraformaldehyde PBS, eBioscience) for 30 minutes. The fixed cells were further suspended in permeabilization buffer (product name Pemiabilization Buffer, eBioscience) and intracellular staining was performed with staining reagents (APC-Cy7-conjugated anti-IFN-γ antibody, PE-conjugated anti-IL-4 antibody, FITC-conjugated anti-IL-17A antibody) for 30 minutes. These cells were analyzed using a flow cytometer (product name FACS canto II, Becton Dickinson).

[0163] Figure 6 shows the flow cytometry results of the alloantigen-specific Th2 cell line. Figure 6 shows a plot of cell density by cytokine staining. From left to right, Figure 6 shows the CD3-positive / CD4-positive population, the IFN-γ-positive / IL-4-positive population, the IL-4-positive / IL-17A-positive population, and the IFN-γ-positive / IL-17A-positive population. As shown in Figure 6, it was confirmed that the alloantigen-specific Th2 cell line contains cells including Th1 (a cell population that produces only IFN-γ), Th0 (a cell population that produces both IFN-γ and IL-4), and Th2 (a cell population that produces only IL-4), as indicated by the arrows. From these results, it was found that alloreactive T cells usually tend to become a cell population that produces only IFN-γ, but by maintaining culture in the IL-4 / IL-2 / anti-IL-12 antibody / anti-IFN-γ antibody-containing Th2 differentiation medium, a cell population that produces IL-4 can be induced in this embodiment.

[0164] Although the present invention has been described above with reference to embodiments and examples, the present invention is not limited to the above embodiments and examples. Various modifications to the configuration and details of the present invention can be understood by those skilled in the art within the scope of the present invention. [Industrial applicability]

[0165] According to the present invention, the respiratory sensitization potential of a test substance can be determined by using a three-dimensional co-culture system of a dendritic cell layer derived from the monocyte cell line or the monocyte-like cell line and a bronchial epithelial cell layer, and by detecting the mRNA expression of a specific gene marker in the dendritic cell layer. [Explanation of symbols]

[0166] 10-cell stack 11 Lower layer 12 Upper layer 20 inserts 30 wells 40 culture medium

Claims

1. The process of preparing a three-dimensional co-culture system of dendritic cell layer and bronchial epithelial cell layer, A step of adding the test substance to the bronchial epithelial cell layer of the three-dimensional co-culture system and incubating it. A step of detecting mRNA expression of a gene marker in the dendritic cell layer of the three-dimensional co-culture system, and The process includes determining the respiratory sensitization potential of the test substance based on the mRNA expression level of the gene marker; In the preparation step described above, the dendritic cell layer is a layer of immature dendritic cells derived from a monocyte cell line or a monocyte-like cell line. The aforementioned determination step is a step of determining the mRNA expression level by comparing the detected mRNA expression level with the criteria for determining mRNA expression levels according to respiratory sensitization. The gene marker is at least one selected from the group consisting of TNFSF4, a subunit of the TSLP receptor, a subunit of the IL-25 receptor, and a subunit of the IL-33 receptor, or a combination thereof. A method for determining respiratory sensitization, characterized by the following features.

2. The TSLP receptor subunit is either CRLF2 or IL-7Rα. The IL-25 receptor subunit is IL-17RA or IL-17RB. The determination method according to claim 1, wherein the subunit of the IL-33 receptor is ST2 or IL-1RAP.

3. The aforementioned monocyte cell line, A cell line in which the c-MYC gene, BMI1 gene, and BCL-2 gene have been introduced into monocyte cells, or The determination method according to claim 1 or 2, wherein the cell line is one in which the c-MYC gene, the BMI1 gene, and the BCL-2 gene have been introduced into monocyte cells induced from induced pluripotent stem cells.

4. The determination method according to claim 1 or 2, wherein the monocyte-like cell line is an acute monocytic leukemia cell line.

5. The determination method according to any one of claims 1 to 4, wherein the monocyte cell line is a human monocyte cell line, and the monocyte-like cell line is a human monocyte-like cell line.

6. The determination method according to any one of claims 1 to 5, wherein in the three-dimensional co-culture system, the lower layer is the dendritic cell layer, the upper layer is the bronchial epithelial cell layer, and the test substance is added to the bronchial epithelial cell layer of the upper layer.

7. The aforementioned criteria are the criteria for determining respiratory non-sensitization. The criteria for determining respiratory nonsensitization is the mRNA expression level of the gene marker when a respiratory nonsensitizing substance is used. In the determination process described above, If the detected mRNA expression level is greater than the criteria for determining respiratory non-sensitization, the test substance is determined to be respiratory sensitizing. The determination method according to any one of claims 1 to 6, wherein the test substance is determined to be non-respiratory sensitizing if the detected mRNA expression level is equal to or lower than the criteria for determining respiratory non-sensitization.

8. The aforementioned criteria are the criteria for determining respiratory sensitization, The criterion for determining sensitization is the mRNA expression level of the gene marker when a respiratory sensitizing agent is used. The determination method according to any one of claims 1 to 7, wherein in the determination step, if the detected mRNA expression level is equal to or greater than the criteria for determining respiratory sensitization, the test substance is determined to be respiratory sensitizing.

9. In the process of preparing the three-dimensional co-culture system, Immature dendritic cells are prepared by stimulating the monocyte cell line or monocyte-like cell line with interleukin 4 using a culture medium containing GM-CSF and M-CFS. The determination method according to any one of claims 1 to 8, comprising preparing the immature dendritic cell layer by culturing the immature dendritic cells.

10. moreover, After the incubation step, a step of collecting the dendritic cell layer from the three-dimensional co-culture system, The step of adding antigen-specific T cells to the dendritic cell layer and incubating it in the presence of antigen-specific T cells, A process for detecting cytokine markers in cell supernatant, and The process includes determining the respiratory sensitization potential of the test substance based on the amount of the cytokine marker; The aforementioned antigen-specific T cells are protein antigen-specific T cells, The cytokine marker is at least one or a combination of multiple markers selected from the group consisting of IL-4, IFN-γ, and IL-2. The determination method according to any one of claims 1 to 9, wherein the determination step is a step of determining by comparing the detected cytokine marker amount with a determination criterion for the amount of cytokine marker according to respiratory sensitization.

11. The determination method according to claim 10, wherein the subset of T cells is at least one selected from the group consisting of Th0 cells, Th1 cells, and Th2 cells.

12. The determination method according to claim 10 or 11, wherein the protein antigen for the protein antigen-specific T cells is an alloantigen.

13. The determination method according to any one of claims 10 to 12, wherein the protein antigen for the protein antigen-specific T cells is at least one selected from the group consisting of pollen, dust mites, house dust, serum albumin, antibody immunoglobulin, and keratin.

14. The determination method according to any one of claims 10 to 13, wherein the detection of the cytokine marker amount is the detection of a protein.

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