Evaluation method of immune response to test substance of cell group

Abstract

To solve a problem that it is necessary to immobilize a T-cell via a membrane protein of the T-cell in a conventional method for evaluating immune response to a test substance of a cell group, and influence to a result of its inspection or evaluation cannot be eliminated.SOLUTION: An evaluation method of immune response of a cell group to a test substance has the steps of: immobilizing a cell membrane modifier constituted of polymers having a hydrophobic chain and a hydrophilic chain onto a substrate; introducing a calcium fluorescent indicator into the cell group; immobilizing the cell group onto the substrate via the cell membrane modifier; adding the test substance to the cell group; and detecting fluorescence derived from the calcium fluorescent indicator of the cell group by a detector. Further in the present invention, a large number of cells can be simultaneously imaged in a lump by using a device capable of capturing an image in a wide viewing angle.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method and a test kit for evaluating the immune response of a cell group to a test substance. [Background technology]

[0002] Animals, including humans, have an immune system that defends against foreign substances that invade the body by inactivating them, such as pathogens, toxins, and cancer cells. The immune system has two types of response: innate immunity, which is an initial response that nonspecifically recognizes and rapidly reacts to foreign substances, and antigen-specific immune responses (also called acquired immunity or adaptive immunity), which specifically recognize and react to foreign substances or parts of them as "antigens."

[0003] Antigen-specific immune responses involve immune cells such as T cells and B cells, which have a mechanism for remembering information about antigens they have encountered in the past and responding quickly when they encounter the same antigen again. Therefore, evaluating the antigen-specific immune responses of immune cells is useful for testing and researching diseases such as infectious diseases, cancer, allergies, and autoimmune diseases, as well as for evaluating drugs that act on the immune system as antigens, such as vaccines.

[0004] A representative method for evaluating an antigen-specific immune response of cells is known, which involves stimulating T cells or other immune cells with an antigen and detecting immune effector molecules (e.g., cytokines such as IFN-γ) secreted as a result of the immune cell response to the antigen. For example, the ELISpot (Enzyme-Linked ImmunoSpot) assay uses an antibody against an immune effector molecule bound to a solid surface to capture immune effector molecules secreted from cells cultured on the solid surface, and after removing the cells and washing the solid surface, visualizes the molecules using a labeled antibody, thereby counting the cells that secrete the molecules. The FluoroSpot assay is a method for evaluating antigen-specific immune responses using a mechanism similar to that of the ELISpot assay, except that a fluorescently labeled antibody is used to visualize the immune effector molecules, but multiple types of immune effector molecules can be detected simultaneously by using multiple labeled antibodies with different fluorescent wavelengths. The ELISpot assay and the FluoroSpot assay are highly sensitive and can be evaluated at the single cell level, and are therefore widely used in medical testing and pharmaceutical development. For example, they are used to test for the presence or absence of tuberculosis infection and to confirm the effectiveness of candidate vaccine materials.

[0005] On the other hand, the ELISpot assay and FluoroSpot assay require complicated operations and require proficiency in the work. Furthermore, cells need to be cultured until sufficient amounts of immune effector molecules are secreted for visualization, and the test time usually takes 1 to 2 days or more. Therefore, in cases where rapid testing is required for infectious diseases, or when a large number of candidate substances or samples need to be evaluated in drug development, a simpler and faster evaluation method to replace the ELISpot assay and FluoroSpot assay has been desired.

[0006] It is known that T cell receptors (TCRs) stimulated by antigen-MHC complexes immediately increase calcium concentration in T cells (Non-Patent Document 1). By adding a test substance to peripheral blood mononuclear cells (PBMCs) containing T cells from a subject and detecting the increase in intracellular calcium concentration, the number of T cells responsive to the test substance can be measured within a short period of time within a few minutes after stimulation with the test substance. In order to detect calcium concentration changes in each cell in order to further improve measurement accuracy, it is recommended to observe the cells before and after antigen stimulation or over time by immobilizing the cells during observation. A method of immobilizing cells and measuring them has also been reported in Non-Patent Document 2. According to Non-Patent Document 2, Poly-L-Lysine (PLL) is immobilized on an observation substrate, anti-Leucocyte Function Associated Antigen-1 (LFA-1) monoclonal antibody is immobilized via this PLL, and the immobilized antibody immobilizes T cells on the substrate via the LFA-1 molecules present on the surface of the T cells. However, the LFA-1 molecule is involved in the immune synapse formed by the binding of the TCR and antigen-MHC complex, and there are concerns that binding antibodies to this LFA-1 molecule on T cells may affect T cell function. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Gillian Grafton,Leanne Thwaite, Calcium channels in lymphocytes. Immunology. 2001 Oct; 104(2): 119-126. [Non-Patent Document 2] Nadia Anikeeva et. Al., Evaluating frequency and quality of pathogen-specific T cells. Nat. Commun. 2016 Oct 27;7:13264. Summary of the Invention [Problem to be solved by the invention]

[0008] Thus, although changes in intracellular calcium concentration are a promising indicator that can rapidly reflect the response of the cells to a test substance, in the past, observations were performed by immobilizing the cells via protein molecules on the cell surface, and it was not possible to eliminate the possibility that this could affect the evaluation results in medical tests and drug development. The object of the present invention is to provide a simple and rapid method and test kit for evaluating the antigen-specific immune response of a cell group, which solves the above problems. [Means for solving the problem]

[0009] Provided is a method for evaluating the immune response of a cell group to a test substance, comprising: a comparison of first autofluorescence information, which is autofluorescence information of a first sample obtained by adding the test substance to the cell group, with second autofluorescence information, which is autofluorescence information of a second sample that serves as a control for the first sample; Effect of the Invention

[0010] By applying a cell membrane modifying agent consisting of a polymer with hydrophobic and hydrophilic chains to a suitable density, the cell group can be quickly immobilized on the substrate, enabling observation over time without changes in cell position. This immobilization also has little effect on cell function. The immune response of the immobilized cell group to the test substance could be evaluated using an intracellular calcium fluorescent indicator and a fluorescence microscope. By using high-resolution wide-field imaging, a measurement system that can simultaneously capture images of many cells can also be used. Compared to prior art cell immobilization methods that use antibodies, in which cells are immobilized via cell membrane proteins whose expression levels can vary from cell to cell, the cell membrane modifying agent binds directly to the cell membrane, making it possible to immobilize any type of cell, including antigen-presenting cells and B cells other than T cells, in the same way as T cells, thereby providing a more suitable method for evaluating the interactions of various cells. [Brief description of the drawings]

[0011] [Figure 1] A conceptual diagram showing an example in which a group of cells immobilized on a substrate with a solid phase of a cell membrane modifying agent reacts specifically to a test substance and emits fluorescence derived from a calcium fluorescent indicator. (a) is a conceptual diagram of cells immobilized on a substrate, and (b) is a conceptual diagram of an enlarged dotted line part in (a). [Diagram 2] FIG. 1 is a conceptual diagram illustrating an embodiment of the present invention. [Diagram 3] FIG. 1 is a diagram showing a system configuration of a wide-field image acquisition device. [Figure 4] FIG. 4 is a diagram showing a functional configuration of a wide-field image acquisition device 1A section in FIG. [Diagram 5] FIG. 4 is a diagram showing a functional configuration of a wide-field image acquisition device 2A section in FIG. [Figure 6] FIG. 1 is a schematic diagram of image analysis in which information is extracted, plotted, and visualized using an image processing device from images for extracting cell regions and fluorescent images captured by a wide-field image acquisition device. [Figure 7] Bright field images of PBMCs immobilized on BAM-modified glass-bottom dishes. [Figure 8] 8 is a fluorescent image of the fixed cells shown in FIG. 7 before addition of calcium ionophore. [Figure 9] 8 is a fluorescent image of the fixed cells shown in FIG. 7 after addition of calcium ionophore. [Figure 10] The results of comparing the time course of the number of cells having a fluorescence intensity equal to or greater than an arbitrarily set threshold with or without antigen stimulation are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] More specific configuration examples are described below, but the present invention is not limited to the following methods.

[0013] As one embodiment, the present invention provides a method for evaluating an immune response of a cell group to a test substance, comprising the steps of: A step of immobilizing a cell membrane modifying agent composed of a polymer having a hydrophobic chain and a hydrophilic chain on a substrate; introducing a calcium fluorescent indicator into said cell population; immobilizing the cell population on the substrate via the cell membrane modifying agent; adding a test substance to the cell population; and detecting fluorescence from the calcium fluorescent indicator in the cell group with a detector; The present invention provides a method having the following structure:

[0014] 1(a) and (b) show examples of cell groups immune-responsive to a test substance. The cell groups in FIG. 1 include T cells 1000 and antigen-presenting cells 1001. A calcium fluorescent indicator 1007 is introduced into these cell groups. Each cell is immobilized on a substrate 1006 via a cell membrane modifying agent 1005. A test substance 1004 is presented on a major histocompatibility complex (MHC) 1003 on the antigen-presenting cells 1001. When the T cells 1000 have a T cell receptor (TCR) 1002 specific to the complex of the test substance 1004 and the major histocompatibility complex 1003, they bind to each other and the T cells 1000 are activated. As a result, the calcium ion concentration in the T cells 1000 increases, and the calcium fluorescent indicator 1007 emits fluorescence. This is detected by a detector (not shown) to evaluate the T cell immune response of the cell groups to the test substance.

[0015] A conceptual diagram of this embodiment is shown in Figure 2. That is, the method of this embodiment comprises the steps of immobilizing a cell membrane modifying agent on a substrate, introducing a calcium fluorescent indicator into a cell population, immobilizing the cell population on the substrate via the cell membrane modifying agent, adding a test substance to the cell population, and detecting the fluorescence derived from the calcium fluorescent indicator with a detector. The steps of immobilizing a cell membrane modifying agent on a substrate and introducing a calcium fluorescent indicator into the cell population may both be performed before immobilizing the cell population, and may be performed before or after these steps.

[0016] (Cell membrane modifying agents) The cell membrane modifying agent is composed of a polymer having a hydrophobic chain and a hydrophilic chain. The hydrophilic chain acts as an anchor to the cell membrane, and the hydrophilic group enhances water solubility. The hydrophobic chain is selected from a saturated or unsaturated hydrocarbon chain, a lipid and a complex lipid chain constituting the cell membrane, preferably selected from the group consisting of saturated or unsaturated hydrocarbon chains having 6 to 22 carbon atoms, and the most preferred hydrophobic chain is an oleyl group. The hydrophilic chain is at least one selected from the group consisting of proteins, oligonucleotides, polymers or copolymers of derivatives of glycolic acid, lactic acid and P-dioxane, oligopeptides, polypeptides, polyamides, polyalkylene glycols, and polysaccharides, and is preferably polyethylene glycol (PEG) (polyoxyethylene structure). The PEG chain may have a molecular weight of 1 to 10 KDa, and preferably has a molecular weight of 4 KDa. Furthermore, the hydrophilic chain may have a functional group, and preferably has an active ester group at the end of the hydrophilic chain.

[0017] A molecule represented by the following formula (1) can be preferably used as a cell membrane modifying agent. This molecule is commonly called Biocompatible Anchor for Membrane (BAM). [ka] In formula (1), X is a reactive group, and n is an integer of 20 to 200. Preferably, -X is a group represented by formula (2). [ka]

[0018] Specifically, as a cell membrane modifying agent, for example, a molecule represented by the following formula (3) (sometimes referred to as BAM-NHS in this specification) can be used. [ka] In formula (3), n is an integer of 20 or more and 200 or less.

[0019] The cell membrane modifying agent can fix the cells on the substrate without damaging them by inserting a hydrocarbon chain into the cell membrane of the cells in the cell group to become an anchor. In this embodiment, the cells of the cell group preferably include T cells, for example, PBMCs are used. However, T cells and PBMCs are floating cells, and if they are not fixed on the substrate, the cells move during observation, making observation difficult, but this can be prevented by using a cell membrane modifying agent. Furthermore, antigen presentation by antigen-presenting cells is necessary for T cell immune response, as shown in FIG. 1. To generate an immune response, it is preferable that the individual cells of the cell group are fixed in a state of contact with each other. This can be achieved by using a cell membrane modifying agent.

[0020] (Substrate bound to cell membrane modifying agent) The substrate is preferably made of a highly transparent material suitable for cell observation, and examples of the material that can be used include glass or transparent resins such as polystyrene, polycarbonate, polyethylene terephthalate, and acrylic. The cell membrane modifying agent can be bound to the substrate surface via a functional group, preferably an amino group, on the substrate surface. In order to provide the substrate surface with a functional group, at least one of a protein, a peptide, a silane coupling agent, and a polymer having a functional group can be bound to the substrate surface. An example of a protein that can be bound to the substrate to provide the substrate surface with a functional group is bovine serum albumin (BSA).

[0021] As an example of creating a substrate bound to a cell membrane modifying agent, BSA was physically adsorbed onto the glass portion of a glass-bottom dish, and BAM-NHS was successfully bound to the BSA bound to this substrate. When BSA is physically adsorbed onto the glass part of a glass-bottom dish, a BSA solution concentration of 1% or more is often used, but as long as BSA can be sufficiently bound, any BSA concentration can be used. When immobilizing PBMCs, PBMCs are characterized by their small cell diameter, and it was necessary to examine the BAM modification conditions suitable for PBMC immobilization. As a result, it was found that a BAM / PBS solution with a concentration range of 30 to 900 μM was added (10 μL) to a BSA-coated glass-bottom dish and left to react at room temperature for 15 minutes to form a cell membrane modifier binding substrate on which PBMCs can be immobilized. Furthermore, the BAM / PBS solution concentration suitable for immobilization at a cell density suitable for T cell evaluation is 100 to 900 μM, and preferably 300 μM.

[0022] (cell group) The cell population may contain cells to be measured, and preferably contains T cells. As a cell group that can be used in clinical evaluation, PBMCs collected from human blood can be used as they are, or PBMCs with an increased lymphocyte ratio can be used by pre-culturing PBMCs, removing adherent cells, and recovering non-adherent lymphocytes. Alternatively, a cell population with an increased proportion of mature T cells (CD3 positive cells) or CD8 positive T cells or CD4 positive T cells purified from human blood by existing methods can be used. Alternatively, a cell group including stem cells such as iPS cells, or cells induced from blood stem cells can also be used. When evaluating CD4 positive T cells, it is effective to also include antigen-presenting cells that have MHC class 2 molecules as a cell population. Other cell populations including non-human cells and cell lines, T cells, and T cell lines may also be used.

[0023] (Calcium fluorescent indicator) Any calcium fluorescent indicator can be used as long as the changes in intracellular calcium concentration can be observed with the desired detector or microscope, such as Fluo-4, Fura-2, Quin 2, Fura 8, Fluo 3, Indo 1, Rhod 2, Rhod 3, Cal-500, Cal-520, Cal-590, Cal-630, Cal-670, Cal-770, Cal Red R525 / 650 AM, etc.

[0024] (Test substance) The test substance may be an antigen, an antigen protein, an antigen peptide, a vaccine, or a pathogen, or a peptide derived from such a test substance. The MHC class 1 molecule bound to a peptide consisting of 8-10 amino acids derived from the test substance can stimulate the T cell receptor (TCR) of CD8 positive T cells. A peptide derived from the test substance that binds to a MHC class 1 molecule prepared in advance can be used as the test substance. Another type of test substance can be a test substance composed of a protein or polypeptide, which can be taken up into a cell, processed within the cell, and bound to an MHC molecule by the peptide derived from the test substance, and used for evaluation. Yet another type of test substance can be a test substance having antigen genetic information (e.g., mRNA vaccine, etc.). This test substance can be taken up into a cell, protein expression and processing can be performed within the cell, and bound to an MHC molecule, and used for evaluation. MHC class 2 molecules bound to peptides consisting of 12-24 amino acids derived from the test substance can stimulate the TCR of CD4 positive T cells. In the evaluation of CD4 positive T cells, peptides derived from the test substance that bind to MHC class 2 molecules prepared in advance can be used, and test substances composed of proteins or polypeptides or test substances containing antigen genetic information can also be used as described above. However, since MHC class 2 molecules are expressed only in antigen-presenting cells, the coexistence of cells expressing them is required.

[0025] (Detector) Any detector can be used as long as it can simultaneously measure the position, time, and fluorescence intensity information of multiple cells in a fixed cell group. For example, a general-purpose microscope such as an inverted fluorescence microscope, a confocal microscope, or a multiphoton excitation microscope for acquiring a fluorescence image can be used. Alternatively, an image acquisition device with a wider field of view than a normal microscope can be used. The wider field of view here means that the area range that can be photographed in one shot is preferably 1 to 220 mm 2 , more preferably 5 to 220 mm 2 For example, an image acquisition device using a digital camera can be used. The digital camera can be, for example, an EOS R5 (product name) manufactured by Canon Inc.

[0026] In this embodiment, there are cases where a large number of cells must be observed at once. For example, the number of T cells that respond to a test substance contained in PBMC is expected to be about 0.1 to 1%. If we assume that the number is 0.1%, then the number of T cells that respond to a test substance must be at least 10 5 By observing approximately 10 cells simultaneously, responses of approximately 100 T cells can be detected. 5 To observe about 10 cells simultaneously, a 50 mm 2 In one embodiment, a wide-field image acquisition device capable of observing an area of ​​about 100 nm can be preferably used.

[0027] (Configuration of wide-field image acquisition device) 3 shows an example of the overall configuration of an imaging analysis device 100, which is a detector that acquires fluorescence information over a wide field of view. The device acquires images of cells and analyzes the changes in fluorescence intensity of each cell by analyzing the acquired images. 3, the imaging analysis device 100 is configured by connecting an image acquisition device 1A and an image processing device 2A via an interface such as a cable 3A so as to be able to transmit and receive data. The method of connecting the image acquisition device 1A and the image processing device 2A is not particularly limited. For example, the image acquisition device and the image processing device may be connected via a LAN (Local Area Network) or wirelessly. The image acquisition device 1A acquires images of cells on a cell culture vessel placed on a mounting stage, and transmits the images to the image processing device 2A.

[0028] As shown in FIG. 4, the image acquisition device 1A is configured with an irradiation unit 11, an imaging unit 12, an image capture unit 13, a control unit 14, a communication I / F 15, an operation unit 16, etc., and each unit is connected via a bus 17. The irradiation unit 11 is configured with a light source filter for fluorescence observation and a white light source for cell morphology observation, and irradiates light onto the cultured cells placed on the mounting stage. The image capture unit 12 is configured with a lens, a filter, etc., and forms an image of transmitted light, reflected light, or fluorescence emitted from the cells on the cell culture vessel by the irradiated light. The image capture unit 13 is a camera equipped with a CMOS (Complementary MOS) sensor, etc., and captures an image formed on the image formation surface by the imaging unit to generate digital image data (image data of R, G, B) of the image. The control unit 14 is configured with a CPU (Central Processing Unit), RAM (Random Access Memory), etc., and executes various processes in cooperation with various programs installed inside. The communication I / F 15 transmits image data of the generated image to the image processing device 2A. In this embodiment, the image acquisition device 1A is provided with a bright field unit that combines an illumination means and an imaging means suitable for bright field observation, and a fluorescent unit that combines an illumination means and an imaging means suitable for fluorescent observation, and it is possible to switch between bright field and fluorescent observation by switching the units.

[0029] The image processing device 2A is a medical image processing device that analyzes the image transmitted from the image acquisition device 1A, calculates features that quantitatively indicate the expression level of a specific biological substance in cells on the cell culture container being observed, and outputs the calculated features. An example of the functional configuration of an image processing device is shown in Fig. 5. The image processing device 2A includes a control unit 21, an operation unit 22, a display unit 23, a communication I / F 24, a storage unit 25, etc., and each unit is connected via a bus 26. The control unit 21 is configured with a CPU (Central Processing Unit), RAM (Random Access Memory), etc., and executes various processes in cooperation with various programs stored in the storage unit 25, and generally controls the operation of the image processing device. For example, the control unit 21 executes image analysis processing (see FIG. 6) in cooperation with the programs stored in the storage unit 25, and realizes functions as an extraction means, a fluorescent spot extraction means, an area estimation means, a feature amount calculation means, and a determination means. The operation unit 22 is configured with a keyboard equipped with letter input keys, number input keys, and various function keys, etc., and a pointing device such as a mouse, and outputs a press signal of a key pressed on the keyboard and an operation signal by the mouse to the control unit 21 as input signals. The display unit 23 is configured to include a monitor such as a CRT (Cathode Ray Tube) or an LCD (Liquid Crystal Display), and displays various screens according to instructions of a display signal input from the control unit 21. In this embodiment, the display unit 23 functions as an output means for outputting the calculated feature amount. The communication I / F 24 is an interface for transmitting and receiving data between the image acquisition device 1A and other external devices. The communication I / F 24 functions as an input unit for the bright field image and the fluorescent image. In the present embodiment, the communication I / F 24 functions as an input unit. The storage unit 25 is configured, for example, with a hard disk drive (HDD) or a non-volatile semiconductor memory. As described above, various programs and various data are stored in the storage unit 25. For example, the storage unit 25 stores various data including a magnification table used in the image analysis process described later. Additionally, the image processing device 2A may be provided with a LAN adapter, a router, etc., and may be configured to be connected to an external device via a communication network such as a LAN.

[0030] The image processing device 2A in this embodiment performs analysis using the image for extracting a cell region and the fluorescent image transmitted from the image acquisition device 1A. A fluorescent image is an image obtained by irradiating a cell having a certain fluorescent activity with excitation light of a predetermined wavelength in the image acquisition device 1A to cause a fluorescent substance derived from an intracellular calcium indicator to emit light, and then enlarging and photographing the fluorescence through a cut filter having a wavelength equal to or greater than the light source wavelength. The image for extracting a cell region is an image from which individual cell regions can be extracted by image processing. Examples include a bright-field image obtained by magnifying and imaging in a bright field with the image acquisition device 1A, and a fluorescent image obtained by irradiating a fluorescent substance in a cell with excitation light of a specific wavelength to cause the fluorescent substance to emit light, and magnifying and imaging the fluorescence.

[0031] A flow chart for performing image analysis processing using the obtained image is shown in Fig. 6. The following processing in Fig. 6 is all performed within the image processing device 2A. First, in step P1, an image for cellular region extraction from the image acquisition device 1A is input by the communication I / F 24. Then, in step P2, a cellular region is extracted from the image for cellular region extraction, a labeling process is performed, and a label is assigned to each cell. Meanwhile, in step P3, the fluorescent image from the image acquisition device 1A is input by the communication I / F 24. If necessary, in step P4, the fluorescent image can be separated into RGB components and analyzed. In step P5, each pixel of the image for extracting a cell region is added to each pixel of the fluorescent image, and in the following step P6, the fluorescent color information in each cell region and the feature amount related to the cell region are calculated from this added image. Furthermore, in step P7, the feature amount calculated by the above processing is output for each cell. This output result can be displayed on the display unit 23 of the image processing device 2A.

[0032] (Bright-field image acquisition) A method for acquiring bright-field images to identify the position of cells involves irradiating the lens with a white light source of a visible light wavelength or a mixture of these wavelengths parallel to the optical axis of the lens, or at an angle that is not parallel but not perpendicular to the optical axis, and detecting the reflected light from the cells or the diffracted light due to birefringence, and the interference between them.

[0033] (Fluorescence image acquisition) The method of acquiring a fluorescent image involves irradiating a light source having a central wavelength of a monochromatic light ranging from ultraviolet to visible light as the fluorescence excitation light source wavelength at an angle parallel to the optical axis of the lens, or at an angle that is not parallel but not perpendicular to the optical axis, to excite the fluorescence of the sample, and the excited fluorescence is detected via a cut filter installed in front of or behind the lens on the observation side of the lens, etc. The wavelength cut filters for both the excitation light and the fluorescence observation cut filter are selected so that part of the excitation light source wavelength does not pass through the fluorescence cut filter on the observation side.

[0034] (Analysis / Visualization) The analysis and visualization method involves visualizing individual cell information, such as the brightness information of each color and cell size information, on one axis, as one-, two-, or three-dimensional graphs. For example, visualization can be done using histograms for one-dimensional data, or scatter plots for two- or three-dimensional data. Instead of histograms and scatter plots, information on individual cells can be visualized using probability density function plots such as kernel density estimation or heat maps.

[0035] (Brightness information) The luminance information is color information obtained by describing the obtained fluorescence information in a certain color space, and is a coordinate in the color space. As an example of the color space, the luminance information can be described using any of the R, G, and B information in the RGB color space, or the Lab color space or the HSV color space, but the luminance information may also be described using a color space other than these.

[0036] (Cell shape information) The cell shape information is information on parameters that can describe the shape of a cell. Examples include information on cell size, cell shape, and cell thickness, but other information that can be obtained from an image may also be used.

[0037] (Evaluation method) Upon addition of a test substance, the number of cells that emit fluorescence from the calcium fluorescent indicator is counted. If this number is equal to or exceeds a certain number or percentage, the cell group can be evaluated as having an immune response to the test substance. The number of cells that react to the test substance is counted by distinguishing cells that have a fluorescence intensity equal to or higher than a cutoff value set in the imaging field and counting the number of cells. The image processing and measurement used here can be analyzed using general-purpose image analysis software. As an example of the analysis, a method is used in which an arbitrary threshold is set in the differential fluorescent image obtained by subtracting the fluorescent image information before antigen stimulation from the fluorescent image information after an arbitrary time after antigen stimulation, and the number of corresponding cells is counted.

[0038] (Test kit) As a further embodiment, the present invention provides a test kit for evaluating the immune response of a cell population to a test substance, the kit including a substrate on which a cell membrane modifying agent composed of a polymer having hydrophobic and hydrophilic chains is immobilized, and a calcium fluorescent indicator. The cell membrane modifying agent may be a molecule represented by the following formula (1). The kit may include, as the test substance, any one selected from an antigen, an antigen protein, an antigen peptide, a vaccine, and a pathogen test substance. The kit may include, without being limited to any particular one, reagents, containers, etc. that are required when introducing the calcium fluorescent indicator into the cell population, when fixing the cell population to the substrate, or when detecting the fluorescence derived from the calcium fluorescent indicator. Furthermore, the kit may include a case for containing these reagents, containers, etc., and an instruction manual. [ka] In formula (1), X is a reactive group, and n is an integer of 20 or more and 200 or less. EXAMPLES

[0039] An embodiment of the present invention will now be described. In the following examples, a specific example is given in which PBMCs are used as a cell group to evaluate an immune response to a test substance, but the reagents and reaction conditions described in the following examples can be modified, and such modifications are within the scope of the present invention. Therefore, the following examples are intended to aid in understanding the present invention, and do not limit the scope of the present invention in any way.

[0040] Example 1 Preparation of BAM-modified glass-bottom dishes 200 μL of 1% BSA / PBS solution that had been sterilized by filtration was dropped onto the glass part of a 3.5 cm glass bottom dish (glass bottom is a circle with a diameter of 1.6 mm) and left to stand overnight in an incubator at 37°C. After washing the glass part three times with 200 μL of sterile water, the glass bottom dish was air-dried in a clean bench. 6 mg of BAM (Yuka Sangyo Co., Ltd., product number SUNBRIGHT OE-040CS) was added to 50 μL of DMSO to prepare a 30 mM BAM / DMSO solution. 10 μL of a solution (final concentration 300 μM) obtained by diluting the 30 mM BAM / DMSO solution with PBS at 1 / 100 was added to the BSA-coated glass bottom dish and left to stand at room temperature for 15 minutes. After washing three times with 200 μL of sterile water, the surface of the glass bottom dish was air-dried. This BAM-modified glass bottom dish was sealed with a desiccant and stored at 4°C until use.

[0041] The state of PBMC immobilized on this BAM-modified glass-bottom dish is shown in Figure 7. The immobilization method was as described in Example 2. In Figure 7, it can be seen that PBMCs were immobilized in a dense state at the BAM-modified position. In order to observe the reactivity of T cells to a test substance, it is necessary for antigen-presenting cells and T cells that recognize that antigen to have an opportunity to come into contact with each other, and so the PBMCs need to be immobilized densely enough that individual cells come into contact with each other. It was confirmed that the cells were immobilized to meet this condition. In addition, to confirm whether calcium-derived fluorescence could be observed using these immobilized cells, calcium ionophore (A23187) was added to a final concentration of 1 μg / mL, and fluorescence was confirmed in almost all of the immobilized PBMCs. Figure 8 shows the fluorescence image before the addition of calcium ionophore, and Figure 9 shows the fluorescence image after its addition. In this example, the fluorescent images were captured using an inverted microscope (TE200-U, manufactured by Nikon), a CCD camera (EM-CCD DIGITAL CAMERA / C9100-13, manufactured by Hamamatsu Photonics), and a filter set (excitation 450-490 DM 505 fluorescence 520).

[0042] Example 2 Preparation of Fluo-4 stained PBMC cells (1) Thawing and pre-culturing cells The cells used were PBMC (Peripheral Blood Mononuclear Cells) (manufactured by CTL). PBMCs frozen and stored in a liquid nitrogen tank were quickly thawed in a water bath at 37°C, and 10 mL of 1xCTL Anti-Aggregate Wash solution at 37°C was added while stirring. The cells were centrifuged at 300G for 8 minutes. The supernatant was removed, and 10 mL of 1xCTL Anti-Aggregate Wash (trademark) solution at 37°C was added again, and the cells were centrifuged at 300G for 8 minutes, after which the supernatant was removed. The cell pellet was suspended in 3 mL of CTL Test medium, and the entire amount was seeded in one well of a 6-well plate, and cultured overnight at 37°C in a 5% CO2 incubator. The 1x CTL Anti-Aggregate Wash solution was prepared by diluting CTL Anti-Aggregate Wash, 20x (manufactured by CTL) at 1 / 20 with RPMI 1640 medium, and the CTL Test medium was prepared by adding 1 / 100 of the amount of 200 mM L-glutamine solution.

[0043] (2) Fluo-4 staining of cells Floating PBMCs were collected from the PBMCs cultured overnight, 7 mL of PBS was added, and the cells were centrifuged at 300 g for 8 minutes. The supernatant was removed, the cells were suspended in 10 mL of PBS, and the number of cells was counted. The cell suspension was centrifuged again at 300 g for 8 minutes, the supernatant was removed, and the PBMCs were stained with Fluo-4 using Calcium Kit-Fluo4 (Dojindo Chemical, product number CS22) according to the manufacturer's manual. After staining, the cell concentration was 1 × 10 7 The cells were suspended in 37°C Probenecid-added recording medium (a mixture of 5 ml of Recording Medium (2X), 5 mL of pure water, and 50 μL of 250 mmol / l Probenecid, a component reagent of the Calcium Kit-Fluo4) to a concentration of 100 cells / mL (referred to as Fluo4-stained PBMCs).

[0044] (3) Preparation of Fluo-4-stained PBMC immobilized on a substrate with a cell membrane modifier 80 μL of Fluo4 stained PBMCs were added to the BAM-modified portion of the BAM-modified glass bottom dish and left to stand for 15 minutes. Approximately 200 μL of Probenecid-added recording medium was gently added, and the dish was left to stand in a 37°C, 5% CO2 incubator until measurement. The cells were fixed as shown in Figure 7.

[0045] Example 3 Calcium imaging For the fixed PBMCs stored in a CO2 incubator, the Probenecid-added recording medium was removed immediately before measurement, and 100 μL of Probenecid-added recording medium warmed to 37°C was newly added. Fluorescence observation of the fixed cells was performed using an inverted microscope (TE200-U, Nikon), a CCD camera (EM-CCD Digital Camera / C9100-13, Hamamatsu Photonics), and a filter set (excitation 450-490 nm, DM 505 nm, emission 520 nm or more). One fluorescence image was taken before adding the test substance, then 100 μL of the test substance solution was added, and fluorescence images were taken at any time thereafter. The test substance used here was 2X CMV pp65 (manufactured by CTL) (concentration: 10 nM). This test substance was confirmed in advance by the manufacturer in an ELISPOT assay to be a peptide that reacts very strongly with the PBMC used in this example.

[0046] Analysis method and results Analysis was performed using fluorescence images taken before and immediately after the addition of the test substance, and 100, 200, and 300 seconds after the addition of the test substance. The number of cells with average fluorescence intensity in the threshold range of 50 to 255 (number of positive cells) was counted in each image. The results are shown in Figure 10. When the test substance was added, a significant increase in the number of positive cells was observed over time. On the other hand, when the test substance was not added, no significant increase in the number of positive cells was observed. From these results, it was confirmed that the intracellular calcium concentration of the fixed PBMC increased within a few minutes due to the immune response to the stimulation by the addition of the test substance, and that the reactivity to the added test substance can be measured in a short period of time.

[0047] The embodiments of the present invention include the following configurations and methods. [Method 1] A method for evaluating an immune response of a cell group to a test substance, comprising the steps of: A step of immobilizing a cell membrane modifying agent composed of a polymer having a hydrophobic chain and a hydrophilic chain on a substrate; introducing a calcium fluorescent indicator into said cell population; immobilizing the cell population on the substrate via the cell membrane modifying agent; adding the test substance to the cell population; and detecting fluorescence from the calcium fluorescent indicator in the cell group with a detector; The method according to claim 1, [Method 2] The method according to method 1, wherein the cell membrane modifying agent has a saturated or unsaturated hydrocarbon chain having 6 to 22 carbon atoms as the hydrophobic chain. [Method 3] The method according to method 1 or 2, wherein the cell membrane modifying agent has polyethylene glycol as the hydrophilic chain. [Method 4] The method according to method 1, wherein the cell membrane modifying agent is represented by formula (1). [ka] In formula (1), X is a reactive group, and n is an integer of 20 or more and 200 or less. [Method 5] The method according to Method 4, wherein -X in formula (1) is a group represented by formula (2). [ka] [Method 6] 6. The method according to any one of methods 1 to 5, wherein said cell population is PBMCs (peripheral blood mononuclear cells). [Method 7] The method according to any one of methods 1 to 6, wherein the cell population is floating lymphocytes in PBMCs (peripheral blood mononuclear cells). [Method 8] 8. The method according to any one of methods 1 to 7, wherein the test substance is an antigen, an antigenic protein, an antigenic peptide, a vaccine, or a pathogen. [Method 9] A method according to any one of methods 1 to 8, wherein the test substance is an antigen peptide capable of binding to an MHC class 1 molecule. [Method 10] The detector is 1 to 220 mm2 10. The method according to any one of methods 1 to 9, wherein the camera is capable of photographing an area of ​​in one shot. [Configuration 1] A test kit for evaluating the immune response of a cell group to a test substance, the kit comprising a substrate on which a cell membrane modifying agent composed of a polymer having hydrophobic and hydrophilic chains is immobilized and a calcium fluorescent indicator. [Configuration 2] The kit according to embodiment 1, further comprising a test substance selected from the group consisting of an antigen, an antigen protein, an antigen peptide, a vaccine, and a pathogen test substance. [Configuration 3] The kit according to embodiment 1 or 2, wherein the cell membrane modifying agent is represented by formula (1). [ka] In formula (1), X is a reactive group, and n is an integer of 20 or more and 200 or less. [Explanation of symbols]

[0048] 1000 T cells 1001 Antigen presenting cells 1002 T cell receptor (TCR) 1003 Major histocompatibility complex (MHC) 1004 Test substance 1005 Cell membrane modifying agents 1006 Board 1007 Calcium fluorescent indicator 100 Imaging analysis device 1A Image acquisition device 11 Irradiation unit 12 Imaging section 13 Imaging unit 14 Control section 15 Communication I / F 16 Control section 17 Bus 2A Image processing device 21 Control section 22 Control section 23 Display section 24 Communication I / F 25 Memory section 26 Bus 3A Cable

Claims

1. A method for evaluating the immune response of a group of cells to a test substance, A process of immobilizing a cell membrane modifier, which is composed of a polymer having hydrophobic and hydrophilic chains, onto a substrate. A step of introducing a calcium fluorescent indicator into the aforementioned cell group, A step of immobilizing the cell group onto the substrate via the cell membrane modifier, The steps of adding the test substance to the cell group, A step of detecting fluorescence from the aforementioned cell group, derived from the calcium fluorescent indicator, using a detector. A method of having.

2. The method according to claim 1, wherein the cell membrane modifier has a saturated or unsaturated hydrocarbon chain with 6 to 22 carbon atoms as the hydrophobic chain.

3. The method according to claim 1, wherein the cell membrane modifier has polyethylene glycol as the hydrophilic chain.

4. The method according to claim 1, wherein the cell membrane modifier is represented by formula (1). 【Chemistry 1】 In equation (1), X is a reactive group, and n is an integer between 20 and 200.

5. The method according to claim 4, wherein -X in formula (1) is the group shown in formula (2). 【Chemistry 2】

6. The method according to claim 1, wherein the cell group is PBMC (peripheral blood mononuclear cells).

7. The method according to claim 1, wherein the cell group is a suspension lymphocyte in PBMC (peripheral blood mononuclear cells).

8. The method according to claim 1, wherein the test substance is an antigen, an antigenic protein, an antigenic peptide, a vaccine, or a pathogen.

9. The method according to claim 1, wherein the test substance is an antigen peptide capable of binding to an MHC class 1 molecule.

10. The detector is 1 to 220 mm ２ The method according to any one of claims 1 to 9, wherein the camera is capable of capturing the area in a single shot.

11. A test kit for evaluating the immune response of a cell population to a test substance, comprising a substrate on which a cell membrane modifier composed of polymers with hydrophobic and hydrophilic chains is immobilized, and a calcium fluorescent indicator.

12. The kit according to claim 11, further comprising, as a test substance, any of the following selected from antigens, antigenic proteins, antigenic peptides, vaccines, and pathogens.

13. The kit according to claim 11 or 12, wherein the cell membrane modifier is represented by formula (1). 【Transformation 3】 In equation (1), X is a reactive group, and n is an integer between 20 and 200.

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