Evaluation method of immune response of cell populations to test substances

Abstract

To provide an evaluation method of immune response of cell populations to test substances capable of simply and swiftly evaluating immune responses in cell populations by solving the conventional problem that, in order to determine vaccine effectiveness, it takes a long time (24-48 hours) for inspection using the ELISpot assay in which human peripheral blood mononuclear cells (PBMCs) are mixed with antigens; however, the long time required for inspection not only results in poor throughput but also raises concerns that the properties of cells may change due to long-term culture.SOLUTION: The evaluation method of immune response of cell populations is an evaluation method of the immune response of cell populations to a test substance based on a comparison of first autofluorescence information which is the autofluorescence information of the first sample obtained by adding the test substance to the cell group and second autofluorescence information which is the autofluorescence information of the second sample as a control for comparison with the first sample.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a method for evaluating the immune response of a cell population to a test substance, an information method, an information processing device, an information processing program, and a system for evaluating the immune response of a cell population 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 phase surface to capture immune effector molecules secreted from cells cultured on the solid phase, and after removing the cells and washing the solid phase surface, visualizes the molecules using a labeled antibody to count the cells that secrete the molecules (Non-Patent Document 1). In addition, the FluoroSpot assay is a method for evaluating antigen-specific immune responses using a mechanism similar to that of the ELISpot, 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 (Non-Patent Document 2). 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. [Prior art documents] [Patent documents]

[0005] [Non-Patent Document 1] J.Immunol.Methods,128,65-73,1990 [Non-Patent Document 2] Cells,3(4),1102-1115,2014 Summary of the Invention [Problem to be solved by the invention]

[0006] As mentioned above, the ELISpot assay and FluoroSpot assay are excellent methods for evaluating antigen-specific immune responses of cells, but the operations are complicated and require skill. 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, when 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 is needed. For example, in the past, to determine the effectiveness of a vaccine, antigens were mixed with human peripheral blood mononuclear cells (PBMCs) and the test was performed over a long period of time, 24-48 hours, using an ELISpot assay. However, the long test time not only results in low throughput, but also raises concerns that the properties of the cells may change due to long-term culture.

[0007] An object of the present invention is to solve these problems and provide a simple and rapid method for evaluating the immune response of a cell group to a test substance. [Means for solving the problem]

[0008] The present inventors have searched for a cellular change that appears earlier than the secretion of immune effector molecules and can be easily detected after stimulating cells with an antigen. As a result, they have found that the autofluorescence of cells, that is, the fluorescence emitted from endogenous fluorescent substances possessed by the cells when the cells are irradiated with excitation light of a specific wavelength, changes shortly after the cells are stimulated with an antigen. Furthermore, the present inventors have found that this change correlates with the antigen-specific immune response of the cells, and have thus achieved the present invention.

[0009] Specifically, the present invention provides a method for evaluating the immune response of a cell group to a test substance, the method comprising comparing first autofluorescence information, which is autofluorescence information of a first sample to which the test substance has been added 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] When evaluating the immune response of a cell group to a test substance, results can be obtained in about one hour after adding the test substance to the cell group, allowing testing to be performed in a shorter time than conventional methods. Furthermore, the reactivity of a cell group to a test substance can be evaluated by a simple procedure of adding the test substance, leaving it to stand, and then acquiring images using an imaging analysis system. Since endogenous fluorescent substances are measured, no staining procedures for visualizing intracellular molecules are required, and measurements can be made without applying unnecessary stimuli to the cells. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram illustrating a cell group that specifically reacts to a test substance. [Diagram 2] FIG. 1 is a conceptual diagram illustrating an embodiment of the present invention. [Diagram 3] FIG. 1 is a diagram illustrating a case where an immune response is evaluated in an embodiment of the present invention. [Figure 4] FIG. 1 is a diagram showing a system configuration of an imaging analysis system. [Diagram 5] FIG. 5 is a diagram showing a functional configuration of an imaging analysis system 1A section in FIG. [Figure 6] FIG. 5 is a diagram showing a functional configuration of an imaging analysis system 2A section in FIG. [Figure 7] FIG. 1 is a schematic diagram of image analysis in which information is extracted, plotted, visualized, and clustered by an image processing device using images for extracting cell regions and autofluorescence images captured by an imaging analysis system. [Figure 8] This is an example of a bright-field image of PBMC cells taken using an imaging analysis system. [Figure 9] This is an example of an image of PBMC cells photographed using an imaging analysis system. [Figure 10]This is an example of the measurement of the FAD / NADH ratio calculated from the results of autofluorescence imaging one hour after test substances with different reactivities were added to PBMCs. [Figure 11] This is an example of the measurement of the FAD / NADH ratio 1 hour and 4 hours after adding a strongly reacting test substance. [Figure 12] This is an example of measurement of the change in the FAD / NADH ratio over time up to 24 hours after the addition of a strongly reacting test substance. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] An embodiment of the present invention provides a method for evaluating the immune response of a cell group to a test substance, based on a comparison of first autofluorescence information, which is autofluorescence information of a first sample to which the test substance has been added to the cell group, and second autofluorescence information, which is autofluorescence information of a second sample that serves as a control for the first sample.

[0013] An example of a cell group that immune-responds to a test substance is shown in Figure 1. The cell group in Figure 1 includes T cells 1000 and antigen-presenting cells (APCs) 1001. 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.

[0014] A conceptual diagram of this embodiment is shown in FIG. According to the method of this embodiment, the immune response of a cell group to a test substance is evaluated based on a comparison of first autofluorescence information, which is autofluorescence information of a first sample to which a test substance has been added, and second autofluorescence information, which is autofluorescence information of a second sample that serves as a control for the first sample. The autofluorescence information is the brightness value of blue autofluorescence (I B ) and the green autofluorescence intensity value (I G ) information about the The autofluorescence information was calculated by the blue autofluorescence intensity value (I B ) and the green autofluorescence intensity value (I G ) ratio value (I G / I B ) of each cell in each cell population. G / I B The first autofluorescence information and the second autofluorescence information can be compared based on the distribution of

[0015] Based on this comparison, for example, as illustrated in FIGS. 3A to 3D, it can be evaluated that the cell group has an immune response to the test substance, or that the immune response is stronger than that of the control, in the following cases. First sample of cells I G / I B the mean, median, or mode of the first sample is smaller than that of the second sample (Figure 3A), or the first sample is multimodal and the second sample is unimodal (Figures 3B and 3D), or both the first and second samples are multimodal but have different shapes (Figure 3C). In addition, when examining the above difference, the time lapse after the addition of the test substance and the concentration of the test substance can be taken into consideration. Furthermore, the strength of the immune response can be evaluated based on the above difference, the time lapse, and the concentration of the test substance. The second sample can be prepared by not adding the test substance to the cell group, by adding the test substance to the cell group in an amount different from that of the first sample, by adding an alternative substance to the test substance to the cell group, or by adding the test substance to the cell group but for a different amount of time after addition than the first cell group.

[0016] The first and second samples do not necessarily have to be prepared separately from the first sample; the second sample can be the first sample prepared before the addition of the test substance, or the first sample prepared after insufficient time has passed since the addition of the test substance. A specific configuration example of this embodiment will be described below, but the present invention is not limited to the following method.

[0017] (cell group) The cell group to be subjected to the evaluation method of this embodiment is not particularly limited as long as antigen-specific immune cells can be evaluated. The cell group preferably includes at least one of T cells, B cells, and other lymphocytes. Peripheral blood mononuclear cells (PBMCs) can be particularly preferably used as the cell group. Alternatively, PBMCs with an increased lymphocyte ratio may be used by pre-culturing the PBMCs, removing adherent cells, and recovering floating lymphocytes. In this way, there is no restriction on the use of cells obtained by temporarily culturing immune cells extracted from a living body. Furthermore, any type of cell, for example, a specific immune cell such as CD8 positive cells, CD4 positive cells, or B cells, may be extracted from PBMCs by existing methods and used.

[0018] The number of cells or cell concentration of the cell group subjected to the evaluation method of this embodiment is set within a suitable range depending on the sample used. For example, when evaluating the antigen-specific immune response of T cells in PBMCs, in order to stimulate the T cells with a test substance, contact with cells presenting antigen sites derived from the test substance is necessary, and therefore a cell density at which the cells come into contact with each other is required. When stimulating by static culture, specifically, a cell density of 1×10 6 cells / cm 2 It is preferable that the temperature is about 100° C. or so. Alternatively, since lymphocytes such as T cells can be cultured in a suspended state, the cells can be brought into contact with each other even at a lower cell density by adjusting the conditions that promote cell movement, for example, the shaking conditions in the case of shaking culture. Alternatively, by using a culture vessel that does not have a flat bottom, such as a V-bottom or U-bottom microplate, it is also possible to culture the cells while keeping them in contact with each other regardless of the cell density. For example, a test substance is added to a group of cells such as PBMCs, and after about one hour, autofluorescence derived from reduced nicotinamide adenine dinucleotide (NADH) and / or reduced nicotinamide adenine dinucleotide phosphate (NADPH) in the cells and oxidized flavin adenine dinucleotide (FAD +The autofluorescence from the FAD / NADH cells is measured, and the responsiveness of the cell group to a test substance can be evaluated based on the change in the fluorescence ratio (FAD / NADH ratio).

[0019] (Test substance) The test substance may be an antigen, an antigen protein, an antigen peptide, a vaccine, a pathogen, or a peptide derived from these test substances. MHC class 1 bound to a peptide consisting of 8-10 amino acids derived from the test substance can stimulate the TCR of CD8 positive T cells. A peptide derived from the test substance that binds to MHC class 1 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 used for evaluation by binding the peptide derived from the test substance to the MHC molecule. Yet another type of test substance can be a test substance having antigen genetic information (e.g., messenger RNA vaccine, etc.). This test substance can be taken up into a cell, protein expression and processing can be performed within the cell, and binding to MHC can be used for evaluation. MHC class 2 bound to a peptide 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, a peptide derived from the test substance that binds to MHC class 2 prepared in advance can be used, and similarly to the above, a test substance composed of a protein or polypeptide or a test substance containing antigen genetic information can also be used. However, since MHC class 2 is expressed only in antigen-presenting cells, the coexistence of cells expressing MHC class 2 is required.

[0020] (Autofluorescence and its measurement methods) The cellular autofluorescence measured in this embodiment includes NADH and / or NADPH (hereinafter sometimes abbreviated as NAD(P)H), flavins (FAD +Examples of the fluorescence emitted by endogenous fluorescent substances produced within cells include those derived from endogenous fluorescent substances such as erythrocytes, collagen, fibronectin, tryptophan, and folic acid, but fluorescence emitted by endogenous fluorescent substances other than these may also be used. In the examples, glucose metabolism activity is evaluated by measuring the autofluorescence of endogenous NAD(P)H and flavins (FAD, etc.). For example, when the glucose metabolism of T cells contained in PBMCs is activated by an antigen-specific immune response, the NAD(P)H produced from the glycolysis pathway and the citric acid cycle increases, and an increase in autofluorescence is observed. In addition, the state of glucose metabolism changes over time, and as the electron transport system works, NAD(P)H decreases and FAD + In this way, the change in the state of glucose metabolism, which indicates the activation state of cells, increases the amount of FAD in the cells. + This can be measured as the difference or change in autofluorescence due to changes in the amount of FAD or NAD(P)H, or the difference or change in the ratio of these autofluorescences (FAD / NADH ratio). Autofluorescence can be measured by any means, including existing means such as conventional fluorescence microscopes and flow cytometers, as long as the means can measure the autofluorescence of the cells described above. Means that can simultaneously and minimally invasively measure the autofluorescence of a large number of cells as described above are preferable. In addition, for example, FAD + Since the wavelengths of fluorescence emitted by NAD(P)H are different, a means capable of simultaneously measuring fluorescence of multiple wavelengths is preferable. As such an autofluorescence measuring means, for example, a fluorescence measuring device and a fluorescence image acquiring method described below can be suitably used.

[0021] When measuring autofluorescence, cells are irradiated with light of a wavelength capable of exciting the endogenous fluorescent substance of interest, and the fluorescence generated is measured. For example, if the endogenous fluorescent substance of interest is NAD(P)H, the cells are irradiated with excitation light with a central wavelength in the range of 320 nm to 370 nm, and the generated blue fluorescence of 400 nm or more, preferably in the range of 420 nm to 500 nm, is measured. In addition, if the endogenous fluorescent substance of interest is FAD, +In this case, it is advisable to irradiate the cells with excitation light having a central wavelength in the range of 360 nm to 490 nm and measure the generated green fluorescence of 500 nm or more, preferably in the range of 500 nm to 580 nm, and more preferably in the range of 520 nm to 580 nm. For example, a test substance is added to a group of immune cells to be evaluated, such as PBMCs, and after about one hour, autofluorescence derived from reduced nicotinamide adenine dinucleotide (NADH) and / or reduced nicotinamide adenine dinucleotide phosphate (NADPH) in the cells and oxidized flavin adenine dinucleotide (FAD + The autofluorescence from the FAD / NADH cells is measured, and the reactivity of the immune cell population to a test substance can be evaluated based on the change in the fluorescence ratio (FAD / NADH ratio).

[0022] (Analysis / Visualization) Methods for analyzing and visualizing the acquired cell autofluorescence information include, for example, visualizing individual cell information, such as autofluorescence brightness information for each wavelength and cell size information, as one-, two-, three- or more-dimensional graphs, with each axis representing that information. More specifically, 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. Here, the cell shape information refers to information of parameters that can describe the shape of a cell. Examples include information on cell size, cell shape, and cell thickness, but other information may also be used.

[0023] (Evaluation or Judgment Method) The following are examples of methods for determining the immune response of a cell group to a test substance. For example, a cell sample is divided into two, and each is treated with the addition of a test substance (first sample) and without it (control: second sample), and the autofluorescence is measured after a certain period of time. The ratio of FAD-derived autofluorescence to NADH-derived autofluorescence (FAD / NADH ratio) under both conditions is compared, and the presence or absence and strength of an antigen-specific immune response is evaluated or judged based on the presence or absence and magnitude of the difference between the samples. Also, for example, for cells treated in the same manner as above, the FAD + And / or, the presence or absence of an antigen-specific immune response and its strength may be evaluated or determined based on the magnitude of the difference in brightness of autofluorescence caused by NAD(P)H. A specific example of such determination is described in the Examples. Here, the certain period of time is not limited as long as it allows evaluation of immune cells.

[0024] When the test substance directly binds to MHC, it can rapidly stimulate immune cells, and in this case, the certain period of time is, for example, 10 minutes to 24 hours, preferably 30 minutes to 4 hours. It is known that intracellular signals are transmitted to antigen-stimulated T cells within a few seconds to a few minutes, so in this evaluation method, too, it is considered that changes start several minutes after the cells are treated with the test substance. Furthermore, in cases where a test substance is first taken up into an antigen-presenting cell, processed within the cell, and then presented on the cell surface, an optimal processing time should be set for each test substance.

[0025] As a further embodiment, the present invention provides an imaging analysis system for evaluating the immune response of a cell group to a test substance, the imaging analysis system comprising: an image acquisition device including an irradiation unit that irradiates excitation light of 320 nm to 490 nm; and an image processing device including a display unit, the image acquisition device having means for acquiring an autofluorescence image of a first sample in which the test substance has been added to the cell group, and an autofluorescence image of a second sample that serves as a control for the first sample, the image processing device having means for generating first autofluorescence information that is autofluorescence information of the first sample and second autofluorescence information that is autofluorescence information of the second sample from the autofluorescence image, and the display unit provides an imaging analysis system that displays a comparison result between the first autofluorescence information and the second autofluorescence information. A specific configuration example of this embodiment will be described, but the present invention is not limited to the following method.

[0026] (Configuration of an imaging analysis system that is a detector for acquiring autofluorescence information) 4 shows an example of the overall device configuration of imaging and analysis system 100 according to this embodiment. The imaging and analysis system acquires images of cells, calculates changes in autofluorescence with different wavelengths by analyzing the acquired images, and displays the autofluorescence information. 4, the imaging analysis system 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.

[0027] As shown in FIG. 5, 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 a group of cells placed on a 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 fluorescent light emitted from the cells 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 capture plane 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.

[0028] The image processing device 2A is an 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 the cells being observed, and outputs the calculated features. An example of the functional configuration of an image processing device is shown in Fig. 6. 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 comprehensively controls the operation of the image processing device. For example, the control unit 21 executes image analysis processing (see FIG. 7) 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 LCD (Liquid Crystal Display), and displays various screens according to instructions of a display signal input from the control unit 21. In the present embodiment, the display unit 23 outputs the calculated feature amount, and further functions as an output means for outputting the comparison result. 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. 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.

[0029] The image processing device 2A in this embodiment performs analysis using the image for extracting a cellular region and the autofluorescence image transmitted from the image acquisition device 1A. An autofluorescence image is an image obtained by irradiating a cell with excitation light of a specific wavelength in the image acquisition device 1A to cause fluorescent substances inherent in the cells to emit light, and then enlarging and photographing this fluorescence through a cut filter with 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 photographed image obtained by magnifying and photographing a bright-field image in the image acquisition device 1A, and a fluorescent photographed image obtained by irradiating an unstained sample with excitation light of a predetermined wavelength to cause a fluorescent substance intrinsic to the cell to emit light, and then magnifying and photographing the fluorescence.

[0030] A flow chart for performing image analysis using the obtained images is shown in Fig. 7. The following processes in Fig. 7 are 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 autofluorescence image from the image acquisition device 1A is input via the communications I / F 24. Then, in step P4, the autofluorescence image is separated into its RGB components. The following processing is performed on each of the separated R, G, and B components. In step P5, each pixel of the image for extracting a cell region is added to each pixel of the autofluorescence 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. In this embodiment, furthermore, in step P8, feature amounts for each cell are obtained for the first and second samples in a similar manner, the first and second autofluorescence information are calculated, and the comparison results are output and displayed on the display unit 23.

[0031] (Fluorescence image acquisition method) 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 perpendicular to the optical axis even if it is not parallel, 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, 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.

[0032] (Bright-field image acquisition method) One method for acquiring bright-field images to identify the position, size, shape, etc. of cells is to irradiate 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 detect the reflected light from the target sample or the diffracted light due to birefringence, and the interference between them.

[0033] (Information obtained from images) The luminance information obtained from the acquired cell image is color information obtained by describing the fluorescence information obtained by the above-mentioned method 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 information of the red component (R component), green component (G component) and blue component (B component) in the RGB color space, or the Lab color space or the HSV color space. For example, the G component is used as information on green fluorescence and the B component is used as information on blue fluorescence, and the luminance information is expressed as the above-mentioned FAD + or NAD(P)H autofluorescence brightness information. Note that brightness information may be described using color spaces other than these. Furthermore, the cell shape information obtained from the acquired cell image includes information such as cell size, cell shape, and cell thickness, but other information that can be obtained from an image may also be used.

[0034] As a further embodiment, the present invention provides an information processing method for evaluating the immune response of a cell population to a test substance, the information processing method including the steps of acquiring first autofluorescence information that is autofluorescence information of a first sample to which the test substance has been added to the cell population, acquiring second autofluorescence information that is autofluorescence information of a second sample that serves as a control for the first sample, and comparing the first autofluorescence information with the second autofluorescence information to evaluate the immune response of the cell population to the test substance. The present invention also provides an information processing device that executes the information processing method, and an information processing program that causes the information processing device to execute the information processing method. EXAMPLES

[0035] In the following examples, a specific example of evaluating cells responding to antigen stimulation using PBMC will be given, but the reagents and reaction conditions described in the following examples can be modified, and such modifications are intended to be included within the scope of the present invention. Therefore, the following examples are intended to aid in the understanding of the present invention, and are not intended to limit the scope of the present invention in any way. The test substances used in the following two examples are peptides that directly bind to MHC.

[0036] Example 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 thawed according to the manufacturer's instructions. The thawed cells were suspended in 3 mL of CTL Test medium (manufactured by CTL), the entire amount was seeded into one well of a 6-well plate, and cultured overnight in a 37°C, 5% CO2 incubator. However, the CTL test medium used was supplemented with 200 mM L-glutamine solution in an amount of 1 / 100 of the medium volume.

[0037] (Antigen stimulation method) After overnight culture, the cells were harvested, 7 mL of PBS was added, and the cells were centrifuged at 300 G for 8 minutes. The supernatant was removed, and the cells were resuspended in CTL Test medium at a cell concentration of 2.5 × 10 6 After suspending the cells at 100 μL / mL, 100 μL of each peptide solution was seeded onto a U-bottom 96-well plate. The peptide solutions Flu Matrix 1 (58-66) Peptide, Influenza A PA (46-54) Peptide, and CMVpp65 (495-503) peptide (all peptide concentrations 10 nM) manufactured by CTL were used as test substances, and 100 μL of each peptide solution was added to the wells containing the cells. The PBMCs used in this experiment reacted strongly to the Flu Matrix 1 (58-66) peptide, barely to the Influenza A PA (46-54) peptide, and weakly to the CMVpp65 (495-503) peptide. This information was previously confirmed by the manufacturer using an ELISpot assay. For the control cell group without the addition of the test substance, 100 μL of CTL test medium was added instead of the peptide solution. After antigen stimulation, the cells were left to stand in a 5% CO2 incubator for 1 hour. 200 μL of cell suspension was collected from each well and centrifuged at 300G for 5 minutes. After centrifugation, the supernatant was removed and the cell pellet was suspended in 700 μL of PBS. 100 μL of the cell suspension was seeded on the cover glass part (diameter 16 mm) of a 35 mm glass bottom dish and used for bright field observation and autofluorescence observation.

[0038] (Bright-field image acquisition) A 525 nm LED (manufactured by Asahi Spectroscopy Co., Ltd.) was irradiated, and a bright field image was captured by the imaging analysis system 100. An EOS R5 (product name) manufactured by Canon Inc. was used as the imaging unit 13, and images were acquired at a shutter speed of 1 / 400 and an ISO sensitivity of 100. An example of bright-field imaging is shown in Figure 8. The field of view size is approximately 18 mm × 12 mm.

[0039] (Autofluorescence image acquisition) The object to be observed was excited by irradiating a 365 nm LED (manufactured by Asahi Spectroscopy Co., Ltd.) light source through a 400 nm short pass filter, and then imaged through a 430 nm long pass filter (blue autofluorescence image). The object was excited by irradiating a 395 nm LED (manufactured by Asahi Spectroscopy Co., Ltd.) light source through a 400 nm short pass filter, and then imaged through a 430 nm long pass filter (green autofluorescence image). The imaging analysis system 100 was used for both images. An EOS R5 was used as the imaging unit 13, and images were acquired at an ISO sensitivity of 12800. An example of a blue autofluorescence image is shown in Figure 9. A portion of the blue autofluorescence image was cut out, and the field of view size was approximately 6.6 mm × 5.25 mm. 7,642 cells were observed in Figure 9.

[0040] (Method for extracting and analyzing color information from autofluorescence images) The cell area was obtained from the bright field image and labeled. After obtaining the blue component (B component) from the blue autofluorescence image and the green component (G component) from the green autofluorescence image, the cell area obtained from the bright field image was overlaid to obtain the brightness value of each cell. The obtained B component was NADH, and the G component was FAD. + The FAD / NADH ratio was calculated for each cell by dividing the G component value of the green autofluorescence image by the B component value of the blue autofluorescence image.

[0041] (Example of evaluation of reactivity of T cells in PBMC to peptide antigens) The results of measuring PBMC stimulated for 1 hour with three types of test substances with different reactivity are shown in Figures 10A to 10C. Figures 10A to 10C are graphs with the vertical axis representing frequency (cell count) and the horizontal axis representing the FAD / NADH ratio. Figure 10A shows the results using a cell group to which a strongly reactive test substance was added, Figure 10B shows a cell group to which a weakly reactive test substance was added, and Figure 10C shows a cell group to which a non-reactive substance was added. When comparing the cell group added with the test substance (Flu Matrix 1 (58-66) Peptide) that strongly reacts with PBMC used in this Example, and the cell group without the test substance, it was found that the FAD / NADH ratio was smaller in the former. Furthermore, the degree of this difference depended on the strength of the reactivity of PBMC to the test substance. In other words, it was found that the strength of the reactivity of immune cells to the test substance can be known by investigating the difference in the FAD / NADH ratio between the cell group without antigen and the cell group with antigen added. Figures 11A and B show the results of PBMCs stimulated with a strongly reactive test substance, comparing the results 1 hour (Figure 11A) and 4 hours (Figure 11B). A decrease in the FAD / NADH ratio due to stimulation with the test substance was observed 1 hour after stimulation, but by 4 hours, the difference had almost disappeared. From this, it was found that when using this test substance, in order to examine the responsiveness of the cells, it is necessary to examine the reaction at least for less than 4 hours, and it is preferable to examine the reaction over about 1 hour.

[0042] Example 2 (Thawing cells) Here, PBMCs with different reactivity to the test substance from that in Example 1 were used. PBMCs manufactured by CTL Inc. were used and thawed according to the manual provided by the manufacturer. Unlike Example 1, the PBMCs were immediately subjected to a test to examine the reactivity to the test substance without pre-culture.

[0043] (Antigen stimulation method) Thawed PBMCs were diluted in CTL Test medium at a cell concentration of 7.2 × 10 6The cells were suspended in 1000 μl / mL of the cells, and 250 μl of each were seeded onto a 24-well plate. As a test substance, a peptide solution, CMVpp65 (495-503) peptide (all peptide concentrations 10 nM) manufactured by CTL was used, and 250 μl of this peptide solution was added to the wells containing the cells. The PBMCs used in this experiment were cells that strongly reacted with the CMVpp65(495-503) peptide, and this information had been previously confirmed by the manufacturer using an ELISpot assay. For the control cell group without the addition of the test substance, 250 μL of CTL test medium was added instead of the peptide solution. After antigen stimulation, the cells were left to stand in a 5% CO2 incubator for 1 to 24 hours. The entire cell suspension was collected from each well and centrifuged at 300G for 5 minutes. After centrifugation, the supernatant was removed and the cell pellet was suspended in 1000 μL of PBS. The cell suspension was further diluted 5 times with PBS, and 100 μL of the diluted cell suspension was seeded on the cover glass part (diameter 16 mm) of a 35 mm glass bottom dish and used for bright field observation and autofluorescence observation.

[0044] (Autofluorescence image acquisition, etc.) Thereafter, the bright field image acquisition method, the autofluorescence image acquisition method, and the color information extraction and analysis method of the autofluorescence image were performed in the same manner as in Example 1.

[0045] (Example of evaluation of reactivity of T cells in PBMC to peptide antigens) The results of measuring PBMCs stimulated with a highly reactive test substance for 1 to 24 hours over time are shown in Figures 12A to 12C. Figures 12A to 12C are also graphs with the frequency (cell count) on the vertical axis and the FAD / NADH ratio on the horizontal axis. Figure 12A shows the results 1 hour after stimulation with the test substance, Figure 12A shows the results 4 hours after stimulation with the test substance, and Figure 12A shows the results 24 hours after stimulation with the test substance. When the cells were stimulated for 1 hour with the test substance (CMVpp65(495-503) peptide) used in this experiment, which reacts strongly with this PBMC, the FAD / NADH ratio was reduced as in Example 1, compared to when no test substance was added. After 4 hours of stimulation with the test substance, almost no decrease in the FAD / NADH ratio was observed as in Example 1. Furthermore, the change in the FAD / NADH ratio was examined up to 24 hours after stimulation, and almost no decrease in the FAD / NADH ratio was observed from 4 hours after stimulation with the test substance until 24 hours after stimulation.

[0046] 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: first autofluorescence information, which is autofluorescence information of a first sample obtained by adding the test substance to the cell group; Second autofluorescence information, which is autofluorescence information of a second sample that is a control of the first sample. A method for evaluating the immune response of the cell group to the test substance based on a comparison of the immune response of the cell group to the test substance. [Method 2] The autofluorescence information is the luminance value of blue autofluorescence (I B ) and the green autofluorescence intensity value (I G ) The method according to method 1, comprising information regarding [Method 3] The autofluorescence information is the intensity value (I B ) and the brightness value of the green autofluorescence (I G ) ratio value (I G / I B ) information, I of individual cells of each sample G / I B 3. The method according to claim 1 or 2, wherein the first autofluorescence information and the second autofluorescence information are compared based on a distribution of the first autofluorescence information and the second autofluorescence information. [Method 4] The second sample comprises: The test substance is not added to the cell group; adding the test substance to the cell group in an amount different from that of the first sample; Adding a substitute for the test substance to the cell population; or adding the test substance to the cell group, provided that the time elapsed after the addition is different from that of the first cell group; 4. The method according to any one of methods 1 to 3, wherein the compound is prepared by: [Method 5] The blue autofluorescence is autofluorescence observed when irradiated with excitation light having a central wavelength in the range of 320 nm to 370 nm, The green autofluorescence is observed when irradiated with excitation light having a central wavelength in the range of 360 nm to 490 nm. The method according to method 2 or 3. [Method 6] The blue autofluorescence is autofluorescence observed in the range of 420 nm to 500 nm, and the green autofluorescence is autofluorescence observed in the range of 500 nm to 580 nm. A method according to any one of methods 2, 3 and 5. [Method 7] The autofluorescence information is irradiating the sample with excitation light having a central wavelength in the range of 320 nm to 370 nm and acquiring a blue fluorescent image of the sample; Irradiating the sample with excitation light having a central wavelength in the range of 360 nm to 490 nm and acquiring a green fluorescent image of the sample; and analyzing the obtained blue and green fluorescent images to obtain autofluorescence information for each cell; obtained by a process comprising: 7. The method according to any one of methods 1 to 6. [Method 8] 8. The method of any one of methods 1 to 7, wherein the population of cells comprises at least one of T cells, B cells, and other lymphocytes. [Method 9] The method according to any one of methods 1 to 8, wherein said cell population is PBMCs (peripheral blood mononuclear cells). [Method 10] 10. The method according to any one of methods 1 to 9, wherein the test substance comprises at least one selected from an antigen, an antigenic protein, an antigenic peptide, a vaccine, and a pathogen. [Method 11] 1. A method for evaluating an immune response of a cell population to a test substance, comprising: acquiring first autofluorescence information, which is autofluorescence information of a first sample obtained by adding the test substance to the cell group; obtaining second autofluorescence information of a second sample that is a control of the first sample; and comparing the first autofluorescence information with the second autofluorescence information to evaluate the immune response of the cell group to the test substance; An information processing method comprising: [Configuration 1] An information processing device that executes the information processing method described in method 11. [Configuration 2] An information processing program for causing an information processing device to execute the information processing method according to method 11. [Configuration 3] An imaging analysis system for evaluating an immune response of a cell group to a test substance, comprising: An image acquisition device including an irradiation unit that irradiates excitation light of 320 nm to 490 nm, and An image processing device including a display unit, The image acquisition device includes: an autofluorescence image of a first sample in which the test substance is added to the cell group; and An autofluorescence image of a second sample that is a control of the first sample. and a means for acquiring The image processing device includes: first autofluorescence information, which is autofluorescence information of a first sample from the autofluorescence image; and second autofluorescence information that is autofluorescence information of a second sample; means for generating The display unit displays a comparison result between the first autofluorescence information and the second autofluorescence information. Imaging analysis system. [Explanation of symbols]

[0047] 100 Imaging Analysis System 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 1000 T cells 1001 Antigen presenting cells 1002 T cell receptor (TCR) 1003 Major histocompatibility complex (MHC) 1004 Test substance

Claims

1. A method for evaluating the immune response of a group of cells to a test substance, The first autofluorescence information is the autofluorescence information of the first sample obtained by adding the test substance to the aforementioned cell group, The second autofluorescence information is the autofluorescence information of the second sample, which is a control sample for the first sample. A method for evaluating the immune response of the cell group to the test substance based on a comparison of the following.

2. The autofluorescence information is the luminance value (I) of the blue autofluorescence. Ｂ ) and the brightness value of green autofluorescence (I Ｇ The method according to claim 1, which includes information relating to ).

3. The autofluorescence information is the brightness value (I) of the blue autofluorescence of individual cells in each sample. Ｂ ) and the brightness value of green autofluorescence (I Ｇ The ratio value of (I Ｇ / I Ｂ ) Includes information, I of individual cells in each sample Ｇ / I Ｂ The method according to claim 1, wherein the first autofluorescence information and the second autofluorescence information are compared based on the distribution of the first autofluorescence information.

4. The second sample mentioned above is, The test substance is not added to the aforementioned cell group. The test substance is added to the cell group in an amount different from that of the first sample. A substitute substance for the test substance is added to the cell group, or The test substance is added to the cell group, provided that the time elapsed after addition is different from that of the first sample. The method according to any one of claims 1 to 3, which is prepared by...

5. The aforementioned blue autofluorescence is autofluorescence observed when excitation light with a central wavelength in the range of 320 nm to 370 nm is irradiated. The aforementioned green autofluorescence is autofluorescence observed when irradiated with excitation light having a central wavelength in the range of 360 nm to 490 nm. The method according to claim 2 or 3.

6. The blue autofluorescence is observed in the range of 420 nm to 500 nm, and the green autofluorescence is observed in the range of 500 nm to 580 nm. The method according to claim 2 or 3.

7. The aforementioned autofluorescence information is, A step of irradiating the sample with excitation light having a central wavelength in the range of 320 nm to 370 nm and obtaining a blue autofluorescence image of the sample. A step of irradiating the sample with excitation light having a central wavelength in the range of 360 nm to 490 nm and obtaining a green autofluorescence image of the sample, and The process involves analyzing the obtained blue and green autofluorescence images to acquire autofluorescence information for each cell. Obtained by a process that includes, The method according to any one of claims 1 to 3.

8. The method according to any one of claims 1 to 3, wherein the cell population includes at least one of T cells, B cells, and other lymphocytes.

9. The method according to any one of claims 1 to 3, wherein the cell group is PBMC (peripheral blood mononuclear cells).

10. The method according to any one of claims 1 to 3, wherein the test substance comprises at least one selected from an antigen, an antigenic protein, an antigenic peptide, a vaccine, and a pathogen.

11. A method for processing information to evaluate the immune response of a group of cells to a test substance, A step of acquiring first autofluorescence information, which is the autofluorescence information of a first sample obtained by adding the test substance to the aforementioned cell group. A step of obtaining second autofluorescence information, which is the autofluorescence information of a second sample that serves as a control for the first sample, and A step of comparing the first autofluorescence information and the second autofluorescence information to evaluate the immune response of the cell population to the test substance, Information processing methods including

12. An information processing apparatus that performs the information processing method described in claim 11.

13. An information processing program for causing an information processing device to execute the information processing method described in claim 11.

14. An imaging and analysis system for evaluating the immune response of a group of cells to a test substance, An image acquisition apparatus including an irradiation unit that irradiates excitation light in the range of 320 nm to 490 nm, and It has an image processing device including a display unit, The image acquisition device is Autofluorescence image of the first sample obtained by adding the test substance to the aforementioned cell group, and Autofluorescence image of a second sample, which serves as a control for the first sample. Having means to acquire, The aforementioned image processing device is From the autofluorescence image, the first autofluorescence information is obtained from the first sample, and the second autofluorescence information is obtained from the second sample. Having means for generating, The display unit displays the comparison result between the first autofluorescence information and the second autofluorescence information. Imaging and analysis system.

Images