Determination of immune cell immune stimulation responsiveness, determination of immune cell immune synapse formation ability, and cell analysis device

Abstract

The present invention relates to the determination of immune cell immunostimulatory responsiveness, the determination of immune cell immunosynapse formation ability, and a cell analysis device. The method for determining the immunostimulatory responsiveness of immune cells comprises: (i) a step of bringing the immune cells of the measurement object into contact with an immunostimulatory factor; (ii) a step of making the immune cells of the measurement object form a contact surface with a substance different from the immune cells of the measurement object; (iii) a step of bringing the immune cells of the measurement object into contact with a capture body that binds to a surface antigen of the contact surface and can generate an optical signal; (iv) a step of detecting the optical signal generated by the capture body; and (v) a step of determining whether the immune cells of the measurement object that have released the contact surface before detecting the optical signal have immunostimulatory responsiveness based on the detected optical signal.

Description

[0001] This application is a divisional application of the invention patent application of the same name 201810314681.7 filed on April 10, 2018. [Technical field]

[0002] The present invention relates to a method for measuring the immunostimulatory responsiveness of immune cells, a method for determining the immunological synapse forming ability of immune cells, and a cell analysis device. [Background technology]

[0003] Immunity is the general term for the cellular and humoral biological reactions that organisms carry out to identify self and non-self in the body and eliminate non-self. The immune system is a very important physiological mechanism in life activities. The cells of the immune system participate in the activation and inhibition of the immune system by secreting proteins called cytokines or exerting cell-killing functions.

[0004] The immune system is closely related to the rejection of foreign cells, which can determine the success of transplantation therapy. In recent years, the usefulness of immunotherapy, which utilizes the immune system to treat human diseases, has been demonstrated, and the importance of understanding the status of the immune system has been increasing.

[0005] As an analysis method for the state of the immune system, an analysis method based on cytokine secretion is known. This analysis method requires culturing immune system cells after immunostimulation, and it takes a long time (tens of hours) and labor from immunostimulation to cytokine detection.

[0006] In recent years, attention has been paid to the structure formed in the immune cell at its contact surface when the immune cell contacts the target cell. Since the structure is similar to the synaptic structure formed by nerve cells in function and morphology, it is called immune synapse. It has been suggested that the immune synapse is related to the increase of the immunostimulatory signal, and the known immune synapse formation ability is related to the ability to secrete cytokines (non-patent literature 1). In addition, the formation of the known immune synapse occurs in a short time (tens of minutes) compared with the secretion of cytokines (non-patent literature 1).

[0007] As a method for measuring immune synapses, for example, the following method is known: immune cells are stimulated using an immunostimulatory factor on a glass substrate, and the immune synapses of T cells are measured using a total internal reflection fluorescence microscope while maintaining the contact surface with the glass substrate (non-patent document 2). In addition, the following method is known: while maintaining the contact surface formed with the target cell, the immune synapses of T cells are measured using an imaging flow cytometer (non-patent document 3). In non-patent document 2, the detection of immune synapses is performed using the costimulatory molecule CD28 as an indicator; in non-patent document 3, the detection of immune synapses is performed using CD3 as an indicator. In both non-patent document 2 and non-patent document 3, immune cells are observed while maintaining the contact surface with the object.

[0008] [Prior art literature]

[0009]

Non-patent literature

[0010] Non-patent document 1: Carlin LM. et al., Blood, vol. 106, p. 3874-3879, 2005

[0011] Non-patent document 2: Yokosuka T. et al., Immunity, vol. 29, p. 589-601, 2008

[0012] Non-patent document 3: Hosseini BH. et al., Proc Natl Acad Sci USA, vol. 106, p. 17852-17857, 2009 [Summary of the invention]

[0013] [Problems to be solved by the invention]

[0014] It is very important to quickly and easily understand the state of the immune system, that is, to understand the responsiveness of immune system cells to various immune stimuli in performing various treatments involving the immune system.

[0015] Conventionally, analysis methods using cytokine secretion as an indicator have been used to analyze the responsiveness of immune system cells to immune stimulation. However, this analysis method requires culturing immune cells from immune stimulation until cytokine secretion, which is cumbersome and time-consuming.

[0016] When a total internal reflection fluorescence microscope is used to measure the responsiveness of immune system cells to various immune stimuli, the number of cells that can be measured is small and the measurement is laborious, so the measurement cannot be performed easily and is not suitable for automated equipment.

[0017] Therefore, in this field, there is a need to quickly and easily grasp the responsiveness of cells of the immune system to various immune stimuli.

[0018] In order to solve the above-mentioned problems, the inventors focused on the immune synapse, which changes in a short time compared to cytokine secretion and has been shown to be related to the increase in immune stimulation signals. However, the immune synapse is a loose bond formed in the immune cell at the interface with the target cell (Non-patent Document 3), so there is a concern that it will dissociate before and during the measurement. In fact, to date, the immune synapse has not been used as an indicator of the responsiveness of the immune cell to immune stimulation in a state where the contact surface between the target cell and the immune cell is released.

[0019] The present inventors have repeatedly studied various factors known to be related to the formation of immune synapses, focusing on a method for rapidly detecting the responsiveness of immune cells to immune stimulation. As a result, they have found that when surface antigens such as co-stimulatory molecules are used as indicators among various factors, the ability of immune cells to form immune synapses can be determined even if the contact surface between immune cells and an object is released, and have completed the present invention.

[0020]

Solutions to Solve the Problem

[0021] The first embodiment of the present invention provides a method for measuring the immunostimulatory responsiveness of immune cells, which comprises: (i) a process of bringing the immune cells of the measurement object into contact with an immunostimulatory factor; (ii) a process of causing the immune cells of the measurement object to form a contact surface with a substance different from the immune cells of the measurement object; (iii) a process of bringing the immune cells of the measurement object into contact with a capture body that can bind to a surface antigen on the contact surface and can generate an optical signal; (iv) a process of detecting the optical signal generated by the capture body; and (v) a process of determining, based on the detected optical signal, whether the immune cells of the measurement object that have released the contact surface before detecting the optical signal have immunostimulatory responsiveness.

[0022] The second aspect of the present invention provides a method for measuring the immunostimulatory responsiveness of immune cells, which comprises: (i) a step of bringing the immune cells of the measurement object into contact with an immunostimulatory factor; (ii) a step of making the immune cells of the measurement object form a contact surface with a substance different from the immune cells of the measurement object; (iii) a step of bringing the immune cells of the measurement object into contact with a capture body that can bind to the co-stimulatory molecules on the contact surface and can generate an optical signal; (iv) a step of detecting the optical signal generated by the capture body; and (v) a step of automatically determining whether the immune cells of the measurement object have immunostimulatory responsiveness based on the detected optical signal.

[0023] The third aspect of the present invention provides a method for determining the ability of immune cells to form immune synapses, which comprises: (i) a step of bringing the immune cells of the object of measurement into contact with an immunostimulatory factor; (ii) a step of causing the immune cells of the object of measurement to form a contact surface with a substance different from the immune cells of the object of measurement; (iii) a step of bringing the immune cells of the object of measurement into contact with a capture body that can bind to a surface antigen on the contact surface and can generate an optical signal; (iv) a step of detecting the optical signal generated by the capture body; and (v) a step of determining the local presence of the surface antigen in the immune cells of the object of measurement that has released the contact surface before detecting the optical signal based on the detected optical signal.

[0024] The fourth aspect of the present invention provides a cell analysis device, comprising: an introduction section, which introduces a complex, wherein the complex is a complex of an immune cell of a measurement object that has been subjected to immune stimulation and a capture body that can bind to a surface antigen of the aforementioned immune cell and can generate an optical signal, the aforementioned capture body being bound to a surface antigen of a contact surface between the aforementioned immune cell and a substance different from the aforementioned immune cell; a photographing section, which photographs the complex supplied by the introduction section; and an analysis section, which determines whether the immune cell of the measurement object, whose contact surface has been released before photographing, is responsive to immune stimulation based on the image photographed by the photographing section.

[0025] The fifth embodiment of the present invention provides a cell analysis device, which includes: an introduction section, which introduces a complex, wherein the complex is a complex of an immune cell of a measurement object that has been subjected to immune stimulation and a capture body that can bind to a surface antigen of the aforementioned immune cell and can generate an optical signal, the aforementioned capture body binds to a surface antigen of a contact surface between the aforementioned immune cell and a substance different from the aforementioned immune cell; a detection section, which irradiates light on the complex supplied by the introduction section and detects the generated optical signal from the complex; and an analysis section, which determines, based on the detected optical signal, whether the immune cell of the measurement object that has released the contact surface before the optical signal is detected has immune stimulation responsiveness.

[0026] [Effects of the invention]

[0027] According to the present invention, the responsiveness of immune cells to various immune stimuli can be analyzed based on the ability to form an immune synapse.

Brief Description of the Drawings

[0028] Figure 1 This is a schematic diagram of steps (i) to (iii) of the embodiment.

[0029] Figure 2 This is a diagram showing the configuration of a cell analysis device according to Embodiment 1.

[0030] Figure 3 This is a diagram showing the structure of the measuring device according to the first embodiment.

[0031] Figure 4 This is a diagram showing the configuration of an imaging unit according to the first embodiment.

[0032] Figure 5 Schematic diagram showing the area on the light receiving surface of the camera according to the first embodiment.

[0033] Figure 6 This is a diagram showing the configuration of an information processing device according to Embodiment 1.

[0034] Figure 7 This is a flow chart showing the measurement process and analysis process of the subject according to the first embodiment.

[0035] Figure 8 This is an image showing the measurement data of the first embodiment.

[0036] Fig. 9 This is a schematic diagram showing a measurement process based on measurement data in the first embodiment.

[0037] Fig.10 This is a flow chart showing the analysis process in the first embodiment.

[0038] Fig.11 2D scattergrams showing the analysis processing of Embodiment 1 (a, b, and c) and Embodiment 2 (d).

[0039] Fig.12 It is a schematic diagram for explaining pulse data of fluorescence signals obtained from cells having different distribution states of costimulatory molecules.

[0040] Fig.13 This is a schematic diagram for explaining a 2D scatter plot of analytical parameters obtained from pulse data of a fluorescence signal.

[0041] Fig.14 The transmitted light image, the fluorescent image, and their superimposed image show the distribution of CD28 in T cells without stimulation (a) and with stimulation (b).

[0042] Fig.15 The images and 2D scatter plots show the distribution of CD28 in T cells. The differential interference image (DIC), fluorescence image (CD28) and their superimposed image (Merge) of T cells without stimulation (a) and with stimulation (c). The 2D scatter plots show the area showing the fluorescence signal in the cells (X axis) and the size of the cells (Y axis) without stimulation (b) and with stimulation (d).

[0043] Fig.16 It is a graph showing the correlation between cytokine secretion in T cells and the formation of immune synapse.

[0044] Fig.17 Fluorescence images showing the distribution of CD3 and CD40L in T cells without stimulation (a and c) and with stimulation (b and d).

[0045] Fig.18 The images and 2D scatter plots show the distribution of CD3 in T cells. The transmitted light image, fluorescent image, and superimposed image (Merge) of T cells without stimulation (a) and with stimulation (c). The 2D scatter plots show the total fluorescence signal intensity (X axis) and the area showing the fluorescence signal (Y axis) of the cells without stimulation (b) and with stimulation (d).

[0046] Fig.19 The images and 2D scatter plots showing the distribution of CD40L in T cells are shown in Figure 1. The transmitted light image, fluorescent image, and superimposed image (Merge) of T cells without stimulation (a) and with stimulation (c). The 2D scatter plots showing the total fluorescence signal intensity (X axis) and the area showing the fluorescence signal (Y axis) of cells without stimulation (b) and with stimulation (d).

[0047] Fig. 20 2D scatter plots showing an example of measurement processing and analysis processing in Example 2. a is a 2D scatter plot showing the size (X axis) and aspect ratio (Y axis) of cells. b is a 2D scatter plot showing the total fluorescence signal intensity (X axis) and count (Y axis) of cells.

[0048] Fig.21 The images are superimposed images of transmitted light images and fluorescent images showing the distribution of CD3, CD28, CD40L and OX40 in T cells without stimulation (a, c, e and g) and with stimulation (b, d, f and h).

[0049] Fig. 22 The images and 2D scatter plots show the distribution of CD3 in T cells. The transmitted light image, fluorescent image, and superimposed image (Merge) of T cells without stimulation (a) and with stimulation (c). The 2D scatter plots show the total fluorescence signal intensity (X axis) and the area showing the fluorescence signal (Y axis) of the cells without stimulation (b) and with stimulation (d).

[0050] Fig.23 The images and 2D scatter plots show the distribution of CD28 in T cells. The transmitted light image, fluorescent image, and superimposed image (Merge) of T cells without stimulation (a) and with stimulation (c). The 2D scatter plots show the total fluorescence signal intensity (X-axis) of cells and the area showing the fluorescence signal (Y-axis) without stimulation (b) and with stimulation (d).

[0051] Fig.24 The images and 2D scatter plots showing the distribution of CD40L in T cells are shown in Figure 1. The transmitted light image, fluorescent image, and superimposed image (Merge) of T cells without stimulation (a) and with stimulation (c). The 2D scatter plots showing the total fluorescence signal intensity (X axis) of cells and the area showing the fluorescence signal (Y axis) without stimulation (b) and with stimulation (d).

[0052] Fig.25 The images and 2D scatter plots showing the distribution of OX40 in T cells are shown. The transmitted light image, fluorescent image, and superimposed image (Merge) of T cells without stimulation (a) and with stimulation (c). The 2D scatter plots showing the total fluorescence signal intensity (X axis) and the area showing the fluorescence signal (Y axis) of the cells without stimulation (b) and with stimulation (d). [Specific implementation method]

[0053] One aspect of the present invention relates to a method for measuring the immunostimulatory responsiveness of immune cells. In this aspect, steps (i) and (ii) include: in the presence of an immunostimulatory factor, the immune cells of the subject of measurement are formed into a contact surface with a substance different from the immune cells of the subject of measurement. In this embodiment, after the step (i) of contacting the immune cells of the subject of measurement with the immunostimulatory factor, the step (ii) of forming the contact surface between the immune cells of the subject of measurement and the aforementioned substance can be performed. Alternatively, when there is an immunostimulatory factor on the surface of the aforementioned substance, steps (i) and (ii) can also be performed simultaneously. Even in this case, strictly speaking, step (ii) is performed after step (i). By performing steps (i) and (ii), an immune synapse is formed on the above-mentioned contact surface of the activated immune cell.

[0054] In step (iii), in order to detect the formed immune synapse, a capture body (hereinafter also referred to as "capture body") that can bind to the surface antigen of the contact surface and generate an optical signal is brought into contact with the immune cell having the above-mentioned contact surface. The capture body preferably binds to the molecules constituting the immune synapse in the surface antigen of the contact surface. By performing step (iii), a complex of the molecules constituting the immune synapse and the capture body is formed in the immune cell.

[0055] Then, in step (iv), the optical signal of the capture body contained in the complex on the immune cell is detected. There is no particular limitation on the means for detecting the optical signal, and it is preferably a means capable of analyzing each cell. As the means for detecting such optical signals, for example, flow cytometers and imaging cytometers can be cited. In a preferred embodiment, step (iv) uses a flow cytometer or an imaging cytometer to detect the optical signal generated by the capture body bound to the surface antigen of the immune cell.

[0056] In the later-described step (v), the immune cells whose contact surfaces have been released are to be determined for their immune stimulation responsiveness, so the later-described operation of releasing the contact surfaces can be performed on the immune cells before the detection of the optical signal in step (vi). When a substance of a non-biological material (e.g., a container, a multi-well plate, a glass slide, etc.) described later is used as a substance different from immune cells and step (iv) is performed using a flow cytometer or an imaging cytometer, the method of this embodiment preferably includes a step of releasing the contact surfaces. After releasing the contact surfaces as needed, the immune cells can be fixed using conventional methods. By fixing, the immune cells whose contact surfaces have been released can maintain the local presence of surface antigens. In the fixation of the cells, a fixing solution containing paraformaldehyde, a low-grade alcohol, etc. is preferably used.

[0057] In a further embodiment, it is possible to determine whether the immune cell is an immune cell that has released its contact surface based on the detected optical signal. Thus, the optical signal from the immune cell that has released its contact surface can be extracted for determination in step (v). For example, when allogeneic cells are used as a substance different from immune cells, in a measurement using a flow cytometer, there will be a difference in optical signals related to the size of the cells (e.g., the intensity or pulse width of forward scattered light) between the immune cells that have released their contact surface and the immune cells that maintain contact with the allogeneic cells. In a measurement using an imaging cytometer, immune cells that have released their contact surface and immune cells that maintain contact with the allogeneic cells can be distinguished based on the captured cell images.

[0058] Referring to the schematic diagram of steps (i) to (iii) of this embodiment for the purpose of illustration, Figure 1 a. Figure 1 a shows an example of using an immunostimulatory antibody described later as an immunostimulatory factor and a plate as a substance different from immune cells. The plate corresponds to, for example, the bottom surface of a container used for cell culture, the bottom surface of a well of a multi-well plate, the surface of a slide glass used for microscopic observation, etc. Figure 1 In a, in step (i), the immune cells are contacted with the immunostimulatory antibodies. In this example, the immunostimulatory antibodies are antibodies that bind to co-stimulatory molecules that are one of the surface antigens of the immune cells. After step (i), in step (ii), the immune cells in contact with the immunostimulatory antibodies are contacted with a plate, so that the immune cells form a contact surface with the plate. In step (iii), in order to detect the co-stimulatory molecules in the immune cells, a secondary antibody against the immunostimulatory antibody used in step (i) is used. The secondary antibody is labeled with a substance that can produce an optical signal. In this example, the contact surface has been released in step (iii), but the immune cells can also be contacted with the capture body while maintaining the contact surface. It should be noted that Figure 1a is an illustration of steps (i) to (iii) in one embodiment of the present invention, but the present invention is not limited to this illustration.

[0059] In step (v), it is determined whether the immune cells that have released the contact surface have immunostimulatory responsiveness based on the optical signal detected in step (iv). In the present embodiment, step (v) measures the value reflecting the distribution of surface antigens in immune cells based on the detected optical signal, and compares the measured value with a threshold value, thereby determining whether the immune cells have immunostimulatory responsiveness. Here, as a value reflecting the distribution of surface antigens, when the capture body contains a fluorescent substance, for example, the pulse width of the fluorescent signal from the capture body bound to the surface antigen, the height and pulse area of ​​the fluorescent pulse, etc. can be cited.

[0060] When the capture body contains a fluorescent substance, in step (v), a fluorescent image can also be obtained based on the fluorescent signal detected in step (iv), and a judgment can be made based on the fluorescent image. That is, the area showing the fluorescent signal reflecting the distribution of the surface antigen is measured in the fluorescent image, and the measured value is compared with the threshold value to determine whether the immune cell has immunostimulatory responsiveness. In this specification, "the area showing the fluorescent signal" refers to the number of pixels (pixels) showing a brightness value exceeding a specified threshold in the fluorescent image. In this embodiment, although not limited, when the aforementioned area is smaller than the threshold value, it can be determined that the immune cell has immunostimulatory responsiveness.

[0061] In a further embodiment, step (v) may include: further measuring at least one selected from the group including the total fluorescence signal intensity in the aforementioned region, the size of the immune cells and their aspect ratio, and comparing the measured value with the threshold. In the present specification, the "total fluorescence signal intensity" of the aforementioned region or cell refers to: the integral value of the brightness value of each pixel in the region showing a brightness value exceeding a specified threshold in the fluorescence image. In the present specification, the "size of the cell" refers to: the cell membrane region detected in the transmitted light image showing a brightness value below the specified threshold, reflecting the number of pixels of the image of the cell surrounded by the detected cell membrane. In the present specification, the "aspect ratio" of the cell refers to: the ratio of the longitudinal length to the lateral length (number of pixels) of the image reflecting the cell particles in the transmitted light image.

[0062] In a further embodiment, the determination of whether the immune cells are responsive to immune stimulation based on the detected optical signal includes: measuring at least one selected from the pulse width of the fluorescence signal, the pulse width of the scattered light signal, the value of the area reflecting the fluorescence signal of each cell, and the value reflecting the cell size, and comparing the measured value with a threshold value to determine whether the immune cells are responsive to immune stimulation.

[0063] In this specification, "immune synapse" refers to a molecular complex formed in an immune cell at the contact surface between an immune cell and a target cell, which is a part that plays a role in the activation of immune cells. It is known that the molecular complex is formed by a combination of a receptor (e.g., T cell receptor (TCR) or NK cell receptor), a cofactor of the receptor (e.g., CD3), various co-stimulatory molecules (e.g., CD28), an adhesion molecule (e.g., integrin), and a signaling molecule.

[0064] In this specification, "contact surface" refers to the area where the immune cells of the object of measurement come into contact with a substance different from the immune cells of the object of measurement. Here, the contact between the immune cells of the object of measurement and the substance different from the immune cells of the object of measurement also includes indirect contact through the interaction between molecules present on the surface of the immune cells of the object of measurement and molecules present on the surface of the substance different from the immune cells of the object of measurement. As described above, in the method of this embodiment, an immune synapse is formed at the contact surface. Therefore, the contact surface on the immune cell can also be referred to as the area where the immune synapse is formed. Although not limited, the contact surface can also be formed by the immune cells of the object of measurement spontaneously contacting a substance different from themselves. Alternatively, the contact surface can also be formed by making the immune cells of the object of measurement contact a substance different from the immune cells of the object of measurement.

[0065] In this specification, "immune cells of the measurement object" refers to: a cell type responsible for the mechanism of identifying self and non-self and excluding non-self. Although not limited, the immune cells of the measurement object can be T cells, natural killer (NK) cells, and mixtures thereof. It should be noted that antigen presenting cells are not included in the immune cells of the measurement object described in this specification, but are included in the "substances other than immune cells of the measurement object" described later.

[0066] In this specification, "substance different from immune cells of the measurement object" refers to: a substance that can provide a contact surface to the immune cells when in contact with the immune cells. The substance different from the immune cells of the measurement object can be either a non-biological material or a biological material. Although not limited, the substance as a non-biological material can be a container, a multi-well plate, a slide, beads, etc. Although not limited, the substance as a biological material can be allogeneic cells, antigen-presenting cells, cancer cells, etc. Allogeneic cells, antigen-presenting cells and cancer cells can be cell strains. In addition, allogeneic cells and cancer cells can also be in the form of tissues containing them. "Allogeneic cells" are cells that are not your own, and can be T cells, natural killer (NK) cells and mixtures thereof.

[0067] In the present embodiment, the method of contacting the immune cells of the object of measurement with the substance different from the immune cells of the object of measurement is not particularly limited. For example, the immune cells of the object of measurement can be added to the holes of a container or a multi-well plate together with the culture medium to make them precipitate, so as to make them contact. At this time, the container or multi-well plate added with the immune cells of the object of measurement can also be centrifuged. In addition, the immune cells can also be placed on a slide and made to contact. In a further embodiment, when the substance different from the immune cells of the object of measurement is a granular state such as beads, allogeneic cells, antigen presenting cells, cancer cells, etc., it is also possible to make them contact by adding these substances to the immune cells of the object of measurement. The contact surface can be formed by maintaining the state in which the immune cells of the object of measurement are in contact with the substance different from the immune cells of the object of measurement. As the time for maintaining the contact state, for example, more than 2 minutes and less than 60 minutes, preferably more than 10 minutes and less than 45 minutes, more preferably more than 20 minutes and less than 40 minutes, and particularly preferably 30 minutes.

[0068] In this specification, "surface antigen" refers to proteins, sugar chains, lipids and combinations thereof present on the cell membrane. Regarding the type of surface antigen, there is no particular limitation as long as there is a capture substance that can bind to the surface antigen or such a capture substance can be produced. Preferred surface antigens are molecules that constitute immune synapses of immune cells. As such molecules, for example, TCRα / β, CD3, CD28, CD40L (CD40ligand), OX40, CTLA4 (cytolytic Tlymphocyteassociated antigen-4), PD-1 (programmed cell death-1) and ICOS (inducibleco-stimulatory molecule) can be cited. The co-stimulatory molecule described later is also one of the surface antigens of immune cells.

[0069] In this embodiment, the immune cell of the measurement object is at least one selected from T cells and NK cells. In a further embodiment, the immune cell of the measurement object is a T cell or a NK cell. In a preferred embodiment, the immune cell of the measurement object is a T cell.

[0070] Although not limited, the T cells may be helper T cells (also known as "CD4 + T cells”), suppressor T cells, regulatory T cells, cytotoxic T lymphocytes (CTLs), also known as “CD8 +The term "T cells" refers to effector T cells such as T cells ("T cells"), natural T cells, and genetically modified cells. Although not limited to this, effector T cells may be cells activated in vivo or in vitro. These T cells may be used alone or in the form of T cell subsets containing two or more.

[0071] In this embodiment, the immune cells of the object of measurement can be prepared from a biological sample. For example, immune cells can be separated from blood (e.g., peripheral blood and umbilical cord blood) or bone marrow fluid using a known cell separation method. As such a cell separation method, a method for separating desired cells from a biological sample containing cells based on the size, agglutination and / or specific gravity of the cells can be cited. For example, if a commercially available cell sorter is used, cells can be separated based on the size or agglutination of the cells. In addition, cells can be separated based on the specific gravity of the cells using a specific gravity gradient centrifugation method. In this embodiment, cells separated from a biological sample can be cultured and proliferated according to conventional methods. In a further embodiment, immune cells can be used in the form of a biological sample, such as blood (e.g., peripheral blood and umbilical cord blood) or bone marrow fluid. Although not limited, the biological sample can be a sample obtained from a mammal, such as a human and a non-human mammal (e.g., companion animals such as dogs and cats; livestock animals such as horses and cattle).

[0072] In a further embodiment, the immune cells of the measurement object may be present in the sample. Therefore, this embodiment provides a method for implementing the method of the present invention on a sample containing the immune cells of the measurement object. More specifically, this embodiment provides a method for measuring the immunoresponsiveness of a sample containing immune cells, which comprises: (i) a process of contacting the immune cells in the aforementioned sample with an immunostimulatory factor; (ii) a process of causing the immune cells in the aforementioned sample to form a contact surface with a substance different from the aforementioned immune cells; (iii) a process of contacting the immune cells in the aforementioned sample with a capture body that can bind to the surface antigen of the contact surface and can generate an optical signal; (iv) a process of detecting the optical signal generated by the aforementioned capture body; and (v) a process of determining whether the immune cells in the aforementioned sample that have been released from the contact surface are immunostimulatory responsive based on the detected optical signal. The sample containing immune cells can be the above-mentioned biological sample.

[0073] In this embodiment, step (v) measures the value of the distribution of the surface antigens bound to the captured body in the immune cells based on the optical signal detected in step (iv), and compares the measured value with the threshold value, so that it can be determined whether the aforementioned immune cells have immunostimulatory responsiveness. When the captured body contains a fluorescent substance, in step (v), a fluorescent image can also be obtained based on the fluorescent signal detected in step (iv), and the determination is made based on the fluorescent image. It is also possible to measure the area showing the fluorescent signal reflecting the distribution of the surface antigen in the fluorescent image, and calculate the proportion of immunostimulatory responsive cells in the aforementioned sample based on the area. Then, the proportion is compared with the proportion of immunostimulatory responsive immune cells obtained by standardizing the sample from a healthy person, so as to make a determination. For example, when the aforementioned proportion of the sample is a value higher than the proportion at the time of standardization, it can be determined that the immunoresponsiveness of the sample is high or there is immunoresponsiveness.

[0074] In this specification, "immune stimulation" of immune cells includes: contacting immune cells with immunostimulatory factors; and causing the immune cells to form a contact surface with a substance different from the immune cells. In this embodiment, immunostimulation can be performed by contacting immune cells with a separate immunostimulatory factor. In a further embodiment, immunostimulation can be performed by contacting immune cells with immunostimulatory factors present on the aforementioned substance. In this case, the immune cells are in contact with the aforementioned substance while contacting the immunostimulatory factors.

[0075] In this specification, "immunostimulatory factor" refers to: a substance that acts on an immune cell and can induce the activation of the immune cell. The activation of immune cells refers to, for example, the proliferation of immune cells, the hyperactivity of their motility, or the secretion of cytokines. Although not limited, the immunostimulatory factor can be an immunostimulatory antibody, an immunostimulatory peptide, a major histocompatibility molecule (MHC molecule), and various immunostimulatory factors present on allogeneic cells, antigen-presenting cells, and cancer cells. The immunostimulatory factor can be used alone or in combination of two or more, or can be used in the form of an immunostimulatory factor alone or in the form of a complex with other substances or substances. The immunostimulatory factor can be, for example, in the form of a complex of an MHC molecule and an antigen peptide; in the form fixed on non-biological materials such as beads and containers; in the form presented to an antigen-presenting cell.

[0076] In this embodiment, the immunostimulatory factor is at least one selected from an immunostimulatory antibody, an immunostimulatory peptide, an MHC molecule, and a complex of an MHC molecule and an antigenic peptide. In a further embodiment, the immunostimulatory factor can be an immunostimulatory antibody, an immunostimulatory peptide, an MHC molecule, or a complex of an MHC molecule and an antigenic peptide. In a specific embodiment, the immunostimulatory factor is an immunostimulatory antibody.

[0077] In this specification, "immunostimulatory antibody" refers to an antibody that can specifically stimulate immune cells, thereby inducing the activation of immune cells. Although not limited, the immunostimulatory antibody can be an anti-TCRα / β antibody, an anti-CD3 antibody, an anti-CD40L antibody, an anti-OX40 antibody, an anti-CD2 antibody, an anti-CD28 antibody, an anti-CD137 antibody, an anti-CTLA4 antibody, an anti-PD-1 antibody, an anti-ICOS antibody, and an anti-integrin antibody (anti-CD2 antibody). These immunostimulatory antibodies can be used alone or in combination of two or more. The immunostimulatory antibody can be used in the form of a single antibody, or in a state of being solid-phased on other substances, such as beads, multi-well plates, slides, containers and other non-biological materials.

[0078] In one embodiment, the immunostimulatory antibody is at least one selected from anti-TCRα / β antibodies, anti-CD3 antibodies, anti-CD40L antibodies, anti-OX40 antibodies, anti-CD2 antibodies, anti-CD28 antibodies, anti-CD137 antibodies, anti-CTLA4 antibodies, anti-PD-1 antibodies, anti-ICOS antibodies, and anti-integrin antibodies (anti-CD2 antibodies). In a further embodiment, the immunostimulatory antibody is at least one selected from anti-CD3 antibodies and anti-CD28 antibodies. In a specific embodiment, the immunostimulatory antibody is an anti-CD3 antibody and an anti-CD28 antibody.

[0079] The antibody is not particularly limited and may be either a monoclonal antibody or a polyclonal antibody. In addition, the antibody may be an antibody fragment, a single-chain antibody, or a derivative thereof.

[0080] In the present specification, "immunostimulatory peptide" refers to a peptide that can stimulate immune cells and induce activation of immune cells. The immunostimulatory peptide is, for example, an antigenic peptide (antigenic peptide of tuberculosis bacteria, etc.).

[0081] In this embodiment, the immunostimulatory peptide is an antigenic peptide. One antigenic peptide may be used alone or in combination of two or more antigenic peptides. In a specific embodiment, the antigenic peptide may be used in the form of a complex with an MHC molecule presented on the MHC molecule.

[0082] In this specification, "major histocompatibility molecules (MHC molecules)" refer to molecules of alloantigenicity that are responsible for causing the strongest rejection reaction during allogeneic transplantation of organs, tissues and cells. In humans, HLA molecules are equivalent to MHC molecules, and in mice, H-2 molecules are equivalent to MHC molecules. MHC molecules include class I molecules and class II molecules. MHC class I molecules are expressed in almost all nucleated cells, form complexes with peptides produced in the cells and present them to the receptors of CTLs (CD8 positive T cells). MHC class II molecules are only expressed in so-called antigen presenting cells such as macrophages and B cells, and present peptides from foreign antigens to the receptors of helper T cells (CD4 positive T cells).

[0083] In this embodiment, when the immunostimulatory factor is an MHC molecule or a complex of an MHC molecule and an antigenic peptide, the MHC molecule can be any of an MHC class I molecule and an MHC class II molecule. The MHC molecule or the complex of an MHC molecule and an antigenic peptide can also be used in the form of a cell membrane present in an antigen presenting cell, a cancer cell, etc.

[0084] In this specification, "a capture body that binds to a surface antigen and can generate an optical signal" includes a substance that binds to a surface antigen of an immune cell and a substance that can generate an optical signal. Although not limited to the substance that binds to the surface antigen, examples thereof include antibodies, antibody fragments, single-chain antibodies, and aptamers that bind to the surface antigen of an immune cell. In this embodiment, the capture body includes a substance that can generate an optical signal and an antibody, antibody fragment, single-chain antibody, or aptamer that binds to a surface antigen.

[0085] The substance that is combined with the epitope antigen can be directly combined with the epitope antigen, or it can be indirectly combined with the epitope antigen. In this specification, "indirectly combined with the epitope antigen" means: combined with the epitope antigen via one or more molecules. Although not limited, the substance that is indirectly combined with the epitope antigen is combined with other substances that are combined with the epitope antigen. That is, the substance that is indirectly combined with the epitope antigen is indirectly combined with the epitope antigen via this other substance.

[0086] Although not limited, the substance capable of generating an optical signal may be a fluorescent substance. As the fluorescent substance, known fluorescent pigments, fluorescent proteins, etc. may be used without particular limitation. Although not limited, the substance capable of generating an optical signal may be chemically bound to a capture body bound to a surface antigen. As such a capture body, for example, a fluorescently labeled antibody directly bound to a surface antigen may be cited (see Figure 1 b and c).

[0087] In this embodiment, the capture body that is combined with the surface antigen and can generate an optical signal includes a substance that directly binds to the surface antigen and a substance that can generate an optical signal. In this embodiment, although not limited, the capture body can be an antibody, antibody fragment, single-chain antibody, or aptamer that is combined with a substance that can generate an optical signal and directly binds to the surface antigen.

[0088] Alternatively, the capture body may include a substance that indirectly binds to the surface antigen and a substance that can generate an optical signal. In this embodiment, the capture body may be an antibody, antibody fragment, single-chain antibody, or aptamer that is bound to a substance that can generate an optical signal and is bound to other substances that bind to the surface antigen. Although not limited, for example, an antibody that specifically binds to the surface antigen is used as a primary antibody and is bound to the surface antigen, and an antibody that specifically binds to the primary antibody is used as a secondary antibody and is bound to it. Here, the substance that can generate an optical signal is bound to the secondary antibody. In this example, the secondary antibody becomes a capture body that is bound to the surface antigen and can generate an optical signal (refer to Figure 1 Alternatively, the primary antibody may be an immunostimulatory antibody.

[0089] The surface antigen bound by the above-mentioned capture body can be a co-stimulatory molecule of an immune cell. In this specification, "co-stimulatory molecule" refers to: in addition to being able to give an antigen-specific signal mediated by a T cell receptor (TCR) when an immune cell is activated, it is also possible to give a substance present in a T cell for an auxiliary signal for activating a T cell to a T cell. In this specification, when present in NK cells, "co-stimulatory molecule" includes a substance that can give an auxiliary signal for activating a NK cell to a NK cell in addition to including a ligand-specific signal of a target cell mediated by an activation receptor of a NK cell. Although not limited, co-stimulatory molecules can be CD28, OX40, CD40L, CTLA4, PD-1 and ICOS.

[0090] In this embodiment, the substance different from the immune cell is at least one selected from a biological material, such as an allogeneic cell, an antigen presenting cell, and a cancer cell. In a further embodiment, the substance different from the immune cell is an allogeneic cell, an antigen presenting cell, or a cancer cell. In a specific embodiment, the substance different from the immune cell is an allogeneic cell. In a specific embodiment, the substance different from the immune cell is an antigen presenting cell. In a specific embodiment, the substance different from the immune cell is a cancer cell.

[0091] When using cells other than immune cells such as allogeneic cells, antigen presenting cells, cancer cells, etc. as substances different from immune cells, the immunostimulatory factors in step (i) are various immunostimulatory factors such as MHC molecules present on the aforementioned cells. For example, when the substance different from immune cells is allogeneic cells, although not limited, the immunostimulatory factors at least include MHC molecules. When the substance different from immune cells is antigen presenting cells, although not limited, the immunostimulatory factors at least include a complex of MHC class II molecules and antigen peptides. When the substance different from immune cells is cancer cells, although not limited, the immunostimulatory factors at least include a complex of MHC class I molecules and cancer antigen peptides.

[0092] Step (i) comprises: contacting the immune cells with at least one substance different from the immune cells selected from allogeneic cells, antigen presenting cells and cancer cells, thereby contacting the immune cells with the immunostimulatory factors on the substance. Step (ii) comprises: maintaining the contact state between the immune cells and the substance in step (i), thereby forming a contact surface between the immune cells and the substance.

[0093] In the present embodiment, the material different from the immune cell is preferably a non-biological material, such as an apparatus commonly used in the field of life sciences. As such an apparatus, a container, a porous plate, a slide glass, or a bead, etc. can be cited. As a container, for example, a dish, a bottle, a tube, etc. can be cited. According to this embodiment, the aforementioned substance is a non-biological material, and therefore has the advantages of being easy to handle and being able to be kept for a long time in addition compared with using a biological material as the aforementioned substance. In a specific embodiment, the container, porous plate, slide glass, or beads as the aforementioned substance include an immunostimulatory factor on its surface. For example, when the immunostimulatory factor is an immunostimulatory antibody, the antibody can be solid-phased on the surface of the container, porous plate, slide glass, or bead. Alternatively, the container, porous plate, slide glass, or beads as the aforementioned substance can include an immunostimulatory factor on its surface.

[0094] In the present embodiment, after the immunostimulatory factor is contacted with the immune cell in step (i), the substance different from the aforementioned immune cell in step (ii) may also contain further immunostimulatory factors on its surface. In this embodiment, for the purpose of additional immunostimulation, a substance having an immunostimulatory factor of the same or different species as the immunostimulatory factor of step (i) on its surface may be used in step (ii). For example, antibodies against costimulatory molecules may be used as immunostimulatory factors in step (i), and multiporous plates with anti-CD3 antibodies immobilized on the hole surface may be used as substances different from immune cells in step (ii). Alternatively, the aforementioned substance in step (ii) may also not contain further immunostimulatory factors on its surface.

[0095] In the present embodiment, when an immunostimulatory antibody is used as the immunostimulatory factor of step (i), the immunostimulatory factor can also be shared as the capture body of step (iii). In this embodiment, the immunostimulatory factor is, for example, a complex labeled with a substance capable of generating an optical signal on anti-CD28 antibodies, anti-CD3 antibodies, anti-CD40L antibodies, anti-OX40 antibodies, anti-CTLA4 antibodies, anti-PD-1 antibodies, anti-ICOS antibodies, their antibody fragments, single-chain antibodies, and a combination of more than one of them. If such a labeled immunostimulatory antibody is used, the immunostimulation of immune cells in step (i) and the capture of the surface antigens of the immune cells in step (iii) can be performed simultaneously.

[0096] Therefore, a further embodiment of the present invention provides a method for determining the immunostimulatory responsiveness of immune cells, which comprises: (i) a process of contacting immune cells with immunostimulatory factors; (ii) a process of causing immune cells to form a contact surface with a substance different from the aforementioned immune cells; (iv) a process of detecting an optical signal generated by the aforementioned capture body; and (v) a process of determining whether the aforementioned immune cells have immunostimulatory responsiveness based on the detected optical signal, wherein the aforementioned immunostimulatory factor is an immunostimulatory antibody labeled with a substance capable of generating an optical signal. According to this embodiment, the immunostimulatory factor acts as a capture body that binds to a surface antigen in an immune cell and can generate an optical signal, thereby reducing the number of reagents and the number of processes. Therefore, this embodiment has advantages in terms of cost and labor.

[0097] In the present embodiment, the immunostimulatory factor of step (i) may be present on a substance different from the immune cells in step (ii). In this embodiment, the immunostimulatory factor is preferably a complex of molecules such as immunostimulatory antibodies, immunostimulatory peptides, MHC molecules, or MHC molecules and antigenic peptides. Although not limited, the aforementioned immunostimulatory factor can be immobilized on a container, a multi-well plate, a slide glass, or a bead. For example, when the immunostimulatory factor is an immunostimulatory antibody or an immunostimulatory peptide, the immobilization method is not particularly limited, and for example, it can be physical adsorption. Alternatively, if the immunostimulatory factor is modified with biotin and avidin (or streptavidin) is fixed on the surface of a substance different from immune cells, the immunostimulatory factor can be immobilized on the aforementioned substance by the combination of biotin and avidin (or streptavidin). In addition, when the immunostimulatory factor is a complex of MHC molecules or MHC molecules and antigenic peptides possessed by allogeneic cells, antigen presenting cells, cancer cells, etc., the cells can be adhered or accumulated on a container, a multi-well plate, a slide glass, or a bead. In this embodiment, the immunostimulatory factors on the aforementioned substance can contact with immune cells and induce activation of the aforementioned immune cells. According to this embodiment, step (i) comprises: contacting immune cells with the aforementioned substance having the immunostimulatory factors immobilized on the surface, thereby contacting the aforementioned immune cells with the immunostimulatory factors on the aforementioned substance. Step (ii) maintains the contact state of the aforementioned immune cells and the aforementioned substance in step (i), thereby enabling the aforementioned immune cells to form a contact surface with the aforementioned substance.

[0098] Therefore, a further embodiment of the present invention provides a method for determining the immunostimulatory responsiveness of immune cells, which comprises: (i) a process of contacting immune cells with immunostimulatory factors on a substance different from the aforementioned immune cells; (ii) a process of forming a contact surface between the immune cells and the aforementioned substance; (iii) a process of contacting the aforementioned immune cells with a capture body that binds to a surface antigen and can generate an optical signal; (iv) a process of detecting the optical signal generated by the aforementioned capture body; and (v) a process of determining whether the aforementioned immune cells whose contact surface has been released have immunostimulatory responsiveness based on the detected optical signal. According to this embodiment, only by contacting the immune cells with a substance having immunostimulatory factors solid-phased on the surface, steps (i) and (ii) can be performed substantially simultaneously. Therefore, this embodiment has the advantage of being able to reduce the number of steps.

[0099] Figure 1 b is a diagram schematically showing steps (i) to (iii) of the above embodiment. Figure 1 b shows that the immunostimulatory factor is an immunostimulatory antibody, the substance different from the immune cells is a plate, and the immunostimulatory antibody is immobilized on the plate. Figure 1As shown in FIG. 1 , by bringing immune cells into contact with a plate having an immunostimulatory antibody immobilized thereon, immune stimulation using the antibody (step (i)) and formation of a contact surface (step (ii)) can be substantially performed simultaneously. Next, in step (iii), a capture body that directly binds to the surface antigen and can generate an optical signal is brought into contact with the immune synapse on the immune cell. In this example, the contact surface is released in step (iii), but the immune cell and the capture body may be brought into contact with each other while maintaining the contact surface. Figure 1 b is an illustration of steps (i) to (iii) according to one embodiment of the present invention, but the present invention is not limited to this illustration.

[0100] In a further embodiment, although not limited, the immunostimulatory factors can also be immobilized on beads. For example, if beads immobilized with immunostimulatory antibodies are used, the stimulation of immune cells and the separation of immune cells can be performed simultaneously. When immune cells are contained in a biological sample, if the above-mentioned beads are added to the sample, the immune cells in the sample are captured on the beads by the immunostimulatory antibodies. The biological sample containing the beads is then centrifuged to separate the immune cells from the sample. If the beads are magnetic beads, the biological sample containing the beads can be magnetically separated. At this time, the separated immune cells are immunostimulated by the immunostimulatory antibodies on the beads. In this embodiment, the contact surface between the immune cells and the above-mentioned beads can be released before and during the measurement.

[0101] Figure 1 c is a diagram schematically showing steps (i) to (iii) of the above embodiment. Figure 1 c shows that the immunostimulatory factor is an immunostimulatory antibody, the substance different from the immune cell is a bead, and the immunostimulatory antibody is immobilized on the bead. Figure 1 As shown in Figure 3, by bringing immune cells into contact with beads having an immunostimulatory antibody immobilized thereon, immune stimulation using the antibody (step (i)) and formation of a contact surface (step (ii)) can be substantially performed simultaneously. Next, in step (iii), a capture body that directly binds to the surface antigen and can generate an optical signal is brought into contact with the immune synapse in the immune cell. In this example, the contact surface has not been released in step (iii), but the immune cell whose contact surface has been released can also be brought into contact with the capture body. Figure 1 c is an illustration of steps (i) to (iii) according to one embodiment of the present invention, but the present invention is not limited to this illustration.

[0102] In this specification, "immune cells whose contact surfaces have been released" refers to immune cells in a state where the contact area formed between the immune cells and a substance other than the immune cells has disappeared. In other words, it refers to a state where the immune cells are separated from the substance other than the immune cells. Although not limited to the method of releasing the contact surface, it can be: physical treatment such as pipetting, tapping, stirring, shaking, ultrasonic treatment, etc. of a container containing immune cells and culture medium, etc.; chemical treatment using chemicals such as surfactants; and biochemical treatment using biochemical products such as enzymes.

[0103] In a preferred embodiment, the contact surface is released by physical treatment such as suction, beating, stirring, shaking, ultrasonic treatment, etc. In a specific embodiment, the above-mentioned method is suction. In this embodiment, when the contact surface is released, step (iv) can also use a flow cytometer to detect the optical signal generated by the capture body.

[0104] In this specification, "flow cytometer" refers to: a measuring device for irradiating cells in a water flow with excitation light and measuring the fluorescence and scattered light emitted by each cell, thereby measuring the size of each cell in the cell cluster and the distribution of the expression amount of membrane proteins. Flow cytometers generally have the following advantages: compared with conventional fluorescence microscope observation, it is objectively possible to measure a large number of cells in a short time (up to tens of thousands of cells / second); it can be quantitatively measured with high sensitivity, and multiple parameters can be obtained at the same time. In this specification, the flow cytometer has a flow system that can perform cell measurement in a state where cells are arranged in a liquid in a manner that the cells are measured one by one. This flow system can be realized by a fluid mechanics-based component called a flow cell. The flow cell is usually realized by hydrodynamic focusing, which uses a sheath flow of a sheath fluid such as a sample flow containing a sample liquid, but is not limited to this. For example, it can also be realized by microcapillaries and acoustic focusing.

[0105] In the present invention, commercial products can be used as flow cytometers without particular limitation. In the present specification, flow cytometers include so-called "imaging flow cytometers", which have a shooting unit using a camera and can obtain images of cells flowing in a liquid. As described in Embodiment 1 described later, multiple parameters including the size and aspect ratio of cells can be obtained based on the captured cell images using an imaging flow cytometer. In the present specification, flow cytometers include both imaging flow cytometers and flow cytometers that do not perform shooting. In a preferred embodiment, the flow cytometer is an imaging flow cytometer. In the present invention, flow cytometers can be used without particular limitation as long as they are commercial products.

[0106] In this embodiment, in step (iv), when the substance other than immune cells is at least one selected from allogeneic cells, antigen presenting cells, and cancer cells or beads, the complex of immune cells, substances other than immune cells, and capture bodies can be measured by flow cytometry or imaging cytometry to obtain an optical signal generated by the capture bodies in the complex. Alternatively, when the substance other than immune cells is a container, a multi-well plate, or a slide, the contact surface between the immune cells and the substance other than immune cells can be released, and the complex of immune cells and capture bodies can be measured by flow cytometry or imaging cytometry to obtain an optical signal generated by the capture bodies in the complex.

[0107] In this specification, "imaging cytometer" refers to a cell measurement device with high statistical accuracy that can obtain and quantitatively measure the fluorescence images or scattered light and transmitted light images of a large number of cells (thousands to millions) in a short time (several seconds to several minutes). It is a measurement device that can extract information about each cell through cell image processing. In the present invention, the imaging cytometer can use commercially available products without particular limitation.

[0108] In a further embodiment, a method for evaluating the activation state of the immune system in a subject is provided. This embodiment includes: performing the above-mentioned process of the present invention on a biological sample containing immune cells obtained from the aforementioned subject. That is, the evaluation method of this embodiment includes: (i) a process of contacting the immune cells in the biological sample with an immunostimulatory factor; (ii) a process of causing the immune cells in the biological sample to form a contact surface with a substance different from the aforementioned immune cells; (iii) a process of contacting the immune cells in the biological sample with a capture body that binds to the surface antigen in the immune cells and can generate an optical signal; (iv) a process of detecting the optical signal generated by the aforementioned capture body; and (v) a process of determining whether the aforementioned sample is immunostimulatory responsive based on the detected optical signal.

[0109] Furthermore, the evaluation method of this embodiment may be: the capture body contains a fluorescent substance, and in step (v), a fluorescent image is obtained based on the fluorescent signal detected in step (iv), and the judgment is made based on the fluorescent image. It is also possible to measure the area showing the fluorescent signal reflecting the distribution of the surface antigen in the fluorescent image, and calculate the proportion of immune cells with immunostimulation responsiveness in the aforementioned biological sample based on the area. Then, the proportion is compared with the proportion of immune cells with immunostimulation responsiveness obtained by standardizing the biological sample from a healthy person, so as to make a judgment. In this embodiment, for example, when the aforementioned proportion of the sample is a value higher than the standardized proportion, it can be judged that the immune responsiveness of the sample is high or there is immune responsiveness. Alternatively, in this evaluation method, the proportion of immune cells with immunostimulation responsiveness in the biological sample containing immune cells obtained from the object can also be compared with the proportion of previous biological samples from the same object. For example, when the aforementioned proportion of the sample is a value higher than the previous proportion, it can be judged that the immune responsiveness of the object is improved or there is immune responsiveness.

[0110] Another aspect of the present invention relates to a method for measuring the immunostimulatory responsiveness of an immune cell, using a capture body that binds to a co-stimulatory molecule in the immune cell and is capable of generating an optical signal.

[0111] In the method of this embodiment, a capture body that binds to a co-stimulatory molecule in an immune cell and can generate an optical signal is used as the capture body, and the description of the method of the above embodiment is the same as that of the above embodiment. Examples of the co-stimulatory molecule bound by the capture body include CD28, OX40, CD40L, CTLA4, PD-1, and ICOS.

[0112] Another embodiment of the present invention relates to a cell analyzer for analyzing the activation state of immune cells. The embodiments of this embodiment of the present invention are described with reference to the accompanying drawings. It should be noted that the following description is illustrative rather than restrictive and does not limit the invention described in the accompanying claims.

[0113] [Embodiment 1 of Cell Analyzer]

[0114] Reference Figure 2 The cell analysis device 1 includes a measuring device 2 and an information processing device 3. The measuring device 2 uses a flow cytometer to optically measure a sample of a complex of a capture body and an immune cell that binds to a surface antigen of an immune cell and can generate an optical signal. The information processing device 3 analyzes the measurement results obtained by the measuring device 2 and displays the analysis results on a display unit 320.

[0115] Reference Figure 3The measuring device 2 includes an introduction unit 201, an imaging unit 203, a signal processing unit 210, a CPU 204, a communication interface 205, and a memory 206. The signal processing unit 210 includes an analog signal processing unit 211, an A / D converter 212, a digital signal processing unit 213, and a memory 214.

[0116] In this embodiment, the introduction unit 201 includes a container and a pump (not shown). The sample in the container is supplied to the flow cell 203c of the imaging unit 203 together with the sheath liquid by the pump (see Figure 4 ). The introduction unit 201 supplies the sample to the imaging unit 203 according to the instruction of the CPU 204. In the first embodiment, the measurement sample uses a complex obtained by: after giving immune stimulation including contact with anti-CD28 antibodies to cloned T cells from human peripheral blood, the cells are fixed and stained twice with phycoerythrin (PE)-labeled anti-mouse IgG antibodies bound to the aforementioned anti-CD28 antibodies.

[0117] The imaging unit 203 irradiates the sample supplied from the introduction unit 201 with laser light and images the complex, and outputs the image information of the generated transmitted light image and fluorescent image in the form of electrical signals to the analog signal processing unit 211. The analog signal processing unit 211 amplifies the electrical signal output from the imaging unit 203 according to the instruction of the CPU 204 and outputs it to the A / D converter 212.

[0118] The A / D converter 212 converts the electrical signal amplified by the analog signal processing unit 211 into a digital signal and outputs it to the digital signal processing unit 213. The digital signal processing unit 213 performs predetermined signal processing on the digital signal output by the A / D converter 212 according to the instruction of the CPU 204, and generates measurement data. The generated measurement data is stored in the memory 214.

[0119] The measurement data stored in the memory 214 includes a transmitted light image and a fluorescent image based on the transmitted light and fluorescent light generated when the complex passes through the flow cell 203 c .

[0120] The CPU 204 outputs the measurement data stored in the memory 214 to the communication interface 205. The CPU 204 receives a control signal from the information processing device 3 via the communication interface 205, and controls each unit of the measurement device 2 according to the control signal.

[0121] The communication interface 205 transmits the measurement data outputted from the CPU 204 to the information processing device 3, and receives the control signal outputted from the information processing device 3. The memory 206 is used as a working area of ​​the CPU 204.

[0122] Reference Figure 4The imaging unit 203 includes: light sources 203a and 203b; a flow cell 203c; condenser lenses 203d and 203e; an objective lens 203f; an optical unit 203g; a condenser lens 203h; and a camera 203i.

[0123] In this embodiment, the light source 203a is a semiconductor laser light source. The light irradiated by the light source 203a is a laser with a wavelength of λ1. The focusing lens 203d focuses the light irradiated by the light source 203a and guides it to the sample in the flow cell 203c. The light with a wavelength of λ1 irradiated by the light source 203a irradiates the sample passing through the inside of the flow cell 203c, thereby generating transmitted light with a wavelength of λ1 by the immune cells.

[0124] The light source 203b is a semiconductor laser light source. The light irradiated by the light source 203b is a laser with a wavelength of λ2. In this embodiment, the wavelength λ2 is about 488 nm. The focusing lens 203e focuses the light irradiated by the light source 203b and guides it to the sample in the flow cell 203c. The light with a wavelength of λ2 irradiated by the light source 203b irradiates the sample passing through the inside of the flow cell 203c, thereby generating fluorescence with a wavelength of λ3 by PE.

[0125] The objective lens 203f focuses the transmitted light of wavelength λ1 and the fluorescence of wavelength λ3. The optical unit 203g has a structure in which two dichroic mirrors are combined. The two dichroic mirrors of the optical unit 203g reflect the transmitted light of wavelength λ1 and the fluorescence of wavelength λ3 at different angles to the light receiving surface 203ia (see FIG. 201 ) of the camera 203i described later. Figure 5 ) are separated. The condensing lens 203h condenses the transmitted light of wavelength λ1 and the fluorescence of wavelength λ3. The camera 203i receives the transmitted light of wavelength λ1 and the fluorescence of wavelength λ3, and outputs the image information of the sample in the flow cell 203c to the analog signal processing unit 211 in the form of an electrical signal. The camera 203i can be a TDI (Time Delay Integration) camera. If it is a TDI camera, it can obtain higher-precision image information.

[0126] like Figure 5 As shown, the camera 203i receives the transmitted light of wavelength λ1 and the fluorescence of wavelength λ3 in the light receiving areas 203ib and 203ic on the light receiving surface 203ia, respectively. The light receiving surface 203ia is the light receiving surface of the imaging element such as the CMOS image sensor configured in the camera 203i. The position of the light irradiated to the light receiving surface 203ia moves in the light receiving areas 203ib and 203ic, respectively, as shown by the arrows as the sample moves in the flow cell 203c. Thus, the two beams of light are separated on the light receiving surface 203ia by the optical unit 203g, so that the CPU 204 can extract the signal corresponding to each light from the image signal output by the camera 203i.

[0127] Reference Figure 6 The information processing device 3 is composed of a main body 300, an input unit 310, and a display unit 320. The main body 300 has a CPU 301, a ROM 302, a RAM 303, a hard disk 304, a reading device 305, an input / output interface 306, an image output interface 307, and a communication interface 308.

[0128] The CPU 301 executes computer programs stored in the ROM 302 and computer programs loaded in the RAM 303. The RAM 303 is used to read computer programs recorded in the ROM 302 and the hard disk 304. The RAM 303 can also be used as a work area of ​​the CPU 301 when executing these computer programs.

[0129] The hard disk 304 stores various computer programs such as an operating system and application programs for the CPU 301 to execute, and data used for executing the computer programs. In addition, the hard disk 304 stores measurement data received from the measurement device 2 .

[0130] The hard disk 304 stores: a program for measuring the number of immune cells contained in the sample, the size of the cells, the aspect ratio, the area showing the fluorescence signal, the total fluorescence signal intensity in the aforementioned area, etc., based on the measurement data and performing the sample analysis; a display program for displaying the analysis results on the display unit 320. By storing these programs, the analysis processing and display processing described later are performed. That is, the CPU 301 is given the ability to execute the following programs through the aforementioned programs. Figure 7 b's processing function.

[0131] The reading device 305 is constituted by a CD drive, a DVD drive, etc. The reading device 305 can read computer programs and data recorded on a recording medium such as a CD or a DVD.

[0132] The input / output interface 306 is connected to an input unit 310 composed of a mouse, a keyboard, etc., and a user uses the input unit 310 to input instructions to the information processing device 3. The image output interface 307 is connected to a display unit 320 composed of a display, etc., and outputs an image signal corresponding to the image data to the display unit 320. The display unit 320 displays an image based on the input image signal.

[0133] The information processing device 3 can receive the measurement data transmitted from the measurement device 2 via the communication interface 308 . The received measurement data is stored in the hard disk 304 .

[0134] Figure 7 This is a flow chart showing the control of the CPU 204 of the measurement device 2 and the CPU 301 of the information processing device 3 . Figure 7a is a flowchart showing the control process by the CPU 204 of the measuring device 2, Figure 7 b is a flowchart showing the control process by the CPU 301 of the information processing device 3 .

[0135] For details about the control process of the CPU 301 of the information processing device 3, please refer to Figure 7 b. When the CPU 301 receives a measurement start instruction from the user via the input unit 310 (step S11: YES), it transmits a measurement start signal to the measurement device 2 (step S12). Then, the CPU 301 determines whether measurement data has been received (step S13). If measurement data has not been received (step S13: NO), the process is in a standby state.

[0136] For details on the control process of the CPU 204 of the measuring device 2, please refer to Figure 7 a. When the CPU 204 receives a measurement start signal from the information processing device 3 (step S21: YES), the measurement of the aforementioned sample is performed (step S22). In the measurement process (step S22), the measurement sample containing the complex of the aforementioned capture body and immune cells is supplied from the introduction part 201 to the flow cell 203c together with the sheath liquid, and a sample liquid flow of the measurement sample contained in the sheath liquid is formed in the flow cell 203c. The formed sample flow is irradiated with a laser beam from the light sources 203a and 203b, and a beam spot is formed in the flow cell 203c. When the aforementioned immune cells pass through the beam spot, transmitted light and fluorescence are generated. The transmitted light and fluorescence are respectively photographed by the camera 203i and converted into electrical signals.

[0137] These electrical signals are converted into digital signals by the A / D converter 212, and signal processing is performed by the digital signal processing unit 213. Thus, for each complex passing through the flow cell 203c, measurement data including a transmitted light image and a fluorescent image are obtained. The measurement data is stored in the memory 214. When the measurement of the sample is completed, the CPU 204 transmits the measurement data generated by the measurement processing to the information processing device 3 (step S23), and the processing is completed.

[0138] Refer again Figure 7 b. When the CPU 301 of the information processing device 3 receives the measurement data from the measuring device 2 (step S13: YES), the measurement data is stored in the hard disk 304, and analysis processing is performed based on the measurement data (step S14). Then, the CPU 301 displays the analysis result obtained in S14 on the display unit 320 (step S15). Then, the processing ends.

[0139] Reference Figure 8 to Figure 10 The analysis process performed by the CPU 301 of the information processing device 3 based on the measurement data will be further described. Figure 7b) is an example of step S14). Figure 8 The transmitted light image, the fluorescent image (CD28), and their superimposed image in the measurement data obtained by measuring the above immune cells are shown. Figure 8 As shown in the fluorescent image of a, when no immune stimulation is given, the co-stimulatory molecule CD28 shows the same distribution tendency on the cell membrane of immune cells. Figure 8 As shown in the fluorescent image of b, when immune stimulation is given, the costimulatory molecule CD28 tends to be locally present on the cell membrane of the immune cell. Although it is not intended to be limited in theory, it can be considered that the cell membrane portion showing the local presence of the costimulatory molecule is a contact surface formed in the immune cell with a substance different from the immune cell. That is, it can be considered that the portion showing the local presence of the costimulatory molecule represents an immune synapse formed between the cell and a substance different from it.

[0140] CPU301 is based on Figure 7 The transmitted light image and fluorescent image of all cells captured in step S22 of a are used to measure the analysis parameters ( Figure 7 b) Step S14). Although not limited thereto, the analysis parameters may include the size of the cell (number of pixels), the aspect ratio of the cell, the area in the cell showing the fluorescent signal (number of pixels), the total fluorescent signal intensity in the aforementioned area (total fluorescent signal intensity of the cell), and their ratios. Fig. 9 The CPU 301 of the information processing device 3 performs Figure 8 Metrological processing of the transmitted light image and the fluorescence image shown in b ( Figure 7 The obtained measurement data is stored in the hard disk 304. It should be noted that the meanings of the terms such as cell size, aspect ratio, area showing fluorescent signal, and total fluorescent signal intensity are as described above.

[0141] Fig.10 3 is a flowchart showing the analysis process in step S15 by the CPU 301 of the information processing device 3. The CPU 301 allocates the analysis parameters measured in step S14 to the X-axis and the Y-axis to create a two-dimensional histogram (2D scattergram), extracts and analyzes the measurement data of cells corresponding to points existing in a predetermined range.

[0142] In this embodiment, the CPU 301 first assigns the cell size to the X axis and the aspect ratio of the cell to the Y axis to create a 2D scatter plot ( Fig.11a), extract the measurement data of cells in the specified range (R1) (step S51). With respect to the range (R1), in the cell size (X axis), a gate is set to a specified range reflecting the size of a single cell; and in the aspect ratio (Y axis), a gate is set to a specified range reflecting the existence of a single cell. In this way, data from substances such as cell fragments smaller than cells and cell aggregates larger than a single cell are excluded.

[0143] Each point shown in the 2D scatter plot represents the measurement value of each cell. CPU 301 extracts the measurement data of the cell in the image that shows the measurement value located in the area shown by R1. It should be noted that in this embodiment, the points shown in the 2D scatter plot are mapped to Figure 7 When the user designates a point shown in the 2D scattergram, the CPU 301 reads the measurement data (transmitted light image and / or fluorescent image) of the cell corresponding to the point from the hard disk 304 and displays it on the display unit 320 .

[0144] For the measurement data of the cells extracted in step S51, the CPU 301 assigns the total fluorescence signal intensity of the cells to the X-axis and the sharpness of the fluorescence image (the clarity of the image) to the Y-axis to create a 2D scatter plot ( Fig.11 b), further extracting the measurement data of cells within the specified range (R2) (step S52). With respect to the range (R2), in the total fluorescence signal intensity of the cells (X axis), a gate is set in a range reflecting the expression amount of the specified amount of the co-stimulatory molecule CD28; in the sharpness (Y axis), a gate is set in a range reflecting the specified image sharpness. Thus, the data of cells expressing the specified amount of the co-stimulatory molecule CD28 are extracted, and the data of cells not expressing the specified amount of the co-stimulatory molecule CD28 are excluded.

[0145] Then, for the measurement data of the cells extracted in step S52, the CPU 301 allocates the area showing the fluorescence signal to the X axis and allocates the size of the cell to the Y axis to create a 2D scattergram ( Fig.11 c), count the number of points located in the specified range (R4) (step S53). With respect to the range (R4), in the area (X-axis) displaying the fluorescent signal, a gate is set to a specified range reflecting the local presence of the co-stimulatory molecule CD28; and in the cell size (Y-axis), a gate is set to a specified range reflecting the size of a single cell. By counting the number of points located within the range (R4), the number of immune cells responsive to immune stimulation is counted. In this embodiment, the cells corresponding to the points located in the range (R4) have a tendency to form immune synapses, and the cells are determined to be responsive to the immune stimulation in this embodiment.

[0146] CPU301 further Fig.11 c The number of points shown in the whole (with Fig.11 The number of points in the range (R2) of b is counted, and the ratio of the number of points in the range (R4) to the number of points in the range (R2) is calculated ([the number of points in the range (R4)] / [the number of points in the range (R2)]) (step S54). Thus, the ratio of immune cells that are responsive to immune stimulation among the immune cells expressing the specified amount of the costimulatory molecule CD28 is calculated.

[0147] The analysis results regarding the number of points and the ratio in each range obtained by the above analysis process are displayed on the display unit 320 .

[0148] By comparing with the ratio of immune cells with immune stimulation responsiveness obtained by, for example, standardization of biological samples from healthy people, the analysis result can be further analyzed. In this further analysis, for example, when the aforementioned ratio of the sample is a value higher than the standardized ratio, it can be determined that the immune responsiveness of the sample is high. In addition, by comparing with the ratio of immune cells with immune stimulation responsiveness of previous biological samples from the same object, the analysis result can be further analyzed. In this further analysis, for example, when the aforementioned ratio of the sample is a value higher than the previous ratio, it can be determined that the immune responsiveness of the object is improved.

[0149] Thus, in one embodiment, a cell analysis device is provided, which includes: an introduction part, which introduces a complex; a shooting part, which shoots the complex supplied by the introduction part; and an analysis part, which determines whether the immune cell is immunostimulatory responsive based on the image shot by the shooting part, wherein the complex is a complex of an immune cell and a capture body that binds to a surface antigen in the immune cell and can generate an optical signal. In a specific embodiment, the analysis part obtains a value reflecting the distribution of the surface antigen based on the image, and compares the obtained value with a threshold value to determine whether the immune cell is immunostimulatory responsive. In another embodiment, a cell analysis device is provided, wherein the capture body includes a fluorescent substance, the image includes a fluorescent image, and the value reflecting the distribution of the surface antigen includes an area of ​​a fluorescent signal reflecting the distribution of the surface antigen based on the fluorescent image, and when the area is smaller than the threshold value, it is determined that the immune cell is immunostimulatory responsive. In another embodiment, a cell analysis device is provided, wherein the image includes a transmitted light image, the analysis unit obtains a value reflecting the size of the immune cell based on the transmitted light image, and determines whether the immune cell is responsive to immune stimulation based on the value reflecting the size of the immune cell and the value reflecting the distribution of surface antigens.

[0150] The analysis process described above is merely an example of analysis process based on measurement data, and a different analysis process may be used.

[0151] [Embodiment 2 of cell analysis device]

[0152] As an alternative to step S53 of the analysis process of embodiment 1, for the measurement data extracted in step S52, CPU 301 may assign the total fluorescence signal intensity of the cells to the X-axis and the area showing the fluorescence signal to the Y-axis to create a 2D scatter plot ( Fig.11 d), and count the number of measured data within the specified range (R3). For range (R3), in the total fluorescence signal intensity of the cell (X-axis), a gate is set to reflect the range of the specified expression amount of the co-stimulatory molecule CD28 of each cell; and in the area showing the fluorescence signal (Y-axis), a gate is set to reflect the specified range of the local presence of the co-stimulatory molecule CD28. By counting the measured data in the range (R3), the number of immune cells responsive to immune stimulation is counted. In this embodiment, the cells corresponding to the points in the range (R3) have a tendency to form immune synapses, and it is determined that the cells are responsive to the immune stimulation in this embodiment.

[0153] In this embodiment, as a substitute for step S54, CPU 301 may further Fig.11 d The number of points shown in the whole (with Fig.11 b) is counted, and the ratio of the number of points in range (R3) to the number of points in range (R2) is calculated ([number of points in range (R3)] / [number of points in range (R2)]). Thus, the ratio of immune cells responsive to immune stimulation among the immune cells expressing a predetermined amount of the costimulatory molecule CD28 is calculated.

[0154] [Embodiment 3 of the cell analysis device]

[0155] As an alternative to step S53 of the analysis process of embodiment 1, for the measurement data of the cells extracted in step S52, the CPU 301 can assign the group value (class value) of the area showing the fluorescence signal to the x-axis and the degree to the Y-axis to make a histogram, and display it on the display unit 320. Further as an alternative to step S54, the CPU 301 can calculate the average value, central value, and the most frequent value of the area showing the fluorescence signal of the measurement data extracted in step S52 from the histogram. The average value, central value, and the most frequent value obtained can be compared with the average value, central value, and the most frequent value obtained by standardizing the area showing the fluorescence signal in the immune cell obtained by, for example, a biological sample from a healthy person. In this further analysis, for example, when the group value (area showing the fluorescence signal) of the sample indicating the highest frequency value is less than the standardized group value (area showing the fluorescence signal), it indicates that the co-stimulatory molecule CD28 is locally present, and the result can be determined as the sample is highly responsive to immune stimulation.

[0156] As shown in Embodiment 3, in the analysis of measurement data, the immune responsiveness of immune cells can be analyzed without creating a 2D scatter plot. However, as described in Embodiment 1, the points shown in the 2D scatter plot can map the measurement data of each immune cell. For example, by specifying each point, the measurement data including the transmitted light image and the fluorescent image of the immune cell can be displayed. Therefore, in terms of being able to perform additional detailed image-based analysis on each cell, analysis using a 2D scatter plot is a preferred analysis processing method.

[0157] [Embodiment 4 of the cell analysis device]

[0158] In the above analysis process, analysis is performed based on the captured image data, but immune cells responsive to immune stimulation may also be analyzed based on measurement data other than the captured image data. Figure 4) are used in place of the optical unit 203g and the camera 203, dichroic mirrors 203j, 203k and a forward scattered light receiving unit 203l and a fluorescence light receiving unit 203m. Light of wavelength λ1 irradiated by the light source 203a is irradiated to the sample passing through the inside of the flow cell 203c, thereby generating forward scattered light of wavelength λ1 by the immune cells. Light of wavelength λ2 irradiated by the light source 203b is irradiated to the sample passing through the inside of the flow cell 203c, thereby generating fluorescence of wavelength λ3 by the PE. It should be noted that the sample in embodiment 4 is the complex used in embodiment 1. The forward scattered light of wavelength λ1 is reflected by the dichroic mirror 203j and received by the forward scattered light receiving unit 203l. The fluorescence of wavelength λ3 is transmitted through the dichroic mirror 203j, reflected by the dichroic mirror 203k and received by the fluorescence light receiving unit 203k. The forward scattered light receiving unit 203l and the fluorescence light receiving unit 203m are photomultiplier tubes. In this example, Figure 7 The optical signal obtained in step S22 of a is a forward scattered light signal and a fluorescent signal. Figure 7 In step S14 of b, the “pulse width (w)”, “pulse area (A)” and “pulse height (H)” as analysis parameters are obtained. Fig.12 These analysis parameters are described below.

[0159] Fig.12 As an example, the cells in which the costimulatory molecule CD28 is uniformly distributed on the cell membrane are shown. Fig.12 a) and cells showing localization on the cell membrane ( Fig.12 b) Schematic diagram of the fluorescence signal detected while passing through the fluorescence signal detection area (not shown) along the arrow direction ( Fig.12 c and Fig.12 d).

[0160] In this manual, Fig.12 c and Fig.12 In the pulse of fluorescence intensity shown in d, the time when the fluorescence signal is obtained after exceeding the baseline as the threshold is called "pulse width (w)", and the fluorescence intensity when the peak of the pulse is shown in the fluorescence intensity is called "pulse height (H)". "Pulse area (A)" refers to the area between the baseline and the fluorescence signal intensity curve.

[0161] In this example, Fig.12 a cells and Fig.12 The cells in b express the same amount of co-stimulatory molecule CD28 on their cell membranes. Fig.12 c and Fig.12 d has the same pulse area (A). Here, the pulse width (w) and pulse height (H) are different due to the distribution pattern of the co-stimulatory molecules. Compared with the case where the co-stimulatory molecules are evenly distributed on the cell membrane ( Fig.12a), when the co-stimulatory molecules are localized on the cell membrane ( Fig.12 b), the pulse width (w) becomes narrower, while the pulse height (H) becomes higher ( Fig.12 c and 12d).

[0162] Fig.13 This is a schematic diagram of the case where a plurality of immune cells are measured and a 2D scattergram is prepared in this embodiment. Fig.13 a is a 2D scatter plot in which the pulse height (H) is assigned to the X-axis and the pulse width (w) is assigned to the Y-axis. Fig.13 b is a 2D scatter plot in which the pulse height (H) is assigned to the X-axis and the pulse area (A) is assigned to the Y-axis.

[0163] As mentioned above, when the co-stimulatory molecules are evenly distributed on the cell membrane ( Fig.12 a), compared with when the co-stimulatory molecules are localized on the cell membrane ( Fig.12 b), the pulse width (w) shows a tendency to become wider, and the pulse height (H) shows a tendency to become lower ( Fig.12 c and d of 12). Therefore, in Fig.13 The 2D scatter plot shown in a shows the following tendency: the data in the area surrounded by the dotted circle are data reflecting the cells in which the costimulatory molecules are uniformly distributed on the cell membrane, while the data in the area surrounded by the solid square are data reflecting the cells in which the costimulatory molecules are locally present on the cell membrane. Fig.13 The 2D scatter plot shown in b shows the following tendency: the data entering the area surrounded by the dotted squares are data reflecting cells in which the costimulatory molecules are uniformly distributed on the cell membrane, while on the other hand, the data entering the area surrounded by the solid squares are data reflecting cells in which the costimulatory molecules are locally present on the cell membrane.

[0164] Thus, in one embodiment, a cell analysis device is provided, which includes: an introduction part, which introduces a complex; a detection part, which irradiates light to the complex supplied by the introduction part and detects the optical signal generated from the complex; and an analysis part, which determines whether the immune cell is responsive to immunostimulation based on the detected optical signal, wherein the complex is a capture body that binds to the surface antigen in the immune cell and can generate an optical signal. In a specific embodiment, a cell analysis device is provided, wherein the capture body includes a fluorescent substance, the detection part detects the fluorescent signal generated by the complex, the analysis part obtains a value reflecting the distribution of the surface antigen based on the fluorescent signal, compares the obtained value with a threshold value, and thereby determines whether the immune cell is responsive to immunostimulation, wherein the value reflecting the distribution of the surface antigen includes at least one of the pulse width of the fluorescent signal from the complex, the height of the fluorescent pulse, and the pulse area.

[0165] As shown in the Examples, cells showing that the surface antigens and co-stimulatory molecules bound to the capture body are localized on the cell membrane can be judged as immune cells showing immune responsiveness. Fig.13 a and Fig.13 In b, cells showing data entering the area surrounded by a solid square can be determined as cells showing immune responsiveness. As exemplified in this embodiment, it can be understood that immune cells responsive to immune stimulation can be determined not only based on the analysis and processing of the captured image data, but also by the analysis and processing described above.

[0166] It should be noted that the analytical parameters and analytical processing shown here are only examples, and different analytical parameters can be measured by the measurement data, and different analytical processing can also be performed on the measured analytical parameters. For example, the pulse area can also be an approximate value as long as it is a value reflecting the area under the time curve of the pulse, and is not limited to the time integral value. The pulse area can be the product of the pulse width and the peak height, or it can be the area of ​​a triangle obtained by the pulse width and the peak height. In addition, in the method of measuring the time integral value, the base can also be set appropriately instead of the baseline. For example, the value exceeding a specified threshold relative to the baseline can also be used as the base.

[0167] The following specific embodiments are provided to illustrate preferred embodiments of the present invention and are not intended to limit the invention described in the appended claims. Equivalent solutions or changes, modifications or variations that are easily identified from the specific embodiments, materials, compositions and methods described in this specification are intended to be included within the scope of the present invention.

[0168] [Example]

[0169] [Example 1: Measurement of immunostimulatory responsiveness of immune cells using CD28 as an indicator]

[0170] (1) Preparation of cloned T cells

[0171] 5×10 6 CD4-positive cells (Stemcell Technologies: ST-70026) and CD8-positive cells (Funacosi: 0508-100, Cosmo Bio: PB08C-1) were inoculated into Yssel's medium (Gemini Bio-Products: 400102) supplemented with 1 μg / mL LEAF (trademark) purified anti-human CD3 antibody (Biolegend: 317315), 10 ng / mL recombinant human IL-2 (rhIL-2) (R&D systems: 202-IL-500), 0.2 μg / mL phytohemagglutinin-L (PHA) (SIGMA: L4144-5MG) and 2% human serum (SIGMA: H4522-100ML), and 1×10 6 Peripheral blood mononuclear cells and 1×10 4 After 14 days, T cells were seeded into 96-well plates at 0.3, 1.0, and 3.0 cells / well by limiting dilution and cultured until 1×10 4 The peripheral blood mononuclear cells are cultured until colonies are formed. T cells are collected from the wells where colonies have formed to obtain cloned T cells. The obtained T cells are hereinafter referred to as "prepared cloned T cells".

[0172] (2) Immobilization of Antibodies

[0173] A solution (0.01 mg / ml) of anti-CD3 antibody (BioLegend #300414 Clone: ​​UCHT1) was added to each well of a 24-well plate at 300 μl to immobilize the anti-CD3 antibody on the well surface. The resulting plate is hereinafter referred to as an "anti-CD3 antibody immobilized plate."

[0174] (3) Stimulation and determination of immune cells

[0175] Approximately 5 × 10 5The cloned T cells were prepared by adding cells in the wells at a final concentration of 0.01 mg / ml. Anti-CD28 antibody (BioLegend#302914Clone: ​​CD28.2) was then added to the cells in the wells at a final concentration of 0.01 mg / ml. The plate was centrifuged (200G, 30 seconds) and incubated at 37°C for 30 minutes. The cloned T cells were recovered from the surface of the wells by pipetting. The recovered T cells were fixed with a fixative containing 1% paraformaldehyde (PFA). The fixed T cells were immunostained with PE-labeled anti-mouse IgG antibody (BioLegend#405307). For the stained T cells (5×10 5 cells / 20 μl) and measured using Imaging FCM (Image Stream X Mark III Imaging Flow Cytometer: Amnis).

[0176] For comparison, the case where no immune stimulation was given to the cells was also studied, wherein the immune stimulation refers to the immune stimulation caused by contact with antibodies (anti-CD28 antibodies and anti-CD3 antibodies) that bind to surface antigens of T cells and contact with different substances (wells of the plate). The cloned T cells prepared in (1) above were fixed with the above-mentioned fixative without the above-mentioned immune stimulation, and then stained with anti-CD28 antibodies. The fixed T cells were stained twice with PE-labeled anti-mouse IgG antibodies and measured.

[0177] When the above immune stimulation is not given to T cells, CD28 is evenly distributed on the cell membrane in T cells (refer to Fig.14 a). When the contact surface between the T cells and the wells was released after the above immune stimulation, it was shown that CD28 was locally present on the cell membrane of the T cells (refer to Fig.14 b). Thus, even in a state where the contact surface between the T cell and the well is released after the above-mentioned immune stimulation, the immune synapse can be detected by using the costimulatory molecule CD28 as an indicator.

[0178] (4) Data analysis

[0179] The measurement data of the sample containing the above-mentioned non-immune stimulated T cells measured using Imaging FCM ( Fig.15 a and b) and the measurement data of the sample containing the above-mentioned immune stimulated T cells ( Fig.15 c and d) were subjected to multi-parameter analysis.

[0180] The detailed measurement and analysis processing has been described in Embodiment 1. In brief, image analysis is performed based on the transmitted light image and the fluorescent image (CD28) measured using Imaging FCM ( Fig. 9), measure the cell size (number of pixels), the cell aspect ratio, the area showing the fluorescence signal in the cell (number of pixels), and the total fluorescence signal intensity in the aforementioned area (total fluorescence signal intensity of the cell). First, the cell size is assigned to the X-axis and the cell aspect ratio is assigned to the Y-axis to create a 2D scatter plot, and the measurement data of the cell corresponding to the point located in the specified range (R1) is extracted ( Fig.10 Then, for the measurement data within the range (R1), the measurement data corresponding to the point where the total fluorescence signal intensity of the cell is within the specified range (R2) is further extracted ( Fig.10 Step S52). For the measurement data within the range (R2), the measurement data within the region indicated as R4 ( Fig.15 Count the number of points in b and d) of 15 ( Fig.10 Step S53). In addition, Fig.15 The number of points on the scatter plot of b and d of 15 (the number of points within the range (R2)) is counted. Then, the ratio of the number of points within the range (R4) to the number of points within the range (R2) is calculated using the following formula (1): Fig.10 The results are summarized in the following Table 1.

[0181] [Ratio (R4 / R2)] = {[Number of points in range (R4)] / [Number of points in range (R2)]} × 100 ... Formula (1)

[0182]

Table 1

[0183] Analysis and processing steps No immune stimulation ( Fig.15 a and 15b) Immune stimulation ( Fig.15 c and 15d) S52 Range (R2) [Count] 1503 2219 S53 Range (R4) [Count] 138 1799 S54 range (R4 / R2) [%] 9.2 81.1

[0184] According to the examples of this specification, when immune cells are given immune stimulation including contact with immune stimulatory factors and formation of contact surfaces with different substances, if the costimulatory molecules are used as indicators to detect the immune synapse that can be formed, even when the contact surface is not maintained, the immune responsiveness information of the immune cells can be accurately obtained. This is a surprising result considering that the immune synapse is a loose combination formed on the immune cell at the binding surface with the target cell.

[0185] Thus, according to the present invention, the immunostimulation responsiveness of immune cells can be measured based on the immunological synapse, and it can be understood that the activation state of immune cells can be measured quickly and simply compared to the conventional immune cell responsiveness analysis method based on cytokine production.

[0186] It has been confirmed in the embodiments of this specification that: since the immunostimulatory responsiveness of immune cells can be measured using an imaging flow cytometer, multiple cells can be measured, and the immunostimulatory responsiveness measurement with high statistical accuracy can be performed. In the above-mentioned embodiments, an imaging flow cytometer is used to measure the immunostimulatory responsiveness of immune cells, but it can be understood that: the imaging cytometer that can measure multiple cells in the same manner as the measuring device used in the embodiments can also be used to measure. In addition, it can be understood that: as shown in the above-mentioned embodiment 4, the immunostimulatory responsiveness of multiple immune cells can also be measured in the same manner as the measuring device used in the embodiments using a flow cytometer that is not photographed. Thus, according to the present invention, the activation state of immune cells can be measured quickly and easily with multiple cells, so it can be understood that: it is useful in the evaluation of drug efficacy with high statistical accuracy.

[0187] The above-mentioned embodiment uses an imaging flow cytometer, so in the analysis of the measurement data of each cell, the image data of each cell can be mapped to each measurement data. Therefore, after the cells are screened by the prescribed analysis processing based on the measurement data, the image data of the cells can be further merged to evaluate the immune stimulation responsiveness. For example, in the analysis processing for screening cells with a high tendency to form immune synapses in the above-mentioned embodiment, there are about 81% of cells falling into the range (R4) in the sample with immune stimulation (Table 1). Here, in Fig.15 By specifying the point corresponding to the cell falling within the range (R4) in d and displaying the image data of the cell, and merging the image data for evaluation, the responsiveness of the immune cell to the immune stimulation can be evaluated in more detail. It is understood that this further evaluation can also be evaluated using an imaging cytometer that captures cell images in the same way as an imaging flow cytometer.

[0188] According to the measuring method and cell analysis device of the present invention, for example, when using immunostimulatory antibodies as immunostimulatory factors, the activation state of immune cells in the body of an object related to cancer immunity, autoimmune diseases, etc. can be quickly and easily evaluated; when using specific antigen peptides as immunostimulatory factors, the activation state of immune cells in the body of an object related to infectious diseases, allergic diseases, etc. can be quickly and easily evaluated; further, when using allogeneic cells or allogeneic MHC as immunostimulatory factors, the immune responsiveness of the object to transplantation medicine and regenerative medicine can be quickly and easily evaluated, so it is useful. As described above, it can be understood that imaging flow cytometers and imaging cytometers that can further utilize image data of each cell in the analysis process have further advantages.

[0189] [Reference example: Correlation between immune synapse formation and cytokine production in immune cells]

[0190] (1) Determination of cytokine-producing cells

[0191] In the same manner as in Example 1, an anti-CD3 antibody was immobilized in each well of the plate to prepare an anti-CD3 antibody immobilized plate. In addition, cloned T cells were prepared from human peripheral blood in the same manner as in Example 1. Approximately 5×10 5 The cloned T cells were added in the form of individual cells. Then, anti-CD28 antibody (BioLegend #302914Clone: ​​CD28.2) was added to the cells in the wells at a final concentration of 0.01 mg / ml and incubated at 37°C for 4 hours. Then, brefeldin A was added to each well at a final concentration of 10 μg / ml and incubated for 2 hours. Then, the plate was left to stand on ice and the cells in each well were recovered. The recovered T cells were fixed with a fixative containing 1% PFA and then treated for membrane permeability with a PBS solution containing 0.5% saponin and 1% BSA. The treated cells were immunostained with Alexa Fluor (registered trademark) 488-labeled anti-IFN-γ antibody (BioLegend #502517Clone: ​​4S.B3). The stained T cells were measured using a flow cytometer (BD biosciences: Accuri (trademark)). The measurement data were analyzed using conventional methods to obtain the proportion of IFN-γ positive cells in the measured T cells.

[0192] (2) Determination of cells that have formed immune synapses

[0193] Using the same T cells inoculated from the wells as the T cells used in (1) above, the cells that have formed an immune synapse are measured using CD28 as an index. The measurement data are analyzed to obtain the ratio of cells that have formed an immune synapse in the measured T cells. It should be noted that the measurement and data analysis are performed in the same manner as in Example 1.

[0194] (3) Results

[0195] The data were plotted with the vertical axis representing the ratio (%) of IFN-γ positive cells and the horizontal axis representing the ratio (%) of immune synapse forming cells. Fig.16 .Depend on Fig.16 It was found that a correlation was observed between the formation of immune synapse and the production of INF-γ in cloned T cells subjected to immune stimulation (R 2 =0.8371). Therefore, it was shown that by detecting the immune synapse of immune cells whose contact surfaces have been released, immune cells activated by immune stimulation can be detected in the same manner as the conventional method based on the production of cytokines.

[0196] [Example 2: Determination of the immunostimulatory responsiveness of immune cells using CD3 and CD40 as indicators]

[0197] (1) Stimulation and determination of immune cells

[0198] In the same manner as in Example 1, an anti-CD3 antibody was immobilized in each well of the plate to prepare an anti-CD3 antibody immobilized plate. In addition, cloned T cells were prepared from human peripheral blood in the same manner as in Example 1. Approximately 5×10 5 The prepared cloned T cells were added in the form of cells. Then, anti-CD28 antibody (BioLegend#302914Clone: ​​CD28.2) and PE-labeled anti-CD40L antibody (BD biosciences#340477Clone: ​​89-76) were added to the cells in the wells at a final concentration of 0.01 mg / ml and 0.01 mg / ml, respectively. The plate was centrifuged (200G, 60 seconds) and incubated at 37°C for 30 minutes. After removing the supernatant in the wells, the prepared cloned T cells were dispersed and recovered from the surface of the wells by aspiration. The recovered T cells were fixed with a fixative containing 1% PFA. The fixed T cells were immunostained with APC-labeled anti-CD3 antibody (BioLegend#300411Clone: ​​UCHT1). It should be noted that since the T cells were immunostimulated with PE-labeled anti-CD40L antibody, they were also in a state of being immunostained with anti-CD40L antibody. The stained T cells (5×10 5 cells / 20 μl) for determination.

[0199] For comparison, the case where the cells were not subjected to immune stimulation due to contact with anti-CD40L antibody, anti-CD28 antibody and anti-CD3 antibody and contact with the wells of the plate was also studied. The cloned T cells prepared in (1) above were fixed on ice with the above fixative without the above immune stimulation. The fixed T cells were immunostained with APC-labeled anti-CD3 antibody and PE-labeled anti-CD40L antibody and measured with Imaging FCM.

[0200] When the above immune stimulation is not given to the preparation of cloned T cells, CD3 and CD40L are evenly distributed on the cell membrane in T cells (refer to Fig.17 a and c, Fig.18 a and b, Fig.19 a and b). It shows that: after the above immune stimulation is given and the contact surface between the T cells and the well is released, both CD3 and CD40L are locally present on the cell membrane of the T cells (refer to Fig.17 b and d, Fig.18 c and d, Fig.19 c and d). Thus, even after the immune stimulation, when the T cells and the wells are no longer in contact with each other, the immune synapse can be detected by using CD3 or CD40L, which is a surface antigen of the T cells, as an indicator.

[0201] (2) Data Analysis

[0202] The measurement data of the sample containing the above-mentioned non-immune stimulated T cells measured by Imaging FCM ( Fig.18 a and b) and the measurement data of the sample containing the above-mentioned immune stimulated T cells ( Fig.18 c and d) to perform multi-parameter analysis. An example of the measurement and analysis process in Example 2 is shown in Fig. 20 In short, based on the transmitted light image and fluorescent image (CD3 and CD40L) measured by Imaging FCM, image analysis was performed to measure the cell size (number of pixels), the aspect ratio of the cell, the area showing the fluorescent signal in the cell (number of pixels), and the total fluorescent signal intensity in the area (total fluorescent signal intensity of the cell). First, the cell size was assigned to the X-axis and the aspect ratio of the cell was assigned to the Y-axis to create a 2D scatter plot (see Fig. 20 a), extract the measurement data of cells corresponding to the points located in the specified range (Cells). Then, for the measurement data within the range (Cells), further extract the measurement data corresponding to the points whose total fluorescence signal intensity of the cells is located within the specified range (Tcells) (refer to Fig. 20 b). For the measurement data within the range (Tcells), the areas indicated as IS_CD3 and IS_CD40L (refer to Fig.18 c and d, Fig.19 The number of points in c and d) is counted. Fig.18 c and d, Fig.19 The number of points on the scatter plots of c and d (the number of points within the range (Tcells)) was counted. Then, the ratio of the number of points within the range (IS_CD3 or IS_CD40L) to the number of points within the range (Tcells) was calculated by the following formula (2). The results are shown in the following Tables 2 and 3. It should be noted that IS is the abbreviation for Immune Synapse.

[0203] [Ratio (IS-forming cells / T cells)] = {[Number of points in the range (IS_CD3 or IS_CD40L)] / [Number of points in the range (T cells)]} × 100 (2)

[0204]

Table 2: CD3

[0205] Analyze the data No immune stimulation Immune stimulation T cells [count] 7269 4481 IS-forming cells [count] 750 795 Ratio (IS-forming cells / T cells) [%] 10.32 17.74

[0206]

Table 3: CD40L

[0207] Analyze the data No immune stimulation Immune stimulation T cells [count] 7269 4481 IS-forming cells [count] 292 1463 Ratio (IS-forming cells / T cells) [%] 4.02 32.65

[0208] As shown in Tables 2 and 3, it was shown that the proportion of T cells that formed immune synapses in T cells stimulated with immunostimulatory antibodies significantly increased compared to when no immune stimulation was performed. Therefore, it was shown that the responsiveness of immune cells to immune stimulation with immunostimulatory antibodies can be measured by immune synapse detection using CD3 or CD40L as an index.

[0209] [Example 3: Measurement of immune cells responding to immune stimulation with allogeneic cells]

[0210] (1) Allogeneic cells and cloned T cells

[0211] As allogeneic cells, retinal pigment epithelial cells (RPE cells) prepared from iPS cells (allogeneic iPS cells) from a predetermined donor were used. RPE cells were provided by Healios Co., Ltd. Approximately 5×10 cells / well were plated in a 24-well plate. 5 RPE cells were seeded at 37°C and 5% CO 2 Incubate at 4 °C for 7 days. Thus, a plate with RPE cells adhered to the bottom of the wells was obtained. Cloned T cells were prepared by the following method. 5 × 10 6 CD4-positive cells (Stemcell Technologies: ST-70026) and CD8-positive cells (Funakoshi: 0508-100, Cosmo Bio: PB08C-1) were seeded in Yssel's medium (Gemini Bio-Products: 400102) supplemented with 1 μg / mL LEAF (trademark) purified anti-human CD3 antibody (Biolegend: 317315), 10 ng / mL recombinant human IL-2 (rhIL-2) (R&D systems: 202-IL-500), 0.2 μg / mL phytohemagglutinin-L (PHA) (SIGMA: L4144-5MG) and 2% human serum (SIGMA: H4522-100ML), and 1.2×10 5 After 14 days, T cells were seeded into 96-well plates at 0.3, 1.0, and 3.0 cells / well by limiting dilution method and cultured until 1×10 4 The RPE cells were cultured until colonies were formed. T cells were collected from the wells where colonies were formed to obtain cloned T cells.

[0212] (2) Stimulation and determination of immune cells

[0213] Approximately 5 × 10 cells were seeded into each well of the plate containing RPE cells. 5 The prepared cloned T cells were added in the form of 100 cells. The plate was centrifuged (200G, 120 seconds) and incubated at 37°C for 30 minutes. After removing the supernatant in the wells, the prepared cloned T cells were dispersed and recovered from the surface of the wells by aspiration. The recovered T cells were fixed with a fixative containing 1% PFA. The fixed T cells were immunostained with APC-labeled anti-CD3 antibody (BioLegend#300411Clone:UCHT1), anti-CD28 antibody (BioLegend#302914Clone:CD28.2), PE-labeled anti-CD40L antibody (BD biosciences#340477Clone:89-76) or PE-labeled anti-OX40 antibody (BioLegend#350003Clone:Ber-ACT35). The T cells immunostained with the anti-CD28 antibody were further immunostained with a PE-labeled anti-mouse IgG antibody (BioLegend#405307). The stained T cells (5×10 5 cells / 20 μl) for determination.

[0214] For comparison, the case where the cells were not given immune stimulation due to contact with allogeneic cells was also studied. The cloned T cells prepared in (1) above were fixed on ice with the above fixative without contact with allogeneic cells. The fixed T cells were immunostained with APC-labeled anti-CD3 antibody, anti-CD28 antibody, PE-labeled anti-CD40L antibody or PE-labeled anti-OX40 antibody and measured with Imaging FCM.

[0215] When the prepared cloned T cells are not given immune stimulation caused by contact with allogeneic cells, CD3, CD28, CD40L and OX40 are evenly distributed on the cell membrane in T cells (refer to Fig.21 a, c, e and g, Fig. 22 a and b, Fig.23 a and b, Fig.24 a and b, Fig.25 a and b). It shows that: after the above immune stimulation is given and the contact surface between T cells and allogeneic cells is released, CD3, CD28, CD40L and OX40 are locally present on the cell membrane of T cells (refer to Fig.21 b, d, f and h, Fig. 22c and d, Fig.23 c and d, Fig.24 c and d, Fig.25 c and d). Thus, even in a state where the contact surface between T cells and the allogeneic cells is released after immune stimulation due to contact with allogeneic cells, immune synapses can be detected using CD3, CD28, CD40L or OX40, which are surface antigens of T cells, as indicators.

[0216] (3) Data analysis

[0217] The measurement data of the sample containing the above-mentioned non-immune stimulated T cells measured by Imaging FCM ( Fig.18 a and b) and the measurement data of the sample containing the above-mentioned immune stimulated T cells ( Fig.18 c and d) for multi-parameter analysis. The measurement and analysis processes are the same as those in Example 2. In short, based on the transmitted light image and the fluorescent image (CD3, CD28, CD40L or OX40) measured using Imaging FCM, image analysis is performed to measure the size of the cell (number of pixels), the aspect ratio of the cell, the area in the cell showing the fluorescent signal (number of pixels), and the total fluorescent signal intensity within the area (total fluorescent signal intensity of the cell). First, the cell size is assigned to the X-axis and the aspect ratio of the cell is assigned to the Y-axis to make a 2D scatter plot, and the measurement data of the cells corresponding to the points within the specified range (Cells) are extracted. Then, for the measurement data within the range (Cells), the measurement data corresponding to the points whose total fluorescent signal intensity of the cells is within the specified range (T cells) are further extracted. For the measurement data within the range (T cells), for the areas represented as IS_CD3, IS_CD28, IS_CD40L and IS_OX40 (reference Fig. 22 b and d, Fig.23 b and d, Fig.24 b and d, Fig.25 The number of points in b and d) of the scatter plots was counted respectively. In addition, the number of points located on each scatter plot (the number of points located in the range (T cells)) was counted. Then, the ratio of the number of points in the range (IS_CD3, IS_CD28, IS_CD40L, and IS_OX40) to the number of points in the range (T cells) was calculated by the following formula (3). The results are shown in the following Tables 4 to 7.

[0218] [Ratio (IS-forming cells / T cells)] = {[Number of points in the range (IS_CD3, IS_CD28, IS_CD40L or IS_OX40)] / [Number of points in the range (T cells)]} × 100 (3)

[0219]

Table 4: CD3

[0220] Analyze the data No immune stimulation Immune stimulation T cells [count] 2758 1905 IS-forming cells [count] 161 471 Ratio (IS-forming cells / T cells) [%] 5.84 24.72

[0221]

Table 5:CD28

[0222] Analyze the data No immune stimulation Immune stimulation T cells [count] 2758 1881 IS-forming cells [count] 75 114 Ratio (IS-forming cells / T cells) [%] 2.72 6.06

[0223]

Table 6:CD40L

[0224] Analyze the data No immune stimulation Immune stimulation T cells [count] 1872 1052 IS-forming cells [count] 46 64 Ratio (IS-forming cells / T cells) [%] 2.46 6.08

[0225]

Table 7: OX40

[0226] Analyze the data No immune stimulation Immune stimulation T cells [count] 2758 1881 IS-forming cells [count] 75 114 Ratio (IS-forming cells / T cells) [%] 2.72 6.06

[0227] As shown in Tables 4 to 7, it is shown that the proportion of T cells that formed immune synapses in T cells that were immune stimulated with allogeneic cells significantly increased compared to when no immune stimulation was performed. Therefore, immune synapse detection using CD3, CD28, CD40L or OX40 as an index can measure the responsiveness of immune cells to immune stimulation with allogeneic cells.

[0228] This manual also includes the following contents:

[0229] 1. A method for determining the immunostimulatory responsiveness of immune cells, comprising:

[0230] (i) a step of contacting immune cells of the measurement object with an immunostimulatory factor;

[0231] (ii) a step of causing the immune cells of the measurement object to form a contact surface with a substance different from the immune cells of the measurement object;

[0232] (iii) a step of bringing the immune cells of the measurement object into contact with a capture body that binds to the surface antigen of the contact surface and can generate an optical signal;

[0233] (iv) a step of detecting an optical signal generated by the capture body; and

[0234] (v) a step of determining, based on the detected optical signal, whether the immune cells of the measurement object that have released the contact surface before the optical signal is detected have immunostimulatory responsiveness.

[0235] 2. A method according to embodiment 1, wherein the immunostimulatory factor of step (i) is shared as a capture body of step (iii) that binds to the surface antigen of the contact surface and can generate an optical signal, and steps (i) and (iii) are performed simultaneously.

[0236] 3. The method according to embodiment 1 or 2, wherein the immunostimulatory factor of step (i) is present on the substance of step (ii) that is different from the immune cells to be measured.

[0237] 4. The method according to any one of embodiments 1 to 3, further comprising a step of releasing the contact surface before detecting the optical signal.

[0238] 5. The method according to embodiment 4, wherein the contact surface is released by stirring, ultrasonic treatment or chemical treatment.

[0239] 6. The method according to any one of embodiments 1 to 5, further comprising: a step of separating the immune cells to be measured from a sample containing cells based on the size, degree of agglutination and / or specific gravity of the cells.

[0240] 7. The method according to any one of embodiments 1 to 6, wherein the surface antigen comprises at least one of TCRα / β, CD3, CD40L, OX40, CD28, CTLA4, PD-1 and ICOS.

[0241] 8. The method according to any one of embodiments 1 to 7, wherein the surface antigen is a co-stimulatory molecule.

[0242] 9. The method according to any one of embodiments 1 to 8, wherein the capture body comprises: a substance capable of generating an optical signal; and an antibody, an antibody fragment, a single-chain antibody or an aptamer that binds to a surface antigen of the contact surface.

[0243] 10. The method according to any one of embodiments 1 to 9, wherein the immunostimulatory factor comprises at least one of an immunostimulatory antibody, an immunostimulatory peptide, a major histocompatibility molecule (MHC molecule), and a complex of an MHC molecule and an antigenic peptide.

[0244] 11. The method according to any one of embodiments 1 to 10, wherein the substance different from the immune cell to be measured is an allogeneic cell, an antigen-presenting cell, a cancer cell, a container, a multi-well plate, a slide, or a bead.

[0245] 12. The method according to any one of embodiments 1 to 11, wherein the immune cell to be measured is a T cell.

[0246] 13. A method according to any one of embodiments 1 to 12, wherein in step (v), a value of the distribution of surface antigens to which the capture body is bound on the contact surface is measured based on the optical signal, and the measured value is compared with a threshold value to determine whether the immune cells of the measured object are responsive to immune stimulation.

[0247] 14. The method according to embodiment 13, wherein:

[0248] The capture body contains a fluorescent substance,

[0249] In step (iv), a fluorescent signal generated by the capture body is detected,

[0250] In step (v), a fluorescent image is obtained based on the detected fluorescent signal, an area showing a fluorescent signal reflecting the distribution of the surface antigen in the fluorescent image is measured, and the area is compared with a threshold value to determine whether the immune cells of the measurement object are responsive to immunostimulation.

[0251] 15. A method according to embodiment 14, wherein in step (v), when the area is smaller than a threshold value, it is determined that the immune cells of the measurement object are responsive to immunostimulation.

[0252] 16. The method according to any one of embodiments 1 to 15, wherein

[0253] When the substance different from the immune cell to be measured is an allogeneic cell, an antigen presenting cell, a cancer cell, or a bead, the step (iv) is performed on the complex of the immune cell to be measured, the substance different from the immune cell to be measured, and the capture body using a flow cytometer or an imaging cytometer; or

[0254] When the substance different from the immune cells of the object of measurement is a container, a multi-well plate or a slide, the contact surface between the substance different from the immune cells of the object of measurement and the immune cells of the object of measurement is released, and the step (iv) is performed on the complex of the immune cells of the object of measurement and the capture body separated from the substance different from the immune cells of the object of measurement using a flow cytometer or an imaging cytometer.

[0255] 17. A method for determining the immunostimulatory responsiveness of immune cells, comprising:

[0256] (i) a step of contacting immune cells of the measurement object with an immunostimulatory factor;

[0257] (ii) a step of causing the immune cells of the measurement object to form a contact surface with a substance different from the immune cells of the measurement object;

[0258] (iii) a step of bringing the immune cell of the measurement object into contact with a capture body that binds to the co-stimulatory molecule on the contact surface and can generate an optical signal;

[0259] (iv) a step of detecting an optical signal generated by the capture body; and

[0260] (v) A step of automatically determining whether or not the immune cells of the measurement object have immunostimulatory responsiveness based on the detected optical signal.

[0261] 18. A method for determining the immune synapse formation ability of immune cells, comprising:

[0262] (i) a step of contacting immune cells of the measurement object with an immunostimulatory factor;

[0263] (ii) a step of causing the immune cells of the measurement object to form a contact surface with a substance different from the immune cells of the measurement object;

[0264] (iii) a step of bringing the immune cells of the measurement object into contact with a capture body that binds to the surface antigen of the contact surface and can generate an optical signal;

[0265] (iv) a step of detecting an optical signal generated by the capture body; and

[0266] (v) a step of determining, based on the detected optical signal, that the localization of the surface antigen in the immune cell of the measurement target on the contact surface is eliminated before the optical signal is detected.

[0267] 19. A cell analysis device, comprising:

[0268] An introduction part for introducing a complex, wherein the complex is a complex of an immune cell of a measurement object that has been immunostimulated and a capture body that binds to a surface antigen of the immune cell and can generate an optical signal, wherein the capture body binds to a surface antigen of a contact surface between the immune cell and a substance different from the immune cell;

[0269] an imaging unit configured to image the complex supplied from the introduction unit; and

[0270] An analyzing unit determines whether or not the immune cells of the measurement object that have been released from the contact surface before the imaging is performed have immunostimulatory responsiveness based on the image captured by the imaging unit.

[0271] 20. The device according to embodiment 19, wherein the analysis unit determines whether the immune cells of the measurement object are responsive to immune stimulation by obtaining a value reflecting the distribution of surface antigens of the contact surface based on the image and comparing the obtained value with a threshold value.

[0272] 21. The device according to embodiment 19 or 20, wherein:

[0273] The capture body contains a fluorescent substance,

[0274] The image comprises a fluorescent image,

[0275] The value reflecting the distribution of the surface antigen includes an area showing a fluorescent signal reflecting the distribution of the surface antigen based on the fluorescent image,

[0276] When the area is smaller than the threshold value, it is determined that the immune cell to be measured has immunostimulatory responsiveness.

[0277] 22. The device according to embodiment 20 or 21, wherein:

[0278] The image comprises a transmitted light image,

[0279] The analysis unit obtains a value reflecting the size of the immune cell of the measurement object based on the transmitted light image, and determines whether the immune cell of the measurement object is responsive to immune stimulation based on the value reflecting the size of the immune cell of the measurement object and the value reflecting the distribution of the surface antigen.

[0280] 23. A cell analysis device, comprising:

[0281] An introduction part for introducing a complex, wherein the complex is a complex of an immune cell of a measurement object that has been immunostimulated and a capture body that binds to a surface antigen of the immune cell and can generate an optical signal, wherein the capture body binds to a surface antigen of a contact surface between the immune cell and a substance different from the immune cell;

[0282] a detection unit that irradiates the complex supplied from the introduction unit with light and detects a generated optical signal from the complex; and

[0283] The analyzing unit determines, based on the detected optical signal, whether the immune cells of the measurement object that have released the contact surface before the optical signal is detected have immunostimulatory responsiveness.

[0284] 24. The device according to embodiment 23, wherein:

[0285] The capture body contains a fluorescent substance,

[0286] The detection unit detects a fluorescent signal generated by the complex,

[0287] The analyzing unit obtains a value reflecting the distribution of the surface antigen based on the fluorescent signal, and compares the obtained value with a threshold value to determine whether the immune cells of the measurement object have immunostimulatory responsiveness.

[0288] The value reflecting the distribution of the surface antigen includes at least one of a pulse width of a fluorescent signal from the complex, a height of the fluorescent pulse, and a pulse area.

Claims

1. A method for labeling immune cells, comprising: (i) a step of contacting immune cells of the measurement object with an immunostimulatory factor; (ii) forming a contact surface on the immune cell of the measurement object by contacting the immune cell with a substance different from the immune cell of the measurement object; (iii) a step of releasing the contact; and (iv) contacting the immune cells of the measurement object with a capture body that binds to the surface antigen on the contacted surface and can generate an optical signal, wherein the substance different from the immune cell to be measured is a substance that, when in contact with the immune cell, can provide a contact surface for the immune cell, and The immunostimulatory factor is a substance that acts on the immune cells and can induce the activation of the immune cells.

2. The method according to claim 1, wherein: The immunostimulatory factor in step (i) is present on the substance different from the immune cell to be measured in step (ii).

3. The method according to claim 1, wherein: The contact surface is released by stirring, ultrasonic treatment or chemical treatment.

4. The method according to claim 1, wherein: The method further comprises the step of separating the immune cells to be measured from a sample containing cells based on the size, degree of aggregation and / or specific gravity of the cells.

5. The method according to claim 1, wherein: The surface antigen comprises at least one of TCRα / β, CD3, CD40L, OX40, CD28, CTLA4, PD-1 and ICOS.

6. The method according to claim 1, wherein: The surface antigen is a co-stimulatory molecule.

7. The method according to claim 1, wherein: The capture body comprises: A substance capable of producing an optical signal; and An antibody, antibody fragment, single chain antibody or aptamer that binds to a surface antigen on the contact surface.

8. The method according to claim 1, wherein: The immunostimulatory factor comprises at least one of an immunostimulatory antibody, an immunostimulatory peptide, a major histocompatibility molecule, and a complex of a major histocompatibility molecule and an antigenic peptide.

9. The method according to claim 1, wherein: The substance different from the immune cell to be measured is an allogeneic cell, an antigen presenting cell, a cancer cell, a container, a multi-well plate, a slide, or a bead.

10. The method according to claim 1, wherein: The immune cells to be measured are T cells.

Images