JP2022037058A - Method for measuring immune stimulating response of immune cells, method for determining the formation ability of immune synapse in immune cells, and cell analysis apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for measuring immune stimulating response of immune cells and a cell analysis apparatus.

SOLUTION: A method for measuring immune stimulation response of immune cells includes: (i) a step for contacting immune cells and immune stimulation factors of the measuring object; (ii) a step for forming a contact surface in the immune cells of the measuring object different from the substance of the measuring object; (iii) a step for joining the immune cells of the measuring object to the prehension object which combines with the surface antigen in the contact surface and can generate an optical signal; (iv) a step for detecting the optical signal which has been generated from the prehension object; and (v) a step for judging whether the immune cells of the measuring object have an immune stimulation response in which the contact surface was canceled by the time they detected the optical signal based on the detected optical signal.

SELECTED DRAWING: Figure 1

COPYRIGHT: (C)2022,JPO&INPIT

Description

The present invention relates to a method for measuring the immune stimulus response of an immune cell, a method for determining the ability to form an immune synapse in an immune cell, and a cell analyzer.

Immunity is a general term for cellular and humoral biological reactions performed by a living body to identify self or non-self and eliminate non-self. The immune system is a very important physiological mechanism in life activity. The cells of the immune system are involved in the activation and suppression of the immune system by secreting proteins called cytokines and exerting cytotoxic functions.

The immune system is deeply involved in rejection of allogeneic cells, which can determine the success or failure of transplantation therapy. In recent years, the usefulness of immunotherapy that treats diseases in humans using the immune system has been shown, and it is becoming more and more important to understand the state of the immune system.

As a method for analyzing the state of the immune system, an analysis method based on cytokine secretion is known. In this analysis method, it is necessary to culture the cells after immune stimulation of the cells of the immune system, and it takes a lot of time (several tens of hours) and labor from the immune stimulation to the detection of cytokines.

In recent years, attention has been paid to a structure formed in an immune cell on the contact surface when an immune cell and a target cell come into contact with each other. This structure is called an immunological synapse because it is functionally and morphologically similar to the synaptic structure formed by nerve cells. It is suggested that the immunological synapse is involved in the amplification of the immunological synapse, and it is known that the ability to form the immunological synapse correlates with the ability to secrete cytokines (Non-Patent Document 1). It is also known that the formation of immunological synapses occurs in a shorter time (several tens of minutes) than that of cytokine secretion (Non-Patent Document 1).

As a method for measuring the immunological synapse, for example, the immunological synapse in the T cell is transferred to a total internal reflection fluorescence microscope while the immune cell is stimulated by using an immunostimulatory factor on the glass substrate and the contact surface with the glass substrate is maintained. The method of measuring is known (Non-Patent Document 2). Further, there is known a method of measuring an immunological synapse in T cells by imaging flow cytometry while maintaining a contact surface formed with a target cell (Non-Patent Document 3). In Non-Patent Document 2, the immunological synapse is detected using the co-stimulator molecule CD28 as an index, and in Non-Patent Document 3, the immunological synapse is detected using CD3 as an index. Non-Patent Document 2 and Non-Patent Document 3
In each case, the immune cells are observed in a state where the contact surface with the object is maintained.

Carlin LM. et al., Blood, vol.106, p.3874-3879, 2005 Yokosuka T. et al., Immunity, vol.29, p.589-601, 2008 Hosseini BH. et al., Proc Natl Acad Sci USA, vol.106, p.17852-17857, 2009

Understanding the state of the immune system, that is, quickly and easily grasping the responsiveness of cells of the immune system to various immune stimuli, is very important for performing treatments related to various immune systems.

Conventionally, in order to analyze the responsiveness of cells of the immune system to immune stimulation, an analysis method using cytokine secretion as an index has been used. However, in this analytical method, after immune stimulation, immune cells had to be cultured until cytokines were secreted, which was complicated and time-consuming.

When a total internal reflection fluorescence microscope is used to measure the responsiveness of cells of the immune system to various immune stimuli, the number of cells that can be measured is small and it takes time to measure, so it is not possible to measure easily, and an automated device. Was not suitable for.

Therefore, there has been a demand in the field for quickly and easily grasping the responsiveness of cells of the immune system to various immune stimuli.

In order to solve the above-mentioned problems, the present inventors focused on immunological synapses, which were suggested to be involved in the amplification of immune stimulating signals by causing changes in a shorter time than cytokine secretion. However, since the immunological synapse is a loose binding formed on the immune cell at the binding surface with the target cell (Non-Patent Document 3), there is a concern that it may dissociate before and during the measurement. In fact, the immunological synapse has not been used as an index of the responsiveness of immune cells to immune stimulation when the contact surface between the target cell and the immune cell is eliminated.

The present inventors have repeatedly investigated a method for rapidly detecting the responsiveness of immune cells to immune stimuli, focusing on various factors known to be involved in the formation of immune synapses. As a result, it was found that when a surface antigen such as a co-stimulator molecule is used as an index among various factors, the ability to form an immunological synapse in the immune cell can be determined even if the contact surface between the immune cell and the object is eliminated. The present invention has been made.

The first aspect of the present invention is (i) a step of contacting an immune cell to be measured with an immunostimulatory factor, and (ii) contacting the immune cell to be measured with a substance different from the immune cell to be measured. The steps of forming a surface, (iii) contacting the immune cell to be measured with a capture body that binds to a surface antigen on the contact surface and can generate an optical signal, and (iv) an optical signal generated from the capture body. And (v) based on the detected optical signal, a step of determining whether or not the immune cell to be measured whose contact surface has been eliminated by the time the optical signal is detected has an immune stimulus responsiveness. Provided are methods for measuring the immune stimulus responsiveness of immune cells, including.

The second aspect of the present invention is (i) a step of contacting an immune cell to be measured with an immunostimulatory factor, and (ii) contacting the immune cell to be measured with a substance different from the immune cell to be measured. The steps of forming a surface, (iii) contacting the immune cell to be measured with a trap that binds to a costimulatory molecule on the contact surface and can generate an optical signal, and (iv) the optics generated from the trap. Immune stimulus response of immune cells, including a step of detecting a signal and (v) a step of automatically determining whether or not the immune cell to be measured has an immune stimulus responsiveness based on the detected optical signal. Provides a method for measuring sex.

The third aspect of the present invention is (i) a step of contacting the immune cell to be measured with an immunological synapse, and (ii) contacting the immune cell to be measured with a substance different from the immune cell to be measured. The steps of forming a surface, (iii) contacting the immune cell to be measured with a capture body that binds to a surface antigen on the contact surface and can generate an optical signal, and (iv) an optical signal generated from the capture body. (V) Based on the detected optical signal, the immunity includes a step of determining the localization of a surface antigen in the immune cell to be measured whose contact surface has been eliminated by the time the optical signal is detected. Provided is a method for determining the ability to form an immune synapse in a cell.

A fourth aspect of the present invention is a complex of an immune cell to be measured that has been immune-stimulated and a capture body that can bind to the surface antigen of the immune cell and generate an optical signal, wherein the capture body is a complex. An introduction unit that introduces the complex bound to a surface antigen on the contact surface between the immune cell and a substance different from the immune cell, and an imaging unit that images the complex supplied from the introduction unit. A cell analyzer including an analysis unit that determines whether or not an immune cell to be measured has an immune stimulus responsiveness, the contact surface of which has been eliminated by the time the contact surface is removed, based on the image captured by the imaging unit. I will provide a.

A fifth aspect of the present invention is a complex of an immune cell to be measured that has been immune-stimulated and a capture body that can bind to the surface antigen of the immune cell and generate an optical signal, wherein the capture body is the complex. , The introduction part that introduces the complex bound to the surface antigen on the contact surface between the immune cell and a substance different from the immune cell, and the complex supplied from the introduction part are irradiated with light to generate. Whether or not the detection unit that detects the optical signal from the complex and the immune cell to be measured whose contact surface is eliminated by the time the optical signal is detected have immune stimulus responsiveness based on the detected optical signal. Provided is a cell analyzer including an analysis unit for determining.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to analyze the responsiveness of immune cells to various immune stimuli based on the ability to form immune synapses.

It is a schematic diagram of steps (i)-(iii) which concerns on embodiment. It is a figure which shows the structure of the cell analyzer which concerns on Embodiment 1. FIG. It is a figure which shows the structure of the measuring apparatus which concerns on Embodiment 1. FIG. It is a figure which shows the structure of the image pickup part which concerns on Embodiment 1. FIG. It is a schematic diagram which shows the region on the light receiving surface of the camera which concerns on Embodiment 1. FIG. It is a figure which shows the structure of the information processing apparatus which concerns on Embodiment 1. FIG. It is a flowchart which shows the measurement process and analysis process of the sample which concerns on Embodiment 1. FIG. It is an image which shows the measurement data which concerns on Embodiment 1. It is a schematic diagram which shows the measurement process based on the measurement data which concerns on Embodiment 1. FIG. It is a flowchart which shows the analysis process which concerns on Embodiment 1. It is a 2D scattergram which shows the analysis process which concerns on Embodiment 1 (a, b and c) and Embodiment 2 (d). It is a schematic diagram for demonstrating the pulse data of the fluorescence signal obtained from the cell which the distribution state of a co-stimulation molecule is different. It is a schematic diagram for demonstrating the 2D scattergram of the analysis parameter obtained from the pulse data of a fluorescent signal. It is a transmitted light image, a fluorescent image, and a superposed image thereof showing the distribution of CD28 in T cells in the case of no stimulus (a) and the case of stimulus (b). Images and 2D scattergrams showing the distribution of CD28 in T cells. It is a differential interference contrast image (DIC), a fluorescence image (CD28) and a superposition image (Merge) of T cells in the case of no stimulus (a) and the case of stimulus (c). It is a 2D scattergram showing an area showing a fluorescence signal in a cell (X-axis) and a cell size (Y-axis) in the case of no stimulation (b) and the case of with stimulation (d). It is a graph which shows the correlation between the cytokine secretion in a T cell and the formation of an immune synapse. 8 is a fluorescence image showing the distribution of CD3 and CD40L in T cells without stimulation (a and c) and with stimulation (b and d). Images and 2D scattergrams showing the distribution of CD3 in T cells. It is a transmitted light image, a fluorescent image, and a superposed image (Merge) of T cells in the case of no stimulus (a) and the case of stimulus (c). It is a 2D scattergram showing the total fluorescence signal intensity (X-axis) and the area (Y-axis) showing the fluorescence signal of the cells in the case of no stimulation (b) and the case of with stimulation (d). Images and 2D scattergrams showing the distribution of CD40L in T cells. It is a transmitted light image, a fluorescent image, and a superposed image (Merge) of T cells in the case of no stimulus (a) and the case of stimulus (c). It is a 2D scattergram showing the total fluorescence signal intensity (X-axis) and the area (Y-axis) showing the fluorescence signal of the cells in the case of no stimulation (b) and the case of with stimulation (d). 2D is a 2D scattergram showing an example of measurement processing and analysis processing in Example 2. a is a 2D scattergram showing the cell size (X-axis) and aspect ratio (Y-axis). b is a 2D scattergram showing the total fluorescence signal intensity (X-axis) and count (Y-axis) of the cell. Transmitted light 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). It is a superposed image of. Images and 2D scattergrams showing the distribution of CD3 in T cells. It is a transmitted light image, a fluorescent image, and a superposed image (Merge) of T cells in the case of no stimulus (a) and the case of stimulus (c). It is a 2D scattergram showing the total fluorescence signal intensity (X-axis) and the area (Y-axis) showing the fluorescence signal of the cells in the case of no stimulation (b) and the case of with stimulation (d). Images and 2D scattergrams showing the distribution of CD28 in T cells. It is a transmitted light image, a fluorescent image, and a superposed image (Merge) of T cells in the case of no stimulus (a) and the case of stimulus (c). It is a 2D scattergram showing the total fluorescence signal intensity (X-axis) and the area (Y-axis) showing the fluorescence signal of the cells in the case of no stimulation (b) and the case of with stimulation (d). Images and 2D scattergrams showing the distribution of CD40L in T cells. It is a transmitted light image, a fluorescent image, and a superposed image (Merge) of T cells in the case of no stimulus (a) and the case of stimulus (c). It is a 2D scattergram showing the total fluorescence signal intensity (X-axis) and the area (Y-axis) showing the fluorescence signal of the cells in the case of no stimulation (b) and the case of with stimulation (d). Images and 2D scattergrams showing the distribution of OX40 in T cells. It is a transmitted light image, a fluorescent image, and a superposed image (Merge) of T cells in the case of no stimulus (a) and the case of stimulus (c). It is a 2D scattergram showing the total fluorescence signal intensity (X-axis) and the area (Y-axis) showing the fluorescence signal of the cells in the case of no stimulation (b) and the case of with stimulation (d).

One aspect of the invention relates to a method of measuring the immune stimulus response of immune cells. In this embodiment, steps (i) and (ii) include causing the immune cell to be measured to form a contact surface with a substance different from the immune cell to be measured in the presence of an immunostimulatory factor. .. In this embodiment, following the step (i) of bringing the immune cell to be measured into contact with the immunostimulatory factor, a step (ii) of forming a contact surface with the substance on the immune cell to be measured is performed. May be good. Alternatively, if an immunostimulatory factor is present on the surface of the substance, step (i) and step (ii) may be performed at the same time. Even in this case, strictly speaking, the step (ii) is performed after the step (i). By performing steps (i) and (ii), immune synapses are formed on the contact surface of the activated immune cells.

In step (iii), in order to detect the formed immunological synapse, a capture body capable of binding to a surface antigen on the contact surface and generating an optical signal (hereinafter, simply referred to as simply) to the immune cells having formed the contact surface described above. Also referred to as a "capturer"). The trap is preferably bound to a molecule constituting the immunological synapse among the surface antigens on the contact surface. By performing step (iii), a complex of a molecule constituting an immune synapse and a trap is formed in an immune cell.

Then, in step (iv), an optical signal from a trap contained in the complex on immune cells is detected. The means for detecting the optical signal is not particularly limited, but a means capable of analyzing individual cells is preferable. Examples of the means for detecting such an optical signal include a flow cytometer and an imaging cytometer. In a preferred embodiment, step (iv) uses a flow cytometer or imaging cytometer to detect an optical signal generated from a trap bound to a surface antigen of immune cells.

In the step (v) described later, the immune stimulus responsiveness is determined for the immune cells whose contact surface has been eliminated. Therefore, before detecting the optical signal in the step (vi), the contact surface described later is applied to the immune cells. You may perform an operation to eliminate the above. When a substance of a non-biomaterial (for example, a container, a multi-well plate, a slide, etc.) described later is used as a substance different from the immune cells and step (iv) is performed using a flow cytometer or an imaging cytometer, the present embodiment. The method of the above preferably includes a step of eliminating the contact surface. If necessary, the immune cells may be fixed by a conventional method after the contact surface is eliminated. Immobilization can maintain the localization of surface antigens in immune cells whose contact surface has been eliminated. For cell fixation, it is preferable to use a fixative containing paraformaldehyde, lower alcohol and the like.

In a further embodiment, it may be determined based on the detected optical signal whether the immune cell is an immune cell whose contact surface has been eliminated. As a result, the optical signal derived from the immune cell whose contact surface has been eliminated can be extracted and used for the determination in step (v). For example, when allogeneic cells are used as a substance different from immune cells, in the measurement by a flow cytometer, between the immune cells whose contact surface is eliminated and the immune cells whose contact with the allogeneic cells is maintained, Differences occur in optical signals regarding cell size (eg, forward scattered light intensity or pulse width). In the measurement by the imaging cytometer, it is possible to distinguish between the immune cell whose contact surface is eliminated and the immune cell whose contact with the allogeneic cell is maintained from the image of the captured cell.

For purposes of illustration, reference is made to FIG. 1a schematically showing steps (i) to (iii) according to the present embodiment. FIG. 1a shows an example in which an immunostimulatory antibody described later is used as an immunostimulatory factor and a plate is used as a substance different from immune cells. The plate corresponds to, for example, the bottom surface of a container used for cell culture, the well bottom surface of a multi-well plate, the surface of a slide used for microscopic observation, and the like. In FIG. 1a, the immune cells are brought into contact with the immune stimulating antibody in step (i). In this example, the immunostimulatory antibody is an antibody that binds to a co-stimulatory molecule, which is a type of surface antigen of immune cells. Following step (i), in step (ii), the immune cells in contact with the immunostimulatory antibody are brought into contact with the plate to cause the immune cells to form a contact surface with the plate. In step (iii), a secondary antibody against the immunostimulatory antibody used in step (i) is used to detect the co-stimulatory molecule in the immune cell. This secondary antibody is labeled with a substance that can generate an optical signal. In this example, although the contact surface is eliminated in step (iii), the immune cells may be brought into contact with the capture body while the contact surface is maintained. Note that FIG. 1a is an example of steps (i) to (iii) according to one embodiment of the present invention, and the present invention is not limited thereto.

In step (v), based on the optical signal detected in step (iv), it is determined whether or not the immune cells whose contact surface has been eliminated have immunostimulatory responsiveness. In the present embodiment, in step (v), a value reflecting the distribution of the surface antigen in the immune cell is measured based on the detected optical signal, and the measured value is compared with the threshold value so that the immune cell is immunized. It may be determined whether or not it has stimulus responsiveness. Here, as the value reflecting the distribution of the surface antigen, for example, when the capture body contains a fluorescent substance, the pulse width of the fluorescence signal from the capture body bound to the surface antigen, the height of the fluorescence pulse, the pulse area, etc. Can be mentioned.

When the capture body contains a fluorescent substance, in step (v), a fluorescent image may be acquired based on the fluorescent signal detected in step (iv), and a determination may be made based on the fluorescent image. That is, in the fluorescence image, the area showing the fluorescence signal reflecting the distribution of the surface antigen is measured, and the measured value is compared with the threshold value to determine whether or not the immune cells have immunostimulation responsiveness. As used herein, the term "area showing a fluorescence signal" means the number of pixels showing a luminance value exceeding a predetermined threshold value in a fluorescence image. In this embodiment, it may be determined that the immune cells have immune stimulus responsiveness when the area is smaller than the threshold value, without limitation.

In a further embodiment, step (v) further measures at least one selected from the group comprising the total fluorescence signal intensity within the area, the size of immune cells and their aspect ratio, and the measured value is used as a threshold value. It may include comparison. As used herein, the "total fluorescence signal intensity" of the area or cell means the integrated value of the luminance value of each pixel in the area showing the luminance value exceeding a predetermined threshold value in the fluorescence image. In the present specification, "cell size" refers to the number of pixels of an image in which a region of a cell membrane showing a brightness value lower than a predetermined threshold value is detected in a transmitted light image and reflects a cell surrounded by the detected cell membrane. means. As used herein, the "aspect ratio" of a cell means the ratio of the length (number of pixels) of the image that reflects the cell particles in a transmitted light image.

In a further embodiment, the determination of whether an immune cell is immunostimulatory responsive based on the detected optical signal is determined by determining the pulse width of the fluorescent signal, the pulse width of the scattered light signal, and the fluorescent signal region per cell. By measuring at least one selected from the reflected value and the value reflecting the cell size and comparing the measured value with the threshold value, it is determined whether or not the immune cell has immunostimulatory responsiveness. Including doing.

As used herein, the term "immune synapse" means a molecular complex that is formed in an immune cell at the contact surface between the immune cell and the target cell and is a portion that plays a role in activating the immune cell. This molecular complex can be a receptor (eg, T cell receptor (TCR) or NK cell receptor), a cofactor for the receptor (eg CD3), various costimulatory molecules (eg CD28), an adhesion molecule (eg CD28). It is known that integrin) and signal molecules are assembled and formed.

As used herein, the term "contact surface" means a region in which an immune cell to be measured is in contact with a substance different from the immune cell to be measured. Here, the contact between the immune cell to be measured and the substance different from the immune cell to be measured is on the surface of the molecule existing on the surface of the immune cell to be measured and the substance different from the immune cell to be measured. It also includes indirect contact through interaction with the molecules present in. As described above, in the method of this embodiment, immunological synapses are formed on the contact surface. Therefore, it can be said that the contact surface on immune cells is a region where immune synapses are formed. The contact surface may be formed by spontaneous contact of the immune cell to be measured with a substance different from the self, although the contact surface is not limited. Alternatively, the contact surface may be formed by contacting the immune cell to be measured with a substance different from the immune cell to be measured.

As used herein, the term "immune cell to be measured" means a cell type responsible for a mechanism that distinguishes between self and non-self and eliminates non-self. The immune cells to be measured may be, but are not limited to, T cells, natural killer (NK) cells and mixtures thereof. The antigen-presenting cells are not included in the immune cells to be measured as used herein, but are included in the "substance different from the immune cells to be measured" described later.

As used herein, the term "substance different from the immune cell to be measured" refers to a substance that can provide a contact surface for the immune cell when it comes into contact with the immune cell. The substance different from the immune cell to be measured may be a non-biological material or a biological material. Substances that are non-biomaterials may be, but are not limited to, containers, multi-well plates, slides, beads and the like. The substance that is a biological material may be, but is not limited to, an allogeneic cell, an antigen-presenting cell, a cancer cell, or the like. The allogeneic cells, antigen-presenting cells and cancer cells may be cell lines. In addition, allogeneic cells and cancer cells may be in the form of tissues containing them. "Allogeneic cells" are non-self cells and may be T cells, natural killer (NK) cells and mixtures thereof.

In the present embodiment, the method of contacting the immune cell to be measured with a substance different from the immune cell to be measured is not particularly limited. For example, the immune cells to be measured may be contacted by placing them in a container or a well of a multi-well plate together with a medium and precipitating them. In this case, the container or multi-well plate containing the immune cells to be measured may be centrifuged. In addition, immune cells may be placed on a slide and brought into contact with each other. In a further embodiment, if a substance different from the immune cell to be measured is in the form of particles such as beads, allogeneic cells, antigen presenting cells, cancer cells, etc., the substance is added to the immune cell to be measured. May be contacted by. The contact surface can be formed by maintaining a state in which the immune cell to be measured and a substance different from the immune cell to be measured are in contact with each other. The time for maintaining the contact state is, for example, 2 minutes or more and 60 minutes or less, preferably 10 minutes or more and 45 minutes or less, more preferably 20 minutes or more and 40 minutes or less, and particularly preferably 30 minutes.

As used herein, a "surface antigen" is a protein, sugar chain, lipid or a combination thereof present on the cell membrane. The type of surface antigen is not particularly limited as long as there is a capture substance capable of binding to the surface antigen or such a capture substance can be produced. Preferred surface antigens are molecules that make up the immunological synapse in immune cells. Examples of such molecules include TCRα / β, CD3, CD28, CD40L (CD40 ligand), OX40, CTLA4 (cytolytic T lymphocyte associated antigen-4), PD-1 (programmed cell death-1) and ICOS (inducible). co-stimulatory molecule) and the like. The co-stimulatory molecule described later is also a kind of surface antigen of immune cells.

In this embodiment, the immune cell to be measured is at least one selected from T cells and NK cells. In a further embodiment, the immune cell to be measured is a T cell or an NK cell. In a preferred embodiment, the immune cell to be measured is a T cell.

T cells are, but are not limited to, helper T cells (also referred to as "CD4 ⁺ T cells"), suppressor T cells, regulatory T cells, cytotoxic T lymphocytes (CTL), ". It may be effector T cells such as (also referred to as "CD8 ⁺ T cells"), naive T cells, and genetically modified cells. Effector T cells can be cells activated in vivo and in vitro, without limitation. These T cells may be used alone or as a T cell subpopulation containing two or more species.

In this embodiment, the immune cells to be measured may be prepared from a biological sample. For example, immune cells may be isolated from blood (eg, peripheral blood and cord blood) or bone marrow fluid by known cell separation methods. Such cell separation methods include a method of separating desired cells from a biological sample containing cells based on cell size, cohesion and / or specific density. For example, using a commercially available cell sorter, cells can be sorted based on the size or degree of cohesion of the cells. In addition, cells can be separated based on the specific gravity of the cells by the specific gravity gradient centrifugation method. In this embodiment, cells isolated from a biological sample may be cultured and proliferated according to a conventional method. In a further embodiment, the immune cells may be used in the form of biological samples such as blood (eg peripheral blood and cord blood) or bone marrow fluid. The biological sample may be, but is not limited to, a sample obtained from a mammal, for example, a human and a non-human mammal (for example, a companion animal such as a dog or a cat, or a domestic animal such as a horse or a cow).

In a further embodiment, the immune cells to be measured may be present in the sample. Therefore, this embodiment provides a method of carrying out the method of the present invention on a sample containing an immune cell to be measured. More specifically, in this embodiment, (i) the step of contacting the immune cells in the sample with an immunostimulatory factor, and (ii) the immune cells in the sample are charged with a substance different from the immune cells. The step of forming the contact surface of the above, (iii) the step of contacting the immune cells in the sample with the capture body that binds to the surface antigen on the contact surface and can generate an optical signal, and (iv) from the capture body. It includes a step of detecting the generated optical signal and (v) a step of determining whether or not the immune cell whose contact surface has been eliminated in the sample is immune stimulus responsive based on the detected optical signal. , Provide a method for measuring the immune responsiveness of a sample containing immune cells. The sample containing immune cells may be the above-mentioned biological sample.

In this embodiment, in step (v), based on the optical signal detected in step (iv), a value reflecting the distribution of the surface antigen to which the capture agent is bound in the immune cell is measured, and the measured value is used as a threshold value. It may be determined whether or not the immune cell has an immune stimulus responsiveness by comparing with. When the capture body contains a fluorescent substance, in step (v), a fluorescent image may be acquired based on the fluorescent signal detected in step (iv), and a determination may be made based on the fluorescent image. In the fluorescence image, an area showing a fluorescence signal reflecting the distribution of the surface antigen may be measured, and based on the area, for example, the proportion of cells having immunostimulation responsiveness in the sample may be calculated. Then, the determination may be made by comparing the ratio with the ratio of immune cells having standardized immunostimulatory responsiveness to a sample derived from a healthy person. For example, if the proportion of the sample is higher than the standardized proportion, the sample may be determined to be immune responsive or immune responsive.

As used herein, "immune stimulation" to an immune cell includes contacting the immune cell with an immunostimulatory factor and causing the immune cell to form a contact surface with a substance different from the immune cell. In this embodiment, immunostimulation may be performed by contacting immune cells with an immunostimulator alone. In a further embodiment, the immunostimulation may be performed by contacting the immune cells with an immunostimulator present on the substance. In this case, the immune cells come into contact with the immune stimulator and at the same time come into contact with the substance.

As used herein, the term "immune stimulating factor" means a substance that can act on an immune cell to induce activation of the immune cell. Activation of immune cells means, for example, proliferation of immune cells, increased motility thereof, or secretion of cytokines. Immunostimulatory factors are present on, but not limited to, immunostimulatory antibodies, immunostimulatory peptides, major histocompatibility molecules (MHC molecules), and allogeneic cells, antigen presenting cells and cancer cells. It may be various immunostimulatory factors. The immunostimulatory factor may be used alone or in combination of two or more, or may be used alone or in the form of a complex with another substance or substance. The immunostimulatory factor may be, for example, a form of a complex of MHC molecule and an antigen peptide, a form immobilized on a non-biological material such as beads or a container, or a form presented on an antigen-presenting cell.

In the present 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 may be an immunostimulatory antibody, an immunostimulatory peptide, an MHC molecule, or a complex of an MHC molecule and an antigenic peptide. In certain embodiments, the immunostimulatory factor is an immunostimulatory antibody.

As used herein, the term "immune-stimulating antibody" means an antibody that can specifically stimulate immune cells to induce activation of the immune cells. Immunostimulatory antibodies are, but are not limited to, anti-TCRα / β antibody, anti-CD3 antibody, anti-CD40L antibody, anti-OX40 antibody, anti-CD2 antibody, anti-CD28 antibody, anti-CD137 antibody, anti-CTLA4 antibody, anti-PD-1. It may be an antibody, an anti-ICOS antibody and an anti-integrin antibody (anti-CD2 antibody). These immunostimulatory antibodies may be used alone or in combination of two or more. The immunostimulatory antibody may be used alone or immobilized on other substances such as beads, multi-well plates, slides, containers and other non-biomaterials.

In one embodiment, the immunostimulatory antibody is 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. , At least one selected from anti-ICOS antibody and anti-integrin antibody (anti-CD2 antibody). In a further embodiment, the immunostimulatory antibody is at least one selected from anti-CD3 antibody and anti-CD28 antibody. In certain embodiments, the immunostimulatory antibody is an anti-CD3 antibody and an anti-CD28 antibody.

The antibody may be either a monoclonal antibody or a polyclonal antibody unless otherwise specified. Further, the antibody may be an antibody fragment, a single-chain antibody or a derivative thereof.

As used herein, "immune-stimulating peptide" means a peptide that can stimulate immune cells and induce activation of the immune cells. The immunostimulatory peptide is, for example, an antigenic peptide (such as an antigenic peptide of Mycobacterium tuberculosis).

In this embodiment, the immunostimulatory peptide is an antigenic peptide. The antigenic peptide may be used alone or in combination of two or more. In certain embodiments, the antigenic peptide may be used in the form of a complex with the MHC molecule presented on the MHC molecule.

As used herein, the term "major histocompatibility complex (MHC molecule)" means a molecule responsible for alloantigenicity that causes the strongest rejection in allogeneic transplantation of organs, tissues and cells. The HLA molecule in humans and the H-2 molecule in mice correspond to MHC molecules. MHC molecules include class I molecules and class II molecules. MHC class I molecules are expressed on almost all nucleated cells, form a complex with intracellularly produced peptides, and present this to the receptor for CTLs (CD8-positive T cells). MHC class II molecules are expressed only on so-called antigen-presenting cells such as macrophages and B cells, and peptides derived from foreign antigens are presented to the receptors of helper T cells (CD4 positive T cells).

In the present embodiment, when the immunostimulatory factor is an MHC molecule or a complex of an MHC molecule and an antigen peptide, the MHC molecule may be either an MHC class I molecule or an MHC class II molecule. The MHC molecule or the complex of the MHC molecule and the antigen peptide may be used in a form existing in the cell membrane of, for example, an antigen-presenting cell or a cancer cell.

As used herein, the "capturer that binds to a surface antigen and can generate an optical signal" includes a substance that binds to the surface antigen of an immune cell and a substance that can generate an optical signal. Examples of the substance that binds to the surface antigen include, but are not limited to, an antibody that binds to the surface antigen of an immune cell, an antibody fragment, a single-chain antibody, and an aptamer. In this embodiment, 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.

The substance that binds to the surface antigen may bind directly to the surface antigen or indirectly to the surface antigen. As used herein, "indirectly binding to a surface antigen" means binding to a surface antigen via one or more molecules. The substance that indirectly binds to the surface antigen is, but is not limited to, binds to other substances bound to the surface antigen. That is, a substance that indirectly binds to the surface antigen binds indirectly to the surface antigen via the other substance.

Examples of the substance that can generate an optical signal include, but are not limited to, a fluorescent substance. As the fluorescent substance, known fluorescent dyes, fluorescent proteins and the like can be used without particular limitation. The substance capable of producing an optical signal may be chemically bound to a trap that binds to a surface antigen, without limitation. Examples of such traps include fluorescently labeled antibodies that directly bind to surface antigens (see traps in FIGS. 1b and 1c).

In the present embodiment, the capture body that binds to 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, the trap is, but is not limited to, an antibody, antibody fragment, single chain antibody, or aptamer that directly binds to a surface antigen, to which a substance capable of producing an optical signal is bound. It may be a thing.

Alternatively, the trap may include a substance that indirectly binds to the surface antigen and a substance that can generate an optical signal. In this embodiment, the trap is an antibody, antibody fragment, single chain antibody, or aptamer that binds to another substance bound to a surface antigen, to which a substance that can generate an optical signal is bound. good. For example, but not limited to, an antibody that specifically binds to a surface antigen is bound to the surface antigen as a primary antibody, and then an antibody that specifically binds to the primary antibody is bound as a secondary antibody. .. Here, the substance that can generate an optical signal is bound to the secondary antibody. In this example, the secondary antibody is a trap that can bind to the surface antigen and generate an optical signal (see the trap in FIG. 1a). Moreover, the primary antibody may be an immunostimulatory antibody.

The surface antigen to which the above-mentioned trap is bound may be a co-stimulatory molecule of an immune cell. As used herein, a "co-stimulatory molecule" provides a co-signal for activating T cells in addition to an antigen-specific signal mediated by the T cell receptor (TCR) when immune cells are activated. It means a substance present in T cells that can be given to cells. As used herein, a "costimulatory molecule", when present in an NK cell, assists in activating the NK cell in addition to a ligand-specific signal of the target cell via the activation receptor of the NK cell. Includes substances that can give signals to NK cells. Co-stimulatory molecules may be, but are not limited to, CD28, OX40, CD40L, CTLA4, PD-1 and ICOS.

In this embodiment, the substance different from immune cells is at least one selected from biological materials such as allogeneic cells, antigen presenting cells, and cancer cells. In a further embodiment, the substance different from immune cells is an allogeneic cell, an antigen presenting cell or a cancer cell. In certain embodiments, the substance that differs from immune cells is allogeneic cells. In certain embodiments, a substance that differs from immune cells is an antigen-presenting cell. In certain embodiments, the substance that differs from immune cells is cancer cells.

When cells other than immune cells such as allogeneic cells, antigen-presenting cells, and cancer cells are used as substances different from immune cells, the immunostimulatory factors in step (i) are various types such as MHC molecules present on the cells. It is an immunostimulatory factor. For example, when the substance different from the immune cell is an allogeneic cell, the immunostimulatory factor contains at least MHC molecule, but is not limited to it. When the substance different from the immune cell is an antigen-presenting cell, the immunostimulatory factor includes at least a complex of an MHC class II molecule and an antigen peptide. When the substance different from the immune cell is a cancer cell, the immunostimulatory factor contains at least a complex of MHC class I molecule and a cancer antigen peptide.

In step (i), the immune cells are brought into contact with a substance different from at least one immune cell selected from allogeneic cells, antigen-presenting cells and cancer cells, whereby the immune cells and immunity on the substances are brought into contact with each other. Includes contact with stimulators. Step (ii) includes making the immune cells form a contact surface with the substance by maintaining the contact state between the immune cells brought into contact with the substance in step (i) and the substance.

In the present embodiment, the substance different from the immune cell is preferably a non-biomaterial, for example, an instrument usually used in the field of life science. Such instruments include containers, multi-well plates, slides, beads, and the like. Examples of the container include dishes, bottles, tubes and the like. According to this embodiment, since the substance is a non-biological material, it has industrial advantages such as easy handling and long-term storage as compared with the case where a biological material is used as the substance. .. In certain embodiments, the substance, a container, a multi-well plate, a slide or a bead, comprises an immunostimulatory factor on its surface. For example, if the immunostimulatory factor is an immunostimulatory antibody, the antibody may be immobilized on the surface of a container, multi-well plate, slide or bead. Alternatively, the substance, such as a container, multi-well plate, slide or bead, does not contain an immunostimulatory factor on its surface.

In the present embodiment, after contacting the immunostimulatory factor with the immune cell in step (i), the substance different from the immune cell in step (ii) may contain an additional immunostimulatory factor on its surface. good. In this embodiment, for the purpose of additional immunostimulation, a substance having an immunostimulatory factor of the same kind or a different kind as the immunostimulatory factor of step (i) on its surface may be used in step (ii). For example, in step (i), an antibody against a co-stimulating molecule was used as an immunostimulatory factor, and in step (ii), a multi-well plate in which an anti-CD3 antibody was immobilized on the well surface was used as a substance different from immune cells. Can be used. Alternatively, the substance in step (ii) may not contain additional immunostimulatory factors on its surface.

In the present embodiment, when an immunostimulatory antibody is used as the immunostimulatory factor of step (i), this immunostimulatory factor may be shared as a trap for step (iii). In this embodiment, the immunostimulatory factor is, for example, an anti-CD28 antibody, an anti-CD3 antibody, an anti-CD40L antibody, an anti-OX40 antibody, an anti-CTLA4 antibody, an anti-PD-1 antibody, an anti-ICOS antibody, an antibody fragment thereof, a single chain. The antibody, and one or more combinations thereof, may be a complex labeled with a substance capable of producing an optical signal. By using such a labeled immunostimulatory antibody, the immune stimulation to the immune cells in the step (i) and the capture of the surface antigen of the immune cells in the step (iii) can be performed at the same time.

Therefore, further embodiments of the present invention include (i) a step of contacting an immune cell with an immunostimulatory factor, and (ii) a step of causing the immune cell to form a contact surface with a substance different from the immune cell. It comprises (iv) a step of detecting an optical signal generated from the capture body, and (v) a step of determining whether or not the immune cell has an immune stimulus responsiveness based on the detected optical signal. Provided is a method for measuring the immunostimulatory responsiveness of an immune cell, which is an immunostimulatory antibody labeled with a substance that can generate an optical signal. According to this embodiment, the immunostimulatory factor also serves as a trap that binds to the surface antigen in the immune cell and can generate an optical signal, so that the number of reagents and the number of steps can be reduced. Therefore, this embodiment has advantages in terms of cost and labor.

In this 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 molecule such as an immunostimulatory antibody, an immunostimulatory peptide, or an MHC molecule, or a complex of an MHC molecule and an antigenic peptide. The immunostimulatory factor may be immobilized on a container, multi-well plate, slide or bead, without limitation. For example, when the immunostimulatory factor is an immunostimulatory antibody or an immunostimulatory peptide, the mode of immobilization is not particularly limited and may be, for example, physical adsorption. Alternatively, if the immunostimulatory factor is modified with biotin and avidin (or streptavidin) is immobilized on the surface of a substance different from the immune cells, the immunostimulatory factor can be obtained through the binding between biotin and avidin (or streptavidin). It can be immobilized on the substance. When the immunostimulatory factor is an MHC molecule possessed by an allogeneic cell, an antigen-presenting cell, a cancer cell, or a complex of an MHC molecule and an antigen peptide, the cell is placed in a container, a multi-well plate, a slide, or a bead. May be adhered or deposited on. In this embodiment, the immunostimulatory factor on the substance can contact the immune cell to induce activation of the immune cell. According to this embodiment, in step (i), the immune cells are brought into contact with the immunostimulatory factor on the substance by contacting the immune cells with the substance on which the immunostimulatory factor is immobilized on the surface. Including letting. In step (ii), the immune cells can be made to form a contact surface with the substance by maintaining the contact state between the immune cells brought into contact with the substance in step (i) and the substance.

Therefore, further embodiments of the present invention include (i) contacting the immune cell with an immunostimulatory factor on a substance different from the immune cell, and (ii) providing the immune cell with a contact surface with the substance. The steps of forming, (iii) contacting the immune cell with a capture body that binds to a surface antigen and can generate an optical signal, and (iv) a step of detecting an optical signal generated from the capture body. (v) Provided is a method for measuring the immune stimulus responsiveness of an immune cell, which comprises a step of determining whether or not the immune cell whose contact surface has been eliminated has an immune stimulus responsiveness based on the detected optical signal. do. According to this embodiment, steps (i) and (ii) can be performed substantially simultaneously by simply contacting the immune cells with a substance on which an immunostimulatory factor is immobilized on the surface. Therefore, this embodiment has an advantage that the number of steps can be reduced.

FIG. 1b is a diagram schematically showing steps (i) to (iii) relating to the above embodiment. In FIG. 1b, the immunostimulatory factor is an immunostimulatory antibody, the substance different from the immune cell is a plate, and the immunostimulatory antibody is immobilized on the plate. As shown in FIG. 1b, by contacting an immune cell with a plate on which an immunostimulatory antibody is immobilized, immunostimulation by the antibody (step (i)) and formation of a contact surface (step (ii)). )) And can be performed substantially at the same time. Then, in step (iii), a trap that directly binds to the surface antigen and can generate an optical signal is bound to the immunological synapse on the immune cell. In this example, although the contact surface is eliminated in step (iii), the immune cells may be brought into contact with the capture body while the contact surface is maintained. FIG. 1b is an example of steps (i) to (iii) according to one embodiment of the present invention, and the present invention is not limited thereto.

In a further embodiment, the immunostimulatory factor may be immobilized on beads, but not limited to. For example, by using beads on which an immunostimulatory antibody is immobilized, stimulation of immune cells and isolation of immune cells can be performed at the same time. When immune cells are contained in a biological sample, when the above beads are added to the sample, the immune cells in the sample are captured by the beads by an immunostimulatory antibody. Then, by centrifuging the biological sample containing the beads, immune cells can be separated from the sample. If the beads are magnetic beads, the biological sample containing the beads may be magnetically separated. At this time, the isolated immune cells are immunostimulated by the immunostimulatory antibody on the beads. In this embodiment, the contact surface between the immune cells and the beads may be eliminated before and during the measurement.

FIG. 1c is a diagram schematically showing steps (i) to (iii) relating to the above embodiment. In FIG. 1c, the immunostimulatory factor is an immunostimulatory antibody, the substance different from the immune cells is beads, and the immunostimulatory antibody is immobilized on the beads. As shown in FIG. 1c, by contacting the immune cells with the beads on which the immunostimulatory antibody is immobilized, the immunostimulation by the antibody (step (i)) and the formation of the contact surface (step (ii)). )) And can be performed substantially at the same time. Then, in step (iii), a trap that directly binds to the surface antigen and can generate an optical signal is bound to the immunological synapse on the immune cell. In this example, the contact surface is not eliminated in step (iii), but the immune cells whose contact surface has been eliminated may be brought into contact with the capture body. FIG. 1c is an example of steps (i) to (iii) according to one embodiment of the present invention, and the present invention is not limited thereto.

As used herein, the term "immune cell whose contact surface has been eliminated" means an immune cell in which the contact region formed between the immune cell and a substance different from the immune cell has disappeared. That is, it means that the immune cells are in a state of being separated from a substance different from the immune cells. The method for eliminating the contact surface is not limited, but is limited to physical treatment such as pipetting, tapping, stirring, shaking, and ultrasonic treatment for a container containing immune cells and a culture solution, and chemistry such as a surfactant. It may be a chemical treatment using a chemical or a biochemical treatment using a biochemical product such as an enzyme.

In a preferred embodiment, the contact surface is eliminated by physical treatment such as pipetting, tapping, stirring, shaking, sonication and the like. In certain embodiments, the technique is pipetting. In the present embodiment, when the contact surface is eliminated, step (iv) may detect the optical signal generated from the capture body by using a flow cytometer.

As used herein, the "flow cytometer" irradiates cells in a stream of water with excitation light and measures the fluorescence and scattered light emitted from the individual cells to measure the size and membrane protein of the individual cells in the cell population. It means a measuring device for measuring the distribution of the expression level of. In general, flow cytometers can objectively measure a large number of cells in a short time (up to tens of thousands of cells / sec) compared to general fluorescence microscope observation, and can perform quantitative measurement with high sensitivity and multiplex at the same time. It has advantages such as being able to acquire parameters. As used herein, the flow cytometer has a flow path system that allows cell measurement with the cells aligned in the fluid so that the cells can be measured individually. Such a flow path system may be realized by a fluid-based component called a flow cell. Flow cells are generally achieved by hydrodynamic focusing using a sheath flow that envelops the sample flow, but is limited to this. is not it. For example, it may be realized by microcapillaries or acoustic focusing.

In the present invention, as the flow cytometer, a commercially available product can be used without particular limitation. In the present specification, the flow cytometer includes a so-called "imaging flow cytometer" which includes an image pickup unit using a camera and can acquire an image of cells flowing in a liquid. As will be described later in the first embodiment, according to the imaging flow cytometer, it is possible to obtain multiple parameters including the cell size and aspect ratio based on the image of the captured cells. As used herein, the flow cytometer includes both an imaging flow cytometer and a flow cytometer that does not perform imaging. In a preferred embodiment, the flow cytometer is an imaging flow cytometer. In the present invention, the flow cytometer can be used without particular limitation even if it is a commercially available product.

In this embodiment, step (iv) includes immune cells if the substance different from immune cells is at least one selected from allogeneic cells, antigen presenting cells, and cancer cells, or beads. , A complex of a substance different from immune cells and a capture body may be measured with a flow cytometer or an imaging cytometer to obtain an optical signal generated from the capture body in the complex. Alternatively, when the substance different from the immune cell is a container, a multi-well plate or a slide, the contact surface between the immune cell and the substance different from the immune cell is eliminated, and the complex of the immune cell and the capture body is formed. Optical signals generated from traps in the complex may be obtained by measurement with a flow cytometer or imaging cytometer.

In the present specification, the "imaging cytometer" acquires fluorescent images, scattered light, and transmitted light images of a large number (thousands to millions) of individual cells in a short time (several seconds to minutes). It means a statistically accurate cell measuring device capable of quantitative measurement. It is a measuring device that can extract information for each cell by cell image processing. In the present invention, as the imaging cytometer, a commercially available product can be used without particular limitation.

In a further embodiment, a method of assessing the activated state of the immune system in a subject is provided. In this embodiment, the above-mentioned invention includes a step of carrying out the above-mentioned invention on a biological sample containing immune cells obtained from the subject. That is, the evaluation method of this embodiment consists of (i) a step of contacting an immune cell in a biological sample with an immunostimulatory factor, and (ii) a substance different from the immune cell in the immune cell in the biological sample. A step of forming a contact surface, (iii) contacting an immune cell in a biological sample with a trap that binds to a surface antigen in the immune cell and can generate an optical signal, and (iv) resulting from the trap. It includes a step of detecting an optical signal and (v) a step of determining whether or not the sample is immunostimulatory responsive based on the detected optical signal.

Further, in the evaluation method of this embodiment, the capture body contains a fluorescent substance, and in the step (v), a fluorescent image is acquired based on the fluorescent signal detected in the step (iv), and the determination is made based on the fluorescent image. You may go. In the fluorescence image, an area showing a fluorescence signal reflecting the distribution of the surface antigen may be measured, and based on the area, for example, the proportion of immune cells having immunostimulation responsiveness in the biological sample may be calculated. Then, the determination may be made by comparing the ratio with the ratio of immune cells having standardized immunostimulatory responsiveness to a biological sample derived from a healthy person. In this embodiment, for example, if the proportion of the sample is higher than the standardized proportion, the sample may be determined to be immune responsive or immune responsive. Alternatively, in this evaluation method, the proportion of immune cells having immunostimulatory responsiveness in the biological sample containing the immune cells obtained from the subject may be compared with the proportion of the conventional biological sample derived from the same subject. For example, if the proportion of the sample is higher than the previous proportion, it may be determined that the subject has improved or immune responsiveness.

Another aspect of the invention relates to a method of measuring the immune stimulus responsiveness of an immune cell using a trap that can bind to a co-stimulator molecule and generate an optical signal in the immune cell.

The method of this embodiment is similar to that described in the method of the above-described embodiment, except that the capture body is a capture body that can bind to a costimulatory molecule in an immune cell and generate an optical signal. Examples of the costimulatory molecule to which the trap binds include CD28, OX40, CD40L, CTLA4, PD-1 and ICOS.

Another aspect of the present invention relates to a cell analyzer for analyzing the activated state of immune cells. An embodiment of the present invention according to this aspect will be described with reference to the accompanying drawings. It should be noted that the following explanations are all illustrative and not restrictive, and do not limit the invention described in the appended claims in any way.

<Embodiment 1 of cell analyzer>
With reference to FIG. 2, the cell analyzer 1 includes a measuring device 2 and an information processing device 3. The measuring device 2 optically measures a sample containing a complex of an immune cell and a trap that binds to the surface antigen of the immune cell and can generate an optical signal with a flow cytometer. The information processing device 3 analyzes the measurement result by the measuring device 2 and displays the analysis result on the display unit 320.

With reference to FIG. 3, the measuring device 2 has an introduction unit 201, an image pickup 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.

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 (see FIG. 4) of the imaging unit 203 together with the sheath liquid by a pump. The introduction unit 201 supplies a sample to the image pickup unit 203 according to the instructions of the CPU 204. In the first embodiment, the measurement sample is a phycoerythrin (PE) that binds to the anti-CD28 antibody by immobilizing the cloned T cells derived from human peripheral blood after immunizing the cloned T cells including contact with the anti-CD28 antibody. A complex secondarily stained with a labeled anti-mouse IgG antibody is used.

The image pickup unit 203 irradiates the sample supplied from the introduction unit 201 with laser light, images the composite, and uses the image information of the generated transmitted light image and the fluorescent image as an electric signal to the analog signal processing unit 211. Output. The analog signal processing unit 211 amplifies the electric signal output from the image pickup unit 203 according to the instruction of the CPU 204, and outputs the electric signal to the A / D converter 212.

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

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

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 part of the measuring device 2 according to the control signal.

The communication interface 205 transmits the measurement data output from the CPU 204 to the information processing device 3, and receives the control signal output from the information processing device 3. The memory 206 is used as a work area of the CPU 204.

With reference to FIG. 4, the imaging unit 203 includes light sources 203a and 203b, a flow cell 203c, a condenser lens 203d and 203e, an objective lens 203f, an optical unit 203g, a condenser lens 203h, and a camera 203i. I have.

In this embodiment, the light source 203a is a semiconductor laser light source. The light emitted from the light source 203a is a laser beam having a wavelength of λ1. The condenser lens 203d collects the light emitted from the light source 203a and guides it to the sample in the flow cell 203c. The light of the wavelength λ1 emitted from the light source 203a irradiates the sample passing through the inside of the flow cell 203c, whereby the transmitted light of the wavelength λ1 is generated from the immune cells.

The light source 203b is a semiconductor laser light source. The light emitted from the light source 203b is a laser beam having a wavelength of λ2. In this embodiment, the wavelength λ2 is about 488 nm. The condenser lens 203e collects the light emitted from the light source 203b and guides it to the sample in the flow cell 203c. The light of wavelength λ2 emitted from the light source 203b irradiates the sample passing through the inside of the flow cell 203c, whereby fluorescence of wavelength λ3 is generated from PE.

The objective lens 203f collects the transmitted light having a wavelength λ1 and the fluorescence having a wavelength λ3. The optical unit 203g has a configuration in which two dichroic mirrors are combined. The two dichroic mirrors of the optical unit 203g reflect the transmitted light of the wavelength λ1 and the fluorescence of the wavelength λ3 at different angles, and are separated on the light receiving surface 203ia (see FIG. 5) of the camera 203i described later. The condenser lens 203h collects the transmitted light having a wavelength λ1 and the fluorescence having a wavelength λ3. The camera 203i receives the transmitted light of the wavelength λ1 and the fluorescence of the wavelength λ3, and outputs the image information of the sample in the flow cell 203c as an electric signal to the analog signal processing unit 211. The camera 203i may be a TDI (Time Delay Integration) camera. With a TDI camera, more accurate image information can be obtained.

As shown in FIG. 5, the camera 203i receives the transmitted light of the wavelength λ1 and the fluorescence of the wavelength λ3 in the light receiving regions 203ib and 203ic on the light receiving surface 203ia, respectively. The light receiving surface 203ia is a light receiving surface of an image pickup element such as a CMOS image sensor arranged in the camera 203i. The position of the light emitted to the light receiving surface 203ia moves within the light receiving regions 203ib and 203ic, respectively, as the sample moves along with the movement of the flow cell 203c, as shown by the arrows. In this way, since the two lights are separated on the light receiving surface 203ia by the optical unit 203g, the CPU 204 can extract the signal corresponding to each light from the image signal output by the camera 203i.

With reference to FIG. 6, the information processing apparatus 3 includes 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.

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

The hard disk 304 stores various computer programs such as an operating system and an application program, and data used for executing the computer programs to be executed by the CPU 301. Further, the hard disk 304 stores the measurement data received from the measuring device 2.

Based on the measurement data, the hard disk 304 measures analysis parameters including the number of immune cells contained in the sample, cell size, aspect ratio, area showing the fluorescence signal, total fluorescence signal intensity in the area, and the like. A program for analyzing a sample and a display program for displaying the analysis result on the display unit 320 are stored. By storing these programs, analysis processing and display processing described later are performed. That is, the CPU 301 is provided with a function of executing the process of FIG. 7b, which will be described later, by the program.

The reading device 305 is composed of a CD drive, a DVD drive, and the like. The reading device 305 can read computer programs and data recorded on a recording medium such as a CD or DVD.

An input unit 310 composed of a mouse, a keyboard, or the like is connected to the input / output interface 306, and the user inputs an instruction to the information processing apparatus 3 using the input unit 310. The image output interface 307 is connected to a display unit 320 composed of a display or the like, and outputs a video signal corresponding to the image data to the display unit 320. The display unit 320 displays an image based on the input video signal.

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

FIG. 7 is a flowchart showing the control of the CPU 204 of the measuring device 2 and the CPU 301 of the information processing device 3. FIG. 7a is a flowchart showing the control process by the CPU 204 of the measuring device 2, and FIG. 7b is a flowchart showing the control process by the CPU 301 of the information processing apparatus 3.

FIG. 7b is referred to with respect to the control process of the CPU 301 of the information processing apparatus 3. When the CPU 301 receives a measurement start instruction from the user via the input unit 310 (step S11: YES), the CPU 301 transmits a measurement start signal to the measuring device 2 (step S12). Subsequently, the CPU 301 determines whether or not the measurement data has been received (step S13). If the measurement data has not been received (step S13: NO), the process is waited for.

FIG. 7a is referred to with respect to the control process of the CPU 204 of the measuring device 2. When the CPU 204 receives the measurement start signal from the information processing apparatus 3 (step S21: YES), the CPU 204 measures the sample (step S22). In the measurement process (step S22), a measurement sample containing the complex of the trap and the immune cells is supplied from the introduction unit 201 to the flow cell 203c together with the sheath liquid, and the sample of the measurement sample wrapped in the sheath liquid in the flow cell 203c is supplied. A stream is formed. The formed sample stream 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 immune cells pass through the beam spot, transmitted light and fluorescence are generated. The generated transmitted light and fluorescence are captured by the camera 203i and converted into an electric signal.

These electric signals are converted into digital signals by the A / D converter 212, and signal processing is performed by the digital signal processing unit 213. As a result, measurement data including a transmitted light image and a fluorescence image can be obtained for each complex that has passed through the flow cell 203c. 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 process to the information processing apparatus 3 (step S23), and ends the process.

Again, see FIG. 7b. When the CPU 301 of the information processing device 3 receives the measurement data from the measurement device 2 (step S13: YES), the CPU 301 stores the measurement data in the hard disk 304 and performs an analysis process based on the measurement data (step S14). Subsequently, the CPU 301 displays the analysis result acquired in S14 on the display unit 320 (step S15). After that, the process ends.

An example of the analysis process (step S14 in FIG. 7b) performed by the CPU 301 of the information processing apparatus 3 based on the measurement data will be further described with reference to FIGS. 8 to 10. FIG. 8 shows a transmitted light image, a fluorescence image (CD28), and a superposed image thereof among the measurement data obtained by measuring the immune cells. As shown in the fluorescent image of FIG. 8a, the co-stimulatory molecule CD28 tends to be uniformly distributed on the cell membrane of immune cells when no immune stimulus is applied. As shown in the fluorescent image of FIG. 8b, the co-stimulatory molecule CD28 tends to be localized on a part of the cell membrane of immune cells when immunostimulated. Although not intended to be bound by theory, it is considered that the cell membrane part where the co-stimulator molecule is localized is the contact surface formed in the immune cell with a substance different from the immune cell. Be done. That is, it is considered that the portion where this co-stimulator molecule is localized indicates the immunological synapse formed between the cell and a different substance.

The CPU 301 measures the analysis parameters based on the transmitted light image and the fluorescence image of all the cells captured in step S22 of FIG. 7a (step S14 of FIG. 7b). The parameters for analysis are not limited, but are the cell size (number of pixels), the aspect ratio of the cell, the area showing the fluorescence signal in the cell (number of pixels), and the total fluorescence signal intensity in the area (total fluorescence of the cell). Signal intensity) and their ratios. FIG. 9 shows a schematic diagram of the measurement processing of the transmitted light image and the fluorescence image (step S14 of FIG. 7b) shown by the CPU 301 of the information processing apparatus 3 in FIG. 8b. The obtained measurement data is stored in the hard disk 304. The meanings of the terms of cell size, aspect ratio, area showing fluorescence signal, and total fluorescence signal intensity are as described above.

FIG. 10 is a flowchart showing the analysis process in step S15 of the CPU 301 of the information processing apparatus 3. The CPU 301 creates a two-dimensional histogram (2D scattergram) in which the analysis parameters measured in step S14 are assigned to the X-axis and the Y-axis, respectively, and measures the measurement data of the cells corresponding to the dots existing in the predetermined range. Extract and analyze.

In this embodiment, the CPU 301 first creates a 2D scattergram with the cell size assigned to the X-axis and the cell aspect ratio assigned to the Y-axis (FIG. 11a), and the cells within a predetermined range (R1). Extract the measurement data (step S51). The range (R1) is a predetermined range that reflects the size of the cell alone in the cell size (X-axis) and reflects the existence of the cell alone in the aspect ratio (Y-axis). Gating with. This excludes data derived from substances such as cell debris smaller than cells and cell aggregates larger than cells alone.

Each of the dots displayed on the 2D scattergram indicates the measured value of each cell. The CPU 301 extracts measurement data of cells indicating measured values in the area displayed as R1 in the image. In this embodiment, the dots displayed on the 2D scattergram are linked with the measurement data of the individual cells measured in step S22 of FIG. 7a. When a dot displayed on the 2D scattergram is specified by the user, the CPU 301 reads out the measurement data (transmitted light image and / or fluorescent image) of the cells corresponding to the dot from the hard disk 304 and displays it on the display unit 320. do.

The CPU 301 creates a 2D scattergram for the measurement data of the cells extracted in step S51, in which the total fluorescence signal intensity of the cells is assigned to the X-axis and the sharpness (image sharpness) of the fluorescent image is assigned to the Y-axis ( FIG. 11b), measurement data of cells within a predetermined range (R2) are further extracted (step S52). The range (R2) is gated in a range that reflects the expression level of a predetermined amount of the co-stimulator molecule CD28 in the total fluorescence signal intensity (X-axis) of the cell, and reflects the sharpness of a predetermined image in the sharpness (Y-axis). Gating in range. As a result, the data of the cells expressing the predetermined amount of the co-stimulatory molecule CD28 are extracted, and the data of the cells not expressing the predetermined amount of the co-stimulatory molecule CD28 are excluded.

Subsequently, the CPU 301 creates a 2D scattergram in which the area showing the fluorescence signal is assigned to the X-axis and the cell size is assigned to the Y-axis for the measurement data of the cells extracted in step S52 (FIG. 11c). The number of dots in the range (R4) of is counted (step S53). The range (R4) is gating in a predetermined range that reflects the localization of the co-stimulator molecule CD28 in the area showing the fluorescent signal (X-axis), and reflects the size of the cell alone in the cell size (Y-axis). Gating within a predetermined range. By counting the number of dots in the range (R4), the number of immune cells responsive to immune stimuli is counted. In this embodiment, the cells corresponding to the dots in range (R4) tend to have immunological synapses, and the cells are determined to be responsive to immune stimuli in this embodiment.

The CPU 301 further counts the number of dots shown throughout FIG. 11c (corresponding to the number of dots in the range (R2) of FIG. 11b) and the range (R4) with respect to the number of dots in the range (R2). The ratio of the number of dots in the range ([number of dots in the range (R4)] / [number of dots in the range (R2)]) is calculated (step S54). As a result, the proportion of immune cells having immunostimulatory response is calculated among the individual immune cells expressing a predetermined amount of the co-stimulator molecule CD28.

The analysis result about the number of dots and the ratio in each range obtained by the above analysis processing is displayed on the display unit 320.

This analysis result may be further analyzed, for example, by comparing a biological sample derived from a healthy person with the proportion of immune cells having a standardized immune stimulus responsiveness. In this further analysis, for example, if the proportion of the sample is higher than the standardized proportion, the sample may be determined to be immune responsive. Alternatively, the results of this analysis may be further analyzed by comparing the proportion of immunostimulatory responsive immune cells to previous biological samples from the same subject. In this further analysis, for example, if the proportion of the sample is higher than the previous proportion, it can be determined that the subject's immune responsiveness has improved.

Thus, in one embodiment, an introduction unit that introduces a complex of an immune cell and a trap that binds to a surface antigen in the immune cell and can generate an optical signal, and the complex supplied from the introduction unit. A cell analyzer is provided that includes an imaging unit that captures an image of an immune cell and an analysis unit that determines whether or not the immune cell has an immune stimulus responsiveness based on the image captured by the imaging unit. In a specific embodiment, the analysis unit acquires a value reflecting the distribution of the surface antigen based on the image, and compares the acquired value with a threshold value so that the immune cell becomes immunostimulatory responsive. Determine if you have it. In another embodiment, the capturer comprises a fluorescent substance, the image comprises a fluorescent image, and a value reflecting the distribution of the surface antigen provides a fluorescent signal reflecting the distribution of the surface antigen based on the fluorescent image. A cell analyzer is provided that includes the area shown and determines that the immune cell is immunostimulatory responsive when the area is smaller than the threshold. In another embodiment, the image includes a transmitted light image, and the analysis unit acquires a value reflecting the size of the immune cell based on the transmitted light image, and reflects the size of the immune cell. A cell analyzer is provided that determines whether or not an immune cell has immunostimulatory responsiveness based on a value and a value that reflects the distribution of the surface antigen.

The analysis process shown above is only an example of the analysis process based on the measurement data, and different analysis processes can be used.

<Embodiment 2 of Cell Analyzer>
Instead of step S53 of the analysis process of the first embodiment, the CPU 301 assigns the total fluorescence signal intensity of the cells to the X-axis and the area showing the fluorescence signal to the Y-axis for the measurement data extracted in step S52. A scattergram may be created (FIG. 11d) to count the number of measurement data within a predetermined range (R3). The range (R3) is gated in a range that reflects a predetermined expression level of the co-stimulator molecule CD28 per cell in the total fluorescence signal intensity (X-axis) of the cell, and is co-located in the area (Y-axis) showing the fluorescence signal. Gating within a predetermined range that reflects the localization of the stimulator molecule CD28. By counting the measurement data in the range (R3), the number of immune cells responsive to immune stimuli is counted. In this embodiment, the cells corresponding to the dots in range (R3) tend to have immunological synapses, and the cells are determined to be responsive to immune stimuli in this embodiment.

In this embodiment, the CPU 301 further counts the number of dots shown throughout FIG. 11d (corresponding to the number of dots in the range (R2) of FIG. 11b) instead of step S54 and counts the range (R2). ) May be calculated as a ratio of the number of dots in the range (R3) to the number of dots in the range (R3) ([number of dots in the range (R3)] / [number of dots in the range (R2)]). As a result, the proportion of immune cells having immunostimulatory response is calculated among the individual immune cells expressing a predetermined amount of the co-stimulator molecule CD28.

<Embodiment 3 of Cell Analyzer>
Instead of step S53 of the analysis process of the first embodiment, the CPU 301 assigns a class value for an area showing a fluorescent signal to the X-axis and a frequency to the Y-axis for the measurement data of the cells extracted in step S52. A histogram may be created and displayed on the display unit 320. Instead of step S54, the CPU 301 may further calculate the average value, median value, and mode value of the area showing the fluorescence signal of the measurement data extracted in step S52 from this histogram. Compare the obtained mean, median, and mode with, for example, the standardized mean, median, and mode of areas showing fluorescent signals in immune cells obtained from biological samples from healthy individuals. By doing so, it may be further analyzed. In this further analysis, for example, if the sample's mode value (area showing fluorescence signal) is smaller than the standardized mode value (area showing fluorescence signal), this is It suggests that the co-stimulator molecule CD28 is localized, and as a result, the sample can be determined to be highly responsive to immune stimuli.

As shown in the third embodiment, the immune responsiveness of immune cells can be analyzed without creating a 2D scattergram in the analysis of measurement data. However, as described in Embodiment 1, the dots represented by the 2D scattergram can link the measurement data of individual immune cells, for example, each dot can be designated to specify a transmitted light image and fluorescence of that immune cell. Measurement data including images can be displayed. Therefore, the analysis by 2D scattergram is a preferable analysis processing method in that detailed analysis based on an image can be additionally performed on individual cells.

<Embodiment 4 of Cell Analyzer>
In the above analysis processing, the analysis based on the captured image data is shown as an example, but the immune cells responsive to immune stimulus can be analyzed from the measurement data other than the captured image data. For example, in the measuring device 2 (FIG. 4) in the first embodiment, instead of the optical unit 203g and the camera 203, a dichroic mirror 203j, 203k, a forward scattering light receiving unit 203l, and a fluorescent light receiving unit 203m are used. The light of wavelength λ1 emitted from the light source 203a irradiates the sample passing through the inside of the flow cell 203c, thereby producing forward scattered light of wavelength λ1 from the immune cells. The light of wavelength λ2 emitted from the light source 203b irradiates the sample passing through the inside of the flow cell 203c, whereby fluorescence of wavelength λ3 is generated from PE. The sample in the fourth embodiment is the complex used in the first embodiment. The forward scattered light having a wavelength λ1 is reflected by the dichroic mirror 203j and received by the forward scattered light receiving unit 203l. The fluorescence of the wavelength λ3 passes through the dichroic mirror 203j, is reflected by the dichroic mirror 203k, and is received by the fluorescence light receiving unit 203k. The forward scattering light receiving unit 203l and the fluorescent light receiving unit 203m are photomultipliers. In this example, the optical signals obtained in step S22 of FIG. 7a are a forward scattered light signal and a fluorescence signal, and in step S14 of FIG. 7b, “pulse width (w)” and “pulse area (A)” are used as analysis parameters. And "Pulse height (H)" is obtained. These analysis parameters will be described with reference to FIG.

As an example, FIG. 12 shows a region in which a cell in which the co-stimulator molecule CD28 is uniformly distributed on the cell membrane (FIG. 12a) and a cell in which the co-stimulator molecule CD28 is localized on the cell membrane (FIG. 12b) detect a fluorescent signal (FIG. 12). It is a schematic diagram which shows the fluorescent signal detected while passing through (not shown) in the direction of an arrow, respectively (FIGS. 12c and 12d).

In the present specification, in the pulse of fluorescence intensity as shown in FIGS. 12c and 12d, the time when the fluorescence signal is obtained beyond the baseline set as the threshold is referred to as "pulse width (w)", and the pulse is in terms of fluorescence intensity. The fluorescence intensity when a peak is shown is called "pulse height (H)". "Pulse area (A)" refers to the area between the baseline and the fluorescence signal intensity curve.

In this example, the same amount of the co-stimulator molecule CD28 is expressed on the cell membrane in the cells of FIG. 12a and the cell of FIG. 12b, and the pasul areas (A) of FIGS. 12c and 12d have the same value. Here, the pulse width (w) and the pulse height (H) differ depending on the distribution pattern of the co-stimulating molecules. The pulse width (w) is narrower when the co-stimulator molecules are localized on the cell membrane (Fig. 12b) than when the co-stimulator molecules are uniformly distributed on the cell membrane (Fig. 12a). On the other hand, the height (H) of the pulse becomes higher (FIGS. 12c and 12d).

FIG. 13 is a schematic diagram when a 2D scattergram is created by measuring a plurality of immune cells in this embodiment. FIG. 13a is a 2D scattergram in which the pulse height (H) is assigned to the X-axis and the pulse width (w) is assigned to the Y-axis, and FIG. 13b assigns the pulse height (H) to the X-axis. , A 2D scattergram in which the pulse area (A) is assigned to the Y-axis.

As described above, when the co-stimulator molecules are uniformly distributed on the cell membrane (Fig. 12a), the pulse width (Fig. 12b) is higher than when the co-stimulator molecules are localized on the cell membrane (Fig. 12b). w) tends to spread and the pulse height (H) tends to decrease (FIGS. 12c and 12d). Therefore, in the 2D scattergram shown in FIG. 13a, the data in the area circled by the dashed line reflects the cells in which the co-stimulator molecules are uniformly distributed on the cell membrane, while the solid line. The data in the area surrounded by the squares tend to reflect the cells in which the co-stimulator molecules are localized on the cell membrane. In the 2D scattergram shown in FIG. 13b, the data in the area surrounded by the dashed square is the data that reflects the cells in which the co-stimulator molecules are uniformly distributed on the cell membrane, while the solid square. The data in the area enclosed by the circles tend to reflect the cells in which the co-stimulator molecules are localized on the cell membrane.

Thus, in one embodiment, an introduction unit that introduces a complex of an immune cell and a trap that binds to a surface antigen in the immune cell and can generate an optical signal, and the complex supplied from the introduction unit. A detection unit that detects an optical signal from the complex generated by irradiating light with light, and an analysis unit that determines whether or not the immune cell has an immune stimulus responsiveness based on the detected optical signal. Including, cell analyzers are provided. In certain embodiments, the trap contains a fluorescent substance, the detection unit detects a fluorescent signal generated from the complex, and the analysis unit detects the distribution of the surface antigen based on the fluorescent signal. By acquiring the value to be reflected and comparing the acquired value with the threshold value, it is determined whether or not the immune cell has an immune stimulus responsiveness, and the value reflecting the distribution of the surface antigen is obtained from the complex. A cell analyzer is provided that comprises at least one of the pulse width of the fluorescent signal, the height of the fluorescent pulse, and the pulse area of the fluorescent signal.

As shown in Examples, a cell in which a surface antigen or a co-stimulator molecule to which a trap is bound is localized on a cell membrane is determined to be an immune cell exhibiting immune responsiveness. Therefore, in FIGS. 13a and 13b, cells showing data that fall into the region surrounded by the solid line square can be determined to be immunoresponsive cells. As exemplified in this embodiment, it is understood that immune cells having an immune stimulus response can be measured not only by an analysis process based on captured image data but also by an analysis process as described above.

The analysis parameters and analysis processes shown here are merely examples, and different analysis parameters may be measured from the measured data, or different analysis processes may be performed on the measured analysis parameters. .. For example, the pulse area may be an approximate value as long as it reflects the area under the time curve of the pulse, and is not limited to the time integral value. The pulse area may be the product of the pulse width and the peak height, or may be the area of a triangle obtained from the pulse width and the peak height. Further, in the form of measuring the time integral value, the base does not have to be the baseline and can be set as appropriate. For example, a value exceeding a predetermined threshold value from the baseline may be used as the base.

Specific examples will be described below, but they show preferred embodiments of the present invention and do not limit the inventions described in the appended claims in any way. It is intended that the equivalents, or modifications, modifications or modifications readily recognized from the specific embodiments, materials, compositions and methods described herein are within the scope of the invention.

Example 1: Measurement of immune stimulus response of immune cells using CD28 as an index
(1) Preparation of cloned T cells
5 × 10 ⁶ CD4 positive cells (Stemcell Technologies: ST-70026) and CD8 positive cells (Funakoshi: 0508-100, Cosmobio: PB08C-1) were cultivated in 1 μg / mL LEAF ™ 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 seeded in Yssel's medium (Gemini Bio-Products: 400102) supplemented with 2% human serum (SIGMA: H4522-100ML), 1 × 10 ⁶ peripheral blood mononuclear cells and 1 × 10 ⁴ JY cells and 14 Co-cultured for days. After 14 days, T cells were seeded on 96-well plates by limiting dilution to 0.3, 1.0 and 3.0 cells / well and cultured until 1 × 10 ⁴ peripheral blood mononuclear cells and colonies were formed. .. T cells were collected from the wells in which the colonies were formed to obtain cloned T cells. Hereinafter, the obtained T cells are also referred to as "prepared clone T cells".

(2) Immobilization of antibody
To a 24-well plate, a solution (0.01 mg / ml) of an anti-CD3 antibody (BioLegend # 300414 Clone: UCHT1) was added in an amount of 300 μl per well, and the anti-CD3 antibody was immobilized on the well surface. Hereinafter, the obtained plate is also referred to as an "anti-CD3 antibody-immobilized plate".

(3) Stimulation and measurement of immune cells To the anti-CD3 antibody-immobilized plate, prepared cloned T cells were added so as to be about 5 × 10 ⁵ cells per well. Then, an anti-CD28 antibody (BioLegend # 302914 Clone: CD28.2) was added to the cells in the well to a final concentration of 0.01 mg / ml. The plates were centrifuged (200 G, 30 seconds) and then incubated at 37 ° C. for 30 minutes. Prepared cloned T cells were harvested from the well surface by pipetting. The recovered T cells were immobilized with a fixative containing 1% paraformaldehyde (PFA). Immobilized T cells were immunostained with PE-labeled anti-mouse IgG antibody (BioLegend # 405307). Stained T cells (5 × 10 ⁵ cells / 20 μl) were measured using Imaging FCM (ImageStreamX MarkII Imaging Flow Cytometer: Amnis).

For comparison, the case where the cells were not stimulated by contact with an antibody (anti-CD28 antibody and anti-CD3 antibody) that binds to the surface antigen of T cells and contact with a different substance (well of the plate) was also examined. The cloned T cells prepared in (1) above were fixed with the above-mentioned fixation solution without performing the above-mentioned immunostimulation, and then stained with an anti-CD28 antibody. The immobilized T cells were secondarily stained and measured using a PE-labeled anti-mouse IgG antibody.

When the above immune stimulation was not applied to T cells, CD28 was uniformly distributed on the cell membrane in T cells (see FIG. 14a). When the contact surface between the T cell and the well was eliminated after the above immunostimulation, CD28 showed localization on the cell membrane of the T cell (see FIG. 14b). As described above, the immunological synapse could be detected by using the co-stimulator molecule CD28 as an index even when the contact surface between the T cell and the well was eliminated after the above-mentioned immune stimulation was given.

(4) Data analysis
Measurement data of the above-mentioned sample containing unstimulated T cells (FIGS. 15a and b) and measurement data of the above-mentioned sample containing immunostimulated T cells (Fig. 15a and b) obtained by measurement using Imaging FCM. Multiple parameter analysis of 15c and d) was performed.

Detailed measurement processing and analysis processing are shown in the first embodiment. Briefly, image analysis is performed based on a transmitted light image measured using Imaging FCM and a fluorescence image (CD28) (Fig. 9), and the cell size (number of pixels), cell aspect ratio, and fluorescence signal in the cell are shown. The area (number of pixels) and the total fluorescence signal intensity in the area (total fluorescence signal intensity of cells) were measured. First, create a 2D scattergram in which the cell size is assigned to the X-axis and the aspect ratio of the cells is assigned to the Y-axis, and the measurement data of the cells corresponding to the dots in the predetermined range (R1) are extracted (Fig. 10). Step S51). Then, with respect to the measurement data in the range (R1), the measurement data corresponding to the dots in which the total fluorescence signal intensity of the cells was in the predetermined range (R2) was further extracted (step S52 in FIG. 10). For the measurement data in the range (R2), the number of dots in the area (FIGS. 15b and 15d) displayed as R4 was counted (step S53 in FIG. 10). In addition, the number of dots on the scattergrams of FIGS. 15b and 15d (the number of dots within the range (R2)) was counted. Then, the ratio of the number of dots in the range (R4) to the number of dots in the range (R2) was calculated by the following equation (1) (step S54 in FIG. 10). The results are summarized in Table 1 below.

[Ratio (R4 / R2)] = {[Number of dots in range (R4)] / [Number of dots in range (R2)]} × 100 ・ ・ ・ Equation (1)

According to the examples herein, the immunological synapses that can be formed when an immune cell is given an immune stimulus, including contact with an immunostimulatory factor and formation of a contact surface with a different substance, are co-stimulatory. It was found that if the detection is performed using a molecule as an index, information on the immune responsiveness of the immune cell can be accurately obtained even if the contact surface is not maintained. This was a surprising result, considering that the immunological synapse is a loose bond formed to the immune cell at the binding surface with the target cell.

Thus, according to the present invention, since the immune stimulus responsiveness of immune cells can be measured based on the immunological synapse, the responsiveness of immune cells can be measured more quickly and easily than the conventional method for analyzing the responsiveness of immune cells based on cytokine production. It is understood that the activation state can be measured.

In the examples of the present specification, since the immune stimulus responsiveness of immune cells can be measured using an imaging flow cytometer, a large number of cells can be measured, and the immune responsiveness can be measured with statistically high accuracy. It was proved to be. In the above example, the immune stimulus response of immune cells was measured using an imaging flow cytometer, but it can also be measured by an imaging cytometer that can measure a large number of cells in the same manner as the measuring device used in the example. Is understood. Further, as described in the fourth embodiment, it is understood that the flow cytometer without imaging can also measure the immune stimulus responsiveness of a large number of immune cells in the same manner as the measuring device used in the examples. Thus, according to the present invention, it is understood that the activated state of immune cells can be measured rapidly and easily with a large number of cells, which is useful for statistically accurate drug efficacy evaluation.

In the above embodiment, since the imaging flow cytometer is used, in the analysis of the measurement data of each cell, the image data of each cell can be linked to each measurement data. Therefore, after selecting cells by performing a predetermined analysis process based on the measured data, the immune stimulus responsiveness can be further evaluated by combining the image data of the cells. For example, in the analysis process for selecting cells having a high tendency for immunological synapses to be formed in the above example, about 81% of the cells in the range (R4) were present in the sample with immunological stimulation (Table 1). ). Here, in FIG. 15d, dots corresponding to the cells in the range (R4) are designated, the image data of the cells are displayed, and the image data is also evaluated, so that the immune cells can be evaluated in more detail. The responsiveness to immune stimuli can be evaluated. It is understood that this further evaluation can be evaluated by an imaging cytometer that captures an image of cells as well as an imaging flow cytometer.

According to the measurement method and cell analyzer according to the present invention, for example, when an immunostimulatory antibody is used as an immunostimulatory factor, the activated state of immune cells in the body of a subject related to cancer immunity or self-disease. When using a specific antigen peptide, the activated state of immune cells in the body of the subject related to infectious diseases or allergic diseases, and when using allogeneic cells or allogeneic MHC, transplantation medicine It is useful because the immune response of the subject to regenerative medicine can be evaluated quickly and easily. As mentioned above, it is understood that the imaging flow cytometer and the imaging cytometer, which can further utilize the image data of individual cells for the analysis process, have an additional advantage.

Reference example: Correlation between immune synapse formation and cytokine production in immune cells
(1) Measurement of Cytokine-Producing Cells In the same manner as in Example 1, an anti-CD3 antibody was immobilized on 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. Clone T cells were added to the anti-CD3 antibody immobilized plate so as to be about 5 × 10 ⁵ cells per well. Then, an anti-CD28 antibody (BioLegend # 302914 Clone: CD28.2) was added to the cells in the well to a final concentration of 0.01 mg / ml, and the cells were incubated at 37 ° C. for 4 hours. Then, Breferdin A was added to each well to a final concentration of 10 μg / ml, and the mixture was incubated for 2 hours. The plate was then allowed to stand on ice and cells in each well were collected. The recovered T cells were fixed with a fixative containing 1% PFA and then subjected to membrane permeation treatment with a PBS solution containing 0.5% saponin and 1% BSA. Treated cells were immunostained with Alexa Fluor® 488-labeled anti-IFN-γ antibody (BioLegend # 502517 Clone: 4S.B3). Stained T cells were measured with a flow cytometer (BD biosciences: Accuri ™). The measured data was analyzed by a conventional method to obtain the proportion of IFN-γ-positive cells in the measured T cells.

(2) Measurement of cells forming an immunological synapse Using T cells seeded from the same well as the T cells used in (1) above, cells forming an immunological synapse were measured using CD28 as an index. The measured data was analyzed to obtain the percentage of cells that formed immunological synapses in the measured T cells. The measurement and data analysis were performed in the same manner as in Example 1.

(3) Results The data were plotted with the vertical axis representing the proportion of IFN-γ-positive cells (%) and the horizontal axis representing the proportion of immunological synapse-forming cells (%). The obtained graph is shown in FIG. As can be seen from FIG. 16, a correlation was observed between immunological synapse formation and INF-γ production in immunostimulated cloned T cells (R ² = 0.8371). Therefore, it was shown that by detecting the immunological synapse in the immune cells whose contact surface has been eliminated, the immune cells activated by immune stimulation can be detected in the same manner as in the conventional method based on cytokine production.

Example 2: Measurement of immune stimulus response of immune cells using CD3 and CD40 as indicators
(1) Stimulation and measurement of immune cells In the same manner as in Example 1, an anti-CD3 antibody was immobilized on 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. Prepared cloned T cells were added to the anti-CD3 antibody immobilized plate so as to be about 5 × 10 ⁵ cells per well. Then, the final concentrations of anti-CD28 antibody (BioLegend # 302914 Clone: CD28.2) and PE-labeled anti-CD40L antibody (BD biosciences # 340477 Clone: 89-76) should be 0.01 mg / ml and 0.01 mg / ml, respectively. Added to cells in the well. The plates were centrifuged (200 G, 60 seconds) and then incubated at 37 ° C for 30 minutes. After removing the supernatant in the well, the prepared cloned T cells were dispersed by pipetting and collected from the well surface. The recovered T cells were immobilized with a fixation solution containing 1% PFA. Immobilized T cells were immunostained with an APC-labeled anti-CD3 antibody (BioLegend # 300411 Clone: UCHT1). Since these T cells were immunostimulated with the PE-labeled anti-CD40L antibody, they were also immunostained with the anti-CD40L antibody. Stained T cells (5 × 10 ⁵ cells / 20 μl) were measured using Imaging FCM (ImageStreamX MarkII Imaging Flow Cytometer: Amnis).

For comparison, the case where the cells were not stimulated by contact with anti-CD40L antibody, anti-CD28 antibody and anti-CD3 antibody and contact with the well of the plate was also examined. The cloned T cells prepared in (1) above were fixed on ice using the above-mentioned fixing solution without performing the above-mentioned immunostimulation. Fixed T cells were immunostained with APC-labeled anti-CD3 antibody and PE-labeled anti-CD40L antibody and measured using Imaging FCM.

When the above-mentioned immunostimulation was not applied to the prepared cloned T cells, both CD3 and CD40L were uniformly distributed on the cell membrane in the T cells (see FIGS. 17a and c, 18a and b, 19a and b). .. When the contact surface between the T cell and the well was eliminated after the above immunostimulation, both CD3 and CD40L showed localization on the cell membrane of the T cell (FIGS. 17b and d, FIGS. 18c and 18c). d, see FIGS. 19c and d). In this way, even in a state where the contact surface between the T cell and the well is eliminated after the above-mentioned immune stimulation is given, the immunological synapse can be obtained by using CD3 or CD40L, which is the surface antigen of the T cell, as an index. I was able to detect it.

(2) Data analysis
Measurement data of the above-mentioned sample containing unstimulated T cells (FIGS. 18a and b) and measurement data of the above-mentioned sample containing immunostimulated T cells (Fig. 18a and b) obtained by measurement using Imaging FCM. Multiple parameter analysis of 18c and d) was performed. An example of the measurement process and the analysis process in Example 2 is shown in FIG. Briefly, image analysis is performed based on transmitted light images and fluorescence images (CD3 and CD40L) measured using Imaging FCM, and areas showing cell size (number of pixels), cell aspect ratio, and fluorescence signal in cells (areas showing fluorescence signals in cells). The number of pixels) and the total fluorescence signal intensity in the area (total fluorescence signal intensity of cells) were measured. First, create a 2D scattergram (see Figure 20a) with the cell size assigned to the X-axis and the cell aspect ratio assigned to the Y-axis, and measure the cells corresponding to the dots in a predetermined range (Cells). Extracted. Then, for the measurement data in the range (Cells), the measurement data corresponding to the dots in which the total fluorescence signal intensity of the cells was in the predetermined range (Tcells) was further extracted (see FIG. 20b). For the measured data in the range (T cells), the number of dots in the area labeled IS_CD3 and IS_CD40L (see Figures 18c and d, Figures 19c and d) was counted, respectively. Also, the number of dots on the scattergrams of FIGS. 18c and d, 19c and d (the number of dots in the range (Tcells)) was counted. Then, the ratio of the number of dots in the range (IS_CD3 or IS_CD40L) to the number of dots in the range (Tcells) was calculated from the following equation (2). The results are shown in Tables 2 and 3 below. IS is an abbreviation for Immuno Synapse.

[Ratio (IS-forming cells / Tcells)] = {[Number of dots in range (IS_CD3 or IS_CD40L)] / [Number of dots in range (Tcells)]} × 100 ・ ・ ・ (2)

As shown in Tables 2 and 3, it was shown that the proportion of T cells that formed immunological synapses was clearly higher in T cells stimulated with an immunostimulating antibody than in the case without immunostimulation. Was done. Therefore, it was shown that the responsiveness of immune cells to immune stimulation by immunostimulating antibodies can be measured by detecting immune synapses using CD3 or CD40L as an index.

Example 3: Measurement of immune cells in response to immune stimulation by allogeneic cells
(1) Allogeneic cells and cloned T cells Retinal pigment epithelial cells (RPE cells) prepared from iPS cells derived from a predetermined donor (allogeneic iPS cells) were used as allogeneic cells. RPE cells were provided by Helios Co., Ltd. RPE cells were seeded on a 24-well plate so as to be about 5 × 10 ⁵ cells per well, and incubated at 37 ° C. and 5% CO ₂ for 7 days. As a result, a plate having RPE cells adhered to the bottom surface of the well was obtained. Clone 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, Cosmobio: PB08C-1) were cultivated in 1 μg / mL LEAF ™ 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) The cells were seeded in Yssel's medium (Gemini Bio-Products: 400102) supplemented with 2% human serum (SIGMA: H4522-100ML) and co-cultured with 1.2 × 105 RPE cells for 14 ^days .
After 14 days, T cells were seeded on 96-well plates by limiting dilution to 0.3, 1.0 and 3.0 cells / well and cultured until 1 × 10 ⁴ RPE cells and colonies were formed. T cells were collected from the wells in which the colonies were formed to obtain cloned T cells.

(2) Stimulation and measurement of immune cells
Prepared cloned T cells were added to the plate inoculated with RPE cells so as to be about 5 × 10 ⁵ cells per well. The plates were centrifuged (200 G, 120 seconds) and then incubated at 37 ° C for 30 minutes. After removing the supernatant in the well, the prepared cloned T cells were dispersed by pipetting and collected from the well surface. The recovered T cells were immobilized with a fixation solution containing 1% PFA. APC-labeled anti-CD3 antibody (BioLegend # 300411 Clone: UCHT1), anti-CD28 antibody (BioLegend # 302914 Clone: CD28.2), PE-labeled anti-CD40L antibody (BD biosciences # 340477 Clone: 89-76) ) Or PE-labeled anti-OX40 antibody (BioLegend # 350003 Clone: Ber-ACT35) for immunostaining. T cells immunostained with anti-CD28 antibody were further immunostained with PE-labeled anti-mouse IgG antibody (BioLegend # 405307). Stained T cells (5 × 10 ⁵ cells / 20 μl) were measured using Imaging FCM (ImageStreamX MarkII Imaging Flow Cytometer: Amnis).

For comparison, we also examined the case where the cells were not stimulated by contact with allogeneic cells. The cloned T cells prepared in (1) above were fixed on ice using the above-mentioned fixing solution without contact with allogeneic cells. 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 using Imaging FCM.

When the prepared cloned T cells were not immunostimulated by contact with allogeneic cells, CD3, CD28, CD40L and OX40 were all uniformly distributed on the cell membrane in the T cells (FIGS. 21a, c, e). And g, FIGS. 22a and b, 23a and b, 24a and b, 25a and b). When the contact surface between T cells and allogeneic cells was eliminated after the above immune stimulation, CD3, CD28, CD40L and OX40 showed localization on the cell membrane of T cells (Fig. 21b, d). , F and h, FIGS. 22c and d, FIGS. 23c and d, FIGS. 24c and d, FIGS. 25c and d). In this way, even when the contact surface between the T cell and the allogeneic cell is eliminated after the immunological stimulation by contact with the allogeneic cell is given, the surface antigens of the T cell, CD3, CD28, By using CD40L or OX40 as an index, immunological synapses could be detected.

(3) Data analysis
Measurement data of the above-mentioned sample containing unstimulated T cells (FIGS. 18a and b) and measurement data of the above-mentioned sample containing immunostimulated T cells (Fig. 18a and b) obtained by measurement using Imaging FCM. Multiple parameter analysis of 18c and d) was performed. The measurement process and the analysis process are the same as those in the second embodiment. Briefly, image analysis is performed based on transmitted light images and fluorescence images (CD3, CD28, CD40L or OX40) measured using Imaging FCM, and the cell size (number of pixels), cell aspect ratio, and fluorescence signal in the cell. The area (number of pixels) showing the above and the total fluorescence signal intensity in the area (total fluorescence signal intensity of cells) were measured. First, a 2D scattergram was created in which the cell size was assigned to the X-axis and the aspect ratio of the cells was assigned to the Y-axis, and the measurement data of the cells corresponding to the dots in a predetermined range (Cells) were extracted. Then, for the measurement data in the range (Cells), the measurement data corresponding to the dots in which the total fluorescence signal intensity of the cells was in the predetermined range (Tcells) was further extracted. Number of dots in the area labeled IS_CD3, IS_CD28, IS_CD40L and IS_OX40 (see Figures 22b and d, Figures 23b and d, Figures 24b and d, Figures 25b and d) for the measured data in the range (Tcells). Was counted respectively. We also counted the number of dots on each scattergram (the number of dots in the range (Tcells)). Then, the ratio of the number of dots in the range (IS_CD3, IS_CD28, IS_CD40L and IS_OX40) to the number of dots in the range (Tcells) was calculated from the following equation (3). The results are shown in Tables 4 to 7 below.

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

As shown in Tables 4 to 7, in T cells immunostimulated by allogeneic cells, the proportion of T cells that formed immunological synapses is clearly higher than that in the case of no immunostimulation. Shown. Therefore, it was shown that the responsiveness of immune cells to immune stimulation by allogeneic cells can be measured by detecting the immunological synapse using CD3, CD28, CD40L or OX40 as an index.

Claims (15)

(i) The process of bringing the immune cells to be measured into contact with the immune stimulator,
(ii) A step of forming a contact surface on the immune cell to be measured by contact with a substance different from the immune cell to be measured.
(iii) A step of physically and / or chemically eliminating the contact,
(iv) The step of contacting the immune cell to be measured with a capture body that binds to a surface antigen on the contacted surface and can generate an optical signal.
The substance different from the immune cell to be measured is a solid phase of the non-biological material and / or a cell different from the immune cell to be measured.
The immune cells to be measured are T cells and / or NK cells.
How to label immune cells. The immunostimulatory factor of step (i) is shared as a trap that can bind to the surface antigen and generate an optical signal in the contacting surface of step (iv).
The method according to claim 1, wherein step (i) and step (iv) are carried out at the same time. The method of claim 1 or 2, wherein the surface antigen comprises at least one of TCRα / β, CD3, CD40L, OX40, CD28, CTLA4, PD-1 and ICOS. The method according to any one of claims 1 to 3, wherein the surface antigen is a co-stimulating molecule. The method according to any one of claims 1 to 4, wherein the trap includes a substance that can generate an optical signal and a substance that directly or indirectly binds to the surface antigen. The method according to claim 5, wherein the substance that directly or indirectly binds to the surface antigen is an antibody, an antibody fragment, a single-chain antibody or an aptamer. (i) The process of bringing the immune cells to be measured into contact with the immune stimulator,
(ii) A step of forming a contact surface on the immune cell to be measured by contact with a substance different from the immune cell to be measured.
(iii) A step of physically and / or chemically eliminating the contact,
(iv) An optical signal of an immunological synapse formed on the contacted surface on the immune cell to be measured, or an immunostimulatory factor bound to the immune cell to be measured on the contacted surface. Including the step of labeling with a trap that can produce
The substance different from the immune cell to be measured is a solid phase of the non-biological material and / or a cell different from the immune cell to be measured.
The immune cells to be measured are T cells and / or NK cells.
How to label immune cells. The method of claim 7, wherein the trap comprises a substance capable of producing an optical signal and a substance that binds to a molecule constituting the immunological synapse or the immunostimulatory factor. The method of claim 8, wherein the molecule constituting the immunological synapse comprises at least one of TCRα / β, CD3, CD40L, OX40, CD28, CTLA4, PD-1 and ICOS. One of claims 1 to 9, wherein the immunostimulatory factor comprises at least one of an immunostimulatory antibody, an immunostimulatory peptide, a major histocompatibility complex (MHC molecule), and a complex of an MHC molecule and an antigenic peptide. The method described in. The method according to any one of claims 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. The method according to any one of claims 1 to 11, wherein the immune cell to be measured is a T cell. The method according to any one of claims 1 to 12, wherein the immunostimulatory factor of step (i) is present on a substance different from the immune cells to be measured in step (ii). The method according to any one of claims 1 to 13, wherein the contact is eliminated by stirring, ultrasonic treatment or chemical treatment. The method according to any one of claims 1 to 14, further comprising a step of separating the immune cells to be measured from a sample containing cells based on the cell size, cohesion and / or specific density. ..

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