JP7165802B2 - Method for measuring immunostimulatory responsiveness of immune cells, method for determining ability of immune cells to form immune synapse, and cell analyzer - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for measuring immunostimulatory responsiveness of immune cells, a method for determining the ability of immune cells to form immune synapses, 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 in the body and eliminate non-self. The immune system is a very important physiological mechanism in life activities. Cells of the immune system are involved in activating and suppressing the immune system by secreting proteins called cytokines and by exerting cytotoxic functions.

The immune system is deeply involved in the rejection of allogeneic cells, which can affect the success or failure of transplantation therapy. In recent years, the usefulness of immunotherapy, in which the immune system is used to treat human diseases, has been shown to be useful, and the importance of understanding the state of the immune system is increasing.

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 cells of the immune system after immunostimulation, and a great deal of time (several tens of hours) and labor is required from immunostimulation to cytokine detection.

In recent years, when immune cells and target cells come into contact with each other, structures formed in immune cells at the contact surface have attracted attention. This structure is called an immune synapse because it is functionally and morphologically similar to the synaptic structure formed by nerve cells. It has been suggested that immune synapses are involved in the amplification of immune stimulation signals, and the ability to form immune synapses is known to correlate with the ability to secrete cytokines (Non-Patent Document 1). In addition, it is known that immune synapse formation occurs in a short time (several tens of minutes) compared to cytokine secretion (Non-Patent Document 1).

As a method for measuring immune synapses, for example, immune cells are stimulated using an immunostimulatory factor on a glass substrate, and immune synapses in T cells are observed under a total internal reflection fluorescence microscope while maintaining the contact surface with the glass substrate. is known (Non-Patent Document 2). Also, a method is known in which immune synapses in T cells are measured by imaging flow cytometry while maintaining contact surfaces formed with target cells (Non-Patent Document 3). In Non-Patent Document 2, immune synapses are detected using the co-stimulatory molecule CD28 as an index, and in Non-Patent Document 3, immune synapses are detected using CD3 as an index. Non-Patent Document 2 and Non-Patent Document 3
In both cases, immune cells are observed in a state in which the contact surface between the object and 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 understanding the responsiveness of cells of the immune system to various immune stimuli is very important for various treatments involving the immune system.

Conventionally, in order to analyze the responsiveness of cells of the immune system to immune stimulation, an analysis method using cytokine secretion as an indicator has been used. However, in this analysis method, immune cells must be cultured until cytokines are secreted after immune stimulation, which is 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 the measurement is time-consuming, making it difficult to measure easily. was not suitable for

Therefore, there is a need in this field for quickly and simply ascertaining the responsiveness of cells of the immune system to various immune stimuli.

In order to solve the above problems, the present inventors have focused on immune synapses, which have been suggested to undergo changes in a shorter period of time than cytokine secretion and to be involved in the amplification of immunostimulatory signals. However, since immune synapses are loose bonds formed in immune cells on the binding surface with target cells (Non-Patent Document 3), there was concern that they would dissociate before and during measurement. In fact, the immune synapse has not been used as an indicator of the responsiveness of immune cells to immunostimulation in the absence of contact surfaces between target cells and immune cells.

The present inventors have repeatedly studied methods for rapidly detecting the responsiveness of immune cells to immune stimulation, focusing on various factors known to be involved in the formation of immune synapses. As a result, it was found that, among various factors, surface antigens such as co-stimulatory molecules can be used as indicators to determine the ability of immune cells to form immune synapses even if the contact surface between the immune cells and the target is eliminated. The present invention has been made.

The first aspect of the present invention includes (i) the step of contacting the immune cells to be measured with an immune stimulator, and (ii) contacting the immune cells to be measured with a substance different from the immune cells to be measured. (iii) contacting an immune cell to be measured with a capturing body capable of binding to a surface antigen at the contact surface and generating an optical signal; (iv) an optical signal generated from the capturing body. and (v) determining, based on the detected optical signal, whether or not the immune cells to be measured, the contact surface of which has been removed by the time the optical signal is detected, have immunostimulatory responsiveness. A method for measuring immunostimulatory responsiveness of an immune cell comprising:

The second aspect of the present invention includes (i) the step of contacting the immune cells to be measured with an immune stimulator, and (ii) contacting the immune cells to be measured with a substance different from the immune cells to be measured. (iii) contacting the immune cell to be measured with a capture body capable of binding to co-stimulatory molecules at the contact surface and generating an optical signal; and (v) automatically determining whether or not the immune cells to be measured have immunostimulatory responsiveness based on the detected optical signal. provide a method for measuring sexuality.

A third aspect of the present invention includes (i) the step of contacting the immune cells to be measured with an immune stimulator, and (ii) contacting the immune cells to be measured with a substance different from the immune cells to be measured. (iii) contacting an immune cell to be measured with a capturing body capable of binding to a surface antigen at the contact surface and generating an optical signal; (iv) an optical signal generated from the capturing body. and (v) based on the detected optical signal, determining the localization of the surface antigen on the immune cell to be measured, the contact surface of which has been resolved by the time the optical signal is detected. A method for determining the ability of a cell to form an immune synapse is provided.

A fourth aspect of the present invention is a complex of an immunostimulated immune cell to be measured and a capture body capable of binding to a surface antigen of the immune cell and generating an optical signal, wherein the capture body is an introduction unit that introduces the complex bound to the surface antigen at the contact surface between the immune cell and a substance different from the immune cell; an imaging unit that captures an image of the complex supplied from the introduction unit; an analysis unit that determines, based on the image captured by the imaging unit, whether or not the immune cells to be measured, the contact surface of which has been resolved before the imaging, has immunostimulation responsiveness. I will provide a.

A fifth aspect of the present invention is a complex of an immunostimulated immune cell to be measured and a capture body capable of binding to a surface antigen of the immune cell and generating an optical signal, wherein the capture body is an introduction section for introducing the complex bound to the surface antigen at the contact surface between the immune cell and a substance different from the immune cell; and irradiating the complex supplied from the introduction section with light to produce a detection unit that detects an optical signal from the complex, and based on the detected optical signal, whether or not the immune cell to be measured, the contact surface of which has been eliminated by the time the optical signal is detected, has immunostimulatory responsiveness and an analysis unit that determines the cell analysis device.

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.

FIG. 4 is a schematic diagram of steps (i) to (iii) according to the embodiment; 1 is a diagram showing the configuration of a cell analysis device according to Embodiment 1. FIG. 1 is a diagram showing the configuration of a measuring device according to Embodiment 1. FIG. 3 is a diagram showing the configuration of an imaging unit according to Embodiment 1; FIG. 4 is a schematic diagram showing regions on the light receiving surface of the camera according to Embodiment 1. FIG. 1 is a diagram showing the configuration of an information processing apparatus according to Embodiment 1; FIG. 4 is a flowchart showing sample measurement processing and analysis processing according to Embodiment 1. FIG. 4 is an image showing measurement data according to Embodiment 1. FIG. 4 is a schematic diagram showing a measurement process based on measurement data according to Embodiment 1. FIG. 4 is a flowchart showing analysis processing according to the first embodiment; 2D is a 2D scattergram showing analysis processing according to Embodiment 1 (a, b and c) and Embodiment 2 (d). FIG. 4 is a schematic diagram for explaining pulse data of fluorescence signals obtained from cells with different co-stimulatory molecule distribution states. FIG. 4 is a schematic diagram for explaining a 2D scattergram of analysis parameters obtained from pulse data of fluorescence signals. FIG. 2 shows transmitted light images, fluorescence images, and superimposed images showing the distribution of CD28 in T cells without stimulation (a) and with stimulation (b). Images and 2D scattergrams showing the distribution of CD28 in T cells. Differential interference contrast images (DIC), fluorescence images (CD28), and superimposed images (Merge) of T cells without stimulation (a) and with stimulation (c). 2D scattergrams showing area (X-axis) showing fluorescent signal in cells and cell size (Y-axis) without stimulation (b) and with stimulation (d). 1 is a graph showing the correlation between cytokine secretion and immune synapse formation in T cells. Fluorescent images showing the distribution of CD3 and CD40L in T cells without (a and c) and with stimulation (b and d). Images and 2D scattergrams showing the distribution of CD3 in T cells. Fig. 2 shows transmitted light images, fluorescence images, and superimposed images (Merge) of T cells without stimulation (a) and with stimulation (c). 2D scattergrams showing the total fluorescent signal intensity (X-axis) and area showing fluorescent signal (Y-axis) of cells without (b) and with (d) stimulation. Images and 2D scattergrams showing the distribution of CD40L in T cells. Fig. 2 shows transmitted light images, fluorescence images, and superimposed images (Merge) of T cells without stimulation (a) and with stimulation (c). 2D scattergrams showing the total fluorescent signal intensity (X-axis) and area showing fluorescent signal (Y-axis) of cells without (b) and with (d) stimulation. 9 is a 2D scattergram showing an example of measurement processing and analysis processing in Example 2. FIG. a, 2D scattergram showing cell size (X-axis) and aspect ratio (Y-axis). b, 2D scattergram showing total fluorescence signal intensity (X-axis) and counts (Y-axis) of cells. Transmitted light and fluorescence 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). is a superimposed image of Images and 2D scattergrams showing the distribution of CD3 in T cells. Fig. 2 shows transmitted light images, fluorescence images, and superimposed images (Merge) of T cells without stimulation (a) and with stimulation (c). 2D scattergrams showing the total fluorescent signal intensity (X-axis) and area showing fluorescent signal (Y-axis) of cells without (b) and with (d) stimulation. Images and 2D scattergrams showing the distribution of CD28 in T cells. Fig. 2 shows transmitted light images, fluorescence images, and superimposed images (Merge) of T cells without stimulation (a) and with stimulation (c). 2D scattergrams showing the total fluorescent signal intensity (X-axis) and area showing fluorescent signal (Y-axis) of cells without (b) and with (d) stimulation. Images and 2D scattergrams showing the distribution of CD40L in T cells. Fig. 2 shows transmitted light images, fluorescence images, and superimposed images (Merge) of T cells without stimulation (a) and with stimulation (c). 2D scattergrams showing the total fluorescent signal intensity (X-axis) and area showing fluorescent signal (Y-axis) of cells without (b) and with (d) stimulation. Images and 2D scattergrams showing the distribution of OX40 in T cells. Fig. 2 shows transmitted light images, fluorescence images, and superimposed images (Merge) of T cells without stimulation (a) and with stimulation (c). 2D scattergrams showing the total fluorescent signal intensity (X-axis) and area showing fluorescent signal (Y-axis) of cells without (b) and with (d) stimulation.

One aspect of the invention relates to a method of measuring immunostimulatory responsiveness of immune cells. In this embodiment, steps (i) and (ii) involve allowing the immune cells to be measured to form a contact surface with a substance different from the immune cells to be measured in the presence of the immunostimulatory factor. . In this embodiment, the step (i) of contacting the immune cells to be measured with the immunostimulating factor is followed by the step (ii) of forming a contact surface with the substance on the immune cells to be measured. good too. Alternatively, step (i) and step (ii) may be carried out simultaneously when an immunostimulating factor is present on the surface of the substance. Even in this case, strictly speaking, step (ii) is performed after step (i). By performing steps (i) and (ii), immune synapses are formed at the contact surfaces of the activated immune cells.

In step (iii), in order to detect the formed immune synapse, a capture body (hereinafter simply (also referred to as a "trapper") is brought into contact. The capturing body preferably binds to a molecule constituting an immune synapse among surface antigens on the contact surface. By carrying out the step (iii), a complex between a molecule that constitutes an immune synapse and a capture substance is formed in an immune cell.

Then, in step (iv), an optical signal from the captured body contained in the complex on the immune cell is detected. The optical signal detection means is not particularly limited, but means capable of analyzing individual cells is preferable. Means for detecting such optical signals include, for example, flow cytometers and imaging cytometers. In a preferred embodiment, step (iv) uses a flow cytometer or an imaging cytometer to detect optical signals generated from capture bodies bound to surface antigens of immune cells.

In the step (v) described below, the immunostimulatory responsiveness of the immune cells whose contact surfaces have been eliminated is determined. You may perform an operation to eliminate the When the non-biological material described below (e.g., container, multiwell plate, slide, etc.) is used as a substance different from immune cells, and step (iv) is performed using a flow cytometer or an imaging cytometer, this embodiment preferably includes the step of eliminating the contact surfaces. If desired, the immune cells may be fixed by standard methods after the contact surface is resolved. Immobilization allows the localization of surface antigens to be maintained in immune cells whose contacts have been resolved. For cell fixation, it is preferable to use a fixative containing paraformaldehyde, lower alcohol, or the like.

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

For purposes of illustration, reference is made to FIG. 1a, which schematically illustrates steps (i)-(iii) according to the present embodiment. FIG. 1a shows an example of using an immunostimulatory antibody described below as an immunostimulator and a plate as a substance different from immune cells. The plate corresponds to, for example, the bottom surface of a container used for cell culture, the bottom surface of a well of a multiwell plate, the surface of a slide used for microscopic observation, and the like. In FIG. 1a, in step (i) the immune cells are contacted with immunostimulatory antibodies. In this example, an immunostimulatory antibody is an antibody that binds to a co-stimulatory molecule, which is a type of immune cell surface antigen. Following step (i), in step (ii), the immune cells in contact with the immunostimulatory antibody are contacted with the plate to allow the immune cells to form an interface between the plate. In step (iii), a secondary antibody against the immunostimulatory antibody used in step (i) is used to detect co-stimulatory molecules in immune cells. This secondary antibody is labeled with a substance capable of producing an optical signal. In this example, the contact surface is eliminated in step (iii), but the immune cells and the capturing body may be brought into contact while the contact surface is maintained. Note that FIG. 1a is an illustration 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 the immune cells whose contact surfaces have been cleared have immunostimulatory responsiveness. In this embodiment, the step (v) measures a value reflecting the distribution of the surface antigen on the immune cell based on the detected optical signal, and compares the measured value with a threshold to determine whether the immune cell is immune. It may be determined whether or not it has stimulus responsiveness. Here, the values reflecting the distribution of the surface antigen include, 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, and the pulse area. is mentioned.

When the capturing body contains a fluorescent substance, in step (v), a fluorescence image may be acquired based on the fluorescence signal detected in step (iv), and determination may be made based on the fluorescence 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 to determine whether or not the immune cells have immunostimulatory 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, without limitation, an immune cell may be determined to have immunostimulatory responsiveness if said area is less than a threshold.

In a further embodiment, the step (v) further measures at least one selected from the group including the total fluorescence signal intensity in the area, the size of immune cells and the aspect ratio thereof, and uses the measured value as a threshold may include comparing. As used herein, the "total fluorescence signal intensity" of the area or of the cell means the integrated value of the brightness values of each pixel in the area showing brightness values exceeding a predetermined threshold in the fluorescence image. As used herein, the term "cell size" refers to the number of pixels in an image that reflects a cell surrounded by a detected cell membrane region in which a cell membrane region exhibiting a luminance value lower than a predetermined threshold value is detected in a transmitted light image. means. As used herein, the “aspect ratio” of a cell means the ratio of the vertical and horizontal lengths (the number of pixels) of an image reflecting cell particles in a transmitted light image.

In a further embodiment, the determination of whether an immune cell has immunostimulatory responsiveness based on the detected optical signal is based on the pulse width of the fluorescent signal, the pulse width of the scattered light signal, the fluorescent signal area per cell. Measure at least one selected from a value reflecting and a value reflecting cell size, and compare the measured value with a threshold value to determine whether immune cells have immunostimulatory responsiveness including doing

As used herein, "immune synapse" refers to a molecular complex formed in an immune cell at the interface between the immune cell and a target cell that is partly responsible for immune cell activation. This molecular complex comprises a receptor (e.g. T-cell receptor (TCR) or NK-cell receptor), a cofactor for the receptor (e.g. CD3), various co-stimulatory molecules (e.g. CD28), adhesion molecules (e.g. integrins) and signaling molecules are known to be formed together.

As used herein, the term “contact surface” means a region where the immune cells to be measured and a substance different from the immune cells to be measured are in contact. Here, the contact between the immune cells to be measured and a substance different from the immune cells to be measured is caused by the molecules present on the surface of the immune cells to be measured and the substances on the surface of the substance different from the immune cells to be measured. It also includes indirect contact through interactions with molecules present in the As described above, in the method of this embodiment, an immune synapse is formed at the contact surface. Therefore, the contact surface on immune cells can be said to be a region where immune synapses are formed. The contact surface is not limited, but may be formed by spontaneous contact of immune cells to be measured with a substance different from self. Alternatively, the contact surface may be formed by contacting the immune cells to be measured with a substance different from the immune cells to be measured.

As used herein, the term “immune cell to be measured” means a cell type responsible for the mechanism of distinguishing between self and non-self and excluding non-self. Immune cells to be measured may be, but are not limited to, T cells, natural killer (NK) cells, and mixtures thereof. Antigen-presenting cells are not included in the immune cells to be measured as used herein, but are included in "substances different from immune cells to be measured" described later.

As used herein, the term "substance different from the immune cells to be measured" refers to a substance that can provide a contact surface to immune cells when contacted with the immune cells. The substance different from the immune cells to be measured may be a non-biological material or a biological material. Substances that are non-biological materials can be, but are not limited to, containers, multi-well plates, slides, beads, and the like. Substances that are biological materials may be, but are not limited to, allogeneic cells, antigen-presenting cells, cancer cells, and the like. Allogeneic cells, antigen-presenting cells and cancer cells may be established cell lines. Allogeneic cells and cancer cells may also be in the form of tissue containing them. "Allogeneic cells" are non-autologous cells and may be T cells, natural killer (NK) cells and mixtures thereof.

In this embodiment, the method of contacting the immune cells to be measured with a substance different from the immune cells to be measured is not particularly limited. For example, the immune cells to be measured may be placed in a container or wells of a multi-well plate together with a culture medium and allowed to settle so that they can be brought into contact. In this case, the container or multiwell plate containing the immune cells to be measured may be centrifuged. Alternatively, the immune cells may be placed on a slide and brought into contact. In a further embodiment, when substances different from the immune cells to be measured are in the form of particles such as beads, allogeneic cells, antigen-presenting cells, and cancer cells, adding these substances to the immune cells to be measured. may be brought into contact with. The contact surface can be formed by maintaining contact between the immune cells to be measured and a substance different from the immune cells to be measured. The time for which the contact state is maintained 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, "surface antigens" are proteins, sugar chains, lipids, and combinations thereof present on cell membranes. 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 immune synapse in immune cells. Such molecules include, for example, 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), etc. The co-stimulatory molecules described below are also a type of immune cell surface antigens.

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

T cells include, 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), " effector T cells, such as "CD8 ⁺ T cells"), naive T cells, and genetically modified cells. Effector T cells can be, but are not limited to, in vivo and in vitro activated cells. These T cells may be used singly or as T cell subpopulations containing two or more types.

In this embodiment, 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, degree of aggregation and/or specific gravity. For example, commercially available cell sorters can be used to sort cells based on cell size or aggregation. Cells can also be fractionated based on their specific gravity by gravity gradient centrifugation. In this embodiment, cells isolated from a biological sample may be cultured and proliferated according to a conventional method. In further embodiments, immune cells may be used in the form of biological samples such as blood (eg, peripheral blood and cord blood) or bone marrow fluid. Biological samples may be, but are not limited to, samples obtained from mammals, including humans and non-human mammals (eg, companion animals such as dogs and cats, and livestock animals such as horses and cows).

In further embodiments, the immune cells to be measured may be present in the sample. Thus, this embodiment provides a method of performing the method of the invention on a sample containing immune cells to be measured. More specifically, this embodiment comprises the steps of: (i) contacting immune cells in said sample with an immune stimulator; and (ii) treating immune cells in said sample with a substance different from said immune cells. (iii) contacting the immune cells in the sample with a capture entity capable of binding to surface antigens at the contact surface and generating an optical signal; (iv) from the capture entity and (v) determining, based on the detected optical signal, whether contact-released immune cells in the sample are immunostimulatory responsive. , provides a method of measuring immunoresponsiveness of a sample containing immune cells. The sample containing immune cells may be the biological sample described above.

In this embodiment, in step (v), based on the optical signal detected in step (iv), a value that reflects the distribution of surface antigens bound by the captured body in immune cells is measured, and the measured value is used as a threshold value. It may be determined whether the immune cells have immunostimulatory responsiveness by comparing with. When the capturing body contains a fluorescent substance, in step (v), a fluorescence image may be acquired based on the fluorescence signal detected in step (iv), and determination may be made based on the fluorescence image. In the fluorescence image, areas showing fluorescence signals reflecting the distribution of surface antigens may be counted, and based on the areas, for example, the percentage of cells having immunostimulatory responsiveness in the sample may be calculated. Then, the determination may be made by comparing the ratio with the ratio of immune cells having immunostimulatory responsiveness standardized for samples derived from healthy subjects. For example, the sample may be determined to be highly or immunocompetent if the percentage of the sample is a higher value than the normalized percentage.

As used herein, "immunostimulation" for immune cells includes contacting immune cells with an immune stimulator, and allowing the immune cells to form a contact surface with a substance different from the immune cells. In this embodiment, immune stimulation may be performed by contacting the immune cells with the immune stimulator alone. In a further embodiment, immunostimulation may be performed by contacting immune cells with immunostimulatory agents present on said material. In this case, the immune cells are in contact with the immune stimulator at the same time they are in contact with said substance.

As used herein, the term "immunostimulatory factor" means a substance that can act on immune cells to induce activation of the immune cells. Activation of immune cells means, for example, proliferation of immune cells, enhancement of their motility, or cytokine secretion. Immunostimulatory factors include, but are not limited to, immunostimulatory antibodies, immunostimulatory peptides, major histocompatibility molecules (MHC molecules), and present on allogeneic cells, antigen-presenting cells and cancer cells. It may be various immune stimulators. The immunostimulatory factor may be used alone or in combination of two or more, or may be used in the form of an immunostimulatory factor alone or in the form of a complex with other substances or substances. Immunostimulatory factors may be, for example, in the form of complexes of MHC molecules and antigenic peptides, immobilized on non-biological materials such as beads and containers, or presented on antigen-presenting cells.

In this embodiment, the immunostimulatory factor is at least one selected from immunostimulatory antibodies, immunostimulatory peptides, MHC molecules, and complexes of MHC molecules and antigenic peptides. In further embodiments, the immunostimulatory agent 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, an immunostimulatory agent is an immunostimulatory antibody.

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

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. , 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 antibodies and anti-CD28 antibodies. In certain embodiments, immunostimulatory antibodies are anti-CD3 and anti-CD28 antibodies.

Antibodies may be either monoclonal antibodies or polyclonal antibodies unless otherwise specified. Antibodies may also be antibody fragments, single-chain antibodies and derivatives thereof.

As used herein, "immunostimulatory peptide" means a peptide that can stimulate immune cells to induce activation of immune cells. Immunostimulatory peptides are, for example, antigenic peptides (such as Mycobacterium tuberculosis antigenic peptides).

In this embodiment, the immunostimulatory peptide is an antigenic peptide. An antigen peptide may be used singly or in combination of two or more. In certain embodiments, antigenic peptides may be used in the form of complexes with MHC molecules presented on MHC molecules.

As used herein, "major histocompatibility molecule (MHC molecule)" means a molecule responsible for alloantigenicity that causes the strongest rejection reaction upon allotransplantation of organs, tissues and cells. HLA molecules in humans and H-2 molecules 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 complexes with intracellularly produced peptides, and present them to CTL (CD8-positive T cell) receptors. MHC class II molecules are expressed only on so-called antigen-presenting cells such as macrophages and B cells, and present foreign antigen-derived peptides to receptors of helper T cells (CD4-positive T cells).

In this embodiment, when the immunostimulatory factor is an MHC molecule or a complex of an MHC molecule and an antigenic peptide, the MHC molecule may be either an MHC class I molecule or an MHC class II molecule. MHC molecules or complexes of MHC molecules and antigen peptides may be used, for example, in the form of being present on the cell membrane of antigen-presenting cells, cancer cells, and the like.

As used herein, the term "capture capable of binding to a surface antigen and generating an optical signal" includes substances that bind to surface antigens of immune cells and substances capable of generating an optical signal. Substances that bind to surface antigens include, but are not limited to, antibodies, antibody fragments, single-chain antibodies, aptamers, and the like that bind to surface antigens of immune cells. In this embodiment, the capture entity 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.

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

A substance capable of generating an optical signal is exemplified by, but not limited to, a fluorescent substance. As the fluorescent substance, known fluorescent dyes, fluorescent proteins, and the like can be used without particular limitation. A substance capable of producing an optical signal may be, but is not limited to, chemically bound to a capture entity that binds to a surface antigen. Such capture entities include, for example, fluorescently labeled antibodies that bind directly to surface antigens (see capture entities in FIGS. 1b and c).

In this embodiment, the capture entity capable of binding to the surface antigen and generating an optical signal includes substances that directly bind to the surface antigen and substances capable of generating an optical signal. In this embodiment, the capture entity 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. can be anything.

Alternatively, the capture entity may include a substance that indirectly binds to surface antigens and a substance that can generate an optical signal. In this embodiment, the capture entity is an antibody, antibody fragment, single-chain antibody, or aptamer that binds to another substance bound to a surface antigen, with an attached substance capable of producing an optical signal. good. For example, without limitation, an antibody that specifically binds to a surface antigen is bound as a primary antibody to the surface antigen, and then an antibody that specifically binds to the primary antibody is bound as a secondary antibody. . Here, a substance capable of producing an optical signal is bound to said secondary antibody. In this example, the secondary antibody becomes a capture entity that can bind to the surface antigen and generate an optical signal (see capture entity in FIG. 1a). A primary antibody may also be an immunostimulatory antibody.

The surface antigens to which the capture bodies are bound may be co-stimulatory molecules of immune cells. As used herein, the term "co-stimulatory molecule" refers to an antigen-specific signal mediated by the T cell receptor (TCR) upon immune cell activation, as well as an auxiliary signal for activating T cells. It means a substance present in T cells that can contribute to the cell. As used herein, a "co-stimulatory molecule", when present on an NK cell, is a ligand-specific signal of the target cell via the activation receptor of the NK cell, as well as a helper to activate the NK cell. It includes substances that can give signals to NK cells. Co-stimulatory molecules can 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 substances that are different from immune cells are allogeneic cells. In certain embodiments, the substance that is different from immune cells is an antigen-presenting cell. In certain embodiments, the substances that are different from immune cells are 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 factor in step (i) is various substances such as MHC molecules present on the cells. Immunostimulatory factor. For example, if the substance different from immune cells is an allogeneic cell, the immunostimulatory factor includes at least MHC molecules, but is not limited thereto. When the substances different from immune cells are antigen-presenting cells, the immunostimulatory factor includes, but is not limited to, complexes of at least MHC class II molecules and antigenic peptides. When the substance different from immune cells is cancer cells, the immunostimulatory factor includes, but is not limited to, complexes of at least MHC class I molecules and cancer antigen peptides.

The step (i) comprises contacting the immune cells with a substance different from at least one kind of immune cells selected from allogeneic cells, antigen-presenting cells, and cancer cells, thereby increasing immunity between the immune cells and the substance. Including contacting with a stimulant. Step (ii) includes allowing the immune cells to form a contact surface with the substance by maintaining the state of contact between the immune cells brought into contact in step (i) and the substance.

In this embodiment, the substance other than immune cells is preferably a non-biological material, such as an instrument commonly used in the life science field. Such devices include vessels, multiwell plates, slides, beads, or the like. Examples of containers 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, compared to the case where a biological material is used as the substance. . In certain embodiments, the material vessel, multiwell plate, slide or bead comprises an immunostimulatory agent on its surface. For example, when the immunostimulatory agent is an immunostimulatory antibody, the antibody may be immobilized on the surface of a container, multiwell plate, slide or bead. Alternatively, said material container, multiwell plate, slide or bead does not contain an immunostimulatory agent on its surface.

In this embodiment, in step (i), after contacting the immunostimulatory factor with the immune cells, the substance different from the immune cells 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 on its surface an immunostimulatory factor homologous or heterologous to the immunostimulatory factor in step (i) may be used in step (ii). For example, in step (i), an antibody against a co-stimulatory molecule is used as an immunostimulatory factor, and in step (ii), a multiwell plate in which an anti-CD3 antibody is immobilized on the well surface as a substance different from immune cells. can be used. Alternatively, the material in step (ii) may not contain additional immunostimulating agents on its surface.

In this embodiment, when an immunostimulatory antibody is used as the immunostimulator in step (i), this immunostimulator may be used as the scavenger in step (iii). In this embodiment, the immunostimulatory agent 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, antibody fragments thereof, single chain It may be a conjugate in which an antibody, or a combination of one or more thereof, is labeled with a substance capable of producing an optical signal. Using such a labeled immunostimulatory antibody, the immunostimulation of immune cells in step (i) and the capture of surface antigens of the immune cells in step (iii) can be performed simultaneously.

Accordingly, a further embodiment of the present invention comprises the steps of: (i) contacting an immune cell with an immune stimulator; (ii) allowing the immune cell to form a contact surface with a substance different from said immune cell; (iv) detecting an optical signal generated from the captured body; and (v) based on the detected optical signal, determining whether or not the immune cell has immunostimulatory responsiveness, wherein Methods are provided for measuring immunostimulatory responsiveness of immune cells, wherein the immunostimulatory agent is an immunostimulatory antibody labeled with a substance capable of producing an optical signal. According to this embodiment, the number of reagents and steps can be reduced because the immunostimulatory factor also serves as a scavenger that can bind to surface antigens on immune cells and generate an optical signal. Thus, this embodiment has cost and labor advantages.

In this embodiment, the immunostimulatory agent in step (i) may be present on a different material than the immune cells in step (ii). In this embodiment, the immunostimulatory agent is preferably an immunostimulatory antibody, an immunostimulatory peptide, a molecule such as an MHC molecule, or a complex such as an MHC molecule and an antigenic peptide. The immunostimulatory factor may be immobilized on, but not limited to, a container, multiwell plate, slide or beads. 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 physical adsorption, for example. Alternatively, if an immunostimulatory factor is modified with biotin and avidin (or streptavidin) is immobilized on the surface of a substance different from immune cells, the immunostimulatory factor can be activated through the binding of biotin and avidin (or streptavidin). It can be immobilized on the substance. In addition, when the immunostimulatory factor is MHC molecules possessed by allogeneic cells, antigen-presenting cells, cancer cells, etc., or complexes of MHC molecules and antigenic peptides, etc., the cells may be placed in containers, multiwell plates, slides or beads. may be adhered or deposited on the In this embodiment, the immunostimulatory agent on the material may contact immune cells and induce activation of said immune cells. According to this embodiment, the step (i) includes contacting the immune cells with the substance on the surface of which the immunostimulatory factor is immobilized, thereby contacting the immune cells with the immunostimulatory factor on the substance. including letting The step (ii) allows the immune cells to form a contact surface with the substance by maintaining the state of contact between the immune cells and the substance brought into contact in step (i).

Accordingly, a further embodiment of the present invention comprises the steps of: (i) contacting an immune cell with an immunostimulatory agent on a material different from said immune cell; (iii) contacting said immune cell with a capture entity capable of binding to a surface antigen and generating an optical signal; (iv) detecting the optical signal generated from said capture entity; (v) determining, based on the detected optical signal, whether said immune cells with the contact surface resolved have immunostimulatory responsiveness. do. According to this embodiment, step (i) and step (ii) can be performed substantially simultaneously only by contacting immune cells with a substance having an immunostimulating factor immobilized on its surface. Therefore, this embodiment has the advantage that the number of steps can be reduced.

FIG. 1b is a diagram schematically showing steps (i)-(iii) for the above embodiment. In FIG. 1b, the immunostimulatory factor is an immunostimulatory antibody, the substance different from immune cells is a plate, and the immunostimulatory antibody is immobilized on the plate. As shown in FIG. 1b, by bringing immune cells into contact with a plate on which an immunostimulatory antibody is immobilized, immunostimulation by the antibody (step (i)) and formation of a contact surface (step (ii) )) can be performed substantially simultaneously. Then, in step (iii), a capture entity capable of directly binding surface antigens and generating an optical signal is bound to immune synapses on immune cells. In this example, the contact surface is eliminated in step (iii), but the immune cells and the capturing body may be brought into contact while the contact surface is maintained. FIG. 1b is an illustration of steps (i)-(iii) according to one embodiment of the present invention, which is not so limited.

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

FIG. 1c schematically illustrates steps (i)-(iii) for the above embodiment. In FIG. 1c, the immunostimulatory factor is an immunostimulatory antibody, the substance different from immune cells is beads, and the immunostimulatory antibody is immobilized on the beads. As shown in FIG. 1c, by bringing immune cells into contact with beads on which an immunostimulatory antibody is immobilized, immunostimulation by the antibody (step (i)) and formation of a contact surface (step (ii)) are performed. )) can be performed substantially simultaneously. Then, in step (iii), a capture entity capable of directly binding surface antigens and generating an optical signal is bound to immune synapses on immune cells. In this example, the contact surface has not been 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 illustration of steps (i)-(iii) according to one embodiment of the invention, but the invention is not so limited.

As used herein, the term “immune cells whose contact surfaces have been eliminated” means immune cells in which the contact area 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 separated from substances different from the immune cells. Techniques for eliminating contact surfaces include, but are not limited to, physical treatments such as pipetting, tapping, stirring, shaking, ultrasonic treatment, and chemical treatments such as surfactants for containers containing immune cells and culture solutions. It may be a chemical treatment using chemicals and a biochemical treatment using biochemicals such as enzymes.

In preferred embodiments, the contact surfaces are eliminated by physical treatments such as pipetting, tapping, stirring, shaking, sonication, and the like. In certain embodiments, said technique is pipetting. In this embodiment, step (iv) may detect optical signals generated from the captors using a flow cytometer when the contact surfaces are resolved.

As used herein, the term “flow cytometer” refers to irradiating cells in a water stream with excitation light and measuring the fluorescence and scattered light emitted from individual cells to determine the size and membrane protein of individual cells in a cell population. 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 period of time (up to tens of thousands of cells/second) compared to general fluorescence microscopy, and can perform highly sensitive and quantitative measurements simultaneously. It has advantages such as being able to acquire parameters. As used herein, a flow cytometer has a channel system that enables cell measurement in a state in which cells are aligned in a liquid so that cells can be individually measured. Such a fluidics system may be realized by a hydrodynamic component called a flow cell. A flow cell is generally realized by hydrodynamic focusing using a sheath flow enveloping a sample flow, but is limited to this. is not. For example, it may be realized by microcapillary or acoustic focusing.

In the present invention, a commercially available product can be used without particular limitation for the flow cytometer. As used herein, a flow cytometer includes a so-called “imaging flow cytometer” that has an imaging unit using a camera and is capable of acquiring images of cells flowing in liquid. As will be described later in Embodiment 1, according to the imaging flow cytometer, multiple parameters including cell size and aspect ratio can be obtained based on the captured image of the cell. As used herein, flow cytometers include both imaging flow cytometers and non-imaging flow cytometers. In preferred embodiments, the flow cytometer is an imaging flow cytometer. In the present invention, even a commercially available flow cytometer can be used without particular limitation.

In this embodiment, in the step (iv), the substance different from immune cells is at least one selected from allogeneic cells, antigen-presenting cells, and cancer cells, or when beads are used, immune cells and Alternatively, 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 cells is a container, multiwell plate or slide, the contact surface between the immune cells and the substance different from the immune cells is eliminated, and the complexes of the immune cells and the trapping bodies are formed, Optical signals generated from capture bodies in the complex may be obtained by measuring with a flow cytometer or an imaging cytometer.

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

In further embodiments, methods of assessing the activation state of the immune system in a subject are provided. In this embodiment, the step of performing the invention described above on a biological sample containing immune cells obtained from said subject is included. That is, the evaluation method of this embodiment includes (i) the step of contacting the immune cells in the biological sample with an immune stimulator, and (ii) the immune cells in the biological sample with a substance different from the immune cells. (iii) contacting an immune cell in the biological sample with a capture entity capable of binding to surface antigens on the immune cell and generating an optical signal; and (v) determining whether the sample is immunostimulatory responsive based on the detected optical signal.

Furthermore, in the evaluation method of this embodiment, the capturing body contains a fluorescent substance, in step (v), a fluorescence image is obtained based on the fluorescence signal detected in step (iv), and determination is made based on the fluorescence image. you can go In the fluorescence image, areas showing fluorescence signals reflecting the distribution of surface antigens may be measured, and based on the areas, for example, the percentage 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 immunostimulatory responsiveness standardized for biological samples derived from healthy subjects. In this embodiment, for example, the sample may be determined to be highly or immunocompetent if the percentage of the sample is a higher value than the normalized percentage. Alternatively, the method of evaluation may compare the percentage of immune cells with immunostimulatory responsiveness in a biological sample containing immune cells obtained from a subject to the percentage for a previous biological sample from the same subject. For example, the subject may be determined to have improved or immunocompetence if the percentage of the sample is a higher value than the previous percentage.

Another aspect of the invention relates to a method of measuring immunostimulatory responsiveness of immune cells using a capture entity capable of binding co-stimulatory molecules in immune cells and generating an optical signal.

This aspect of the method is the same as the method of the above aspect, except that the trapping agent is capable of binding to co-stimulatory molecules in immune cells and generating an optical signal. Co-stimulatory molecules to which the captor binds include, for example, CD28, OX40, CD40L, CTLA4, PD-1 and ICOS.

Another aspect of the present invention relates to a cell analyzer for analyzing the activation state of immune cells. Embodiments according to this aspect of the invention will now be described with reference to the accompanying drawings. It should be noted that the following description is all illustrative and non-limiting, and does not limit the invention described in the appended claims in any way.

<Embodiment 1 of Cell Analyzer>
Referring to FIG. 2 , cell analyzer 1 includes measurement device 2 and information processing device 3 . The measurement device 2 optically measures a sample containing a complex of an immune cell and a capturing body capable of binding to an immune cell surface antigen and generating an optical signal using a flow cytometer. The information processing device 3 analyzes the measurement results obtained by the measurement device 2 and displays the analysis results on the display unit 320 .

With reference to FIG. 3 , measuring device 2 includes introduction section 201 , imaging section 203 , signal processing section 210 , CPU 204 , communication interface 205 and memory 206 . The signal processing section 210 has an analog signal processing section 211 , an A/D converter 212 , a digital signal processing section 213 and a memory 214 .

In this embodiment, inlet 201 comprises 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 the pump. The introduction unit 201 supplies the sample to the imaging unit 203 according to instructions from the CPU 204 . In Embodiment 1, the measurement sample is obtained by subjecting cloned T cells derived from human peripheral blood to immunostimulation including contact with an anti-CD28 antibody, immobilizing the cells, and binding the anti-CD28 antibody to phycoerythrin (PE). Complexes are used that are secondary stained with a labeled anti-mouse IgG antibody.

The imaging unit 203 irradiates the sample supplied from the introduction unit 201 with laser light, images the complex, and transmits the generated image information of the transmitted light image and the fluorescence image to the analog signal processing unit 211 as electrical signals. Output. The analog signal processing unit 211 amplifies the electrical signal output from the imaging unit 203 according to instructions from the CPU 204 and outputs the amplified electrical signal to the A/D converter 212 .

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

The measurement data stored in the memory 214 includes transmitted light images and fluorescence images based on transmitted light and fluorescence generated when the complexes pass through the flow cell 203c.

CPU 204 outputs the measurement data stored in memory 214 to communication interface 205 . The CPU 204 receives control signals from the information processing device 3 via the communication interface 205 and controls each part of the measuring device 2 according to the control signals.

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

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

In this embodiment, light source 203a is a semiconductor laser light source. The light emitted from the light source 203a is laser light with a wavelength 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 wavelength λ1 emitted from the light source 203a is applied to the sample passing through the inside of the flow cell 203c, whereby transmitted light of wavelength λ1 is generated from immune cells.

The light source 203b is a semiconductor laser light source. The light emitted from the light source 203b is laser light with a wavelength of λ2. In this embodiment, wavelength λ2 is approximately 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 is applied to the sample passing through the inside of the flow cell 203c, and fluorescence of wavelength λ3 is generated from the PE.

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

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

Referring to FIG. 6, information processing apparatus 3 is configured with main body 300 , input unit 310 , and 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 computer programs stored in the ROM 302 and computer programs loaded in the RAM 303 . The RAM 303 is used for reading computer programs recorded in the ROM 302 and hard disk 304 . The RAM 303 is also used as a work area for the CPU 301 when executing these computer programs.

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

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

The reading device 305 is configured by a CD drive, a DVD drive, or the like. The reading device 305 can read computer programs and data recorded on recording media such as CDs and DVDs.

The input/output interface 306 is connected to an input unit 310 including a mouse, a keyboard, and the like, and a user uses the input unit 310 to input instructions to the information processing apparatus 3 . The image output interface 307 is connected to a display unit 320 such as a display, and outputs a video signal corresponding to 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 measurement data transmitted from the measurement device 2 through the communication interface 308 . The received measurement data is stored in hard disk 304 .

FIG. 7 is a flow chart showing the control of the CPU 204 of the measuring device 2 and the CPU 301 of the information processing device 3. As shown in FIG. 7a is a flowchart showing control processing by the CPU 204 of the measuring device 2, and FIG. 7b is a flowchart showing control processing by the CPU 301 of the information processing device 3. FIG.

Regarding the control processing of the CPU 301 of the information processing device 3, refer to FIG. 7b. 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 measurement data has been received (step S13). If the measurement data has not been received (step S13: NO), the process is put on standby.

Regarding the control processing of the CPU 204 of the measuring device 2, refer to FIG. 7a. When the CPU 204 receives the measurement start signal from the information processing device 3 (step S21: YES), the sample is measured (step S22). In the measurement process (step S22), a measurement sample containing a complex of the capture body and immune cells is supplied together with the sheath liquid from the introduction part 201 to the flow cell 203c, and the sample of the measurement sample wrapped in the sheath liquid in the flow cell 203c. A stream is formed. The formed sample flow is irradiated with laser beams from the light sources 203a and 203b to form a beam spot on the flow cell 203c. As the immune cells pass through the beam spot, they generate transmitted light and fluorescence. The generated transmitted light and fluorescence are captured by the camera 203i and converted into electrical signals.

These electric signals are converted into digital signals by the A/D converter 212 and subjected to signal processing by the digital signal processing section 213 . Thereby, measurement data including a transmitted light image and a fluorescence image are obtained for each complex that has passed through the flow cell 203c. The measurement data is stored in memory 214 . When the measurement of the sample ends, the CPU 204 transmits the measurement data generated by the measurement process to the information processing device 3 (step S23), and ends the process.

Again, refer to FIG. 7b. When the CPU 301 of the information processing device 3 receives the measurement data from the measurement device 2 (step S13: YES), it stores the measurement data in the hard disk 304 and performs analysis processing 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 processing (step S14 in FIG. 7b) performed by the CPU 301 of the information processing device 3 based on the measurement data will be further described with reference to FIGS. 8 to 10. FIG. FIG. 8 shows a transmitted light image, a fluorescence image (CD28), and a superimposed image thereof among measurement data obtained by measuring the immune cells. In the absence of immunostimulation, the co-stimulatory molecule CD28 tends to be uniformly distributed on the plasma membrane of immune cells, as shown in the fluorescence image of Figure 8a. As shown in the fluorescence image of FIG. 8b, the co-stimulatory molecule CD28 tends to localize partly on the plasma membrane of immune cells upon immune stimulation. Although it is not intended to be bound by theory, it is believed that the cell membrane portion where co-stimulatory molecules are localized is a contact surface formed in the immune cell between substances different from the immune cell. be done. In other words, it is thought that the part where this co-stimulatory molecule is localized shows an immune synapse formed between this cell and a different substance.

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

FIG. 10 is a flowchart showing analysis processing in step S15 of the CPU 301 of the information processing device 3. As shown in FIG. 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 obtains measurement data of cells corresponding to dots existing within a predetermined range. Extract and parse.

In this embodiment, the CPU 301 first creates a 2D scattergram (FIG. 11a) assigning cell size to the X-axis and cell aspect ratio to the Y-axis to determine the number of cells in a given range (R1). Measured data is extracted (step S51). Range (R1) is a predetermined range in cell size (X-axis) that reflects the size of cells alone and a predetermined range in aspect ratio (Y-axis) that reflects the presence of cells alone. gating with This excludes data derived from material such as cell debris that is smaller than a cell or clumps of cells that are larger than a single cell.

Each dot displayed in the 2D scattergram represents an individual cell count. The CPU 301 extracts the measurement data of the cells indicating the measurement values within the region indicated as R1 in the image. In this embodiment, the dots displayed on the 2D scattergram are linked to the measurement data of individual cells measured in step S22 of FIG. 7a. When the user designates a dot displayed on the 2D scattergram, the CPU 301 reads out the measurement data (transmitted light image and/or fluorescence image) of the cell corresponding to the dot from the hard disk 304 and displays it on the display unit 320. do.

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

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

CPU 301 also counts the number of dots shown throughout FIG. The ratio of the number of dots within the range ([the number of dots within the range (R4)]/[the number of dots within the range (R2)]) is calculated (step S54). Thereby, the proportion of immune cells having immunostimulatory responsiveness among individual immune cells expressing a predetermined amount of costimulatory molecule CD28 is calculated.

The display unit 320 displays the analysis results of the number of dots and the ratio within each range obtained by the above analysis processing.

The results of this analysis may be further analyzed, for example, by comparing the percentage of immune cells with immunostimulatory responsiveness normalized for biological samples from healthy subjects. In this further analysis, the sample may be determined to be highly immunoresponsive, for example, if the percentage of the sample is higher than the normalized percentage. Alternatively, the results of this analysis may be further analyzed by comparing the percentage of immunostimulatory responsive immune cells for previous biological samples from the same subject. In this further analysis, it can be determined that the subject has improved immune responsiveness, for example, if said percentage of the sample is a higher value than the previous percentage.

Thus, in one embodiment, an introduction portion for introducing a complex of an immune cell and a capture entity capable of binding to a surface antigen on said immune cell and generating an optical signal, and said complex supplied from said introduction portion and an analysis unit that determines whether or not the immune cells are responsive to immunostimulation based on the image captured by the imaging unit. In a specific embodiment, the analysis unit acquires a value that reflects the distribution of the surface antigen based on the image, and compares the acquired value with a threshold to determine whether the immune cells are immunostimulatory responsive. It is determined whether or not there is. In another embodiment, the capture entity comprises a fluorescent substance, the image comprises a fluorescence image, and the value reflecting the distribution of the surface antigen comprises a fluorescence signal reflecting the distribution of the surface antigen based on the fluorescence image. A cell analysis device is provided comprising an indicated area and determining that the immune cells are immunostimulatory responsive when the area is less than the threshold. In another embodiment, the image includes a transmitted light image, 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 analysis device is provided that determines whether the immune cells are immunostimulatory responsive based on the values and values reflecting the distribution of the surface antigens.

The analysis processing described above is merely an example of analysis processing based on measurement data, and different analysis processing can be used.

<Embodiment 2 of Cell Analyzer>
Instead of step S53 of the analysis processing of Embodiment 1, the CPU 301 assigns the total fluorescence signal intensity of 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 generated (FIG. 11d) and the number of measured data within a given range (R3) may be counted. The range (R3) is gated on a range that reflects a given expression level of the co-stimulatory molecule CD28 per cell in the total fluorescence signal intensity of the cells (X-axis), and in the area showing the fluorescence signal (Y-axis). Gating on a range that reflects the localization of the stimulatory molecule CD28. By counting the measured data in the range (R3), the number of immune cells responsive to immune stimulation is counted. In this embodiment, cells corresponding to dots in range (R3) are likely to have formed immune synapses and are determined to be responsive to immune stimulation in this embodiment.

In this embodiment, CPU 301 also counts the number of dots shown throughout FIG. 11d (corresponding to the number of dots in range (R2) in FIG. ) ([the number of dots in the range (R3)]/[the number of dots in the range (R2)]) may be calculated. Thereby, the proportion of immune cells having immunostimulatory responsiveness among individual immune cells expressing a predetermined amount of costimulatory molecule CD28 is calculated.

<Embodiment 3 of Cell Analyzer>
Instead of step S53 of the analysis process of the first embodiment, the CPU 301 assigns the class value for the area showing the fluorescence signal to the X-axis and the frequency to the Y-axis for the cell measurement data extracted in step S52. A histogram may be created and displayed on the display unit 320 . Further, instead of step S54, the CPU 301 may calculate the average value, median value, and mode value of the area indicating the fluorescence signal in the measurement data extracted in step S52 from this histogram. Comparing the mean, median, and mode obtained to the standardized mean, median, and mode of areas exhibiting fluorescent signals in immune cells, e.g., obtained from biological samples from healthy subjects. may be further analyzed by In this further analysis, for example, if the class value indicating the mode of the sample (area indicating fluorescence signal) is smaller than the class value indicating the standardized mode (area indicating fluorescence signal), this It suggests that the co-stimulatory molecule CD28 is localized, and consequently the sample can be judged highly responsive to immune stimulation.

As shown in Embodiment 3, the immunoreactivity 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 shown in the 2D scattergram can be linked to the measurement data of individual immune cells, for example, each dot can be designated and the transmitted light image and fluorescence image of that immune cell can be linked. Measurement data including images can be displayed. Therefore, analysis by 2D scattergram is a preferable analysis processing method in that detailed analysis based on an image can additionally be performed on individual cells.

<Embodiment 4 of Cell Analyzer>
In the analysis processing described above, an analysis based on captured image data is shown as an example, but immunostimulatory-responsive immune cells can also be analyzed from measurement data other than captured image data. For example, in the measurement apparatus 2 (FIG. 4) of Embodiment 1, the optical unit 203g and the camera 203 are replaced with dichroic mirrors 203j and 203k, a forward scattering light receiving section 203l, and a fluorescence light receiving section 203m. The light of wavelength λ1 emitted from the light source 203a is applied to the sample passing through the inside of the flow cell 203c, whereby forward scattered light of wavelength λ1 is generated from immune cells. The light of wavelength λ2 emitted from the light source 203b is applied to the sample passing through the inside of the flow cell 203c, and fluorescence of wavelength λ3 is generated from the PE. The sample in Embodiment 4 is the composite used in Embodiment 1. The forward scattered light of wavelength λ1 is reflected by the dichroic mirror 203j and received by the forward scattered light receiving section 203l. The fluorescence of wavelength λ3 is transmitted through the dichroic mirror 203j, reflected by the dichroic mirror 203k, and received by the fluorescence light receiving section 203k. The forward scattering light receiving section 203l and the fluorescence light receiving section 203m are photomultipliers. In this example, the optical signals obtained in step S22 of FIG. 7a are the forward scattered light signal and the fluorescence signal, and in step S14 of FIG. and "pulse height (H)" are obtained. These analysis parameters will be described with reference to FIG.

FIG. 12 shows, as an example, cells in which the co-stimulatory molecule CD28 is uniformly distributed on the cell membrane (FIG. 12a) and cells showing localization on the cell membrane (FIG. 12b). 12c and 12d), respectively, showing the fluorescence signals detected during passage through the (not shown) in the direction of the arrows.

12c and 12d, the time at which the fluorescence signal exceeds the threshold baseline is referred to herein as "pulse width (w)". The fluorescence intensity at the peak is called "pulse height (H)". "Pulse area (A)" refers to the area between baseline and fluorescence signal intensity curve.

In this example, the same amount of co-stimulatory molecule CD28 is expressed on the cell membrane in the cells of Figures 12a and 12b, and the pulse areas (A) of Figures 12c and 12d are equal. Here, the pulse width (w) and pulse height (H) differ depending on the distribution pattern of costimulatory molecules. Compared to the case where co-stimulatory molecules are uniformly distributed on the cell membrane (Fig. 12a), the pulse width (w) becomes narrower when co-stimulatory molecules are localized on the cell membrane (Fig. 12b). , while the pulse height (H) increases (FIGS. 12c and 12d).

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

As mentioned above, when co-stimulatory molecules are uniformly distributed on the cell membrane (Fig. 12a), the pulse width ( w) shows a trend towards broadening and the pulse height (H) shows a trend towards lowering (Figs. 12c and 12d). Therefore, in the 2D scattergram shown in FIG. 13a, the data falling within the dashed circled area are data reflecting cells in which co-stimulatory molecules are uniformly distributed on the cell membrane, whereas the solid line data falling within the boxed region of , tend to be data reflecting cells in which co-stimulatory molecules are localized on the cell membrane. In the 2D scattergram shown in Fig. 13b, data falling within the area enclosed by the dashed box are data reflecting cells in which the co-stimulatory molecules are uniformly distributed on the cell membrane, while the solid box The data in the boxed area tend to be data reflecting cells in which co-stimulatory molecules are localized on the cell membrane.

Thus, in one embodiment, an introduction portion for introducing a complex of an immune cell and a capture entity capable of binding to a surface antigen on said immune cell and generating an optical signal, and said complex supplied from said introduction portion A detection unit that irradiates light on the complex and detects an optical signal from the resulting complex, and an analysis unit that determines whether or not the immune cell has immunostimulatory responsiveness based on the detected optical signal. A cell analysis device is provided, comprising: In a specific embodiment, the capturing body contains a fluorescent substance, the detection unit detects a fluorescence signal generated from the complex, and the analysis unit determines the distribution of the surface antigen based on the fluorescence signal. determining whether said immune cells have immunostimulatory responsiveness by obtaining a value reflective and comparing said obtained value to a threshold value, said value reflective of said surface antigen distribution from said complex; A cell analysis device is provided comprising at least one of a pulse width of a fluorescence signal of , a height of the fluorescence pulse, and a pulse area of the fluorescence signal.

As shown in the Examples, cells in which surface antigens and co-stimulatory molecules to which capture bodies are bound are localized on the cell membrane are determined to be immune cells exhibiting immunoreactivity. Therefore, in FIGS. 13a and 13b, cells exhibiting data falling within the solid boxed area can be determined to be cells exhibiting immunocompetence. As exemplified in this embodiment, it is understood that immune cells having immunostimulatory responsiveness can be measured not only by analysis processing based on captured image data, but also by analysis processing as described above.

Note that the analysis parameters and analysis processing shown here are merely examples, and different analysis parameters may be measured from the measurement data, and different analysis processing may be performed on the measured analysis parameters. . For example, the pulse area may be an approximate value that 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. In addition, 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, the base may be a value exceeding a predetermined threshold from the baseline.

Specific examples are set forth below, which illustrate preferred embodiments of the invention and are not intended to limit the invention described in the appended claims in any way. Any readily recognizable equivalents or alterations, modifications or variations from the specific embodiments, materials, compositions and methods described herein are intended to be within the scope of the invention.

Example 1: Measurement of immunostimulatory responsiveness of immune cells using CD28 as an index
(1) Preparation of clone T cells
5×10 ⁶ CD4-positive cells (Stemcell Technologies: ST-70026) and CD8-positive cells (Funakoshi: 0508-100, Cosmo Bio: PB08C-1) were treated with 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 Phytohemagglutin-L (PHA) (SIGMA: L4144-5MG) and 2% human serum (SIGMA: H4522-100ML) in Yssel's medium (Gemini Bio-Products: 400102), 1×10 ⁶ peripheral blood mononuclear cells, 1×10 ⁴ JY cells and 14 co-cultured for 2 days. After 14 days, the T cells were seeded in a 96-well plate at 0.3, 1.0 and 3.0 cells/well by limiting dilution, and cultured with 1×10 ⁴ peripheral blood mononuclear cells until colonies were formed. . T cells were collected from wells in which colonies were formed to obtain clone T cells. Hereinafter, the obtained T cells are also referred to as "prepared clone T cells".

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

(3) Stimulation and Measurement of Immune Cells Prepared clone T cells were added to an anti-CD3 antibody-immobilized plate so that about 5×10 ⁵ cells per well. An anti-CD28 antibody (BioLegend #302914 Clone: CD28.2) was then added to the cells in the wells at a final concentration of 0.01 mg/ml. The plate was centrifuged (200 G, 30 seconds) and incubated at 37°C for 30 minutes. Prepared clonal T cells were collected from the well surface by pipetting. Collected T cells were fixed using a fixative containing 1% paraformaldehyde (PFA). Fixed T cells were immunostained using a 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 cells were not subjected to immune stimulation by contact with antibodies that bind to surface antigens of T cells (anti-CD28 antibody and anti-CD3 antibody) and contact with different substances (plate wells) was also examined. The cloned T cells prepared in (1) above were fixed with the above fixative without the above immunostimulation, and then stained with an anti-CD28 antibody. The fixed T cells were secondarily stained with a PE-labeled anti-mouse IgG antibody and measured.

When T cells were not given the above-mentioned immune stimulation, CD28 was uniformly distributed on the cell membrane of T cells (see FIG. 14a). CD28 localized on the cell membrane of T cells when the contact surface between the T cells and the well was eliminated after the above-described immunostimulation (see FIG. 14b). Thus, immune synapses could be detected by using the co-stimulatory molecule CD28 as an index, even in a state where the contact surface between the T cells and the well was eliminated after the above-described immune stimulation.

(4) Data analysis
Measurement data of the sample containing T cells without the above immunostimulation (Fig. 15a and b) and measurement data of the sample containing T cells with the above immunostimulation obtained by measurement using Imaging FCM (Fig. A multiparameter analysis of 15c and d) was performed.

Detailed measurement processing and analysis processing have been described in the first embodiment. Briefly, image analysis is performed based on transmitted light images and fluorescence images (CD28) measured using Imaging FCM (Fig. 9), showing cell size (number of pixels), cell aspect ratio, and fluorescence signal in cells. The area (number of pixels) and the total fluorescence signal intensity within the area (total fluorescence signal intensity of cells) were measured. First, a 2D scattergram is created in which the X-axis is assigned the cell size and the Y-axis is assigned the aspect ratio of the cell, and the cell measurement data corresponding to the dots in a given range (R1) are extracted (Fig. 10). step S51). Next, for the measurement data within the range (R1), the measurement data corresponding to the dots where the total fluorescence signal intensity of the cells was within the predetermined range (R2) was further extracted (step S52 in FIG. 10). For the measured data within the range (R2), the number of dots within the region labeled R4 (FIGS. 15b and 15d) was counted (step S53 in FIG. 10). Also, the number of dots (the number of dots within the range (R2)) on the scattergrams of Figures 15b and 15d was counted. Then, the ratio of the number of dots within the range (R4) to the number of dots within the range (R2) was calculated by the following formula (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, immune synapses that can be formed when immune cells are subjected to immune stimulation, including contact with immune stimulators and formation of contact surfaces between different substances, are co-stimulatory It was found that if the detection is performed using molecules as indicators, even if the contact surface is not maintained, information on the immune reactivity of the immune cells can be obtained accurately. This was a surprising result, considering that immune synapses are loose connections formed in immune cells at their binding surfaces with target cells.

Thus, according to the present invention, it is possible to measure the immunostimulatory responsiveness of immune cells based on the immune synapse, so that it is faster and easier 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, the imaging flow cytometer was used to measure the immunostimulatory responsiveness of immune cells, which enabled the measurement of a large number of cells and the measurement of immune responsiveness with high statistical accuracy. One thing has been proven. In the above examples, the immunostimulatory responsiveness of immune cells was measured using an imaging flow cytometer, but it is also possible to measure with an imaging cytometer capable of measuring a large number of cells, similar to the measurement device used in the examples. is understood. Moreover, as described in Embodiment 4 above, it is understood that the immunostimulatory responsiveness of a large number of immune cells can be measured with a flow cytometer that does not perform imaging, as with the measurement apparatus used in the examples. Thus, according to the present invention, the activation state of immune cells can be measured quickly and easily using a large number of cells, and thus it is understood that the present invention is useful for statistically highly accurate evaluation of drug efficacy.

Since the imaging flow cytometer was used in the above example, the image data of individual cells can be linked to each measurement data in the analysis of the measurement data of each cell. Therefore, after performing predetermined analysis processing based on the measurement data to select cells, the immunostimulatory responsiveness can be evaluated together with the image data of the cells. For example, in the analysis process for selecting cells with a high tendency to form immune synapses in the above example, about 81% of the cells in the immunostimulated sample fell within the range (R4) (Table 1 ). Here, in FIG. 15d, by designating a dot corresponding to a cell in the range (R4), displaying the image data of the cell, and evaluating the image data together, the immune cell can be identified in more detail. Responsiveness to immune stimulation can be evaluated. It is understood that this further evaluation can be evaluated by an imaging cytometer that takes images of cells as well as an imaging flow cytometer.

According to the measurement method and the cell analysis device according to the present invention, for example, when an immunostimulatory antibody is used as an immunostimulatory factor, the activation state of immune cells in the body of a subject related to cancer immunity, self-disease, etc. , the activation state of immune cells in the subject's body related to infectious diseases and allergic diseases when using specific antigen peptides, and transplantation medicine when using allogeneic cells or allogeneic MHC It is useful because it can quickly and easily evaluate the immune responsiveness of a subject to regenerative medicine. As noted above, it will be appreciated that imaging flow cytometers and imaging cytometers that are more able to utilize individual cell image data for analytical processing have additional advantages.

Reference example: Correlation between immune synapse formation and cytokine production in immune cells
(1) Measurement of Cytokine-Producing Cells 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, in the same manner as in Example 1, clone T cells were prepared from human peripheral blood. Clone T cells were added to the anti-CD3 antibody-immobilized plate at about 5×10 ⁵ cells per well. Then, an anti-CD28 antibody (BioLegend #302914 Clone: CD28.2) was added to the cells in the wells at a final concentration of 0.01 mg/ml and incubated at 37°C for 4 hours. Then, Brefeldin A was added to each well to a final concentration of 10 μg/ml and incubated for 2 hours. After that, the plate was left on ice and the cells in each well were collected. Collected T cells were fixed with a fixative containing 1% PFA and then permeabilized with a PBS solution containing 0.5% saponin and 1% BSA. Treated cells were immunostained using 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 measurement data were analyzed by a conventional method to obtain the percentage of IFN-γ positive cells in the measured T cells.

(2) Measurement of Cells Forming Immune Synapses Using T cells seeded from the same well as the T cells used in (1) above, cells forming immune synapses were measured using CD28 as an indicator. By analyzing the measurement data, the percentage of cells forming an immune synapse in the measured T cells was obtained. Measurement and data analysis were performed in the same manner as in Example 1.

(3) Results Data were plotted with the percentage (%) of IFN-γ-positive cells on the vertical axis and the percentage (%) of immune synapse-forming cells on the horizontal axis. The resulting graph is shown in FIG. As can be seen from FIG. 16, a correlation was observed between immune synapse formation and INF-γ production in immunostimulated clonal T cells (R ² =0.8371). Therefore, it was shown that immune cells activated by immune stimulation can be detected by detecting immune synapses in immune cells whose contact surfaces have been resolved, as in conventional methods based on cytokine production.

Example 2: Measurement of immunostimulatory responsiveness of immune cells using CD3 and CD40 as indicators
(1) Stimulation and Measurement of Immune Cells 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, in the same manner as in Example 1, clone T cells were prepared from human peripheral blood. The prepared clone T cells were added to the anti-CD3 antibody-immobilized plate so that about 5×10 ⁵ cells per well. Then, anti-CD28 antibody (BioLegend #302914 Clone: CD28.2) and PE-labeled anti-CD40L antibody (BD biosciences # 340477 Clone: 89-76) were added to final concentrations of 0.01 mg/ml and 0.01 mg/ml, respectively. Added to cells in wells. The plate was centrifuged (200 G, 60 seconds) and incubated at 37°C for 30 minutes. After removing the supernatant in the wells, the prepared clone T cells were dispersed by pipetting and recovered from the well surface. Collected T cells were fixed using a fixative containing 1% PFA. Fixed T cells were immunostained using 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 subjected to immune stimulation by contact with anti-CD40L antibody, anti-CD28 antibody and anti-CD3 antibody and contact with wells of the plate was also examined. The cloned T cells prepared in (1) above were fixed on ice with the above fixative without the above 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 prepared cloned T cells were not given the above immune stimulation, both CD3 and CD40L were uniformly distributed on the cell membrane in T cells (see FIGS. 17a and c, FIGS. 18a and b, and FIGS. 19a and b). . When the contact surface between the T cells and the well was eliminated after the above-described immunostimulation, both CD3 and CD40L localized on the cell membrane of the T cells (Figures 17b and d, Figure 18c and d, see Figures 19c and d). As described above, even in a state where the contact surface between the T cells and the well is eliminated after the above-described immune stimulation, the immune synapse can be detected by using the T cell surface antigen CD3 or CD40L as an index. detected.

(2) Data analysis
Measurement data of the sample containing T cells without the above immunostimulation (FIGS. 18a and b) and the measurement data of the sample containing T cells with the above immunostimulation (Fig. A multiparameter analysis of 18c and d) was performed. An example of measurement processing and analysis processing 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, cell size (number of pixels), cell aspect ratio, area showing fluorescence signal in cells ( number of pixels) and the total fluorescence signal intensity in the area (total fluorescence signal intensity of cells) were measured. First, a 2D scattergram (see Figure 20a) is created with the cell size assigned to the X-axis and the cell aspect ratio assigned to the Y-axis, and cell measurement data corresponding to dots within a given range (Cells) are generated. Extracted. Then, for the measurement data within the range (Cells), the measurement data corresponding to the dots where the total fluorescence signal intensity of the cells was within the predetermined range (Tcells) was further extracted (see Figure 20b). For the measured data within the range (Tcells), the number of dots within the regions labeled IS_CD3 and IS_CD40L (see Figures 18c and d, Figures 19c and d) were counted, respectively. In addition, the number of dots (the number of dots within the range (Tcells)) on the scattergrams of FIGS. 18c and d and FIGS. 19c and d was counted. Then, the ratio of the number of dots within the range (IS_CD3 or IS_CD40L) to the number of dots within the range (Tcells) was calculated using the following formula (2). The results are shown in Tables 2 and 3 below. Note that IS is an abbreviation for Immune Synapse.

[Percentage (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, the proportion of T cells that formed immune synapses was clearly higher in T cells stimulated with immunostimulatory antibodies than in those without immunostimulation. was done. Therefore, it was shown that the responsiveness of immune cells to immunostimulation by immunostimulatory antibodies can be measured by detecting immune synapses using CD3 or CD40L as an index.

Example 3: Measurement of immune cells responding to immune stimulation by allogeneic cells
(1) Allogeneic Cells and Cloned T Cells As allogeneic cells, retinal pigment epithelial cells (RPE cells) prepared from iPS cells (allogeneic iPS cells) derived from a given donor were used. RPE cells were provided by Helios Co., Ltd. RPE cells were seeded in a 24-well plate at about 5×10 ⁵ cells per well and incubated at 37° C., 5% CO ₂ for 7 days. As a result, a plate with RPE cells attached to the bottom of the wells 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, Cosmo Bio: PB08C-1) were treated with 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 Phytohemagglutin-L (PHA) (SIGMA: L4144-5MG) and Yssel's medium (Gemini Bio-Products: 400102) supplemented with 2% human serum (SIGMA: H4522-100ML), and co-cultured with 1.2×10 ⁵ RPE cells for 14 days.
After 14 days, T cells were seeded in 96-well plates at 0.3, 1.0 and 3.0 cells/well by limiting dilution, and cultured with 1×10 ⁴ RPE cells until colonies were formed. T cells were collected from colony-formed wells to obtain cloned T cells.

(2) Stimulation and measurement of immune cells
Prepared clone T cells were added to the RPE cell-seeded plate at approximately 5×10 ⁵ cells per well. The plate was centrifuged (200 G, 120 sec) and incubated at 37°C for 30 min. After removing the supernatant in the wells, the prepared clone T cells were dispersed by pipetting and recovered from the well surface. Collected T cells were fixed using a fixative containing 1% PFA. Fixed T cells were treated with 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). T cells immunostained with anti-CD28 antibody were further immunostained using 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 cells were not subjected to immune stimulation by contact with allogeneic cells. The cloned T cells prepared in (1) above were fixed on ice with the above 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 subjected to immune stimulation by contact with allogeneic cells, CD3, CD28, CD40L and OX40 were uniformly distributed on the cell membrane in T cells (Fig. 21a, c, e). and g, Figures 22a and b, Figures 23a and b, Figures 24a and b, Figures 25a and b). CD3, CD28, CD40L and OX40 were localized on the cell membrane of T cells when the contact surface between T cells and allogeneic cells was eliminated after the above-mentioned immunostimulation (Fig. 21b, d). , f and h, Figures 22c and d, Figures 23c and d, Figures 24c and d, Figures 25c and d). Thus, even in a state in which the contact surface between the T cell and the allogeneic cell is eliminated after the immunostimulation by contact with the allogeneic cell, surface antigens of the T cell, CD3, CD28, By using CD40L or OX40 as an indicator, immune synapses could be detected.

(3) Data analysis
Measurement data of the sample containing T cells without the above immunostimulation (FIGS. 18a and b) and the measurement data of the sample containing T cells with the above immunostimulation (Fig. A multiparameter analysis of 18c and d) was performed. Measurement processing and analysis processing are the same as 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 cell size (number of pixels), cell aspect ratio, and fluorescence signal in cells are analyzed. The area (the number of pixels) showing and the total fluorescence signal intensity (total fluorescence signal intensity of cells) in the area were measured. First, a 2D scattergram was created in which the X-axis was assigned the cell size and the Y-axis was assigned the cell aspect ratio, and cell measurement data corresponding to dots in a predetermined range (Cells) was extracted. Next, for the measurement data within the range (Cells), the measurement data corresponding to the dots where the total fluorescence signal intensity of the cells was within the predetermined range (Tcells) was further extracted. Number of dots in the regions 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 measured data within range (Tcells) were counted respectively. Also, the number of dots on each scattergram (the number of dots within the range (Tcells)) was counted. Then, the ratio of the number of dots within the range (IS_CD3, IS_CD28, IS_CD40L and IS_OX40) to the number of dots within the range (Tcells) was calculated using the following formula (3). The results are shown in Tables 4-7 below.

[Proportion (IS-forming cells/Tcells)]={[number of dots in range (IS_CD3, IS_CD28, IS_CD40L or IS_OX40)]/[number of dots in range (Tcells)]}×100 (3)

As shown in Tables 4 to 7, the proportion of T cells that formed immune synapses was clearly higher in T cells that had been immunostimulated with allogeneic cells compared to those without immunostimulation. shown. Therefore, it was shown that the responsiveness of immune cells to immune stimulation by allogeneic cells can be measured by detecting immune synapses using CD3, CD28, CD40L, or OX40 as indicators.

Claims (15)

(i) contacting the immune cells to be measured with an immunostimulator;
(ii) contacting the immune cells to be measured with a substance different from the immune cells to be measured to form a contact surface;
(iii) physically and/or chemically breaking said contact;
(iv) contacting the immune cells to be measured with capture bodies capable of binding to surface antigens on the contact surface and generating optical signals;
the substance different from the immune cells to be measured is a solid phase of a non-biological material and/or cells different from the immune cells to be measured;
The immune cells to be measured are T cells and/or NK cells,
A method for labeling immune cells. the immunostimulatory agent of step (i) is used as a scavenger capable of binding to surface antigens on said contacted surface of step (iv) and producing an optical signal;
2. The method of claim 1, wherein steps (i) and (iv) are performed simultaneously. 3. The method of claim 1 or 2, wherein said surface antigen comprises at least one of TCRα/β, CD3, CD40L, OX40, CD28, CTLA4, PD-1 and ICOS. A method according to any one of claims 1 to 3, wherein said surface antigen is a co-stimulatory molecule. A method according to any one of claims 1 to 4, wherein said capture entity comprises a substance capable of producing an optical signal and a substance that binds directly or indirectly to said surface antigen. 6. The method of claim 5, wherein the substance that directly or indirectly binds to the surface antigen is an antibody, antibody fragment, single chain antibody or aptamer. (i) contacting the immune cells to be measured with an immunostimulator;
(ii) contacting the immune cells to be measured with a substance different from the immune cells to be measured to form a contact surface;
(iii) physically and/or chemically breaking said contact;
(iv) an immune synapse formed on the contact surface of the immune cell to be measured, or an immunostimulatory factor bound to the immune cell to be measured on the contact surface by an optical signal; labeling with a capture entity that can give rise to
the substance different from the immune cells to be measured is a solid phase of a non-biological material and/or cells different from the immune cells to be measured;
The immune cells to be measured are T cells and/or NK cells,
A method for labeling immune cells. 8. The method according to claim 7, wherein the capture body comprises a substance capable of producing an optical signal and a substance that binds to the molecule that constitutes the immune synapse or the immune stimulator. 9. The method of claim 8, wherein the molecules that make up the immune synapse comprise at least one of TCRα/β, CD3, CD40L, OX40, CD28, CTLA4, PD-1 and ICOS. 10. Any one of claims 1 to 9, wherein the immunostimulatory factor comprises at least one of an immunostimulatory antibody, an immunostimulatory peptide, a major histocompatibility molecule (MHC molecule), and a complex of an MHC molecule and an antigenic peptide. The method described in . The method according to any one of claims 1 to 10, wherein the substance different from the immune cells to be measured is allogeneic cells, antigen-presenting cells, cancer cells, containers, multiwell plates, slides or beads. The method according to any one of claims 1 to 11, wherein the immune cells to be measured are T cells. 13. The method according to any one of claims 1 to 12, wherein the immunostimulatory factor in step (i) is present on a different substance than the immune cells to be measured in step (ii). A method according to any one of claims 1 to 13, wherein said contact is eliminated by stirring, sonication or chemical treatment. The method according to any one of claims 1 to 14, further comprising the step of sorting the immune cells to be measured from a sample containing cells based on cell size, degree of aggregation and/or specific gravity. .

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