WO2009110614A1 - Procédé de mesure de la fonction de cellules effectrices, kit de mesure et système de mesure - Google Patents

Procédé de mesure de la fonction de cellules effectrices, kit de mesure et système de mesure Download PDF

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WO2009110614A1
WO2009110614A1 PCT/JP2009/054340 JP2009054340W WO2009110614A1 WO 2009110614 A1 WO2009110614 A1 WO 2009110614A1 JP 2009054340 W JP2009054340 W JP 2009054340W WO 2009110614 A1 WO2009110614 A1 WO 2009110614A1
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cell
cells
measurement
measuring
function
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PCT/JP2009/054340
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Japanese (ja)
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泰信 小林
啓司 谷川
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Kobayashi Yasunobu
Tanigawa Keishi
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism

Definitions

  • the present invention relates to a method for measuring cell functions centering on the cytotoxic activity of lymphocytes, and a measurement kit and measurement system therefor.
  • Our body has an immune mechanism that recognizes and eliminates foreign objects.
  • Abnormal cells such as pathogens that enter from the outside and cancer cells generated in the living body are recognized as foreign substances and are excluded from the living body by various immune system mechanisms.
  • Such immune functions are skillfully controlled by leukocytes that circulate throughout the whole body on the flow of blood or lymph.
  • lymphocytes play the main role in the so-called effector phase of cellular immunity.
  • cell groups called so-called effector cells such as natural killer cells (hereinafter sometimes abbreviated as NK cells) and cytotoxic T cells (hereinafter sometimes abbreviated as killer T cells) include, for example, viruses. Detecting abnormalities occurring in their own cells in the body, such as cells infected with cancer and cancer cells, and eliminating them by killing the abnormal cells (for example, Non-Patent Document 1).
  • effector cells kill virus-infected cells and cancer cells, but especially in the case of NK cells and killer T cells, the mechanism of cytotoxic activity via perforin and granzymes plays an important role.
  • effector cells release perforin and granzymes after adhering to the target cells.
  • Perforin makes a hole in the cell membrane of the target cell, and granzyme is injected into the target cell through the hole.
  • Granzyme A is known to induce apoptosis in target cells through at least a caspase-independent pathway, although the detailed mechanism of action is unknown. Simultaneously with perforin and granzyme, granulysin, a lipid-binding protein, is also released extracellularly. Although there are many unclear points regarding the mechanism of action of granulysin and its physiological significance, it has been pointed out that it may induce cell death in the target cell by directly binding to the cell membrane of the target cell (eg, non-patented). Reference 2).
  • LAK therapy is a therapy in which lymphocytes taken out of the body are activated by interleukin 2 or the like so as to increase the ability to kill cancer cells, and the cells are returned to the living body again. It has been reported that when lymphocytes are actually cultured in the presence of interleukin 2, the perforin content of NK cells in lymphocytes is significantly increased (for example, Non-Patent Paper 4).
  • Activated lymphocyte transfer therapy is an immune cell therapy that specifically activates killer T cells in lymphocytes removed outside the body. Like “LAK therapy”, the target cell killing ability by perforin granzyme etc. It is an important factor to enhance the therapeutic effect.
  • therapeutic cells which are not purified, uniform cell populations, but mostly a collection of diverse lymphocytes with different antigen specificities, but at least in the cell population, the desired effector In what ratio the cells are present, in addition to whether the effector cells actually express killing components such as perforin and granzyme B, the level of expression, and the therapeutic cells
  • the immune function inherent in the living body is attracting attention not only from the scene of cancer treatment by immune cell therapy but also from the viewpoint of preventive medicine.
  • the function of NK cells to kill cancer cells is also called NK activity, and is widely recognized as one of the representative indicators of the immune function of the living body.
  • NK activity is generally evaluated by a test called a chromium release test or a cytotoxic activity test by flow cytometry (see, for example, Non-Patent Document 5), but the activity varies depending on age and lifestyle. It has also been reported (for example, see Non-patent Document 6), and it has also been reported that its activity is enhanced by the intake of certain health foods and supplements (for example, see Non-Patent Document 7). That is, NK activity is positioned as an important test item today as a barometer for promoting health and as a means for confirming the effects of drugs, foods, supplements and the like on the immune function of the living body.
  • effector cell functions is an important test item for knowing the immunity of a living body.
  • the activity of effector cells is evaluated by various methods.
  • NK cells The function of NK cells is generally to measure NK activity. That is, the peripheral blood of the subject is used as an effector cell, the myeloid leukemia cell line K562 cell is used as a target cell, and these cells are co-cultured at a ratio uniquely determined by each laboratory, and after a certain time, the target cell The degree of injury of K562 cells is measured. The measurement of cytotoxicity is generally assessed by a chromium release test. As a result, for example, lymphocytes in peripheral blood and K562 cells were mixed at a ratio of 10: 1 and cultured for 4 hours, and at 4 hours after culture, the damaged K562 cells were 60% of the whole. The NK activity is defined as 60%.
  • NK activity the actual test material is a cell population of leukocytes centered on blood or mononuclear cells. And, NK cells are usually contained in these specimens by about a dozen percent or less. In addition, the number of NK cells in the specimen in each test is not constant. In addition, the number of NK cells present in the specimen is considered to be not homogeneous in terms of their function. For example, in the aforementioned non-patent paper 4, it is described that the perforin content of NK cells varies depending on sex and age.
  • NK activity evaluation and its measurement results do not take into account the difference in the number of NK cells in the sample and their quality, but simply represent the final results of cell death occurring in the target cells. Not too much. Therefore, a specimen having a large number of NK cells generally tends to exhibit a higher NK activity value, and a specimen having a small number of NK cells tends to exhibit a low NK activity value.
  • NK activity when there are differences in NK activity when comparing multiple specimens, there are various possibilities whether the difference is due to the difference in the number of NK cells or the function of individual NK cells. However, the details cannot be clarified by the above-described inspection method.
  • Non-Patent Document 5 a cytotoxic activity test using a flow cytometer has also been developed as an alternative method of the chromium release test.
  • this flow cytometer test by appropriately combining a plurality of antibodies and fluorescent dyes, the degree of cytotoxicity generated in the target cells is measured, and at the same time, the ratio of NK cells in the specimen used for the test, It is also possible to measure the frequency of granzyme expression in the NK cells.
  • the data that is stored at the time of measurement and used for the analysis is numerical data such as scattered light and fluorescence intensity detected when the cells flow through the flow path in the device.
  • a statistical analysis process is performed on a plurality of parameters converted into list mode data, and the cell itself is not directly analyzed. Therefore, even if various analysis methods are used in the analysis software, there are limits to the phenomena that can be analyzed and the accuracy of the data.
  • an example is disclosed in which adhesion between NK cells or T cells and target cells is analyzed by a flow cytometer.
  • killer T cells since the number of cells is smaller than that of NK cells, it is very difficult to measure the function of the cells, so that a certain cell population in a living body such as peripheral blood mononuclear cells In many cases, only the frequency of the cells can be measured. In particular, the number of specific killer T cells against a specific tumor antigen detected in the peripheral blood of cancer patients is extremely small. For example, the detection frequency is 0.1% of the total killer T cells, and the peripheral blood. In the mononuclear cell fraction, it is often less than 0.01%.
  • the number of tumor antigen-specific killer T cells that can be collected by normal blood collection is extremely small, and for carrying out the above-described chromium release test or cytotoxic activity test using a flow cytometer, In many cases, even the minimum number of cells cannot be secured, and as a result, the cytotoxic activity against the target cancer cells cannot be directly measured. Therefore, at present, in most cases, only MHC tetramer method, ELISPOT method, etc. are used to limit the measurement of the frequency of specific killer T cells responding to a specific antigen in peripheral blood killer T cells. Absent.
  • the MHC tetramer method is a method in which a complex of an antigenic peptide and an MHC class I-like molecule is labeled with a fluorescent dye, and killer T cells bound to the molecule are detected and analyzed with a flow cytometer.
  • a fluorescent dye a fluorescent dye that detects killer T cells bound to the molecule.
  • killer T cells bound to the molecule are detected and analyzed with a flow cytometer.
  • it is widely used as one of representative techniques for detecting killer T cells that specifically recognize viral antigens.
  • flow cytometry it is generally difficult to ensure the reliability of data, for example, when the amount of measurement is less than 0.01% of cells to be measured.
  • a dot indicating the presence of one cell is a signal derived from a cell to be measured or mixed Even if there is a question about whether this artifact is caused by noise, it is no longer possible to remeasure or reanalyze that single dot.
  • the ELISPOT method is a method for detecting interferon ⁇ (hereinafter sometimes abbreviated as IFN ⁇ ) or granzyme B produced by killer T cells activated with a specific antigen. It is widely used as one of representative techniques for detecting specific killer T cells. However, it is difficult to ensure the reliability of data in low-frequency cell analysis as in the MHC tetramer method.
  • the measurement target is a spot scattered in the membrane, that is, a spot visualized by staining an IFN ⁇ or granzyme B produced by a cell with an enzyme antibody method or the like, and the cell is also a direct measurement target. Absent.
  • the present invention provides a new cell function measurement method for overcoming the disadvantages and problems of the conventional effector cell function measurement method as described above, and a kit for carrying out the measurement and its kit
  • An object is to provide a measurement system. That is, the present invention provides a novel cell function measurement method for measuring the function of an effector cell, such as cytotoxic activity against a target cell, in detail and exhaustively, with high accuracy, a kit for performing the measurement, and its Provide a measurement system.
  • effector cells and target cells are mixed and cultured in an arbitrary time at an arbitrary ratio in a standard microtiter plate.
  • a plurality of molecules are fluorescently labeled antibodies, fluorescent substances, etc. And then measure the expression of multiple molecules simultaneously and as needed using a cell-based assay device capable of acquiring and analyzing individual images of the cells in the plate. And an effector cell evaluation method characterized by qualitatively and quantitatively analyzing the measurement results.
  • the bottom of the standard microtiter plate is desirably a flat bottom, and the number of cells used as a sample may be one or more. Further, when a plurality of cells are used as a sample, it is desirable to arrange them in a single layer on the bottom surface of the flat bottom.
  • the flow cytometer has been used so far, such as the ratio of the desired effector cells in the specimen, the ratio of the effector cells expressing perforin granzyme in the cells, and the content of perforin granzyme in each effector cell.
  • Intracellular granule containing perforin granzyme etc. produced by encountering target cells with the localization of perforin granzyme etc. in individual effector cells The dynamics and other items that have been observed with a fluorescence microscope or a laser confocal microscope until now are measured simultaneously.
  • cell biological phenomena such as adhesion of effector cells to target cells and changes related to apoptosis in target cells are also measured simultaneously in the same sample.
  • the phenomenon when measuring and quantifying the cytotoxicity of a target cell, whether the phenomenon is due to, for example, apoptosis, and how much the effector cell adheres to the target cell.
  • Targeting intracellular granules containing granzyme B in the cytoplasm because a killing component that induces apoptosis in the target cell, such as granzyme B, is expressed, and the effector cell kills the target cell Simultaneously measure and analyze various phenomena such as whether or not the cell is moved to the adhesive surface.
  • the ratio of the effector cells present in the specimen, the ratio of the effector cells expressing granzyme B, and the granzyme B expression level of individual effector cells are simultaneously measured and analyzed. That is, according to the method of the present invention, it is possible to analyze the quantity and quality of effector cells, and their functions in detail, simultaneously and in a multifaceted manner, thereby confirming the specificity and validity of the phenomenon. It becomes possible to do.
  • this image acquisition obtains image data of individual cells and makes the image data subject to analysis, in addition to enabling detailed morphological observation and analysis of cells as described above, data By confirming the image of each cell at the time of analysis, it becomes easy to find foreign substances mixed during sample preparation and measurement, or to find false positive data derived from the foreign substances. And it becomes possible to reanalyze a result only with the true positive data except the said false positive data.
  • Such exclusion of false positive data and reanalysis cannot be performed by conventional methods such as chromium release test, NK activity measurement by flow cytometry, or measurement of killer T cell frequency by MHC tetramer method or ELISPOT method. This is one of the excellent characteristics of the present invention. Therefore, even when a low-frequency cell group such as a killer T cell is used as a measurement target, measurement and analysis with higher reliability can be performed as compared with the conventional method.
  • the present invention even after a plurality of parameters are measured and the analysis is completed, if necessary, another parameter can be added and remeasured, and the data can be analyzed again.
  • another parameter can be added and remeasured, and the data can be analyzed again.
  • the effector cell to be measured is again a different killing component. If you want to investigate whether or not a certain granulysin is expressed, use an appropriate antibody that does not cross immunologically, and can be detected optically and separated from fluorescent dyes used for other parameter measurements.
  • a device for performing the measurement and analysis as described above is not specifically specified, but a fluorescence microscope image of a cell is acquired fully automatically, for example, a cell image analysis device such as Olympus RS-100, Furthermore, it is desirable that the apparatus can obtain statistical data by analyzing and digitizing fluorescence luminance information and cell morphology information. That is, for example, when the measurement target is a cell monolayer fixed to the well bottom of a standard flat bottom 96-well microtiter plate and subjected to various fluorescent labels, the 96-well microtiter plate is used.
  • a cell-based assay device comprising means for directing to a camera and further comprising a computer system for receiving and processing digital data from the digital camera. Then, the entire region of the 96-well microtiter plate is imaged, and this image is analyzed to quantitatively analyze the expression level of surface antigens and intracellular antigens of individual cells.
  • the above-described apparatus is a high-magnification fluorescent optical system apparatus equipped with an objective lens of at least 10 times, preferably 20 times or more, more preferably 40 times or more, for observing the internal structure of a cell in detail. It is preferable that the apparatus is capable of acquiring a microscopic image. For high-sensitivity detection, it is preferable to include a light source and a detector capable of widely detecting many fluorescent molecules having different excitation wavelengths and fluorescence wavelengths. At that time, in order to detect multiple parameters with the same specimen, when performing multiple staining with multiple fluorescent dyes, an optical separation function for accurately and independently measuring the fluorescence intensity of each dye, It is preferable to have an optical filter or the like.
  • the number of fluorescent dyes that can be measured simultaneously and independently is more preferably an apparatus having an optical measurement function capable of simultaneously using at least two kinds, preferably four or more kinds of dyes.
  • the above-mentioned apparatus has computer means for receiving and processing digital data from, for example, a CCD camera, specifically storing the data as an image image and processing the image image as necessary.
  • an image analysis function capable of quantitative analysis is installed, or an apparatus that can process and analyze measurement data in conjunction with an external image analysis apparatus.
  • a function that automatically aligns and focuses at the time of measurement is installed, so that more processing is performed. It is more preferable if the apparatus has a function of automatically acquiring high-speed image image data of cells.
  • Non-Patent Document 5 an analysis is performed in which a virtual cell image is reproduced on a display from a scattered light or fluorescence data using an imaging flow cytometer device, and morphological observation is performed using a microscope image.
  • the method disclosed in this paper is basically a method obtained by modifying the flow cytometry method, and is essentially different from the present invention in which cell image data is analyzed. That is, in the method according to the present invention, an image image of a cell acquired by a CCD camera or the like is stored as a measurement / analysis target, and the image data is converted into digitized data by various image processing to perform statistical analysis or the like.
  • the measurement method in the above paper is the conventional analysis method in flow cytometry, that is, individual cells are generated when light such as laser light is irradiated to cells that continuously flow through a specific flow path.
  • the intensity of scattered light or fluorescence is stored as data together with light emission position information, and the optical data is analyzed by various methods. Therefore, the method described in the non-patent document is essentially a method different from the present invention in which cells are directly observed and the image data is directly analyzed.
  • false positive data cannot be excluded and reanalyzed, and remeasurement and reanalysis with new parameters for the same sample is also possible. It is impossible, and from this point, it is considered that the method is essentially different from the measurement method according to the present invention.
  • a CMOS image sensor can be used in addition to a CCD camera including a CCD image sensor, and light including fluorescence of 300 nm to 800 nm is detected, and light emitted from an object to be photographed is used as a lens.
  • the optical system forms an image on the light receiving plane of the image pickup device, and the light and darkness of the image is photoelectrically converted into an amount of electric charge, which is sequentially read and converted into an electric signal.
  • the apparatus used in the invention is a digital image capturing means for capturing an image of a CCD image sensor, a CMOS image sensor or the like to perform measurement / analysis, a filter for spectrally separating the captured image by wavelength, and spectrally captured imaging data.
  • a memory that stores brightness information, position information, and wavelength information from each other, a central processing unit that obtains and analyzes a graph of the wavelength vs. frequency of the imaging data as light intensity for each wavelength, and a display unit that displays the calculation results It is to be prepared.
  • the present invention provides a method for detecting cell death or a method for detecting apoptosis as a method for detecting injury caused to a target cell.
  • the method for detecting cell death is not particularly limited.
  • PI sodium iodide
  • 7-AAD 7-aminoactinomycin D
  • the method of doing can also be used.
  • the method for detecting apoptosis is not particularly limited.
  • active caspase 3 which is a characteristic molecule expressed in cells at the early stage of apoptosis can be used as a measurement target.
  • Caspase 3 becomes active in the cytoplasm by being cleaved by the action of granzyme B injected into the target cell from, for example, an effector cell. This activated caspase 3 is visualized by immunostaining using a specific antibody.
  • cytokeratin 18 can also be used as an index of apoptosis.
  • Cytokeratin 18 is cleaved by activated caspase 3 (or caspase 7). By detecting the cleaved cytokeratin 18 immunochemically, it is also possible to detect apoptosis occurring in the target cell. Is possible.
  • a fluorescent labeling compound that specifically binds to active caspase 3 or a specific color that develops fluorescence when cleaved by caspase 3 is used.
  • a chemical substance such as a typical substrate may be used to measure the amount of active caspase 3 and its enzyme activity.
  • apoptosis such as nuclear concentration / fragmentation
  • fluorescence-labeled annexin V to detect phosphatidylserine exposed on the cell surface in the early stage of apoptosis, or by staining the cell nucleus with a dye such as DAPI, apoptosis such as nuclear concentration / fragmentation
  • a method of quantitative analysis using morphological changes characteristic of cells as an index may be used.
  • the target cell itself, or a target cell that has been subjected to some kind of injury, selected by various indicators as described above, a target cell that has undergone apoptosis, and / or a characteristic of apoptosis.
  • a method for measuring the function of an effector cell comprising the step of confirming whether or not the effector cell is actually attached to a target cell expressing a molecule. Adhesion between effector cells and target cells is an essential phenomenon for effector cells to kill target cells. Therefore, by including the step of analyzing this phenomenon over time, it is possible to confirm whether or not the effector cell is a functional cell having the ability to actually identify and adhere to the target cell.
  • NK cells characteristic expression on the cell surface of NK cells such as NKp46 and NKp30
  • NKp46 characteristic expression on the cell surface of NK cells
  • NKp30 characteristic expression on the cell surface of NK cells
  • the expression of these molecules may be extremely low, which may make it difficult to identify the cells.
  • MHC tetramer molecules are used. A method of detecting by, for example, is preferable. However, depending on the antigen, the MHC tetramer molecule may not be easily prepared. Therefore, it is possible to use a method of identifying the presence or absence of expression of a plurality of other molecules as an indicator, such as selecting CD3 positive and CD8 positive cells. Good.
  • molecules produced from the antigen-specific killer T cells or the expression level of the antigen-specific killer T cells changes depending on the stimulation of the specific antigen.
  • the expression level of intracellular killer T cells can be used as an index. Specifically, production of cytokines such as IFN ⁇ from killer T cells by stimulation of specific antigens, accumulation of such cytokines in cells, and cell killing components such as perforin, granzyme A, granzyme B, granulysin Increased expression in cells can be used as an index.
  • purified effector cells in addition to the above method, for example, a method of discriminating by directly incorporating the fluorescent marker gene for expressing GFP or the like into the cell.
  • a method may be used in which a specific molecule, for example, bromodeoxyuridine or the like is taken into a cell at the time of cell division and the effector cell is identified using the presence or absence of the expression of the molecule as an index.
  • the target cell does not need to be labeled with a fluorescent molecule or the like as long as it can be distinguished from the effector cell only by its morphological characteristics such as the difference in cell size.
  • fluorescent labeling may be performed using an antibody that recognizes a molecule characteristic of the target cell, similarly to labeling of effector cells.
  • the target cell is a cell line such as the above K562 cell, for example, the target cell is labeled and identified by directly incorporating the fluorescent marker gene for expressing GFP or the like into the target cell.
  • bromodeoxyuridine or the like may be incorporated into a cell and labeled, and the target cell may be identified using the presence or absence of the expression of the molecule as an index.
  • the method for detecting the adhesion between the effector cell and the target cell is not particularly limited. For example, it is easiest to observe the target cells in the image data one by one and measure the ratio of cells to which effector cells actually attach. On the other hand, if the image analysis apparatus has a masking tool, the analysis may be performed using the analysis tool. In that case, for example, the nucleus or cell membrane of the target cell is stained, and the area of the target cell where the lymphocyte adhering to the periphery is included on the outer periphery of the staining site.
  • the effector cell By setting (masking) a region similar to the shape of the cell nucleus or cell membrane outside a certain pixel around the cell membrane, and examining whether there is a fluorescent signal originating from the effector cell in that region, the effector cell It becomes possible to automatically select and measure the target cell to which the cell adheres and the target cell to which the effector cell does not adhere.
  • an effector cell evaluation method comprising the step of confirming the amount of cell killing components in the effector cell and the kinetics thereof.
  • the amounts of perforin, granzyme A, granzyme B, and granulysin to be measured can be quantified by performing immunofluorescence staining using a specific antibody as usual, and measuring the fluorescence intensity. If an image with high magnification and high resolution can be obtained, the number of granules in the cell can be measured and calculated.
  • the method for analyzing the dynamics of these cell killing components in the effector cells is not particularly limited, but, as described above, either the method for directly observing and analyzing the image of the cell or the automatic analysis by the image analyzer.
  • the method can be used. That is, intracellular granules containing these killing ingredients are normally distributed relatively uniformly in effector cells, but when the effector cells and target cells adhere, as a pre-stage that is injected into the target cells, Intracellular cell killing components accumulate near the cell adhesion surface (see, for example, Non-Patent Document 2 above). Using this phenomenon, the intracellular dynamics of the killing component of effector cells are analyzed.
  • the distribution of the intracellular granules is relatively broadly distributed within a certain pixel from the cell nucleus or cell membrane of the target cell. However, if the granules are accumulated on the adhesion surface with the target cells, the distribution is biased closer to the nucleus and the cell membrane than in the case of the uniform case described above.
  • the method for staining the nucleus or cell membrane of the target cell is not particularly limited.
  • DAPI 6-diamino-2-phenylindole
  • the cell membrane may be stained by a method of staining a molecule specifically expressed in the cell membrane of the cell depending on the type of target cell to be used.
  • a method of fluorescently labeling only target cells using a lipophilic fluorescent material such as PKH-26 (manufactured by Sigma) is used. It doesn't matter.
  • kits for measuring changes at various cellular and molecular levels occurring in effector cells and / or target cells as described above.
  • the kit comprises a cell culture vessel used for co-culture of cells and / or measurement of various parameters, and a reagent such as a specific antibody and a fluorescent dye for identifying cells and molecules.
  • the cell culture vessel which is one of the components of the kit, is used for culturing effector cells and target cells, and as a place to arrange cells in a single layer at the time of measurement, both of which are so-called microtiter plates and microplates.
  • Any container can be used as long as it is used for general cell culture and can be used for antibody staining by an immunochemical technique.
  • containers contained in one kit may be at least two different types for culture and measurement.
  • a petri dish, a slide glass, a flask, or the like may be used as long as the measuring device allows.
  • the measurement container is preferably a multi-well microtiter plate or microplate.
  • the number of wells of the microtiter plate or microplate is not particularly limited, however, for example, a 6-, 12-, 24-, 48-, or 96-well microtiter plate generally used for cell culture is preferable.
  • a microtiter plate or a microplate having wells with holes is preferred.
  • the material of the above-mentioned microtiter plate or microplate is not particularly limited.
  • a commercially available standard plastic plate for cell culture or ELISA, a glass plate, or the like can be used.
  • the bottom surface of these plates that is, the material and shape of the surface in contact with the cells, the charged state of the surface, etc. are not particularly limited.
  • Surface treatment with molecules for assisting cell attachment such as proteins such as collagen, polymer compounds such as poly-D-lysine, and other substances, as long as they do not interfere with fluorescence measurement. It can also be used.
  • other containers such as petri dishes, slide glasses, and flasks may be used, not limited to microtiter plates and microplates, but these containers are similarly materials that assist cell adhesion. Or surface-treated ones can be used.
  • the operation of fixing the cells to the bottom surface of the container is indispensable.
  • the method is not particularly limited, for example, when using a microtiter plate or a microplate, after seeding a cell suspension, the plate is centrifuged to submerge the cells, and then dried. Cells can be attached and extended to the bottom of the plate.
  • a microtiter plate or a microplate that has been subjected to a surface processing treatment for assisting the adhesion of cells as described above can be used as long as it does not hinder fluorescence measurement. Even when another container such as a microtiter plate or a microplate, a petri dish, a slide glass, or a flask is used, the cells can be fixed in the same manner.
  • kits for example, commercially available antibodies and reagents can be used as the reagent that is one of the components of the kit.
  • a specific antibody against NKp46 or NKp30, or a combination of CD56 antibody and CD3 antibody (for example, all manufactured by Beckman Coulter) and in the case of MHC tetramer, for example, HLA type is A0201 type
  • reagents such as PE-labeled Her2 / neu (KIFGSLAFL) MHC tetramer (manufactured by MBL) can be used.
  • specific antibodies against commercially available perforin, granzyme A, granzyme B and granulysin can also be used, and commercially available specific antibodies can also be used for cytokine detection.
  • cytokine detection it is possible to use a procedure for adding a protein transport inhibitor to the culture medium and accumulating the cytokine in the cell.
  • the protein transport inhibitor used for this purpose For example, commercially available reagents such as monensin A and brefeldin A can be used.
  • the above-mentioned specific antibody may be an antibody already labeled with a fluorescent dye, but may be a combination of an unlabeled antibody and a commercially available fluorescently labeled secondary antibody that specifically recognizes the antibody.
  • the fluorescent dye at that time is not particularly limited, but for a kit for simultaneously identifying at least three different molecules, for example, a fluorescent dye made by Invitrogen: Molecular Probes, Alexa Fluor (registered trademark) ) 488, Alexa Fluor (registered trademark) 555, Alexa Fluor (registered trademark) 647 can be combined with primary or secondary antibodies labeled with three types of fluorescent dyes.
  • the reagent for discriminating cell death and apoptosis is not particularly limited, but the kit uses PI (propidium iodide), 7-AAD (7-aminoactinomycin D), etc., for example, for the identification of dead cells. be able to.
  • PI sodium iodide
  • 7-AAD 7-aminoactinomycin D
  • a fluorescent labeling reagent that specifically binds to an intracellular active caspase such as APO LOGIX reagent manufactured by Cell Technologies may be used, or a fluorescent molecule is released by the action of the active caspase.
  • a substrate may be used, for example, a reagent such as a BioVision 7-AFC binding substrate.
  • a fluorescent labeling reagent such as FITC-labeled annexin V manufactured by Immunotech can be used for annexin V detection.
  • a commercially available antibody or anti-cytokeratin 18 antibody that specifically recognizes active caspase 3 can be used.
  • the antibody may be an antibody already labeled with a fluorescent dye, or may be a combination of an unlabeled antibody and a commercially available secondary antibody that binds to a fluorescent substance that specifically recognizes the antibody. .
  • the kind of fluorescent dye at that time is not particularly limited.
  • the properties of the effector cells themselves and the phenomenon at the multiple cellular and molecular levels when the cells kill target cells can be simultaneously and / or It becomes possible to measure at any time.
  • Example 1 of this invention It is a schematic diagram of the setting method of the measurement area
  • A It is a schematic diagram which shows selection of the immunostaining image of a cell
  • B Cell nucleus It is a schematic diagram showing recognition of (main object)
  • C is a schematic diagram showing recognition of cell surface antigens
  • D is a schematic diagram showing recognition of barforin
  • Example 1 of the present invention An example of analysis of perforin positive NK cells in PBMC.
  • (A) is a graph which is an example of analysis by the effector cell function measurement method according to the present invention, in which CD56-positive CD3-negative cells (NK cells) are selected and the ratio of perforin-positive cells in the cell group is calculated.
  • (B) A CD56-positive CD3-negative cell is selected by a conventional flow cytometer, and the ratio of perforin-positive cells in the cell group is calculated. It is the graph and imaging of an example of the image analysis of a perforin positive NK cell by the function measuring method of the effector cell which concerns on Example 1 of this invention.
  • An arbitrary dot indicated by an arrow in the dot plot of (A) is an imaging corresponding to an image of a cell in the region indicated by the arrow in (B).
  • FIG. 1 It is an imaging and analysis image which show an example of the analysis of the perforin containing granule of NK cell by the function measuring method of effector cell concerning Example 2 of the present invention.
  • A is one arbitrarily selected NK cell (perforin positive, CD56 positive)
  • B is an analysis image showing an example of a granule detection method when analyzing the number of granules of the cell.
  • It is imaging of an example of the image data of the culture mixture of PBL and K562 cell by the function measuring method of the effector cell which concerns on Example 3 of this invention. Arrows indicate K562 cells, and expression of active caspase 3 was observed in the cytoplasm of some cells.
  • ⁇ indicates a CD56-positive cell (NK cell).
  • ⁇ indicates a CD56-positive NK cell
  • an arrow indicates an active caspase-positive K562 cell.
  • ⁇ indicates a CD56-positive NK cell
  • an arrow indicates an active caspase-positive K562 cell.
  • ⁇ indicates a CD56-positive NK cell
  • an arrow indicates an active caspase-positive K562 cell.
  • ⁇ indicates a CD56-positive NK cell
  • an arrow indicates an active caspase-positive K562 cell.
  • the perforin granules of NK (*) are relatively uniformly distributed in the cytoplasm, but in the other two NK cells, the perforin granules on the cell adhesion surface with K562 cells Accumulation is recognized.
  • a schematic diagram and imaging of an example of analysis of MAGE-3-specific killer T cells in peripheral blood by a conventional method are shown.
  • (A) is an example of an analysis chart of the MHC tetramer method
  • (B) is an example of a result of the ELISPOT method, and shows spots of IFN ⁇ produced by the specific killer T cell group.
  • the schematic diagram and imaging of an example of the analysis of a MAGE-3-specific killer T cell by the method which concerns on Example 4 of this invention are shown.
  • the histogram of (A) shows an example of analysis of intracellular IFN ⁇ positive cells.
  • (B) shows an image of all cells in the range of IFN ⁇ positive in the histogram of (A).
  • the X-marked portion at the bottom left was judged as noise because it did not actually show cell morphology.
  • (C) shows an image corresponding to the portion of the X mark in the image data. The portion that was mistakenly recognized as a cell is indicated by an arrow, and this portion is actually non-specific fluorescence. It was reconfirmed that the noise was radiating.
  • PBMC peripheral blood mononuclear cells
  • a flat-bottom 96-well microplate (Corning 3596) was seeded with 100,000 cells per well, and then the plate was centrifuged at 50 ⁇ g for 10 minutes, and then the supernatant was removed for 30 minutes. Air dried. The cells were then fixed with 4% paraformaldehyde for 30 minutes.
  • cytoplasmic perforin is stained with mouse anti-human perforin monoclonal antibody (ANCELL) and Alexa Fluor (registered trademark) 647-labeled anti-mouse IgG antibody (Invitrogen), and FITC-labeled anti-human CD3 antibody (Beckman Coulter) And R-PE labeled anti-human CD56 antibody (manufactured by Beckman Coulter) were used to label the cell surface antigen of lymphocytes.
  • ANCELL mouse anti-human perforin monoclonal antibody
  • Alexa Fluor registered trademark
  • 647-labeled anti-mouse IgG antibody Invitrogen
  • FITC-labeled anti-human CD3 antibody Beckman Coulter
  • R-PE labeled anti-human CD56 antibody manufactured by Beckman Coulter
  • the cell nuclei were stained with DAPI, and after staining, the ratio of NK cells (CD3 negative, CD56 positive) in PBL and the ratio of perforin positive cells were analyzed using a cell image analyzer (OLYMPUS CELAVIEW RS100).
  • FIG. 1A An outline of a specific method for setting a measurement region in image analysis is shown in FIG. 1.
  • 101 indicates cell surface antigens (CD3 and CD56)
  • 102 indicates perforin
  • 103 indicates a nucleus.
  • a plurality of arbitrary cell images subjected to these staining processes were selected.
  • a region that recognizes the nucleus region 104 that is, a region stained with DAPI, as a main object is set (B).
  • the nuclear region is set by the maximum and minimum values of the area of the DAPI-stained portion and the threshold value of the fluorescence intensity.
  • the watershed algorithm method is used. Regions were set to recognize individual cells by recognizing the constriction between nuclei.
  • a recognized main object that is, a donut-shaped area 105 existing at equal intervals and an area 107 covering all the cells, with each cell nucleus as a center, was created.
  • the innermost start point of the donut from the outermost nuclear line and the end point that is the outermost line of the donut can be freely set.
  • the cell surface antigens CD3 and CD56
  • C When a donut area such as the region 105 in which all or at least a part of the cell membrane 106 is included in the area is set, and the perforin 108 in the cell is a measurement target (D), the cell nucleus 109 A region 107 including the cell membrane 110 was set as a measurement target.
  • D the cell nucleus 109 A region 107 including the cell membrane 110 was set as a measurement target.
  • the region 111 that specifies the cell population by the description factor of the shape of the cell nucleus (main object) such as the area of the nucleus (Area) and the circularity factor is selected as the measurement target
  • noise was eliminated to improve the accuracy of the analysis data (E).
  • the gallery function (F) of the analysis software of the device is used to check the individual images of the cells in the region selected above, and an error such as recognizing the mixed foreign substance 112 or a plurality of cells as one cell. When recognition 113 grade
  • a sample for a flow cytometer is used to detect cell surface antigens of lymphocytes using a PC-5 labeled anti-human CD3 antibody (manufactured by Beckman Coulter) and an R-PE labeled anti-human CD56 antibody (manufactured by Beckman Coulter).
  • NK cells CD3 negative, CD56 positive
  • perforin positive cells in PBL was also analyzed.
  • FIG. 2 and FIG. As a result of analyzing a total of 5,008 PBLs by the method according to the present invention, there were 952 CD56-positive CD3-negative cells, that is, NK cells, and the ratio was 19.0%, and in these 952 NK cells The number of perforin positive cells was 899, and the ratio was 94.4% (FIG. 2 (A)). This result is the result of measuring the same sample with a conventional flow cytometer, that is, the ratio of NK cells is 19.3%, and the perforin positive ratio in the NK cells is 95.2% (FIG. 2 ( B)), and the method according to the present invention can identify and measure specific cells with the same accuracy as the conventional flow cytometer method even if a plurality of cells are mixed. It was confirmed that it was possible.
  • the measurement method according to the present invention stores the analysis data of each cell as an image image, so that each of the graphs in the dot plot analysis is one by one. It was possible to visually confirm whether the dots were actually cells.
  • FIG. 3 shows an example in which an image image corresponding to the arbitrarily selected dot (A) in the CD56 positive CD3 negative region is displayed on the display of (B).
  • (A) was confirmed to be CD56-positive NK cells that actually express perforin granules.
  • a foreign substance or the like is mixed for some reason at the time of sample preparation or measurement, it is difficult to distinguish noise caused by such a foreign substance from cells by the conventional method, and as a result, the noise is excluded during analysis.
  • FIG. 4 shows an example of the analysis.
  • the perforin granules in the original cell image shown in FIG. 4A are recognized as a plurality of spots by the spot detection function, and the total number of the spots is 17.
  • B NK cells in PBL were selected from the image data of the specimen, and the number of intracellular perforin positive granules was measured.
  • Example 2 Culture mixture analysis in mixed culture with K562 cells by healthy human PBL
  • a U-bottom 96-well microplate (Falcon 3077) previously seeded with 20,000 cells / well of K562 cells (human chronic myeloid leukemia cell line) was seeded with 400,000 cells / well of the above PBL. Both cells were co-cultured for a time.
  • the microplate was centrifuged at 50 ⁇ g for 10 minutes, immediately 4% paraformaldehyde was added, and the cells were fixed for 30 minutes. Thereafter, the fixed cells were divided in half, and one was used as a sample for measurement with the cell image analyzer according to the present invention, and the other was used as a sample for flow cytometry analysis, which is a conventional method.
  • NK cells with two antibodies, R-PE labeled anti-human CD56 antibody (manufactured by Beckman Coulter) and FITC-labeled anti-human CD3 antibody (manufactured by Beckman Coulter), or R-PE labeled anti-human CD56 antibody (manufactured by Beckman Coulter) alone Labeled.
  • Samples of NK cells labeled with the latter R-PE-labeled anti-human CD56 antibody alone consist of mouse anti-human perforin monoclonal antibody (manufactured by ANCELL) and Alexa Fluor (registered trademark) 488-labeled anti-mouse IgG antibody (manufactured by Invitrogen). ).
  • a rabbit anti-human active caspase 3 polyclonal antibody manufactured by TREVIGEN
  • Alexa Fluor registered trademark
  • NK cells CD3 negative, CD56 positive
  • PBL cell image analyzer
  • the ratio of NK cells (CD3 negative, CD56 positive) in PBL is analyzed using a cell image analyzer (OLYMPUS CELAVIEW RS100), the expression of perforin granules in NK cells, and The ratio of active caspase 3 positive cells in K562 cells was measured.
  • samples for measurement / analysis with a flow cytometer were subjected to treatment with PC-5 labeled anti-CD3 antibody and R-PE labeled anti-CD56 antibody in the same manner as in [Example 1] to identify NK cells.
  • active caspase 3 was stained using a rabbit anti-human active caspase 3 polyclonal antibody (manufactured by TREVIGEN) and a FITC-labeled anti-rabbit IgG antibody (manufactured by Beckman Coulter). Immediately using Epix XL (manufactured by Beckman Coulter), the ratio of NK cells (CD3 negative, CD56 positive) in PBL and the ratio of active caspase 3 positive cells in K562 cells were measured.
  • the ratio of CD56 positive and CD3 negative NK cells in PBL is almost the same as 8.7% in the measurement / analysis method according to the present invention and 8.6% in the measurement / analysis with the flow cytometer.
  • the ratio of active caspase 3 positive cells in K562 cells was 16.0% in the measurement / analysis method according to the present invention, and 16.9% in the measurement / analysis method using a flow cytometer. It was rate.
  • FIG. 5 shows an example of image data actually used for analysis in the measurement / analysis method according to the present invention, that is, an example of an image of a culture mixture of PBL and K562 after 4 hours of culture.
  • small cells such as lymphocytes
  • large K562 cells are mixed in the culture mixture, and CD56 molecules are expressed exclusively in the cell membrane of some small cells (NK cells).
  • NK cells some small cells
  • the expression of active caspase 3 was limited to the cytoplasm of some large cells (K562 cells). That is, in the measurement / analysis method according to the present invention, morphological observation using the acquired image data is possible, and the validity of the numerical data can be morphologically confirmed.
  • FIG. 6 shows another example of image data actually used in the analysis in Example 2.
  • NK cells expressing CD56 molecules were attached to K562 cells in which apoptosis occurred.
  • 47 active Caspase 3 positive K562 cells were confirmed, of which 13 active caspase 3 It was confirmed that NK cells were attached to positive K562 cells.
  • the number of NK cells adhering to the K562 cells was There were a total of 66, of which the total number of NK cells with perforin granules accumulating on the adhesion surface with K562 cells was 25, 38% of the total NK cells measured.
  • Example 4 Detection of MAGE-3-specific killer T cells in peripheral blood of cancer patients MAGE-3 peptide was administered to colon cancer patients (HLA-A2402) who were positive for tumor antigen MAGE by immunohistochemical staining of surgically excised specimens.
  • the peptide dendritic cell vaccine therapy used was performed. That is, 10 million to 100 million mature autologous dendritic cells previously treated with MAGE-3 peptide (IMPKAGLLI) are inoculated once a week into the skin near the inguinal lymph nodes of the patient. In vivo, MAGE-3-specific killer T cells were induced.
  • peripheral blood of the patient was collected, and the presence / absence of MAGE-3-specific killer T cells in the peripheral blood was analyzed by the measurement / analysis method according to the present invention.
  • the peripheral blood was used as a specimen, and MAGE-3-specific killer T cells in the peripheral blood were similarly analyzed using the conventional MHC tetramer method and ELISPOT method.
  • PBMCs are prepared from the collected peripheral blood by the same method as described above, and some PBMCs are immediately frozen and stored, and some PBMCs are obtained by magnetic microbeads (Miltenyi Biotech). CD14-positive cells or CD8-positive cells were collected and stored frozen, and then thawed and used as necessary.
  • the measurement / analysis according to the present invention was performed according to the following procedure. First, the cryopreserved CD14-positive cells are thawed, and then cultured in the presence of GM-CSF and IL-4 for 5 days according to a conventional method. Further, TNF ⁇ is added and cultured for 2 days to obtain mature dendritic cells. Induced. The mature dendritic cells cultured in AIM-V medium (manufactured by Invitrogen) containing 10 ⁇ g of synthetic MAGE-3 peptide (IMPKAGLLI) per mL were used as antigen-presenting cells.
  • AIM-V medium manufactured by Invitrogen
  • IMPKAGLLI synthetic MAGE-3 peptide
  • these antigen-presenting cells (20,000) and PBMC (200,000) separately thawed were suspended in AIM-V medium (manufactured by Invitrogen) containing 10% human AB serum at a final concentration.
  • AIM-V medium manufactured by Invitrogen
  • monensin A (manufactured by Sigma) was added to a final concentration of 2 ⁇ M, and the culture was further continued for 4 hours, for a total of 24 hours.
  • the cells were fixed with 4% paraformaldehyde for 30 minutes after centrifugation at 50 ⁇ g for 10 minutes, and after washing the fixed cells, the cells were all seeded in a flat-bottom 96-well microplate.
  • the microplate on which the cells were seeded was immediately centrifuged at 450 ⁇ g for 10 minutes and air-dried for 60 minutes to allow the cells to adhere to the plate. Thereafter, intracellular IFN ⁇ is stained with a mouse anti-human IFN ⁇ antibody (BD Farmingen), Alexa Fluor (registered trademark) 647-labeled anti-mouse IgG antibody (Invitrogen), and FITC-labeled anti-human CD3 antibody (Beckman).
  • Killer T cells were labeled using a Coulter) and R-PE labeled anti-human CD8 antibody (Beckman Coulter), and the cell nuclei were stained with DAPI. After staining, the ratio of IFN ⁇ -producing cells in CD3 and CD8 positive killer T cells was analyzed using a cell image analyzer (Olympus CELAVIEW RS100).
  • MHC tetramer method thawed PBMC is used as a material, and the MAGE-3 peptide MHC tetramer for FITC-labeled HLA-A2402 (manufactured by Proimmune) and the PC-5-labeled anti-human CD3 antibody (manufactured by Beckman Coulter) and PE-labeled anti-antibody Fluorescent labeling of PBMC using a human CD8 antibody (manufactured by Beckman Coulter) as usual, and immediately measuring the ratio of MAGE-3-specific killer T cells in the PBMC using Epix XL (manufactured by Beckman Coulter) Analyzed.
  • the ELISPOT method used IFN ⁇ ELISPOT SET (manufactured by BD) and AEC SUBSTRATE SET (manufactured by BD) to detect and measure the spot of IFN ⁇ produced from killer T cells by antigen stimulation according to the procedure described in the kit. Specifically, CD8-positive cells (100,000) that had been sorted and stored in advance and MAGE-3-labeled mature dendritic cells (10,000) prepared by the same procedure as described above were used as antigen-presenting cells. Samples co-cultured for 24 hours were used as specimens, and the number of detected IFN ⁇ spots was counted.
  • Table 2 summarizes the ratios of MAGE-3-specific killer T cells in peripheral blood measured by each test method.
  • the ratio of MAGE-3-specific killer T cells was similar, and was as low as 1% or less of killer T cells (CD8 + T cells).
  • the method according to the present invention enables more detailed data analysis than the conventional method.
  • FIG. 8 shows an example of data of the conventional MHC tetramer method (A) and ELISPOT method (B), but it is impossible to discriminate between cells and noise (nonspecific positive signal) by either method.
  • the presence or absence of false positive data could not be confirmed from either the dot plot in the MHC tetramer method (A) or the spot image in the ELISPOT method (B).
  • FIG. 9 shows an example of data analysis by the measurement method according to the present invention. 16 cells were detected in the range of IFN ⁇ positive cells on the histogram of (A), but images of individual cells (B) As a result, one was determined to be false positive data due to noise, not cells.
  • the present invention provides a new cell function measurement method for measuring details of functions of NK cells and antigen-specific killer T cells, a kit for the measurement, and a measurement system.
  • a new cell function measurement method for measuring details of functions of NK cells and antigen-specific killer T cells
  • a kit for the measurement for example, in cancer immune cell therapy
  • the function of an effector cell in an example of a patient is monitored in detail. It becomes possible to do.
  • it becomes possible to measure the details of the immune response of a subject which could not be analyzed by conventional NK activity, more easily and multifacetedly. It becomes possible to provide the subject with information. It is also an extremely useful assay tool when confirming the effectiveness of foods and drugs that regulate immune function.

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Abstract

L'invention concerne un nouveau procédé de mesure de la fonction de cellules qui surmonte les divers inconvénients et problèmes des procédés antérieurs de mesure de la fonction des cellules effectrices, un kit de mesure et un système de mesure pour la mesure concernée. Par la mesure/analyse des comptages de cellules effectrices, les propriétés et fonctions de celles-ci simultanément ou si nécessaire à l'aide d'un dispositif d'essai à base de cellules apte à une analyse d'images de cellules, cette invention permet d'obtenir des données hautement détaillées et fiables qui sont supérieures aux combinaisons des procédés antérieurs.
PCT/JP2009/054340 2008-03-07 2009-03-06 Procédé de mesure de la fonction de cellules effectrices, kit de mesure et système de mesure WO2009110614A1 (fr)

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Cited By (9)

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JP2011075278A (ja) * 2009-09-29 2011-04-14 Olympus Corp 細胞核を構成する構造体の解析方法、及び細胞核の形態の解析方法
WO2013146841A1 (fr) * 2012-03-30 2013-10-03 コニカミノルタ株式会社 Processeur d'image médical et programme
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WO2017169267A1 (fr) * 2016-03-30 2017-10-05 ソニー株式会社 Dispositif d'observation de cellules, procédé d'évaluation du niveau d'activité de cellules immunitaires, et procédé de régulation de la qualité de cellules immunitaires
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JPWO2017169267A1 (ja) * 2016-03-30 2019-03-14 ソニー株式会社 細胞観察装置、免疫細胞の活性度の評価方法及び免疫細胞の品質管理方法
JP7021633B2 (ja) 2016-03-30 2022-02-17 ソニーグループ株式会社 細胞観察装置及び免疫細胞の品質管理方法
US10690902B2 (en) 2017-10-25 2020-06-23 Olympus Corporation Image processing device and microscope system
WO2023103785A1 (fr) * 2021-12-06 2023-06-15 Wuxi Biologics (Shanghai) Co., Ltd. Bioessais 3d de mesure de cytotoxicité à médiation cellulaire dépendant d'anticorps

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