Secretome detection method and T cell labeling medium

Abstract

To provide a method for culturing a T-cell on a culture medium to which labeling amino acid is added, and detecting T-cell derived secretome labelled by the labeling amino acid, where a sufficient amount of T-cell derived secretome can be detected.SOLUTION: A method for detecting secretome includes: a labeling process of culturing a T-cell by T-cell labeling culture medium which contains labeling amino acid in which at least one kind of essential amino acid for T-cell proliferation is labelled instead of the amino acid, and contains a common γ-chain family cytokine; and a detection process of detecting T-cell derived secretome labelled by the labeling amino acid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a secretome detection method and a T cell labeling medium. More specifically, the present invention relates to a secretome detection method in which T cells are cultured in a medium containing a labeled amino acid and T cell-derived secretomes labeled with the labeled amino acid are detected, and a T cell labeling medium for use in the secretome detection method. [Background technology]

[0002] Cellular immunotherapy using cytotoxic T lymphocytes (CTLs) specific to the antigen associated with the disease is known as a new treatment method for diseases such as malignant tumors and chronic intractable infectious diseases. In recent years, cancer immunotherapy has been attracting attention as an effective cancer treatment method, in which T cells (unmodified T cells) are collected from a patient, genetically modified to enhance their ability to attack cancer, and then these genetically modified T cells are returned to the original patient to treat the cancer.

[0003] Furthermore, because T cells collected from patients have issues such as a tendency for cell exhaustion and difficulty in expansion culture (or expansion culture), development of cancer immunotherapy using T cells derived from iPS cells, which have no limit on the number of cells that can be used, is also underway. However, because the quality and state (e.g., cell proliferation ability) of iPS cell-derived T cells vary depending on the iPS cell line and culture conditions, the quality and state of the obtained T cells must be evaluated to select those suitable for the cancer immunotherapy. Furthermore, the quality and state of the obtained genetically modified T cells must also be evaluated to select those suitable as the desired genetically modified T cells.

[0004] As a method for evaluating the state of a cell, for example, there is a method for detecting and analyzing secretome, which is a protein secreted from a cell or exposed to the outside of the cell from within the cell membrane. A conventionally known method for analyzing secretome is, for example, a method for converting some atoms into deuterium (2 H), 13 C. 15 One example is the SILAC (Stable Isotope Labeling with Amino acids in Cell culture) method, in which cells to be analyzed are cultured in a medium containing isotope-labeled amino acids substituted with a pulsed stable isotope such as N, the isotope-labeled amino acids are incorporated into the cells to be analyzed, and then proteins (secretome) secreted or exposed extracellularly by the cells to be analyzed and labeled with the isotope-labeled amino acids are detected and analyzed (Non-Patent Document 1). Another known method uses amino acid analogs instead of the isotope-labeled amino acids, for example, by culturing cells to be analyzed in a medium containing L-azidohomoalanine (AHA), a structural analog of methionine, and then AHA is incorporated into the cells to be analyzed, and then AHA-labeled proteins (secretome) secreted or exposed extracellularly by the cells to be analyzed are detected and analyzed (Non-Patent Document 2).

[0005] In these methods for detecting and analyzing secretomes, in order to improve detection accuracy, it is necessary to prevent the incorporation of amino acids corresponding to the labeled amino acids, such as the isotope-labeled amino acids and amino acid analogs (e.g., methionine as the amino acid corresponding to AHA), into the cells to be analyzed. Therefore, the culture medium for cell labeling must be free of such amino acids, and typically, dialyzed serum or serum-free medium supplemented with the labeled amino acids is used. However, because delicate cell types such as primary T cells often require serum components, the dialyzed serum or serum-free medium makes it difficult for T cells to grow or survive, resulting in insufficient numbers of T cells, making it difficult to detect a sufficient amount of secretome for analysis. Furthermore, culturing T cells in the dialyzed serum or serum-free medium results in insufficient labeling with the labeled amino acids, making it difficult to accurately detect the secretome. Furthermore, some types of labeled amino acids used are toxic to T cells, which also makes it difficult for T cells to grow or survive, making it difficult to obtain a sufficient number of T cells, resulting in insufficient numbers of secretome for analysis. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Shao-En Ong et al., Molecular & Cellular Proteomics, 1.5, 2002, p.376-386 [Non-patent document 2] Katrin Eichelbaum et al., Nature Biotechnology, Vol. 30, No. 10, 2012, p. 984-990 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made in consideration of the problems associated with the above-mentioned prior art, and aims to provide a secretome detection method that can detect a sufficient amount of T cell-derived secretome in a method of culturing T cells in a medium supplemented with labeled amino acids and detecting T cell-derived secretome labeled with the labeled amino acids, and a T cell-labeling medium to be used therefor. [Means for solving the problem]

[0008] As a result of extensive research aimed at solving the above-mentioned problems, the present inventors have found that, in a method for culturing T cells in a medium supplemented with a labeled amino acid and detecting a T cell-derived secretome labeled with the labeled amino acid, by further adding a common γ chain family cytokine to the medium, cells having a secretome labeled with the labeled amino acid, i.e., cells that grow or survive sufficiently even in a medium containing dialyzed serum or a serum-free medium supplemented with the labeled amino acid, take up the labeled amino acid, and secrete or extracellularly expose the labeled amino acid-labeled secretome, can be obtained more efficiently than in the past. Therefore, the present inventors have found that this method makes it possible to detect a sufficient amount of T cell-derived secretome, thereby enabling more accurate analysis of the secretome, and have completed the present invention.

[0009] That is, the aspects of the present invention are as follows.

[0010] [1] A labeling step of culturing T cells in a T cell labeling medium containing a labeled amino acid in place of at least one essential amino acid for T cell proliferation, and containing a common gamma chain family cytokine; a detection step of detecting the T cell-derived secretome labeled with the labeled amino acid; A secretome detection method comprising:

[0011] [2] The secretome detection method described in [1], wherein the common gamma chain family cytokine is at least one selected from the group consisting of interleukin 2, interleukin 4, interleukin 7, interleukin 9, interleukin 15, and interleukin 21.

[0012] [3] The secretome detection method according to [1] or [2], wherein the content of the common γ chain family cytokine in the T cell labeling medium is 5 to 100 ng / mL.

[0013] [4] The secretome detection method according to any one of [1] to [3], wherein the essential amino acid for T cell proliferation is methionine and the labeled amino acid is L-azidohomoalanine.

[0014] [5] The secretome detection method according to any one of [1] to [4], wherein the content of the labeled amino acid in the T cell labeling medium is 0.001 to 0.02 mmol / L.

[0015] [6] The secretome detection method according to any one of [1] to [5], further comprising a stimulation step of stimulating T cells prior to the labeling step.

[0016] [7] A T cell labeling medium for use in the secretome detection method described in any one of [1] to [6], which contains a labeled amino acid in place of at least one essential amino acid for T cell proliferation, and also contains a common gamma chain family cytokine.

[0017] [8] The T cell labeling medium described in [7], wherein the common gamma chain family cytokine is at least one selected from the group consisting of interleukin 2, interleukin 4, interleukin 7, interleukin 9, interleukin 15, and interleukin 21.

[0018] [9] The T cell labeling medium according to [7] or [8], wherein the content of the common γ chain family cytokine is 5 to 100 ng / mL.

[0019]

[10] The T cell labeling medium according to any one of [7] to [9], wherein the essential amino acid for T cell proliferation is methionine and the labeled amino acid is L-azidohomoalanine.

[0020]

[11] The T cell labeling medium according to any one of [7] to

[10] , wherein the content of the labeled amino acid is 0.001 to 0.02 mmol / L. [Effects of the Invention]

[0021] According to the present invention, it is possible to provide a secretome detection method that can detect a sufficient amount of T cell-derived secretome in a method of culturing T cells in a medium supplemented with labeled amino acids and detecting T cell-derived secretome labeled with the labeled amino acids, and a T cell-labeling medium to be used therefor. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention will be described in detail below based on preferred embodiments thereof.

[0023] The secretome detection method of the present invention comprises: a labeling step of culturing T cells in a T cell labeling medium containing a labeled amino acid in place of at least one essential amino acid for T cell proliferation, and containing a common gamma chain family cytokine; a detection step of detecting the T cell-derived secretome labeled with the labeled amino acid; It includes:

[0024] (Secretome) In the present invention, "secretome (or "T cell-derived secretome")" refers to proteins secreted from T cells or exposed extracellularly from within the cell membrane, and also includes secretory proteins present within cells. Such proteins are not particularly limited, and examples include proteins secreted by T cells, proteins expressed on the surface of T cells, proteins released extracellularly from T cells, proteins retained within T cells, membrane protein fragments, and proteins present in or exposed within exosomes.

[0025] The secretome detected in the present invention may be any one of these or a combination of two or more of them. The following labeled amino acids and their detection methods can be selected depending on the type of secretome of interest.

[0026] (T cells) In the present invention, the origin of the T cells is not particularly limited, and they may be T cells derived from healthy individuals or patients (for example, individuals with weakened immune functions or individuals suffering from malignant tumors, infectious diseases, or autoimmune diseases). Furthermore, the T cells may be T cells induced to differentiate from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells); T cells induced to differentiate from somatic stem cells such as hematopoietic stem cells; or established T cell lines.

[0027] In the case of T cells collected from a human, for example, T cells can be isolated from peripheral blood mononuclear cells (PBMCs) separated from peripheral blood using a conventionally known method or a commercially available kit. Furthermore, the T cells of the present invention may be naive T cells, antigen-stimulated memory T cells, or effector T cells, and the effector T cells may be any of helper T cells, killer T cells, regulatory T cells, and natural killer T (NK / T) cells.

[0028] Furthermore, in the present invention, "T cells" include not only T cells that have not been artificially genetically modified (hereinafter sometimes referred to as "unmodified T cells"), but also "genetically modified T cells" that have been artificially genetically modified from the unmodified T cells. In the present invention, "artificial genetic modification" includes modifying the function of T cells by introducing a gene into the T cells or by gene editing of the T cells. The artificial genetic modification may be a modification of the gene itself that the T cells possess, a deletion of the gene itself that the T cells possess, a modification in which an exogenous gene is introduced, or a combination of two or more of these.

[0029] The artificial gene modification method includes, but is not limited to, conventionally known methods and methods based thereon. Examples of gene transfer into T cells include the introduction of a gene that modifies T cell function (DNA, mRNA, miRNA, antagomir, ODN, etc.) or a vector into which the gene has been inserted (lentivirus vector, γ- or α-retrovirus vector, adenovirus vector, adeno-associated virus vector, herpesvirus vector, equine encephalopathy virus vector, etc.). Examples of gene editing of T cells include editing of T cell genes (genome editing) using site-specific nucleases (meganuclease, zinc finger nuclease, TALEN, PPR, CRISPR-Cas, etc.). In the present invention, the artificial gene modification method may be one of these methods alone or a combination of two or more of them.

[0030] (T cell labeling medium) In the present invention, the term "T cell labeling medium" refers to a medium containing at least a labeled amino acid for culturing and labeling T cells. The T cell labeling medium of the present invention contains a labeled amino acid in place of at least one amino acid essential for T cell proliferation, in which the amino acid is labeled, and also contains a common γ chain family cytokine. The T cell labeling medium is a medium for labeling T cell-derived secretomes with at least the labeled amino acid, but may also be used for proliferation culture (or expansion culture), T cell stimulation, T cell gene modification (viral vector infection), cell isolation, and cell establishment.

[0031] In the present invention, "essential amino acids for T cell proliferation (sometimes simply referred to as "essential amino acids" in the present invention)" refer to amino acids that are essential for T cell proliferation, and more specifically, are valine, isoleucine, leucine, methionine, lysine, phenylalanine, tryptophan, threonine, and histidine.

[0032] The T cell labeling medium of the present invention contains a labeled amino acid in place of at least one of the essential amino acids, i.e., does not contain at least one of the essential amino acids, and the non-contained essential amino acid contains a labeled amino acid. In the present invention, "does not contain an essential amino acid" means that the T cell labeling medium is substantially free of the essential amino acid, and more specifically, the content of the essential amino acid in the T cell labeling medium is 1 x 10 -3 mmol / L or less, 1×10 -4 The essential amino acids excluded from the T cell labeling medium of the present invention (i.e., not substantially contained in the T cell labeling medium) may be any one of the above or a combination of two or more thereof, but among these, methionine is preferred from the viewpoint of efficient incorporation into the protein synthesis pathway.

[0033] In the present invention, the term "labeled amino acid" refers to an essential amino acid that has been partially modified while maintaining its structure, and that can be detected based on the modification. The term "detectable" includes not only direct visual confirmation by coloration (color development), quenching, reflected light, luminescence, fluorescence, etc., but also confirmation by a specific measurement method or device.

[0034] Such labeled amino acids include, for example, amino acids in which some atoms are pulsed with stable isotopes (e.g., 2 H, 13 C. 15 N); structural analogs of amino acids in which a part of the amino acid has been modified with a modifying group such as an azide group or an alkyne group, and any one of these may be used alone or in combination of two or more types corresponding to the essential amino acids.

[0035] For example, when the T cell labeling medium does not contain methionine among the essential amino acids, the labeled amino acid contained in the T cell labeling medium instead of methionine is, for example, isotope-labeled methionine in which some of the atoms constituting methionine are substituted with the stable isotope. 2 H-labeled methionine, 13 C-labeled methionine, 15 N-labeled methionine includes structural analogues of methionine, such as L-azidohomoalanine (AHA), L-homopropargylglycine (HPG), and L-homoallylglycine (HAG), and may be any one of these or a combination of two or more of them.

[0036] The content of the labeled amino acid in the T cell labeling medium (when there are two or more labeled amino acids, the total content of those amino acids; the same applies hereinafter) can be appropriately determined taking into consideration the T cell uptake efficiency, culture efficiency, toxicity to the T cells, etc., and is not particularly limited, but may be, for example, in the range of 0.0001 to 0.1 mmol / L, preferably 0.001 to 0.05 mmol / L, and more preferably 0.001 to 0.02 mmol / L. More specifically, for example, when the labeled amino acid is AHA, the content is preferably 0.0005 to 0.03 mmol / L, more preferably 0.001 to 0.03 mmol / L, even more preferably 0.002 to 0.03 mmol / L, and even more preferably 0.005 to 0.02 mmol / L. If the content of the labeled amino acid exceeds the upper limit, the cells tend to weaken or die, whereas if it is below the lower limit, the number of cells that secrete or extracellularly expose the secretome labeled with the labeled amino acid decreases, and the accuracy of secretome analysis tends to decrease.

[0037] The T cell labeling medium of the present invention further contains a common γ chain family cytokine, which makes it possible to obtain cells expressing a secretome labeled with the labeled amino acid, i.e., cells that grow or survive in the T cell labeling medium, take up the labeled amino acid, and secrete or extracellularly expose the secretome labeled with the labeled amino acid, more efficiently than ever before.

[0038] In the present invention, the term "common γ chain family cytokine" refers to a cytokine that acts via a receptor containing a common γ chain (γ subunit). More specifically, examples include interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 7 (IL-7), interleukin 9 (IL-9), interleukin 15 (IL-15), and interleukin 21 (IL-21) (T cell culture method using interleukin 21: International Publication WO 2018 / 135646). Any one of these cytokines may be used alone, or a combination of two or more of these cytokines may be used. Among these cytokines, interleukin 7, interleukin 15, interleukin 21, and a combination of two or more of these cytokines are more preferred from the viewpoint of maintaining cell survival and proliferation.

[0039] In the T cell labeling medium, the content of the common γ chain family cytokine (when there are two or more common γ chain family cytokines, the total content of those cytokines; the same applies hereinafter) can be appropriately determined taking into consideration the properties of the T cells, etc., and is not limited thereto, but is preferably 1 to 1000 ng / mL, more preferably 5 to 100 ng / mL, and even more preferably 5 to 20 ng / mL. If the content of the common γ chain family cytokine exceeds the upper limit, the effect of including the common γ chain family cytokine tends to be reduced or cell proliferation tends to be inhibited. On the other hand, if the content is less than the lower limit, the number of cells that secrete or extracellularly expose secretome labeled with the labeled amino acid tends to be reduced, and the accuracy of secretome analysis tends to be reduced.

[0040] In order to remove the essential amino acids, the T cell labeling medium of the present invention preferably does not contain serum, or if it does contain serum, it is preferably dialyzed serum from which the essential amino acids have been removed. Furthermore, the T cell labeling medium of the present invention may also contain other additives for cell protection, such as serum albumin and ITS; scaffolding agents such as ECM components; etc. These T cell labeling media or their basal media are not particularly limited as long as they do not contain the essential amino acids of interest, and include basal media such as DMEM, RPMI, αMEM, etc.; and AIM V, a serum-free medium specifically for T cells. TM Culture medium: A known or commercially available culture medium for T cells, such as PRIME-XV T cell Expansion XSFM, can be appropriately used.

[0041] (labeling process) In the labeling step according to the present invention, T cells are cultured in the T cell labeling medium, thereby allowing the T cells to incorporate the labeled amino acid and secrete or extracellularly expose a secretome labeled with the labeled amino acid (T cell-derived secretome).

[0042] The culture method in the labeling step is not particularly limited, and known T cell growth culture conditions can be appropriately adopted, and can be adjusted appropriately taking into account the properties and concentration of the T cells, etc., but examples of such conditions include a culture temperature of 35 to 37.5°C, preferably 36 to 37°C, and a culture time of 2 to 48 hours, preferably 8 to 24 hours. The culture device in the labeling step is not particularly limited, and examples include plates, dishes, columns, flasks, culture bags, etc., and may further include a supply means for supplying the T cell labeling medium, etc., and a discharge means for discharging the medium (sensor, valve, pump, tank, etc.).

[0043] The T cells subjected to the labeling step of the present invention are not particularly limited and may be naive T cells or effector T cells. They may also be T cells stimulated by contact with an antigen or T cells in a stationary state (i.e., unactivated). The effector T cells can be obtained by a stimulation step in which naive T cells (T cells that have never encountered an antigen) are stimulated by contact with an antigen. In the stimulation step and the above-described method for stimulating T cells, the substance that activates T cells can be appropriately selected depending on the purpose of analysis, and includes, but is not limited to, substances that stimulate the T cell receptor (TCR) of T cells (e.g., CD3 / CD28 beads, OKT3, X-ray-irradiated PBMC, PHA), and the like. These substances may be used alone or in combination of two or more. Furthermore, the stimulation step and the above-described method for stimulating T cells are preferably performed in the presence of cytokines. The cytokine can also be appropriately selected depending on the purpose of analysis, etc., and is not particularly limited, and examples thereof include IL-12, IL-6, IL-21, IL-7, IL-15, and IL-2, and may be any one of these or a combination of two or more of these.

[0044] (Detection process) In the detection step according to the present invention, T cell-derived secretome that has been labeled with the labeled amino acid and secreted or exposed to the extracellular space is detected. In the present invention, "detection" includes not only detecting the presence or absence of the T cell-derived secretome, but also quantitating or semi-quantitating the amount of the T cell-derived secretome. The detection step according to the present invention may be performed simultaneously (in real time) with the labeling step.

[0045] The detection method used in the detection step can be appropriately selected depending on the type of labeled amino acid. Such a detection method is not particularly limited, and known methods or methods based thereon can be appropriately selected. When the labeled amino acid is an isotope-labeled amino acid or a structural analogue of an amino acid, the secretome labeled with the labeled amino acid can be detected by, for example, performing mass spectrometry such as gas isotope ratio mass spectrometry, isotope ratio infrared spectrometry, or liquid chromatography mass spectrometry (LC-MS / MS) on the medium after or during the labeling step (i.e., during the culture).

[0046] Furthermore, when the labeled amino acid is a structural analog of an amino acid, it can be detected by staining the modified group specifically. For example, when the modified group is an azide group (e.g., AHA), the azide group can be detected by Click-iT TM The secretome can be stained with a fluorescent reagent such as Alexa Fluor 488 sDIBO Alkyne (Thermo Fisher Scientific), and can be detected using a fluorescence microscope, flow cytometry, imaging cytometry, etc. to detect the secretome labeled with the labeled amino acid.

[0047] The detection results of the T cell-derived secretome labeled with the labeled amino acids can be used for the analysis of the T cell-derived secretome. For example, it is possible to identify and quantify individual secretomes by combining multiple labeled amino acids or by mass spectrometry. [Example]

[0048] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

[0049] (Preparation Example 1) Acquisition and expansion of T cells (1) Human peripheral blood mononuclear cells (hPBMC, Lonza) suspension 1 x 10 7Mitomycin C (MMC, Fujifilm Wako Pure Chemical Industries, Ltd.) was added at 10 μg per 1 mL of the suspension, and the cells were cultured at 37° C. in 5% CO 2 for 3 hours.

[0050] (2) (1) was washed twice with phosphate-buffered saline (PBS(-)) containing no calcium ions or magnesium ions, suspended in 2 mL of α-modified Eagle's minimum essential medium (αMEM, manufactured by Life Technologies) supplemented with 15% (w / v) fetal bovine serum (FBS, manufactured by BioWest), and then seeded onto a 6-well plate and cultured for 6 hours at 37°C and 5% CO2.

[0051] (3) CD8 + T cells were extracted using a T cell isolation kit (Miltenyi Biotec).

[0052] (4) The T cells extracted in (3) above were suspended in growth medium (αMEM supplemented with 15% (w / v) FBS, 5 ng / mL interleukin-7, and 5 ng / mL interleukin-15), and then plated at 1 × 10 5 Cells were seeded at 1000 cells / well.

[0053] (5) 5 × 10 MMC-treated hPBMCs (as described in (2) above) were used as feeder cells. 7 After adding 1000 cells / well to each well of (4) above, 2 μg / mL phytohemagglutinin-P (Fujifilm Wako Pure Chemical Industries, Ltd.) and 10 μmol / L Z-VAD-fmk (R&D Systems), a pan-caspase inhibitor, were added, and the cells were cultured for 2 weeks at 37°C and 5% CO2, with appropriate medium changes in the growth medium (culture method using Z-VAD-fmk: International Publication WO2018 / 135646). The cultured cells were cryopreserved until immediately before use.

[0054] (Examples 1 to 5, Comparative Example 1) Labeling of T cell-derived secretome (1) Cryopreserved T cells were thawed and stimulated using Dynabeads Human T-Activator CD3 / CD28 for T Cell Expansion and Activation (Thermo Fisher Scientific). The T cells were then cultured for 16 days at 37°C in 5% CO with the aforementioned growth medium, with appropriate medium changes.

[0055] (2) The T cells cultured after stimulation in (1) above were washed twice with 2 mL of PBS(-), suspended in 2 mL of medium A listed in Table 1 below, and plated in a 6-well plate at 2 × 10 5 The cells were seeded at 1000 cells / well and cultured at 37°C and 5% CO2 for 1 hour.

[0056] (3) After the culture in (2) above, the medium was replaced with medium B (medium for labeling T cells) listed in Table 1 below, containing L-azidohomoalanine (AHA, manufactured by AnaSpec Inc.) at various concentrations (0 mmol / L (Comparative Example 1), 0.000125 mmol / L (Example 1), 0.000625 mmol / L (Example 2), 0.00125 mmol / L (Example 3), 0.00625 mmol / L (Example 4), 0.0125 mmol / L (Example 5)), and the cells were cultured for one day under conditions of 37°C and 5% CO2.

[0057] [Table 1]

[0058] The descriptions in Table 1 indicate the following: αMEM(-Met): α-modified Eagle's minimum essential medium (without L-methionine), manufactured by Cosmo Bio Co., Ltd.; Dialyzed FBS: BI Biological Industries; ITS-G: Insulin-Transferrin-Selenium, Thermo Fisher Scientific; PAA: L-ascorbic acid, manufactured by Nacalai Tesque; IL-7: Recombinant human interleukin 7, Peprotech; IL-15: recombinant human interleukin-15, Peprotech; AHA: L-azidohomoalanine, manufactured by AnaSpec Inc.

[0059] (4) After culturing as described in (3), the cells were washed twice with 2 mL of PBS(-) containing 1% (w / v) FBS. After washing, each well was added with 30 μM Click-iT TM Alexa Fluor 488 sDIBO Alkyne (Thermo Fisher Scientific) and PBS(-) containing 1% (w / v) FBS were added, and the mixture was allowed to react for 1 hour under conditions of 37°C and 5% CO2.

[0060] (5) After the reaction in (4) above, the wells were washed four times with 2 mL of PBS(-) containing 1% (w / v) FBS. After washing, 2 mL of 4% (w / v) formalin was added to each well and allowed to act for 15 minutes to fix the T cells.

[0061] (6) Each well in which the T cells were fixed in (5) above was washed twice with 2 mL of PBS(-), and then 2 mL of PBS(-) was added. The AHA labeling rate of the T cells was analyzed by flow cytometry (Guava easyCyte, manufactured by Luminex). The labeling rate was calculated using the following formula: Labeling rate (%) = number of AHA-labeled T cells / number of cells counted by flow cytometry (5,000 cells) × 100 The results are shown in Table 2 below.

[0062] [Table 2]

[0063] As shown in Table 2, AHA labeling using medium B (T cell labeling medium) resulted in the detection of many cells with labeled secretomes (Examples 1 to 5). In particular, in Examples 4 and 5, where the AHA concentration was 0.00625 to 0.0125 mmol / L, the labeling rate was particularly high at 85% or more. [Industrial Applicability]

[0064] According to the present invention, it is possible to provide a secretome detection method that can detect a sufficient amount of T cell-derived secretome in a method of culturing T cells in a medium supplemented with labeled amino acids and detecting T cell-derived secretome labeled with the labeled amino acids, and a T cell-labeling medium to be used therefor.

Claims

1. a labeling step of culturing T cells in a T cell labeling medium containing a labeled amino acid in place of at least one essential amino acid for T cell proliferation, and containing a common γ chain family cytokine; a detection step of detecting the T cell-derived secretome labeled with the labeled amino acid; Including, The secretome detection method, wherein the essential amino acid for T cell proliferation is methionine and the labeled amino acid is L-azidohomoalanine.

2. The secretome detection method according to claim 1, wherein the common gamma chain family cytokine is at least one selected from the group consisting of interleukin 2, interleukin 4, interleukin 7, interleukin 9, interleukin 15, and interleukin 21.

3. The secretome detection method according to claim 1 or 2, wherein the content of the common gamma chain family cytokine in the T cell labeling medium is 5 to 100 ng / mL.

4. The secretome detection method according to any one of claims 1 to 3, wherein the content of the labeled amino acid in the T cell labeling medium is 0.001 to 0.02 mmol / L.

5. The secretome detection method according to any one of claims 1 to 4, further comprising a stimulation step of stimulating T cells prior to the labeling step.

6. A T cell labeling medium for use in the secretome detection method according to any one of claims 1 to 5, comprising a labeled amino acid in place of at least one essential amino acid for T cell proliferation, wherein the amino acid is labeled, and the T cell labeling medium also comprises a common gamma chain family cytokine, wherein the essential amino acid for T cell proliferation is methionine, and the labeled amino acid is L-azidohomoalanine.

7. 7. The T cell labeling medium according to claim 6, wherein the common gamma chain family cytokine is at least one selected from the group consisting of interleukin 2, interleukin 4, interleukin 7, interleukin 9, interleukin 15, and interleukin 21.

8. 8. The T cell labeling medium according to claim 6, wherein the content of the common gamma chain family cytokine is 5 to 100 ng / mL.

9. The T cell labeling medium according to any one of claims 6 to 8, wherein the content of the labeled amino acid is 0.001 to 0.02 mmol / L.

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